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TYPE III VIRULENCE EFFECTORS OF PSEUDOMONAS S YRINGAE
PV. TOMA T 0 STRAIN DC3000

By

Sruti Bandyopadhyay

A DISSERTATION

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

DOCTOR OF PHILOSOPHY
Cell and Molecular Biology

2003

ABSTRACT
TYPE III VIRULENCE EFF ECTORS OF
PSEUDOMONAS SYRINGAE PV. T OMAT 0 STRAIN DC3000
By
Sruti Bandyopadhyay

As a group, plant pathogenic bacteria infect virtually all crop plants and cause
significant damage to crop production worldwide. Pseudomonas syringae pathovar
tomato strain DC3000 (Pst DC3000) is a virulent pathogen of both tomato and
Arabidopsis thaliana. The Arabidopsis thaliana-PstDC3000 pathosystem has proven to
be a very useful model system to study plant-pathogen interactions and results are
generally correlated with observations from other systems. During infection in
Arabidopsis, the pathogen multiplies vigorously for two days before the onset of disease
symptoms, which are characterized by water-soaking in the apoplast followed by tissue
necrosis and chlorosis. The hrp gene-encoded type 111 protein secretion system (TTSS) is
essential for Pst DC3000 pathogenesis. The TTSS is highly conserved and secretes
bacterial proteins, collectively referred to as effectors, directly into the host cytoplasm.
Once translocated into the plant host cell, the effectors promote disease presumably by
suppressing host defense responses and promoting the release of nutrients for utilization
by bacteria. My research has identified new effectors and revealed their contribution to
Pst DC3000 virulence on Arabidopsis.

A mutant carrying a deletion of six genes in the Conserved Effector Locus (CEL)
of Pst DC3000 shows a drastic reduction in growth and symptom development on

Arabidopsis plants. HothoM (ORF3) and Sth (ORF4), two of the deleted ORFs, were

found to be sufficient to restore virulence to the ACEL mutant. HothoM is translocated
into plant cells and translocation of HothoM is dependent on its chaperone, Sth. The
first 200 amino acids of HothoM are required for interaction with Sth. The ACEL
mutant was compromised in its ability to suppress basal host immunity characterized by
the deposition of callose-containing papillae in the cell wall in response to pathogen
attack. Activation of papilla formation by the ACEL mutant is dependent on salicylic acid,
whereas papilla formation activated by the hrp mutant is independent of salicylic acid.
The ACEL mutant can be restored to suppress papilla formation by HothoM and Sth,
suggesting that HothoM is a suppressor of an SA-dependent cell wall defense in
Arabidopsis.

Using a gene homology-based method, I identified three new effectors in Pst
DC3000. These were avrPtoB, averhEpm, and averin,o. All three effectors were
demonstrated to be translocated by the TTSS. Using bacterial mutagenesis method, no
contribution to virulence was detected for either averhEpm or averinm. On the other
hand, even though I detected a significant virulence effect for avrPtoB in both tomato and
Arabidopsis, I was unable to complement this mutant. Transgenic expression of
AVTPphEpm in Arabidopsis resulted in symptoms that mirror those seen during Pst
DC3000 infection. AverhEpto plants promoted the growth of the non-pathogenic hrpH
mutant, which does not deliver any type III effectors. In addition, microarray experiments
showed that the expression profile of the TTSS-dependent host gene cluster of
Arabidopsis was 90% similar between plants expressing AverhEp‘L0 and plants infected

with Pst DC3000, further supporting a virulence role of AverhEpto in disease promotion.

C0pyn'ght by
Sruti Bandyopadhyay

2003

To
My Parents

ACKNOWLEDGEMENT

First of all, I would like to thank my thesis advisor, Dr. Sheng Yang He for giving
me an opportunity to conduct research in his lab. It has truly been a wonderful learning
experience. He has always supported me and encouraged me to pursue better and bigger
things despite difficulties. We have had many good discussions and debates.

I would like to thank my committee members, Dr. Jonathan Walton, Dr. Gregg
Howe, Dr. Richard Allison and Dr. Linda Mansfield for their guidance and help through
these years.

I have been very thankful and very lucky for having wonderful people to work
with in the lab: Qiaoling J in, Roger Thilmony, Julie Zwiesler-Vollick, Anne Plovanich-
Jones, Paula Hauck, Elena BraySpeth, Kinya Nomura, Bill Underwood, Ola Kolade,
Yong Hoon Lee and Young Nam Lee. This lab has been like a warm and loving family
which has always inspired and encouraged me through good and bad times in life,
scientifically and otherwise. I am very grateful for the numerous scientific discussions
and all the help that I have received from everyone. I would like to specially thank Kinya
Nomura, for helping me with the work presented in Chapter 2. A very special thanks also
to John Scott Craig, for his help, advice and encouragement through the years.

