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dissertation entitled

IDENTIFICATION AND CHARACTERIZATION OF TOMATO
(SOLANUM LYCOPERSICUM) PROTEINS INVOLVED IN
RESISTANCE TO INSECT HERVIBORES

presented by

ELIANA GONZALES-VIGIL

has been accepted towards fulfillment
of the requirements for the

Doctoral degree in Genetics

 

 

 

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5108 K:/Proj/Aoc&Pres/CIRC/DateDuo.indd

IDENTIFICATION AND CHARACTERIZATION OF TOMATO (SOLAN UM
LYCOPERSIC UM) PROTEINS INVOLVED IN RESISTANCE TO INSECT
HERVIBORES

By

Eliana Gonzales-Vigil

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Genetics

2009

ABSTRACT

IDENTIFICATION AND CHARACTERIZATION OF TOMATO (SOLAN UM
LYCOPERSIC UM) PROTEINS INVOLVED IN RESISTANCE TO INSECT
HERBIVORES

By

Eliana Gonzales-Vigil

In response to wounding or herbivore attack, plants synthesize proteins that
negatively affect the growth and development of arthropod herbivores. Many of these
proteins are induced in plant tissue in response to herbivory and, following ingestion by
the herbivore, target processes involved in insect digestive physiology. The objective of
this thesis research is to identify and characterize plant proteins that impair the ability of
insect herbivores to obtain nutrients from host tissue. To address this objective, liquid
chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify proteins
in tomato (Solanum lycopersicum L.) that are excreted in the insect feces (frass). This
approach is based on the premise that plant anti-insect proteins are stable during passage
of food through the insect digestive system, and therefore enriched in the frass. The
results establish the utility of insect feces as a source of material for proteomic-based
discovery of defensive proteins that target insect digestive processes. Comparative
proteomic analysis of frass from three tomato—reared insect species, including
lepidopteran (Manduca sexta and Trichoplusia ni) and coleopteran (Leptinotarsa
decemlineata) herbivores, provided evidence that the lepidopteran insects digest bulk

tomato leaf protein more efficiently than the coleopteran insect. This study also identified

a subset of tomato leaf proteins that are highly stable in the digestive tract of all three
insect species. Including in this subset were proteins previously shown to have a role in
defense against insect attack. These findings are consistent with the hypothesis that plant
anti-insect proteins are inherently stable in the insect digestive track.

One of the most abundant tomato proteins excreted in the frass from all three
insects was a jasmonate-inducible isoform of threonine deaminase (TD2) that converts
threonine (Thr) to a-ketobutyrate and ammonium. TD2 and other plant TDs contain a C-
terminal regulatory domain that, upon binding isoleucine (Ile), feedback inhibits the N-
terminal catalytic domain. Following ingestion of tomato foliage by lepidopteran insects,
the regulatory domain of TD2 is removed by a chymotrypsin—Iike protease of insect
origin. This processed form of TD2 efficiently degrades Thr in the presence of Ile,
thereby starving the insect of an essential nutrient. The increased growth rate of
Spodoptera exigua larvae on transgenic tomato lines silenced for TD2 expression showed
that this enzyme serves a role in anti-insect defense. Tomato contains a second TD
isoform (TDl) that catalyzes the committed step in the biosynthesis of Ile. Based on the
comparison of the expression pattern and biochemical properties of TDI and TD2, it is
concluded that the two TD isoforms have evolved specialized functions in plant primary

metabolism and anti-insect defense, respectively.

ACKNOWLEDGEMENTS

Many people have contributed one way or another during the course and completion of
this thesis:

Gregg Howe let me join his lab and treat my scoleciphobia (fear of worms). I thought I
wouldn’t have to deal much with insects in the Howe lab. However, I had to rear, touch,
dissect, collect poop and spit from caterpillars throughout my research. Anyway, I think
having such a nice project was worth the high price, and I thank Gregg for that and for
his guidance and support all these years. I have learnt so much from him.

Barb Sears accepted my late application for the Genetics Program, led the writing group,
served as a member on my committee when it was needed, and shared her passion for her
job —something that I wish I could always have.

My guidance committee: Ke Dong and Sheng Yang He listened carefully to my updates
and provided useful comments. Rob Last gave me advice on so many things from
experiments to postdoc positions, and most importantly, he read this document in its
entirety!

A special thanks to the MSU Proteomics Core: Curtis Wilkerson and Doug Whitten let
me inject insect frass in their expensive LTQ-FTICR; and Kevin Carr handled the
bioinforrnatics that made dealing with the data so much easier.

The people in the lab made the Howe lab a great place to work in. Hoo Sun Chung, was a
fiend when I needed it most; Marco Herde, my shitomics partner helped me make this
thesis better through his discussions; I in Ho Kang, the trichome king kept the Genetics

discussions alive; Abe Koo, the senior lab member kept us all on track; Leron Katsir, the

iv

not-so-neat grad student made lab meetings so entertaining; and Chris Bergum, the
undergraduate student who was able to cope with me and still like his job, provided his
technical skills that kept this research going while I was stuck on a computer. Also I want
to thanks the new Howe lab generation, Christine Shyu, Javier Moreno, Lalita Patel and
Tom Cooke; and the former lab members: Hui Chen, Tony Schilmiller, Bonnie McCaig
and Guanghui Liu.

I also received assistance from Rob Larkin and his lab during the protein purification; and
from Nora Bello in the statistical analysis of the performance assays.

My family, and specially my mom, was always supportive even when they had no idea
what exactly I was doing. So many friends made Michigan the lively place that I never
thought it could be. And last but not least, the one that was behind the scenes supporting,
cheering me up, cooking, changing diapers and keeping me a sane person, my loving
husband Erick. This wouldn’t have been possible without you by my side, and now with
our Luciana.

Thanks you all for making these years of fiass collection unforgettable.

 

TABLE OF CONTENTS

 

 

LIST OF TABLES ix
LIST OF FIGURES X
CHAPTER 1
Plant Defenses to Insect Herbivory ...................................................................................... 1
Introduction ....................................................................................................................... 1
Sounding the alarm: the jasmonate pathway .................................................................... 3
Requesting backup: plant volatiles as indirect defenses ................................................... 5
Close combat fighting: defensive compounds that directly target insect herbivores ........ 6
Secondary metabolites with direct roles in plant defense ................................................. 7
Plant anti-insect proteins ................................................................................................... 9
Eliminate the enemy: Toxic proteins ........................................................................... 10
Lectins ....................................................................................................................... 10
Cysteine proteases ..................................................................................................... l 1
Cyclotides ................................................................................................................. 12
Canatoxin .................................................................................................................. 13
Neutralizing the enemy: Anti-nutritional proteins ....................................................... 14
Protease Inhibitors .................................................................................................... 14
Polyphenol oxidase ................................................................................................... 15
Amino acid degrading enzymes ................................................................................ 16
Vegetative Storage Protein ....................................................................................... 19
Plants exploit insect proteases for the activation of defense ........................................ 20
Thesis rationale and overview ........................................................................................ 21
Literature cited ................................................................................................................ 28
CHAPTER 11
Stability of plant defense proteins in the gut of insect herbivores ..................................... 42
Abstract ........................................................................................................................... 43
Introduction ..................................................................................................................... 44
Results ............................................................................................................................. 48
Tomato has two TD genes that are differentially expressed ........................................ 48
Digestion of bulk tomato leaf protein in the gut lumen of M. sexta larvae ................. 50
Proteolytic processing of TD2 in the lepidopteran gut ................................................ 51
Biochemical properties of pTD2 .................................................................................. 53
Identification of plant defensive proteins by shotgun proteomic analysis of insect
frass .............................................................................................................................. 53
Discussion ....................................................................................................................... 56
vi

Functional diversification of two TD isoforms in tomato ........................................... 56

Proteolytic processing of TD2 ..................................................................................... 58
Shotgun proteomic analysis of insect frass .................................................................. 60
Materials and Methods .................................................................................................... 64
Biological material and growth conditions .................................................................. 64
Protein extraction and enzyme assays .......................................................................... 65
Purification of pTD2 from M. sexta frass .................................................................... 66
Cloning and expression analysis of SlTDl and SlTD2 ............................................... 68
Antibody production and western-blot analysis .................................................. 69
LC-MS/MS-based identification of tomato proteins in M. sexta frass ........................ 71
Accession Numbers ..................................................................................................... 73
Acknowledgements ......................................................................................................... 73
Literature Cited ............................................................................................................... 85
CHAPTER III
The defensive function of tomato threonine deaminase 2 is activated by an inset digestive
protease .............................................................................................................................. 93
Abstract ........................................................................................................................... 93
Introduction ..................................................................................................................... 94
Materials and Methods .................................................................................................... 97
Plant material and transformations .............................................................................. 97
Insect rearing and bioassays ......................................................................................... 98
Expression of TDI and TD2 in E. coli ........................................................................ 99
TD enzyme assays ...................................................................................................... 100
TD2 cleavage assays .................................................................................................. 101
Protease fractionation ................................................................................................. 101
Results ........................................................................................................................... 102
Silencing of TD2 expression improves S. exigua performance on tomato ................ 102
Proteolytic cleavage of TD2 by a lepidopteran protease ........................................... 103
TD2 is cleaved by a chymotrypsin-like protease ....................................................... 105
TD] and TD2 exhibit differential stability to insect digestive proteases ................... 106
pH optimum and temperature stability of TDI and TD2 ........................................... 107
Kinetic parameters of TD2 and TD] ......................................................................... 108
Discussion ..................................................................................................................... 109
Defensive function of tomato TD2 ............................................................................ 109
Biodegradative TD in tomato ..................................................................................... 110
Evolution of TD2 ....................................................................................................... 111
Acknowledgements ....................................................................................................... 116
Literature Cited ............................................................................................................. 129
CHAPTER IV
Differential digestion of the Solanum chopersicum leaf proteome by lepidopteran and
coleopteran insect herbivores ........................................................................................... 133
Abstract ......................................................................................................................... 133

vii

Introduction ................................................................................................................... 134

Results ........................................................................................................................... 138
Performance of Leptinotarsa decemlineata and T richoplusia ni on tomato is reduced
by COIl-dependent defenses ..................................................................................... 138
Fate of the tomato leaf proteome in the digestive tract of L. decemlineata ............... 139
Effect of insect gut environment on digestion of tomato leaf proteins ...................... 141
Differential digestion of TD2 in lepidopteran and coleopteran herbivores ............... 143
Stability of JA-inducible proteins in the insect gut .................................................... 144
Discussion ..................................................................................................................... 146
Adaptability of L. decemlineata to tomato defenses .................................................. 146
Keys to the success of L. decemlineata on tomato .................................................... 147
Degradation of Rubisco during herbivory ................................................................. 148
TD2 and other JIPs ..................................................................................................... 150
Trends of stable proteins ............................................................................................ 151
Oxidative stress in the gut .......................................................................................... 152
Plant proteases and protease inhibitors ...................................................................... 153
Materials and Methods .................................................................................................. 156
Plant material and growth conditions ........................................................................ 156
Insect feeding bioassays ............................................................................................. 156
F rass collection, protein extraction, and mass spectrometry ..................................... 157
Identification of tomato proteins by LC-MS/MS ....................................................... 160
Microarray analysis .................................................................................................... 161
Acknowledgements ....................................................................................................... 161
Literature Cited ............................................................................................................. 181
CHAPTER V
Conclusions and Future Perspectives ............................................................................... 191
Literature Cited ............................................................................................................. 198
APPENDICES
Appremdix 1: Protein report ........................................................................................... 199

Appremdix 2: List of cDNA rnicroarray clones that were differentially regulated 23-fold...227

viii

LIST OF TABLES

Table 2.1. Host plant proteins identified in frass of tomato-reared M. sexta larvae ......... 74
Table 4.1. Common proteins identified in the three frass samples ................................. 162
Table 4.2. Abundance of JA-regulated proteins .............................................................. 164
Appendix 1. Protein report .............................................................................................. 199

Appendix 2. List of cDNA microarray clones that were differentially regulated 23-fold
.......................................................................................................................................... 227

ix

LIST OF FIGURES

Figure 1.1. Mode of action of toxic plant proteins ............................................................ 24
Figure 1.2. Mode of action of anti-nutrional plant proteins .............................................. 26
Figure 2.1. Mode of action of anti-nutrional plant proteins .............................................. 75
Figure 2.2. Differential expression of S! T D] and SIT D2 in tomato .................................. 77

Figure 2.3. Digestion of tomato foliar protein during passage through the M. sexta
digestive tract ..................................................................................................................... 79

Figure 2.4. Proteolytic processing of TD2 in the digestive tract of M sexta and T. ni

larvae .................................................................................................................................. 80
Figure 2.5. Purification of pTD2 from M. sexta frass ....................................................... 82
Figure 2.6. Biochemical features of purified pTD2 .......................................................... 83
Figure 2.7. LAP-A is excreted from M sexta as an active enzyme .................................. 84
Figure 3.1. TD activity in flowers of TD2 antisense lines .............................................. 117
Figure 3.2. TD2 reduces the growth rate of Spodoptera exigua larvae .......................... 118
Figure 3.3. TD2 is cleaved by an insect protease ............................................................ 119

Figure 3.4. Comparison of TD2 peptide sequence identified after TD2 processing by
frass proteases and commercial chymotrypsin ................................................................ 120

Figure 3.5. In vivo processing of TD2 ............................................................................ 121

Figure 3.6. Fractionation of digestive proteases excreted in the frass of T. ni reared on

artificial diet ..................................................................................................................... 122
Figure 3.7. TD2 is cleaved by an excreted chymotrypsin-like protease from T. ni ....... 123
Figure 3.8. Degradation of tomato TDs by insect digestive proteases ........................... 124
Figure 3.9. Temperature stability of tomato TDI and TD2 ............................................ 126
Figure 3.10. Sensitivity of tomato TDs to Ile ................................................................. 128
Figure 4.1. Jasmonate defenses reduce T richoplusia ni fitness on tomato ..................... 168

Figure 4.2. Jasmonate defenses reduce Leptinotarsa decemlineata fitness on tomato... 1 70

Figure 4.3. Distribution of proteins identified in the wild-type tomato leaf and frass of L.

decemlineata ................................................................................................................... 1 72
Figure 4.4. Distribution of the proteins identified in the three insect frass samples ....... 174
Figure 4.5. Differential digestion of TD2 in the lepidoptera gut .................................... 176
Figure 4.6. Processing of potato TD2 ............................................................................. 177
Figure 4.7. Identification of JA-inducible proteins ........................................................ 178
Figure 4.8. Distribution of proteins with cofactors ......................................................... 179
Figure 4.9. Subcellular localization of identified proteins .............................................. 180

xi

Figure 5.1. Homology models of tomato TDs ................................................................ 197

xii

Chapter I

Plant Defenses to Insect Herbivory

Introduction

Plants are exposed to various biotic stresses, including competing weeds, pathogens, and
animal pests. It is estimated that 45% of crop yield is lost to these agents. Despite the
increase in pesticide use, these losses have not decreased over the last 40 years (Oerke,
2006). The control of insect pests alone is estimated to cost $10 billion dollars annually.
Moreover, global warming will likely result in greater damage by insect herbivores to
crops and forests (Currano et al., 2008; DeLucia et al., 2008). Insects can reduce plant
fitness directly through removal of photosynthetic tissues, or indirectly by reducing
photosynthetic rates and the plant’s competitive ability (Bernays, 1998; Zangerl et al.,
2002). Most current approaches to controlling insect pests involve the use of chemical
pesticides and synthetic pheromones. However, concerns about the effect of these
chemicals on the environment as well as the development of insecticide-resistant insects

have promoted the search for more environmentally friendly control measures.

One such alternative is the use of transgenic approaches to generate insect
resistant crops. The first generation of insect-resistant transgenic plants was engineered to
express Cry genes encoding so-called Bt toxins from Bacillus thuringiensis. This
approach has proven successful in the development of insect resistance in cotton

(Gossypium hirsutum) and maize (Zea mays) (Gatehouse, 2008). Different Br strains

produce toxins that are effective against specific insects in the orders lepidoptera,
coleoptera, and diptera. However, Cry proteins are not suitable for the control of all
insect pests, and laboratory experiments have demonstrated that several insect pests can

develop resistance to the toxin (Tabashnik et al., 2008).

Another approach for developing insect resistant crop plants has relied on the
overproduction of plant compounds that have anti-insect activity. Because this approach
relies on understanding specific plant defense mechanisms, it has been limited to a few
genes that encode proteins with a role in plant defense, including protease inhibitors,
polyphenol oxidase, or-amylase inhibitor, and lectins (Hilder et al., 1987; Johnson et al.,
1989; Shade et al., 1994; Xu et al., 1996; Gatehouse et al., 1997; Rao et al., 1998; Foissac
et al., 2000; Wang and Constabel, 2004; Thipyapong et al., 2007; Bhonwong et al.,
2009). Transgenic manipulation of genes involved in the biosynthesis of toxic secondary
metabolites, caffeine, hydrogen cyanide, and terpenoids (Tattersall et al., 2001; Aharoni
et al., 2003; Uefuji et al., 2005), successfully enhanced plant resistance to certain insect
pests. A limitation of this approach is that the strength of resistance is relatively weak,
and insects can quickly adapt their physiology to cope with the toxic compounds. Insect
adaptation to these chemical defenses can be retarded by engineering plants with a gene
from a non-host plant of the target insect or combining multiple defense mechanisms

(Jongsma and Bolter, 1997).

A promising alternative approach to controlling insect herbivores is the use of
double stranded RNA (dsRNA) to suppress insect genes. In this case, the crop plant is
transformed with a construct that targets an essential insect gene for suppression. This

technique has been successfully utilized to suppress a cytochrome P450 involved in

gossypol tolerance in cottom bollworrn (Helicoverpa armigera) (Mao et al., 2007), and a
V-type ATPase subunit and B-tubulin from the Western corn rootworm (Diabrotica
virgifera virgifera) (Baum et al., 2007). This approach has the potential to produce the
next generation of insect-resistant crops. A current limitation of this technology is the

identification of insect target genes that would confer plant protection (Price and

Gatehouse, 2008).

Sounding the alarm: the jasmonate pathway

Upon insect attack, the wounded plant perceives signals derived from herbivore oral
secretions and the damaged leaf (Schih'niller and Howe, 2005; Tumlinson and Lait, 2005;
Schmelz et al., 2009). These signals promote calcium ion fluxes, kinase cascades, the
formation of reactive oxygen species (ROS), and the activation of the octadecanoid
pathway leading to synthesis of the plant defense hormone jasmonate (Howe and Jander,
2008; Mithofer and Boland, 2008). The octadecanoid pathway converts linolenic acid to
jasmonic acid (JA) through various steps of oxygenation, dehydration, reduction, and [3-
oxidation (Vick and Zimmerman, 1984). JA and its conjugated forms (collectively
referred as jasmonates) are key players in the activation of plant defense responses to
insect herbivory (Howe and Jander, 2008). This has been demonstrated through genetic
analysis of the wound response (Lightner et al., 1993; Howe and Ryan, 1999; Li et al.,
2001; Li et al., 2004a; Schilrniller and Howe, 2005) and transcriptional profiling

experiments performed with several model plant species, including tomato, tobacco, and

Arabidopsis (Reymond et al., 2000; Halitschke et al., 2003; Reymond et al.,2004;

Devoto et al., 2005).

C011 (Coronatine lnsensitivel) is a key component of the jasmonates signaling
cascade. Mutations in this gene, which have been described in Arabidopsis, tomato
(Solanum lycopersicum), and Nicotiana attenuata, result in insensitivity to jasmonate
(F eys et al., 1994; Li et al., 2004b; Paschold et al., 2007). C011 encodes an F -box protein
that participates in ubiquitin-dependent protein degradation of Jasmonate ZIM-domain
proteins (JAZ) (Thines et al., 2007; Katsir et al., 2008). JAZ proteins repress the
' transcription of JA-responsive genes through interaction with transcription factor such as
MYC2 (Chini et al., 2007). In damaged leaves, increased levels of jasmonoyl-isoleucine
(JA-Ile) promote interaction between C011 and JAZ proteins. Degradation of JAZ
proteins leads to derepression of JA-responsive genes (Katsir et al., 2008). The
expression of JA-responsive genes is observed in the attacked leaf, as well as in
undamaged leaves of the attacked plant. This systemic wound response heightens
resistance to subsequent insect attacks, and is accompanied by large-scale changes in
gene expression. Local and systemic wound responses include downregulation of
photosynthetic and other growth-related processes, and increased expression of various

defensive traits (Kessler and Baldwin, 2002).

Jasmonates provide protection against insect herbivores from different feeding
guilds, including chewers, suckers, and cell-content feeders (Walling, 2000; Thaler et al.,
2002; Browse and Howe, 2008). Jasmonate-mediated defense responses reduce the
fitness of generalist insects that feed on a few plant hosts, as well as specialists that feed

on one of a few closely related species (Reymond et al., 2004). The overall effect of the

jasmonate pathway on host resistance results from the combined action of direct defenses
that slow insect feeding and indirect defenses (e.g., volatiles) that recruit predators or
parasites of the attacking insect (Thaler, 1999; Thaler, 2002; Chen et al., 2006). The
induction of indirect defense responses is thought to be coordinated with the induction of
direct defenses because neither strategy alone is completely effective in targeting
different developmental stages of multiple herbivores (Thaler, 2002). Direct defenses are
mostly effective against hatchling larvae, whereas indirect defenses frequently kill older
herbivores (Cornell et al., 1998). In the absence of a functional JA-signaling pathway,
plants become a host not only to opportunistic herbivores, but also to detrivorous
crustaceans that do not normally feed on living plant material (Kessler et al., 2004;

Farmer and Dubugnon, 2009).

Requesting backup: plant volatiles as indirect defenses

Plant volatile organic compounds (VOCs) released in response to insect herbivory
perform a dual function: they exert direct toxic or repellent effects on the attacker, and
they attract natural enemies of the herbivore. Wound-induced plant VOCs allow
predators and parasitoids to distinguish between infested and uninfested plants, and to
locate their prey (Tumlinson et al., 1993; Takabayashi and Dicke, 1996). The ecological
role of VOCs in tritrophic interactions has been extensively studied in both laboratory

and natural field conditions (Kessler and Baldwin, 2001).

The blend of volatiles released after damage varies according to the attacking

herbivore (Pare and Tumlinson, 1999; Thaler, 2002). VOCs are synthesized by three

different pathways. These pathways include the octadecanoid pathway for rapid wound-
induced production of green leaf volatiles, the mevalonate and nonmevalonate pathways
that synthesize terpenoid volatiles, and the shikimate pathway for production of methyl
salicylate (Kessler and Baldwin, 2002). Nectaries also produce extrafloral nectar that
attract natural enemies of the attacking insect in response to herbivore attack (Heil et al.,
2001). Manipulation of volatile emission in transgenic plants has been used to increase
plant resistance to herbivores (Turlings and Ton, 2006). Increased resistance is due not
only to the role of volatiles as indirect defenses, but also to the important effect of

volatiles in priming plant defenses for firture attack (Baldwin et al., 2006).

Close combat fighting: defensive compounds that directly target insect

herbivores

Direct defenses include not only metabolites and proteins that thwart insect attack
(Kessler and Baldwin 2002), but also physical barriers that impede access to nutritious
tissues (Hanley et al., 2007). Morphological traits such as spines, trichomes, and leaf
toughness are among the direct defenses (Hanley et al., 2007). Trichomes are thought to
have evolved as protection against radiation and water loss, but they are also widely
regarded as the first line of defense against herbivores (Hanley et al., 2007). Trichomes
act as a mechanical barrier to limit access to nutritious tissue, and also as chemical
barriers that produce anti-insect compounds. Trichome-borne defensive compounds
include secondary metabolites and proteins (Yu et al., 1992; Amme et al., 2005;

Simmons and Gurr, 2005; Liu et al., 2006; Schilmiller et al., 2008). Trichomes provide

protection against aphids, whiteflies, chewing insects and leafminers; but are less

effective against larger insects such as grasshoppers (Hanley et al., 2007).

Secondary metabolites with direct roles in plant defense

Secondary metabolites are not only responsible for the plant’s “cry-for—help” as discussed
above, but they can also act as feeding deterrents, repellents, and post-ingestive defenses
against insects. An insect’s choice of host plant is based largely on the content of
secondary metabolites (Hadacek, 2002). Toxic compounds are usually characterized by
their ability to repel herbivores, reduce the fitness of generalist herbivores, and force
specialist herbivores to invest in detoxification mechanisms that incur fitness costs in
growth and development (Kessler and. Baldwin, 2002). Terpenoids, phenolics, and
various nitrogen-containing compounds, including glucosinolates, alkaloids, and
cyanogenic glycosides, have insecticidal properties (Howe and Jander, 2008). The
synthesis of these compounds can be either constitutive or inducible in response to insect

herbivory (Jansen et al., 2009).

Glucosinolates are a classical example of plant anti-insect compounds. Their
production is almost exclusive to the Brassicaceae. Glucosinolates are classified
according to the chemical nature of the amino acid side chain, and include indole
(derived from tryptophan), aliphatic (derived from methionine), and aromatic
glucosinolates (derived from phenylalanine or tyrosine). Additional chemical diversity is
achieved through chain elongation, oxidation, and hydroxylation of the side chain (Fahey

et al., 2001). The enzyme myrosinase catalyzes the breakdown of glucosinolates into

compounds that are toxic and pungent. This breakdown process does not occur in planta
because myrosinase and its glucosinolate substrates are located in separate cellular
compartments. Upon tissue disruption, for example by insect chewing, mixing of
myrosinase with vacuole-stored glucosinolates results in the formation of compounds
such as thiocyanates, isothiocyanates, and isonitriles (Hopkins et al., 2009).
Glucosinolates can act as oviposition and feeding stimulants for a number of insect
specialists. In the case of generalist insects, however, these compounds have potent
insecticidal properties (Barth and Jander, 2006; Schlaeppi et al., 2008). Specialist
herbivores use various mechanisms to cope with glucosinolates, including enzymatic
detoxification, excretion, and sequestration (Mainguet, 2000), as well as with behavioral
responses to avoid consumption of plant tissues that contain high glucosinolate content

(Shroff et al., 2008).

Terpenoids constitute the most diverse group of anti-insect metabolites that serve
roles in direct and indirect defense (Frelichowski and Juvik, 2001). Terpenoid—based
defenses against insect herbivores have been extensively studied in conifers (Keeling and
Bohlmann, 2006). In conifers, terpenoids accumulate in large amounts in oleoresins that
are stored in specialized resin-producing resin cells. The extraordinary diversity of
terpenoids is generated in large part by the action of two enzyme families, namely the
terpenoid synthases and cytochrome P450-dependent monooxygenases (Bohlmann et al.,
1998). Despite the importance of terpenoids in plant defense, little information is

available on the mode of action of terpenoids as direct defenses.

In many plants, most notably oak, tannins have long been thought to serve as a

key chemical defense against insect herbivores (Feeny, 1970). Upon oxidation in the

midgut, tannins produce semiquinone radicals that could potentially lead to oxidative
stress. Recent studies, however, indicate that semiquinones do not contribute to oxidative
stress in the midgut, and consequently do not reduce larval performance (Barbehenn et

al,2009)

Plant anti-insect proteins

Plant proteins are another layer of defense that has received less attention compared to
secondary metabolites. Insect digestion and nutrition are important targets of plant anti-
insect proteins. In the following sections, information on plant proteins that affect post-
ingestive targets in the insect will be reviewed. Plants can also limit the insect’s access to
nutrients prior to ingestion of plant food through fortification of the cell wall and
physico-chemical barriers such as epicuticular waxes (Chen, 2008; Miiller, 2008).
Several genes that encode proteins for cell wall modification are regulated by insect
attack. This group includes pectin esterases, expansins, xyloglucan
endotransglycosylases/hydrolases, and cellulose synthases (Goggin, 2007; Liu et al.,

2007)

Plant proteins that serve a post-ingestive role in defense are typically synthesized in
response to wounding and herbivore attack. This was established early on in tomato
(Solanum lycopersicum) by Ryan and colleagues (Farmer et al., 1992; Constabel et al.,
1995; Bergey et al., 1999; Moura et al., 2001; Diez-Diaz et al., 2004). A protein’s
effectiveness as a post-ingestive defense depends in part on its resistance to inactivation

by insect digestive proteases. Therefore, another common feature of plant anti-insect

proteins is their stability in the gut (Chen et al., 2005). Post-ingestive defenses can be
further subdivided into proteins that directly disrupt the insect digestive system (toxic

proteins) and proteins that limit the access to essential nutrients (anti-nutritive proteins).

