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This is to certify that the
dissertation entitled

UNDERSTANDING THE MOLECULAR BASIS OF
DISEASE SUSCEPTIBILITY OF ARABIDOPSIS TO

PSEUDOMONAS SYRINGAE PV. TOMA T0 DC3000.

presented by

Paula Margaret Hauck

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l1#-.

UNDERSTANDING THE MOLECULAR BASIS OF DISEASE
SUSCEPTIBILITY OF ARABIDOPSIS To PSEUDOMONAS SYRINGAE PV.
TOMATO DC3000.

By

Paula Margaret Hauck

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Genetics Program

2004

Abstract

UNDERSTANDING THE MOLECULAR BASIS OF DISEASE SUSCEPTIBILITY OF
ARABIDOPSIS TO PSEUDOMONAS SYRINGAE PV. TOMA T0 DC3000.

By

Paula Margaret Hauck

Plant diseases are widespread and cause devastating crop losses each year.
However, little is known about the molecular mechanisms of disease susceptibility to
virulent pathogens. Knowledge of disease progression could be vital to designing
improved methods for disease control. Pseudomonas syringae pv. tomato strain
DC3000 (Pst DC3000), an important model for studying plant-pathogen interactions,
causes bacterial speck on tomato and Arabidopsis thaliana. Pst DC3000 enters plants
through natural Openings such as stomata and wounds. It is an extracellular pathogen
that must suppress or evade plant defenses and obtain nutrients from the host to be
successful. Disease progression is typified by bacterial multiplication and development
Of water soaking followed by chlorosis and necrosis in the infected tissues. Pst DC3000
relies on the type III secretion system to deliver protein effectors across the plant cell
wall into the host cell. These effectors are essential for pathogenesis as demonstrated
by the inability of hrp mutants to multiply or cause disease on otherwise susceptible
hosts.

To gain insight into the function of Psi DC3000 effectors in the host cell, I

created transgenic Arabidopsis plants that express avrPto. I showed that transgenic

expression of avrPto repressed a set ofArabidopsis genes that were also repressed
during Pst DC3000 infection. In addition, avrPto plants permitted enhanced
multiplication of a Pst DC3000 hrp mutant, an avirulent derivative of Pst DC3000, and
P. fluorescens (a non-phytopathogenic bacterium). The increased growth of these
bacteria in avrPto plants is not correlated with water-extractible nutrients in the
apoplastic space, but is associated with impaired host extracellular defense and
secretion. avrPto plants were unable to deposit defense-related callose in the cell wall.
Furthermore, several host proteins that are present in the apoplast of wild-type plants
inoculated with an avirulent pathogen were absent in avrPto plants. Based on these and
other results, we postulate that one virulence function of Ath0 in Arabidopsis is to
promote pathogenesis by interfering with host trafficking to the extracellular space.

In addition to determining how AvrPto operates in the plant cell, I investigated
host components that are involved in disease symptom development. An Arabidopsis
mutant screen uncovered a mutant that did not develop disease-associated chlorosis in
response to Pst DC3000 infection. The growth and development of this mutant, nocl
@-§MOrosisl), is not different from wild-type, but the nod plants lose chlorophyll at a
Slower rate than wild-type plants during disease development. Both nod and wild-type
plants had similar increased transcript levels ofAtCIhII (a gene in the chlorophyll
degradation pathway) upon Pst DC3000 infection. The nocI gene is located on the long
arm of chromosome 4.

The information gained from this research may lead to an increased
understanding of the molecular processes that occur during Pst DC3000 infection of

susceptible Arabidopsis.

Acknowledgements

I would first like to thank Dr. Sheng Yang He for accepting me in his lab, his
support, his guidance, and his enlightening discussions throughout my graduate career.

I also would like to thank him for letting me do a six month internship on a project that
was unrelated to his research. I would like to thank Dr. Rebecca Grumet, Dr. Gregg
Howe, and Dr. Jonathan Walton for agreeing to be on my committee, spending their
time with me and giving me their input over the years.

I would like to thank all of the past and current members of the He lab: Jing
Yuan, Wensheng Wei, Anne E. Plovanich-Jones, Julie Zwiesler-Vollick, Wenqi Hu,
Mingbo Lu, Yong Bum Kwack, Qiaoling Jin, Roger Thilmony, Kinya Nomura, Ola
Kolade, Sruti DebRoy, Elena Bray Speth, William Underwood, Yong Hoon Lee, Lori
Imboden, Maeli Melotto, Young Nam Lee, Francisco Uribe, Eliana Gonzales-Vigil. In
addition, I am grateful for all of our undergrads, but especially Nate Pumplin, Katie
McLellen, Marisa Trapp, Beth Rzendzian, Ruth Nantais, and Vusumuzi Ndlovu for help
in the nod project.

I would particularly like to thank Roger Thilmony for collaborating with me I on
the work described in Chapter 2. I would like to thank Elena Bray Speth, Dr. Sheng
Yang He and others for critical reading of this dissertation. I would especially like to
thank Julie Zwiesler-Vollick, Anne E. Plovanich-Jones, Sruti DebRoy, and Elena Bray
Speth for their support, encouragement, advice, and friendship.

I would like to express my gratitude to all Of the support staff at the PRL and the

genetics program. They have really helped me survive graduate school and were

iv

invaluable in answering my questions. I would like to thank Jim Klug for his excellent
care of the growth chambers and helping me keep my plants healthy. I would also like
to thank all of my teachers who have provided me with the knowledge base critical for
understanding science. I would like thank all of the PRL for making my stay at
Michigan State a memorable and rewarding experience. It truly is an environment that
is conducive for research.

Lastly, I appreciate all of the love, encouragement, support, reassurances and
advice I have received from my Family. They have been there for me through it all. I
would also like to thank my husband, Nat, for being patient with me and for tolerating
my tendency for procrastination. I would like to thank him for understanding that when
I say 10 minutes I really mean 40 minutes. I would also like to thank him for critical

reading of this dissertation.

TABLE OF CONTENTS

List of Tables ...................................................................................................................... ix

List ofFigures ................................................. x

Chapter 1: Literature review ............................................................................................... 1
Introduction ................................................................................................................... 2
The pathogen: Pseudomonas syringae pv. tomato strain DC3000 ............................... 3
AvrPto ........................................................................................................................... 6
The host: Arabidopsis thaliana ..................................................................................... 9
Disease resistance mechanisms ................................................................................. 9
Papillae .................................................................................................................... 11
Plant defense signaling hormones: salicylic acid, jasmonic acid and ethylene ...... l4
Rationale ..................................................................................................................... 16

Chapter 2: A Pseudomonas syringae type III effector suppresses cell wall-based

extracellular defense in susceptible Arabidopsis plants .................................................... 29
Abstract ....................................................................................................................... 30
Introduction ................................................................................................................. 3 1
Materials and methods ................................................................................................ 34

Plant grth and bacteria enumeration. .................................................................. 34
Construction Of the COR' hrpS double mutant. ...................................................... 34
Production of AvrPto transgenic plants ................................................................... 35
Microarray experiments. ......................................................................................... 35
Callose staining. ........................................................................ ' .............................. 36
Results ......................................................................................................................... 37
Roles of SA- and ethylene-mediated defense pathways in resistance to
hrp mutants .............................................................................................................. 37
Biased suppression OfArabidopsis genes encoding putatively secreted
cell wall and defense proteins. ................................................................................ 39
Transgenic expression of a single effector, AvrPto, regulates host genes in a
manner similar to that of the Pst DC3000 TTSS. ................................................... 50
The cell wall-based extracellular defense is compromised in Pst DC3000-
infected and AvrPto transgenic plants. .................................................................... 53
Enhanced growth of the hrcC mutant in avrPto transgenic plants. ........................ 55
Discussion ................................................................................................................... 57
References ................................................................................................................... 63

Chapter 3. Further characterization of avrPto plants. ....................................................... 70
Abstract ....................................................................................................................... 71
Introduction ................................................................................................................. 72
Materials and Methods ................................................................................................ 74

Plant grth and bacterial enumeration .................................................................. 74
Callose staining ....................................................................................................... 74
Immunoblotting ....................................................................................................... 75

vi

Secretion assays ...................................................................................................... 75

Results ......................................................................................................................... 77
Enhanced growth of Pseudomonasfluorescens 55 and Pst DC3000 (aerptZ) in
avrPto plants ........................................................................................................... 77
AvrPto does not inhibit the HR triggered by Aerpt2 ........................................... 77
Callose deposition upon Pf 55 and Pst DC3000 (aerptZ) infection is
compromised in avrPto transgenic plants ............................................................... 80
Pst DC3000 (aerptZ) triggers the appearance of several proteins in the IWF of
wild-type plants, but not in avrPto plants ............................................................... 80
The IWF of avrPto plants does not support more bacterial growth than that of
wild-type plants. ...................................................................................................... 83

Discussion ................................................................................................................... 85

References ................................................................................................................... 89

Chapter 4: Characterization of an Arabidopsis thaliana mutant, nocI , with altered
symptom development in response to Pseudomonas syringae pv. tomato DC3000

infection ............................................................................................................................. 91
Abstract ....................................................................................................................... 92
Introduction ................................................................................................................. 93
Methods ....................................................................................................................... 97

Plant material, mutagenesis, and growth conditions ............................................... 97
Screening and isolation of Arabidopsis mutants ..................................................... 97
Bacteria enumeration in infiltrated leaves of nocI mutants and wild-type plants ..98
RNA isolation and northern blotting ....................................................................... 98
Microarray analysis ................................................................................................. 99
Chlorophyll extraction .............................................................. ' .............................. 99
MeJA sensitivity assay .......................................................................................... 100
Ethylene sensitivity assay ..................................................................................... 100
Gene mapping ....................................................................................................... 100
Results ....................................................................................................................... 102
Identification of the nod mutant .......................................................................... 102
The decrease in total chlorophyll level is greater in wild-type plants than in nocI
plants afier infection with Pst DC3000 ................................................................. 102
The expression Of one gene, AtCLHI, in the chlorophyll degradation pathway is
slightly reduced in me] plants .............................................................................. 103
Microarray ............................................................................................................. 108
Sensitivity to JA is similar in me] and wild-type plants ...................................... 110
Sensitivity to ethylene is similar in nod and wild-type plants ............................. 110
The NOCl gene is located on the long arm of chromosome 4 ............................. 113
Discussion ................................................................................................................. 1 15
References ................................................................................................................. 1 1 8

Chapter 5: Summary and future directions ..................................................................... 122
AvrPto ....................................................................................................................... 123
The nocI mutant ........................................................................................................ 128
References ................................................................................................................. 1 3 1

vii

Appendix A: Putative AvrPto interactors ...................................................................... 132

viii

List of Tables

Table 2-1. Arabidopsis genes repressed or induced by Pseudomonas syringae

pv. tomato DC3000 in a type III secretion-dependent manner ............................ 43
Table 2-2. Predicted locations of proteins encoded by TTSS-regulated

Arabidopsis genes ............................................................................... 48
Table 2-3. TTSS-repressed genes that encode proteins predicted to enter the

secretory pathway ................................................................................ 49
Table 3-1. Average callose deposition in wild-type and avrPto plants after

inoculation with Pst DC3000, hrpA mutants, Pf 55, or Pst DC3000 (aerptZ) ......... 81
Table 4-1. Differentially regulation of genes in the nocI mutant compared to
wild-type plants .................................................................................. 109
Table 4-2. Genes of chromosome 4 located near NOCI that encode proteins predicted
to be targeted to the chloroplast ............................................................... 114
Table A-1. Putative interactors of AvrPto .................................................. 135

ix

List of Figures

Images in this dissertation are presented in color.

Figure 2-1. Bacterial populations in wild-type Col-0, ein2, and nahG transgenic

plants ................................................................................................ 38
Figure 2-2. Phenotype of avrPto transgenic plants ........................................... 52
Figure 2-3. Callose depositions in wild-type and avrPto leaves ............................ 54
Figure 2-4. Bacterial populations in wild-type and avrpto transgenic plants ............. 56

Figure 2-5. A hypothetical model of the function of the AvrPto class of type III
effectors ............................................................................................. 60

Figure 3-1. Growth of Pf 55 and Pst DC3000 (aerptZ) in avrPto-expressing plants..78

Figure 3-2. avrPto leaves after infiltration Of Pst DC3000 (aerptZ) ..................... 79
Figure 3-3. Protein profiles of the IWFS from avrPto plants inoculated with water,

Pst DC3000 or Pst DC3000 (aerptZ) .......................................................... 82
Figure 34. Growth of the hrcC mutant in the IWFS from wild-type (WT) and

avrPto plants ........................................................................................ 84
Figure 4-1. The chlorophyll degradation pathway... . . . .. . .. . ... . .. ..- ....................... 96
Figure 4-2. Phenotype of the mo] mutant after Pst DC3000 inoculation. . . . . . . . . 1 04
Figure 4-3. Growth of Pst DC3000 in nocI plants ......................................... 105
Figure 4-4. Total amount of chlorophylls (a and b) in wild-type and nocl leaves

during the course of Pst DC3000 infection ................................................... 106
Figure 4-5. Northern blot analysis OfAtCIhI transcript .................................... 107
Figure 4—6. Wild-type, nocl, and coil seedlings on SOuM MeJA medium. . . 1 11
Figure 4—7. Wild-type (WT), nocl, and ein2 seedlings germinated in the presence

of ethylene ......................................................................................... l 12
Figure A-l. AvrPto interacts with the RabE family ......................................... 144

Chapter 1: Literature review

Introduction

Plants, as photosynthetic organisms, are essential for most other forms of life
because they are able to harness energy from the sun and convert it into a form that can
be used by other organisms. Since plants are so important, it is vital for us to
understand how they work and what factors influence their yield. Being sessile, they
cannot avoid unfavorable conditions, but must cope with environmental stresses. Plants
are in constant contact with viruses, fungi, nematodes, insects, and bacteria. Some of
these organisms have evolved the ability to cause disease on plants by evading or
overcoming the plants’ resistance mechanisms. Diseases caused by these pathogens
result in significant crop losses each year (1).

The interaction between a plant and a pathogen is defined based on its outcome.
In a compatible interaction, in which the host plant is susceptible and the pathogen is
virulent, disease will occur. An incompatible interaction, involving a resistant host and
an avirulent pathogen, leads to resistance.

Despite the rapidly accumulating knowledge of the components and
mechanisms of resistance, the molecular basis of plant susceptibility to pathogen
infection remains largely elusive. In order to study disease development, it is
advantageous to have a model plant-pathogen system, such as Arabidopsis thaliana and
the bacterial pathogen Pst DC3000. Both organisms have sequenced genomes and have
many other attributes (discussed below) that make them good models for studying

plant-pathogen interactions.

The pathogen: Pseudomonas syringae pv. tomato strain DC3000

Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) is an extracellular
bacterial pathogen that causes bacterial speck disease in A. thaliana and tomato plants.
Bacterial speck occurs throughout the world where conditions are cool and wet. The
bacteria are spread by aerosols or splashed by rain and enter leaves through existing
openings such as stomata or wounds. Pst DC3000 is an extracellular pathogen because
it remains outside of the plant cell and multiplies in the leaf apoplastic space. In tomato,
disease symptoms include small black or brown necrotic lesions (specks) that become
surrounded by chlorotic halos caused by the bacterial toxin coronatine. Lesions also
form on both unripe and ripe tomato fruit, causing decreased marketability of the fruit
(2). The symptoms on Arabidopsis are similar to those on tomato; water soaking
develops within two days and necrosis surronded by chlorosis occurs by three days after
infection.

Pst DC3000 is a successful pathogen because of a variety of virulence
mechanisms. The previously mentioned phytotoxin, coronatine, has been shown to be
important for virulence because bacterial mutants that are unable to produce coronatine
are less virulent on wild-type Arabidopsis (3-5). The most important virulence
mechanism, however, is the type III protein secretion system. The type 111 protein
secretion system is encoded by genes found in the 25 kb hrp (hypersensitive response
and pathogenicity) gene cluster. The proteins encoded by these genes are required for
both the HR and pathogenicity, since hrp mutant bacteria (e.g., hrcC and hrpS), which

are defective in type III secretion, do not multiply or cause disease symptoms in host

plants. Other genes in the hrp cluster encode proteins responsible for regulation of the
type III secretion system, several effectors, and genes of unknown function (6).

The type III secretion system is only expressed in planta or in minimal media
(which is thought to mimic in planta conditions) and its transcription is tightly regulated
by hrpR and hrpS. These proteins are members of a two-component regulatory system
that is required for the transcription of the hrpL gene. hrpL encodes an alternative
sigma factor which is thought to bind a particular cis-element in the promoters of hrpRS
regulated genes known as the hrp box. Most effector genes, as well as the hrp genes
themselves, contain a hrp box in their promoter region (7).

The type III-secreted effectors are thought to be translocated directly to the host
cell cytoplasm via the Hrp pilus, and a number of experiments substantiate this idea (6,
8-18). About 40 effectors have been identified in Pst DC3000, and a list of all the
known effectors (Avr and virulence proteins) as well as a guide to commonly used
terminology is available at the Pseudomonas database (http://wwwpseudomonag
sgingaeorg/pst home.html). Although effectors are known to be crucial for virulence,
their mode of action in the plant cell is only beginning to be elucidated. Mutating or
deleting individual effectors has little or no effect on virulence. This is likely due to
either fimctional redundancy or the possibility that each effector has only a small
quantitative effect on virulence. Currently, four approaches are most frequently used to
study the functions of type III effectors. First, a search for sequence homology with
known proteins can reveal possible effector functions that can be tested. Unfortunately,
most effectors have no homology to other genes in the databases. Second, microarray

analysis can be used to study effector function by analyzing transcriptional changes in

the host induced by the wild-type pathogen or various effectors. Third, transgenic
plants that express a single effector can be created and its affect on the host studied.
Fourth, cross-kingdom yeast-two-hybrid screens to identify host proteins that physically

interact with specific effectors can be conducted.

AvrPto

AvrPto is a well studied effector, mainly for its role in avirulence on tomato.
Different races of Pst differ in their virulence on tomato plants. Resistance to Race 0
strains is controlled by a single resistance locus, called Pto (19). Ronald et al. (20)
found that avrPto was responsible for limiting disease on resistant tomato plants
carrying Pto. This gene is present in all the 14 Race 0 strains tested and in none of the
12 Race 1 strains. P. syringae pathogens of radish, bean, pea, and oat all have
sequences homologous to avrPto (20). AvrPto has virulence activity in Pst race T1
when a functional Pto pathway is absent in the host (21, 22).

AvrPto was further analyzed by Salmeron and Staskawicz (23), who found that
the protein is encoded by a single ORF whose predicted translation product is a 164
amino acid protein Of 18.3 kD. Although the protein is mostly hydrophilic, its first 6
amino acids are hydrophobic. Salmeron and Staskawicz (23) also found that avrPto has
a conserved hrp box in its promoter and its expression is coordinately regulated with the
hrp genes. Induction of avrPto occurs within 1 hour after infiltration into either
resistant or susceptible tomato plants (23). As with many gene-for-gene interactions,
Pto-mediated resistance to avrPto-expressing Pst strains is associated with a localized
HR.