A big thank you for all the PRL support staff. They have been a big help in many
different ways. I would also like to thank all the wonderful undergraduate students who
have worked hard to take care of a lot of things that has made our lives less stressful. I
would also like to thank all my fellow graduate students and everyone in the PRL. I am

glad to have been part of this wonderful scientific community.

vi

Finally, I would like to thank my parents. They have always supported me,
believed in me, encouraged me to follow my dreams and given me the courage to make
my own choices in life. A very special thank you to my husband, Ashish, for all his love,
patience, strength, trust, support, belief and encouragement. Thank you for putting up
with all my whims and quirks and helping me to see things in perspective. I would also
like to thank all my fiiends, not all of whom are in East Lansing anymore, for bringing

hope, happiness and warmth and making this world a better place to live in.

vii

TABLE OF CONTENTS

List of Tables ........................................................................................ xi
List of Figures ....................................................................................... xii
Chapter 1
Introduction and Literature Review ................................................................ 1
Introduction ................................................................................... 2
Virulence mechanisms in phytopathogenic bacteria ..................................... 3
P. syringae as a pathogen ................................................................... 4
The type III secretion system (TTSS) ..................................................... 5
Gene organization .................................................................... 6
Regulation ............................................................................ 7
The type III apparatus and the Hrp pilus ........................................ 9
Secreted proteins ................................................................... 10
Harpins .............................................................................. 1 1
Type III virulence effectors ...................................................... 12
Virulence role of effector proteins ............................................... 15
Type III chaperones ............................................................... 19
Phytotoxins .................................................................................. 20
Additional virulence factors ............................................................... 22
Project summary ........................................................................... 24
References .................................................................................. 28
Chapter 2

HothoM is a type HI virulence protein of Pseudomonas syringae pv. tomato DC3000
that suppresses salicylic acid-dependent activation of cell wall-based extracellular host

defenses in Arabidopsis thaliana .................................................................. 44
Abstract ...................................................................................... 45
Introduction ................................................................................. 46
Materials and Methods ..................................................................... 49

Bacterial strains and media ....................................................... 49
Recombinant DNA techniques .................................................. 49
Construction of complementation plasmids .................................... 50
Plant growth and bacteria enumeration ......................................... 51
Secretion assays ................................................................... 52
Type III translocation analysis ................................................... 52
Yeast two hybrid analysis ......................................................... 53
Generation of transgenic plants .................................................. 54
Dexamethasone-induction of transgenic expression. . . . . . . . . . . . . . . . . . ....55
Generation of antibody against HothoM and Sth ......................... 55
Irnmunoblotting .................................................................... 56
Northern blot analysis ............................................................. 56
Callose assay ....................................................................... 57

viii

Results ....................................................................................... 58
The Pst DC3000 ACEL mutant can be restored by a fragment containing

ORF3 and ORF4 ................................................................... 58
DRE 3, but not ORF4, is secreted in culture by the Hrp system ............. 64
ORF3 is translocated into plant cells by the TTSS in an ORF4-dependent
manner ............................................................................... 66
ORF4 physically interacts with ORF3 in the yeast two hybrid
system .............................................................................. 68
HothoM transgenic plants exhibit a distinct seedling and growth
phenotype ........................................................................... 70
Arabidopsis thaliana plants expressing HothoM exhibit chlorosis and
necrosis upon induction .......................................................... 71
Expression of HothoM in planta allows the growth of the CEL deletion
mutant .............................................................................. 73

The ACEL mutant is impaired in suppressing cell wall-based defenses...77
The ACEL mutant is compromised in eliciting callose formation in NahG

and eds5 plants ..................................................................... 78
NahG plants support growth of the ACEL mutant ............................ 79
Discussion ................................................................................... 83
References ................................................................................... 90

Chapter 3
Homology-based identification of type III effectors in Pseudomonas syringae pv. tomato
DC3000 and characterization of transgenic Arabidopsis thaliana plants expressing

averhEpw ............................................................................................. 95
Abstract ...................................................................................... 96
Introduction ................................................................................. 97
Materials and Methods .................................................................... 100