Eliminate the enemy: Toxic proteins

Lectins

Proteins are classified as lectins if they possess at least one non-catalytic domain that
binds reversibly to specific carbohydrates. This relaxed definition allowed the grouping
of diverse lectin-like proteins into seven families of structurally and evolutionary related
proteins. Several lectins are induced by jasmonate treatment or insect attack (Zhu-
Salzman et al., 1998; Williams et al., 2002). Insecticidal activity of these proteins was
deduced from their ability to bind glycosylated proteins and chitin, a polymer present in
the peritrophic membrane that protects the delicate midgut cells, in the insect gut (Van
Damme, 2008). Lectins are highly stable under harsh conditions, including the proteolytic
environment in the gut, and were reported to cross the gut epithelium to reach the
hemolymph (Peumans and Van Damme, 1995; Zhu-Salzman and Salzrnan, 2001; F itches
et al., 2002). The anti-insect role of lectins was demonstrated with the use of transgenic
plants expressing foreign lectins, and in vitro assays in which purified lectins are added to
artificial insect diet (Carlini and Grossi-de-Sa, 2002). The exact mechanism of the
toxicity of lectins remains to be determined. However, there is evidence to indicate that
some lectins impair formation of the peritrophic membrane by binding to glycoproteins in

midgut epithelial cells, or by binding to glycosylated insect proteins involved in food

10

digestion (Figure 1.1.A) (Peumans and Van Damme, 1995). The harmful effects of
lectins on the peritrophic membrane was verified in the European corn borer (Ostrinia
nubilalis) fed with the lectin wheat germ agglutinin (W GA). In contrast to a continuous
peritrophic membrane protecting the midgut epithelial cells in control larvae, WGA-fed
larvae showed a multilayered and disorganized peritrophic membrane, and disintegration
of the midgut microvilli resulting from abrasive food particles and microorganisms that
penetrated the peritrophic membrane (Harper et al., 1998) (Figure 1.1.A). The utility of
transgenically expressed lectins for insect control has been limited by the high level of
expression required to inhibit insect growth, as well as the fact that some lectins can be

toxic to mammals (Estruch et al., 1997; Carlini and Grossi-de-Sa, 2002).

Cysteine proteases

Cysteine proteases are defined by the presence of a cysteine residue at the catalytic site
(Shindo and Van der Hoorn, 2008). The insecticidal activity of cysteine proteases has
been studied in several plant-insect interactions. Papain and ficin are cysteine proteases
found in the latex of the Papaya tree (Carica papaya) and fig (Ficus virgata),
respectively. The toxicity of latex from these plants is abolished by the cysteine-specific
protease inhibitor E-64, indicating that cysteine proteases are responsible for the toxic
effects on lepidopterans (Konno et al., 2004). Mirl-CP (Maize inbred resistance 1) from
maize (Zea mays) is one of the most thoroughly characterized cysteine proteases. The
anti-insect role of this protease was established by overexpression of the mirI gene in

maize callus, which resulted in growth reduction of the fall armyworm (Spodoptera

11

fiugiperda) that fed on the callus (Pechan et al., 2000). The Mir] gene of maize is
upregulated in response to insect feeding. The mode of action of Mir-CP involves
disruption of the peritrophic membrane (Figure 1.1.B) (Pechan et al., 2002). Perforation
of the peritrophic membrane by Mir-CP increases the membrane’s permeability to toxic
proteins and microorganisms, resulting in damage to the midgut microvilli (Mohan et al.,
2006). This mechanism of toxicity is comparable to that of the Bt toxin Bt-CryIIA

(Mohan et al., 2008).

C yclotides

Cyclotides are insecticidal peptides (28-37 amino acids in length) identified in species
from the Violaceae, Rubiaceae, Cucurbitaceae, and Apocynaceae (Gruber et al., 2008).
These disulphide-rich compounds contain a series of cyclic peptide bonds and are
stabilized by six cysteine residues that form a cystine knot (Craik et al., 1999).
Cyclotides are highly stable at extreme pH and temperatures, and are resistant to the
activity of proteases (Colgrave and Craik, 2004). These structural features of cyclotides
allow them to tolerate multiple mutations in the backbone sequence as long as the
disulfide bonds are maintained, giving rise to a large diversity of variants. Discovered as
the active component in Oldenlandia aflinis, cyclotides have a range of medicinal
properties, including anti-HIV, uterotonic, haemolytic, and cytotoxic activity (Craik et
al., 2004). Nevertheless, their primary role in plants appears to be protection from insects
(Jennings et al., 2001). The insecticidal properties of the cyclotides kalata B1 and B2

were demonstrated by incorporation of the peptides in the diet of two Helicoverpa

12

species (Jennings et al., 2005). The growth retardation effect on Helicoverpa armigera
was caused by disruption of midgut epithelial cells (Figure 1.1.C). Ingestion of cyclotides
causes thickening of the epithelial cells caused by blebbing of cell fi'agments into the gut
lumen, and swelling and lysis of the columnar cells that form the microvilli. The
peritrophic membrane in larvae reared on cyclotide-containing diet was highly
degenerated. Similar to Bt toxins, cyclotides are hypothesized to form pores in the plasma

membrane of epithelial cells (Barbeta et al., 2008).

C anatoxin

Canatoxin (CNTX) was identified in the seed of jackbean (Canavalia enszformis). The
widespread occurrence of CNTX-like proteins in F abaceae seeds and their accumulation
pattern during seed maturation suggested a protective role in the plant (Carlini et al.,
1988; Barcellos et al., 1993). Subsequent studies demonstrated that CNTXs have
fungicidal and insecticidal properties (Carlini et al., 1997; Oliveira, 1999). CNTX is a
variant of the enzyme urease. Although urease also displays insecticidal activity, it is not
as potent as CNTX (Follmer et al., 2004). CNTX is lethal to insects that use cathepsin B-
and D-type proteases as their main digestive enzymes; insects with trypsin-based
digestive systems are not affected by CNTX. The insect target and mode of CNTX action
remain to be determined. Interestingly, however, CNTX-mediated toxicity requires
proteolytic activation of CNTX by insect cathepsins. Differential proteolytic digestion of
CNTX in different insects may account for the toxic effect of CNTX on some insects but

not on others (Carlini and Grossi-de-Sa, 2002; Staniscuaski et al., 2005).

13

Neutralizing the enemy: Anti-nutritional proteins

Protease Inhibitors

In 1972, Green and Ryan reported for the first time that wounding of tomato and potato
causes the systemic accumulation of Protease Inhibitors (PI) (Green and Ryan, 1972).
Since then, Pls have been widely used as markers for the plant wound responses, and
extensively studied for their role in defense against herbivores (Schilmiller and Howe,
2005). PIs accumulate constitutively in seeds, tubers, and flowers, but are massively
produced in leaves after insect attack. PIs that are active against each of the four main
classes (serine, cysteine, aspartic, and metalloproteases) of proteases have been found in

plants.

Digestive proteases release amino acids and peptides from dietary protein. PIs work in
the gut by binding to proteases and blocking their activity (Figure 1.2.A). Insects
overproduce proteases in response to P15 in the diet in an attempt to compensate for
reduced protease activity. Overproduction of proteases depletes the availability of amino
acids needed for the synthesis of other insect proteins, which results in reduced growth
rates (Jongsma and Bolter, 1997; Schilmiller and Howe, 2005). Serine and cysteine PIs
frequently have deleterious effects when fed to lepidoptera and coleoptera, respectively.
The effects of dietary Pls range from reduced fecundity and decreased weight to
increased mortality and severe developmental defects (Gruden et al., 1998; Wilhite et al.,

2000)

14

Reduced growth of insect larvae reared on Pl-containing diets is not always observed,
even in the case of larvae reared on transgenic plants expressing high Pl levels (Gruden et
al., 2004). This phenomenon is explained by the adaption of insects to P15. In response to
Pls in the diet, insects can induce the synthesis of proteases that break down the P15 (Giri
et al., 1998; Gruden et al., 1998; Yang et al., 2009). In addition, many insect have the
capacity to synthesize novel proteases that are insensitive to dietary Pls (Jongsma et al.,
1995; Volpicella et al., 2000; Gruden et al., 2004; Bayes et al., 2005). A third insect
adaptive strategy to dietary P13 is simply to increase food consumption (Cloutier et al.,
2000; Winterer and Bergelson, 2001). As part of the evolutionary “arms race” between
plants and insects, plants have expanded the diversity of PI genes to target the multitude
of insect digestive proteases (Baldwin and Karban, 1997; Jongsma and Beekwilder,
2008). Transgenic expression of P15 containing domains targeting different proteases may
reduce the ability of insects to adapt to the existing complement of Pls in a given host
plant (Outchkourov et al., 2004). This approach has the additional advantage of
protecting the PIs from degradation by insect proteases, an effect known as cross-

protection (Jongsma and Bolter, 1997).

Polyphenol oxidase

PPOs are copper metalloenzymes that catalyze 02-dependent oxidation of mono- and o-
diphenols to o-diquinones. The high reactivity of the quinone products leads to secondary
reactions that cause damage to proteins, lipids, and DNA, thereby reducing the nutritional

quality of the tissue (Felton et al., 1992; Duffey and Stout, 1996). The wound inducibility

15

of PPO expression has been reported for several species including tomato, potato, and
hybrid Poplar (Constabel et al., 2000; Thipyapong etal., 2007). A defensive role for PPO
was demonstrated with the use of transgenic plants altered in the expression of PPO
(Wang and Constabel, 2004; Mahanil et al., 2008; Bhonwong et al., 2009). Results of
experiments performed with tomato suggest that PPO can act defensively against
lepidoptera and coleopteran insects, which have very different gut physiologies
(Thipyapong et al., 2007). The mechanisms involved in FPO-mediated plant resistance to
insects are not fully understood. The quinone products of PPO may be directly toxic to
insects, participate as signaling molecules that activate other plant defenses, or may
promote cell wall fortification. A leading hypothesis is that highly reactive quinones
alkylate dietary protein and, as a consequence, reduce the nutritional quality of the plant
tissue (Felton, 1989). Recently, the role of PFC as a post-ingestive defense has been
questioned on the grounds that the anaerobic environment and high ascorbate content of
the lepidopteran gut severely limit the activity of PPO and other oxidases (Barbehenn et
al., 2007; Barbehenn et al., 2008). This finding suggests that the effect of PFC on
herbivore performance is likely caused by a pre-ingestive effect (Constabel and
Barbehenn, 2008). In this context, it is worth noting that glandular trichomes of tomato
and potato contain high levels of PPO (Yu et al., 1992). Upon disruption of the trichome
gland, PPO can rapidly oxidize phenolic substrates that are also stored in the gland
(Kennedy, 2003). Reactive quinones formed in this manner could be responsible for

repelling or deterring insect herbivores prior to tissue ingestion.

Amino acid degrading enzymes

16

The low protein content of plant tissue represents a major challenge for herbivorous
insects whose rapid growth rate depends on the assimilation of relatively high levels of
amino acids from dietary protein (Bemays, 1998). This challenge is exacerbated by the
presence of plant defense enzymes that degrade essential amino acids in the insect gut
(Chen et al., 2005; Felton, 2005). Proteomic analysis of the fate of leaf proteins in the M
sexta gut showed that the tomato enzymes arginase and threonine deaminase, which
previously were thought to be involved in primary metabolism, serve a role in restricting

the availability of essential amino acids (Chen et al., 2005).

Arginase catalyzes the conversion of L-arginine (Arg) to urea and omithine. An
important form of storage nitrogen, free Arg and protein-bound Arg accounts for a large
proportion of nitrogen reserves in storage organs (Pollaco and Holland, 1993). During
seed germination, arginase activity is implicated in the breakdown of Arg to release
nitrogen (Goldraij and Polacco, 1999, 2000). Arg also is a substrate for putrecsine
biosynthesis. Tomato has two arginase-encoding genes designated ARGI and ARGZ.
ARGZ gene expression is induced in response to wounding, JA treatment, and the
Pseudomonas syringae-derived toxin coronatine. ARGZ is expressed to high levels in
reproductive tissues under basal conditions. ARGI is expressed is tissues throughout the
plant and is not induced by stress. Despite these differences in expression pattern and
mode of regulation, the two isoforms exhibit very similar substrate specificity, pH
optimum, and kinetic parameters (Chen et al., 2004). The high pH optimum of ARG2
(Chen et al., 2008) suggested that this wound-inducible isoform might be active in the
alkaline environment of the lepidopteran midgut. Support for this hypothesis came from

the finding that total arginase activity in the midgut of tomato-reared M sexta larvae was

17

inversely proportional to Arg levels (Chen et al., 2005). Moreover, transgenic tomato
plants overexpressing ARGZ were more resistant to attack by M sexta larvae. This
information, together with the accumulation of ARG2 in the M sexta gut, are consistent
with a post-ingestive role for tomato ARG2 in degrading the essential amino acid Arg

from the insect midgut (Figure 1.2.8) (Chen et al., 2005).

It has been speculated for several reasons that the anti-insect function of ARG2 in the
lepidopteran gut may be facilitated by leucine aminopeptidase (LAP—A), a tomato
exopeptidase that releases Arg from protein and peptide substrates (Figure 1.2.8) (Chen
et al., 2005; Felton, 2005). First, the expression of LAP-A in tomato leaves is co-
regulated with ARG2 in response to wounding and JA treatment (Hildmann et al., 1992;
Pautot et al., 1993; Chao et al., 1999). Second, LAP-A has a high pH and temperature
optimum (Gu et al., 1999), and the enzyme is highly stable during passage of tomato leaf
tissue through the insect gut (Chen et al., 2005). Finally, transgenic tomato plants that
either over- or underexpress LAP-A exhibit decreased and increased resistance,
respectively, to attack by M sexta larvae (Fowler et al., 2009). Although these
observations are consistent with a role for LAP-A in insect resistance, direct evidence

that LAP-A provides Arg substrate for ARG2 is lacking.

Threonine deaminase (TD), which catalyzes the conversion of threonine (Thr) to or-
ketobutyrate and ammonia, is another example of a host plant enzyme that accumulates in
the gut of tomato-reared M sexta (Chen et al., 2005). This reaction constitutes the first
step in the biosynthesis of isoleucine (Ile) and is negatively regulated by Ile. In
solanaceous species, TD is expressed constitutively to high levels in floral organs. In

leaves, however, TD expression is induced via the JA/COIl signaling pathway in

18

response to mechanical wounding and insect herbivory (Samach et al., 1991; Samach et
al., 1995; Li et al., 2004b; Kang et al., 2006a). Plant and bacterial TDs consist of an N-
terminal catalytic domain and a C-terminal regulatory domain. The ability of tomato TD
to efficiently degrade Thr in the insect gut is associated with proteolytic removal of the
regulatory domain. This post-translational modification renders TD insensitive to
feedback inhibition by Ile, thereby allowing the enzyme to efficiently deplete Thr from
the midgut (Figure 1.2.C) (Chen et al., 2005). This hypothesis is supported by the finding '
that the midgut of M sexta larvae reared on TD-expressing tomato leaves contained
lower Thr levels (and higher ammonia levels) than larvae reared on TD-deficient leaves
(Chen et al., 2005). Additional evidence for the role of TD in plant defense against M
sexta attack was obtained through analysis of transgenic Nicotiana attenuata plants that

were silenced in the expression of TD (Kang et al., 2006b).

Vegetative Storage Proteins

Vegetative storage proteins (VSPs) accumulate to high levels in storage organs of
vegetative tissues and seeds. Based on this expression pattern, it was suggested that VSPs
may function as an amino acid reserve that is utilized during seed germination (Staswick
et al., 1994). This hypothesis was not supported, however, by transgenic studies showing
that VSP-deficient soybean (Glycine max) lines do not exhibit phenotypes related to seed
development or seedling establishment (Staswick et al., 2001). Arabidopsis VSPs share
overall sequence similarity with soybean VSP. Recent insight into the physiological

function of VSPs has come from studies performed in Arabidopsis thaliana. Similar to

19

the expression pattern of VSP genes in soybean, VSP expression in A. thaliana is induced
by methyl-IA, wounding, insect feeding, osmotic and nutritional stress, and phosphate
starvation (Utsugi et al., 1998; Gong et al., 2001; Berger et al., 2002; Reymond et al.,
2004; Liu et al., 2005). This pattern of expression, together with high level accumulation
of VSPs in the vacuole (Franceschi et al., 1983), is consistent with a role for these
proteins in insect resistance. Direct support for this idea came from experiments showing
that recombinant VSPs from A. thaliana are highly toxic to coleopteran and dipteran
insect species that have an acidic midgut (Liu et al., 2005). Interestingly, the acid
phosphatase activity exhibited by these VSPs is required for the insecticidal property of
the proteins. It has been proposed that VSPs may interfere with phosphate metabolism in

the gut of target insects (Zhu-Salzman et al., 2008).

Plants exploit insect proteases for the activation of defense

Limited proteolysis of plant proteins in the insect gut adds another level of complexity to
plant-insect interactions. Defensive enzymes such as TD are presumably kept latent in the
plant and subsequently activated upon exposure to insect digestive proteases. Full activity
of PFC from hybrid Poplar (Populus trichocarpa x Populus deltoides) requires treatment
with detergent or proteases (Constabel et al., 1995; Constabel et al., 2000). Treatment
with trypsin, for example, effectively activates PPO, which appears to be latent inside the
chloroplast. These findings are consistent with the observation that poplar PPO is
activated upon passage through the insect gut (Wang and Constabel, 2004). Biochemical

studies have shown that this phenomenon involves removal of an inhibitory peptide from

20

the active site of the enzyme (Gandia-Herrero et al., 2005). Cry toxins provide another
remarkable example of insecticidal proteins that are activated by insect digestive
proteases. The Cry pro-toxin is activated by removal of an N-terminal peptide that blocks
access of the protein to its receptor on the membrane of the midgut epithelium cells
(Bravo et al., 2007). Binding of the proteolytically actived Cry protein to the receptor

facilitates insertion of the toxin into the membrane.

Recent studies have also shown that peptide products derived from digestion of
plant proteins in the insect gut can function as signals for activation of plant defense
responses. The disulfide-bridged peptide inceptin, which was isolated from oral
secretions of the fall armyworm (Spodoptera fi-ugiperda) fed on cowpea (Vigna
unguiculata), elicits the production of ethylene, JA, and salicylic acid (Schmelz et al.,
2006). Inceptins are derived from the regulatory domain of the chloroplastic ATP
synthase 7 subunit (Schmelz et al., 2006). Interestingly, digestion of intact ATP synthase
by insect gut proteases is required to release the active peptide signal (Schmelz et al.,
2007). Because inceptin is a plant-derived signal whose production requires the action of
insect proteases, this mechanism of elicting plant defense responses is consistent with the

guard hypothesis of plant immunity (Dangl and Jones, 2001; Schmelz et al., 2006).

Thesis rationale and overview

The work presented here expands our current knowledge of the mechanisms of
plant defense against insect herbivores. Most previous research aimed at understanding

the chemical basis of plant-insect interactions has been focused on plant secondary

21

metabolites. More recently, however, there is growing emphasis on understanding
insecticidal proteins that exert toxic or anti-nutritional effects on insect pests. Modern
proteomic technologies have greatly facilitated the identification of these proteins (Chen
et al., 2005). Tomato was used for all the experiments in this research because it has been
extensively used as a model system for the study of plant-insect interactions. In addition,
cultivated tomato is a host for a large number of arthropod herbivores that attack roots,
leaves, and fruit (Lange, 1981). The results from experiments described in this thesis may
help in the design of effective pest control measures. Because the genes identified are of
plant origin, these genes may be used to develop insect-transgenic plants with good

public acceptance since the genes are of plant origin.

Chapter 2 describes the use of insect feces (frass) as a source of enriched plant
proteins with potential roles in defense. The use of fi'ass for proteomic analyses is based
on the premise that midgut-active defense proteins are stable during passage of plant food
through the insect and thus are excreted in the frass. This study identified a TD isoform
(TD2) of tomato that likely serves an antinutritional role in defense. The expression
pattern of TD2 and its closely related paralog TDl is also described in this chapter.
Chapter 3 examines the role of tomato TD2 as an herbivore defense in more detail. The
results provide direct evidence for the contribution of TD2 to insect resistance, as well as
new insight into the mechanism by which TD2 is activated in the gut by proteolysis. This
chapter also compares the biochemical properties of tomato TDI and TD2, and provides
evidence for functional specialization of these two enzymes. Chapter 4 describes the

results of a comparative proteomics study of frass from three different insect species

22

reared on tomato. The results obtained provide insight into how physicochemical

conditions in the gut may affect the stability and digestibility of dietary protein.

23

Figure 1.1. Mode of action of toxic plant proteins.

(A) Lectins cause the formation of a multi-layered peritrophic membrane that allows
abrasive food particles to access the midgut epithelium.

(B) The cysteine protease Mirl-CP impairs nutrient utilization by degrading the
peritrophic membrane and allowing access of microorganisms and toxic proteins to the
microvilli.

(C) Cyclotides form pores at the membrane of the midgut epithelium, which causes
blebbing, swelling and, ultimately, rupture of the cells.

GL: gut lumen; PM: peritrophic membrane; MV: microvilli of the midgut epithelial cells

24

 

   

.- : Chitin
.- = Glycosylated membrane protel s

= Glycosylated digestive enzymes
\ = Food particles

 

 

B) GL

0 = Toxic protein
0 = Microorganisms

 

 

 

 

C) GL PM

Cyclotldes

 

0 = Blebbling cells

 

Figure 1.2. Mode of action of anti-nutritional plant proteins.

(A) Protease inhibitors (PI) induce a compensatory mechanism in insects to
overproduce proteases, which depletes amino acid pools for the synthesis of other insect
proteins.

(B) Leucine aminopeptidase-A (LAP-A) and Arginase2 (ARG2) may work
synergistically to deplete arginine (Arg) from the midgut. LAP-A releases Arg from the
N-terminus of polypeptides, whereas ARG2 catabolizes the resulting free Arg.

(C) Threonine deaminase (TD) is activated by removal of the regulatory domain that
mediates inhibition of TD activity by isoleucine (Ile). Upon processing, TD degrades the
essential amino acid threonine (Thr).

GL: gut lumen; PM: peritrophic membrane; MV: microvilli of the midgut epithelial cells

26

 

A) GL PM

0 =Amino acid A
. = Pl-sensitive proteases ‘

“t; = Pl-insensitive proteases

 

 

B) GL PM

 

@ _) Ornithlne
+ Urea

O = Amino acid

09 =Arg

 

 

 

 

C) GL PM
/ —) / K)

,_ REG ..
LBJ U

Q o
a-ketobutyrate
u ‘o—’ + NH:
/ O

0:1,th U

U =l|e

 

 

27

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41

Chapter 11

Chen H*, Gonzales-Vigil E*, Wilkerson CG, Howe GA (2007) Stability
of plant defense proteins in the gut of insect herbivores. Plant Physiol

143: 1954-1967

* These authors contributed equally to this work and are listed in alphabetical order.

42

Abstract

Plant defense against insect herbivores is mediated in part by enzymes that impair
digestive processes in the insect gut. Little is known about the evolutionary origins of
these enzymes, their distribution in the plant kingdom, or the mechanisms by which they
act in the protease-rich environment of the animal digestive tract. One example of such
an enzyme is threonine deaminase (TD), which in tomato (Solanum lycopersicum) serves
a dual role in Ile biosynthesis in planta and Thr degradation in the insect midgut. Here,
we report that tomato uses different TD isozymes to perform these functions. Whereas
the constitutively expressed TDI has a housekeeping role in Ile biosynthesis, expression
of TD2 in leaves is activated by the jasmonate signaling pathway in response to herbivore
attack. Ingestion of tomato foliage by specialist (Manduca sexta) and generalist
(T richoplusia ni) insect herbivores triggered proteolytic removal of TD2’s C-terrninal
regulatory domain, resulting in an enzyme that degrades Thr without being inhibited
through feedback by Ile. This processed form (pTD2) of TD2 accumulated to high levels
in the insect midgut and feces (frass). Purified pTD2 exhibited biochemical properties
that are consistent with a post-ingestive role in defense. Shotgun proteomic analysis of
frass from tomato-reared M sexta identified pTD2 as one of the most abundant proteins
in the excrement. Among the other tomato proteins identified were several jasmonate-
inducible proteins that have a known or proposed role in anti-insect defense. Subtilisin-
like proteases and other pathogenesis-related proteins, as well as proteins of unknown
fimction, were also catalogued. We conclude that proteomic analysis of frass from insect
herbivores provides a robust experimental approach to identify hyperstable plant proteins

that serve important roles in defense.

43

Introduction

The optimal grth of leaf-eating insects depends on their ability to acquire essential
amino acids from dietary protein. The low protein content of plant tissue, however, poses
a major nutritional challenge to phytophagous insects; protein is both the major
macronutrient and the most commonly limiting nutrient for insect growth (Mattson, 1980;
Bernays and Chapman, 1994). In addition to factors affecting protein quantity, evidence
indicates that dietary protein quality also has a significant impact on plant-insect relations
(Broadway and Duffey, 1988; Felton, 1996). Insect diets containing nutritionally
unbalanced proteins pose a serious impediment to herbivory and may also influence
patterns of host plant utilization among insect herbivores (Moran and Hamilton, 1980;
Karowe and Martin, 1989; Haukioja et al., 1991; Berenbaum, 1995). The idea that
variation in protein quality has evolved as a plant defense is supported by studies
showing that certain classes of allelochemicals, such as tannins and phenolic resins,
impair herbivore performance by interfering with the digestibility of dietary protein
(Feeny, 1976; Rhoades, 1976).

Plants also produce defensive proteins that disrupt nutrient acquisition and other
aspects of insect digestive physiology. Proteinase inhibitors (PIs) that impair the activity
of digestive proteases are perhaps the best example of this type of post-ingestive defense
(Green and Ryan, 1972; Ryan, 1990). Because PIs are not catalytic, their capacity to slow
herbivore grth is dependent on accumulation to relatively high concentrations inside
the gut lumen. Enzymes have the potential to exert defensive effects at much lower
concentrations, but this hypothesis has received relatively little attention until recently

(Duffey and Stout, 1996; Felton, 1996; Chen et al., 2005; Felton, 2005). Research on

44

midgut-active plant enzymes has focused mainly on polyphenol oxidase (PPO) and other
oxidative enzymes that covalently modify dietary protein, thus reducing the digestibility
of plant food (Constabel et al., 1995; Duffey and Stout, 1996; Felton, 1996; Wang and
Constabel, 2004). Other defensive proteins directly target structural components of the
insect digestive apparatus. Members of the cysteine protease family of enzymes, for
example, are thought to disrupt the integrity of the peritrophic membrane that protects the
gut epithelium (Pechan et al., 2002; Konno et al., 2004; Mohan et al., 2006). These
collective studies indicate that enzymes play a pivotal role in host plant defense, and thus
broaden the traditional view that secondary metabolites are the major determinants of
host plant utilization and specialization (F raenkel, 1959; Berenbaum, 1995).

Many plant anti-insect proteins are synthesized in response to wounding and
herbivore attack. Induced expression of the vast majority of these proteins is regulated by
the jasmonate signaling pathway (Walling, 2000; Gatehouse, 2002; Kessler and Baldwin,
2002; Howe, 2004; Schilmiller and Howe, 2005). Examples of jasmonate-inducible
proteins (JIPs) that have a confirmed or proposed role in post-ingestive defense include
PPO, arginase, leucine amino peptidase A (LAP-A), lipoxygenase, and a battery of P18
(Duffey and F elton, 1991; F elton et al., 1994; Constabel et al., 1995; Felton, 1996; Chen
et al., 2005; Walling, 2006; Lison et al., 2006). A JA-inducible acid phosphatase (V SP2)
in Arabidopsis was recently shown to exert insecticidal activity against coleopteran and
dipteran insects (Liu et al., 2005). These observations indicate that a primary function of
the jasmonate signaling pathway is to promote the expression of proteins that act post-
ingestively to impair the growth and development of insect herbivores (Chen et al.,

2005)

45

Biosynthetic threonine deaminase (TD) is a pyridoxal phosphate-dependent
enzyme that converts L-threonine (Thr) to a-ketobutyrate and ammonia. Plant TDs
function in the chloroplast to catalyze the committed step in the biosynthesis of Ile. The
enzyme contains an N-terminal catalytic domain and a C-terminal regulatory domain and
is subject to negative feedback regulation by Ile (Gallagher et al., 1998). The
physiological importance of TD in plant growth and development was demonstrated by
studies of TD-deficient mutants of Nicotiana plumbaginifolia and, more recently, N.
attenuata (Sidorov et al., 1981; Colau et al., 1987; Kang et al., 2006). TD expression in
leaves of several solanaceous plants is massively induced by the jasmonate signaling
pathway in response to wounding and herbivory (Hildmann et al., 1992; Samach et al.,
1995; Hermsmeier et al., 2001; Li et al., 2004). In contrast to this expression pattern, TD
is constitutively expressed to high levels in reproductive organs (Hildmann et al., 1992;
Kang and Baldwin, 2006). TD is reported to be the most abundant protein in tomato
flowers (Samach et al., 1991). The high level of TD expression in reproductive tissues is
similar to the expression pattern of HS and other JIPs that impair insect growth.