Pto encodes a hydrophilic 321 amino acid protein that was identified as a serine-
threonine protein kinase (24). There has been a considerable amount of research
conducted on Pto (16, 25-46); for a review, see Pedley and Martin (2). By Southern

analysis, Pto-like sequences are present in potato, tobacco, Arabidopsis, bean, soybean,

pea, rice, maize, barley, wheat, and sugarcane (24). However, to date, none of these
Pto-like proteins have been shown to have recognition specificity for AvrPto (2).

AvrPto acts within the plant cell, as demonstrated by Agrobacterium-mediated
transient expression of avrPto in tobacco leaves (15, 47). The HR was observed in Pto-
expressing leaves but not in leaves lacking Pto. This confirms that AvrPto acts alone,
without additional Pseudomonas proteins, inside the plant cell to elicit HR in a Pto-
specific fashion. In addition, AvrPto interacts directly with Pto in the yeast-two-hybrid
system (15, 47, 48). Alterations of AvrPto or Pto that disrupt the interaction in yeast
also abolish disease resistance in plants (15). Currently, the interaction of Pto and
AvrPto has yet to be demonstrated in vivo (2). Several studies investigated the effect of
point mutations (22, 49, 50) and deletions (15, 47) in avrPto on its ability to interact
with Pto. Although the majority of mutations did not affect the interaction, alterations
to several residues disrupted binding to Pto. In all of the cases where the mutation in
AvrPto disrupted binding to Pto, HR was abolished as well. Only one study (22)
evaluated the effect of avrPto mutations on virulence. Shan et a1. (22) found three
mutations that affected binding with Pto. However, these mutations did not decrease
the virulence activity of AvrPto. In fact, there are no known mutations that code for
stably expressed AvrPto proteins in Pseudomonas that have been shown to affect
virulence.

AvrPto contains a putative myristylation site at the N-terminus (50, 51). AvrPto
is associated exclusively with the plant plasma membrane (50). A G2A mutation of the
myristylation motif abolished this localization (50). Although this mutation did not

affect type 111 protein secretion in bacteria, the interaction with Pto in the yeast-two-

hybrid system, or the stability in plant cells, the mutant protein failed to exhibit
avirulence activity in tomato and tobacco. These findings suggest that association with
the host plasma membrane is critical for recognition by Pto ”(50).

Bogdanove and Martin (48) screened a tomato cDNA library for proteins that
interact with Pto in an AvrPto-dependent fashion. They found a catalase, two
serine/threonine kinases, a large hydrophilic protein and Pti2 (a proteasome alpha
subunit). They also looked for proteins that interacted with AvrPto and found a stress-
related protein, an N-myristyltransferase and 2 small Ras-related GTP binding proteins.
At the time of this publication, the authors had not established whether any of these
proteins were actually involved in resistance or virulence. The functional Significance of

these interactions remains to be determined.

The host: Arabidopsis thaliana

A. thaliana has proven to be a valuable model for plant research. It has a short
life cycle, many plants can be propagated in a limited space because of its small size,
and it has a small, sequenced genome (115Mb, >25,500 genes). A. thaliana is easily
transformable via Agrobacterium-mediated transformation. Many resources are
available to the scientific community, including: the Arabidopsis Information Resource
(hips/NWWArabidopsis.0232/), the TIGR Arabidopsis thaliana Database
http://wmutigr.org/tdb/e2kl/ath l/ , the Monsanto Arabidopsis polymorphism and Ler
sequence collection (thM/www.Arabidopsis.org/Cereonfl, insertion knock-out
collections (http://signal.salk.edu/cgi-bin/tdnaeigpress and http://nasc. nott. ac. uk/ ), and
the Massively Parallel Signature Sequencing site (http://mnss. udel.edu/at/iava.html ),
which contains expression data for genes in different tissues under different
experimental conditions. Arabidopsis and other plants exhibit similar defense
responses; therefore, components of the plant-pathogen interaction that are identified in

Arabidopsis will likely have similar counterparts in crop species (52).

Disease resistance mechanisms

In addition to pre-existing defenses, such as the plant cell wall, cutin, wax, and
other structural components, which provide the first line of defense, there are many
types of induced resistance responses that protect the plant against pathogenic
microorganisms. The different classes of resistance to microbes include gene-for-gene

resistance, systemic acquired resistance, non-host resistance and basal defenses. These

forms of resistance often overlap and work cooperatively to prevent the growth of
pathogens.

Flor’s gene-for-gene hypothesis (53, 54) states that the genetic interaction
between a pathogen avirulence gene product (Avr) and the corresponding host plant
resistance gene product (R) leads to resistance. Gene-for-gene resistance is often
accompanied by the hypersensitive response (HR) and up-regulation of local defenses.
One example Of a gene-for-gene interaction is described in this section. aerptZ is an
avirulence gene from P. syringae pv. tomato strain JL1065 that causes the HR in
Arabidopsis cells that express RPSZ (the corresponding R gene). RPSZ physically
interacts with a protein called RIN4, whose presence is eliminated by Aerpt2 in an
RPS2-independent manner (55). , Aerpt2 is a cysteine protease (56). RPSZ initiates
resistance signaling in response to the disappearance of RIN4, rather than by direct
recognition of Aerpt2 (55, 57).

Systemic acquired resistance (SAR) provides resistance throughout the plant
against a wide range of pathogens for an extended period of time (58). Local infection
leads to systemic resistance against subsequent challenge with potential pathogens.
Necrogenic fungal, bacterial, and even viral pathogens or elicitors can all trigger this
resistance mechanism (59). SAR is accompanied by elevated expression of
pathogenesis-related (PR) genes.

Non-host resistance is the most durable and common form of plant resistance in
nature (60). Very little is known about the molecular mechanism of non-host resistance,
but there is one Arabidopsis gene, non-host resistance (NHOI), which plays significant

role in non-host resistance to bacteria and fungi. It encodes a glycerol kinase and is

10

required for wild-type Arabidopsis resistance to Botrytis cinerea and P. syringae
isolates from bean or tobacco, which are normally not pathogenic on Arabidopsis (61 ,
62). The expression of NHOI is suppressed by virulent P. syringae (61). Interestingly,
hrp mutants, saprophytes, and avirulent strains of bacteria are all able to multiply in
nhol mutants (62). nhol plants are capable of responding with an HR to avirulent P.
syringae strains. This result is interesting because HR usually signals up-regulation Of a
successful defense response.

Gene-for-gene resistance, SAR, and non-host resistance are all elicited by
pathogens. There is another type of resistance, called basal defense, which is elicited by
both pathogenic and non-pathogenic bacteria. Basal defenses involve up-regulation of
several defense/stress genes (e.g. phenylalanine ammonia lyase, chalcone synthase and
chitinase) and the production of phytoalexins (63, 64), and is elicited by molecules that
are conserved in both plant pathogens and non-plant pathogens. Flg22 and flng, two
peptides corresponding to the most conserved domain of eubacterial flagellin, for
example, elicit this basal defense (65). The receptor for these peptides is FLS-l/F L82
(flagellin sensing 1 or 2). Treatment OfArabidopsis leaves with these peptides caused
the rapid release of active oxygen species, papillae formation (to be discussed in the
next section), and strongly inhibited bacterial multiplication (65). Papillae are elicited

by flg22 and flng, and are discussed in more detail in the next section.

Papillae

Deposition of papillae at the site of contact with bacteria or attempted
penetration by fungal hyphae is an integral part of most forms of plant resistance to

microbial pathogens and non-pathogens (66). Papillae form beneath infection sites

11

between the cell wall and the plasma membrane and are composed of callose, phenolics,
hydroxyproline-rich glycoproteins (HRGPs) (e.g., extensins), and other materials. Their
formation involves the synthesis and directed deposition of these compounds to the site
of the interaction (67). Callose is an exception in that it is synthesized at the site of
infection. Callose is a B-l,3-glucan with some 1,6 branches (68). Callose is a
convenient marker for papillae. It is easily stained with aniline blue and can be
visualized by fluorescence microscopy. Although papillae normally contain callose, it
is important to note that the formation of papillae lacking callose is possible (69).

Although the precise function of papillae during microbial attack has not been
demonstrated unequivocally, it has been postulated that they act as physical barriers.
According to this interpretation, papillae impede microbial penetration (69) or
immobilize the invading microbe and potentially expose it to anti-microbial compounds
(67), such as wall-degrading enzymes, phytoalexins, and active oxygen species. Callose
may also contribute to host defenses by impeding nutrient transfer from the host to the
pathogen or possibly by delaying pathogen growth long enough for other host defenses
to become active (70). There are several studies that demonstrate the importance of
callose and papillae (69, 71-73), as well as studies that demonstrate that callose is not
important for defense against pathogens (69, 74, 75).

Papillae are deposited much more quickly in response to an avirulent strain than
to a hrp mutant (76). The first response to avirulent bacteria is the apparent convolution
of the plasma membrane adjacent to bacterial cells (67, 76, 77) and, within three to five
hours after inoculation, lightly stained fibrillar materials accumulate between the

convoluted membrane and the plant cell wall (76). The early stages of papilla

l2

formation are frequently associated with the presence of Golgi and ER in the underlying
cytoplasm. Immunogold labeling revealed that callose is present at all stages of papillae
development, but not in the cell wall before inoculation (67). Deposits increase in
thickness and complexity between 3-8 h. The plasma membrane can be detached from
developing deposits during plasmolysis (76). As papillae develop, distinct proliferation
and swelling of the endoplasmic reticulum occurs in the majority of challenged cells.
Smooth vesicles and multivesicular bodies (MVBS) become visible within the
cytoplasm and near sites of deposition. In some cases, the MVBs appear to fuse with
the plasma membrane, discharging vesicles out of the cell. As deposits increased in
complexity, an electron-translucent material appears throughout the fibrillar matrix,
which contains layers of irregularly shaped osmiophillic particles and vesicles.
Histochemical studies indicate that the earliest deposits contain HRGPS and that the
initial matrix becomes impregnated with phenolics and finally callose (76).

hrp mutant strains ofXanthomonas campestris pv. vesicatoria induce the
formation of large papillae in pepper regardless of whether they are inactivated by
antibiotic treatment before inoculation or not (77). In contrast, wild-type pathogenic
strains do not elicit papillae formation unless they are inactivated by chloramphenicol or
heat-killed before inoculation. However, if antibiotic treatment of the wild-type strain
is delayed until 8 hr afier inoculation, no large papillae are produced. These
experiments Show that the wild-type strain actively suppresses the deposition of papillae

in a hrp gene-dependent manner (77).

13

Plant defense signaling hormones: salicylic acid, jasmonic acid and ethylene

The various forms of plant resistance just described require one or more defense
hormones, including salicylic acid (SA), jasmonic acid (JA) and ethylene. Hormone
signaling in response to pathogens is complex and depends on the plant-pathogen
system. In many cases, antagonistic or synergistic cross-talk between the SA, JA and
ethylene pathways has been described (78-85).

The Arabidopsis response to bacterial pathogens is strongly dependent on SA.
Transgenic plants that constitutively express nahG, which encodes an enzyme,
salicylate hydroxylase, that degrades SA to catechol, do not have detectable levels of
SA (86), and are hyper-susceptible to a variety of pathogens, including Pst DC3000.
The expression of PR genes has proven to be a good marker for SA-based defenses
because SA is required for PR gene induction to occur; however, there is little evidence
for their role in inhibiting bacterial growth.

Ethylene, traditionally known for its role in a wide variety of physiological
processes including seed germination, cell elongation, epinasty and various forms of
senescence, including fruit ripening, has been shown to mediate responses to pathogen
infection as well (79, 80, 87-89). The Arabidopsis ein2 (ethylene i_nsensitive) mutant
has reduced symptom development upon Pst DC3000 infection, without a reduction in
bacterial growth (90). ein2 is an integral membrane protein that acts downstream of the
ethylene receptors and upstream of the gene transcription changes associated with the
ethylene response (87).

JA is essential in flower development because JA biosynthetic and perception

mutants are sterile (91). JA is structurally similar to the phytotoxin coronatine,

14

produced by Pst DC3000 and several other strains of P. syringae. Besides causing
chlorosis, coronatine causes stunted roots as well as other physiological changes in a
broad variety of plants (92). The coil (mronatine-insensitive) Arabidopsis mutant was
isolated in a screen for mutants that exhibit normal root growth in the presence of
coronatine (91, 93). coil plants are also insensitive to JA, resistant to infection by
bacterial pathogens (91, 94), and more susceptible than wild-type plants to some fungal
pathogens (84, 95). C011 encodes an F-box protein (93, 96-98), which regulates
expression of JA-responsive genes, possibly by targeted ubiquitination of a histone

deacetylase and other factors (96).

15

Rationale

Currently, one pathogen control is the use of expensive and toxic chemicals. An
environmentally safer method of control involves genetic modification, such as
breeding R genes into crops. The problem with the latter method is that monoculture
and genetic uniformity create a significant selection pressure for pathogens to overcome
host resistance mechanisms. For example, a mutation in the corresponding avr gene
could be sufficient for the pathogen to evade recognition by a newly introduced R gene.
One way for plant breeders to cope with this problem is to combine several R genes
within a single cultivar so that multiple avr genes would have to be mutated in order to
avoid detection. This process, called “pyramiding”, along with crop rotation has helped
reduce crop losses due to successful pathogens. However, there are a limited number of
R genes available, and pathogens may evolve and eventually overcome all available R
genes. Therefore, new methods other than R gene-mediated resistance and increased
chemical use are needed to prevent the yield losses caused by plant pathogens.

The ultimate goal of this research is to determine the molecular basis of
susceptibility ofArabidopsis to Pst DC3000. It is hoped that a basic knowledge of the
mechanisms underlying disease progression can help the development of alternative

disease control strategies. I focused my Ph.D. research on two specific questions.

1.) What is the virulence function of AvrPto in the compatible interaction
between Arabidopsis and Pst DC3000?
2.) What is the molecular basis of symptom development during disease caused

by Pst DC3000?

l6

I attempted to achieve these objectives by two different approaches. I created
transgenic plants that express avrPto and evaluated these avrPto plants to gain an
understanding of the role of AvrPto in virulence. I also characterized an Arabidopsis

mutant that has altered symptom development in response to pathogen infection.

17

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28

Chapter 2: A Pseudomonas syringae type III effector
suppresses cell wall-based extracellular defense in susceptible
Arabidopsis plants

This chapter has been previously published in Proceedings of the National Academy of
Sciences USA.

HAUCK, P.*, THILMONY, R.* & HE, S. Y. (2003) A Pseudomonas syringae type III
effector suppresses cell wall-based extracellular defense in susceptible Arabidopsis
plants, Proc Natl Acad Sci U S A, 100, 8577-82.

* These authors contributed equally to this work.

I would like to acknowledge Roger Thilmony for contribution of Figure 2-1, Table 2-1,
Table 2-2 and Table 2-3.

29

Abstract

Bacterial effector proteins secreted through the type III secretion system (TTSS)
play a crucial role in causing plant and hmnan diseases. Although the ability of type III
effectors to trigger defense responses in resistant plants is well understood, the disease-
promoting functions of type III effectors in susceptible plants are largely enigmatic.
Previous microscopic studies suggest that in susceptible plants the TTSS of plant-
pathogenic bacteria transports suppressors of a cell wall-based plant defense activated
by the TTSS-defective hrp mutant bacteria. However, the identity of such suppressors
has remained elusive. We discovered that the Pseudomonas syringae TTSS down-
regulated the expression of a set OfArabidopsis genes encoding putatively secreted cell
wall and defense proteins in a salicylic acid-independent manner. Transgenic expression
of AvrPto repressed a similar set of host genes, compromised defense-related callose
deposition in the host cell wall, and permitted substantial multiplication of a hrp mutant
AvrPto is therefore one of the long postulated suppressors of a salicylic acid-

independent, cell wall-based defense that is aimed at non-pathogenic bacteria.

30

Introduction

Many plant pathogenic bacteria, such as Pseudomonas syringae, carry a type III
secretion system (TTSS), which delivers effector proteins into the plant cell (1-5).
Translocation of these effectors is required for bacterial pathogenesis. The TTSS also
plays a crucial virulence role in bacterial diseases of mammals (3, 4, 6, 7). However,
mammalian and plant pathogenic bacteria appear to produce largely distinct sets of type
III effectors, possibly reflecting their different lifestyles and unique host cellular
structures (8-13). For intracellular mammalian pathogenic bacteria, such as Salmonella
and Shigella, a key function of type III effectors is the regulation of host cytoskeleton
dynamics, which aids the invasion of bacteria into the host cell (6). Most plant
pathogenic bacteria, such as P. syringae, however, are noninvasive, extracellular
pathogens; they colonize the host intercellular space outside the plant cell wall, a
structure absent in animal cells. TTSS-defective bacteria do not usually multiply or
cause disease symptoms in otherwise susceptible plants. The inability of TTSS mutants
to multiply in the plant intercellular space is similar to that of saprophytic bacteria found
in nature.

In plant pathogenic bacteria, the TTSS is encoded by hrp (hypersensitive
reaction and pathogenicity) genes (1, 5). We are using P. syringae pv. tomato strain Pst
DC3000 (Pst DC3000), which infects Arabidopsis and tomato (14, 15), to elucidate the
virulence function of the TTSS in bacterial pathogenesis in plants. In Arabidopsis, Pst
DC3000 multiplies aggressively for 2 days before the onset of disease symptoms, which
is characterized by water soaking in the apoplast, followed by tissue necrosis and

chlorosis (14, 15). We have shown (16, 17) that the ability of Pst DC3000 to infect

31

Arabidopsis depends on the TTSS because hrp mutants [e.g., hrpS and hrcC (formerly
hrpH) mutants] of Pst DC3000 do not multiply or cause disease in Arabidopsis. The
TTSS of Pst DC3000 is believed to secrete and/or translocate >30 effector proteins into
the host cell (8-13). Cumulatively, these effectors alter host cellular processes and
promote disease development through largely unknown mechanisms. Although the
primary function of type III effectors is to promote plant susceptibility, some effectors
may be recognized by the corresponding plant disease resistance proteins in resistant
plants and trigger defense responses, including the hypersensitive response (HR) (18,
19). In fact, many type III effector genes in P. syringae were discovered based on their
ability to trigger the HR in resistant plants and have been named avr (for avirulence)
genes (20). For example, the type III effector, AvrPto, was identified based on its
avirulence activity in plants (21-23). Although the ability of type III effectors to trigger
defense responses in resistant plants is well understood, the mechanism by which type
III effectors, as a group, enable plant pathogenic bacteria to proliferate in the
intercellular space of a susceptible plant remains enigmatic. In addition to type III
effectors, Pst DC3000 also produces the phytotoxin coronatine (COR), which is
required for full virulence in Arabidopsis (24-26).

A decade ago, Jakobek and coworkers (27, 28) showed that in bean, general
defense genes encoding phenylalanine ammonialyase, chalcone synthase, and chalcone
isomerase, which are involved in the biosynthesis of antimicrobial phytoalexins, are
induced by the hrp mutants of a non-host bacterium, P. syringae pv. tabaci, and
saprophytic bacteria, but not by the wild-type virulent P. syringae pv. phaseolicola.

Ultra-structural studies have illustrated that hrp mutants OfXanthomonas campestris pv.