Bacterial strains and media ...................................................... 100
Recombinant DNA techniques ................................................. 100
DNA gel blots .................................................................... 101
Cosmid library screening for detection of effector orthologs .............. 101
Cloning and sequencing full-length effector genes. . .......103
Type III translocation analysis using an Aerpt2 fusion ................... 103
Transposon mutagenesis and marker exchange .............................. 104
Plant growth and bacteria enumeration ....................................... 105
Geneartion of transgenic plants ................................................ 106
Production of antibody .......................................................... 107
Irnmunoblotting ................................................................... 107
DEX induction of transgene expression ...................................... 108
Microarray analysis .............................................................. 108
Results ...................................................................................... 110
Identification of putative effector genes in Pst DC3000 that are
homologous to known avr genes ............................................... 110
Isolation of full-length clones of the three putative effector genes ....... 113
Translocation analysis of the identified orthologs ........................... 114

ix

Generation of mutants by transposon mutagenesis .......................... 1 17

Analysis of the mutants on Arabidopsis and tomato plants ................ 117
In planta expression of AverhEpto ........................................... 123
Growth of non-pathogenic hrpH mutant in averhEpm plants ............ 126
Expression profiling of plants expressing AverhEpto ...................... 132
Discussion ................................................................................. 136
References ................................................................................. 143
Chapter 4
Conclusions and future perspectives ............................................................ 150
References ................................................................................. 160
Appendix A
Supplementary material for Chapter 2 .......................................................... 162
Appendix B
Supplementary material for Chapter 3 ........................................................... 167

LIST OF TABLES

Table 3-1. List of avr genes used for identification of orthologs in Pst DC3000. . . . . ....l 11

Table B-1.Arabidopsis genes repressed or induced in a type III secretion-dependent
manner by Pst DC3000 infection and AverhEpt0 expression (30uM DEX). . ...168

Table B-2. Arabidopsis genes repressed or induced in a type III secretion-dependent
manner by Pst DC3000 infection and AverhEpto expression (0.1 uM DEX). . ..173

Table B-3. Arabidopsis proteins that interact with AverhEpto in yeast. . . . . . . . . . ....176

xi

LIST OF FIGURES

Images in this dissertation are presented in color.

Fig. 2-1. Disease symptoms and growth in planta of Pst DC3000 and the ACEL mutant
following vacuum-infiltration with an inoculurn of 106 cfu/ml ....................... 59

Fig. 2-2. Schematic representation of the complementation of the ACEL mutant ......... 60
Fig. 2-3 Schematic representation of further complementation of the ACEL mutant... . .62

Fig. 2-4. Restoration of the ability of the ACEL mutant to multiply and elicit disease

symptoms in Arabidopsis .................................................................. 63
Fig. 2-5. Analysis of type III-dependent secretion of ORF3 (HothoM) and DRE 4

(Sth) ....................................................................................... 65
Fig. 2-6. Type III translocation analysis of ORF 3 and ORF4 in Arabidopsis. . . . . . . . . .67
Fig. 2-7. Physical interaction between ORF3 and ORF4 in the LexA two-hybrid

system ....................................................................................... 69
Fig. 2-8. Expression of HothoM in planta causes disease-like symptoms ................. 72

Fig. 2-9. Complementation of the ACEL mutant multiplication in plants expressing
HothoM ..................................................................................... 74

Fig. 2-10. Growth of the ACEL mutant in hothoM transgenic plants is accompanied by
the development of symptoms similar to those of Pst DC3000 infection .......... 75

Fig. 2-11. The multiplication of the ACEL mutant is not complemented in uninduced
hothoM transgenic plants ................................................................ 76

Fig. 2-12. The ACEL mutant triggers the callose response in Arabidopsis .................. 80

Fig. 2-13. The ACEL mutant is compromised in the ability to activate callose response in
leaves of the SA-deficient NahG plant .................................................. 81

Fig. 2-14. The ACEL mutant proliferates and causes symptoms more aggressively in
NahG plants than in C01 glI plants ........................................................ 82

Fig. 2-15. Schematic representation of the pathways that trigger callose deposition in

Arabidopsis plants in response to the wildtype Pst DC3000, the ACEL mutant and
the non-pathogenic hrpA mutant .......................................................... 89

xii

Fig. 3-1. Southern blot analysis of genomic DNA from Pst DC3000 for the presence of

putative orthologs of known avr genes ................................................. 112
Fig. 3-2. Type III translocation analysis of identified effectors in Arabidopsis. . . . . . . 1 16
Fig. 3—3. Schematic representation of the strategy employed to obtain insertion mutants of

the identified Pst DC3000 effectors genes ............................................. 119
Fig. 3-4. Symptoms of Pst DC3000 mutants in A. thaliana Col g1] plants. . . . . . . ..........120
Fig. 3-5: Bacterial proliferation in A. thaliana Col g1] plants ............................... 121
Fig. 3-6. Bacterial proliferation in tomato Castlemart 11 plants ............................. 122
Fig. 3-7. Phenotype of averhEpm transgenic plants, lines 342 and 422 ................... 125
Fig. 3-8. Bacterial proliferation in uninduced averhEpm transgenic plants ............... 128
Fig. 3-9. Multiplication of the hrpH mutant in averhEpm transgenic plants ............ 129
Fig. 3-10. Growth ofthe hrpH in A. thaliana Col g1] and averhEpm plants... . . . . . . . ....130