Direct evidence for the hypothesis that TD has a role in anti-insect defense came
initially from studies showing that the enzyme accumulates in the midgut of tomato-
reared Manduca sexta larvae (Chen et al., 2005). TD activity in the midgut was correlated
with reduced levels of free Thr, which is a dietary requirement for phytophagous insects.
A jasmonate-insensitive mutant (jail) of tomato that fails to express TD is more
susceptible to attack by M sexta larvae. Because this mutant is defective in all jasmonate-
signaled processes, however, decreased resistance of jail plants could not be linked

directly to loss of TD function (Li et al., 2004; Chen et al., 2005). A recent study by Kang

46

et a1. (2006) showed that mutants of N. attenuata engineered specifically for TD
deficiency are compromised in resistance to M sexta larvae. Supplementation of N.
attenuata leaves with Thr led to increased larval performance, indicating that Thr
availability in the leaf diet is limiting for larval growth. The Ile deficiency in TD-silenced
N. attenuata plants also resulted in decreased production of jasmonoyl-Ile (JA-Ile), which
is an important signal for induced defense responses to pathogens (Staswick et al., 1998)
and insects (Kang et al., 2006). Thus, TD’s defensive firnction in N. attenuata was
attributed both to its involvement in JA-Ile synthesis and its role in post-ingestive defense
(Kang et al., 2006).

TD’s dual function in primary metabolism and defense makes it an attractive
subject for research aimed at understanding the evolutionary origins of plant enzymes
that exert toxic or antinutritional effects on insect herbivores. Here, we show that tomato
has two TD genes (designated TD] and TD2) whose differential expression pattern is
consistent with functional divergence of the two isoforms. Second, we show that
ingestion of tomato foliage by specialist and generalist herbivores triggers proteolytic
removal of the TD2 regulatory domain, resulting in an enzyme (pTD2) that effectively
degrades Thr in the lepidopteran gut. Third, we show that the biochemical properties of
pTD2 are consistent with a post-ingestive role in defense. Finally, we employed a
“shotgun” proteomic approach to demonstrate that pTD2 is one of the most abundant
proteins in frass from tomato-reared M sexta larvae. Nineteen additional tomato proteins
were cataloged in M sexta frass. Among these were JIPs that have a known role in
defense against insect herbivores, pathogenesis-related proteins, and proteins of unknown

function. These findings provide new insight into the evolution of plant anti-insect

47

proteins and establish a robust experimental approach to identify hyperstable proteins that

serve important roles in plant protection against biotic stress.

Results

Tomato has two T D' genes that are differentially expressed
Previous studies of TD-encoding genes in tomato and potato have focused on a single
orthologous gene whose expression in leaves is induced by various stress conditions
including wounding and jasmonate treatment (Samach et al., 1991; Hildmann et al., 1992;
Samach et al., 1995; Schaller et al., 1995; Strassner et al., 2002; Li et al., 2004). We
previously reported that the jail mutant of tomato, which is defective in all known
jasmonate responses as a consequence of a null mutation in Coil, fails to express this TD
gene but nevertheless does not exhibit symptoms (e.g., stunted growth) of Ile deficiency
(Li et al., 2004). This observation led us to test the hypothesis that tomato uses a different
TD isozyme for Ile biosynthesis. Indeed, searches of the tomato EST database provided
evidence for a second expressed TD gene. The corresponding full-length cDNA is
predicted to encode a 606-amino-acid protein with a molecular mass of 66,182 Da
(Figure 2.1A). The recombinant protein expressed in E. coli converted Thr to a-
ketobutyrate in a manner that was inhibited by exogenous Ile (data not shown), indicating
that the enzyme is an authentic TD. For reasons explained below, we henceforth refer to
this previously uncharacterized gene as SlTDI, and refer to the JA-inducible gene
initially described by Samach et a1. (1991) as SlTD2.

SlTDl and SlTD2 share 48% amino acid sequence identity (Figure 2.1A). Both

proteins contain a predicted chloroplast-targeting sequence, as well as canonical catalytic

48

and regulatory domains found in other plant and bacterial TDs. Phylogenetic analysis
showed that plant TD sequences cluster into two major groups (Groups 1 and 2; Figure
2.1B). SlTDl was more similar to TDs from Arabidopsis (66% identity), poplar (68%
identity), and rice (69% identity) than it was to SlTD2. Because Arabidopsis, poplar, and
rice each contain a single TD gene, this finding supports the idea that SlTDl performs a
housekeeping role in Ile biosynthesis. JA-inducible isozymes from tomato (SlTD2) and
potato (StTD2) comprised a distinct subgroup of proteins that, interestingly, were closely
related to a TD sequence from chickpea. The JA-inducible TD from N. attenuata, which
has a dual role in Ile biosynthesis and post-ingestive defense (Kang et al., 2006),
occupied an intermediate position in the phylogeny and thus was not assigned to either
group.

We used RNA blot analysis to compare the developmental and stress-induced
expression patterns of SlTDI and SlTDZ. SITDI was constitutively expressed in all
tissues examined (Figure 2.2A). In contrast, SIT D2 transcripts accumulated to very high
levels in immature buds and unopened flowers, but were not detected in unstressed leaves
and other vegetative tissues. Expression of SIT D2 in leaves was massively induced in
response to methyl-JA (MeJA) application, as previously reported (Hildmann et al., 1992;
Samach et al., 1995; Li et al., 2004) (Figure 2.2B). MeJA had little or no effect on SIT D1
transcript levels. SlTD2 expression was induced locally and systemically in response to
mechanical wounding, whereas SlTDI mRNA levels were not affected by wounding
(Figure 2. 2C). Taken together, these findings provide strong support for the hypothesis

that SlTDI and SIT D2 serve distinct physiological roles.

49

Digestion of bulk tomato leaf protein in the gut lumen of M. sexta larvae

As a prelude to studying the fate of TD2 in the M sexta digestive system, we used SDS-
polyacrylamide gel electrophoresis (PAGE) to qualitatively assess changes in bulk
tomato leaf protein during passage through the insect. These studies were facilitated by
analysis of caterpillars raised either on wild-type (WT) plants or on mutants that are
affected in the IA signaling pathway. These mutants included a transgenic line (35S::PS)
that constitutively expresses high levels of JA-inducible proteins (JIPs) (McGurl et al.,
1994; Bergey et al., 1996), and the jaiI mutant that fails to express TD2 and other JIPs
(Li et al., 2004). A phenol-based protein extraction procedure was used to isolate total
protein from three sources: 1) insect-darnaged leaves; 2) midgut content from actively
feeding larvae; and 3) larval feces (i.e., frass). The polypeptide profile of total leaf protein
was much different from that of protein isolated from midgut content or frass (Figure
2.3A). One conspicuous difference was the large subunit (RbcL) of ribulose-1,5
bisphosphate carboxylase-oxygenase (Rubisco). As the most abundant soluble protein in
tomato leaves, RbcL is a major source of amino acids for phytophagous insects and a
convenient marker for bulk leaf protein (Sheen, 1991; Bemays and Chapman, 1994; RF,
1994; Felton, 1996). In contrast to the high level of RbcL in herbivore-damaged leaves,
midgut and frass contained very little intact RbcL. Efficient digestion of chloroplast
proteins within the midgut was confirmed by western blot analysis with antibodies
against the chloroplast outer envelope protein Toc75 (Figure 2.3B)(Reumann et al.,
' 2005). We also found that a peroxisomal matrix protein, acyl-CoA oxidaselA (AcxlA),

accumulated in leaves but not in the midgut content or frass (Figure 2.3C). These results

50

demonstrate that bulk tomato leaf protein is efficiently degraded during passage through
the M sexta digestive system.

Numerous discretely sized polypeptides exhibiting a wide range of molecular
weights were present in frass extracts (Figure 2.3A). The polypeptide profile of frass
from larvae reared on the various genotypes exhibited several reproducible differences.
For example, frass extracts from larvae grown on jail plants contained more discretely
sized, higher-molecular-weight polypeptides in comparison to the WT and 35S::PS frass
samples (Figure 2.3A). A second host genotype-specific difference was a protein of ~40-
kDa that accumulated in WT and 35S::PS frass but not in frass from jail -reared larvae
(Figure 2.3A, arrow). Differential accumulation of this protein was also observed in the
midgut content, but not in herbivore-damaged leaves. This pattern of accumulation

suggests that the 40-kDa polypeptide is a JIP that is stable in the M sexta gut.

Proteolytic processing of TD2 in the lepidopteran gut

We previously reported mass spectrometry evidence indicating that a form of TD2
lacking the enzyme’s regulatory domain accumulates in M sexta midgut content and
frass (Chen et al., 2005). The predicted size of this TD2 variant was consistent with its
identity as the above-mentioned 40-kDa protein (Figure 2.3A). To test this hypothesis, a
gel slice containing the 40-kDa protein was digested with trypsin and the resulting
peptides were sequenced by LC-MS/MS. Among 18 unique peptides that were
confidently identified (P<0.05), all showed an exact match to the catalytic domain of

TD2 (Figure 2.4A). The predicted molecular weight of the protein defined by LC-MS/MS

51

(Met52 to K418) was 38,890 Da, which was in good agreement with the size observed by
SDS-PAGE.

Mature TD2 isolated from tomato tissues has an apparent molecular mass of 55
kDa (Samach et al., 1991; Samach et al., 1995). We used western blot analysis to
determine whether there is a product-precursor relationship between the truncated TD2
variant, designated pTD2, and the 55-kDa protein. Anti-TD2 antibodies cross-reacted
with a ~55-kDa protein in herbivore-damaged WT and 35S::PS leaves (Figure 2.48). The
absence of this polypeptide in jail leaves, in which TD2 is not expressed, confirmed the
specificity of the antibody for TD2. In contrast to leaf tissue, pTD2 was the predominant
form of the protein in midgut and frass extracts. These results indicate that TD2 is
proteolytically processed to pTD2 following ingestion of foliage by M sexta. A small
amount of unprocessed TD2 in midgut extracts was observed upon prolonged
development of western blots (Figure 2.4B, asterisks). Shorter exposure times showed
that pTD2 migrates as a doublet, suggesting heterogeneity in the size of the processed
protein.

M sexta is highly specialized for feeding on tomato and other solanaceous plants.
To determine whether proteolytic processing of TD2 occurs in the gut lumen of a
generalist herbivore, we analyzed the TD2 content in frass from T richoplusia ni (cabbage
looper) caterpillars that were raised on tomato foliage. Western blot analysis showed that
T. ni fiass contained a form of TD2 that co-migrated with pTD2 from M sexta frass
(Figure 2.4C). The absence of this polypeptide in frass from jail-reared T. ni larvae
confirmed that the cross-reacting protein is derived from TD2. TD activity was detected

in frass from T. ni larvae grown on WT plants (Figure 2.4D). Consistent with a

52

processing event that removes the regulatory domain, this activity was insensitive to
feedback inhibition by 10 mM Ile. We conclude that ingestion of tomato foliage by both
specialist (M sexta) and generalist (T. ni) insect herbivores results in proteolytic removal

of the regulatory domain of TD2.

Biochemical properties of pTD2

To investigate the biochemical properties of pTD2 in more detail, we purified the enzyme
from M sexta frass. An aqueous buffer system effectively extracted active pTD2 from
frass (Figure 2.5A). The 40-kDa polypeptide co-purified with Ile-insensitive TD activity
during subsequent purification steps (data not shown). Following the final stage of
purification by gel filtration chromatography, we estimated that pTD2 was at least 90%
pure as determined by SDS-PAGE (Figure 2.5B). The purified enzyme was active against
L-Thr and L-Ser. Kinetic analysis showed that the apparent Km of L-Thr and L-Ser was
2.3 and 3.0 mM, respectively. The Vmax for L-Thr was ~5000 umol/mg protein/hr,
which was about 1.5 times higher than the Vmax for L-Ser. The enzyme was highly
active in an alkaline pH range that matches that of the lepidopteran midgut; little or no
activity was observed at pH values below 6.0 (Figure 2.6A). pTD2 was also active over a
wide range of temperatures. Optimal enzyme activity against L-Thr was observed at 58°C

(Figure 2.6B).

Identification of plant defensive proteins by shotgun proteomic analysis of insect

frass

53

Excretion of pTD2 as an active enzyme from M sexta and T. ni led us to hypothesize that
insect frass may be a useful source of material in which to identify other defense-related
proteins. To test this idea, we used a shotgun proteomic approach to catalogue and
quantify tomato proteins in frass from M sexta caterpillars reared on tomato foliage. The
total protein content of frass was digested with trypsin and the resulting peptide mixture
was subjected to LC-MS/MS. Protein identifications were considered positive if at least
two peptides derived from the same protein were confidently detected in searches of the

MS/MS data against the tomato EST database. These stringent criteria resulted in

identification of 20 distinct tomato proteins with probability scores of P<1 0'4 (Table 2.1).

Wound- and jasmonate-inducible proteins comprised the largest group of tomato
proteins in M sexta frass (Table 2.1). Among the proteins previously implicated in
defense against lepidopteran insects were TD2, LAP-A (Gu et al., 1999; Chen et al.,
2005), cathepsin D inhibitor (CD1) (Lison et al., 2006), and a germin-like protein (GLP)
similar to a GLP isozyme fi'om N. attenuata (Lou and Baldwin, 2006). Two stress-
inducible proteins of unknown function were also identified. One of these is a member of
a plant-specific group of stress-related proteins that contain a lipoxygenase homology
(LH) domain (Coker et al., 2005). The second uncharacterized protein is a chloroplast-
targeted member of the highly conserved ngF family of proteins (Leitner-Dagan et al.,
2006). We previously showed that the gene encoding this protein, which is annotated in
the tomato EST database as a protein translation inhibitor, is regulated by the jasmonate
signaling pathway (Li et al., 2004).

Proteins implicated in plant defense against pathogens were also identified in

M sexta frass (Table 2.1). Among the pathogenesis-related (PR) proteins identified were

54

the P69A and B members of the subtilisin-like family of endoproteases (PR-7) (Tornero
et al., 1996, 1997), B-l,3-glucanase (PR-2) (Domingo et al., 1994), lignin-forrrring
peroxidase (PR-9) (Vera et al., 1993), and a hevein-like protein P2 (PR-4) (Linthorst et
al., 1991). A xyloglucan-specific fungal endoglucanase inhibitor protein previously
reported from tomato, potato, and tobacco (N aqvi et al., 2005) was also identified. All of
these proteins contain an N-terminal signal peptide for targeting to the secretory pathway
(Table 2.1), and most have been shown to be expressed in response to pathogen infection
or wounding.

All other tomato proteins identified in M sexta frass, with the exception of
mitochondrial malate dehydrogenase, were chloroplastic metalloproteins (Table 2.1).
These included plastocyanin, ferredoxin, superoxide dismutase, and carbonic anhydrase.
Given that TD2, LAP-A, and the ngF-related protein (see above) are also plastid-
localized, it would appear that chloroplast proteins are highly represented in the frass.
The failure to identify peptides corresponding to Rubisco in this experiment argues
against the possibilty that this phenomenon results from passage of the intact plastids
through the insect digestive tract.

The number of mass spectral counts obtained for a given protein by LC-MS/MS
provides a quantitative measure of the protein’s abundance in the extract (Old et al.,
2005; Gilchrist et al., 2006). Based on this information, pTD2 and LAP-A were among
the most abundant tomato proteins in the frass (Table 2.1). LAP activity assays were
performed to determine whether LAP-A, like pTD2, is excreted as an active enzyme.
Both LAP and TD activity was detected in frass from larvae grown on WT plants (Figure

2.7). The lack of activity in frass from jail -reared larvae indicated that the activity was

55

specific for the JA-inducible isozymes LAP-A and pTD2. LAP activity in frass from
insects reared on 35S::PS plants was significantly greater than that in the WT frass,
which is consistent with the fact that LAP-A expression in tomato foliage is upregulated
by systemin (Chao et al., 1999). These findings indicate that LAP-A, like TD2, is

excreted from M sexta as an active enzyme.

Discussion

Functional diversification of two TD isoforms in tomato

The role of TD in producing Ile for protein synthesis is essential for all aspects of plant
growth and development. Ile is also required for the synthesis of JA-Ile, which is an
important signal for activation of jasmonate-based defenses (Staswick et al., 1998; Kang
et al., 2006). The broad distribution of jasmonates in the plant kingdom indicates that
TD’s participation in JA-lle synthesis is likely conserved in all plants. In contrast, TD’s
function as a post-ingestive defense against insect herbivores appears to be restricted to
certain plant lineages. Here, we provide evidence that tomato employs different TD
isozymes to fulfill distinct roles in Ile biosynthesis in planta and post-ingestive defense.
This contrasts the situation in native tobacco, which uses a single TD isoform to perform
both functions (Kang et al., 2006).

Several observations lead us to conclude that SlTDl performs a role in Ile
biosynthesis. First, the deduced amino acid sequence of SlTDl is more similar to TDs in
plants such as Arabidopsis, rice, and poplar, which all harbor a single “housekeeping”
form of the enzyme, than it is to SlTD2. Second, constitutive expression of TD] in all

tissues is consistent with a general role in amino acid biosynthesis. Third, recombinant

56

TDI expressed in E. coli exhibits Ile-sensitive TD activity. Finally, the jail-l mutant,
which lacks detectable TD2 expression in leaves (Figure 2.48), does not exhibit chlorosis
or other signs of Ile deficiency (Li et al., 2004). This finding provides functional evidence
that TDl can produce Ile pools that are utilized for normal grth and development in
the absence of TD2.

A specialized role for TD2 in post-ingestive defense is supported by the fact that
this isozyme accumulates in the midgut and frass of tomato-reared caterpillars (Figures
2.3 and 2.4). The gut-accumulating form of the enzyme (i.e., pTD2) has biochemical
features that presumably facilitate its action in the midgut environment. These features
include protease-resistance, an alkaline pH optimum, and the capacity to degrade Thr in
the presence of high concentrations of Ile. The high temperature optimum of pTD2
indicates that the enzyme would be active at elevated body temperatures, which for M
sexta caterpillars in natural field conditions can easily exceed 35°C (Casey, 1976). The
expression pattern of TD2 also supports a role in anti-insect defense. T D2 is coordinately
induced with other defensive genes in response to wounding and JA treatment (Hildmann
et al., 1992; Samach et al., 1995; Li et al., 2004). In reproductive tissues, TD2 is
expressed constitutively at extraordinarily high levels (Samach et al., 1991; Samach et al.,
1995). Many other JA-regulated defensive proteins including Pls, arginase, LAP-A, and
AtVSP2 exhibit a similar expression pattern (Hildmann et al., 1992; Utsugi et al., 1998;
Chao et al., 1999; Chen et al., 2004). These observations support the idea that
accumulation of TD2 and other JIPs in floral tissues protects reproductive structures from
insect herbivores. Induction of TD2 expression by diverse types of biotic and abiotic

stress (Hildmann et al., 1992; Zhao et al., 2003) raises the possibility that the enzyme

57

performs other physiological roles in planta. For example, it is possible that a
proteolytically processed form of TD2 accounts for the biodegrative TD activity observed
in senescing tomato leaves (Szamosi et al., 1993).

Functional divergence of two TD isozymes in tomato raises interesting questions
about the evolutionary origins of plant TD’s that participate in post-ingestive defense. It
is reasonable to assume that SlTD2 arose from a gene duplication event, and that
selective pressure imposed by insect herbivores led to the evolution of this isoform as a
defensive enzyme. A key feature acquired by both SlTD2 and N. attenuata TD during
evolution was regulation via the jasmonate signaling pathway. Whether or not these
enzymes evolved novel biochemical or structural features that enhance their ability to
impair insect digestive physiology is unclear. Future studies aimed at comparing the
structure, stability, and activity of SlTDl and SlTD2 promise to provide insight into this
question. The dual role of N. attenuata TD in Ile synthesis and post-ingestive defense
(Kang et al., 2006) is consistent with the intermediate position of this protein in the TD
phylogenetic tree (Figure 2.1A). The evolution of N. attenuata TD as a midgut-active
enzyme may be constrained, however, by its essential role in Ile biosynthesis. Tomato
TD2 is presumably not subjected to such constraint, and thus may be better adapted to

function in the lepidopteran gut.

Proteolytic processing of TD2
Our results confirm and extend previous evidence (Chen et al., 2005) indicating that TD2
is proteolytically processed following ingestion of foliage by the herbivore. LC-MS/MS

and other biochemical data demonstrated that the midgut-active form of the enzyme

58

(pTD2) contains the entire catalytic domain, but lacks the C-terrninal regulatory domain.
That very little unprocessed TD2 was observed in midgut content suggests that the
processing reaction occurs rapidly upon maceration of leaf tissue by the caterpillar. Based
on the structural organization of TD2 into distinct catalytic and regulatory domains, we
propose that processing involves an endoprotease that cleaves the neck region between
the two domains. Additional work is needed to test this hypothesis and to determine
whether the processing enzyme is of plant or insect origin. The finding that TD2 is
processed to an Ile-insensitive enzyme in the digestive tract of the generalist caterpillar T.
ni indicates that the processing phenomenon likely occurs in a broad range of tomato-
insect interactions.

An important consequence of proteolytic removal of the regulatory domain is loss
of feedback inhibition by Ile. The rrridgut content of M sexta larvae reared on tomato
plants contains levels of Ile (~2.5 mM) that are sufficient to inhibit TD2 activity (HC and
GAH, unpublished data). Thus, proteolytic cleavage of TD2 is required to activate the
enzyrne’s ability to degrade Thr in the amino acid-rich environment of the midgut. This
interpretation is consistent with the fact that the Thr content in the midgut of M sexta
larvae reared on TD2-containing tomato foliage is much less than that in the gut of
insects grown on TD2-deficient foliage (Chen et al., 2005). Given that dietary Thr is
limiting for M sexta growth on native tobacco (Kang et al., 2006), these results strongly
support the notion that post-ingestive processing of TD2 has evolved as a plant defense to
deplete Thr levels in the midgut. It is also possible that the defensive function of pTD2 is
related to its ability to produce ammonia, which at alkaline pH is highly toxic to

biological systems (V isek et al., 1984).

59

Herbivore-induced processing of TD2 provides support for the more general
concept that proteolysis of dietary protein is part of the plant’s overall defense response
against insect attack. Other examples of plant defensive proteins that are activated by
digestive proteases include polyphenol oxidase (Wang and Constabel, 2004) and urease
(Ferreira—DaSilva et al., 2000). Schmelz and co-workers (Schmelz et al., 2006) identified
a peptide elicitor from the oral secretion of insect herbivores that promotes the expression
of plant defense responses. Interestingly, this elicitor is a proteolytic fragment of the 7-
subunit of chloroplastic ATP synthase. It was thus pr0posed that proteolysis of dietary
proteins by insect digestive proteases can generate peptide signals that are introduced to
the host plant via insect oral secretions. The accumulation of tomato LAP-A and P69
proteins in M sexta frass leads us to suggest that plant proteases, in addition to insect

proteases, play a role in the digestion of dietary protein in the insect gut.

Shotgun proteomic analysis of insect frass

Anal droppings of insect herbivores are a rich repository of biological information
(Gangwere, 1993; Weiss, 2006). It is well established, for example, that frass is an
important source of compounds involved in host selection by insect parasitoids (Vinson,
1976). To our knowledge, the composition of plant proteins excreted by insect herbivores
has not been previously described. The proteomic analysis reported herein shows that
insect frass is enriched in pTD2 and other hyperstable plant proteins that serve defense-
related firnctions. Many of these proteins were previously shown to accumulate in the M
sexta midgut (Chen et al., 2005) and, significantly, have an established role in anti-insect

defense. CDI is a Ser PI (rather than an Asp PI) that exerts potent growth-inhibiting

60

effects on lepidopteran caterpillars (Lison et al., 2006). The frass-accumulating GLP is
closely related to a MeJA-inducible GLP from N. attenuata that has a role in resistance to
M sexta attack (Lou and Baldwin, 2006). Detection of this protein in frass raises the
possibility that GLPs exert defensive effects (e.g., H202 production) in the herbivore gut.
A role for LAP-A in post-ingestive defense is supported by the stability of the protein in
the lepidopteran digestive tract (Chen et al., 2005; this study), the enzyme’s high pH
optimum (Gu et al., 1999), co-expression with other midgut-active defensive proteins (Li
et al., 2004; Chen et al., 2005), and the increased susceptibility of LapA-silenced plants to
herbivory (Walling, 2006). There is also evidence indicating that LAP-A performs a
signaling role in jasmonate-induced expression of defensive proteins (Walling, 2006).

Shotgun proteomic analysis also identified proteins that had not previously been
implicated in plant defense. These included stress-inducible isoforms of an LH2 domain
protein that may participate in lipid metabolism (Coker et al., 2005), and a member of the
ngF family of proteins that is conserved in bacteria, yeast, animals, and plants. In the
context of TD function, it is noteworthy that ngF and related proteins have been
implicated in the regulation of Ile biosynthesis and Thr degradation (Datta et al., 1987;
Kim et al., 2001; Parsons et al., 2003). A recent study (Leitner-Dagan et al., 2006)
showed that the ngF-related tomato protein accumulates in chloroplasts of stressed
leaves, and is required for optimal photosynthetic function. Additional work is needed to
test the hypothesis that this protein has a role in defense against insects.

The expression of many tomato proteins identified in M sexta frass is promoted
by the jasmonate signaling pathway. Genes encoding these JIPs tend to be among the

most highly induced following wounding or jasmonate treatment. For example, a DNA

61

microarray study identified T D2 and LapA as the most highly expressed JA-responsive
genes among all elements on the array (Li et al., 2004), whereas proteomic analysis
showed that TD2 and LAP-A are two of the most abundant tomato proteins in the midgut
and frass of tomato-reared M sexta larvae (Chen et al., 2005; this study). Thus, there is a
strong correlation between the level of induced mRNA accumulation in leaves and
protein accumulation in the insect gut. Similar correlations hold for the ngF-related
protein, CD1, and other PIs. We suggest that jasmonate-induced accumulation of
defensive proteins in leaves, together with the stability of these proteins in the gut lumen,
provide complementary mechanisms to maximize the effectiveness of post-ingestive
plant defense. The correlation between gene and protein expression suggests that
microarray data can be used as a starting point to identify novel anti-insect proteins.
Several tomato PR proteins were excreted in M sexta frass. Nearly all of these
proteins have been shown to be highly expressed in response to pathogen infection or
wounding, and secreted into the extracellular space where they presumably interact
directly with invading pathogens (van Loon et al., 2006). The biological significance of
PR protein accumulation in frass is unclear. It is possible that these proteins accumulate
in frass simply because they are highly resistant to proteolysis. This idea is supported by
studies showing that PR proteins are extremely stable (Ferreira et al., 2001; Flamini and
De Rosso, 2006; van Loon et al., 2006). It is also possible that PR proteins perform a
physiological role in the digestive system of insect herbivores. The alkaline pH optimum
of subtilisin-like P69 proteases (Vera and Conejero, 1988), which appear to be the most
abundant PR proteins in M sexta frass, indicates that these extracellular proteases may be

activated upon entry of macerated leaf tissue into the lepidopteran gut. Studies of cysteine

62

proteases establish a precedent for the role of plant proteases in post-ingestive defense
against insect herbivores (Pechan et al., 2002; Konno et al., 2004).