32

vesicatoria and P. syringae pv. phaseolicola, as well as a saprophytic bacterium, cause
the plant cell wall to thicken, forming a papilla (29-31). Papillae are cell wall
appositions composed of callose, phenolics, hydroxyproline—rich glycoproteins (e.g.,
extensins), and other materials. The type III secretion-competent wild-type X.
campestris pv. vesicatoria, on the other hand, does not induce papillae formation (30).
These experiments led to the attractive hypothesis that TTSSS of plant pathogenic
bacteria secrete one or more suppressors of this hallmark cell wall-based plant defense
response elicited by nonpathogenic bacteria (e. g., hrp mutants and saprophytic bacteria).
However, the identity of such a suppressor has remained elusive. Similarly, the plant
defense response that is aimed at hrp mutant bacteria, but is overcome by the TTSS, is
also poorly defined at the molecular level.

In this chapter, we used a combination of large-scale host gene expression
profiling, transgenic expression of a Pst DC3000 effector, and cytological examination
to identify AvrPto as a suppressor of the papilla-associated cell wall defense.
Furthermore, we Show that the TTSS of Pst DC3000 is involved in highly biased
suppression of a set of Arabidopsis genes that encode putatively secreted cell wall and
defense proteins in a salicylic acid (SA)-independent manner. This research provides a
much needed guide for further progress on the elucidation of the virulence functions of

type III effectors in susceptible plants.

33

Materials and methods

Plant growth and bacteria enumeration.

Arabidopsis thaliana accession Col-0 gll plants were grown in soil in growth
chambers with a day/night cycle of 12 h/12 h, a light intensity of 100 BE, and a constant
temperature of 20°C. Four- to 5-week-old plants were used for experiments. Bacteria
were grown in low-salt Luria—Bertani broth (14, 32) to the mid- to late-logarithmic
phase at 30°C. Bacterial cultures were centrifuged to recover bacteria, which were
resuspended in sterile water to a final OD600 Of 0.002 [equivalent to 1 x 106 colony-
forming units (CF U)/ml]. Fully expanded leaves were infiltrated with bacterial
suspensions, and bacteria were enumerated as described by Katagiri et al. (14). The
mean values of the bacterial populations are plotted with the SD displayed as error.
Plants analyzed in Figure 2-4 were sprayed daily with a 30-uM dexamethasone solution
containing 0.02% Silwet L-77 (Osi Specialties, Friendship, WV). Bacterial suspensions
were infiltrated into leaves 1 day after the first dexamethasone treatment. The
regulation-defective hrpS mutant and the secretion-defective hrcC mutant used in this

article were described (17).

Construction of the COR' hrpS double mutant.

The COR“ hrpS double mutant was generated by introducing a reported (25)
Tn5Sp-disrupted hrpS gene into the chromosome ofDC3118 (COR' mutant) through
marker exchange mutagenesis. The COR' mutant causes a normal HR in tobacco, but
slightly reduced and delayed disease symptoms in Arabidopsis, suggesting a virulence

role of COR in Pst DC3000—Arabidopsis interaction The COR' hrpS mutant does not

34

elicit an HR in tobacco or cause disease in Arabidopsis. The wild-type hrpS gene
carried on pHRPRSZ (33) restored the ability of the COR’ hrpS mutant to elicit an HR

in tobacco and cause disease symptoms in Arabidopsis.

Production of AvrPto transgenic plants.

avrPto was amplified by PCR from Pst DC3000 (not strain JL1065) genomic
DNA using the following primers: sense primer 5'-
CCGCTCGAGACCATGGGAAATATATGTGTC-3' and anti-sense primer 5'-
GACTAGTTCATTGCCAGTTACGGTACG-3'. The avrPto fragment was cloned into
pTA7002 under the control of the dexamethasone-inducible promoter (34, 35) and
confirmed by sequencing. AvrPto transgenic plants were produced after a protocol that
was described (36). Seven independent avrPto transforrnants were analyzed and all

exhibited characteristics similar to those of lines 76 and 129 reported here.

Microarray experiments.

Four- to 5-week-old A. thaliana accession Col-0 gll leaves were vacuum-
infiltrated with bacterial suspensions containing 1 x 10‘5 CFU/ml bacteria (14). For
microarray analysis, infiltrated leaves were collected at 12, 24, and 36 h post-
inoculation, before the appearance of water-soaking symptoms (at ~48 h) and necrosis
and chlorosis (at ~72 h). Total RNA was isolated from each leaf sample and equal
amounts of RNA from different time points were pooled for DNA microarray analysis
according to the protocol described (37). The first two microarray experiments were
performed by using the Arabidopsis Functional Genomic Consortium's (Michigan State

University) microarray slides, each containing ~7,200 unique genes (37). Subsequent

35

experiments were performed by using a subarray enriched for Pst DC3000-regulated
genes (RT. and S.Y.H., unpublished data).

Genes with a .>_2-fold expression difference (a ratio of 50.5 for repressed genes or
a ratio of 22.0 for induced genes) in at least two of the three biological replicates of the
Pst DC3000/hrpS mutant comparison in Col-0 Arabidopsis plants (I-A, I-B, and I-C) are
described in Table 2-1.

Gene clustering analysis shown in Figure 2-ZB was performed by using the
CLUSTER and TREEVIEW programs (38). The predicted protein locations were
determined using TARGETP analysis conducted on the Arabidopsis genome by the
Munich Information Center for Protein Sequences (Neuherberg, Germany), which can

be accessed at http://mips.gsf.de/prOi/thal/db/tables/tabIes menu.html (39).

Callose staining.

Arabidopsis leaves were sprayed with 30 11M dexamethasone and then infiltrated
24 h later with a bacterial suspension of OD600 = 0.2 (1 x 108 CF U/ml). Leaves were
harvested 12 h after bacterial infiltration, cleared, and stained with aniline blue for
callose as described (40). Leaves were examined with a Leica DM RA2 microscope
with an A4 fluorescence cube. The number of callose depositions was determined with
QUANTITY ONE software (Bio-Rad). More than 10 adjacent fields of view along the
length of the leaf (not including the mid-vein or leaf edge) were analyzed and averaged.
The values in Figure 2-3B are the average and SD of more than five independent leaves

for each treatment.

36

Results

Roles of SA- and ethylene-mediated defense pathways in resistance to hrp mutants.

Recently, several P. syringae type III effectors, most notably AvrPtoB, VierhA,
VierhF, Aerpt2, and Aerme have been shown or suggested to modulate the HR or
SA defense (41-45). To test the hypothesis that it is host defense that prevents efficient
multiplication of the TTSS-defective mutants in the intercellular space, we examined
the multiplication of the Pst DC3000 hrcC mutant in nahG (46) and ein2 (47) plants,
which are defective in two major defense pathways effective against avirulent and/or
virulent strains of P. syringae: the SA-mediated pathway and the ethylene-mediated
pathway, respectively (48). We found that the hrcC mutant reached a slightly higher
population in nahG plants, compared with wild-type control plants (Figure 2-1).
However, the 5-fold population increase was small compared with the >10,000-fold
increase of the Pst DC3000 population in wild-type leaves (Figure 2-1). No significant
increase in multiplication was observed for the hrcC mutant population in the ein2
plants, compared with that in wild-type plants (Figure 2-1). Thus, abrogation of the
SA- or ethylene-mediated defense pathway is not sufficient for a TTSS-defective mutant
to multiply efficiently in the Arabidopsis intercellular space. These observations argue
against a primary role of the SA- or ethylene-mediated resistance in preventing the

growth of the nonpathogenic hrp mutants in Arabidopsis.

37

 

 

 

 

 

 

 

 

Figure 2-1. Bacterial populations in wild-type Col-0, ein2, and nahG transgenic plants.
hrcC mutant growth in Col-0 (black bars), ein2 (dark gray bars) and nahG (light gray
bars) leaves. Pst DC3000 growth in Col-0 (white bars) is shown for comparison.
Graph was contributed by Roger Thilmony.

38

Biased suppression of Arabidopsis genes encoding putatively secreted cell wall and
defense proteins.

To date, a host gene expression signature that marks the virulence function of the TTSS
has not been identified in any plant pathogenic bacterium. To gain molecular insight
into the enigmatic virulence functions of Pst DC3000 type III effectors, we used a
cDNA microarray to examine the expression of ~7,200 randomly chosen Arabidopsis
genes in pre-symptomatic leaves inoculated with Pst DC3000 or hrp mutants (Table 2-
1).

In initial experiments we compared the gene expression profiles in leaf tissues
inoculated with DC3000, the hrpS regulatory mutant, or the hrcC secretory mutant (17).
Comparison of gene expression profiles using DC3000 and the hrpS mutant enabled us
to identify 385 genes that are differentially regulated at >2-fold (a ratio <0.5 for
repressed genes or >2.0 for induced genes in at least two of the three biological
replicates; R.T., E. Bray-Speth, and S.Y.H., unpublished results). A similar profile was
obtained using DC3000 and the hrcC mutant. Surprisingly, we found many jasmonic
acid (JA)-response genes among DC3000-regulated genes. The TTSS was recently
found to influence the production of the phytotoxin coronatine (COR), a molecular
mimic of JA, in DC3000 (9, 49). Further analysis using the DC3118 COR' mutant
((25); defective in the production of COR) led to identification of a large number of
COR-responsive genes. In order to identify TTSS-regulated host genes, we compared
leaf tissues inoculated with the COR' mutant (defective in only COR production) to that
of tissues inoculated with the COR‘ hrpS double mutant (defective in both COR

production and type III secretion). Using this comparison, we selected genes that were

39

differentially expressed at >2.0-fold in two biological replicates Of experiment I and at
>1 .8-fold in both biological replicates of experiment II. That analysis identified the 117
genes contained in Table 2-1. The differential expression patterns in experiments I and
II are globally similar. The quantitative difference between experiments I and 11 may
suggest an additive contribution of COR toxin to the regulation of at least some of these
host genes. To examine the reproducibility of our microarray results, we also
conducted RNA blot analysis of 10 selected genes (At2g38540, Atl g72610, At1g12090,
At2g10940, At1g03870, Atlg29670, At3g16240, At2g17500, At5g26340, and

At4g023 80), all independently confirming their TTSS-dependent expression (R.T., E.
Bray-Speth, and S.Y.H., unpublished results). Of the 117 genes whose expression was
associated with the fimctions of the Pst DC3000 TTSS (Table 2-1), 53 were repressed
and 64 were induced.

Examination of the Arabidopsis genes repressed by the Pst DC3000 TTSS
revealed that a surprisingly large percentage of the genes encode putatively secreted
proteins. In fact, 42% of repressed genes are predicted to encode proteins that enter the
plant secretory pathway, compared with only 17% of the whole genome and 16% of
genes on the microarray used in this article (Table 2-2). On the other hand, the proteins
encoded by the TTSS-induced genes exhibited no obvious bias toward secreted proteins.
This result is in contrast to the moderately enriched chloroplast-targeted proteins in both
TTSS-repressed and TTSS-induced gene sets (Table 2-2). Interestingly, we Observed
relatively little type III effector-mediated repression of genes involved in primary
metabolic pathways in the cytoplasm, nucleus, or mitochondria, suggesting that in the

first 36 h post-infection, host cells had not yet undergone global, nonspecific

4O

deterioration. This result is expected because we used pre-symptomatic tissues for RNA
isolation.

The strong bias of TTSS-repressed genes toward those encoding secreted
proteins can best be explained by suppression of extracellular plant defense. Indeed, we
found that the majority of TTSS-repressed genes are apparently associated with plant
cell wall functions including hydroxyproline-rich proteins or extensins, which are
known components of papillae; and at least four genes which share sequence similarities
with genes encoding known extracellular defense-associated proteins: a germin-like
protein (50, 51), a nonspecific lipid transfer protein (52, 53), and two acid phosphatases
((54); see Table 2-3). Interestingly, germin-like proteins have also been shown to be
associated with papillae (50, 51). Overall, the biased repression of genes encoding
secreted proteins appears to provide a molecular explanation for the type III secretion-
dependent suppression of papillae formation observed using microscopic analysis (29,
30) as well as additional extracellular host responses that are not microscopically
visible.

The TTSS of Pst DC3000 induced the expression of several SA-dependent
putative defense genes, including PR1 (Table 2-1). This finding supports earlier
observations (55, 56) that virulent P. syringae strains induce these genes in susceptible
Arabidopsis plants, albeit with slower kinetics and at lower levels compared with those
in resistant plants. Because we compared a bacterial strain that was able to secrete type
III effector proteins to a strain that was type III secretion deficient, we can now
conclude that type III effectors are responsible for the induction of these genes in

Arabidopsis. Yet, Pst DC3000 multiplies aggressively under these conditions,

41

suggesting that this level of SA-dependent defense is not effective at limiting Pst

DC3000 multiplication or symptom development.

42

 

 

 

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47

Table 2-2. Predicted locations of proteins encoded by TTSS-regulated Arabidopsis

 

 

genes
Predicted Repressed Induced Microarray Genome-wide
location genes genes

Secreted 42% 20% 16% 17%
Chloroplast 28% 23% 1 8% 14%
Mitochondria 2% 9% 10% l 1%
Others 28% 47% 56% , 58%

 

Predicted locations of proteins encoded by the 53 TTSS-repressed and 64 TTSS-
induced Arabidopsis genes (Table 2-1) were analyzed by TargetP
(http://www.ch.dtu.dk/services/TargetP/) and compared to those of the 25,534 genes in
the Arabidopsis genome and the 7,155 genes present on the microarray used in this
study. This table was contributed by Roger Thilmony.

48

Table 2—3. TTSS-repressed genes that encode proteins predicted to enter the secretory

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

pathway.
BLASTP TargetP
Locus Gene homology, species, accession number 8 value score
At2g38540 Cell wall-localized nonspecific lipid transfer protein 1 (LT?! ), Arabidopsis thaliana, 042589 4e-43 0.98
Atl g72610 Germin-like protein 1 (AtGLPl), cell wall localized, Arabidopsis thaliana, P94040 3e-94 0.64
At1g12090 Extensin-like protein, Arabidopsis thaliana, T5171? 4e-35 0.89
At2g10940 Proline-rich protein, extensin-like, Pinus (aeda, AAF75825 le-15 0.73
At1g03870 Arabinogalactan-protein 9, fasciclin-like, Arabidopsis thaliana, AAK20861 le-l 12 0.91
At3g45970 Expansin-like protein 1, AtEXPLl, Arabidopsis thaliana, Q9LZT4 le-l43 0.85
At5g15350 Cell wall-localized phytocyanin, Firms taeda, AAF75824 le-l3 0.93
At] g29660 Proline-rich protein APG-like, extracellular GDSL-motif lipase/hydrolase-like. Arabidopsis
thaliana, AAK30016 le-44 0.93
At1g29670 Proline-rich protein APG-like, extracellular GDSL-motif lipase/hydrolase-like, Arabidopsis
thaliana, AAK30016 4e-52 0.99
At5g45950 Proline-rich protein APG-like, extracellular GDSL-motif lipase/hydrolase-Iike, Arabidopsis
thaliana, AAK30018 2e-56 0.93
At4g23820 Polygalacturonase PG] , Glycine max, AAD46483 2e-23 0.99
Atlg68560 Alpha-xylosidase XYL1,Arabidopmrhaliana, AAD05539 0.0 0.98
At3gl 6370 Proline-rich protein APO-like, extracellular GDSL-motif lipase/hydrolase-like, Arabidopsis
thaliana, AAK30016 4e-70 0.98
At5g44020 Acid phosphatase 1, Lycopersicon esculentum, "1‘06587 2e—47 0.84
Atl g04040 Acid phosphatase l, Lycopersicon esculenrum, 1‘06587 2e-45 0.94
At2g37450 ' Nodulin MtN2l. Medicago truncarula, CAA75575 6e-4l 0.90
At4g08950 Phosphate-induced protein 1 (phi-1), Nicotiana tabacum, BAA33810 le-121 0.87
At4g34260 Putative large secreted protein, Streptomyces coelicolor, NP_624665 le-136 0.90
At3g07460 Hypothetical protein Se-94 0.99
At4gl 7340 Aquaporin water channel protein, Helianthm amus, T14000 le-88 0.71
At3g16240 Delta tonoplast integral water channel protein, Arabidopsis thaliana, AAC49281 le-102 0.81
At2g19860 Hexokinase 2(Atl-D(1(2),Arabidopsis thaliana. P93834 I 0.0 0.78

 

 

 

 

This table was contributed by Roger Thilmony.

49

 

Transgenic expression of a single effector, AvrPto, regulates host genes in a
manner similar to that of the Pst DC3000 TTSS.

To provide further evidence that the regulation of TTSS-associated genes was
caused by the action of type III effectors, we decided to examine host gene expression
in response to type III effectors expressed in plants. We expressed AvrPto, a type III
effector well known for its avirulence activity in plants (21-23), in susceptible
Arabidopsis under the control of the glucocorticoid-inducible promoter (34, 35). In
these transgenic plants, the expression of AvrPto was induced to a level detectable by
western blotting 24 h after spraying with 30 uM dexamethasone (Figure 2-2A). _ Leaves
became chlorotic after 4 days of daily induction with dexamethasone. However, no
disease-associated water soaking or necrosis developed. Two independent lines of
AvrPto transgenic Arabidopsis plants, AvrPto-76 and AvrPto-129, were further
analyzed by microarray. Remarkably, AvrPto alone regulated as80%'of the TTSS-
regulated genes, including those that encode putatively secreted cell wall and defense
protein genes, in the same manner as Pst DC3000 (Figure 2-2B). These results confirm
that type III effector-associated genes are indeed regulated directly by at least the type
III effector AvrPto. The striking similarity between the TTSS- and AvrPto-regulated
host gene expression profiles demonstrates that AvrPto expression in transgenic
Arabidopsis globally mimicked the Pst DC3000 TTSS functions at the molecular level.

We also found that the repression of TTSS/AvrPto-regulated Arabidopsis
secreted cell wall and defense protein genes in nahG plants was not reproducibly
different from that in wild-type plants (see columns III-A and III-B in Table 2-1). Thus,

the TTSS- and AvrPto-targeted cell wall-based defense is largely SA-independent. This

50

result suggests that the AvrPto-suppressed cell wall-based defense is fundamentally
different from that suppressed by AvrPtoB, VierhA, AverhF, Aerpt2, or Aerme,
which target HR cell death or SA-mediated defenses (41 -45). Consistent with this
conclusion, AvrPto-expressing plants still responded to Pst DC3000 (aerpt2) with an
HR (data not shown).

The TTSS of Pst DC3000 secretes >30 effector proteins (10, 11). Because
mutations in individual effector genes often give only a subtle virulence phenotype or
none at all, it is widely believed that the virulence functions of individual effector
proteins, at the concentrations delivered by bacteria, are redundant or additive (57, 58).
Consistent with this hypothesis, we show that AvrPto is only one of the Pst DC3000
effectors that modulate TTSS-associated Arabidopsis genes because an AvrPto deletion
mutant (59) still regulated Arabidopsis gene expression (see columns VI-A and VI-B in
Table 2-1) in a manner similar to Pst DC3000. This result provides molecular evidence

from the host side for the functional redundancy of Pst DC3000 type III effectors.