Fig. 3-11. Growth of the hrpH mutant in averhEpm transgenic plants is accompanied by
the development of symptoms similar to those of Pst DC3000 infection. . . . . ....l31

Fig. 3-12. Cluster analysis of the expression profiles of 117 TTSS-regulated genes
(colored bars) after Pst DC3000 infection and transgenic expression of
AverhEpt0 with 30 uM DEX ........................................................... 134

Fig. 3-13. Cluster analysis of the expression profiles of 117 TTSS-regulated genes
(colored bars) afier Pst DC3000 infection and transgenic expression of
AverhEpt0 with 0.1 uM DEX .......................................................... 135

Fig. A-l. Physical interaction between ACSlO and HothoM in the LexA two-hybrid
system ...................................................................................... 163

Fig. A-2. Multiplication of Pst DC3000, and the ACEL mutant in wildtype Arabidopsis
and ein2 mutant plants afier treatment with ACC or AVG .......................... 164

Fig. A-3. Growth of Pst DC3000 and the ACEL mutant in Arabidopsis Col g1] and
ACSI 0 knockout lines .................................................................... 165

Fig. A-4. Symptom development on wild-type Arabidopsis and ACSI 0 knockout
plants ....................................................................................... 166

xiii

Fig. B-l. Predicted structural domains of At5 g20480, the receptor-like protein kinase that
interacts with AverhEpt0 ................................................................ 179

Fig. 8-2. Northern blot analysis of total RNA isolated from Arabidopsis plants infected

with Pst DC3000, the non-pathogenic hrpRS mutant, and the ACEL mutant with
(A) At3g51650 and (B) At2g27210 as probes ........................................ 180

xiv

Chapter 1
Introduction and Literature Review

Virulence in phytopathogenic Pseudomonads

Introduction

Plants are the primary carbon source for nearly all terrestrial non-photosynthetic
organisms, including ourselves. Many species seek to tap this phytosynthate, and plants
are continuously resisting their strenuous and intimate advances. Plants are constantly
being challenged by a wide variety of pathogenic organisms including bacteria, viruses,
fungi, nematodes and protozoa. As a group, they infect virtually all crop plants and cause
significant damage to crop production worldwide. The worldwide loss in major crops due
to pathogens was estimated at $76.9 billion for 1988-1990 (Baker et al., 1997). Prevalent
practices of pathogen control involve widespread use of pesticides. Not only is such
extensive usage of these control agents a large expense, but also a source of
environmental damage and toxicity. Breeding and cultivation of disease resistant and
high-yield varieties of crop plants have helped combat pathogens and increase
productivity. However, large-scale genetic uniformity serves as an evolutionary pressure
for adaptive changes in pathogens, which can render enormous areas susceptible to
devastation. An understanding of the molecular mechanisms of pathogen infection and
plant defense response could be instrumental in developing novel technologies to battle
these epidemics.

Bacterial diseases of plants are difficult to control and require a combination of
approaches. The most devastating bacterial phytopathogens belong to the gram-negative
genera of Agrobacterium, Erwinia, Xanthomonas, Ralstonia and Pseudomonas. Lately,
another gram-negative bacteria, Xylella fastidiosa, has become a major cause for concern
for grape vines (Hopkins, 1981) and citrus fruits (Simpson et al., 2001). The gram-

positive genera of plant pathogens include Clavibacter and Streptomyces. Members of

each of these genera cause a wide variety of spots, blights, cankers, scabs, Wilts, soft rots
and other diseases on a wide variety of host plants. Each symptom can be caused by
members of different genera and each genera may contain pathogens that can cause
different types of diseases. Members belonging to different genera have also been
developed as model systems to further our understanding of the molecular mechanisms

that promote pathogenesis in divergent hosts.

Virulence mechanisms in phytopathogenic bacteria

Despite the wide and diverse nature of bacterial phytopathogens and the diseases
they cause, they have maintained striking similarities in the mechanisms they utilize to
overcome their respective hosts to cause parasitism and pathogenesis. One common
feature shared by most bacteria is the extracellular localization of the pathogen during
infection. Thus, pathogens cause the movement of water and nutrients from the host cells
into the apoplastic space. The apoplast is considered to be a nutritionally poor
environment that requires enrichment during pathogenesis in order to support the large
population levels that are typically attained by invading bacteria. This is also thought to
cause dilution of antimicrobial compounds that may be present in the apoplast.

Bacterial plant pathogens have several methods to subvert their hosts including
secretion of enzymes, production of toxins, and injection of bacterial proteins into the
host. The genomes of many bacterial phytopathogens have been sequenced and their
analysis is providing new insight into the mechanisms of pathogenesis. In this chapter, I
will discuss the virulence mechanisms employed by pathogenic bacteria of the genus

Pseudomonas with a greater emphasis on the type III protein secretion system.