An important conclusion from this and previous (Chen et al., 2005) work is that
foliar proteins have a wide range of stability in the gut lumen of phytophagous insects.
Whereas bulk dietary protein (e.g., Rubisco) is efficiently degraded in the M sexta
midgut, other plastidic proteins such as TD2 and LAP-A remain active following passage
through the gut. The most straightforward interpretation of these results is that midgut-
active defensive proteins are highly resistant to digestive proteases and, as a consequence,
are selectively enriched during passage of the food bolus through the animal. The
biophysical properties that allow pTD2 and LAP-A to accumulate and function in the
extreme environment of the lepidopteran gut remain to be determined. In this context, it
is worth noting that hyperstable (as well as alkaliphilic) enzymes are of significant
commercial interest for their use as industrial biocatalysts (Hough and Danson, 1999).
Although research in this area has focused mainly on extremophilic bacteria and Archaea,
our results suggest that frass-accumulating plant proteins can be exploited as a new
source of hyperstable enzymes.

Seminal work by Green and Ryan (1972) introduced the idea that wound-
inducible plant proteins act directly in the insect gut as a defense. The recent discovery of
TD, arginase, VSP, and proteases as anti-insect proteins extends this concept to include
plant enzymes that impair digestive physiology (Pechan et al., 2002; Chen et al., 2005;
Liu et al., 2005; Kang et al., 2006). Proteomic-based technologies provide a powerful tool
to address the question of how variation in the quantity and quality of dietary protein

influences plant-insect relations. Only recently have these approaches been used to assess

63

the effects of herbivory on large-scale changes in plant protein content (Francis et al.,
2006; Giri et al., 2006; Lippert et al., 2007). Our results demonstrate that proteomic
analysis of midgut content (Chen et al., 2005) and frass (this study) can be used to track
the fate of the plant proteome during passage through the insect digestive tract, thus
providing insight into how the plant proteome interacts with components of the insect
gut. Additional work is needed to determine the limitations of this approach for
identifying midgut-active plant defense proteins. For example, it conceivable that some
defensive proteins are degraded by microbial flora in the insect gut or frass, or that
covalent modification of dietary polypeptides in the insect gut (Felton, 1996) prevents
protein identification by MS. These limitations notwithstanding, we conclude that
proteomic analysis of frass has general utility for large-scale identification of plant
defensive proteins in virtually any plant-insect interaction for which appropriate sequence

databases are available.

Materials and Methods

Biological material and growth conditions

Solanum lycopersicum cv Castlemart was used as the wild type (WT) for all experiments
except where otherwise noted. 35S.°:PS and jail mutant lines and conditions for plant
growth were previously described (Chen et al., 2005). M sexta eggs were obtained from
the Department of Entomology, North Carolina State University (Raleigh, NC). Newly

hatched larvae were transferred directly to three-week-old tomato plants. Larval midguts

were dissected from cold-anesthetized larvae (4th - 5th instar) that were actively feeding

64

at the time of collection. Total midgut content was isolated by removing the food bolus
from the dissected midgut. Care was taken to avoid mixing the midgut content with insect

tissue. Midgut content from three to five larvae was pooled and frozen at -20°C until

. . . (1 th .
further use for protern extraction. For collection of M sexta frass, 3r to 4 instar larvae

were transferred to a Tupperware box containing cut leaves from ~6-week-old tomato
plants that were heavily damaged by M sexta feeding. The petiole of the cut leaf was
inserted through the closed cap (in which a hole was punctured) of a 1.5 ml plastic
microcentrifuge tube containing water. Cut leaves were replaced on a daily basis. Frass
was collected at least once daily, and stored at -20°C until needed for protein extraction.
The use of host genotypes that are non-inducible (jail) or constitutively induced
(35S::PS) helped to control for possible effects of wounds that were generated by leaf
cutting. Care was taken to avoid contamination of frass with intact leaf tissue.

T. ni eggs were obtained from Benzon Research (Carlisle, PA) and hatched at

30°C. Within 8 h of hatching, larvae were transferred to three-week-old tomato plants.
. th th .
Frass pellets were collected daily from 4 to 5 instar larvae grown on cut tomato

leaves. Leaves were replaced daily. Pelletswere stored at -20°C until further use.

Protein extraction and enzyme assays

A modified version of a phenol-based protein extraction method (Constabel et al., 1995)
was used to isolate total protein for SDS-PAGE (Figure 2. 3A) and immunoblot analysis.
Frozen leaf tissue, midgut content, and frass were ground in liquid nitrogen to fine
powder. One volume equivalent of the ground tissue was mixed with two volumes of

protein extraction buffer [0.7 M sucrose, 100 mM Tris-HCI (pH 6.8), 20 mM EDTA, 100

65

mM KCl, 2% (v/v) 2-mercaptoethanol, and 1 mM phenylmethylsulfonyl fluoride].
Following the final protein precipitation step (Constabel et al., 1995), protein pellets were
resuspended in solution containing 9.5 M urea, 2% (v/v) Nonidet detergent, and 5% 2-
mercaptoethanol. Samples were heated to 65°C for 5 min prior to SDS-PAGE.

An aqueous buffer system (Chen et al., 2005) was used to prepare frass extracts
for TD and LAP enzyme assays, purification of pTD2, and shotgun proteomic analysis.
TD activity measurements in the presence or absence of Ile were as previously described
(Chen et al., 2005). LAP activity was measured as previously described (Gu et al., 1999;
Nampoothiri et al., 2005) with some modifications. Briefly, the reaction mixture

contained 1 ml of a 2.5-mM solution of L-leucine-p-nitroanilide substrate in 100 mM

NaOH-glycine buffer (pH 8.5), 1 ml 0.5 mM MnC12 in 100 mM NaOH-glycine buffer

(pH 8.5), and 0.5 ml H20. Reactions were initiated by the addition of 5 pl of protein

extract prepared from frass. Following 30 min incubation at 37°C, the reaction was
stopped by the addition of 1 ml glacial acetic acid. The absorbance was measured at 405
nm against a mock reaction devoid of enzyme. A standard curve was prepared with p-
nitroaniline. L-leucine-p-nitroanilide and p-nitroaniline were purchased from Sigma (St.

Louis, MO).

Purification of pTD2 from M. sexta frass

Frass obtained from tomato-reared M sexta larvae was ground in liquid nitrogen to a fine
powder. Ten g of powder was extracted with approximately 2 volumes of 100 mM Tris
buffer (pH 7.5) containing 1 mM EDTA, 1% (vol/vol) 2-mercaptoethanol, and 0.1 mM

PMSF. The mixture was centrifuged at 20,000g for 10 min and the resulting supernatant

66

was filtered through a 0.45-um filter (Millipore). Protein in the supernatant was brought
to 30% (w/v) saturation with ammonium sulfate and stirred for 2 h at 4°C. Precipitated
proteins were discarded following centrifugation at 20,000g for 15 min. The supernatant
was adjusted to 65% saturation with ammonium sulfate and stirred for 4 h at 4°C.
Following centrifugation at 20,000g for 15 min, the supernatant was discarded. The
protein precipitate was dissolved in 15 mM Tris buffer (pH 7.5) and then desalted on a
Sephadex G-25 colurrm (Amersham Pharmacia Biosciences) that was equilibrated with
the same buffer. The desalted extract was applied to a DEAE-cellulose (DE52;
Maidstone, England) column (30 x 1.5 cm i.d.) that was equilibrated with the same
buffer. Proteins were eluted from the column with a linear gradient of 0 to 0.5 M NaCl in
15 mM Tris-HCI (pH 7.5). Fractions (1.5 ml) were collected with a Gilson fraction
collector (model FC-203B; Middletown, WI). Fractions containing the bulk of TD
activity (~2/3 of the activity peak height) were pooled and concentrated with a 10-kDa
molecular weight cut-off Amicon centrifugal filter (Millipore, Bedford, MA).
Concentrated enzyme preparation (0.2 ml) was loaded on a Superose-12 gel filtration
column (Pharmacia, Piscataway, NJ) that was pre-equilibrated with 50 mM Tris-HCl (pH
7.5) containing 100 mM NaCl. Proteins were eluted with the same buffer at a flow rate of
0.6 ml/min on a Waters HPLC system equipped with a model 600 pump, a 996
photodiode array detector, and a 717-plus autosampler. Fractions (1.0 ml) were collected
manually and assayed for TD activity. The specific activity of TD increased ~30-fold
during the purification procedure. Protein concentrations were determined by the
Bradford method, using bovine serum albumin as a standard. The relative purity of

protein samples was assessed by SDS-polyacrylamide gel electrophoresis and staining of

67

gels with Coomassie Brilliant Blue R-250. A Superose-12 gel filtration column
equilibrated with a 50 mM Tris-HCI (pH 7.5) solution containing 100 mM NaCl was

used to estimate the native molecular weight of purified pTD2.

Cloning and expression analysis of SITDI and SlTD2

A search of the tomato EST database (version 11.0, released on June 21, 2006) at The
Gene Index Project (http://compbio.dfci.harvard.edu/tgi/plant.html) identified a tentative
consensus sequence (TC176654) annotated as a TD. This sequence, which we designated
as SITDI, was distinct from the published tomato TD sequence (Samach et al., 1991).
Three cDNA clones (cTOF22A12, cTOC4022, and cTOD22M4) corresponding to SIT DI
were obtained from the Boyce Thompson Institute and sequenced in their entirety.
Overlapping regions between the clones showed that the three cDNAs corresponded to
the same transcript. The assembled full-length cDNA sequence of SlTDl, deposited in
GenBank as accession number EF 026088, has an 1821-bp open reading fi'ame, a 91-bp 5’
UTR upstream of the ATG initiation codon, and a 235-bp 3’ UTR excluding poly(A)
residues.

A full-length SlTD2 cDNA was obtained by RT-PCR (DuraScriptTM, Sigma) of
total RNA isolated from leaves of tomato plants (cv Castlemart) that were treated with
MeJA for 24 h. The PCR primers for the cDNA amplification step were TD5 (forward)
5’-ATGGAATTCCTTTGTTTAGCCCCA-3’ and TD3 -2 (reverse) 5’-
GCCATTACATTACATTGGATACAT-3’. The resulting PCR product was cloned into
the pGEM-T Easy vector (Promega) to yield pGEM-SIT D2. The sequence of the cDNA

insert perfectly matched that of the TD sequence reported by Samach et a1. (1991).

68

RNA blot experiments were performed with total RNA isolated from WT tomato
plants (cv Micro-Tom), as previously described (Howe et al., 2000). Roots, stems,
petioles, and leaves were collected from 3-week-old plants. Floral tissues were harvested
from six-week-old plants. Wound and MeJA treatments were performed according to

published methods (Howe et al., 2000; Li and Howe, 2001). RNA blots were probed with

32P-labeled SlTDI and SIT D2 cDNAs, or with a cDNA for eIF 4A as a loading control.

Antibody production and western-blot analysis

The pET30TD plasmid for expression of AtTD was kindly provided by Dr. Renaud
Dumas (Wessel et al., 2000). This vector, which is derived from pET30a+, was digested
with NdeI and SalI to release the AtT D cDNA. The resulting linearized vector was ligated
to a PCR product containing a modified SIT DZ cDNA. This cDNA was prepared by PCR
amplification of pGEM-SITDZ with the following primer sets. The forward primer (5’-
TGATTAATAIQATGTCACCAATTGTTTCTGTG-3’) was designed with an Asel
restriction site that is compatible with the Ndel site on the vector. The underlined ATG
sequence in the forward primer represents the initiation codon in the resulting
recombinant protein. This ATG codon replaces the first amino acid (Ly552) of the mature
protein, thus eliminating the 51-amino-acid chloroplast-targeting sequence (W essel et al.,
2000). The reverse primer was designed with a TGA stop codon upstream of the SalI site
(5’-ATGTCGACTCACTCACTTACTACAAGGAA-3’). The amplified PCR product
was cloned into the pET30 vector (described above) to yield a plasmid called pET30-
TD2. This expression vector produces a truncated form of SlTD2 that lacks 51 amino

acids corresponding to the N-terminal chloroplast-targeting sequence.

69

TD2 expression and purification were performed as described previously (Wessel
et al., 2000) with minor modifications. A 750-mL log-phase culture of E.coli BL21
(DE3) cells containing the pET30-TD2 plasmid grown at 37°C was induced by the
addition of IPTG to a final concentration'of 0.5 mM. The induced cells were incubated
with agitation at 28°C for 15 h, and then harvested by centrifugation at 20,000g for 15
min. The cell pellet was resuspended in buffer A [50 mM Hepes (pH 7.5) and 1 mM
EDTA] containing 0.1 mM PMSF and 1 mM dithiothreitol (DTT). The cell suspension
was treated with lysozyme (1mg/mL) for 30 min at 30°C, followed by sonication. Cell
debris was removed by centrifugation at 20,000g for 15 nrin and the supernatant (crude
lysate) was saved. The crude lysate was brought to 30% (w/v) saturation with ammonium
sulfate and stirred for 1 h at 4°C. The precipitated proteins were discarded, and the
solution was adjusted to 65% saturation with ammonium sulfate. The precipitate was
collected by centrifirgation at 20,000g for 15 min, resuspended in buffer A, and applied to
a Sephadex G-25 column, and eluted with buffer A. The eluate was collected and loaded
onto a Whatrnan DEAE-cellulose DE52 column (30 x 1.5 cm) equilibrated with buffer A.
The column was eluted with 600 mL of a 0-400 mM KCl gradient. Elution was
monitored by absorbance at 280 nm and enzyme activity. Fractions containing TD
activity were pooled, concentrated with an Amicon Ultra-15 30-kDa filter (Millipore),
and subjected to further purification on a Pharmacia Biotech AKTA FPLC system.
Specifically, the concentrate was applied to a HiLoad26/60 Superdex 200 column
(Amersham Pharmacia Biotech) previously equilibrated with buffer A. The column was
eluted with 1.5 column volumes of buffer A containing 150 mM KCI. Fractions

containing TD activity were pooled and loaded directly into a HiPrep 16/10 column

70

(Pharmacia Biotech) equilibrated in buffer A. The column was eluted with 20 column
volumes of a 0-400 mM KCl gradient. The enzyme was concentrated with an Amicon
Ultra-15 30-kDa filter to a concentration of 4.5 mg/mL. The purified enzyme was
determined to be >95% pure as determined by SDS-PAGE. Rabbit polyclonal antibodies
against purified TD2 antigen were produced by a commercial vendor (Cocalico
Biologicals, Reamstown, PA) according to their standard protocol, using 0.5 mg of the
purified protein as antigen.

Western blot analysis was performed as previously described (Schilmiller et al.,
2007), using anti-SlTD2 antibodies that were diluted 1:2000 in TTBS (Tris-buffered
saline with 0.1% Tween 20) containing 1% nonfat milk. Blots were washed 3 times with
TTBS and then incubated with a peroxidase-conjugated anti-rabbit secondary antibody
(1:10,000 dilution; Sigma, St. Louis, MO). TD2 protein-antibody complexes were
visualized with the SuperSignal West Pico Chemiluminescent substrate (Pierce,
Rockford, IL) according to the manufacturer’s instructions. Anti-AcxlA antibodies
(Schilmiller et al., 2007) were used at a 1:1,000 dilution. Toc75 antibodies (Tranel et al.,

1995) were used at a 1:2,000 dilution.

LC-MS/MS-based identification of tomato proteins in M. sexta frass

(1 th .
Frass was collected from M sexta larvae (3r to 4 instar) that were grown on WT (cv

Castlemart) tomato plants as described above. Frass was frozen in liquid nitrogen, ground
to a fine powder, and extracted with 100 mM Tris buffer (pH 7.5) containing 1 mM
EDTA, 1% (v/v) B-mercaptoethanol, and 0.1 mM PMSF. Extracts were centrifuged at

20,000g for 10 min at 4°C. Approximately 60 pg protein was electrophoresed through a

71

4% SDS-polyacrylamide stacking gel (1.5 cm) and ~l cm into a 12% resolving gel. Gels
were stained with Coomassie Blue and the protein-stained region of the gel was excised.
Proteins within the gel piece were reduced and alkylated, followed by digestion with
trypsin as previously described (Chen et al., 2005).

The extracted peptides were automatically injected by a Michrom Paradigm

Endurance Bio-Cool Autosampler onto a Paradigm Platinum Peptide Nanotrap (C18,
0.15 x 50 mm) and washed for 5 min. The bound peptides were eluted onto a 10 cm x 75
pm New Objectives Picofrit column packed with Microm Magic C18 AQ packing
material. Peptides were eluted from this column over 90 min with a gradient of 5% B to
90% B, with constant 10% C in 76 min using a Michrom Paradigm MDLC (Buffer A,
100% water; Buffer B, 100% acetonitrile; Buffer C, 1% formic acid), at a flow rate of
300 nL/min. Eluted peptides were analyzed with a ThermoElectron LTQ Linear Ion trap
mass spectrometer (Thermo Electron Crop, San Jose CA). The top five ions in each
survey scan were subjected to data-dependent zoom scans followed by low-energy
collision-induced dissociation (CID). The resulting MS/MS spectra were converted to
peak lists using BioWorks Browser v 3.2.

The X!-tandem algorithm (Craig and Beavis, 2003, 2004) was used to search
MS/MS spectra against the tomato EST database from the TIGR Gene Indices
(MM/compbiodfci.harvard.edu/tgi/). Protein identifications were considered positive if
tWO or more peptides from the same protein were identified, each with a probability score
0f P S 0.01 (Eriksson and Fenyo, 2004). Of the 20 tomato proteins cataloged by this

Procedure, 18 proteins were identified on the basis of two or more unique peptides.

72

Accession Numbers

The GenBank accession number for the SIT DI cDNA sequence is EF 026088.

Acknowledgements

We thank Doug Whitten of the Michigan Proteome Consortium for assistance with
proteomic experiments and bioinformatic analysis of LC-MS/MS data. Guanghui Liu and
Rob Larkin (MSU) are acknowledged for assistance with the expression and purification
of recombinant TD. John Froehlich (MSU) and Leron Katsir are acknowledged for the
anti-Toc75 antibody and assistance with constructing SlTD homology models,
respectively. Bonnie St. John provided helpful assistance with immunoblot analysis. We
also thank R. Dumas for the pET30-TD expression vector for A. thaliana TD. Tomato

EST clones were obtained from the Boyce Thompson Institute at Cornell University.

73

Table 2.1. Host plant proteins identified in frass of tomato-reared M sexta larvae.

 

 

Protein identity or best BLAST hit1 Access. No. Unigene2 No. Peptides Local.3
Jasmonate- and wound-inducible proteins
Threonine deaminase (pTD2) AAA34171 U312574 25 CP
Leucine aminopeptidase (LAP-A) AAC49456 U312377 23 CP
Trypsin inhibitor-like protein AAA80497 U3 1 33 84 SP
Cathepsin D inhibitor (CD1) CAC00536 U312623 4 SP
ngF family protein4 BT013249 U313029 CP
Stress-induced LH2 domain protein 81209796 U315202 3 SP
Aspartic protease inhibitor B1929912 U312622 2 SP
Germin-like protein CN384576 U318102 2 SP
Pathogenesis-related proteins
P69B (PR-7) CAA71234 U313775 20 SP
P69A (PR-7) CAA64566 U3 13772 7 SP
Lignin-fornring peroxidase (PR—9) CAA50597 U321126 10 SP
B—l ,3-Glucanase (PR-2) CAA52872 U314382 7 SP
Endoglucanase inhibitor protein AAN 87262 U3 14071 3 SP
PR protein P2 (PR-4) CAA41439 U316008 2 SP
Other proteins
Plastocyanin CAA32121 U312690 16 CP
Malate dehydrogenase AAU29198 U313128 7 MT
Ferredoxin B193 1 178 U312380 3 CP
Superoxide dismutase AAQ09007 U315384 2 CP
Carbonic anhydrase AW093720 U319550 2 CP
Chlorophyll a/b binding protein CAA84525 U312438 2 CP 1 In
cases

where the BLAST hit did not perfectly match a known tomato protein, the best BLAST hit is
listed.

Unigene indicates the tomato gene nomenclature provided by the SOL Genomics Network at
http://www.sgn.comell.edu/index.pl.

Local. denotes the predicted protein location. The presence of an N-terrrrinal signal peptide (SP)
for protein secretion was analyzed with the SignalP software
(http://www.cbs.dtu.dk/scrvices/SignalP). Proteins having a signal peptide probability score >
0.75 are indicated. CP, chloroplast-targeted protein; MT, mitochondrial-targeted protein.

4
Annotated in the tomato EST database (http://compbio.dfci.harvard.edu/tgi/plant.html) as a
protein translation inhibitor (Li et al., 2004).

74

Figure 2.1. Tomato has two distinct TD isoforms.

(A) Comparison of the deduced amino acid sequence of SlTDl (TDI) and SlTD2 (TD2).
Amino acids that are either identical (black) or similar (gray) between the two sequences
are indicated. The inverted triangle denotes the site of cleavage (between Leu51 and
Ly552) of the plastid—targeting peptide on TD2 (Samach et al., 1991). The dotted line
denotes the “neck” region that connects the N-terminal catalytic and C-terrninal

regulatory domains, based on homology modeling with E. coli TD (Gallagher et al.,
1998)

(B) Phylogenetic relationship of SlTDl and SlTD2 to TDs from other plants. Shown is an
unrooted neighbor-joining tree constructed with MEGA 3.1 (Kumar et al., 2004) from the
following sequences: maize (ZmTD; CO446428); sugarcane (SoTD; CA208490); rice
(OsTD; NP_001051069); poplar (PtTD; estExt__fgenesh4_pg.C__280257); Arabidopsis
(AtTD; NP_187616); Aquilegia formosa x pubescens (AfT D; DT735861); tomato
(SlTDl; ABK20067) (SlTD2; P25306); potato (StTDl; BI436101) (StTD2; X67846); N.
attenuata (NaTD; AAX22214); chickpea (CaTD; Q39469). GenBank accession numbers
are given in parentheses. The indicated bootstrap values (% of 1000 repeated tree
reconstructions) show the reliability of each branch of the inferred tree. The two major
subgroups of the tree are designated “Group 1” and “Group 2”.

75

)2 (TD2).
equences
:u51 and
med line
terminal
at et al.,

.wn isan
from the
90): riCe
bidopslS

tomato
846); N
rumbefS
ted tree
0 major

TDl
TD2

TDl
TD2

TDl
TD2

TDl
TD2

TDl
T02

T01
TD2

TDl
TD2

TDl
TD2

TDl
TD2

TDl
TDZ

TDl
TD2

TDl
TD2

TDl
TD2

1 FTA NSC SSVIVPI
1 LAP FS--T SIPSDHTS

51
48

101
91

151
140

201
190

251
240

301
289

351
339

401
389

451
439

501
489

551
539

601
589

  
 

 
 

  
 

S KS AK
S F- QN GST PL
E LS TVTE S SLQCE
IVSVP PVEN ------- T D
GGVTA S
SDEL- I
' ‘ S S
- S S

 

 

 

HTI
DKES

 

Figure 2.2. Differential expression of SITDI and SITDZ in tomato.

(A) Five ug total RNA from root (R), stem (S), petiole (P), leaf (L), immature flower bud
(B), unopened flower (UF), and opened flower (OF) was immobilized to a membrane and
hybridized to full-length cDNA probes for SITDI and SlTD2. A duplicate blot (lower
panel) was stained with ethidium bromide to visualize rRN A, as a means to determine the
quality and quantity of the loaded RNA.

(B) Expression of SIT DI and SIT D2 in response to treatment with MeJA. F our-week-old
plants were exposed to MeJA vapor for the indicated length of time (h) in an enclosed
box, after which leaves were harvested for RNA extraction. RNA isolated from untreated
plants (0 hr time point) was analyzed as a control. RNA gel blots were hybridized to
cDNA probes for SITDI and SlTD2. Blots were also hybridized to an elF 4A probe as a
loading control.

(C) Expression of SIT DI and SIT DZ in response to mechanical wounding. Leaflets on the
2nd and 3rd firlly expanded leaves (counted from the oldest leaf) of four-week-old plants
were wounded three times with a hemostat, perpendicular to the main vein. Total RNA
was isolated separately from the lower wounded (local) and the upper unwounded
(systemic) leaves at various times (h) after wounding. RNA was also isolated from
unwounded plants (0 h time point) as a control. RNA gel blots were hybridized to cDNA
probes for SIT DI and SIT D2. Blots were also hybridized to an eIF 4A probe as a loading
control.

77

A RSPLBUFOF
TD1l..- .ufi

T02

 

  

rRNA

 

 

 

    

B
TD1
TDZ
eIF4
C Local Systemic
012481224 012481224
TD1
TDZ

eIF4

78

A Leaf Midgut Frass
jai1 WT PS jai1 WT PS jai1 WT PS

 

 

100

 

 

 

 

Acx1 A

 

 

 

Figure 2.3. Digestion of tomato foliar protein during passage through the M. sexta
digestive tract.

(A) M sexta larvae were reared to the 4th instar on jail, WT, or 35S:.'PS (PS) tomato
plants. Total protein was extracted fi'om three different sources of material: tomato leaves
that were heavily damaged by the herbivore (Leaf); the midgut content of actively
feeding, 4 -instar larvae (Midgut); fecal droppings from actively feeding, 4th-instar
larvae (Frass). Forty pg of protein from each sample was analyzed on a 10% to 18%
polyacrylamide gradient gel, which was stained with Coomassie blue. The position of
protein standards (kDa) is shown on the left, as is the major polypeptide corresponding to
the large subunit of Rubisco (RbcL). The arrowhead denotes a 40-kDa polypeptide
observed in midgut and frass of larvae reared on WT and 35S::PS plants.

(B and C) Western blot analysis of the protein samples shown in (A) with polyclonal
antibodies raised against the chloroplast outer envelope protein Toc75 (B) or the
peroxisomal matrix protein AcxlA (C).

79

Figure 2.4. Proteolytic processing of TD2 in the digestive tract of M. sexta and T. ni
larvae.

(A) MS-based identification of a truncated form (pTD2) of TD2. Underlined letters
denote the amino acid sequence of TD2 that was identified by LC-MS/MS analysis of a
gel slice containing the 40-kDa protein. The chloroplast-targeting peptide of TD2 is
denoted by lowercase italicized letters. The “neck” region that connects the N-terminal
catalytic and C-terrninal regulatory domains is indicated by bold letters.

(B) Proteolytic processing of TD2 in M sexta. Protein (10 pg per lane) isolated from
tomato leaf, M sexta midgut content, and M sexta frass was separated by SDS-PAGE.
The gel was subjected to Western blot analysis with an anti-TD2 antibody. See Figure 2.
3A for a description of the samples. The cross-reacting polypeptide labeled “TD2”
corresponds to the 55-kDa mature form of the protein that accumulates in herbivore-
damaged leaves. The polypeptide labeled “pTD2” is the proteolytically processed form of
TD2 that lacks the C-terminal regulatory domain. Asterisks denote faint bands
corresponding to incompletely processed TD2.

(C) Proteolytic processing of TD2 in the digestive tract of T. ni larvae. Protein was
analyzed by Western blot analysis as described in (B). Protein was isolated from the
following material: lane 1, herbivore-damaged WT leaves; lanei2, frass from M sexta
larvae reared on WT plants; lane 3, frass from T. ni larvae reared on WT plants; lane 4,
frass from T. ni larvae reared on jail plants.

(D) TD activity in fiass fiom T. ni larvae reared on WT tomato plants. Frass extracts were
assayed for TD activity in the absence or presence of 10 mM Ile. Data indicate the mean
and SD of measurements from four different pools of frass.