51

A Col-0 AvrPto-76 AvrPto-129

 

 

Fold expression

Figure 2-2. Phenotype of avrPto transgenic plants. (A) Western blot analysis of
AvrPto expression in leaves of wild-type and AvrPto transgenic plants 24 h after
spraying with 30 uM dexamethasone. (B) Cluster analysis of the expression profiles of
117 TTSS-regulated genes (colored bars) following Pst DC3000 infection and
transgenic expression of AvrPto. Rows LA and LB represent Pst DC3000 TTSS-
regulated genes from two independent biological replicates (Columns I-A and I-B;
Supporting Information Table A-l). Rows IV and V represent gene expression in
AvrPto-129 and AvrPto-76 transgenic plants, respectively, 24 h after dexamethasone
induction (Columns IV and V; Supporting Information Table A-l).

52

The cell wall-based extracellular defense is compromised in Pst DC3000-infected
and AvrPto transgenic plants.

Because the germin-like proteins and hydroxyproline-rich cell wall proteins
repressed by the Pst DC3000 TTSS and AvrPto are associated with the papilla-
associated cell wall defense, we suspected that AvrPto is one of the long postulated
suppressors of extracellular defense elicited by hrp mutant bacteria (30). We examined
this possibility by treating leaves with aniline blue to stain callose, a major component
of papillae (29, 30). Indeed, we found that the hrcC mutant (positive control) induced a
large number of highly localized callose deposits in leaves of wild-type plants (Figure
2-3). A significantly lower level of callose deposition was found in Pst DC3000-
infected wild-type leaves (Figure 2-3), demonstrating that the TTSS of Pst DC3000 is
involved in the suppression of callose-associated cell wall modifications in Arabidopsis.
This result establishes that the Arabidopsis- Pst DC3000 system can be used to identify
the suppressor of the papilla-associated plant defense.

We next examined the ability of the hrcC mutant to induce callose deposition in
AvrPto-expressing plants. We found that AvrPto-expressing plants were compromised
in mounting an active papilla-based response to the hrcC mutant (Figure 2-3). The
number of callose deposits in hrcC-inoculated AvrPto leaves was only ~5% of that in
hrcC-inoculated wild-type leaves. As expected, Pst DC3000 also did not induce a
significant level of callose deposition in AvrPto-expressing leaves (Figure 2-3). Thus,
transgenic expression of AvrPto functionally mimicked the TTSS of Pst DC3000 not
only in regulating Arabidopsis gene expression, but also in effectively suppressing the

papilla-associated plant cell wall defense.

53

AvrPto- 1 29

    

. 1

Number of callose deposits per 0.58 mm2

Col-0 g1 AvrPto-76 AvrPto-129
hrcC mutant 459.4 i 46.9 15.9 i 6.5 21.6 i 0.4
Pst DC3000 35.2 i 8.1 51.2 3: 28.4 46.3 i 25.1

Figure 2-3. Callose deposits in wild-type and avrPto leaves. (A) Portions of wild-type
and AvrPto transgenic leaves stained with aniline blue for callose (white dots in these
images) afier inoculation with the hrcC mutant or Pst DC3000. Scale bar, 100 um. (B)
Average number of callose depositions per field of view (0.58 mmz) with standard
deviation displayed as error.

54

Enhanced growth of the hrcC mutant in avrPto transgenic plants.

The ability of AvrPto to mimic Pst DC3000 in the regulation of host gene
expression and the suppression of callose deposition prompted us to examine the
susceptibility of the AvrPto transgenic plants to the hrcC mutant. We found that
expression of AvrPto alone was sufficient to allow substantial multiplication of the hrcC
mutant in the transgenic plants (up to SOD-fold, which was 2:1 O-fold lower than the
levels reached by Pst DC3000 in these experiments; Figure 2-4). Unlike Pst DC3000,
however, hrcC-inoculated leaves did not exhibit typical water soaking or extensive
necrosis, suggesting a requirement of other effectors for wild-type levels of bacterial
multiplication and symptom production. Transgenic expression of AvrPto did not
significantly affect Pst DC3000 multiplication because Pst DC3000 multiplied similarly

in the AvrPto plants and wild-type Columbia plants (Figure 2-4).

55

Log CFU/ cm2

 

 

Log CFU/ cm2

 

 

 

Figure 2-4. Bacterial populations in wild-type and avrPto transgenic plants. (A) hrcC
mutant growth in wild-type (black bars) and AvrPto-76 (dark gray bars) plants. Pst
DC3000 growth in wild-type (light gray bars) and AvrPto-76 (white bars) plants. (B)
hrcC mutant growth in wild-type (black bars) and AvrPto-129 (dark gray bars) plants.
Pst DC3000 growth in wild-type (light gray bars) and AvrPto-129 (white bars) plants.

56

Discussion

The hypothesis of a suppressor of a HR-independent cell wall-based plant
defense was formulated almost a decade ago (28). However, the identity of such a
suppressor remained elusive. Using a combination of large-scale host gene expression
profiling, transgenic expression of AvrPto, and cytological examination, we have now
demonstrated that AvrPto is a suppressor of this defense response in susceptible
Arabidopsis. In addition, our TTSS-specific host gene expression analysis provided
global insight into the collective virulence functions of Pst DC3000 type III effectors in
Arabidopsis, revealing an SA-independent, plant cell wall-based extracellular defense as
a major target for Pst DC3000 type III effectors.

The ability of AvrPto to globally mimic the TTSS modulation of host gene
expression, to effectively suppress the papilla-associated cell wall response, and to
substantially enhance multiplication of non-pathogenic hrp mutant bacteria provides
important insights into two long-standing questions in plant-microbe interactions: First,
why do the vast majority of nonpathogenic microbes (e. g., saprophytic bacteria) in
nature fail to colonize plants? Second, what is the role of the TTSS in the evolution of
bacterial pathogenicity? One possibility is that the SA-independent papilla-associated
cell wall defense is a critical part of the still poorly defined plant basal defense system
that prevents multiplication of saprophytic bacteria. In this scenario, acquisition of the
TTSS and the AvrPto class of type III effectors, which may vary in different bacteria, by
a saprophytic ancestor may have enabled it to down regulate this cell wall-based defense,
allowing it to multiply substantially in the plant intercellular space. The acquisition of

suppressors could therefore represent a milestone in the evolution of P. syringae as a

57

virulent pathogen of higher plants. Effector interference with the plant cell wall-based
defense also provides a possible explanation for the production of a largely distinct set
of type III effectors by extracellular plant pathogenic bacteria, compared with
intracellular mammalian pathogenic bacteria (10-12). Down-regulation of the
coordinated extracellular host defenses may be especially important for plant pathogenic
bacteria (such as P. syringae) and reflects the need for this group of bacteria to
overcome the unique host cell wall-based defense of plants. Future research is needed
to further define the exact extracellular defense compounds and structures that are
modulated by P. syringae type III effectors to overcome plant resistance. Such research
will provide critical information for comparative studies of the common and unique
functions of type III effectors produced by plant pathogenic bacteria and mammalian
pathogenic bacteria, some of which also inhibit host defense (60, 61).

Our identification of AvrPto as a suppressor of papilla-associated extracellular
responses is intriguing because in tomato, AvrPto interacts with two Ras-related small G
proteins, Rab proteins, which are involved in vesicular trafficking (62). Previous ultra-
structural studies (29, 30) showed that papilla formation was accompanied by
accelerated extracellular trafficking, as illustrated by an increased abundance of host
endoplasmic reticulum and membrane vesicles. One of the AvrPto-interacting Rab
proteins shows sequence similarity with Rab8, which in mammalian systems is involved
specifically in extracellular secretion (63). Therefore, one mechanism by which
AvrPto could act to suppress cell wall-based plant defense would be to inhibit an
extracellular vesicle trafiicking pathway (Figure 2-2). This inhibition may indirectly

lead to feedback repression of genes encoding secreted proteins that are transported

58

through this particular trafiicking pathway. It is also possible that AvrPto interacts with
a component of a signal transduction pathway to inhibit the expression of the cell wall-
based extracellular defense. A recent proposal hypothesizes that the tomato Pto kinase,
with which AvrPto interacts to trigger resistance responses in tomato, may be a
virulence target of AvrPto guarded by the resistance protein Prf (48, 64). If this
hypothesis is true, AvrPto could interact with a Pto-like kinase in Arabidopsis to
directly down-regulate a signal transduction pathway leading to the activation of a SA-
independent, host cell wall-based defense and other associated genes (Figure 2-5).
Whereas the AvrPto class of effectors appears to play a key role in overcoming a
largely SA-independent cell wall-based extracellular defense, we hypothesize that an
additional class of effectors in Pst DC3000 could have evolved to optimize bacterial
virulence in specific plant genotypes by blocking gene-for-gene resistance, HR-type
programmed cell death, and/or SA-dependent responses. The gene-for-gene resistance
and/or SA-dependent responses could result either from plant recognition of certain
effectors as Avr proteins, or from cellular perturbation caused by the virulence action of
other effectors. Effectors modulating these particular defense responses are exemplified
by AvrPtoB, VierhA, AverhF, Aerpt2, and Aerme (41-45, 65, 66). This class of
effectors would be especially relevant to battling the ever-evolving host recognition
system and may account for the presence of a large number of effector genes in the P.
syringae genome. It is apparent that plants use type III effectors as a main source of
recognition to activate innate defense and turn virulence-intended effector proteins into

avirulence proteins. To remain a successful pathogen, P. syringae must evade

59

Nutrients : ,_ ._

 

AvrPto-type

Golgi

 

ER

    

Figure 2-5. A hypothetical model of the function of the AvrPto class of type III
effectors. (A) The plant cell responds to a hrp mutant bacterium with a papilla-based
extracellular defense by production and transport of cell wall and defense proteins
through the secretory pathway. Extracellular defense compounds (blue triangles) and a
large papilla beneath the hrp mutant bacterium inhibit the bacterial metabolism and/or
produce a ‘desolate zone’ that isolates the hrp mutant bacterium from access to
nutrients/water. Wavy lines above the cell wall (CW) indicate nutrients/water. Golgi:
Golgi apparatus. ER: Endoplasmic reticulum. (B) Wild-type (WT) DC3000 delivers
the AvrPto class of type III effectors (red circles) into the plant cell. Mechanism 1:
Effectors suppress the extracellular secretory pathway, which could lead to feedback
repression of the genes encoding secreted cell wall and defense proteins that enter this
particular secretory pathway. Mechanism 2: Effectors directly inhibit the transcription
of the genes encoding cell wall and defense proteins that are components of the papilla-
based defense.

recognition by mutating these avr genes or evolve additional effector genes that mask
avr gene recognition. It is possible that various defense mechanisms, as well as the
actions of various effectors, may be interconnected at some level. However, the two
classes of effectors appear to target different plant defenses. Therefore, elucidating the
functions of both of these classes will be essential to our understanding of P. syringae
pathogenesis and the different stages of virulence evolution in P. syringae.

,The study of the functions of the 30 or more Pst DC3000 effectors has been
thwarted by the typically weak contributions they individually make to virulence.
Deletion of a single effector gene does not often lead to a noticeable loss of virulence.
In most cases, the virulence contribution, as measured by attenuation of symptoms and
bacterial growth, is subtle, which supports the concept that type III effectors, at the
concentrations delivered by bacteria, contribute to virulence in a subtle or partially
redundant manner (57, 58). Therefore, to efficiently study the functions of most type III
effectors in P. syringae and other plant pathogenic bacteria, methods other than the
traditional ones that measure bacterial populations or assess disease symptoms must be
developed. Despite the apparent functional subtlety and redundancy of type III effectors
when delivered by bacteria, we show here that transgenic expression of AvrPto alone,
which likely results in a higher level of AvrPto in the plant cell than that delivered by
bacteria during infection, could effectively substitute for the redundant/additive
functions of a class of effectors in Pst DC3000 to modulate host gene expression, to
effectively suppress the papilla-associated cell wall response, and to substantially
enhance multiplication of nonpathogenic hrp mutant bacteria. Because TTSS

suppression of cell wall-based defense is likely to be a common feature in plant

61

pathogenic bacteria (29, 30), we believe that the global host gene expression, cytological
examination, and transgenic expression methods used to identify AvrPto as a suppressor
of this host defense will facilitate the functional study of type III effectors not only in P.

syringae but also in other plant pathogenic bacteria.

Acknowledgements

We thank J. Walton and members of our laboratory for critical reading of the
manuscript; R. Schaffer, J. Landgraf, M. Larson, C. Wilkerson, and E. Wisman for help
with microarray and bioinformatic analyses; K. Osteryoung and T. Sanderfoot for
assistance with callose analysis; and K. Bird for assistance in preparation of this paper.
This work was supported by grants from the US. Department of Energy (to S.Y.H.), the
US. National Science Foundation (to S.Y.H.), and the US. Department of Agriculture

(to RT.)

62

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69

Chapter 3. Further characterization of avrPto plants.

70

Abstract

Over-expression of AvrPto, an effector from Pseudomonas syringae pv. tomato
DC3000 (Pst DC3000), in Arabidopsis compromises defense-related callose deposition
in the host cell wall. In this chapter, I describe results that Show that over-expression of
AvrPto enhances multiplication Of several other bacterial strains, including Pst DC3000
(aerpt2). Pst DC3000 (aerptZ) normally triggers the hypersensitive response (HR)
and resistance and induces the appearance of several proteins in the apoplast of
Arabidopsis ecotype Col-0 plants. avrPto-expressing plants still undergo the HR
Similary tO wild-type plants when inoculated with Pst DC3000 (aerptZ). They do not,
however, have the same proteins in their apoplastic space as Col-0 plants. These results
suggest that HR is not sufficient to confer resistance to avirulent P. syringae in avrPto
plants and that AvrPto interferes with secretion/leakage of host proteins induced by

avirulent P. syringae.

71

Introduction

Formation of papillae, localized production Of reactive oxygen species, and
increased synthesis Of compounds such as phytoalexins and extracellular pathogenesis-
related (PR) proteins are known defense responses, but their efficacy in limiting
pathogen multiplication is currently unknown. For a pathogen tO be successful, it must
be able to suppress or evade these defenses and to release nutrients from host cells. The
process by which Pseudomonas syringae pv. tomato strain DC3000 (Pst DC3000)
overcomes plant defenses and Obtains nutrients is not well understood. Pst DC3000
transfers a large number of effector proteins, via the type III secretion system, into host
cells. In an incompatible host, some Of these effectors are recognized by cognate R
genes and this recognition leads to up-regulation of plant defenses and the
hypersensitive response (HR). The HR is defined as a rapid and localized host cell
death and is thought to restrict pathogen growth.

Several lines of evidence suggest that effectors are essential for virulence in
compatible hosts. First, hrp mutants, which do not secrete type III effectors, do not
multiply or cause disease symptoms in host plants. Second, involvement of several
effectors in Virulence has been demonstrated by various methods (1-4). However, the
exact mechanisms by which effectors enable plant pathogenic bacteria to proliferate in
the intercellular space of plant leaves and cause disease remains enigmatic.

The microarray analyses described in Chapter 2 showed that repression Of host
genes by type III effectors was biased towards host genes that encode secreted proteins.
In fact, 42% Of the repressed genes were predicted to be targeted for secretion. This

biased repression suggests that host extracellular secretion is a target Of Pst DC3000

72

type III effectors. Furthermore, expression of a single effector, AvrPto, in transgenic
Arabidopsis plants, resulted in repression of 85% Of those same genes. This result
suggests that AvrPto may be involved in disrupting host secretion. In this chapter, I
describe further characterization of avrPto plants to gain insights into the mechanism by

which avrPto over-expression leads to increased bacterial growth.

73

Materials and Methods

Plant growth and bacterial enumeration

Arabidopsis thaliana accession Col-O gll plants and Col-0 gl avrPto transgenic
plants (Chapter 2) were grown in soil in growth chambers with a day/night cycle Of 12
h/12 h, a light intensity Of 100 uE, and a constant temperature of 20°C. Four- to five-
week-old plants were used for experiments. Bacteria were grown in low-Salt Luria—
Bertani broth (5, 6) to the mid- to late-logarithmic phase at 30°C. Bacterial cultures
were centrifuged to recover bacteria, and the pellets were re-suspended in sterile water
to a final OD600 Of 0.002 [equivalent to l x 106 colony-forrning units (CFU)/ml]. Fully
expanded leaves were infiltrated with bacterial suspensions, and bacteria were
enumerated as described by Katagiri et al. (5). The mean values of the bacterial
populations are plotted with the standard deviation displayed as error. Plants analyzed
in Figure 3-1 were sprayed daily with a 30-uM dexamethasone solUtion (7, 8)
containing 0.02% Silwet L-77 (Osi Specialties, Friendship, WV). Bacterial suspensions

were infiltrated into leaves 24 h after the first dexamethasone treatment.

Callose staining

Arabidopsis leaves were sprayed with 30 uM dexamethasone (7, 8) and then
infiltrated 24 h later with a bacterial suspension Of OD600 = 0.2 (1 x 108 CFU/ml).
Leaves were harvested 6 h after bacterial infiltration, cleared, and stained with aniline
blue for callose as previously described (9). Leaves were examined with a Leica DM
RA2 microscope with an A4 fluorescence cube. The number of callose deposits was

determined with QUANTITY ONE software (Bio-Rad). More than 10 adjacent fields

74

of view along the length of the leaf (not including the mid-vein or leaf edge) were
analyzed and averaged. The values in Table 1 are the average and standard deviation of

more than five independent leaves for each treatment.

Immunoblotting

Approximately 9 mg of tissue was homogenized in 90 ul 2 x loading buffer
(0.125M Tris-HCl pH 6.8, 4% SDS, 20% glycerol, 10% B-mercaptoethanol) and
denatured at 100°C for 10 minutes. An equal volume Of each sample was separated on
a 15 % SDS-polyacrylamide gel (6) and proteins were transferred onto lmmobilon-P
membrane (Millipore #IPVHOOOIO Bedford, MA) using a semi-dry apparatus (SEMI
PHOR, Hoefer Scientific Instruments, San Francisco, CA). Immunoblotting was
carried out using PR1 and PR5 antisera and anti-rabbit alkaline phosphatase conjugate.

Protein bands were visualized by SIGMA FAST (Sigma B5655 St. Louis, MO)

Secretion assays

Arabidopsis leaves were sprayed with 30 uM dexamethasone (7, 8) and then
infiltrated 24 h later with a bacterial suspension Of OD600 = 0.2 (1 x 108 CFU/ml).
Plants were then incubated for 6 h under low humidity, excised from pots, and vacuum-
infiltrated with water containing 0.004% Silwet L-77 (Osi Specialties, Friendship, WV).
Excess water was removed from leaves and the tissue was centrifuged at 400 g for 20
minutes. Intercellular wash fluid (IWF) was collected and stored at -20°C. Samples

were mixed with 2 x loading buffer, denatured at 100°C for 10 minutes. Protein

75

samples were then separated on a 15% SDS-PAGE gel, which was then stained with

Coomassie Blue.