P. syringae as a pathogen

Pseudomonas belongs to the gamma subgroup of proteobacteria that contains
animal pathogens such as Yersinia, Salmonella, Shigella and Escherichia and plant
pathogens such as Erwinia, Xanthomonas, Pantoea and Xyllela. The various strains of P.
syringae are noted for their diverse and host-specific interactions and can be classified
into at least 40 pathovars based on host specificity (Gardan et al., 1999). Individual P.
syringae strains often exhibit a high degree of host specificity towards a few plant species
or only a few cultivars within a plant species. In nature, P. syringae often lives initially
on the surface of leaves as an epiphyte, and later in the intercellular spaces of the plant as
a pathogenic endophyte. As a pathogen, P. syringae generally gains access into the leaf
tissue of plants through stomata, multiplies vigorously in the apoplastic space, and
eventually produces necrotic lesions that are surrounded by chlorotic halos (Hirano and
Upper, 2000).

P. syringae pv. tomato (Pst) strain DC3000 has emerged as an important model
organism in molecular plant pathology because of its genetic tractability, its
pathogenicity on both tomato and Arabidopsis thaliana, and its ability to deliver

virulence proteins into host cells via the type III secretion system.

The type III secretion system (TTSS)

Bacterial pathogens of plants, animals and humans cause very different diseases,
ranging from bubonic plague to leaf blights and cankers. One of the amazing discoveries
made in the last two decades is the presence of a protein secretion system in both plant
and animal bacterial pathogens. This system was classified as type III. The unique

property of this system is its remarkable ability to transport bacterial proteins directly into

the cytoplasm of the eukaryotic host cell. This system is broadly conserved among
phytopathogenic bacteria like Erwinia, Xanthomonas, Ralstonia and Pseudomonas and
enteric bacterial pathogens of animals like Yersinia, Shigella, Escherichia and Salmonella.
Among phytopathogenic bacteria, the type HI secretion system was first identified
in P. syringae pv. phaseolicola (Lindgren et al., 1986) and P. syringae pv. syringae
(Niepold et al., 1985). Mutants of P. s. phaseolicola were isolated that were unable to
elicit either the hypersensitive response in the non-host tobacco or disease in the normal
host, bean. These were named hrp mutants (for hypersensitive response and
pathogenicity). These hrp genes were later found to encode the TTSS. Since then, hrp
genes have been found in other pathovars of Pseudomonas, and in Erwinia, Xanthomonas
and Ralstonia species (Bonas et al., 1991; Beer et al., 1991; Gough et al., 1991).
Interestingly, even though the TTSS was initially identified as a major
determinant of pathogenicity, the TTSS is not restricted to pathogenic bacteria. The TTSS
has been detected in several nitrogen-fixing symbiotic bacterium including Rhizobium
species NGR234, Bradyrhizobium japonicum, Sinorhizobium fredii USDA257 and
Mesorhizobium loti MAFF303099. The TTSS in these bacteria is involved in the
secretion of proteins called Nops (modulation outer proteins) (Krause et al., 2002;
Krishnan et al., 2003; Marie et al., 2003). Sodalis glossinidius is a maternally transmitted
secondary endosymbiont residing intracellularly in tissues of the tsetse flies. It was found
to harbour homologues of the inv/spa genes that encode the Salmonella/Shigella TTSS
and to utilize them to parasitize their host (Dale et al., 2001). The function of the inv/spa
genes in maintaining symbiosis has also been demonstrated in Sitophilus zeamais, a

mutualistic bacterial endosymbiont of grain weevils (Dale et al., 2002).

Gene organization

In plant pathogenic bacteria, the TTSS (also called the Hrp secretion pathway or
system) is encoded by hrp genes (He, 1998; Lindgren , 1997; Lindgren et al., 1986). The
P. syringae hrp genes are encoded by a single locus that spans a 25 Kb chromosomal
region that forms part of a pathogenicity island. The hrp cluster contains 27 genes that are
arranged into six operons. There seems to be at least three kinds of genes in the hrp
cluster.

The largest group of hrp genes encode various components of the Type III
secretion apparatus. The core apparatus is likely composed of 13 proteins. Nine of these
hrp genes have been renamed hrc (for hrp genes conserved) because of their broad
conservation among all bacteria that harbor type HI protein secretion systems, and the
flagellar assembly system. The second class consists of hrp genes that are responsible for
the expression of all type III-associated genes in planta or in hrp-inducing minimal
medium. The third class encodes proteins that are secreted by the TTSS, including some
extracellular components of the type III secretion apparatus.