80

A
mefl cl aptrsfs tnpkl tksipsdh ts t tsriftyqnmrgs tmrplalp
lKflSPIVSVPDITAPVENVPAILPKVVPGELIVNKPTGGDSDELFQYLVD
ILASPVYDVAIESPLELAEKLSDRLGVNFYIKREDKQRVFSFKLRGAYNM
MSNLSREELDKGVITASAGNHAQGVALAGQRLNCVAKIVMPTTTPQIKID
AVRALGGDVVLYGKTFDEAQTHALELSEKDGLKYIPPFDDPGVIKGQGTI
GTEINRQLKDIHAVFIPVGGGGLIAGVATFFKQIAPNTKIIGVEPYGAAS
MTLSLHEGHRVKLSNVDTFADGVAVALVGEYTFAKCQELIDGMVLVANDG
ISAAIKDVYDEGRNILETSGAVAIAGAAAYCEFYKIKNENIVAIASGANM
DFSKLHKVTELAGLGSGKEALLATFMVEQQGSFKTFVGLVGSLNFTELTY
RFTSERKNALILYRVNVDKESDLEKMIEDMKSSNMTTLNLSHNELVVDHL
KHLVGGSANISDEIFGEFIVPEKAETLKTFLDAFSPRWNITLCRYRNQGD
INASLLMGFQVPQAEMDEFKNQADKLGYPYELDNYNEAFNLVVSE

3 Leaf Midgut Frass
jai1 WT PS jai1 WT PS jai1 WT PS

 

 

 

 

 

 

 

 

 

Tnz-v ~ 5
*
pTDZ"

C 1 2 3 4 D
193- 3300
118— g 250

_ 0
99 7 3.9.
Q.
54- '3 g 150
.9?
e 100
38_ E
. 1 50
29— v
0

 

 

 

 

 

- Ile + Ile

81

 

 

 

 

A jai1 wr PS B 1 2 3
95‘ 95‘ «H. I..- ’7
fr . , 3- J
52‘ -. * on
52- 37-‘% _ “T
_ Q ' _., t
—t 1" f : ‘
29_ .
19—

 

 

 

Figure 2.5. Purification of pTD2 from M. sata frass.

(A) Frass pellets collected from M sexta larvae grown on the indicated host plant
genotype were extracted with an aqueous buffer to maintain TD activity. The resulting
protein (~60 pg) was separated by SDS-PAGE, and the gel was stained with Coomassie
blue. The arrow indicates pTD2, which accumulates in fiass from larvae grown on WT
and 35S::PS plants, but not in frass fiom jail-reared larvae. Migration position of
molecular weight markers (kDa) are shown on the left.

(B) Frass from M sexta larvae grown on WT tomato foliage was used as the starting
material for purification of pTD2. The Coomassie-stained gel shows pTD2 at various
steps of the purification procedure: Lane 1, 65% (NH4)ZSO4 cut; Lane 2, pooled TD-
containing fractions from DEAE-cellulose chromatography; Lane 3, pooled TD-
containing fractions from Superose-12 gel filtration chromatography. Migration position
of molecular weight markers (kDa) are shown on the left. The arrow indicates the
polypeptide corresponding to pTD2.

82

>
D

 

 

 

 

 

 

 

 

 

§100 $100+
3‘
E 80' :5 30,
T5 1 ’8'
‘° 601 a 60.
E J .—
“>’ 401 3’3 40
'5 "" "
s 1 s
o
a: 20, a: 20-
0 . . . . 0 ' f. . ' . a T
2468101214 20 40 60 80
pH of assay buffer Temperature (C)

Figure 2.6. Biochemical features of purified pTD2.

(A) pH optimum of pTD2 activity assayed against L-Thr in the following buffer systems:
Na-citrate (closed circles); NaPO4 (inverted triangles); glycine (closed squares); KH2P04
(open diamonds). The data are expressed relative to the activity at pH 9.0.

(B) Effect of temperature on pTD2 activity. Enzyme activity was assayed against L-Thr
at the indicated temperature (°C). The data are expressed relative to the activity at 58°C.

83

 

9??

A
h

mm

TD activity
(pmol/mg protein/h
o 8 3 8 8 8 8

 

LAP activity

(pmol/mg proteln/hr)
8

 

 

jai1 WT 35S::PS

Figure 2.7. LAP-A is excreted from M. sexta as an active enzyme.

(A) TD activity was measured in frass collected from M sexta larvae grown on the
indicated host plant genotype. Data represent the mean and SD of three independent
measurements.

(B) LAP activity in the same extracts used in (A). Data represent the mean and SD of
three independent measurements.

84

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Chapter III

The defensive function of tomato threonine deaminase 2 is activated by

an insect digestive protease

Abstract

Threonine deaminase (TD) is a pyridoxal phosphate-dependent enzyme that catalyzes the
dehydration of threonine (Thr) to or-ketobutyrate and ammonia, which is the committed
step in the biosynthesis of isoleucine (Ile). All plant and bacterial TDs consist of an N-
terminal catalytic domain and a C-terminal regulatory domain that binds the allosteric
inhibitor Ile. Solanum lycopersicum (cultivated tomato) has two TD-encoding genes,
designated TD] and TD2. TDI likely serves a primary metabolic role in Ile synthesis
because the gene is constitutively expressed in all tissues and is homologous to T BS from
plants such as rice and Arabidopsis that contain a single TD gene. In contrast, TDZ
expression is strictly regulated in an inducible manner by the plant stress hormone
jasmonate. Accumulation of TD2 protein in frass (feces) of insect herbivores fed on
tomato leaves led to the hypothesis that TD2 is a defensive enzyme that acts post-
ingestively to deplete Thr in the insect gut, thereby reducing the nutritional quality of
ingested plant material. Interestingly, ingestion of tomato foliage by lepidopteran insects
results in proteolytic removal of the TD2 regulatory domain, resulting in a TD2 variant
(pTD2) that degrades Thr without being inhibited by Ile. Here, we employ in vitro TD2-

cleavage assays to show that the protease responsible for removal of the regulatory

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domain is most likely a chymotrypsin-like protease of insect origin. Incubation of TD2
with a crude mixture of lepidopteran digestive proteases resulted in production of pTD2
whereas the same protease mixture rapidly degraded and catalytically inactivated TDI. In
addition to differential susceptibility to digestive proteases, TD2 exhibited high
temperature stability in comparison to TDl. Lepidopteran larvae performed better on
tomato TD2 antisense plants compared to wild-type plants, indicating that TD2 serves a
role in anti-insect defense. These findings provide evidence for functional divergence of
tomato TD isoforms, and further indicate that TD2 evolved structural and catalytic

properties that facilitate its post-ingestive role in plant defense.

Introduction

The growth and development of insect herbivores depends on their ability to acquire
essential amino acids from dietary protein (Nation, 2002). The essential amino acids for
insects are arginine, threonine, isoleucine, leucine, lysine, histidine, methionine,
phenylalanine, tryptophan, and valine (Nation, 2002; Chang, 2004). Insect herbivores are
faced with the challenge of acquiring these amino acids from plant tissues that, unlike
animal tissue, contain low protein levels (Mattson, 1980; Bemays and Chapman, 1994;
Bemays et al., 2004). Plants have evolved several defensive strategies to impair the
ability of insects to digest dietary protein. One strategy involves host plant secondary
metabolites such as phenolics and tannins that act as digestibility reducers (Cates and
Rhoades, 1977). Plants also synthesize various proteins that act postingestively to disrupt

amino acid acquisition from dietary protein. These include proteinase inhibitors (PIs) that

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impair insect digestive enzymes, polyphenol oxidases (PPOs) that covalently modify
dietary protein, and enzymes such as threonine deamidase (TD) and arginase that degrade
essential amino acids (Felton et al., 1994; Duffey and Stout, 1996; Bolter and Jongsma,
1997; Murdock and Shade, 2002; Pechan et al., 2002; Wang and Constabel, 2004; Chen
et al., 2005), These plant defense proteins are thought to act coordinately and
synergistically to starve herbivores of key nutrients (Felton, 2005; Zhu-Salzman et al.,

2008)

The identification of an inducible isoform (TD2) of tomato TD as a highly
abundant protein in the midgut and feces of tomato-reared lepidopteran larvae led to the
suggestion that this enzyme has a post-ingestive role in depletion of Thr (Chen et al.,
2005; Chen et al., 2007). In plants and microorganisms, TD (EC 4.2.1.16) catalyzes the
dehydration/deamination of Thr to yield or-ketobutyrate and ammonia. This reaction
constitutes the first step in the biosynthesis of isoleucine (Ile) and is tightly regulated by
feedback inhibition (Hatfield and Umbarger, 1970). The enzyme consists of an N-
terrninal catalytic domain containing the cofactor pyridoxal phosphate and a C-terminal
regulatory domain that binds Ile. The X-ray crystal structure of E. coli TD shows that the
catalytic and regulatory domains are connected by a neck-like region, and that the

holoenzyme assembles as a tetramer (Gallagher et al., 1998).

Tomato contains two TD genes that have presumably evolved under different
selective pressures. TB] is constitutively expressed in all tissues, whereas TDZ
expression is dependent on the plant hormone jasmonate (JA) (Chen et al., 2007).
Phylogenetic analysis of plant TDs showed that there are two distinct groups: the first

group includes tomato TDI and TDs from rice, Arabidopsis, and poplar, which serve a

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primary metabolic role in Ile synthesis (Garcia and Mourad, 2004; Joshi et al., 2006;
Chen et al., 2007). The second cluster is represented by TD2, the potato ortholog of TD2,
and a TD isoform from chickpea (Chen et al., 2007). Nicotiana attenuata, which has one
TD gene (Kang et al., 2006), occupies an intermediate position in the phylogenetic tree.
The first clue that TD2 serves a role in plant defense was provided by Hildmann and
coworkers (1992), who showed that potato TD2 is highly expressed in response to
mechanical wounding or treatment with ABA and methyl-JA (MeJA). Expression of the
TD2 ortholog in tomato, as well as N. attenuata TD, is also strongly induced by JA
(Samach et al., 1995; Hermsmeier et al., 2001; Strassner et al., 2002; Li et al., 2004; Chen
et al., 2007). The overall pattern of expression of TD2 in tomato is very similar to that of

P15 and other JA-inducible proteins, and is indicative of a role in anti-insect defense.

The anti-nutritional role of TD2 appears to be enhanced by proteolytic cleavage of
the enzyme’s regulatory domain following ingestion of tomato leaves by insect larvae.
Removal of the regulatory domain results in an enzyme (designated pTD2) that is
insensitive to feedback inhibition by Ile and, as a consequence, highly efficient in
degrading Thr in the Ile-rich environment of the lepidopteran gut (Chen et al., 2005).
This finding indicates that whereas TD2 may function in planta as a biosynthetic enzyme
that is regulated by Ile-mediated feedback inhibition, proteolytic processing within the
insect gut converts TD2 to a degradative enzyme (pTD2) that depletes Thr (Chen et al.,

2005; Chen et al., 2007).

The mechanism by which TD2 is converted to pTD2 during ingestion of plant
tissue by lepidopteran insects is not known. It is possible that proteolytic cleavage is

catalyzed by a plant protease that gains access to TD2, which is located in the chloroplast

96

(Samach et al., 1995), during ingestion of leaf tissue by the insect. An alternative
hypothesis is that TD2 processing is catalyzed by an insect protease. Here, we report the
results of experiments designed to discriminate between these two possibilities and to test
the putative role of TD2 in anti-insect defense. First, we show that transgenic tomato
lines silenced for the expression of TD2 are compromised in resistance to Spodoptera
exigua larvae. Second, we demonstrate that the regulatory domain of TD2 is removed in
the lepidopteran midgut in the absence of plant proteins by a chymotrypsin-like protease
of insect origin. Finally, we show that TDI and TD2 have unique biochemical features,
including protease- resistance and thermostability. These findings suggest that these two
isoforms are specialized for their respective roles in primary metabolism and plant

defense.

Materials and Methods

Plant material and transformations

The full-length TD2 cDNA was obtained by PCR with the primers 5’-
ATCTCGAGATGGAATTCCTTTGTTTAGCCCCA-3’ and 5’-
ATGGATCCGCCATTACATTACATTGGATACAT-3’ that contain restriction sites for
cloning into the XhoI and BamHI sites of the binary vector pBIlZl (Clontech). The
resulting vector contains T DZ in antisense orientation under the control of the CaMV 358
promoter, and was used to transform tomato (Solanum lycopersicum cv. Microtom)
cotyledons with Agrobacterium strain AGLO as previously described (Li et al., 2005).

Kanamycin-resistant explants were screened by PCR with the primers 35S-G 5’-

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CTATCCGCAAGACCC-3’ and TD5 5’- ATGGAATTCCTTTGTTTAGCCCCA-3’to
confirm the presence of the transgene. A secondary screen for TD2-deficient
transforrnants took advantage of the fact that TD2 is constitutively expressed to very high
levels in tomato flowers (Samach et al., 1995). Fully open flowers were ground in 200 pL
of extraction buffer (100 mM Tris-HCI ,pH 6.8, 20 mM EDTA,100 mM KCl, 2% (v/v) 2-
mercaptoethanol, 1 mM PMSF). The mixture was centrifuged for 20 min at 20,000g and
the supernatant used for activity assays. Two antisense lines, TDAs7 and TDAslS,
exhibited low levels of TD activity in flowers and thus were selected for subsequent
experiments. Insect bioassays were conducted with T3 generation plants obtained from a
TDAs7 homozygous line, as well as with segregating progeny from a TDAs15 line that
was hemizygous for the transgene; transgene-containing progeny from this line were
identified by PCR screening. Conditions for plant growth have been described previously

(Chen et al., 2005).

- Insect rearing and bioassays

M sexta (Linne') eggs were obtained from the Entomology Department at North Carolina
State University. T. ni (Hfibner) and S. exigua (Hiibner) eggs were obtained from Benzon
Research. All eggs were hatched at 30°C. Tobacco hornworrn diet was obtained from
Carolina Biological Supply. The diet for the noctuids was composed of enriched soybean
fluor, wheat germ, sucrose, mineral salt and vitamin mix, agar, methyl paraben, sorbic
acid, aureomycin, and calcium propionate (Southland Products Inc.). The diet was

supplemented with raw linseed oil for rearing T. ni. For the bioassay of S. exigua on

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antisense T D2 plants, larvae were reared on diet for 72 h prior to transfer to plants. These
larvae were pre-selected to have a uniform weight at the start of the feeding trial.
ANOVA was used to test for significant differences in weight between larvae reared on
the wild-type and transgenic plants, and larval weight data was log-transformed to‘meet
ANOVA assumptions. Untransformed data were used in figures. Differences between
treatments were assessed with the Least Significance Difference test. Statistical analysis
was performed with SAS® software, Version 9.1.3 of the SAS System for Windows
(Copyright © 2002-2003). For the experiments where TD was added to the food, the diet
was prepared according to the manufacturer’s instructions and allowed to cool to 55°C
before adding recombinant TD2. The diet was poured on plates, cut into pieces and

placed in plastic cups containing one insect each.

Expression of TD] and TD2 in E. coli

The vector (pET30TD) for expression of Arabidopsis thaliana TD (AtTD) was kindly
provided by R. Dumas (Wessel et al., 2000). Vectors for expression of tomato TDs were
constructed by excising the AtTD cDNA from pET30TD with Ndel and Sal], with
subsequent replacement with the tomato TD cDNAs. Prior to cloning of a T D1 coding
sequence lacking the N-terminal chloroplast targeting signal into these sites, the cDNA
clone cTOD22M4 was site-directed mutagenized to remove two Ndel sites from the T D]
coding region. This manipulation did not alter the amino acid sequence of TDI. The
forward primer (5’-CGCAT_AiG_TCATCGCCAGCTACG-3’) was designed to contain an

Ndel restriction site and to replace the first amino acid (Leu-55) of the mature protein

99

with a Met (underlined). The reverse primer (5’-
CGCTCGAGTC_AATGCATTATGAGCTG-3’) contains a stop codon (underlined) and
an Xhol site that is compatible with the vector SalI site. Cloning and functional
expression of TD2 was performed as previously described (Chen et al., 2007), except that
Ile (1 mM) was added to the extraction buffer (50 mM Tris, pH 7.5, 1 mM EDTA) and all
buffers used for purification except the final resuspension buffer. The purity of
recombinant enzymes was determined by SDS-polyacrylamide gel electrophoresis and

estimated to be above 95%.

TD enzyme assays

The protein concentration of purified TDI and TD2 was calculated based on its amino
acid composition and Beer’s law. TD activity was determined by measuring the
formation of keto acids as initially described by Hatfield and Umbarger (Hatfield and
Umbarger, 1968). The reaction mixture (250 pl) contained 150 mM Tris-HCI, pH 9, 10
mM Thr, 12 mM KCl. Reactions were initiated by addition of enzyme, incubated at 30°C

for 30 min, and terminated by the addition of TCA. Keto acid formation was monitored

by absorbance at 505 nm on a Beckman Spectrophotometer. For Km and Vmax
calculations, reactions were initiated by addition of a range (0 to 40 mM) of substrate
(Thr) concentrations. Reactions were performed in triplicate and the data fitted with a

non-linear regression model using Prism 5 for Windows, trial version 5.02 (GraphPad

Software).

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TD2 cleavage assays

For use as protease sources for the TD2 cleavage assay, total protein was extracted with
an aqueous extraction buffer (250 mM Tris-HCI, pH 8, 2.5 M NaCl) from leaves
wounded by T. ni, and frass collected from the same insect reared either on tomato or
artificial diet. The cleavage test was performed by incubating 0.2-0.4 pg of TD2 at 37°C
with 0.25 pg of the protease in assay buffer (150 mM Tris-HCl, pH 9.0, 2 mM CaClz, 0.5
mM DTT). The reaction product was loaded on a 10% SDS-polyacrylamide gel for
visualization of TD2 cleavage by western blot analysis or Coomassie blue staining.
Protease inhibitors were obtained from GBiosciences and used at 2X concentration,

except for TPCK (obtained from Sigma) and used at 250 pM.

Protease fractionation

The TD2-cleaving protease was partially purified from T. ni frass as follows. Frass (2
grams fresh weight) was collected fi'om first- to fifth-instar-T. ni larvae reared
individually on artificial diet, and frozen at -20°C until further use. Frozen frass was
ground in liquid nitrogen to a fine powder and homogenized in the extraction buffer
described above. After centrifugation for 30 min at 3,210g, the supernatant was brought
to 25% saturation with ammonium sulfate and stirred for 1 h at 4°C. The precipitated
protein was collected by centrifugation and saved (0-25% fraction). The supernatant was
adjusted to 50% ammonium sulfate, and the insoluble fraction collected as before (25-
50% fraction). Once again, the supernatant was taken to 75% saturation and the pellet

saved (SO-75% fraction). The fractions were resuspended in extraction buffer and

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dialyzed overnight against 500 volumes of extraction buffer at 4°C before being

quantified with a Bradford assay.

Results

Silencing of TD2 expression improves S. wrigua performance on tomato

To test the hypothesis that TD2 has a role in anti-insect defense, Agrobacterium-mediated
transformation was used to generate transgenic tomato lines that express an antisense
TD2 cDNA under the control of the Cauliflower mosaic virus (CaMV) 358 promoter.
Regenerated plants expressing the 35S-TD2-As transgene were screened for TD2
deficiency by measuring TD activity in flowers, which constitutively express TD2 to very
high levels (Samach et al., 1995). 3SS-TDZ-As lines TDAs7and TDAslS were identified
among several transgenic lines with reduced TD2 expression (Figure 3.1). The severely
reduced expression of TD2 in these lines did not result in any morphological or
developmental phenotypes related to Ile deficiency, in contrast to the observations in
Nicotiana species (Sidorov et al., 1981; Colau et al., 1987; Kang et al., 2006). This
observation is consistent with the idea that tomato TDl is responsible for synthesis of Ile

for use in general metabolism, whereas TD2 serves a specialized role in defense (Chen et

aL,2005;2007)

The TDAs7and TDAslS lines were used for their low levels of enzyme activity
for bioassays with S. exigua. After 4 days of feeding, larvae growing on TDAs7 plants
were significantly heavier than larvae on growing on wild-type plants (t-test: 5.26, p-

value < 0.0001). Afier 7 days of feeding, the difference in larval weights was more

102

pronounced (t-test: 6.58, p-value < 0.0001) (Figure 3.2.A and B). A significant difference
in the weight of larvae grown on TDAslS and wild-type plants was observed at the 7-day
time point (t-test: 3.14, p-value: 0.0021) (Figure 3.2.3). Increased caterpillar performance
on TDAs7 and TDAslS lines was inversely correlated with TD2 protein levels in S.
exigua-challenged leaves (Figure 3.2.C). The accelerated growth of S. exigua on both
lines was also correlated with increased leaf consumption (data not shown). The
experiments were repeated three times with similar results. We conclude that tomato TD2

has a role in defense against attack by S. exigua.

Previous studies have shown that TD2 is proteolytically processed to a lower-
molecular-weight, Ile-insensitive form of the enzyme (pTD2) during digestion of tomato
leaves by M sexta (Chen et al., 2005), and that pTD2 is excreted as a stable and active
enzyme in the frass of tomato-reared M sexta and T. ni (Chen et al., 2007). To determine
whether this is also the case for S. exigua, we used immunoblot analysis to determine the
TD2 content of frass collected from S. exigua grown either on wild-type or TDAs7
plants. The results in Figure 3.2.D show that TD2 is processed to pTD2 during passage of
tomato leaves through S. exigua. The absence of immunodetecable pTD2 in frass fi'om
larvae reared on TDAs7 plants indicates that the anti-TD2 antibody does not cross-react

with proteins of insect origin.

Proteolytic cleavage of TD2 by a lepidopteran protease

We developed an in vitro processing assay to investigate the identity of the protease

responsible for removal of TD2’s regulatory domain in the lepidopteran gut. This assay

103

used recombinant TD2 as a substrate for processing by candidate proteases in plant and
insect protein extracts. Formation of the ~40 kDa pTD2 product from the higher
molecular weight (~55 kDa) TD2 precursor was assessed by separation of reaction
products by SDS-PAGE and visualization of the cleavage product by Coomassie staining
or irnmunoblotting. As an initial test to determine whether TD2 processing results from
the action of a host plant- or insect-derived protease, recombinant TD2 was incubated at
pH 9 (to simulate conditions in the lepidopteran gut) with crude protein extract from
insect-damaged tomato leaves or frass collected from larvae (T richoplusia. ni) grown on
tomato leaves. As shown in Figure 3.3, TD2 was efficiently processed to a ~40 kDa
product in the presence of the frass extract but not upon addition of either the leaf extract
or a mock control. The identity of the 40 kDa protein as pTD2 was verified by LC—
MS/MS (Figure 3.4), as well as by measurement of Ile—insensitive TD activity (see
below). To exclude the possibility that TD2 processing results fiom the presence of a
tomato protease excreted in the frass, we conducted processing assays with frass
collected from larvae grown on artificial diet. The results showed that fiass from artificial
diet-reared larvae, which had not encountered tomato proteins, contained a protease that

efficiently processes TD2 to pTD2 (Figure 3.3).

We next addressed the question of whether TD2 is processed by an insect
protease during passage of food through the gut. Artificial diet containing recombinant
TD2 was fed to T. ni larvae that had previously been reared on diet lacking TD2.
Proteolytic cleavage of TD2 was then assessed by immunoblot analysis of frass protein.
The appearance of pTD2 was detected in frass fiom diet-reared larvae (Figure 3.5.A),

thus demonstrating that TD2 is processed in the T. ni digestive system in the absence of

104

any other tomato protein. Repetition of this experiment with M sexta larvae, whose
relatively large size facilitates dissection of the gut into its component foregut, midgut,
and hindgut compartments, showed that dietary TD2 was completely processed in the
midgut, hindgut, and frass, whereas the predominant form of TD2 was the 55 kDa protein
and only trace amounts of pTD2 were found in the foregut (Figure 3.5.B). These findings

indicate that the midgut is the major site of TD2 processing.

TD2 is cleaved by a chymotrypsin-like protease

To further investigate the nature of the excreted insect protease that cleaves TD2, frass
protein obtained from T. ni larvae reared on artificial diet was fractionated by ammonium
sulfate precipitation (Figure 3.6.A and B). The protein fraction precipitating between 50-
75% ammonium sulfate saturation contained a protease activity that cleaves the
regulatory domain from TD2. This protein fraction, referred to hereafier as the frass
protease, was largely deficient in the protease responsible for generating a slightly lower
molecular weight cleavage product (pTD2*) that presumably results from further
processing of pTD2. The partially purified fiass protease was insensitive to inhibitors of
aspartic (i.e., pepstatin), metallo (i.e., EDTA), cysteine (i.e., E-64) and amino (i.e.,
bestatin) peptidases (Figure 3.7.A). Among several serine protease inhibitors tested, the
frass protease was insensitive to inhibition by aprotinin but sensitive to PMSF. We also
tested N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK) and chymostatin, which
are specific inhibitors of chymotrypsin, to determine whether the protease might be a

trypsin- or chymotrypsin-like serine protease. The results showed that cleavage of TD2

105

was partially inhibited by TPCK and strongly inhibited by chymostatin. Chymostatin
inhibited TD2 processing by the frass protease in a dose-dependent manner (Figure
3.7.B). These results indicate that TD2 is cleaved by a chymotrypsin-like protease that is

excreted from T. ni larvae.

To further test the hypothesis that TD2 is cleaved in the insect by a chymotrypsin-
like protease, we used LC-MS/MS to compare the TD2 proteolytic fragments produced
by the frass protease to those generated by digestion of the substrate with commercial
chymotrypsin (Figure 3.4.B). For this purpose, the pTD2 product generated by the two
proteases was excised from an SDS-PAGE gel and subjected to in-gel digestion with
trypsin and subsequent analysis by LC-MS/MS. As shown in Figure 3.4.A, peptide
fragments identified after cleavage of TD2 with the T. ni protease completely overlapped
with the peptides identified from TD2 cleavage by commercial chymotrypsin. Taken
together, these results provide evidence that removal of the TD2 regulatory domain
during passage of the enzyme through the lepidopteran gut is catalyzed by a

chymotrypsin-like protease of insect origin.

TDI and TD2 exhibit differential stability to insect digestive proteases

The existence in tomato of separate genes encoding “housekeeping” (TDI) and defense-
related (TD2) TD isoforms provides an opportunity to study how enzymes involved in
primary metabolism have adopted novel roles in defense. We first addressed this question
by comparing the effect of T. ni digeStive (excreted) proteases on TDI and TD2. As

expected, incubation of TD2 with T. ni digestive proteases resulted in accumulation of

106

the pTD2 fragment that is highly resistant to proteases (Figure 3.8). That the pTD2
product formed in this reaction corresponds to the enzyme’s catalytic domain was
confirmed by enzymatic assays showing that processing is accompanied by an increase in
Ile-insensitive TD activity (Figure 3.8.A and B). Incubation of TDI with the frass
protease fraction resulted in rapid disappearance of the substrate (Figure 3.8.A) and a
corresponding disappearance in TD activity (Figure 3.8.C). In the absence of frass
proteases, TDl activity was completely inhibited by the addition of 10 mM Ile, and
formation of an Ile-insensitive form of TDI was not observed. We conclude that TDI and
TD2 are highly susceptible and resistance, respectively, to degradation by insect digestive

proteases.

pH optimum and temperature stability of TD] and TD2

Among the biochemical properties that likely facilitate TD2’s ability to degrade Thr in
the insect gut are activity at alkaline pH and resistance of the catalytic domain to insect
digestive proteases. Previous experiments have shown that pTD2 is active at high pH and
has a high temperature optimum (Chen et al., 2007). We performed experiments to
determine whether these features are shared by TDl. Both recombinant enzymes
exhibited a pH optimum between 8 and 10 (data not shown), indicating that the
alkaliphilic nature of TD2 is not specific for this isoform. The temperature stability of the
two proteins was assessed by measuring TD activity over a range of temperatures
between 16 and 80°C (Figure 3.9.A). TD2 was active over a broad range of temperatures,

with optimal activity observed around 60°C. This finding is consistent with the

107

temperature optimum of 58°C reported for pTD2 (Chen et al., 2007). In contrast to TD2,
TDI was active over a range of lower temperatures. Maximal TDl activity was observed
at 16°C and no activity was detected at temperatures above 55°C. The heat lability of
TDI was confnmed by incubating the enzyme at 55°C for various times prior to
measuring enzyme activity (Figure 3.9.8). Incubation of TDI at 55°C for 1 min was
sufficient to completely inactivate the enzyme. The same incubation condition (i.e., 1 min
at 55°C) resulted in a 7% decrease in TD2 activity. Following 30 min incubation at 55°C,
the activity of TD2 was 76% of that observed in a control reaction maintained at 30°C
(Figure 3.7A). These results demonstrate that the thermostability of TD2 is unique to this

isoform.

Kinetic parameters of TD2 and TD]

Purified recombinant enzymes were used to compare the kinetic parameters of the tomato

TDs. Using Thr as a substrate, the apparent Km of TDI and TD2 was 5.7 i 0.6 and 1.0 i
0.1 mM, respectively. The Vmax of TDI (18,759 i 746 pmoles or-KB/mg protein/h),
however, was 7.5-fold greater than the Vmax of TD2 (2,494 i 61 pmoles or-KB/mg
protein/h). The turnover number (kau) for TDl was approximately 8-fold higher than for

TD2. The keg/Km ratio was 36% larger for TDI, indicating that TDl is more efficient in

catalysis. We also determined the sensitivity of the enzymes to various concentrations of
Ile (Figure 3.10). The activity of TDI was completely inhibited by 1 mM and higher

concentrations of Ile. TD2 activity was strongly but not completely inhibited by 1 mM

108

Ile. Even in the presence of 10 mM Ile, a low level TD2 activity (~5% of the control) still

remained.