76

Results

Enhanced growth of Pseudomonasfluarescens 55 and Pst DC3000 (aerptZ) in
avrPto plants

It was previously shown that over-expression Of AvrPto in Arabidopsis plants
allowed the growth Of the hrcC mutant (Chapter 2). hrc/hrp mutants behave similarly
to the vast majority Of non-plant pathogenic bacteria found in nature. TO determine if a
similar result would be Obtained with a non-phytopathogenic bacterium, the growth of a
saprophyte, Pseudomonasfluarescens 55 (Pf 55), was assayed in avrPto plants. Figure
3-la shows that this strain was able to multiply almost 600-fOld in avrPto plants,
whereas the population decreased in wild-type plants. Next, I examined whether an
avirulent strain Of Pst DC3000 would be able to multiply tO higher levels in avrPto
plants than in wild-type plants. Indeed, avrPto plants allowed over 900-fold more

grth Of Pst DC3000 (aerpt2) than did wild-type plants (Figure 3-1b).

AvrPto does not inhibit the HR triggered by AerptZ

The resistance OfArabidopsis to Pst DC3000 (aerpt2) is mediated by the
corresponding R gene RPSZ, and is associated with the HR. I investigated whether the
HR was altered in avrPto plants, which have a functional RPS2 gene. There was little
difference in the HR Of avrPto and wild-type plants tO Pst DC3000 (aerpt2) (Figure 3-

2), although avrPto plants collapsed slightly earlier than wild-type plants.

77

 

 

 

 
    

 

 

6
5 I WT plants
D AvrPto
N plants
5 4
U)
D
(”5
3 Lil—'7
2 _
O 3
Day
B.
71
6‘ I WT plants
E] AvrPto
5 , plants
8
Q
m
:> 4
LL.
0
3 .
2 0 3 _i
Day

Figure 3-1. Growth of Pf 55 and Pst DC3000 (aerptZ) in avrPto-expressing plants.
Multiplication of Pf 55 (A) and Pst DC3000 (aerpt2) (B) in wild-type (WT) plants and
avrPto-expressing plants. The growth of Pf 55 and Pst DC3000 (aerpt2) was
significantly greater (approximately 500-fold and 900-fold, respectively) in avrPto-
expressing lines than in wild-type (WT) plants.

78

Wild-type leaves

 

avrPto leaves

 

Figure 3-2. avrPto leaves afier infiltration of Pst DC3000 (aerptZ). Leaves from
wild-type and avrPto plants 7 hours afier infiltration with 2x106 CFU/ml of Pst DC3000
(aerptZ). HR is evident in both the wild-type and avrPto leaves.

79

Callose deposition upon Pf 55 and Pst DC3000 (aerptZ) infection is compromised
in avrPto transgenic plants

In Chapter 2, it was shown that avrPto plants are unable to deposit callose in
response to the hrcC mutant. Here I wanted to examine whether avrPto plants would be
compromised in the callose response to Pf 55 and Pst DC3000 (aerptZ). As shown in
Table 3-1, the hrpA mutant, Pf 55, and Pst DC3000 (aerpt2), elicited 180-fold, 34.2-
fold 74.3-fold, respectively, more callose deposits in wild-type plants than in avrPto
plants. As expected, Pst DC3000 elicited very low numbers of callose deposits in either

wild-type or avrPto plants.

Pst DC3000 (aerptZ) triggers the appearance of several proteins in the IWF of
wild-type plants, but not in avrPto plants

As described in Chapter 2, a large percentage of genes that were expressed at
lower levels in avrPto plants compared to wild-type plants encode proteins predicted to
be secreted. The proteins in the IWF of wild-type and avrPto plants were examined by
separation on SDS-PAGE gels and staining with Coomassie Blue. It was discovered
that Pst DC3000 (aerptZ), but not Pst DC3000, elicited the appearance of several
proteins in the IWF of wild-type plants (Figure 3-3a). Furthermore, these proteins were
not detected in the IWF of avrPto plants (Figure 3-3a). To determine whether these
apoplastic proteins were PR proteins, immunoblot analyses using PR1 and PR5
antibodies were conducted. There was no difference in the amount of PR-l or PR-S in

the apoplastic fluid from wild-type and avrPto plants (Figure 3b and c).

80

Table 3-1. Average callose deposition in wild-type and avrPto plants 6 hours after
inoculation with Pst DC3000, hrpA mutants, Pf 55, or Pst DC3000 (aerptZ).

 

Wild-type 1 avrPto plants 1
Pst DC3000 9.9 :t 3.4 2.3 :t 0.8
hrpA mutant 253.3 :t 35.5 1.4 i 0.7
Pf55 273.4 i 40.1 8.0 i 5.5
Pst DC3000 (aerptZ) 207.9 2‘.- 91.6 2.8 d: 1.6

' Average callose depositions per field of view with standard deviation
displayed as error.

81

Pst

    

DC3000
(aerptZ)
A' Pst
Water DC3000
avr avr avr
. WT Pto WT Pto 4 WT Pto
56 RD . . ,.
33 kD
30 kD I ‘—

6""

B ‘ _..,
PR5 am: at! M «a.

C l
PR1 m mull. .-

" sT‘Ww ; an ~— 7'1'" T"

Figure 3-3. Protein profiles of the IWFs from avrPto plants inoculated with water,
Pst DC3000 or Pst DC3000 (aerptZ). A. Coomassie stained SDS-PAGE gel showing
that Pst DC3000 (aerptZ), but not Pst DC3000, induces the appearance of host
proteins (indicated by arrows) in the IWF of wild-type (WT) plants. Neither strain
triggers the appearance of these proteins in avrPto plants. Western blots of B. PR5 and
C. PR1 show that PR expression in wild-type and avrPto plants is similar and thus,
cannot be the proteins whose appearance is triggered by Pst DC3000 (aerptZ). The
level of expression induced by different bacterial strains is variable between different
experiments.

82

The IWF of avrPto plants does not support more bacterial growth than that of
wild-type plants.

To determine whether the IWF of avrPto plants contains more nutrients and can,
therefore, support bacterial growth, the IWFS from these plants were collected and
inoculated with the hrcC mutant. The hrcC mutant grew over 1,600-fold more in the
IWF from wild-type plants than in water. This level of growth is comparable, only 6-
fold less than the amount of growth observed in LB (Figure 3-4a). The grth of hrcC

was similar in the IWFs from wild-type and avrPto plants (Figure 3-4b).

83

 

1010

 

 

 

hrcC growth (CFU/ml)
hrcC growth (CF U/ml)

 

 

 

 

 

Start water WT LB Start WT avrPto
cone. IWF conc. IWF IWF

Figure 3-4. Growth of the hrcC mutant in the IWF from wild-type (WT) and avrPto
plants. Growth was assayed afier ~ 24 hr incubation. A. The growth of the hrcC

mutant in the IWF from WT plants was significantly greater than in water and
comparable to that in LB media. B. The growth of the hrcC mutant in the IWF from

WT plants and avrPto plants was not significantly different.

84

Discussion

In Chapter 2 I demonstrated that the hrcC mutant is able to multiply
significantly higher in avrPto plants than in wild-type plants. In this study, I wanted to
determine if other non-virulent bacteria would multiply in avrPto-expressing plants as
well. I show that, besides hrcC, avrPto plants also allowed a significant increase in
growth of the non-phytopathogenic bacterium Pf 55, and the normally avirulent strain
Pst DC3000 (aerptZ). I examined several possible mechanisms for the increased
grth of Pst DC3000 (aerptZ) in avrPto plants. avrPto plants may be compromised
in the HR, cell-wall based defenses, or the secretion of defense proteins, and/or avrPto
plants may be leaking nutrients into the apoplastic space.

To test the hypothesis that AvrPto may suppress the HR, inoculated avrPto
plants with Pst DC3000 (aerptZ). Pst DC3000 (aerptZ) caused a normal HR when
inoculated into avrPto plants. Thus HR was uncoupled from resistance in avrPto plants.
My finding adds to a growing list of studies that show uncoupling of HR from disease
resistance in different pathosystems (2, 10-12). I conclude that the increased
susceptibility of avrPto plants to Pst DC3000 (aerptZ) is not correlated with loss of
the HR.

In contrast, I found a correlation of increased growth with suppression of callose
deposition and the absence of several extracellular proteins in the apoplast of avrPto
plants. I observed that the hrpA mutant, Pf 55 and Pst DC3000 (aerptZ) elicited high
levels of callose deposition in wild-type plants, but not in avrPto plants. Callose
deposition is a marker for papilla formation, which is believed to require an intact host

secretion system. In addition, I found that four protein bands (between 21 and 30 kD)

85

were present in the IWF of wild-type plants infected with Pst DC3000 (aerptZ).

These proteins were neither detected in the IWF from avrPto plants after treatment with
Pst DC3000 (aerptZ), nor from wild-type plants treated, with Pst DC3000. These
results support the hypothesis that AvrPto disrupts extracellular secretion in the host.

A previous study showed that the apoplastic fluid of plants treated with a
salicylic acid analog, 2, 6-dichlorolisonicotinic acid (INA), contains 3 proteins: PR-l
(16 kD), PR-S (26 kD), and PR-2 (37 kD). These bands were absent in the apoplastic
fluid from control plants treated with water (13). Since the sizes of PR proteins are
similar to those proteins found in the apoplast of Pst DC3000 (aerptZ) inoculated
wild-type plants, western blot analyses were conducted to assay for the presence of PR
proteins. There were no differences in the abundance of PR-l or PR-S in the IWF of
wild-type plants compared to that of avrPto plants. It is possible that there are multiple
protein secretion pathways in the Arabidopsis cell and the one involved in PR protein
secretion may be different from the one responsible for secreting the proteins elicited by
Pst DC3000 (aerptz). The PR secretion pathway may not be affected by AvrPto.

The identity of the proteins found in the IWF of wild-type plants inoculated with
Pst DC3000 (aerptZ) is being determined. Preliminary results indicate that these
proteins include plastocyanin, calmodulin, and an oxygen-evolving enhancer protein 3
(data not shown). It is unknown, yet, if these apparently chloroplastic proteins are
involved in the resistance response. In can be concluded, however, that the appearance
of these proteins in the apoplast is not necessary for the HR.

Alternatively, it is possible that Pst DC3000 (aerptZ) causes non-specific

leakage in both wild-type and avrPto plants, but the expression of these proteins could

86

be lower in avrPto plants than in wild-type plants. In this case, the proteins were
detected only in the Pst DC3000 (aerptZ) inoculated wild-type plants because they
were more abundant there. This possibility is being explored using antibodies against
plastocyanin (kindly provided by R. B. Klosgen). However, the background bands in all
treatments appeared to be similar, arguing against a general non-specific leakage.
Furthermore, RabE, an intracellular protein, was not detected in our IWF by
immunoblotting (E. Bray Speth, unpublished data).

Besides blocking secretion of potential defense compounds, AvrPto could cause
leakage of nutrients into the apoplast, thus promoting bacterial multiplication. To test
this hypothesis, 1 inoculated the IWF from non-inoculated plants with the hrcC mutant.
This strain was able to grow equally well in the IWF from avrPto and wild-type plants.
Therefore, I can conclude that there are abundant water-extractable nutrients available
in the apoplast of Arabidopsis leaves and that the level is the same in both wild-type and
avrPto plants. It is important to note that nutrients in the leaf could be inaccessible to
the bacteria under natural conditions, but are released to the fluid during our
experimental procedure. In addition, the apoplastic fluid assay does not address
whether water is limiting in the leaf. avrPto plants may cause leakage of water and this
may be sufficient to allow growth of non-virulent strains of bacteria. However, I did
not observe any water soaking in uninoculated avrPto plants. Lastly, these IWF
experiments suggest that there is no difference in effective and stable, water-extractable,
antimicrobial compounds in the IWF from avrPto plants or wild-type plants.

In summary, my microarray, callose staining, and IWF experiments conducted

with avrPto plants support the hypothesis that AvrPto contributes to virulence by

87

interfering with a host’s extracellular secretion system. In addition, yeast-two-hybrid
results (Appendix A) revealed that AvrPto interacts with RabE family members. Rabs
are small GTPases that are putatively involved in extracellular protein secretion. Future
experiments using transgenic plants that over-express constitutively active, dominant-
negative, and wild-type versions of RabE will further test the hypothesis that AvrPto
contributes to virulence by disrupting a secretion pathway necessary for successful plant

resistance.

Acknowledgements:

I thank Ralph Bernd Klosgen (Martin-Luther—Universitat Halle-Wittenberg, Germany

for providing plastocyanin antibody.

88

References

CHANG, J. H., RATHJEN, J. P., BERNAL, A. J., STASKAWICZ, B. J. & MICHELMORE,
R. W. (2000) avrPto enhances growth and necrosis caused by Pseudomonas
syringae pv. tomato in tomato lines lacking either Pto or Prf. Mol Plant Microbe
Interact, 13, 568-571.

CHEN, Z., KLOEK, A. P., BOCH, J ., KATAGIRI, F. & KUNKEL, B. N. (2000) The
Pseudomonas syringae aerptZ gene product promotes pathogen virulence from
inside plant cells. Mol Plant Microbe Interact, 13, 1312-1321.

CHEN, Z., KLOEK, A. P., CUZICK, A., MOEDER, W., TANG, D., INNES, R. W.,
KLESSIG, D. F., MCDOWELL, J. M. & KUNKEL, B. N. (2004) The Pseudomonas
syringae type III effector Aerpt2 functions downstream or independently of
SA to promote virulence on Arabidopsis thaliana. Plant J, 37, 494-504.

SHAN, L., HE, P., ZHOU, J. M. & TANG, X. (2000) A cluster of mutations disrupt
the avirulence but not the virulence function of AvrPto. Mol Plant Microbe
Interact, 13, 592-598.

KATAGIRI, F., THILMONY, R. & HE, S. Y. (2002) The Arabidopsis thaliana-
Pseudomonas syringae interaction. The Arabidopsis Book, 1.

SAMBROOK, J ., FRITSCH, E. F. & MANIATIS, T. (1989) Molecular cloning: a
laboratory manual (Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory
Press).

AOYAMA, T. & CHUA, N. H. (1997) A glucocorticoid-mediated transcriptional
induction system in transgenic plants. Plant J, 11, 605-612.

MCNELLIS, T. W., MUDGETI', M. B., LI, K., AOYAMA, T., HORVATH, D., CHUA,
N. H. & STASKAWICZ, B. J. (1998) Glucocorticoid-inducible expression of a

bacterial avirulence gene in transgenic Arabidopsis induces hypersensitive cell
death. Plant J, 14, 247-257.

ADAM, L. & SOMERVILLE, S. C. (1996) Genetic characterization of five powdery
mildew disease resistance loci in Arabidopsis thaliana. Plant J., 9, 341-356.

89

10.

11.

12.

13.

YU, I. C., PARKER, J. & BENT, A. F. (1998) Gene-for-gene disease resistance

without the hypersensitive response in Arabidopsis dndI mutant. Proc. Natl.
Acad. Sci. U SA, 95, 7819-7824.

CENTURY, K. S., HOLUB, E. B. & STASKAWICZ, B; J. (1995)NDR1, a locus of
Arabidopsis thaliana that is required for disease resistance to both a bacterial
and a fungal pathogen. Proc. Natl. Acad. Sci. U S A, 92, 6597-6601.

LU, M., TANG, X. & ZHOU, J. M. (2001) Arabidopsis NHOI is required for
general resistance against Pseudomonas bacteria. Plant Cell, 13, 437-447.

UKNES, S., MAUCH-MANI, B., MOYER, M., POTTER, S., WILLIAMS, S., DINCHER,
S., CHANDLER, D., SLUSARENKO, A., WARD, E. & RYALS, J. (1992) Acquired
resistance in Arabidopsis. Plant Cell, 4, 645-656.

90

Chapter 4: Characterization of an Arabidopsis thaliana
mutant, noel, with altered symptom development in response
to Pseudomonas syringae pv. tomato DC3000 infection

91

Abstract

Very little is known about the molecular basis of the development of specific
disease-associated symptoms in plants. In this study, approximately 10,000
ethylmethane sulfonate-mutagenized A. thaliana ecotype Columbia gll plants were
screened for reduced disease symptom development in response to Pseudomonas
syringae pv. tomato DC3000 (Pst DC3000) infection. A recessive mutation, nocI (no
chlorosis), caused a defect specifically in chlorosis development, while Pst DC3000
multiplication and necrosis development remained normal. In wild-type plants, the
abundance of chlorophyll a and b decreased after infection with Pst DC3000. The total
amount of chlorophyll in the nod mutant, however, remained relatively unchanged
after infection with Pst DC3000. Although jasmonic acid (JA) and ethylene have been
implicated in chlorosis, the nocI mutant was not impaired in its response to JA or
ethylene. Linkage mapping revealed that the mutation was located in a 619-kb region

between At4g22340 and At4g24050 on the long arm of chromosome 4.

92

Introduction

Understanding plant-pathogen interactions is vital for our future ability to
improve resistance in crop plants. Pseudomonas syringae pv. tomato strain DC3000
(Pst DC3000) causes speck disease in Arabidopsis and tomato, characterized by
bacterial multiplication and the progressive appearance of symptoms in the infected
leaves. Typically, the appearance of water-soaking is followed by chlorosis (yellowing
of the tissue) and ultimately necrosis (cell death) in the infected leaves. The molecular
basis for these symptoms is unknown.

Chlorosis is caused by chlorophyll breakdown, one of the events accompanying
plant senescence. Chlorophyll can be degraded via two pathways: an oxygen-dependent
(or oxidative bleaching) pathway and an oxygen-independent pathway (1). The
existence of the oxygen-dependent pathway is controversial (2) and thus will not be
discussed here. The oxygen-independent pathway (Figure 4-1) is the generally accepted
pathway and is also known as the “chlorophyllase pathway” because the first step is
catalyzed by chlorophyllase, which converts chlorophyll into chlorophyllide. Mg-
dechelatase converts chlorophyllide into pheophorbide, which is then either converted
into pyropheophorbide by pheophorbidase or into a red chlorophyll catabolite by
pheophorbide oxygenase. Cleavage of the chlorophyll tetrapyrrolic ring by
pheophorbide oxygenase causes the loss of green color in downstream products. Only
one gene encoding an enzyme upstream of pheophorbide oxygenase has been cloned
from Arabidopsis. Expression of this gene, AtCLHI (chlorophyll-chlorophyllido
hydroxylase) (3), is up-regulated by ethylene (4), wounding, methyl jasmonate (MeJA)

or jasmonic acid (JA), and coronatine, which is a toxin produced by Pst DC3000 (5).

93

“Stay-green” plants are traditionally considered delayed-senescence variants, in
which degradation of chlorophyll and the photosynthetic apparatus is partially or
completely prevented (6). There are at least four different categories of stay-green
plants (6, 7). In one type, senescence is initiated late, but then proceeds at a normal rate.
In the second type, senescence is initiated on time, but proceeds at a slower rate. In the
third class, senescence proceeds normally, but chlorophyll is retained indefinitely. In
the fourth class, plants stay green when they are killed rapidly by freezing, boiling, or
drying (6, 7). Some stay-green plants are a result of a combination of these types. Stay-
green cereals are economically important because they carry out photosynthesis for a
longer period of time, which results in an increase in yield (7).

For unknown reasons, most stay-green mutants impaired in chlorophyll
catabolism are deficient at the ring-opening step catalyzed by pheophorbide oxygenase
[i.e. sid from F estuca pratensis (8) and the stay-green mutant of Lolium temulentum (9)].
Stay-green mutants not affected in chlorophyll catabolism have been described as well.
The cause of one soybean stay-green phenotype is a maternally inherited cth mutation,
which renders chlorophyll b more stable than chlorophyll a (10).