The hrp clusters from the four major phytopathogens have been sequenced and
characterized. Based on gene organization, sequence relatedness and regulatory systems,
the hrp clusters have been divided into two groups. Pseudomonas and Erwinia belong to
Group 1, whereas Xanthomonas and Ralstonia belong to Group 11.

Recent studies conducted in three different P. syringae strains suggest that the P.
syringae pv. syringae strain 61 and P. syringae pv. tomato DC3000 hrp clusters and
flanking sequences constitute a pathogenicity island (PAI). Pathogenicity islands are

defined as gene clusters that include many virulence genes, are present selectively in

pathogenic strains, have a different G+C content compared to the host bacterial DNA,
occupy large chromosomal regions, are often flanked by direct repeats, are bordered by
tRNA genes and/or cryptic mobile genetic elements, and are unstable (Alfano et al.,
2000). The hrp PAI has a tripartite mosaic structure consisting of the hrp/hrc cluster, an
exchangeable effector locus (EEL) and a conserved effector locus (CEL). The EEL
begins downstream of 12er and contains four ORFs, two of which have the hrp box and
are putative hrp-regulated genes. The Pst DC3000 EEL also contains mobile genetic
elements. The CEL contains ten ORF3, three of which have been previously identified as
aer, aer (Lorang and Keen, 1995) and hrpW (Charkoswki et al., 1998). The CEL has

no known mobile genetic elements. (Alfano et al., 2000).

Regulation

The expression of P. syringae hrc/hrp genes is under tight transcriptional control.
In nature, the expression of hrc/hrp genes is induced only when the bacteria enter the host
apoplast. Most hrp genes are expressed at a very low level in standard, nutrient-rich
medium, but high levels of hrp gene expression are observed in infected plant tissues or
in artificial hrp-inducing minimal media (Rahme et al., 1992; Xiao et al., 1992). The hrp-
inducing medium is acidic, lacks complex nitrogen sources and somehow mimics the
conditions that P. syringae experiences in the plant apoplast. Transcriptional regulation
of the TTSS involves three positive regulators — HrpR, HrpS and HrpL. hrpR and hrpS
are transcribed from a single operon and belong to the NtrC family of response regulators
which are c 54-dependent enhancer-binding proteins (Grimm et al., 1995; Hutcheson et

al., 2001). These proteins are members of two-component regulatory systems which

control transcription in bacterial systems (Stock et al., 2000). hrpl. encodes an alternative
sigma factor which shares homology with the ECF family of sigma factors (Xiao et al.,
1994). It is believed that HrpR and HrpS are involved in the activation of hrpL expression
in response to a signal in host tissue or in hrp-inducing minimal medium. HrpL then
recognizes a conserved sequence motif called the hrp box, which is present in the
promoter region of all TTSS-associated genes (Xiao and Hutcheson, 1994).

Although the transcriptional regulation of the hrpRS operon is not well
understood, recent studies showed that the HrpR protein is posttranscriptionally regulated
via the Lon protease (Bretz et al., 2002). The HrpR protein of Pst DC3000 has a shorter
half life when the bacteria are cultured in rich medium than in hrp-inducing medium, but,
the half life is unaffected by the kind of medium in Ion' mutants. The increased stability
of HrpR in hrp-inducing conditions would favour the expression of the TTSS-associated
genes. How the Lon protease affects the stability of HrpR is not yet known. Two hrp
genes have been shown to function as negative regulators of the TTSS in P. syringae. In
hrp-inducing minimal medium, overexpression of the hrpV gene down-regulates hrp/hrc
gene expression, whereas a hrpV mutant is elevated in hrp/hrc gene expression (Preston
et al., 1998). Overexpression of both hrpV and hrpRS results in normal levels of hrp gene
transcripts. In addition to hrp V, the hrpA gene also plays a key role in secretion of TTSS-
associated proteins. Deletion of hrpA, the major component of the hrp pilus, was found to
down regulate the expression of all examined TTSS-associated genes. Again,
overexpression of hrpRS can compensate for the lack of hrpA. Further study is required

for a complete understanding of this regulatory circuit.

Unlike Group I, the Group II hrp operons are activated by a member of the AraC
family, which is designated HrpB in R. solanacearum and HrpX in X. campestris

(Wengelnik and Bonas, 1996; Oku et a1, 1995; Genin et el, 1992)

The type III apparatus and the Hrp pilus

Structurally, the type III apparatus consists of an intracellular membrane-
embedded basal body and an extracellular appendage. The core apparatus was initially
predicted to be similar in structure to the flagellum based on the high level of sequence
similarity between eight hrc genes and the flagellar assembly genes. The type III basal
body has been isolated from several mammalian pathogens. It consists of an inner
membrane ring structure and an outer membrane ring structure with a periplasmic rod-
like component connecting them. The type III basal body has not been isolated from any
of the plant pathogenic bacteria yet.