Discussion

Defensive function of tomato TD2

Several lines of evidence indicate that tomato TD2 performs a role in plant defense
against lepidopteran insects. First, the T D2 gene is strongly upregulated in tomato, potato
and N. attenuata in response to leaf wounding (Hildmann et al., 1992; Samach et al.,
1995; Dammann et al., 1997; Hermsmeier et al., 2001; Schittko et al., 2001; Kang et al.,
2006). Second, the protein is enzymatically active in the frass of lepidopteran larvae
reared on tomato leaves (Chen et al., 2005; Chen etal., 2007). And third, previous studies
with JA mutants of tomato established a correlation between loss of TD2 expression and
reduced resistance to arthropod herbivores (Li et al., 2002; Li et al., 2004). The latter
genetic evidence was inconclusive because these mutants are compromised in many JA-
regulated defensive traits. Direct evidence for an anti-insect role of TD was provided by
Kang and coworkers (2006), who showed that silencing TD expression in N. attenuata
increased the growth rate of M sexta larvae. This study also showed that supplementation
of N. attenuata leaves with exogenous Thr improved insect performance, indicating that
Thr is likely a limiting nutrient for caterpillars feeding on wild-type plants. Silencing of
NaT D also caused reduced accumulation of Ile and, as a consequence, decreased
production of the bioactive jasmonate jasmonoyl-L-isoleucine (JA-Ile) (Staswick et al.,

1998; Kang et al., 2006; Katsir et al., 2008). The increased susceptibility of NaTD-

109

silenced plants to insects was thus attributed mainly to reduced signaling through the JA
pathway, although a post-ingestive fimction in Thr degradation is also possible (Kang et
al., 2006). Because TD is encoded by a single gene in N. attenuata, it is likely that the
essential function of this enzyme in branched-chain amino acid synthesis has constrained

its ability to evolve as an anti-nutritional enzyme.

In contrast to N. attenuata, our results provide evidence that two distinct TD
isoforms in tomato, TDI and TD2, serve separate roles in Ile synthesis and post-ingestive
Thr degradation, respectively. This conclusion is supported by the finding that TDZ-
silenced transgenic plants are compromised in resistance to the generalist insect S.
exigua. In the most strongly silenced lines (TDAs7), TD2 activity was severely reduced
(e.g. ~30-fold in flowers) without obvious negative effects on plant growth and
development. This observation, together with the normal vegetative growth habit of jail
plants that fail to express detectable levels of TDZ (Li et al., 2004), implies that TDl
provides sufficient amounts of Ile for growth and development. The fact that other JA-
regulated defenses (e.g. PIs) are expressed normally in TD2-silenced lines also suggests
that TD2 is not required for the synthesis of JA-Ile. Previous studies suggested that TD2
is one of multiple JA-inducible proteins in tomato that act synergistically to reduce the
nutritional quality of the plant food (Chen et al., 2005; Felton, 2005; Chen et al., 2007).
The measurable effect of silencing TD2 expression on S. exigua growth provides direct

evidence that TD2 is an important component of this induced defense response.

Biodegradative TD in tomato

110

Szamosi and coworkers (1993) described a biodegradative TD activity in senescing
tomato leaves that is insensitive to feedback inhibition by Ile. Enzymatic studies
differentiated this biodegradative isoform from an Ile-sensitive TD activity that decreased
with leaf age. Interestingly, the Ile-insensitive enzyme had a lower molecular weight and
higher affinity for Thr than did the Ile-sensitive enzyme. The authors concluded that
tomato contains distinct biodegradative (Ile-insensitive) and biosynthetic (Ile-sensitive)
TDs that are likely encoded by two different genes. Our results are consistent with the
hypothesis that the biosynthetic isoform reported by Szamosi et a1 (1993) corresponds to
TDl, whereas the biodegradative isoform corresponds to an active proteolytic fragment
of TD2 that has lost the regulatory domain. Among the many metabolic changes that
occur during leaf senescence is disintegration of chloroplasts and the upregulation of
proteases (BuchananWollaston, 1997). Such proteases may be capable of processing TD2
upon its release from plastids in senescing tissues. We did not observe processing of
endogenous TD2 in insect-damaged tomato leaves, nor did we obtain evidence for
cleavage of recombinant TD2 by proteases in crude protein extracts from damaged (but
non-senescing) tomato leaves. Future studies aimed at determining whether TD2 is

processed during tomato leaf senescence are warranted.

Evolution of TD2

As is the case for plant secondary metabolites (Fraenkel, 1959), the restricted occurrence
of T DZ to a subgroup of Solanum spp (Chen et al., 2007) suggests that this gene evolved

from TD], which serves an essential role in plant primary metabolism. One obvious

111

hypothesis is that duplication of T D1 and subsequent neofunctionalization produced a
novel isoform (TD2) that functions in plant anti-insect defense. If this idea is correct,
several key features that distinguish TD2 from TDl must have evolved over time,
presumably in response to selection pressure imposed by insect herbivores whose growth
is limited by Thr availability. One of these features is the regulation of T D2 expression by
the JA signaling pathway (Chen et al., 2007). cis-acting elements responsible for JA-
induced TD expression have been identified (Samach et al., 1995; Kang and Baldwin,
2006). It is possible that a gene duplication event placed TD2 under the control of such
cis-acting elements that pre-existed in the tomato genome. Gene duplication may be a
common strategy in the Solanaceae for recruitment of anti-nutritional defenses from
enzymes involved in primary metabolism. In support of this idea, tomato contains two
genes (designated ARGl and ARGZ) encoding the arginine-degrading enzyme arginase.
Whereas ARC] is constitutively expressed in all tissues, ARG2 is co-expressed with T D2
in response to wounding and JA treatment (Chen et al., 2004). ARG2 was also shown to
have a post-ingestive role depleting Arg in the lepidopteran midgut (Chen et al., 2005).
Another example is the enzyme leucine aminopeptidase-N (LAP-N). While all plants
appear to contain a constitutively expressed LAP-N gene, a wound- and JA-inducible
isoform (LAP-A) is found only in the Solanaceae family (Chao et al., 2000). The high
stability and activity of LAP-A in M sexta larvae reared on tomato suggests a role for
this peptidase in anti-insect defense (Chen et al., 2007). A recent study indicates that
tomato LAP-A also plays a role in modulating the JA response pathway (Fowler et al.,

2009)

112

The post-ingestive defensive function of TD2 depends on the ability of the
enzyme to efficiently degrade Thr in the insect gut. Accumulation of the protein in the
digestive tract is facilitated by wound-induced expression of TD2 in insect-damaged
leaves, as well as the inherent stability of the enzyme. Amino acid sequence determinants
that confer resistance of pTD2 to digestive proteases remain to be identified. In this
context, it is noteworthy that tomato TDI and TD2 are 51% identical (74.5% similar) at
the amino acid sequence level (Chen et al., 2007). Major blocks of sequence conservation
in TDs from E. coli, Saccharomyces cerevisiae, and several higher plants are also
conserved in the two tomato TDs (Taillon et al., 1988; Garcia and Mourad, 2004). That
both tomato enzymes are active at high pH suggests that the alkaliphilic property of TD2
did not evolve in response to the environment of the lepidopteran gut. Rather, the
inherent ability of TDI, as well as other TDs, to catabolize Thr at alkaline pH may have

provided a starting point for the evolutionary specialization of TD2 as a defense protein.

Another important biochemical feature of TD2 as an anti-insect protein is its high
stability. Our results show that TD2 has a temperature optimum around 60°C and is active
over a wide range of temperatures. This property would presumably allow the enzyme to
function at the elevated body temperatures experienced by insect larvae in natural
conditions (Casey, 1976). In addition to thermostability, the excretion of catalytically
active pTD2 from tomato-reared larvae indicates that this activated form of the enzyme is
remarkably resistant to digestive proteases (Chen et al., 2007). In contrast to TD2, TDl is
inactivated by elevated temperatures and readily degraded by lepidopteran digestive
proteases. It thus appears that TD2 has acquired unique biochemical features that are

consistent with its role in defense. Because stable proteins better tolerate mutations than

113

unstable proteins, protein stability can promote the evolution of novel biochemical
functions (Bloom et al., 2006). It has also been shown that proteins tend to be only
marginally more stable than is required by their environment (Bloom et al., 2006). These
considerations lead us to speculate that after duplication of T D], subsequent mutations
conferred increased stability to TD2 and increased capacity of the enzyme to degrade Thr
in the insect gut. Strong selection pressure imposed by insect herbivores presumably
facilitated this process of firnctional specialization. Structural biological approaches
aimed at comparing TDI and TD2 may help to elucidate sequence determinants that
confer protease resistance and thermostability to TD2, and provide insight into how these

determinants evolved.

Plant TDs, as their counterparts in bacteria, are feedback inhibited upon binding
of Ile to allosteric sites in the regulatory domain (Sharma and Mazumder, 1970; Wessel
et al., 2000; Halgand et al., 2002; Garcia and Mourad, 2004). Amino acid residues that
mediate Ile binding to the regulatory domain of Arabidopsis TD have been identified and
found to be highly conserved among TDs from monocots and dicots, including tomato
TDl (Wessel et al., 2000; Garcia and Mourad, 2004). Interestingly, two of these
conserved residues (R499 and H542 in Arabidopsis TD) are not conserved in TD2 from
tomato and potato. These changes may explain the residual activity of recombinant TD2
in the presence of high levels of Ile (Figure 3.8). Relaxed selection pressure on these
residues in the TD2 regulatory domain would be consistent with the hypothesis that
feedback inhibition by Ile is less important for the function of TD2 than it is for the

function of the TD] biosynthetic isoform.

114

In fact, a key biochemical feature of TD2 as an antinutritional protein is that
proteolytic removal of the entire regulatory domain (~150 amino acids) allows the
enzyme to efficiently degrade Thr in the presence of high concentrations of Ile (2.5 mM)
present in the midgut (Chen et al., 2005; 2007). In this study, we demonstrate that
conversion of TD2 into the biodegradative pTD2 variant occurs in the midgut of
lepidopteran herbivores. This proteolytic processing event can be catalyzed by a
chymotrypsin-like protease of insect origin. TD2 can thus be viewed as a proenzyme that
is activated by proteolytic removal of the C-terminal regulatory domain. Other examples
in which plant defense proteins are activated by insect digestive proteases have been

described (Carlini et al., 1997; Wang and Constabel, 2003, 2004; Schmelz et al., 2006).

Our results support a scenario in which TD2 accumulates in the chloroplast in
response to wound stress or other conditions that activate the JA signaling pathway. This
unprocessed form of enzyme is most likely subject to feedback inhibition by Ile pools in,
the chloroplast. Although it remains to be determined whether TD2 performs a role in
amino acid biosynthesis in planta, the complete lack of TD2 expression in the jail -1
mutant (Li et al., 2004) indicates that the enzyme is not required for normal plant growth
and development. Upon ingestion of induced leaf tissue by a lepidopteran insect, TD2 is
cleaved by a chymotrypsin-like digestive protease, presumably after release of the
substrate from the chloroplast. Because lepidopterans depend on chymotrypsin for food
digestion, it would appear that tomato has exploited this fundamental aspect of insect
digestive physiology as part of a complex antinutritional defense strategy that includes
protease inhibitors and amino acid-degrading enzymes. Chymotrypsin-like proteases in

lepidopteran insects are encoded by a large gene family (Srinivasan et al., 2006; Broehan

115

et al., 2008). Our results indicate that numerous chymotrypsin-like proteases from T. ni
are excreted in the frass (E Gonzales-Vigil and G Howe, unpublished results). The broad
substrate specificity of chymotrypsin (Kraut, 1977; Peterson et al., 1995) suggests that
more than one of these enzymes is capable of cleaving TD2. If this is the case, it would
be difficult for T. ni or other lepidopterans to adapt to TD2-mediated defense by blocking
the processing of TD2. Other mechanisms by which insects may adapt to TD2-mediated
Thr degradation include activation of transport systems that efficiently sequester dietary
Thr and proteolytic destruction of pTD2. X-ray crystallography studies promise to

provide insight into the structural basis of pTD2 resistance to digestive proteases.

Acknowledgements

The experiments described here would not have been possible without the technical

assistance of Christopher Bergum.

116

 

‘1
O

as

258

Activity (pmoles- a KB/mg prot/h)
A J}
O O

 

 

 

0 .
TDAs7 TDAs15 TDAsZO TDA321 WI"

Figure 3.1. TD activity in flowers of TD2 antisense lines.

Open flowers from individual (TI generation) transgenic plants were collected and fi'ozen
in liquid nitrogen. The tissue was ground in liquid nitrogen and 100 mg of the resulting
powder was homogenized in 600 pL aqueous extraction buffer (see Methods). Following
centrifugation, the supernatant was used for TD assays. The bars represent the mean t SE
(n=10 for TDAs7; n= 12 for TDAslS; n=15 for TDAsZO; n=8 for TDAle; n=7 for wild-

type).

117

 

larval weig ht (mg) cu

 

 

   

C WT TDAstS TDAs7 WT TDAsY
dai047047047 dai047frass047frass

 

 

 

Figure 3.2. TD2 reduces the growth rate of Spodoptera exigua larvae.

Three-day old larvae were transferred from artificial diet to 4-week old wild-type or 35S-
TD2As transgenic lines (TDAslS or TDAs7). One larva was caged per plant as described
in the Methods section. At the indicated time points after challenge (days after
infestation), larvae were weighted and returned to their plant of origin.

(A) Representative picture of larvae recovered 7 days after infestation (dai) of wild-
type and TDAs7 plants.

(B) Weight gain of S. exigua on wild-type and two independent 355- T D2As transgenic
lines. Values indicate the mean 1 SE before infestation (0), and at 4 and 7 dai. Means
with the same italicized letter are not significantly different at a p-value of 0.01
(Differences of Least Squares Means adjusted with the Bonferrroni inequality).

(C) Immunoblot analysis of TD2 protein accumulation in undamaged control (before
infestation, 0 in figure) and damaged tomato leaves at 4 and 7 dai.

(D) Immunoblot analysis of TD2 accumulation in the damaged leaves from wild-type
and TDAs7 plants. The lane marked “frass” represents protein extracted from frass of S.
exigua larvae reared on wild-type and TDAs7 transgenic plants.

118

T.ni f rass
Leaf Leaf Diet

15’ 30’ 15’ 30’ 15' 30'

  
   

 

 

195 kDa

117
97

50

38
29

 

Figure 3.3. TD2 is cleaved by an insect protease.

Recombinant TD2 (40 pg) was incubated at 37°C (pH 9.5) for the indicated time (min)
with crude protein extract (0.25 pg protein) from T. ni-damaged tomato leaves (Leaf),
frass from T. ni reared on tomato (T. ni frass, Leaf), or frass from T. ni reared on artificial
diet (T. ni frass, Diet). As a control, TD2 was incubated at 37°C with extraction buffer
only (Mock). Arrows denote the unprocessed form of recombinant TD2 (~55 kDa) and
the pTD2 processed form (~40 kDa) lacking the C—terminal regulatory domain.

119

E MEFLCLAPTRSFSTNPKLTKSIPSDHTSTI’SRIFTYQNMRGSTMRPLALPL

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

KMSPIVSVPDITAPVENVPAILPKVVPGELIVNKPTGGDSDELFQYLVDILASPWDVAIES
g PLELAEKLSDRLGVNFYIKREDKQREKLRGAYNMMSNLSREELDKGVITASAGNHA
é QGVALAGQRLNCVAKIVMPITIPQIKIDAVRALGGDVVLYGKTFDEAQTHALELSEKDGL
:3: KYIPPFDDPGVIKGQGTIGTEINRQLKDIHAVFIPVGGGGLIAGVATFFKQIAPNTKIIGVEP
YGAASMTLSLHEGHRVKLSNVDTFADGVAVALVGEYTFAKCQELIDGMVLVANDGISAAI
KDWDEGRNILETSGAVAIAGAAAYCEFYKIKNENIVAIASGANMDFS
f3 K_LHKVTELAGLG
z ....................
E SGKEALLATFMVEQQGSFKTFVGLVGSLNFTELTYRFTSERKNALILYRVNVDKESDLEK i
o — I
:5 MIEDMKSSNM‘ITLNLSHNELWDHLKHLVGGSANISDEIFGEFIVPEKAETLKTFLDAFSP
E’ RWNITLCRYRNQGDINASLLMGFQVPQAEMDEFKNQADKLGYPYELDNYNEAFNLWSEE
B 1 2 3
TD2
pTD2

 

Figure 3.4. Comparison of TD2 peptide sequence identified after TD2 processing by
frass proteases and commercial chymotrypsin.

(A) Sequencing of proteolytic processing products obtained afler incubation of TDZ with an extract
from T. ni frass (dotted line) or commercial chymotrypsin (solid line). Processing reactions were conducted
as described in the legend to Figure 3.2. Cleavage products (~40 kDa) were excised from polyacrylamide
gels and subjected to in-gel trypsin digestion and LC-MS/MS. Peptides identified were mapped to the
various domains of TD2: TP (chloroplast transit peptide), Catalytic domain, Regulatory domain.

(B) Western blot analysis of TD2 proteolysis products obtained afier incubation of recombinant TD2

with partially purified proteases excreted from T. ni (lane 1), and commercial bovine chymotrypsin (lane
2). As a control, TD2 was incubated without addition of proteases (lane 3).

120

B .
Diet Frass D'et F9lJt MQUt HQUt Frass

{—TDZ (— T02

pTD2

   

<— pTD2

Figure 3.5. In vivo processing of TD2.

Insects were reared on artificial diet until they reached the 3rd (M sexta) or 4th instar (T.
ni), at which time larvae were starved for 16 h before being transferred to TD2-
containing artificial diet. Following a 24 h period of feeding on TD2-supplemented diet,
insect frass and the remaining diet were collected. The actively feeding larvae were
frozen at -80°C for 10 min and then dissected. Protein extracts from the various dissected
gut regions were analyzed by Western blotting for the presence of TD2.

(A) T. ni larvae were fed with artificial diet containing 0.01% TD2. Each lane was
loaded with the following amount of total protein: diet, 0.5 pg; frass, 1 pg.

(B) M sexta fed larvae were fed with artificial diet containing 0.0075% TD2. Each
lane was loaded with the following amount of total protein: diet, 1 pg; foregut (Fgut in
the figure), 60 pg; midgut (Mgut); 120 pg; hindgut (ngt), 30 pg; frass, 30 pg.

121

 

 

 

Figure 3.6. Fractionation of digestive proteases excreted in the frass of T. ni reared
on artificial diet.

(A) T. ni larvae were grown on artificial diet until fiflh instar. At this stage, feces were
collected and stored at -20°C until further use for protein extraction. Protein was
extracted in an aqueous buffer (see Methods) and subsequently fractionated by
ammonium sulfate precipitation. Fractions were dialyzed against extraction buffer prior
to use in the TD2 processing assay. The cleavage assay was performed with recombinant
TD2 (0.2 pg) and one of the following protein fractions (0.25 pg fiass protein): Lane 1,
total soluble protein extracted from frass; lane 2, 0-25% ammonium sulfate cut; lane 3,
25-50% ammonium sulfate cut; lane 4, 50-75% ammonium sulfate cut; 1ane5, soluble
protein afier precipitation with 75% ammonium sulfate. TD2 cleavage products were
separated by SDS-PAGE and the gel was stained with Coomassie Blue.

(B) Coomassie Blue-stained gel showing the TD2 cleavage products generated at
various times after incubation of TD2 with the 50-75% ammonium sulfate fraction
described in panel (A).

122

 

B Chymostatin
(11M): 0 10 20 50 100 200 Mock

 

Figure 3.7. TD2 is cleaved by an excreted chymotrypsin-like protease from T. ni.

(A) Effect of protease inhibitors on TD2 processing. The indicated protease inhibitors
were incubated with a protein fraction containing the T. ni frass protease for 15 min at
25°C, at which time 0.2 pg TD2 substrate was added. Cleavage reactions were incubated
at 37°C for l h and then stopped by addition of SDS-containing loading buffer and
incubation for 10 min at 100C. Formation of pTD2 was assessed by Western blot analysis
with an anti-TD2 antibody.

(B) Dose-dependent effect of chymostatin on TD2 processing by an excreted protease
fiom T. ni. Chymostatin (at the indicated concentration) was incubated with the fiass
protease for 15 min prior to addition of 0.4 pg TD2 substrate. Reactions were incubated
and processed as described in panel (A). Reaction products were separated by SDS-
PAGE and the resulting gel was stained with Coomassie Blue. A reaction containing TD2
without the frass protease or chymostatin was included as a control (Mock).

123

Figure 3.8. Degradation of tomato TDs by insect digestive proteases.

Recombinant TD substrates (0.2 pg) were incubated with the frass protease fraction (0.25
pg) for the indicated time at 37°C. As a control, the substrates were incubated at 37°C in
the absence of frass protease (Mock).

(A) Analysis of cleavage reactions by SDS-PAGE and Coomassie staining of the gel.

(B) TD2 enzymatic activity assayed after the cleavage reaction. Activity was
measured either in the absence (black bars) or presence (gray bars) of 10 mM Ile.

(C) TDl enzymatic activity assayed after the cleavage reaction. Activity was
measured either in the absence (black bars) or presence (gray bars) of 10 mM Ile.

124

% remaining T02 activity

% remaining TD1 activity

T02 TD1

 

Frass Frass
MOCk proteases MOCk proteases

0' 60' 15' 30' 60' 0' 60’ 15' 30' 60'

 

 

 

 

 

 

 

 

on. W C. out
- DO Ile
100 - 10mM lie
80
60
40
20
0
0' 60’ 15’ 30’ 60’
Mock Frass proteases
_ no Ile
100 -10mM Ile

 

 

 

 

 

Mock Frass proteases

125

Figure 3.9. Temperature stability of tomato TD1 and TD2.

(A) Differential temperature optimum of tomato TD1 and TD2. Reaction mixtures
containing 0.20 pg recombinant TD1 (closed circles) or TD2 (open circles) were
incubated at the indicated temperature for 30 min prior to measuring TD activity. Activity
is expressed relative to the activity observed at the temperature optimum 16°C and 60°C
for TD1 and TD2, respectively.

(B) Differential heat inactivation of TD1 and TD2. Recombinant proteins (0.20 pg)
were incubated at 55°C for the indicated time (1 min to 30 min) prior to measuring TD
activity at 30°C for 30 min. Activity is expressed relative to a control reaction that was
not pre-incubated at 55°C.

126

 

:-
§.

80‘

% remaining activity

 

 

 

20 40 60
Temperature (CD)

81

 

+TD1
10° -o-TD2

 

% remaining activity
8

 

 

 

20‘
(rice—e e r +

0 5 10 15 20 25 31
Incubation time at 55°C (min)

 

 

127

 

 

1001 -TD2
arm
a 30‘
IE
8'
U) 604
.5
.5
3401
e
.s
20-
0.

 

 

   

0.51 5 10
lle(mM)

0_ 0.0

Figure 3.10. Sensitivity of tomato TDs to Ile.

Enzyme activity assays were performed in the presence of various concentrations (0 to 10
mM) of the allosteric inhibitor Ile. Enzyme activity is expressed as a percentage of the
activity measured in a control reaction that did not contain the inhibitor.

128

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Chapter IV

Differential digestion of the Solanum lycopersicum leaf proteome by

lepidopteran and coleopteran insect herbivore;

Abstract

The growth of phytophagous insects is constrained by the low protein content of plant
tissue and by the plant defensive compounds, including proteins, which impair the ability
of herbivores to acquire amino acids from the diet. In Solanum lycopersicum (cultivated
tomato) and many other plants, anti-nutritional defense responses are regulated in an
inducible manner by the jasmonate (JA) signaling pathway. Here, we used LC-MS/MS to
investigate the fate of tomato leaf proteins during passage through the gut of three insect
herbivores whose grth is negatively affected by the JA signaling pathway: the
lepidopteran generalist T richoplusia ni that consumes a wide variety of plant species, the
lepidopteran specialist Manduca sexta, and the coleopteran specialist Leptinotarsa
decemlineata that only thrive on Solanaceae species. Over 200 tomato proteins were
identified in the feces from one or more of the three herbivores. Comparative analysis of
the proteomic data showed that JA-inducible and other defense-related proteins were
highly stable in all three insects, and also revealed insect order-specific differences in the

enrichment of certain classes of dietary proteins. One of the most abundant tomato

 

*

In chapter IV, the experiments were conducted by the author of this thesis and Dr. Youfu Zhao. Dr. Zhao
was responsible for the nricroarray data and analysis.

133

proteins excreted by all three insects was an isoformof threonine deaminase (TD2) that
efficiently degrades Threonine following proteolytic removal of the enzyme’s C-terminal
regulatory domain. TD2 was proteolytically activated in the gut of both lepidopteran
insects but not in the gut of L. decemlineata. The results show that the protein content in
plant food is differentially digested by different herbivore species, and suggest that these

differences contribute to the outcome of plant-insect interactions.

Introduction

Herbivorous insects pose a significant threat to the reproductive fitness of plants in
natural and agricultural ecosystems. Plants have evolved myriad constitutive and
inducible self-protection mechanisms to thwart these attackers. Inducible defenses are
initiated upon generation of signals at the plant-insect interface that trigger de novo
synthesis of the plant defense hormone jasmonic acid (JA) (Schilmiller and Howe, 2005;
2005). IA is conjugated to isoleucine (Ile) to form the bioactive jasmonoyl-isoleucine,
which is recognized by the Coronatine Insensitive l-JAZ receptor system that
orchestrates the expression of anti-insect defenses (Browse and Howe, 2008; Howe and
Jander, 2008; Katsir et al., 2008; Yoshida et al., 2009). Mutants defective in the C011
component of the JA receptor have been described in Arabidopsis, Solanum lycopersicum
(cultivated tomato), and Nicotiana attenuata (Feys et al., 1994; Li et al., 2004; Paschold
et al., 2007). These mutants have been invaluable for studies aimed at defining the global
contribution of the IA pathway to plant defense against arthropod herbivores and other

plant aggressors (Browse and Howe, 2008; Howe and Jander, 2008; Browse, 2009).

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Gene expression profiling experiments have shown that tissue damage inflicted by
wounding or herbivore attack results in large-scale, JA-mediated changes in gene
expression (Hildmann et al., 1992; Reymond et al., 2000; Halitschke et al., 2001;
Hermsmeier et al., 2001; Strassner et al., 2002; Reymond et al., 2004; Devoto et al.,
2005; Sasaki-Sekimoto et al., 2005; Uppalapati et al., 2005). These studies provide
insight into how plant defense responses are spatially and temporally regulated, but have
been less useful for identifying specific proteins or other plant compounds that directly
deter insect feeding. LC-MS/MS analysis of plant proteins that accumulate in the insect
gut provides an alternative approach for identifying proteins that impair insect
performance (Chen et al., 2005; Chen et al., 2007). This idea is based on the fact that
many plant defense proteins are highly expressed in response to herbivory and, in
addition, are highly resistant to insect digestive proteases. Proteinase inhibitors (PIs),
polyphenol oxidases (PPOs), lectins, protease/peptidase, and enzymes involved in amino
acid degradation are among the plant proteins that have a known or proposed role in anti-
insect defense and that are stable after passage through the lepidopteran gut (Green and
Ryan, 1972; F elton et al., 1992; F elton et al., 1994; Duffey and Stout, 1996; Murdock and

Shade, 2002; Pechan et al., 2002; Chen et al., 2005).

The success of shotgun proteomic analysis depends in part on the availability of
samples that are enriched for the target proteins. Several considerations indicate that
insect frass provides an excellent source of protein for these analyses. Protease-resistant
plant proteins that escape digestion in the insect gut are highly enriched in the frass, thus
facilitating their identification by LC-MS/MS. Whereas dietary protein extract from the

insect gut may be partially digested because of non-uniform exposure to digestive

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proteases, host proteins excreted in the frass tend to be subject to homogeneous digestive
conditions by virtue of their complete passage through the animal. Chen et al (2007)
found that most of the tomato proteins that accumulate in the M sexta midgut are also

excreted in the frass, thus validating the choice of this material for proteomic analysis.