Oh et al. (11) conducted a screen to find Arabidopsis mutants with delayed
senescence. They found eleven mutants that exhibited delayed loss of chlorophyll
content (oreI-I 1). Loss of photochemical efficiency, however, was only delayed in
are], 2, 3 and 9, but not in ore10 or 11. ore2 and ore3 were found to be allelic to a
previously isolated mutant, ein2 (ethylene-i_nsensitive). EIN2 is an integral membrane
protein that acts downstream of the ethylene receptors and upstream of the gene

transcription changes associated with the ethylene response (12). 0RE9 encodes an F-

94

box protein that has been suggested to target negative regulators of senescence for
ubiquitin-mediated degradation (13). In ore10 and are] 1 mutants, all chlorophyll-
containing protein complexes [LHCI (light harvesting complex I), PSI (photosystem 1)
reaction center, PSII (photosystem II) reaction center] except for LHCII are degraded.
This result suggests that chlorophyll stability in these non-photosynthetic stay-green
mutants is due to defects in their proteolytic pathways (14). The response of the stay-
green and are mutants to Pst DC3000 infection is not known.

In this study, an Arabidopsis mutant, m-ghlorosis 1 (nocI), showing altered
chlorosis development following inoculation with Pst DC3000, was isolated and
characterized. Infected nocI leaves do not develop chlorosis; they do, however,
develop normal disease-associated necrosis and permit normal levels of Pst DC3000
multiplication. Uncoupling of chlorosis and necrosis has not previously been described.
Characterization of this mutation should lend insight into the molecular basis of disease-

related chlorosis, which is common to several plant diseases.

95

. I , Nonfluorecent
(.hloroph} ll chlorophyll catobolite
A
Chlorophyllase l A

A

Primary fluorecent

 

Chlorophyllidc chlorophyll catobolite
Red chlorophyll
Mg-dechelatase l catabolite
reductase

Pheophorbide
. oxygenase Red chlorophyll
PheophwlmdC > catabolite

Pheophorbidase l

 

 

   

P)’ ropheophorbide

Figure 4-1. The chlorophyll degradation pathway. Simplified model of the steps
involved in chlorophyll catabolism in higher plants. Products upstream of the ring
cleavage step catalyzed Pheophorbide oxygenase are green and indicated as green boxes.

96

Methods

Plant material, mutagenesis, and growth conditions

Approximately 1g of Arabidopsis thaliana ecotype Columbia gll seeds was
mixed with 100 ml of distilled water and 250 pl of ethylmethane sulfonate (EMS). _The
mixture was incubated overnight at room temperature in the dark with gentle agitation.
The seeds were washed six times with 500 ml of distilled water, resuspended in 300 ml
of 0.1% agarose and sown onto a soil mixture (equal portions of Bacto high porosity
professional plant mix, perlite, and vermiculite, covered with a thin layer of fine
vermiculite). The flats were covered with lids and incubated in the dark at 4°C for three
days. The flats were then transferred to a grth chamber [20°C with 12 hours of
fluorescent light (100 uEinsteins/mz/sec) and 12 hours of darkness] until the end of the
life cycle. The plants were self-fertilized for two generations to create a population of

M2 plants.

Screening and isolation of Arabidopsis mutants

Four to six-week-old M2 plants were dipped in a 1x108 CFU/ml suspension of
Pst DC3000 and 0.05% Silwet L-77 (Osi Specialties, Friendship, WV) for two to three
seconds. The inoculated plants were incubated in high (80-90%) humidity conditions

for 96 hours and screened for a lack of symptom development.

97

Bacteria enumeration in infiltrated leaves of nocl mutants and wild-type plants

Four- to five-week-old plants were used for bacteria enumeration. Pst DC3000
was grown in low-salt Luria—Bertani broth (15, 16) to the mid- to late-logarithmic
phase at 30°C. Bacterial cultures were pelleted and resuspended in sterile water to a
final ODeoo of 0.002 [equivalent to 1 x 106 colony-forming units (CFU)/ml]. Fully
expanded leaves were vacuum-infiltrated with bacterial suspensions, and bacteria were

enumerated as described by Katagiri et al. (15).

RNA isolation and northern blotting

RNA isolation from leaf tissue and Northern blotting were conducted as
described by DebRoy et al. (17). Tissue was collected at 0, 24, and 48 hours-post-
infiltration with Pst DC3000 and frozen in liquid nitrogen. RNA was isolated using the
Promega RN Agents kit (Cat#ZSl 10) according to manufacturer’s instructions. About
20 ug of RNA was denatured with two volumes of loading buffer (500 pl forrnamide,
170 pl formaldehyde, 100 pl 10X MOPS buffer, and 10 ul of 1 mg/ml etlridiurn
bromide) for 10 minutes at 65°C. The RNA was separated on a formaldehyde agarose
gel and transferred onto a nylon membrane (Hybond N+; Amersham Pharmacia Biotech
#RPN303B) via capillary transfer (16). The membrane was hybridized overnight at
60°C in 20 ml Church’s buffer [1% crystalline BSA (fraction V), 1 mM EDTA, 0.5 M
NaHPO4, pH 7.2, 7% SDS] and 600 [.11 denatured salmon sperm DNA. Approximately
100 ng of AtCLHI DNA was labeled with 32F CTP and purified using a BIORAD
column (Cat #732-6223) according to the manufacturer’s instructions. The radiolabeled
DNA probe was then added to the Church’s buffer and the membrane was incubated

with the probe for 16 hours at 60°C. Membranes were washed to a stringency of 0.5X

98

SSC (10 minutes at 60°C) and exposed to X-ray film (Kodak Scientific Imaging film X-

OMAT AR#1651454).

Microarray analysis

Microarray analysis was conducted as described by Hauck et al. (18). RNA
from 5-week-old wild-type and nocl plants, taken at 24 and 48 hours post infiltration
with 2x106 cells/mL, were pooled. The custom microarray slides used were printed with
approximately 600 non-redundant A. thaliana ESTs, found to be reproducibly
differentially regulated during the compatible A. thaliana-Pst DC3000 interaction
(Thilmony and He, unpublished data), were used. The subarray was derived from the
Arabidopsis Functional Genomic Consortium’s microarray slides, which contain about

7,200 unique genes (19).

Chlorophyll extraction

Chlorophyll abundance assays were performed using leaf tissue infiltrated with
2x106 CFU/mL Pst DC3000 and samples were collected at 0, 24, 48, 72 and 96 hours
post-inoculation. All chlorophyll extraction steps were conducted in near darkness.
Leaf disks from four separate leaves at each time point were frozen in liquid nitrogen
and stored at —80°C. The frozen tissue was homogenized in 600 pl of 80% acetone.
The tubes were centrifuged at 500 x g for three minutes at 4°C and the supernatant was
transferred to a new tube and kept on ice. The absorbance of four dilutions (1 :10, 1:5,
1:3, and 122.5) of each sample was determined using a spectrophotometer. The amount

of chlorophyll was calculated as previously described (20).

99

MeJA sensitivity assay

Seeds were vapor-sterilized by incubation in a sealed container with a beaker
containing 100 ml of bleach (6.15% sodium hypochlorite) and 3 ml of concentrated HCl
for 16 hours. The seeds were then sown on Arabidopsis germination media [4.3 g/L
Murashige-Skoog salts (Invitrogen), 30 g/L sucrose (J .T. Baker), 0.5 g/L MES (Sigma),
pH 5.7] containing 50 pM MeJA (21). Seeds were placed in the dark at 4°C for three
days and then transferred to the growth chamber. After one week, the seedlings were

analyzed for their response to Me] A.

Ethylene sensitivity assay

Seeds were sown on 3 mm Whatrnan paper (Whatman International Ltd.
Maidstone, England) moistened with distilled water, and incubated in the dark at 4°C
for three days. The seedlings were then placed in a desiccator with 10 ul/L of ethylene

and incubated in continuous darkness at 20 °C for four days (22).

Gene mapping

Mapping of the nod gene was conducted as described by Lukowitz, Gillmor
and Scheible (23). Initial genome-wide screening was conducted using an array of
primers (Invitrogen Carlsbad, CA) to detect simple sequence length polymorphisms

(SSLPs) from each chromosome. Additional SSLPs identified in the Monsanto

100

Arabidopsis Polymorphism and Ler sequence collection (St. Louis, M0) were used to

further define the region containing the nocI mutation (24).

101

Results

Identification of the nod mutant

Approximately 10,000 EMS-mutagenized A. thaliana ecotype Columbia gll
plants were screened for altered symptom development after dipping the plants in a
suspension of Pst DC3000. One mutant isolated from this screen, we] (m-ghlorosis),
was found to be defective in chlorosis development. nocI leaves remained green while
wild-type leaves began to show chlorosis between 48 and 72 hours after inoculation
(Figure 4-2). The severity and timing of water soaking and necrosis (24 hours and 96
hours post inoculation, respectively) were similar in both nod and wild-type plants.
There were no noticeable differences between wild-type and nocI plants in size,
morphology, growth, or development, and the initiation or rate of senescence. Although
me] has altered symptom development, bacterial multiplication in nocI plants was not
statistically different from that in wild-type plants (Figure 4-3). This result indicates
that in the noc] plants, bacterial growth and chlorosis symptom production were

uncoupled.

The decrease in total chlorophyll level is greater in wild-type plants than in and
plants after infection with Pst DC3000

To determine whether the chlorotic response to Pst DC3000 infection was due to
chlorophyll degradation, a chlorophyll abundance assay was conducted using tissue
infiltrated with 2x106 CFU/mL of Pst DC3000 and collected at 0, 24, 48, 72 and 96
hours post-inoculation. The results from one representative experiment are shown in

Figure 4-4. Prior to inoculation with Pst DC3000, nocI and wild-type plants had

102

approximately equal amounts of total chlorophyll (27.1 mg/cm2 and 28.5 mg/cmz,
respectively). Wild-type plants began to lose chlorophyll by 24 hours post-inoculation
and continued through 96 hours post-inoculation. nocl plants, on the other hand, did
not begin to lose chlorophyll until after 48 hours post-inoculation. At 72 hours, nocI
plants contained more than twice as much total chlorophyll as wild-type plants (26.8
mg/cm2 in me] plants vs. 12.4 mg/cm2 in wild-type plants). This experiment
demonstrates that wild-type plants lose chlorophyll faster than nocl plants after Pst

DC3000 infection.

The expression of one gene, AtCLHI, in the chlorophyll degradation pathway is
slightly reduced in nocI plants

One possible explanation for the greater amount of chlorophyll in nocl is a
block in the chlorophyll degradation pathway. The first enzyme in the oxygen-
independent chlorophyll degradation pathway is AtCLHl (3). AtCLHI, also called
A T HCOR I (_A. thaliana c_o_ronatine-induced) was shown to be induced by the Pst
DC3000-produced phytotoxin coronatine, which causes chlorosis in tomato (25). Since
this gene encodes an enzyme in the chlorophyll degradation pathway, a mutation in
AtCLHI could explain the absence of chlorosis in nocI upon infection with Pst DC3000.
Alternatively, a difference in expression could indicate altered flux through the
chlorophyll degradation pathway. Northern blot analysis was performed to determine
whether AtCLHI gene expression upon infection with Pst DC3000 was altered in the
nod mutant. The transcript level of AtCLHI was only slightly reduced in nocI plants

compared to that in wild-type plants (Figure 4-5).

103

 

Figure 4-2. Phenotype of the nod mutant afier Pst DC3000 inoculation. noc1 mutant
(right), and wild- (lefi) developed symptoms three days after vacuum infiltration of Pst
DC3000 at 1 x 10 CFU/ml.

104

31

    

Log (CF U/cm2

-+- wild-type

-l- nocI

 

 

Days post inoculation

Figure 4-3. Growth of Pst DC3000 in nocI plants. Plants were inoculated with l x 106
CFU/ml of Pst DC3000. The mean values of the bacterial populations in wild-type (red)
and nocI (green) plants are plotted with the standard deviation displayed as error.

105

 

 

Chlorophyll (mg/cmz)

 

 

—l— nocI

 

 

+ wild-type

 

 

 

Days post infiltration

Figure 4—4. Total amount of chlorophylls (a and b) in wild-type and nocI leaves
during the course of Pst DC3000 infection. Wild-type (red) and nocl (green) leaves were
inoculated with Pst DC3 000 at a concentration of 1 x 106 CFU/ml.

106

O 24 48 hrs post infiltration

 

wt nocl wt nocI wt nocI

yr '- -. 11:" .4-u~.xl-‘._‘o 1. 1.“.“ I‘m.

_' .w ,, AtClhl
'c _ . ; v.

...!

ribosomal RNA
loading control

_. .
I
. . J
, i , ' .. , ..

 

Figure 4-5. Northern blot analysis ofAtClhI transcript. AtClhI transcript abundance in
wild-type (WT) was compared to nocl leaves during the course of Pst DC3000 infection.

107

Microarray

Microarray analysis was performed to examine Arabidopsis gene expression
differences between nod and wild-type plants during Pst DC3000 infection. AtClhI is
represented on the microarray slides used in these experiments. We found that
transcript abundance was 1.37-fold higher in wild-type plants than in the nod mutant.
This result independently confirms the Northern result. Genes for which there was
greater than a two-fold difference between nod and wild-type were considered to be
differentially regulated (Table 4-1). No JA/coronatine, salicylic acid, or ethylene-
responsive genes were differentially regulated. The only gene that showed more than a
2-fold (2.5-fold) higher level of expression in nocI than in wild-type plants was alanine:
glyoxylate arninotransferase 2 homologue (At2g38400). Only 12 genes had more than
a 2-fold lower level of expression in nocI compared to wild-type plants. The
Arabidopsis ATP-dependent Clp protease (At3 g48 870), which is associated with
chloroplasts and may play a role in protein degradation in the chloroplast (26), was

expressed at a 2.5-fold lower level in me] than in wild-type plants.

108

Table 4-1. Differentially regulation of genes in the nocl mutant compared to

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

wild-type plants.
Ratio At Locus Description
0.41 At2g3 8400 AGT2 alaninezglyoxylate aminotransferase 2 homolog
2.07 At4g23820 put. polygalacturonase, similar to genes induced by nematodes and senescence
2.08 At3gl4210 lipase acylhydrolase, myrosinase assoc
2.12 At4g01310 L5P family of plastid ribosomal proteins
2.20 At] g52400 Beta glucosidase, suppressed by salt, ER localized
2.22 At3g54050 Fructose-1,6-Bisphosphatase
2.26 Atl g72610 germin-like protein
phosphoribulokinase/Ribulose-S-phosphate kinase, Phosphopentokinase,
2.36 Atl g32060 chloroplast, Calvin cycle, light regulated via thioredoxin by reversible
oxidation/reduction of sulflrydryl/disulfide groups
2.31 At2g02950 PKSl phytochrome kinase substrate 1, modulates light signaling
sulfate transporter Sultrl , high affinity sulfate transporter, root l-1+/Sulfate
2'38 At5g13550 cotransporter for sulfate uptake
2.42 At4g15440 HPL hyperoxide lyase
2.47 At3g28290 Atl4a, similarity to integrins
AtClpC Arabidopsis ATP-dependent Clp protease, ATP-binding subunit,
2’55 At3g48870 dgrades proteins in the chloroplast

 

Arabidopsis genes that were differential expressed (at least 2-fold difference) upon Pst DC3000
infection. Ratio represents wild-type/nocl.
RNA was isolated from plants 24 and 48 hours post infiltration with Pst DC3000.

109

 

Sensitivity to JA is similar in two] and wild-type plants

The bacterial toxin coronatine has been shown to induce chlorosis in tomato
plants (27). Coronatine is structurally and functionally similar to MeJA. In addition,
both coronatine and MeJA induce similar biological responses in Arabidopsis seedlings,
including inhibition of root elongation and stimulation of ethylene production (21).
Coronatine insensitive (coil) plants are resistant to Pst DC3000 (28). To determine
whether the nod mutant is affected in its sensitivity to MeJA, nocl, coil, and wild-type
seeds were grown on Arabidopsis germination media containing MeJA. As shown in
Figure 4-6, both the wild-type and nocl seedlings had short roots and stunted growth.
coil seedlings, on the other hand, had long roots and normal growth. This result shows

that the root response to JA was not altered in the nocl mutant.

Sensitivity to ethylene is similar in nocl and wild-type plants

ein2 mutants show decreased symptom development after infection with Pst
DC3000 without a reduction in bacterial growth (29). To determine whether nocl was
deficient in ethylene perception, dark-grown seedlings were treated with ethylene and
observed for the triple response. Wild-type and me] seedlings both had tight apical
hooks and short hypocotyls. ein2 seedlings, however, had elongated hypocotyls (Figure
4-7). Based on these results, the nocl mutation does not affect perception and response

to ethylene.

110

 

Figure 4-6. Wild-type, nocl, and coil seedlings on SOuM MeJA medium. Wild-type
(WT), nocl and coil seedlings were germinated on medium containing 50 [.LM MeJA (Scale
bar, 3 mm).

111

 

Figure 4-7. Wild-type (WT), nocl, and ein2 seedlings germinated in the presence of
ethylene. Seedlings were germinated in complete darkness with 10 111/L of ethylene
(Scale bar, 3 mm).

112

The NOCl gene is located on the long arm of chromosome 4

The nocl mutation shows normal Mendelian genetics and is recessive. nocl
was crossed with Ler plants and the F1 progeny were selfed to create an F2 population
for mapping. Bulked segregant analysis was used to analyze a pool of approximately
100 F2 individuals that showed the mutant phenotype (homozygous for the nocl
mutation). One marker, NGA1107, showed linkage to the mutation. This marker is
located on the long arm of chromosome 4.

F2 individuals were tested using additional SSLP markers from the Monsanto
Arabidopsis Polymorphism and Ler sequence collection (St. Louis, M0) to further
pinpoint the mutation on chromosome 4. Currently, the mutation is mapped between
At4g22340 and At4g24050, a region that includes 193 genes. A mutation in any one of
many different pathways may results in the nocl phenotype, but if the function of
NOCl is to destabilize the chloroplasts in some manner, it is probably targeted to the
chloroplast. Of the 193 genes, 24 encode proteins predicted to be targeted to the
chloroplast. These included 11 expressed proteins, and 2 hypothetical proteins. The
other chloroplast targeted genes are listed in Table 4-2. Fine mapping is currently being

conducted.

113

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114

Discussion

The molecular basis of plant susceptibility to pathogen infection remains elusive
in spite of the intense effort to understand bacterial pathogenesis in plants. We isolated
an Arabidopsis mutant (nocI) that has altered symptom development upon Pst DC3000
infection. nocI plants undergo water-soaking and necrosis similarly to wild-type plants,
but do not exhibit chlorosis. To our knowledge, this is the first report of a mutant in
which chlorosis and necrosis have been uncoupled. Despite the altered symptom
development, Pst DC3000 multiplication in wild-type plants and in the nod mutant are
comparable. These results suggest that we have identified a host mutation that
specifically affects the development of disease chlorosis.