The extracellular appendage in mammalian pathogens is very short and has been
called the needle. It is about 8 nm in diameter and < 100 nm in length (Kubori et al.,
1998). Plant pathogenic bacteria assemble a type III-dependent appendage known as the
Hrp pilus. Hrp pili are much longer (several micrometers long) than the needle, but have
the same diameter (~8nm). The Hrp pilus has been reported in Erwinia amylovora (J in et
al., 2001) and Ralstonia solanacearum (Van Gijsegem et al., 2000). In Pst DC3000 the
major structural constituent of this pilus is HrpA. Non-polar hrpA mutants do not
assemble the pilus, do not secrete effector proteins in culture, and are unable to cause
disease in host plants or elicit the HR in non-host plants (Roine et al., 1997; Wei et al.,

2000). This suggests that the Hrp pilus plays an important role in the type 111 protein

secretion process. An in situ immunogold labeling procedure has been used to
demonstrate that secretion of type III effectors occurs only at the site of the Hrp pilus
assembly and the secreted proteins are localized specifically along the Hrp pilus, but not
along the flagellum or randomly in the intercellular space (Jin et al., 2001; Brown et al.,
2001). Further immunogold-labeling experiments were used to visualize the extrusion of
newly synthesized effector protein from the tip of the Hrp pilus, providing direct
evidence that the Hrp pilus functions as a conduit for protein delivery (J in and He, 2001;
Li et al., 2002). The assembly mechanism of the Hrp pilus in Pst DC3000 has also been
established. The Hrp pilus has been shown to elongate by the addition of the new HrpA
subunit at the distal end, fiirther supporting the mechanistic similarity between the type

IH system and the flagellar system (Li et al., 2002; Emerson et al., 1970).

Secreted proteins

There are two kinds of proteins that are secreted in a hrp-dependent manner. They
are classified based on their localization in plants. The first class of proteins, exemplified
by the harpins, are secreted in the medium when bacteria are cultured under hrp-inducing
conditions. The other class of proteins consist of the type III effectors. This group of
proteins travel through the hrp system and are delivered directly into the cytoplasm of the

plant cell.

Harpins

Harpins are a family of type III secreted proteins that are unique to plant

pathogenic bacteria. The first harpin, HrpN, was identified in Erwinia amylovora (Wei et

10

al., 1992). The Hrp secretion system of Pseudomonas syringae has been shown to secrete
two harpins - HrpZ and HrpW (He et al., 1993; Yuan and He, 1996; Charkowski et a1,
1998). These are heat stable proteins that are rich in glycine and proline, lack cysteine,
and elicit an HR-like response when injected into the intercellular space of plant leaves.
Their precise function in pathogenesis remains elusive. It has been observed that under
certain conditions, HrpZ is required for avr genes to trigger an R gene-dependent HR.
This suggests that HrpZ assists in the delivery of Avr proteins (Gopalan et al., 1996;
Alfano et al., 1996). Recently, purified HrpZ has been demonstrated to associate with
lipid bilayers to form cation-conducting pores in vitro and bind to plant protoplast
membranes (Lee et al., 2001; Lee et al., 2001). HrpW also associates with the plant cell
wall. HrpW has a C-terminal pectate lyase domain that binds to calcium pectate, a major
plant cell wall component (Charkowski et a1, 1998). These proteins could be involved in
assisting the penetration of the TTSS pilus through the plant cell wall even though there
is no direct evidence of such a function yet. Analysis of the recently completed Pst
DC3000 genome revealed the presence of other HrpW-like proteins in P. syringae and
Ralstonia (Petnicki-chieja et al., 2002 ; Guttman et al., 2002 ; Zwiesler-Vollick et al.,

2002 ; Salanoubat et al., 2002).

Type III virulence effectors

This group of type III secreted proteins is by far the most interesting and is
currently the focus of intense research. Although type III effectors are very diverse, as a
group, these proteins have several characteristics: i) unlike harpins, their site of action

has been demonstrated to be inside the plant cell (Gopalan et al., 1996); ii) genes

11

encoding type III effectors in a given pathogen are scattered within the genome, being
encoded on the chromosome as well as on plasmids. While some are within or physically
associated with the hip cluster, others are known to be located elsewhere in the genome
but tend to be in clusters (Buell et al., 2003); iii) they are hydrophilic proteins and often
do not share sequence similarities with any genes of known function; iv) infiltration of
purified effectors into leaf apoplast does not elicit any response; v) distribution of these
genes within different pathovars of a single species is scattered; vi) the virulence
contribution of a given effector is often specific to certain pathogen/plant genotypes and
the same effector has been demonstrated to function differently in different cultivars of
the same host; vii) deletion of a single effector often has subtle or no impact on
virulence as measured by attenuation of disease symptoms and bacterial growth; viii)
they are often flanked by mobile genetic elements indicating horizontal movement among
pathovars and possibly across species; and ix) they are the prime targets for host
recognition to activate plant defense responses.