The low level of protein in plant tissue (Mattson, 1980) poses a major nutritional
challenge to phytophagous insects that must acquire essential amino acids fiom dietary
protein. The physicochemical properties of the insect digestive tract play a major role in
determining how protein-derived amino acids and other nutrients are obtained from plant
tissue. Perhaps not surprisingly, insects have evolved feeding and digestive strategies to
optimize nutrient acquisition from diverse diets. Insight into the mechanisms by which
insect herbivores cope with variation in dietary protein has come mainly from studies
performed with “model” proteins, such as casein or ribulose bisphosphate carboxylase,
incorporated into artificial diets (Martin et al., 1987; Simpson and Simpson, 1989;
Bemays and Chapman, 1994). These studies have shown that herbivores can respond to
low protein diet by increasing their rate of food ingestion or retention, a process known as
compensatory feeding (Lundberg and Palo, 1993; Yang and Joern, 1994, 1994; Lee et al.,
2004). However, because artificial diets do not accurately reflect the protein composition
of plant tissue, it is unclear whether compensatory feeding is a widespread phenomenon
in plant-insect interactions. It was recently shown, for example, that induction of
compensatory feeding by Manduca sexta in response to high PI levels in N. attenuata
was reduced by the presence of a toxic metabolite (nicotine) in the leaf diet (Steppuhn

and Baldwin, 2007). The availability of modern proteomic technologies to monitor

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hundreds of proteins in a single sample provides an attractive opportunity to better

understand the dual role of the plant proteome in host defense and insect nutrition.

Protein digestion by insect herbivores is influenced not only by the chemical
composition of the diet, but also by differences in the gut environment of different insect
herbivores. The physicochemical properties of the coleoptera and lepidoptera digestive
system, for example, differ in pH, redox potential, ionic strength, as well as in the type of
proteases used for dietary protein digestion (Dow, 1984; Johnson and Felton, 1996;
Bolter and Jongsma, 1997; Krishnan et al., 2007). Whether or not these order-specific
differences in gut physiology affect protein digestion in natural diets remains unclear. In
this study, we used LC-MS/MS to investigate the extent to which the protein content in
tomato leaves is digested by insect herbivores that have different host ranges and gut
physiologies. L. decemlineata (Colorado potato beetle), which belongs to the order
coleoptera, specializes in the nightshade family and is the most destructive potato pest in
North America, Europe and Asia (Franca et al., 1994; Hitchner et al., 2008). M sexta is
also highly specialized for feeding on solanaceous plants but belongs to the lepidoptera.
We also studied the interaction of tomato with a second lepidopteran, T richoplusia ni,
which has a wider host range that includes tomato, potato, lettuce, beans, maize, and
cotton (V ogel et al., 2007). Our results show that proteins involved in defense (i.e. JA-
inducible proteins, proteases and protease inhibitors) are enriched in the frass of the three
insects studied here. However, there were some insect order specific differences.
Additionally, we found a correlation between the levels of expression in the leaf and the
abundance of the protein in the excreta of the insects. We also show that JA-regulated

defenses confer resistance of tomato to the beetle specialist L. decemlineata and the

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generalist caterpillar T. ni, and that this form of induced defense affects different fitness

components of the two insect species.

Results

Performance of Leptinotarsa decemlineata and T richoplusia ni on tomato is reduced

by C011-dependent defenses

To assess the effect of JA-regulated defenses on the solanaceous specialist Leptinotarsa
decemlineata (Colorado Potato Beetle) and the generalist T richoplusia ni (cabbage
looper), first-instar larvae were reared on either wild-type tomato plants or on the jail -1

mutant that fails to perceive JA (Li et al., 2004). T. ni larvae gained significantly more
weight on jail-I plants than on the wild-type host (F1,180=842.08, P<0.0001) (Figure
4.1a and b). The weight of surviving larvae was increasingly affected with feeding time
(F2,130=78.78,P<0.0001); three days after initiating the feeding trial jail -I -reared larvae

were 3.4-fold heavier than larvae grown on wild-type plants, and this difference increased
to 11.6- and 17.4-fold at the 6- and 8-day time points, respectively. At the end of the

feeding trial, the survival rate of T. ni reared on jail -1 plants was significantly increased
in comparison to caterpillars grown on wild-type plants (F 1,23=15.05,P=0.0006) (Figure

4.10).

L. decemlineata larvae also gained more weight on jai l -1 than on wild-type plants

(F 1,160=13.0, P=0.0004) (Figure 4.2a and b). In contrast to T. ni, however, the largest

138

 

difference (1.9-fold) in L. decemlineata larval weight gain was observed 2 days after
feeding. This difference was generally maintained throughout the time course of the
experiment (Figure 4.2a). The number of L. decemlineata larvae recovered from wild-

type plants at the end of the feeding trial was only 50% of that recovered from jail -1

plants (F1,18=54.l9,P<0.0001) (Figure 4.2c), indicating that C011-dependent defenses

have a major effect decreasing the survival of L. decemlineata. The experiments to assess
the effect of jasmonate defenses on larval weight were repeated twice with similar results.
These results demonstrate that the JA signaling pathway confers resistance of tomato to
both T. ni and L. decemlineata, and provide evidence that the major effect of this defense
pathway on T. ni and L. decemlineata fitness is related to larval growth rate and

survivorship, respectively.

Fate of the tomato leaf proteome in the digestive tract of L. decemlineata

Induced resistance of tomato to lepidopteran insects results, at least in part, from the
action of JA-regulated proteins that accumulate in and interfere with the insect’s digestive
system (Green and Ryan, 1972; Felton, 2005; Browse and Howe, 2008). In contrast to the
wealth of information concerning the biochemical basis of JA-mediated resistance to
caterpillars, very little is known about the nature of induced traits that confer resistance to
coleopteran pests such as L. decemlineata (Felton et al., 1992). To investigate this
question, we used LC-MS/MS to identify tomato leaf proteins that are excreted in the
frass of tomato-reared L. decemlineata larvae. Discovery of these stable host proteins was

facilitated by comparison of the most abundant proteins in L. decemlineata-challanged

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tomato leaves to the most abundant proteins recovered in frass from larvae fed on these

leaves (Figure 4.3a).

A total of 319 tomato proteins were identified in at least one of the two (i.e., leaf
and frass) samples. As shown in 4.3a, 216 proteins were leaf-specific (group A), whereas
52 proteins were found in both the leaf and frass (group B). To gain insight into why
group B proteins accumulate in the frass, proteins were assigned to a functional category
according to the FunCat classification scheme (Ruepp et al., 2004) (Figure 4.3b). The
larger classes of group B proteins were “perception/response to stimuli”, which was the
largest protein class among the leaf-specific proteins (group A), or as proteins involved in
energy-generating processes. The latter group included highly abundant chloroplast
proteins such as the large subunit of ribulose-1,5-bisphosphate carboxylase (RbcL) and
the a and [3 subunits of the CFl portion of ATP synthase. These results suggest that the
occurrence of group B proteins in the frass may be explained by their high level of
accumulation in leaves, and not necessarily by the resistance of the protein to insect
digestive proteases. The third group (group C) of tomato proteins identified in this
experiment was exclusive to the L. decemlineata frass (Figure 4.3; Table 4.1). The two
most prominent functional categories represented by group C prOteins was
perception/response to stimuli (45%), which includes proteins involved in defensive
processes; and protein degradation (11%). Tomato proteins having a defense role are
enriched relative to less stable dietary proteins during passage of the leaf diet through L.

decemlineata.

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Effect of insect gut environment on digestion of tomato leaf proteins

The ability of phytophagous insects to digest dietary proteins may be influenced by the
physiochemical features of the insect’s digestive system (Terra, 1987, 1990; F elton et al.,
1992; Johnson and Felton, 1996, 1996) or by the degree of host specialization of the
insect. To test these hypotheses, we used LC-MS/MS to compare the composition of
tomato leaf proteins excreted by larvae of L. decemlineata (coleopteran; solanaceous
specialist), M sexta (lepidopteran; solanaceous specialist), and T. ni (lepidopteran;
generalist) reared on tomato. A total of 212 unique proteins were identified in at least one
of the three frass samples (Appendix I). As shown in Figure 4.4a, 103 proteins were
catalogued in both the L. decemlineata and T. ni frass samples and 141 proteins were
identified in M sexta frass. One hundred eighteen (56% of all proteins) proteins were
specific to a single frass sample. The remaining proteins were identified either in all three
samples (41 proteins) or in two of the samples (53 proteins). Inspection of the latter group
of 53 proteins indicated that the frass proteomes of the two lepidopteran species are more
similar to each other than they are to the L. decemlineata sample, and that the L.
decemlineata proteome shared more proteins with the specialist M sexta than it did with
the generalist T. ni. These observations were supported by the distribution of proteins
within various firnctional categories (Figure 4.4b). The general distribution of proteins
identified in the frass of T. ni and M sexta were very similar to each other, and

distinguishable from that of the L. decemlineata frass.

The list of proteins identified in frass from the three insect herbivores revealed
several noteworthy trends. For example, proteins involved in photosynthesis, protein

degradation, protease inhibition and other defense-related processes were highly

141

represented in the group of 41 proteins that were common to all three frass samples
(Table 4.1). Among these were several well characterized wound-inducible proteins
implicated in plant anti-insect defense, including leucine amino peptidase-A (LAP-A),
arginase 2 (ARG2), Thr deaminase 2 (TD2), TC121 and other P13, and polyphenol
oxidase-F (PPO-F) (Green and Ryan, 1972; Chao et al., 1999; Wang and Constabel,
2004; Chen et al., 2005; Lison et al., 2006; Chen et al., 2007). Pathogenesis-related
proteins (PR proteins) were also found in the frass. Several PR proteins were identified
exclusively in the L. decemlineata sample. Host plant proteases, including several types
of endoproteinases (metallo-, serine-, aspartic- and cysteine-proteases) and exoproteases
(carboxypeptidases and aminopeptidase) were also highly represented in frass from all
three insects. Members of the subtilisin-like protease family (Vera and Conejero, 1988;
Tornero et al., 1996; Meichtry et al., 1999; Rautengarten et al., 2005) comprised the most
abundant class of proteases as determined by number of spectral counts (Table 4.1).
Based on the observation that peptides corresponding to only one subtilisin-like protease
were identified in the leaf sample, it would appear that these proteins are highly stable
and therefore enriched during passage of leaf tissue through the insect. The P69 members
of the tomato subtilisin-like protease family have been classified as PR proteins (PR-7)

(van Loon et al., 2006).

Overrepresentation of energy-related proteins was observed in the L.
decemlineata frass sample (Figure 4.4.b). RbcL, the [3 subunit of ATPase and other
proteins involved in the production of energy contribute to 29% of the total spectral
counts identified in this sample. This is 2 to 3 times higher than the spectral counts from

the same class of proteins in T. ni and M sexta (14 and 10%, respectivelY), and more

142

'similar to the ratio observed in the tomato leaf (36%). This could indicate differential

processing of bulk leaf protein in L. decemlineata compared to the lepidopterans.

Differential digestion of TD2 in lepidopteran and coleopteran herbivores

Proteolytic removal of the C-terminal regulatory domain of TD2 produces an enzyme
variant (called pTD2) whose ability to degrade Thr in the insect gut is not impaired by
Ile, a negative allosteric regulator of TD catalytic activity (Chen et al., 2007). To further
test the hypothesis that lepidopteran and coleopteran insects differentially digest tomato
leaf protein, we investigated the fate of TD2 after passage of leaf tissue through M sexta,
T. ni, and L. decemlineata. Analysis of LC-MS/MS data showed that the peptide
coverage of TD2 in M sexta and T. ni frass was limited to the catalytic domain. In
contrast, the peptide coverage observed for tomato leaf and L. decemlineata frass samples
included both the catalytic and regulatory domains (Figure 4.5a). This observation
suggested that TD2 processing does not occur, or occurs inefficiently, in L. decemlineata.
To test this idea further, we used immunoblotting to analyze the extent of TD2 processing
in fiass samples obtained from the three insects reared either on wild-type plants or, as a
control for antibody specificity, jail -1 plants. The results showed that TD2 is efficiently
processed in the two lepidopteran insects but, interestingly, remained unprocessed in L.
decemlineata frass (Figure 4.5b). This observation was extended to the potato TD2

(Figure 4.6).

RbcL, which is one of the most abundant soluble proteins in tomato leaves, is a

major source of amino acids for insect herbivores and a suitable marker for bulk leaf

143

protein (Bemays and Chapman, 1994; F elton, 1996; Chen et al., 2007). We performed
immunoblot analysis with an anti-RbcL antibody to determine the extent to which bulk
tomato leaf protein was digested in the same protein samples used for analysis of TD2
processing. Cross-reactive RbcL polypeptides were not detected in frass from either of
the two lepidopteran insects, whereas a lower molecular-weight form of RbcL
(presumably a stable degradation product) was detected in L. decemlineata frass (Figure
4.5b). This finding is consistent with the proteomic analysis showing that RbcL-derived
peptides are present in the beetle frass. We conclude that both nutritional (e.g., RbcL) and
defense-related (TD2) host proteins are digested less efficiently by L. decemlineata in

comparison to the lepidopteran insects.

Stability of JA-inducible proteins in the insect gut

We previously reported that several tomato JIPs, including TD2 and various PIs,
accumulate in the midgut and frass of M sexta larvae reared on tomato plants (Chen et
al., 2005; 2007). To systematically investigate the fate of JIPs during passage of leaf
tissue through different tomato-reared insects, we searched the list of tomato proteins
identified in each frass sample against a list of tomato genes whose expression in leaves
is differentially regulated in response to methyl-JA (MeJA) treatment. JA-regulated genes
were identified by hybridizing mRNA from MeJA-treated wild-type and jai1-1 leaves to
the TOMI cDNA array that contains 12,899 ESTs corresponding to ~8500 unique genes
(Van der Hoeven et al., 2002; Alba et al., 2004). We identified 292 genes that are

differentially regulated at least three-fold by the JA/COII pathway. Of these, 239 genes

144

were upregulated (and thus potentially encode a JIP), whereas 53 genes were down-

regulated.

Merging of the transcriptomics and proteomics data showed that 45 proteins in
either the tomato leaf or insect frass samples are encoded by genes whose expression was
determined to be differentially regulated in the microarray experiment (Table 4.2). Genes
encoding 33 of these proteins were upregulated by the JA/COIl pathway and could thus
be classified as putative J IPs. We used spectral count data, which is a reliable indicator of
protein abundance in label-free shotgun proteomic analyses (Zhang et al., 2006), to
estimate the abundance of each putative JIP in the various protein samples and the
proportion of total spectra represented by JIPs. As shown in Figure 4.6a (and Table 4.2),
JIPs accounted for 12% of the total spectral counts obtained for the herbivore-induced
tomato leaf sample. In frass samples obtained from T. ni, M sexta, and L. decemlineata,
spectra corresponding to JIPs accounted for 43, 31, and 23%, respectively, of the total
spectra. Therefore, the abundance of proteins annotated as JIPs (on the basis of gene

expression data) appeared to be enriched in frass.

A breakdown of the most abundant JIPs in the leaf and frass samples is shown in
Figure 4.7. These results are consistent with previous studies showing that LAP-A, TD2,
and various serine PIs are among the most abundant tomato proteins in midgut and frass
of tomato-reared M sexta (Chen et al., 2005; Chen et al., 2007). The silver leaf whiteflyl
(SLW-l) and ngF proteins, which were previously described as JA-inducible inducible
(van de Ven et al., 2000; Li et al., 2004), were also found in all frass and leaf samples. As
shown in Table 4.2 and in previous studies (Hildmann et al., 1992; Pautot et al., 1993;

Chen et al., 2004; Li et al., 2004; Uppalapati et al., 2008; Ishiga et al., 2009), the

145

.-m

.131 3-1-
\ ._.. ‘

expression of genes encoding the high abundance JIPs is strongly induced by the JA
pathway. Enrichment of these JIPs in insect frass likely reflects the combined effects of

high expression in insect-damaged leaves and protein stability in the insect gut.

Discussion

Adaptability of L. decemlineata to tomato defenses

Our results indicate that JA-regulated defenses impair the grth of both T. ni and L.
decemlineata, but that different components of insect fitness are affected by C011-
dependent defenses. These defenses affected the development of T. ni larvae more than
that of L. decemlineata, which might be expected for a generalist insect that is not well
adapted to tomato defenses. The growth of T. ni was affected by C011-dependent
defenses for extended periods of larval development, whereas the growth of L.
decemlineata was mainly affected at an early time point during the feeding trial. In
contrast to this effect on larval development, JA-based defenses had a greater effect on
the survivorship of L. decemlineata (50% mortality) than T. ni (34% mortality). These
results provide evidence that JA-regulated defenses have differential effects on the
mortality and grth of different insect herbivores. The decreased growth of T. ni on
wild-type plants correlated with an enrichment of JIPs in T. ni frass. One interpretation of
this observation is that T. ni, as a generalist herbivore, is less adapted than solanaceous
specialists such as M sexta and L. decemlineata for digesting JIPs that have anti-insect

activity.

146

‘1 T". {TEA—"m -

Previous studies have compared the growth of L. decemlineata larvae on Solanum
tuberosum (potato) plants that were elicited with MeJA treatment or that were impaired
in the IA pathway as a result of transgenic manipulation (Bolter and Jongsma, 1995;
Royo et al., 1999). Consistent with these studies, we observed that the JA pathway has
only a minor effect on the growth of L. decemlineata larvae. However, we found that the
JA signaling pathway is responsible for high mortality of L. decemlineata larvae on
tomato. The effect of JA-dependent defenses on weight gain after two days of feeding
was not accentuated over time, implying that the surviving L. decemlineata larvae grew
at the same rate on the wild-type and jail -1 plants. These observations suggest that JA-
dependent defenses are mostly effective against L. decemlineata during early larval
development and that the surviving larvae adapt quickly to the host defenses. Although L.
decemlineata has only recently expanded its host range from the wild Solanum rostratum
(Hitchner et al., 2008), it seems to adapt easily to other Solanum species (Gruden et al.,
2004; Lyytinen et al., 2007). Mechanisms involved in the adaptability of L. decemlineata
to different host plants may facilitate the insect’s ability to rapidly develop resistance

against pesticides (Roush et al., 1990).

Keys to the success of L. decemlineata on tomato

In comparison to the lepidopteran insects, frass from tomato-reared L. decemlineata
contained relatively high levels of energy-related proteins, including RbcL, several
subunits of ATP synthase and photosystem components. One interpretation of this

observation is that incomplete digestion of plant material by L. decemlineata reflects a

147

strategy to minimize exposure to plant defense proteins. Limited protein digestion could
result from a short transit time of plant material in the digestive tract. However, despite

the voracity of the beetle, the transit time of food through L. decemlineata larvae (150

min) is reported to be similar to that of 5th instar M sexta (120-160 min) (Martin et al.,

1987; Krishnan et al., 2007). Another potential explanation for undigested protein in L.
decemlineata frass is incomplete breakdown of chloroplasts or leaf tissue. Excretion of
unprocessed TD2 in the frass of L. decemlineata could also be explained by protection of
TD2 inside chloroplasts. However, the absence of intact RbcL in L. decemlineata frass
argues against this idea. Additionally, some of the most abundant proteins identified in
the leaf were Rubisco activase 1 and a chloroplastic phosphoglycerate kinase, which
together account for 6.8% of the identified spectra from the leaf. That these proteins were
not identified in the frass of L. decemlineata indicates that bulk chloroplastic protein is

efficiently digested.

Our data support the idea that L. decemlineata is less efficient than Lepidoptera
insects at digesting tomato leaf protein, which may reflect the different chemical
properties of the coleopteran and lepidoptem digestive sytems. It has been suggested that
the acidic gut of L. decemlineata may be protected from the action of PPO and other

oxidative enzymes that are active at alkaline pH (F elton et al., 1992).

Degradation of Rubisco during herbivory

Because of its high abundance in plants leaves, Rubisco is a major source of amino acids

for insect herbivores. Immunoblot analysis showed that breakdown of RbcL is initiated in

148

the herbivore-damaged leaf. Degradation of leaf Rubisco occurs in response to a wide
range of environmental stresses, including exposure to low temperature, heavy metals,
and ozone (Hajduch et al., 2001; Agrawal et al., 2002; Yan et al., 2006). Oxidative stress
caused by these adverse conditions is thought to be an important factor in promoting
Rubisco degradation (Desimone et al., 1996). An active mechanism for degrading
Rubisco in response to wounding may indirectly allow the plant to recycle amino acids
for use in the synthesis of defense-related proteins before the tissue is consumed by

herbivores.

Chen and colleagues (2007) reported that RbcL is quickly degraded in the gut of
M sexta. It has also been shown that L. decemlineata and M sexta gut fluids gratituously
degrade Rubisco within minutes of exposure to digestive proteases (Martin et al., 1987;
Brunelle et al., 1999). The use of more sensitive LC-MS/MS techniques allowed us to
identify RbcL-derived peptides in each of the leaf and frass samples analyzed.
Immunoblot analysis confirmed the presence of an RbcL degradation product in the L.
decemlineata frass sample. The extent to which Rubisco and other bulk dietary protein is
digested may be affected by interactions between insect proteases and plant PIs or other
plant compounds that affect the solubility and digestibility of dietary protein (Johnson
and Felton, 1996; McNabb et al., 1998). Interestingly, we identified an aspartic protease
(CND41) in the lepidopteran frass samples that were highly depleted in RbcL. Because
CND41 was previously shown to be involved in the degradation of RbcL during
senescence (Kato et al., 2004), we speculate that this protease may also play a role in

RbcL turnover during tomato-lepidopteran insect interactions.

149

TD2 and other JIPs

JIPs having a known or proposed defensive function against lepidopteran insects were
identified in the frass of L. decemlineata, and included LAP-A, PPO-F, TD2, and TC121.
The acidic gut environment of L. decemlineata, together with its unique complement of
digestive proteases, may render the defensive function of these host proteins inactive.
TD2 is a good example of this. Lack of proteolytic processing in L. decemlineata is
predicted to restrict the enzyme’s ability to efficiently degrade Thr because of negative
feedback inhibition by Ile, which presumably is present in the coleopteran gut. The lack
of TD2 processing indicates that either the cleavage of the regulatory domain is inhibited
in the L. decemlineata gut or that the protease responsible for cleaving TD2 is not present
in the beetle gut. Identification of the protease responsible for processing TD2 will

facilitate the testing of these hypotheses.

Detection of host defense proteins in insect frass does not necessarily imply a
defensive role for the protein in the source insect because the activity of plant defensive
proteins is often tailored to the gut physiological conditions of specific insects. For
example, it has been suggested that the alkalophilic enzymes LAP-A, ARG2, and TD2
may act synergistically to deplete essential amino acids in the high pH environment of the
lepidopteran gut (Chen et al., 2005; F elton, 2005). The relatively low activity of these
enzymes under acidic conditions would likely limit their effectiveness in the acidic gut of

L. decemlineata (Gu et al., 1999; Chen et al., 2004; Chen et al., 2007).

Only a subset of the proteins that were annotated as JIPs on the basis of the

microarray data were identified in the frass. Because many JA-inducible genes encode

150

 

transcription factors and other low abundance proteins involved in signal transduction,

this result is to be expected.

Trends of stable proteins

Tomato proteins that were enriched in the frass compared to the leaf proteome tended to
belong to one of several defense-related categories, including perception and response to
stimuli, protein degradation, and protease inhibitor activity. We also found that proteins
(i.e., J IPs) encoded by JA-regulated genes were among the most abundant proteins in the
frass samples. Taken together, these findings support the idea that defense-related plant
proteins have been selected for increased stability. As is the case for PIs that impair insect
digestive enzymes, high stability and resistance to proteolysis is expected to be a requisite

feature of plant proteins that act in the insect gut.

The list of plant proteins identified in insect frass may provide insight into the
biochemical determinants of protein stability. For example, disulfide bonds are known to
confer stability and proteolytic resistance to P13 (Betz, 1993). Metals and cofactors can
also contribute to protein stability (Woo et al., 2000; Mukhopadhyay and Lecomte, 2004;
Bertini, 2007; Yogavel et al., 2008). Annotations based on information provided by
UniProt, Expasy, and Percudani and Peracchi (2003) provide evidence that many tomato
proteins excreted in the frass bind cofactors and metals. Pyridoxal phosphate-binding
proteins were the most represented cofactor-containing proteins in the leaf sample.
Because fewer pyridoxal phosphate-containing proteins were identified in the frass,

binding of pyridoxal phosphate does not appear to be sufficient to confer stability (Figure

151

4.8). A similar trend was observed for Fe/S and metal (Ca2+, K+, Na+, Mg2+) containing

. . . . . 2+ 2+ +
proteins. Proteins containing heme and various heavy metals (Cu , Fe , Zn ) were

equally abundant in the leaf and frass, suggesting that these proteins may be more

resistant to digestion by insect gut proteases.

Extracellular proteins tend to be more stable than cytosolic proteins (Xia et al.,
2007). Because the apoplastic space is rich in hydrolytic enzymes, extracellular proteins
are generally more resistant to proteolysis (Kusumawati et al., 2008). We observed a
significant increase in the proportion of extracellular and cell wall-associated proteins in
the frass compared to the leaf (Figure 4.9). A significant proportion (26%) of the plant
proteins exclusively identified in L. decemlineata fiass were annotated as pathogenesis-
related (PR) proteins. PR proteins are typically targeted to either vacuoles or the
apoplastic space (Fernandez et al., 1997), both of which have a pH comparable to that of
the beetle gut (Rayle and Cleland, 1992; Lodish, 2000). The inherent stability of PR
proteins in acidic and protease-rich environments may explain, at least in part, why they
are so stable in the L. decemlineata digestive tract. The high stability of two PR proteins,
PR-l and gerrnin, can be attributed to a compact quaternary structure that is stabilized by
hydrogen bonds and hydrophobic interactions (Van Loon and Van Strien, 1999; Woo et

aL,2000)

Oxidative stress in the gut

152

Plants often respond to biotic invasion with a rapid increase in enzyme-mediated
production of reactive oxygen species (Levine et al., 1994). Increased levels of hydrogen
peroxide and other reactive oxygen species have been negatively correlated with insect
performance (Ramputh et al., 2002; Lou and Baldwin, 2006). Reactive oxygen species -
generating enzymes are thought to decrease the palatability of plant tissues and impair
insect digestive processes (Bi and Felton, 1995). Plants also produce secondary
metabolites, including phenols and alkaloids that participate in reactive oxygen species
production in the lepidoptera gut, thus reducing protein quality. It is possible that the
activity of oxidative stress enzymes persist in the gut, exacerbating reactive oxygen

species accumulation.

We identified peptides corresponding to tomato superoxide dismutase, catalase,

ferredoxin, and peroxidases in the frass samples. Germin and germin-like proteins that

generate H202, were among the most abundant proteins in the frass (Lane, 2000; Bernier

and Berna, 2001). We also identified enzymes involved in the detoxification of oxidative
stress: dehydroascorbate reductase (DHAR) and glutathione reductase (GR). It has been
suggested that the resistance of oxidative stress enzymes to harsh environments is linked
to their ability to generate ROS (Xia et al., 2007). It remains to be determined whether
the tomato proteins identified here are active in the insect gut and, if so, whether they
contribute to ROS generation or dissipation in the insect, or serve a role in reducing

herbivore performance.

Plant proteases and protease inhibitors

153

 

.1

 

Plant proteases appear to play several roles in anti-insect defense, including
remobilization of amino acids within plant tissue, processing of plant proteins to release
signaling peptides (Moura et al., 2001), and destruction of the peritrophic membrane that
protects the midgut epithelium (Mohan et al., 2006). The latter group of proteins are
cysteine proteases that exert toxic effects on lepidopteran insects (Pechan et al., 2002;
Konno et al., 2004). Several tomato proteases that we identified in insect frass belong to
the subtilisin-like family of serine proteases. In tomato, 15 subtilases have been classified
into five subfamilies that exhibit distinct patterns of expression (Meichtry et al., 1999).
The function of the large majority of these proteases in tomato and other plants remains
unknown. We identified only one subtilisin-like protease in the leaf sample, whereas
multiple isoforms (13 in M sexta, 8 in T. ni , and 6 in L. decemlineata) were identified in
the frass. The stability of these proteins during passage through the insect gut is
consistent with their high thermostability conferred by the stabilizing effect of Ca2+
binding (Siezen et al., 1991). The fact that many subtilisin-like proteases from plants
have an alkaline pH optimum (Hamilton et al., 2003) suggests that they may be

catalytically active in the lepidopteran gut.