We demonstrated that the decreased chlorosis seen in this mutant is associated
with a greater amount of chlorophyll compared with the wild-type plants. We do not
currently know whether the increased abundance of chlorophyll is caused by a defect in
the chlorophyllase-mediated degradation pathway or in other cellular pathways.
However, we have provided evidence that transcription of chlorophyllase is still up-
regulated, albeit to a slightly lower level, in me] plants after infection with Pst DC3000.
Therefore, the chlorophyll degradation pathway is at least partially intact in the nod
mutant. Whether the mutation affects a step downstream of chlorophyllase in this
degradation pathway remains to be determined.

The microarray analysis reveals that, in fact, very few genes are differentially
regulated in this mutant. Although mutants that are deficient in the perception of
certain phytohormones (ethylene and JA) also have decreased symptom development

after bacterial infection, not all phenotypes of these mutants are shared with me] . Both

115

ethylene and JA are perceived by nocl plants. This is consistent with the microarray
results which showed that ethylene and JA responsive genes were not differentially
regulated during infection. Thus it is unlikely that these pathways are altered in nocl
plants.

From preliminary physical mapping, we know that the mutated gene is located
on the long arm of chromosome 4. The delimited region contains 193 genes and,
although this number is too large to apply a candidate gene approach, there are several
interesting possibilities. One candidate is a tyrosine arninotransferase (At4g23600),
which was later reclassified as a cysteine lyase. Cysteine lyases may be involved in
sulfur metabolism, glucosinolate biosynthesis, or indole acetic acid metabolism. This
gene is an interesting candidate because it is induced by the phytotoxin coronatine,
which is known to cause chlorosis in tomato plants (30). There is also a gene that is '
annotated as a senescence-associated family protein (At4g23410). The physiological
role of this protein has not been studied. Although nocl does not have delayed or
altered senescence (data not shown), it is possible that a senescence-related gene is
altered in the mutant.

Theoretically, nocl may be allelic to one of the senescence mutants found in the
previous screen by Oh et al. (11). However, we] plants are sensitive to ethylene,
suggesting that nocI cannot be allelic to ore2, ore3, or ein2, which are all ethylene
insensitive. In addition, nocI cannot be allelic to ein2, are], or ore9 because they are
located on different chromosomes (ein2 (12) and are] (11) are on chromosome 5; ore9
is on chromosome 2 (11)). The physical map locations for oreIO and 11 have not yet

been published and it is possible that MCI is allelic to one of these two mutants. nocl

116

is not allelic to AtCLHI or AtCLHZ because those genes are located on chromosomes 1
and 5, respectively (3). Interestingly, AtCLHI anti-sense lines are not distinguishable
from wild-type plants at a whole-plant phenotype level (5). However, the authors did
not report on the phenotype of the anti-sense lines during pathogen infection or during
senescence.

It is feasible that the nocl mutation causes the gain-of-function of a gene that
inhibits chlorosis. Two examples that lead to delayed senescence and a stay-green
phenotype are over-expression of GF14A, a 14-3-3 protein (31), and over-expression of
cytokinin (32, 33). Whether cytokinin perception or signaling is altered in nocl
mutants remains to be determined.

Fine mapping and cloning of me] is currently underway. Determining the
identity of the gene responsible for the nod phenotype will be an important step toward
increasing our understanding of the processes involved in disease symptom

development during Pst DC3000 infection of Arabidopsis.

Acknowledgements-

I would like to thank R. Nantais, V. Ndlovu, and A. E. Plovanich—Jones for conducting
the original mutant screen and isolating the nod mutant; N. Pumplin, Y. B. Kwack, K.
McLellen, and M. Trapp for help with scoring the F2 population; and N. Raikhel for use

of the initial mapping primer set from Invitrogen, Carlsbad, CA.

117

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121

Chapter 5: Summary and future directions

122

The overall goal of the described research was to increase our understanding of
the compatible plant-pathogen interaction. Although we know that bacterial type III
effectors are important for pathogenesis, their specific functions in the plant cell are just
beginning to be elucidated. Studying susceptibility is technically challenging. Bacterial
mutagenesis is not sufficient to explore the role of effectors because effectors are
functional redundant to each other or each effector only has a small effect on virulence.
The tools used to measure symptom development or disease progression are often not
sensitive enough to discern subtle differences in bacterial growth or symptom
development. To gain further understanding of pathogenesis, alternative approaches
need to be explored. I applied two different approaches to study the compatible A.
thaliana-P. syringae interaction: first, A. thaliana plants that over-express the avrPto
effector were characterized; second, an A. thaliana mutant (nocI) that has reduced

disease-associated symptom development was isolated and studied. .

AvrPto

AvrPto, one effector from Pseudomonas syringae pv. tomato JL1065, has been
well characterized as an avirulence protein, but its virulence function, until now, has
only minimally been addressed. In fact, only a few reports have investigated the
function of AvrPto in susceptible hosts. These studies demonstrate the addition of
avrPto to certain Pst strains increases their virulence (1, 2). Although avrPto mutants
do not have a virulence phenotype, this result may be due to functional redundancy
and/or the lack of sensitivity in the methods currently used to assay virulence.

I have chosen to study the virulence role of the Pst DC3000 effector, AvrPto, by

creating Arabidopsis plants that over-express this effector driven by an inducible

123

promoter (Chapters 2 and 3). After induction of the transgene, plants become paler than
wild-type plants within 24 hours. This paleness progresses into chlorosis by 3 days
after induction.

In Chapter 2, I showed that over-expression of avrPto causes gene expression
changes in the host similar to those induced by Pst DC3000. Remarkably, 78% and
85% of the genes induced by, or repressed by Pst DC3000, respectively, are regulated
in a similar fashion in avrPto plants. If the transcriptional profile in avrPto plants
mimics the response to Pst DC3000 infection so closely, then maybe the need for other,
presumably functionally redundant, type III effectors has been bypassed and rendered
unnecessary in avrPto plants. Indeed, a hrp mutant which cannot secrete any type III
effectors into the host cell and therefore, is not pathogenic, was able to multiply in
avrPto plants up to SOD-fold more than in wild-type plants. In addition, avrPto plants
also allowed Pseudomonasfluorescens 55 (Pf 55), which is a non-pathogenic bacterium,
and an avirulent strain, Pst DC3000 (aerptZ), to grow over 600- and 900- fold,
respectively, more than in wild-type plants (Chapter 3).

The biased down-regulation OfArabidopsis genes that encode putatively
secreted proteins suggests that AvrPto may be disrupting extracellular protein secretion
in the host. One process that is believed to require functional protein secretion is papilla
formation. Papillae form between the plasma membrane and the cell wall. They are
composed of hydroxy proline-rich glycoproteins, phenolic compounds, and callose, and
are thought to be structural barriers against invading microbes. Papilla deposition is
elicited by hrp mutants, but not by the wild-type pathogen, Pst DC3000. In order to

determine if AvrPto alters secretion in the host, I assayed for papilla formation in

124

response to various bacterial strains. I found that avrPto plants suppress papilla
deposition in response to two different hrp mutants (hrpA and hrcC), Pf 55, and Pst
DC3000 (aerptZ) (Chapters 2 and 3). One concern using ~callose deposition as a
marker for secretion is that callose is synthesized at the plasma membrane. Our lab is
currently trying to find other, more reliable, markers for secretion.

Results from yeast-two-hybrid experiments (Appendix A) to find host
interactors of AvrPto also seem to support the theory that AvrPto may be inhibiting
protein secretion. AvrPto interacts with members of the Arabidopsis RabE family.
Rabs are small GTPases that are involved in protein transport from the Golgi to the
plasma membrane. Possibly, AvrPto is preventing protein secretion by binding to RabE
and either interfering with proper RabE function or localization. Currently, our lab is
studying the effects of wild-type, dominant-negative, and constitutively active RabE
expression in transgenic Arabidopsis plants on resistance.

Another interesting candidate identified from the AvrPto yeast-two-hybrid
screen was a kinase. This protein is interesting because Pto, the corresponding R gene
in tomato, is also a kinase. In addition, a kinase has been shown to interact with Rab8,
the rat homologue of RabE. Confirmation of the interaction of this kinase with AvrPto,
as well as characterization of T-DNA insertion lines is presently underway.

An alternative method for assaying host protein secretion is to measure the
abundance of proteins in the apoplast. The intercellular wash fluid (IWF) was collected
from wild-type plants after infection with high concentrations of Pst DC3000 and Pst
DC3000 (aerptZ). At least four protein bands were present in the fluid from the Pst

DC3000 (aerpt2)-inoculated tissue but absent in fluid from the Pst DC3000-inoculated

125

tissue. There are at least three possibilities that would account for this result: 1) Pst
DC3000 (aerptZ) could be causing leakage of these proteins, perhaps as part of the
cell death process; 2) Pst DC3000 (aerptZ) is inducing the specific secretion of these
proteins; 3) or Pst DC3000 (aerptZ) causes higher expression levels of these proteins
and then the same amount of leakage or secretion occurs in both Pst DC3000- and Pst
DC3000 (aerpt2)- inoculated tissue. Interestingly, these proteins are absent in the
apoplastic fluid from avrPto plants inoculated with Pst DC3000 (aerptZ). This result
indicates that AvrPto is either interfering with the leakage, secretion or expression of
these proteins. Future experiments will test the above hypotheses by comparing the
abundance of these proteins in whole leaf extracts under the different treatments. If the
protein concentration is the same in all the treatments, then I propose to distinguish
between leakage and secretion by using the above mentioned dominant negative RabE
transgenic plants or inhibitors of secretion. If I do not detect the same proteins in the
IWF of these plants or after treatment with the inhibitors, then I will conclude that these
proteins are secreted into the apoplastic space.

It was previously shown that Arabidopsis secretes several PR proteins into the
apoplast during SAR induced by 2, 6-dichloroisonicotinic acid (INA) (3). I conducted
western blots on the IWFS and found that PR proteins were detectable in all the
different treatments, probably due to the infiltration process. I believe that avrPto
plants were able to secrete PR proteins. This result suggests that PR protein secretion is
not affected by AvrPto.

To test whether avrPto over-expression leads to excessive leakage of nutrients, I

inoculated the IWF from these plants and wild-type plants with the hrcC mutant. The

126

hrcC mutant grew to similar levels in the IWF from either wild-type or avrPto plants.
From this result, we can conclude that at least water-extractable nutrients are not
limiting in the apoplastic space. However, it is possible that these nutrients may not be
available to the bacteria under physiological conditions in wild-type plants, but are
available in avrPto plants.

It is important to note that my results were obtained from over-expressing
avrPto in plants. Therefore, at this time, I cannot exclude the possibility that I observed
a gain-of-function effect. Shan et al. (4) showed that mutation of the myristylation site
disrupts AvrPto function in resistant tomato plants and that the plasma membrane
localization is essential for this avirulence function. An N—terminal 6x His-tagged
version of avrPto was expressed in Arabidopsis. In contrast to wild-type avrPto plants,
the 6x His-avrPto plants do not undergo chlorosis or allow growth Of hrp mutants (K.
Nomura and W. Underwood, unpublished results). The 6x His tagged AvrPto is no
longer localized to the host plasma membrane (K. Nomura, unpublished results)
suggesting that not only the production, but host membrane localization is also

necessary for AvrPto to carry out its virulence function in Arabidopsis.

127

The nocI mutant

Ng-ghlorosis (nocl) was identified from a screen to identify Arabidopsis
mutants that had decreased symptom development in response to Pst DC3000. Water
soaking, necrosis and bacterial growth are normal in this mutant, but it lacks chlorosis
development in response to infection with Pst DC3000. TO our knowledge, this is the
first instance in which disease-associated bacterial growth, necrosis, and chlorosis have
been uncoupled.

To test the hypothesis that there was more chlorophyll in nocl leaves than in
wild-type leaves, I assayed chlorophyll abundance. I showed that the stay-green
phenotype Ofnoc1 was due to higher amounts of chlorophylls a and b in the nod leaves
compared to those in wild-type plants afier Pst DC3000 infection.

There are at least two possibilities that could explain the nod phenotype: the
rate of chlorophyll synthesis exceeds the rate of chlorophyll breakdown, or there is a
defect in chlorophyll degradation. I assayed the transcript abundance of AtClhl, which
is the first gene in a chlorophyll degradation pathway, to address this question. I found
that this gene is up-regulated in the nod mutant afier Pst DC3000 infection, albeit to a
slightly lower level than in wild-type plants. This result suggests that signaling leading
up to the chlorophyllase degradation pathway is not drastically disrupted in the me]
mutant. Transcript abundance of downstream genes of this step could not be assayed
because they have not yet been identified.

Microarray results also confirm that the AtClhI gene is expressed similarly in
nod and wild-type plants. In fact, there were very few genes that were differential
regulated in the nocI plants compared to wild-type plants. Salicylic acid (SA),

jasmonic acid (JA), and ethylene are signaling molecules in plants that are essential for

128

the Arabidopsis response to pathogens. There are several mutants that have altered
perception of these compounds that exhibit some phenotypes in common with the nocl
mutant. However, there were no SA, JA or ethylene responsive genes that were
differentially expressed in nocI plants, even though the microarray was enriched with
SA, JA and ethylene response genes.

Seedling growth in the presence of these hormones confirms the microarray
results. The ein2 mutant is also interesting with respect to the nod mutant because ein2
plants have reduced symptom development in response to Pst DC3000 infection
without decreased growth. Because nocI plants also have altered symptom
development without altered levels of growth, we tested nocl plants for insensitivity to
ethylene. Etiolated wild-type and me] seedlings had tight apical hooks, thickened
hypocotyls, and reduced hypocotyl elongation, whereas the ein2 mutant does not exhibit
this triple response upon exposure to ethylene. This result suggests that the nocl
mutant is not affected in ethylene perception. .

Coronatine, a toxin produced by Pst DC3000, has been shown to elicit chlorosis
on tomatoes and has structural Similarity to MeJ A. Therefore, altered perception of
MeJA could explain the lack of chlorosis in the nod mutant. However, when nocl,
coil (which are resistant to Pst DC3000), and wild-type seeds were germinated on
MeJA plates, the roots of nod and wild-type seedlings were stunted, while those of the
coi1 seedlings were not. This result demonstrates that nocI plants were able to perceive
MeJ A.

The nocI mutation was mapped to a 619-kb region on the long arm of

chromosome 4, which contains 193 genes. Only a few of these genes were present on

129

the microarray slide mentioned above and none of them were differentially regulated in
the nod mutant compared to wild-type plants. Of the 193 genes, 24 encode proteins
predicted to be targeted to the chloroplast These proteins are potential candidates
because we] plants have more chlorophyll than wild-type plants and chlorophyll is
located in the chloroplast, but I cannot exclude the possibility that NOCl is not
localized to the chloroplast. Future work will focus on the fine mapping of the nocl
mutation and cloning of the gene.

We are a long way from knowing how to prevent disease without chemicals or R
genes, but we are steadily getting the information we need. Once we understand the
role of bacterial effectors and how bacterial disease progresses, we will then be able to

design strategies that will allow increased protection of crop plants.

130

References

CHANG, J. H., RATHJEN, J. P., BERNAL, A. J ., STASKAWlCZ, B. J. & MICHELMORE,
R. W. (2000) avrPto enhances growth and necrosis caused by Pseudomonas
syringae pv. tomato in tomato lines lacking either Pto or Prf. Mol Plant Microbe
Interact, 13, 568-571.

SHAN, L., HE, P., ZHOU, J. M. & TANG, X. (2000) A cluster of mutations disrupt
the avirulence but not the virulence function of AvrPto. Mol Plant Microbe
Interact, 13, 592-598.

UKNES, S., MAUCH-MANI, B., MOYER, M., POTTER, S., WILLIAMS, S., DINCHER,
S., CHANDLER, D., SLUSARENKO, A., WARD, E. & RYALS, J. (1992) Acquired
resistance in Arabidopsis. Plant Cell, 4, 645-656.

SHAN, L., THARA, V. K., MARTIN, G. B., ZHOU, J. M. & TANG, X. (2000) The
Pseudomonas AvrPto protein is differentially recognized by tomato and tobacco
and is localized to the plant plasma membrane. Plant Cell, 12, 2323-2338.

131

Appendix A: Putative AvrPto interactors
Supplementary Material for Chapter 3

132

Proteins that are able to interact with AvrPto were screened by using the LexA-
based yeast-two-hybrid system (Clonetech Laboratories, Inc. Palo Alto, CA).
Bogdanove and Martin (I) conducted a similar screen using a tomato cDNA library.
While Bogdanove and Martin (1) used avrPto from Pst JL1065, my screen was
conducted using avrPto from Pst DC3000. The two versions of avrPto differ from each
other in just four bases that cause four amino acid changes. There may be new
information gained from my screen.

avrPto was amplified by PCR and cloned into pNLexA using the sense primer
5’ GCGAATTCCGAACCATGGGAAATATATGTGTC 3’ and the antisense primer, 5’
GCCTCGAGAT'TGCCAGTTACGGTA 3’. The construct was transformed into the
EGY48 strain carrying the lacZ reporter plasmid. The construct was tested for
autoactivation and protein expression (data not shown). Two independent Arabidopsis
cDNA libraries (kindly provided by J. Jones) made using infected and uninfected
Landsberg erecta plants were screened.

Originally, 203 blue colonies were identified. Of these, 147, retained their blue
color and grew on plates lacking leucine. These clones were then PCR-amplified using
pB42AD primers and the products were digested with HaeIII. The clones were grouped
into classes based on the digestion pattern. Representatives from 27 classes were sent
for sequencing. Only 21 sequences were readable and Blast analysis of these sequences
revealed a total 15 different genes (listed in Table A-l). As yet, autoactivation and
confirmation of AvrPto interaction of these clones has not been conducted. In addition,

it is possible that more than one plasmid could be retained in some yeast colonies.

133

AvrPto is localized to the plasma membrane of the host. Therefore, the predicted
targeting of these proteins is pertinent to this study and is also listed in Table A-1.
Both the Bogdanove and Martin (1) and my screen found a Rab8 homologue in
tomato and Arabidopsis, respectively. This small GTP binding protein is thought to be
involved in host cell trafficking from the Golgi apparatus to the plasma membrane.
Representatives of different Rab families (A-G) were then investigated further by
transforming yeast containing AvrPto as the bait. Figure A-l shows that AvrPto
interacts with only the five RAB8 homologues (RabE in plants) and none of the other

Rab families in Arabidopsis.

134

Table A-1. Putative interactors of AvrPto.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

# 0f Predicted
clones At Locus Description 1 . ,, Score
ocatlons
found
1 AT1(L2_870 expressed protein M 0.646
1 AT1G1129O PentaFI’FOPCPtid? (PPR) “pea“ C 0.661
containing protein
1 AT] 629930 light-harvesting chlorophyll a/b binding C 0.778
protein
1 A_T2.LG3 5260 expressed protein C 0.954
1 AT2G46220 expressed protein C 0.637
1 AT3G26600 expressed protein -
l AT3G50700 zinc finger protein C 0.845
1 AT5G16840 RRM-containinggotein -
2 AT] G719§ histidinol-phosphate arninotransferase C 0.957
GCNS-related N-acetyltransferase
2 AT4G28030 (GN AT) family C 0.843
auxin-responsive protein IAA7
3 AT3GZ3OSO (Indoleacetic acid-induced protein 7) '
3 AT4G0432_0 malonyl-COA decarboxylase -related M 0.669
4 AT5G59840 Ras family GTP-binding protein -
ubiquitin-conjugating enzyme, putative,
strong similarity to SP|P50550
6 AT3G57870 Ubiquitin-like protein SUMO-1 M 0.700
conjugating enzyme (Ubiquitin-
conjugating enzyme chE2A)
8 AT4G11890 protein kinase family -

 

* TARGETP-predicted locations (http://www.cbs.dtu.dk/services/TargetP/)

M = Mitochondrion; C = Chloroplast; - = other, location unknown

135

 

Rahs: trafficking and secretion

A typical plant cell contains 5000 to 10,000 different polypeptide sequences and
billions of individual protein molecules. For cells to function properly, it must direct
these proteins to specific places within the cell (2). Protein trafficking is crucial for all
cellular processes (3). It is central to the interaction of cells with their environment
because it is the route by which components of the extracellular matrix and secreted
enzymes are released into the surrounding milieu (2). Although crucial for a number of
many other house-keeping functions in plants, such as the formation of the
phragmoplast during cell division, cell polarization during development, general protein
metabolism, and plant wall biogenesis, the secretory pathway is also involved in
specialized plant processes such as responses to abiotic and biotic stresses (4).