We identified the SLWl-like protein (also known as Drought Inducible Protein-1,
DIP-1) an abundant protein in the frass of L. decemlineata. The tomato protein shares
66% and 69% amino acid identity with SLWl from squash and DIP-l from watermelon,
respectively. The genes encoding the cucurbit proteins are induced by silverleaf whitefly
feeding, MeJA and ethylene treatment, and drought stress (Kawasaki et al., 2000; van de
Ven et al., 2000; Yokota et al., 2002). SLWl and DIP-l are cytosolic proteins belonging

to the M203 family of proteases that have broad substrate specificity. Members of the

154

family have been implicated in deacetylation of peptides and amino acids and as
carboxypeptidases (van de Ven et al., 2000). In watermelon it has been involved in the
production of citrulline from glutamate under drought conditions (Kawasaki et al., 2000).
The bacterial counterpart of these proteases has a neutral pH optimum (Javid-Majd and
Blanchard, 2000). The role of the SLWl-like protein in plant-insect interactions, if any,

remains to be determined.

Tomato leaves produce a wide range of P15 in response to wounding and insect
herbivory. These defensive compounds are well characterized for their ability to inhibit
digestive proteases in the insect gut (Ryan, 1990). Our proteomic analysis of insect frass
identified PIs belonging to three mechanistic classes (serine-, aspartic-, and cysteine-
type) proteases. Serine PIs were the most abundant class of PI identified in the frass of
the two lepidopteran insects. This finding is consistent with the fact that most
lepidopteran digestive proteases are serine-type proteases and are sensitive to serine Pls
(Christeller et al., 1992). Genetic and biochemical evidence indicates that the serine Pls
identified in our study serve a role in defense against lepidopteran insects and other biotic
stresses (Zavala et al., 2004; Herrnosa et al., 2006; Lison et al., 2006). Serine PIs also
accounted for the major class of P15 identified in the frass of L. decemlineata. Cathepsin
D-like proteases have been reported to be important for the initiation of protein digestion
in L. decemlineata, whereas cysteine- and serine-proteases participate in later stages of
protein hydrolysis (Brunelle et al., 1999). A cysteine protease inhibitor (multicystatin)
was also detected in the lepidopteran frass, despite the fact that lepidopterans do not use

cysteine proteases for digestion (Christeller et al., 1992) .

155

 

One rationale for cataloging plant dietary proteins in insect feces is to identify
candidate proteins that impair insect nutrition. We successfully identified known tomato
proteins that target insect digestive processes, including PIs, TD2, arginase, and cysteine
proteases. Other excreted proteins, including SLWl-like, Wound Inducible
Carboxypeptidase, and the ngF family protein, were known to be JA-inducible but have
unknown functions. Our findings raise the possibility that these proteins have a role in
plant defense related to disruption of an insect digestive process. Transgenic approaches
or feeding assays performed with heterologously expressed protein incorporated into

artificial diet could be used to test this hypothesis.

Materials and Methods

Plant material and growth conditions

Wild-type and jail -I tomato plants (Solanum lycopersicum cv. Castlemart) were grown
on soil as described previously (Chen et al., 2005). Leptinotarsa decemlineata (Say) eggs
were obtained from the Phillip Alampi Beneficial Insect Laboratory at the New Jersey
Department of Agriculture. T richoplusia ni (Hiibner) eggs were obtained from Benzon
Research. Manduca sexta (Linnaeus) eggs were obtained from the North Carolina'State
University Entomology Insectary. All eggs were hatched at 27°C (17 hours of light and 7

hours of dark).

Insect feeding bioassays

156

 

 

 

Fifteen newly hatched L. decemlineata larvae were placed on each of ten 5-week-old
jail -I and wild-type tomato plants arranged in a randomized design, with each plant
being the unit of replication. Movement of the larvae between the genotypes was
prevented by bagging the pots inside a 8xl6x18 in spun-polyester pot sleeve closed at the
top (Hummert International, Earth City, MO, USA). Larvae were kept on plants at 17 h
of light at 27°C and 7 hours of dark at 18°C. At each time point, three larvae were
randomly selected from each pot and weighed. These readings were considered repeated
measurements from the same experimental unit. After weighing, insects were returned to
their plant of origin. At the end of the feeding trial, all larvae were collected and counted
to determine the larval survivorship on each genotype. The conditions for the T. ni

experiment were similar except that 10 larvae were used to infest 15 plants of each

genotype.

Analysis of Variance was used to determine whether the mean weight and
survivorship were significantly different between host genotypes. Larval weight data was
log-transforrned to obtain homogeneity of variance, and analyzed with two-way ANOVA
with repeated measures. Both the effect of host genotype and time (afier infestation) were
evaluated. Untransformed data were used in figures. Survivorship, expressed as the
percentage of the total number of larvae recovered at the end of the trial, was analyzed
using one-way ANOVA. The statistical analysis was made with SAS® software, Version

9.1.3 of the SAS System for Windows (Copyright © 2002-2003).

Frass collection, protein extraction, and mass spectrometry

157

Four- to five-week—old wild-type tomato plants were used to feed L. decemlineata larvae.
Twice a day, third and fourth instar insects were removed from the plants and transferred
to Petri dishes for one hour. Feces were collected fi'om the Petri dishes and the insects
were returned to the plants. This method facilitated the collection of the watery feces of
L. decemlineata and also minimized contamination of collected feces with plant material.
F rass (approximately 200 mg) collected over a period of five days was pooled. Damaged
leaves from the L. decemlineata feeding trial were collected and used for protein

extraction.

In the case of T. ni and M sexta, cut leaves from five-week-old tomato plants

were used to feed the larvae. Cut leaves were replaced daily with fresh leaves. Frass

pellets were collected from 4th to 5th instar larvae over a period of three to four days.

Frass samples were stored at -20°C until subsequent use in protein extraction.

Proteins were extracted with a modified phenol-based extraction method
described elsewhere (Chen et al., 2007) and quantified with a Bradford assay. For mass
spectrometry analysis, one hundred mg of total protein were electrophoresed 1 cm into a
10% denaturing polyacrylamide gel. The gel was stained with Coomassie Brilliant Blue
and destained overnight. Gel pieces containing the protein were excised and subjected to
in-gel trypsin digestion. Briefly, the proteins were reduced with dithiothreitol, alkylated
with iodoacetamide and digested with trypsin (Promega) (Jensen et al., 1999). Proteins
were extracted with a modified phenol—based extraction method described elsewhere

(Chen et al., 2007) and quantified with a Bradford assay.

158

For mass spectrometry analysis, one hundred mg of total protein were run 1 cm
into a 10% denaturing polyacrylamide gel. The gel was stained with Coomassie Brilliant
Blue and destained overnight. The piece of gel containing the proteins was excised and
subjected to in-gel trypsin digestion. Briefly, the proteins were reduced with
dithiothreitol, alkylated with iodoacetamide and digested with trypsin (Promega) (Jensen
et al., 1999). The extracted peptides were re-suspended in a solution of 2%
Acetonitrile/0.1% Trifluoroacetic Acid to 20 pl. From this, 10 pl were automatically
injected by a Waters nanoAcquity Sample Manager and loaded for 5 minutes onto a
Waters Symmetry C18 peptide trap (5 pm, 180 pm x 20 mm) at 4pL/min in
2%ACN/0.1%Formic Acid. The bound peptides were then eluted onto a Waters BEH
C18 nanoAcquity column (1.7 pm, 100 pm x 100 mm) and eluted over 240 minutes with
a gradient of 2% B to 30% B in 200 min at a flow rate of 0.45 pI/min. Separations were
done using a Waters nanoAcquity UPLC (Buffer A = 99.9% Water/0.1% Formic Acid,
Buffer B = 99.9% Acetonitrile/O.l% Formic Acid) and sprayed into a ThermoFisher
LTQ-FTICR mass spectrometer using a Therrno nano-spray source. Survey scans were
taken in the FT (25000 resolution at m/z 400) and the top ten ions in each survey scan
were then subjected to automatic low energy collision induced dissociation (CID) in the
LTQ. The resulting MS/MS spectra were converted to peak lists using BioWorks
Browser v3.2 (ThermoFisher) using the default LTQ-F T parameters and searched against
tomato Transcript Assembly (TA) database (Release 2) found at TIGR Plant Transcript
Assemblies website (http://plantta.tigr.org/cgi-bin/pjanttatreleasepl ), using the Mascot
searching algorithm (Matrix Science, version 2.1). The Mascot output was then analyzed

using Scaffold (Proteome Software Inc., Portland, OR) to probabilistically validate

159

 

l. I un—A. 9 >.._ rlJf‘L‘fij
‘ .

 

protein identifications using the ProteinProphet computer algorithm (Nesvizhskii et al.,
2003). Assignments validated above the Scaffold 95% confidence filter were considered

true.

Identification of tomato proteins by LC-MS/MS

Proteins were identified using a translated database constructed from the tomato
Transcript Assembly (TA) database (Release 2) at the TIGR Plant Transcript Assemblies
website (http://plantta.tigr.org/cgi-bin/planttafreleasepl ). The following criteria were
used for protein identification: the protein probability was 295% as determined by the
Protein Prophet algorithm and at least two individual peptides per protein were identified.
For protein quantification using spectral count data (Table 4.1 and Figure 4.7), we
imposed the more stringent criterion of accepting peptides only if their probability of

identification was 295%.

Identified tomato proteins were classified into functional categories according to
the FunCat classification scheme (Ruepp et al., 2004). To do this, BLASTX was used to
identify the best hit in the TAIR8 protein set for the TIGR Tomato Transcript
Assemblies. Categories associated with the Arabidopsis top hit were then assigned by
inheritance to the TIGR Tomato TA and thus represent putative functional assignments.
In the few cases were no match was found but the protein has a known function (i.e.

polyphenol oxidase F), the functional category was manually assigned.

160

 

 

Microarray analysis

Wild-type and jai1-1 plants were treated with vaporous MeJA for either 8 or 24 h as
previously described (Li et al., 2004). Equal amounts of total RNA from the 8 and 24 h
time points were pooled prior to RNA labeling with Cy5 or Cy3 dUTP as previously
described (Zhao et al., 2003). Three biological replicate RNA samples were used for
hybridization experiments with Cy5-labeled WT RNA and Cy3-labeled jai1-1 RNA that
were mixed in equal molar ratios prior to hybridization. Labeled RNA was hybridized to
the TOMl cDNA array that contains 12,899 ESTs corresponding to ~8500 genes, which

represents ~25% of all tomato genes (Van der Hoeven et al., 2002).

We used the following procedure to merge the microarray and proteomics data.
Clone IDs for each element on the array were updated to determine the current Unigene
cluster membership (SGN-U). The sequence of each SGN-U ID was used to perform

BLASTN searches against the tomato Transcript Assembly .(Solanum lycopersicum

release 2) with an. E-value cutoff of le'loo. The list of TA accessions identified in all

proteomic experiments was used to search the BLAST output to identify SGN-U

sequences that match the identified proteins.

Acknowledgements

We would like to thank Doug Whitten for doing the LC-MS/MS analysis, and Kevin Carr

for the bioinforrnatics work. Nora Bello assisted us with the statistical analysis.

161

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166

Figure 4.1. J asmonate defenses reduce Trichoplusia ni fitness on tomato.

Ten newly hatched larvae were transferred to fifteen S-week old wild-type (WT) or jail-
1 plants.
(a) Weight gain of T. ni was measured at 3, 6 and 8 days afier infestation (dai) of the
indicated host genotype. Values are the mean weight i standard error. F2,130=78.78,
P<0.0001 for the interaction between feeding time and host genotype. F1,130=842.08, P
<0.0001 for host genotype. F1,130=122.63, P<0.0001 for larval weight at 3 days;
F1,130=467.68, P<0.0001 for 6 days; F 1,130=709.58, P<0.0001 for 8 days.

(b) Photograph of representative larvae recovered at the 8-day time point.

(c) Percent recovery of larvae at the end of the experiment (8 days) as a measure of

survivorship. Values are the mean percentage of recovery fl: standard error. F1,23=15.05,
P=0.0006.

167

 

 

 

 

 

 

 

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168

 

Figure 4.2. J asmonate defenses reduce Leptinotarsa decemlineata fitness on tomato.

F ifieen newly hatched larvae were transferred to ten 5-week old wild-type (W T) or jail -1
plants.

(a) Weight gain of L. decemlineata was measured at 2, 4, 6 and 7 days after
infestation (dai) of the indicated host genotype. Values are the mean weight d: standard

error. F1,160=l3.0, P=0.0004 for host genotype. F3,160=0.98, P=0.4025 for interaction
between time and host genotype.

(b) Photograph of representative larvae recovered at the 7-day time point.

(e) Percent recovery of larvae at the end of the experiment (7 days) as a measure of

survivorship. Values are the mean percentage of recovery :t standard error. F1,13=54.19,
P<0.0001.

169

tomato.

orjml-l

335 after
standmd

iteractiofi

easure of

l8:54.19.

(a)

  

% survivorship

Larval weight (mg)

 

 

‘°' WT
-o- 'ai1-1

  

 

 

 

 

170

 

 

Figure 4.3. Distribution of proteins identified in the wild-type tomato leaf and frass
of L. decemlineata.

(a) The Venn diagram shows the intersection of sets of tomato proteins identified in
wild-type leaf damaged by L. decemlineata and the frass of tomato-reared L.
decemlineata larvae. The diagrams were drawn to scale (proportional area) with 3Venn
Applet.

(b) Distribution of proteins by functional category according to FunCat. The
cumulative chart displays the percentage that each category contributes to the total. The
identity of each functional category is shown in the legend (listed from top to bottom).

A, proteins identified in the leaf only; B, proteins identified in both leaf and frass; and C,
proteins identified only in the L. decemlineata frass sample.

171

fed in
rd 1..
Venn

llie

1d C.

(a) WT leaf damaged by
L. decemlineata

(b)

  
  

L. decemlineata
frass

  

 

100%—
90%—
80%‘ gga'
70%—
60%-
50%- ..
40%—
30%4
20%1

10%-

 

 

 

 

 

 

 

E Amino acid metabolism

[22] C-oompound and
carbohydrate metabolism

I Secondary metabolism

B Other metabolism

EEl Energy

I Cell cycle. transcription
and protein synthesis

Q Protein degradation

Protein fate

[I] Protease inhibitor activity

D Cellular transport

Perception and response
to stimuli

a Developmental processes

Other categories

EB Unknown

 

0%—

 

172

 

Figure 4.4. Distribution of the plant proteins identified in the three insect frass
samples.

(a) Venn diagram showing the plant proteins identified in the frass. The diagrams
were drawn to scale (proportional area) with 3Venn Applet .

(b) Distribution of identified proteins among functional categories according to
FunCat. The cumulative chart displays the percentage that each category contributes to
the total. The identity of each functional category is shown in the legend (listed from top
to bottom).

L. dec: Leptinotarsa decemlineata; T. ni: T richoplusia ni; and M. sex: Manduca sexta

173

mg lo
Lies ID
in top

la

(a)

L. decemlineata
frass

(b)

100%-

\\
:77 \:
/

80%‘

70% ‘

A

b4

60%-
50% _ . .
4o%— ._-.
30% - :-:-§

20% - E}:

 

10% - 3:. -:-E—

 

0% J
WT leaf L. dec 72 ni
damaged frass frass

 
 

M. sexta
frass

  

 

 

E Amino acid metabolism
E] C-compound and
carbohydrate metabolism
Secondary metabolism
HI] Other metabolism
[El Energy
I Cell cycle. transcription
and protein synthesis
E Protein degradation
El Protein fate
|]] Protease inhibition
[2] Cellular transport
[3 Perception and response
to stimuli
Developmental processes
Q Other categories

E Unknown

 

M. sex
frass

174

 

Figure 4.5. Differential digestion of TD2 in the lepidopteran gut.

(a) Peptide coverage of TD2 in wild-type (WT) leaf and frass samples. The peptides
identified by LC-MS/MS were mapped to the different regions of the protein: transit
peptide (TP); catalytic domain (CAT); regulatory domain. (REG) of TD2.

(b) Total protein was extracted from tomato leaves that were damaged by L.
decemlineata (leaf), and feces of M. sexta, T. ni, and L. decemlineata reared on tomato
(frass). Protein extracts (20 pg) were analyzed on a 10% SDS-polyacrylamide gel and
stained with Coomassie blue. The same samples were used for Western blot analysis with
an anti-TD2 and anti-RbcL antibodies

175

: peptides
in: mil

ed by L
)n tomato
le gel and
lysis Willi

 

(a) .

l l i l l *1
0 100 200 300 400 500 600 aa

Hi CAT _

WTlear — -—-—- — — -—

 

M. sexta frass - — — - __ _
T. ni frass - — _ - _ __
L. decemlineata frass - — — - _ _ -

(b) Leaf Frass
M. sex 72 ni L. dec

 

 

jai1 WT jai1 WT jai1 WT jai1 WT

97
CB stain

50

38
29

21
Anti-TD

Anti-rbcL

 

176

Frass

 

Leaf M. sex T. ni L. dec

 

Figure 4.6. Processing of potato TD2.

Larvae from M sexta, T ni were grown on artificial diet until third to fourth instar. At
that stage, larvae were transferred to potato plants (Solanum tuberosum cv White Red
Craigg Royal) and frass was collected two days after infestation. L. decemlineata
neonates were grown on potato until third instar. At that point they were transferred to
new plants and frass collected two days later. Leaves from plants damaged by L.
decemlineata were included as a control (Leaf). An antibody raised against tomato TD2
was used for immunoblotting.

L. dec: Leptinotarsa decemlineata; T. ni: T richoplusia ni; and M. sex: Manduca sexta

177

(a) WT leaf damaged by L. decemlineata
12% of total spectral
counts

PIS. 25”.;

     
  
 

LAP—A.
13%

YlgF . 3%

ARGZ, 6"/
° SLW1-like,
10%

(c) 1'. nl frass
43% of total spectral
counts

_ Others.
YlgF . 5% 11%

    
  

ARGZ, 2%

SLW1-like,
11%

(b) L. decemlineata frass

23% of total spectral
counts

ngF.6°/o Pie. 21“..

LAP-A. 7%

   

SLW1-like.
26%

(d) M. sextafrass

31% of total spectral
counts

Others.

   

ngF , 9%

ARGZ. 7%

SLW1-like,
3%

Figure 4.7. Identification of JA-inducible proteins.

The relative abundance of JA-inducible proteins (JIPs) identified in each of the four
samples was estimated using the number of spectral counts.

PIs, protease inhibitors; LAP—A, Leucine aminopeptidase A, TD2: Threonine deaminase
2; SLW1-like, silverleaf whitefly-induced protein 1-like; ARGZ, Arginase 2.

 

J\FeIS/L

 

 

 

 

 

 

 

 

 

 

 

eta
TTT W
i P}! L

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 4.8. Distribution of proteins with cofactors.

The width of the arrow is proportional to the number of proteins containing the cofactor
identified in the sample. Solid pattern: Wild-type leaf damaged by L. decemlineata; grid:
L. decemlineata frass; stripped: T. ni frass; checkered: M. sexta frass. PLP: pyridoxal-
phosphate.

179

1 00%
90%
80%
70%
60%
50%
40%
30%
20%
1 0%

0%

 

l

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

WT leaf L. dec frass

damaged

T. ni frass M. sex frass

Figure 4.9. Subcellular localization of identified proteins.

For all of the TIGR Tomato TAs (Solanum lycopericum Release 2) the most similar
Arabidopsis protein (TAIR v.8) was identified by using BLASTX (Tomato TA
nucleotide translated vs. TAIR 8 proteins). The set of Arabidopsis proteins identified as
best matches to the identified Tomato TAs were analyzed using the TargetP, ChloroP,
SignalP programs which search for known target signal peptides at the N-terminus of a
protein sequence.(SP: Secretory pathway, mTP: Mitochondrial transit peptide, cTP:
Chloroplast transit peptide, L. dec: Leptinotarsa decemlineata; T. ni: T richoplusia ni; and

M. sex: Manduca sexta)

180

El Other
I SP
a mTP
I cTP

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190

Chapter V

Conclusions and Future Perspectives

At the beginning of this thesis research, studies of tomato TD had focused on a single
gene (T D2) that was reported to encode the most abundant protein in tomato flowers
(Samach et al., 1991). Subsequent studies showed that the potato ortholog of this gene is
strongly induced by wounding and JA treatment (Hildmann et al., 1992; Dammann et al.,
1997). The extraordinary level of expression of this gene in floral organs and wound-
stressed leaves was not consistent with a role in primary metabolism (i.e., Ile
biosynthesis). To investigate the possibility that tomato and potato have multiple
isoforms of TD, a search of the tomato EST database was performed, which led to the
identification of a second TD gene in these species. This uncharacterized gene was called

T D] , to differentiate it from the jasmonate-inducible gene TD2.

The expression pattern and biochemical properties of tomato TDs are consistent
with functional divergence of the two isoforms. Results described in this thesis indicate
that TD] likely serves a primary metabolic role in Ile synthesis because the gene is
constitutively expressed in all tissues and is orthologous to TDs from plants such as rice
and Arabidopsis that contain a single TD gene. In contrast, TD2 expression is strictly
regulated in an inducible manner by the jasmonate pathway (Chen et al., 2007).
Moreover, TD2 is highly abundant in the frass of M. sexta, T. ni, and L. decemlineata
larvae reared on tomato. These findings led to the hypothesis that TD2 performs a post-

ingestive defensive role in depleting the essential amino acid Thr from the insect’s diet.

191

The increased weight of S. exigua larvae growing on transgenic tomato plants silenced in
TD2 expression provides direct evidence for this hypothesis. Duplication and subsequent
neofimctionalization of genes involved in primary metabolism may be a recurrent
strategy in the evolution of plant defense proteins that impair the ability of arthropod
herbivores to acquire nutrients fiom plant tissue. Similar examples are available in the
biosynthesis of secondary metabolites (Bohlmann et al., 1996; Ober, 2005). For example,
in Ruta graveolens a tryptophan —sensitive anthranilate synthase enzyme is responsible
for amino acid synthesis, whereas a tryptophan-resistant anthranilate synthase has been

recruited for the biosynthesis of anthranilate-derived alkaloids (Bohlmann et al., 1996).

Our results also show that TD2’s role in insect resistance is associated with
certain biochemical properties that optimize the enzyme’s capacity to function in the
extreme environment of the insect gut. The Thr-degrading activity of TD2 in the insect
gut is enhanced by proteolytic cleavage of the enzyme’s regulatory domain following
ingestion of tomato leaves by insect larvae. TD2 is presumably maintained in a latent
state inside the chloroplast, and is subsequently activated by an insect chymotrypsin-like
protease in the midgut of lepidopteran herbivores. Removal of the regulatory domain
results in an enzyme that is insensitive to feedback inhibition by Ile and, as a
consequence, is highly efficient in degrading Thr in the Ile-rich environment of the

lepidopteran gut.

Another key feature of TD2 is its thermostability and resistance to insect digestive
proteases. Further studies are needed to determine the structural features that underlie
these properties. Answers to this question promise to provide basic insight into why plant

proteins accumulate in the digestive tract, and may also facilitate the rationale design of

192

hyperstable proteins that are useful as pesticides or other commercial applications. The
TD1 and TD2 isoforms provide an attractive comparative model system in which to study
this question. As an initial step toward this goal, the structure of TD1 and TD2 was
modeled using the X-ray structure of E. coli TD (Gallagher et al., 1998). These models
suggest that the overall structure of TD1 and TD2 are very similar (Figure 5.1), despite
the sequence divergence of the two isoforms. Superimposition of the structures, however,
revealed an a-helix in the regulatory domain of TD1 that is not present in TD2. This
observation suggests that the regulatory domain of TD2 may not be subject to the
functional constraints of the regulatory domain of TD1 and other biosynthetic-type TDs.
More detailed information is needed to establish the structural basis of the differential
stability of TD1 and TD2. Efforts to determine the X-ray crystal structure of the two
isoforms are underway in collaboration with Chris Bianchetti and George Phillips at the

University of Wisconsin.

A significant part of this thesis is devoted to the analysis of the frass proteome of
different insects reared on tomato. These results show that shotgun proteomic analysis of
the insect feces provides a rapid and robust approach to identify hyperstable proteins that
may have a role in plant defense. The approach was validated through the identification
of well characterized proteins that impair insect digestion, including a variety of protease
inhibitors, cysteine protease, a germin-like protein, and the amino acid-degrading
enzymes arginase and TD2. Other JA-inducible proteins with unknown roles were also
identified. Arriong these were a member of the highly conserved ngF family of proteins
and a silverleaf whitefly-l like protein (van de Ven et al., 2000; Leitner-Dagan et al.,

2006). Plant proteins that are both stable in the insect gut and regulated by the JA

193

pathway are good candidates for additional testing as anti-insect compounds using

transgenic approaches.

The comparison of the tomato leaf proteome before and after passage through L.
decemlineata indicated that most leaf proteins are digested in the insect. Nevertheless,
enrichment of a subset of leaf proteins, including defense-related and/or JA-inducible
proteins, in the insect gut was observed. This finding suggests that plant anti-insect
proteins have been selected during evolution for increased stability and resistance to
proteolysis. The fact that many gut-accumulating tomato proteins are encoded by JA-
responsive genes indicates that herbivore-induced transcriptional reprogramming is an
important part of the plant’s defensive strategy to limit nutrient acquisition. This idea is
supported by the observation that the generalist insect T. ni, whose growth is severely
affected by JA-based defenses, excreted a higher proportion of JA-inducible proteins

compared to the Solanaceae specialists.

Proteomic analysis indicated that both nutritional (e. g., RbcL) and defense-related
(TD2) host proteins are digested less efficiently by L. decemlineata in comparison to the
lepidopteran insects. Reduced digestion of dietary protein by L. decemlineata may
represent a counteradaptive strategy to minimize exposure to host defense proteins.
Pathogenesis-related (PR) proteins also appeared to be more stable in the gut of L.
decemlineata compared to the lepidopterans. Although it is widely accepted that PR
proteins serve important roles in plant resistance to pathogens, the biological firnction of
many PR proteins remains unknown (van Loon et al., 2006). The potential function of PR

proteins as a post-ingestive defense against insects deserves further investigation.

194

For the vast majority of stable tomato proteins identified in this thesis research,
additional work is needed to determine whether they have a role in anti-insect defense.
This goal could be accomplished by altering the expression of the corresponding gene in
transgenic plants, or by incorporating the purified protein in insect artificial diet. Because
of its amenability to reverse genetic analysis, genetic approaches may be facilitated with
the use of Arabidopsis thaliana as a host plant. Proteomic analysis frass from T. ni and S.
exigua fed on Arabidopsis showed that the most abundant Arabidopsis protein excreted
by both insects is a cystine lyase encoded by locus At4g23600 (E Gonzales-Vigil and G
Howe, unpublished results). RNA interference technology was subsequently used to
silence the expression of this gene. Insect feeding assays performed with At4g23600-
silenced lines showed a small but consistent effect of the gene in promoting Arabidopsis
resistance to S. exigua (E Gonzales-Vigil and G Howe, unpublished results). These
preliminary results provide additional evidence that shotgun proteomic analysis of insect
frass is a valid approach for identifying plant genes that mediate resistance to insect

herbivores.

Restricted occurrence in a subset of species within the Solanaceae is an
interesting feature of TD2 as a JA-regulated gene (Chen et al., 2007). Thus, despite the
conservation of the JA-signaling pathway in the plant kingdom, some JA-inducible
defenses (e.g., TD2) are plant-lineage specific, whereas others (e.g., Pls) are widespread.
This observation indicates that the discovery of novel plant anti-insect proteins may be

facilitated by extending proteomic-based surveys to other plant species.

In summary, this thesis research has generated new insight into the role of plant

proteins in defense against insect herbivores. Based on the results obtained, further

195

experiments will be designed to understand the mechanisms underlying the role of these
proteins in plant defense. The ultimate goal of this work is to develop transgenic crops

with increased resistance to arthropod herbivores.

196

 

   

catalytic
domain

neck

regulatory
domain

 

 

 

Figure 5.1. Homology models of tomato TDs.

The X-ray structure of E. coli TD (Gallagher et al., 1998) was used as a template to
construct the homology models with SWISS-MODEL. Images were rendered with
PyMol.

197

 

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198

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