Proteins traverse the secretory system in vesicles that bud off one compartment
and dock, then fuse with the next. Within the secretory pathway, proteins travel from
the endoplasmic reticulum (ER) to the Golgi, and through the Golgi into the trans-Golgi
network (TGN). At the TGN, secreted proteins are sorted from vacuolar proteins and
packaged into secretory vesicles (5). The proteins then travel to the plasma membrane
for secretion or to the tonoplast for delivery to the vacuole. There is also the endocytic
pathway in which proteins are internalized into early endosomes, and transported by
means of late endosomes to the lysosome, or vacuole. Anterograde transport carries
membrane and cargo proteins through the exocytotic and endocytotic pathways.
Retrograde transport retrieves “resident” proteins and membrane components, returning
them to their original compartments (6). The large number of different transport

intermediates in cells and the nmnerous potential targets for those carriers seem to

136

demand a means of ensuring that the carriers fuse at the appropriate destination and
time (7). If docking and fusion were unregulated, all of the organelles in the cytoplasm
might become stuck together as part of a giant sandwich (‘8). Two classes of proteins
have emerged as specific and essential players in many vesicle transport processes. One
class, the SNARES {soluble N-ethylmaleirnide-sensitive fusion factor (NSF) attachment
protein receptors} is composed of integral membrane proteins which serve as receptors
for soluble factors that are necessary for docking and fusion. The other class is
comprised of a branch of the RAS superfamily of small GTPases, called Rab proteins
(RAS-related in brain) (9). Rab proteins are thought to determine the fusion competence
of membranes (10) and have been primarily implicated in vesicle docking as regulators
of SNARE pairing (9).

Rab functions are conserved across all eukaryotes (10) and they control cellular
events ranging from secretion and endocytosis to signal transduction and development
(11). Each Rab protein has a characteristic distribution on cell membranes and, with
only a few exceptions, participates in a specific trafficking step of vesicular transport
(9). The specificity of Rab localization is provided by structural determinants unique to
each family member that appear to be recognized by distinct sets of proteins on
organelle surfaces (8).

A newly synthesized Rab is recognized in the cytoplasm by a REP (Rab escort
protein) which presents the Rab to the geranylgeranyl transferase (12). The reversible
membrane localization of Rabs depends on this post-translational modification of a
cysteine motif at the very carboxyl terminus with one or two highly hydrophobic

geranylgeranyl groups (12). Mutant mono-prenylated Sec4 proteins in yeast are unable

137

to localize to the correct subcellular compartment and as a result are non-functional (11).
REP then functions as a chaperone which keeps the Rab soluble and delivers it to the
appropriate membrane (12).

Rab proteins exist in two states: an active form, which is bound to GTP, and an
inactive form, which is bound to GDP. REP and GDI (GDP dissociation inhibitor) show
a marked preference for GDP-bound Rabs (11). GDI has structural similarity to REP
and like REP, GDI can present geranylgeranylated, GDP-bound Rabs to specific
membranes (12). Despite the sequence conservation, however, GDI cannot replace REP
in the prenylation reaction. REP is therefore a specialized GDI that plays a dual role in
Rab prenylation and membrane association (9).

Membrane delivery is catalyzed by a GDF (GDI displacement factor) (11, 12),
which recruits Rabs to Specific donor membranes (3). GDP is secretory-compartment-
specific and at least has specificity for a subgroup of Rabs (6). It may play a role in the
regulation of trafficking by controlling GDI dissociation and the shift to GTP
association (6). Membrane delivery precedes the exchange of GDP with GTP (11). The
exchange of GDP for GTP results in a conformational change that releases REP and
exposes an isoprenoid lipid anchor attached to the C-terrninal cystein residue. This
hydrophobic tail allows Rabs to bind to membranes. Rab proteins exchange GDP for
GTP with the help of GEF (guanine nucleotide exchange factor) (2). Since GEFS are
specific for particular Rabs, they make an important additional contribution to the
fidelity of Rab targeting (11). Alternatively, Rabs can be maintained in the inactive state
by GDI (2). GDI prevents indiscriminant membrane binding and contributes to the

process of recruitment to the membrane (9).

138

Although they demonstrate a low level of intrinsic ATPase activity, Rab proteins
must interact with a GAP (GTPase activating protein) to hydrolyze GTP effectively (2).
GAPS may also be specific for some Rab family members (9), but, although newly
identified GAPS Show some substrate preference, they do not exhibit absolute
specificity in vitro, suggesting that they might also be promiscuous in vivo (3).

Upon GTP hydrolysis, the Rab may be released from the membrane due to a
conformational change that facilitates the efficient dissociation of Rab-GDP from the
membrane (2). This is mediated by GDI, which is capable of retrieving the
geranylgeranylated, GDP-bound Rab from intracellular membranes (12). Current
models propose that GDI extracts Rabs from target membranes after the vesicle fusion
event (6) and recycles them back to their membranes of origin (8). GDI, which is more
abundant than REP, thus serves as a recycling factor that allows several rounds of
membrane association and retrieval of the Rabs (12). The (GTP/GDP) cycle imposes
temporal and spatial regulation to membrane transport, with the Rabs acting like timers
whose clocks are set depending on the intrinsic and catalyzed rates of nucleotide
exchange and hydrolysis (13). Their on/Off regulatory functions are restricted to the

membrane compartments where they are localized (13).

As previously mentioned, SNARES also play a role in protein trafficking.
SNARES, initially defined as a category of proteins that bind a-SNAP (soluble NSF
attachment protein), has come to denote a more general class of vesicle trafficking
proteins. Thus SNARE proteins may not bind a-SNAP, but they do possess the

hallmark characteristics of being relatively small and compartment-specific (14). In

139

general, SNARES are C-terrninally anchored integral membrane proteins with most of
their mass present in the cytoplasm (7) in order to engage in protein trafficking
interaction (14). All SNARES bear an approximate 60-65 amino acid residue ‘SNARE
motif in the membrane proximal region. Conserved in this motif are hydrophobic
heptad repeats indicating a propensity to form a-helical coiled-coil structures (7).
Specific complex formation between SNARES on opposing membranes is required for
membrane fusion reactions (14). The assembled trans-SNARE complex consists of a
bundle of four helices, one of which is supplied by the v-SNARE (vesicle SNARES
which are anchored on the cargo vesicle) and the other three by the t-SNARE (target
SNARES which reside on the target membrane) (5, 15). The t-SNARES always include
a syntaxin, which contributes one helix, whereas the remaining two helices are either
from a single SNAP 25-type protein or from two separate t-SNARE light chains (16).
Trimeric SNARE complexes involving a SNAP25 homologue have been described in
plasma membrane fusion events, whereas tetrameric SNARE complexes prevail in
endomembrane fusion processes (15). Membrane docking is mediated by the formation
of this four helical bundle of SNARE proteins (16). The matching of v- and t-SNARES
is believed to provide specificity to membrane fusion reactions (8).

SNARES cannot be the sole membrane targeting determinants because some
SNARES act at multiple steps in vivo and can be found in more than one SNARE
complex. Also, non-cognate sets of SNARES can form complexes in vitro and non-
cognate complexes exhibit comparable stabilities. Lastly, although plasma membrane t-
SNAREs are dispersed over a wide area, secretion only occurs in well defined

subdomains (7). Any given organelle will contain SNARE complexes that must remain

140

inactive unless they are at their specific place of function (13). Thus targeting
specificity cannot be determined solely by the specific localization of t-SNARES and
the relative affinities of v/t-SNARE interactions (7). Genetic experiments implicate
Rabs in the processes by which transport vesicles recognize their cognate fusion targets
(8). Rather than being mere regulators of SNARE protein complexes, Rab GTPases and
their effectors are primary determinants of compartrnental specificity in the organelles
of eukaryotic cells (13).

Early in the targeting process Rabs mediate the tethering of an incoming vesicle
to the correct target organelle (7, 13). It is not clear whether tethering is completely
independent of all SNARE functionality. If tethering is ignorant of the downstream
SNARES, then it would be the critical targeting event in membrane traffic (7).
Alternatively, Rabs may coordinate tethering with downstream catalysis of cognate
SNARE complex assembly (7). This tethering proposal differs from current models
which hold that Rabs act though the tethering factor (for example by simply conferring
tether localization or by acting as physical components of the tether). The Rab protein
may coordinate tethering with SNARE assembly by interacting either directly or
indirectly with t-SNARE complex on the target membrane (7). Rabs, in their GTP -
bound conformations, may recruit transport step-specific docking factors from the
cytosol that facilitate v-/ t-SNARE pairing. Thus Rabs act either directly or indirectly to
facilitate SNARE complex formation but are not core elements of such complexes (8).
If the initial Rab recruitment onto a nascent transport vesicle is coupled (or quickly
followed by) its conversion to Rab GTP, only functional transport vesicles will recruit

docking factors. Therefore, docking will only take place between transport vesicles and

141

their targets, rather than between entire organelles (8). Rabs may also act by stabilizing
the v-/t-SNARE interaction (17). Regardless, vesicle docking is the accepted role of

Rabs in vesicular transport (6).

The t-SNARE membrane and v-SNARE membrane fusion event is traditionally
assigned to SNARES. Once docked, the coiled-coil motifs of the v- and t-SNAREs
interact in a head to head manner, bringing the vesicle and target membranes into close
contact (2). This interaction is thought to be a key step in the reaction leading to vesicle
fusion. Experimental support that SNARES can directly mediate membrane fusion
comes from studies of liposomes containing purified SNARES (18). This work shows

'that liposomes containing v-SNARES are able to fuse with t-SNARE bearing liposomes.
Another study with isolated vacuolar SNARE proteins and liposomes also showed that
fusion automatically follows after trans-SNARE pairing unless a special mechanism
exists to prevent it, as in regulated exocytosis (19). In other words, pairing of these
SNARE proteins by itself results in spontaneous and efficient bilayer fusion. Studies in
permeabilized cells also Show that trans-SNARE complex assembly is coincident with
membrane fusion and the ability of SNARES to fuse membranes is roughly proportional
to their binding affinities, as reflected by their thermal stabilities (7).

Alternatively, in an in vitro system that reconstitutes vacuole fusion, the vast
majority of v/t-SNARE complexes could be enzymatically disassembled by NSF, yet
membrane fusion could still take place (7). It is important to note that whereas NSF
disrupts cis-SNARE complexes, under the same conditions, the trans-SNARE

complexes engaged in fusion are resistant to NSF (19). Whether a small amount of

142

fusion competent trans-SNARE complexes persisted to catalyze fusion could not be
ruled out by this approach (7). Experiments with sea urchin eggs also suggest that the
fiision of secretory vesicles can proceed in the absence of SNARE complexes (7). Thus,
the role of SNARES in fusion is still controversial.

After fusion, the complex is disassembled by NSF and a-SNAP. a -SNAP binds
the v- / t-SNARE complex and recruits NSF to it (2). NSF uses the energy of ATP
hydrolysis to unravel the coiled-coil interaction between the helical domains of the
SNARE proteins (20) fleeing the v-SNAREs and t-SNARES for subsequent fusion
events (2). The requirement for NSF-mediated reactivation of SNARES may also allow
the cell to control when and where membranes fuse (20). Although NSF and SNAP are
required to sustain continuous fusion, they are not required for bilayer fusion per se (19).

Lastly, the v-SNARE can recycle back to the donor membrane (7).

143

 

 

 

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Figure A-l. AvrPto interacts with the Rab E family. AvrPto was fused to the DNA
binding domain in pNLexA and the Rabs were fused to the transcriptional activation
domain in pB42AD. (A) Irnmunoblot of each Rab probed with antibody raised against
the hemagglutinin epitope. Yeast strains were grown in media containing glucose (-) or
galactose (+). Galactose is necessary for protein expression. (B) Physical interaction
between AvrPto and Rab proteins in the LexA two hybrid system. Yeast strains were
grown at 30°C for 5 days on galactose X-gal plates. Blue color indicates interaction

whereas white color indicates no interaction. AvrPto interacts with members of the
RabE family.

144

This next section will focus specifically on the last step of the secretion pathway,
from the trans-Golgi network to the plasma membrane. The specific class of Rabs
involved at this step includes Rab8 in mammals, Sec4 in yeast, and RabE in plants.
While there is abundant information available for this class of Rabs in mammals in
yeast, there is very little information about them in plants.

Although there is some evidence that some Rabs function in vesicle budding,
Rab8 has not been shown to be involved in this step of vesicle transport. It has been
shown, on the other hand, to play a role in the motility of vesicles as suggested from
both in vivo and in vitro studies. Specifically, ectopic expression of wild-type or
activated Rab8 in cells resulted in reorganization of the cytoskeleton and changes in cell
morphology (6, 21). Rab8 was Shown to be able to promote the reorganization of actin
and microtubules (9). In addition, genetic interactions have been uncovered in yeast
between sec4, and the myosin heavy chain Myo2p, indicating a possible mechanism
whereby vesicles are propelled by motor proteins along polarized actin cables towards
the site of exocytosis (13).

Hattula et al. (22) found a novel human protein, called Rabin8, which binds only
to Rab8 when it is bound to GDP. They show that both Rabin8 and Rabin3 (from rat)
are GEFS that facilitate the release of GDP from Rab8, and GTP association. They also
found that a non-Rab8-binding region of Rabin8’s carboxy terminus is essential for
targeting Rab8 vesicles to the cell surface. Rabin8 co-localized with cortical actin.
However, they were not able to demonstrate a direct in vitro association between
purified actin and Rabin8. Rabin8 localizes to the plasma membrane, but when co-

expressed with the dominant negative form of Rab8 (Rab8 T22N), Rab8 T22N

145

relocalized from the perinuclear region to numerous vesicles and Rabin8 was
translocated from the plasma membrane to the Rab8-T22N containing vesicles. Hattula
et al. (22) conclude that Rab8’s activation takes place on'intracellular vesicles (the
donor compartments). They acknowledge that Rabin8-mediated activation of Rab8
might also occur on the plasma membrane. Lastly, they demonstrated that Rabin8’s
carboxy terminus is essential for Rabin8 to move to Rab8-containing vesicles and for
polarized delivery of these vesicles to the cell surface (22). In yeast, it is has been
shown that Sec2, Rabin8, and Rabin3 are GEF ’s for Sec4 (6), and genetic studies
suggest that Gypl acts as a GAP for Sec4 (13).

The Exocyst complex is an effector for Sec4 (3). It plays a role in fusion of
trans-Golgi vesicles with the plasma membrane (6) at a point upstream from the
SNARES (7). The Exocyst marks the sites of exocytosis on the plasma membrane and
mediates vesicle targeting (13). The Exocyst is a large (1000- to 2000-kD (6)) 19.58
particle complex that contains Sec3, Sec5p, Sec6p, Sec8p, SeclOp, Sec15p, and exo70p
(8). One of the Exocyst components, Sec3, stably associates with the secretion-active
site on the plasma membrane, even in the absence of membrane trafficking (7, 8), which
suggests that Sec3 may serve as a spatial landmark for this site (3, 6, 7). Another
Exocyst subunit, Sec15, can bind to the GTP-bound form of Sec4p, which localizes to
secretory vesicles (6, 7). Interestingly, incorporation of Sec3p into the Exocyst requires
Sec4p Rab function, suggesting that the Rab may regulate assembly of the Exocyst (7).

Rab 8-interacting protein (Rab8ip) interacts with the GTP-bound form of Rab8
(17). It is serine/threonine protein kinase that is a component of the stress-activated

protein kinase pathway in humans. Although Rab8ip was primarily found in the cytosol,

146

substantial amounts of the kinase were associated with sedimentable membranes in a
salt-sensitive linkage (17). Irnmunofluorescence microscopy showed that it was
concentrated in the plasma membrane and the Golgi region (similarly to Rab8) (17).
Protein phosphorylation-dephosphorylation events are known to be required for many
stages in intracellular protein traffic. Protein kinases have been implicated in the
generation of secretory vesicles in polarized secretion. One possible mechanism is that
the kinase serves to regulate Rab8 by selectively phosphorylating active Rab8
molecules and, consequently, modulating their function in vesicular transport. However,
Ren et al. (17) found that, in vitro, Rab8 is not a substrate for Rab8ip. An alternative
model is that rab8 regulates the kinase activity of Rab8ip, which serves as its effector. It
iS even possible that this phosphorylation may render v-, t-SNARES and/or v-/t-SNARE
complexes competent for vesicle targeting and/or fusion (1 7). It is well established that
phosphorylation can prevent the assembly of t-SNAREs and dephosphorylation may
relieve such inhibitory effects on membrane fusion (19). If Rab8 affects the kinase
activity of this effector, then Rabs may modulate secretion in response to stress stimuli
or Rab regulated protein phosphorylation may be important for vesicle targeting and
fusion (6).

Rabs may also play a role in plant defense against pathogens. The expression of
AtSNAP33, a homolog of the t-SNARE SNAP 25 in Arabidopsis, is induced after
inoculation with pathogens in inoculated leaves as well as in systemic leaves (5). The
local induction is partially SA-independent whereas the systemic induction requires SA.
SA increases the level of expression ofAtSNAP33 in the absence of a pathogen.

Interestingly, AtSNAP33 is induced in nprl (non-expressor of 3 genes) plants afier

147

pathogen infection. Wick et al. (5) hypothesize that after pathogen attack, increased
vesicle fusion may be required to permit increased secretion of PR proteins. In addition,
increased vesicle fusion may be required for repair of damage to the plasma membrane
provoked by reactive oxygen species. Reactive oxygen species are produced afler
pathogen attack and mechanical stimulation. It has been shown in sea urchin eggs that
SNAP-25 is required for membrane rescaling after injury (5). Ethylene, another plant
hormone that plays a role in plant defense, rapidly and transiently increased expression
and GTP binding activity of Rab8 (23).

PENl is a syntaxin that is localized to the plasma membrane and is required for
resistance OfArabidopsis to Blumeria graminis hordei (24). It was later shown that
PENl is required for timely papillae association (25). Bogdanove and Martin (1)
screened a tomato cDNA library for proteins that interact with the bacterial effector
protein, AvrPto. They suggest that one of the interactors, RabE, might regulate the
polarized secretion of antimicrobial compounds and/or components involved in

mounting cellular responses to attack by bacterial pathogens.

148

10.

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