Historically, a large number of type III effectors have been identified by their
ability to elicit host defense responses, including the HR, in plant genotypes that carry
cognate disease resistance genes. These type III effectors have been named avirulence
(avr) genes. While avr genes are naturally present in avirulent strains, cloned avr genes
can convert virulent strains into avirulent strains if the test host contains the
corresponding R gene (Leach and White, 1996). This gain-of-function property has been
used to identify several avr genes (Staskawicz et al., 1984). Analyses of many bacterial
pathovar-plant species combinations has established the idea that avr gene activity can

play a major role in restricting the host range of plant pathogenic bacteria (Dangl et al.,

12

1992; Debener et al., 1991; Innes et al., 1993a ,b; Jenner et al., 1991; Ritter and Dangl,
1994; Vivian et al., 1989; Whalen et al., 1991). It was later found that all avr genes
require a functional hrp cluster for phenotypic expression of race-specific resistance and
that the site of action of avr gene products is inside the plant cell (Gopalan et al., 1996;
Keen et al 1990; Pirhonen et al., 1996; He , 1997; Kjemtrup et al., 2000; Huynh et al.,
1989; Huang et al., 1988; Leister et al., 1996.; Tang et al., 1996; Van der Ackerveken et
al., 1996). It has long been an enigma why bacterial populations should maintain factors
that have a negative effect on pathogen fitness (Dangl, 1994). Certain avr genes, although
characterized by their ability to induce HR, have been shown to play a role in virulence in
the absence of the interacting R gene These include avrBsZ of Xanthomonas campestris
pv. vesicatoria (Kearney and Staskawicz, 1990), the pthA gene of X. citri (Swarup et al.,
1991; Swarup et al., 1992), avrBs6 from X. campestris pv. maculicola (Yang et al.,
1996), and avaa7 from X. oryzae pv. oryzae (Choi, 1993). Among P. syringae ,
aerme from P. syringae pv. maculicola (Dangl, 1994; Ritter and Dang], 1995), and
avrA and aer from P. syringae pv. tomato strain PT23 (Lorang et al., 1994) have been
demonstrated to contribute to pathogen aggressiveness or fitness.

This has led to the following question: how many effectors are present in a single
bacterium? Several studies have aimed at addressing this question using different
approaches. One of the earliest attempts to identify effectors utilized the fact that type III
effectors were secreted in hrp-inducing minimal medium. This approach has been used to
identify many effectors from animal pathogens. However, the only phytobacterial
proteins identified in this manner have been HrpZ, HrpW, and HrpA (Yuan and He,

1996). The main limitation of this method is that only abundantly secreted proteins can

13

be detected. P. syringae and other plant pathogens produce very low quantities of
effectors that are below the level of detection on SDS-PAGE gels by coomassie-staining.

Boch et a1. (2002) used a modified in vivo expression technology (IVET)
approach to identify type III effectors of Pst DC3000. This methodology was based on
the fact that all known effector genes are co-ordinately regulated by the hrp system,
which is induced in planta. This study identified several known and potential virulence
genes, including hrp/hrc, avr and coronatine biosynthesis genes as well as several genes
with unknown function that may encode novel virulence factors.

The recent release of the sequence of the Pst DC3000 genome has facilitated
several independent surveys for type HI effectors. These studies were based on the
presence of the hrp box motif in the promoter, induction of gene expression in hrp-
inducing medium, and secretion and translocation assays (Fouts et al., 2002; Guttman et
al., 2002; Petnicki-chieja et al., 2002; Zwiesler-Vollick et al., 2002). Proteins secreted
by the TTSS were designated Hops for hrp outer proteins. From these studies, it was
concluded that Pst DC3000 has more than 30 putative effector genes. These studies have
also revealed specific biophysical properties of the N—terminal region of type III effectors
that might be a secretion signal that dictates these proteins to travel the TTSS (Guttman et
al., 2002; Petnicki-chieja et al., 2002). This signal may be similar to that found in type
III effectors of bacterial pathogens of animals (Anderson et al., 1997, 1999; Lloyd et al.,

2001).

14

Virulence role of effector proteins

Recent studies show that a single virulence factor often shows virulence and
avirulence functions in different host cultivars. This phenomenon is described most
extensively in the P. syringae pv phaseolicola—bean pathosystem, where two effectors
have been shown to have cultivar-specific avirulence and virulence functions. These are
vierhA and averhC. VierhA is the first virulence factor from P. s. phaseolicola. P.
syringae pv. phaseolicola (Pph) strains harbor a large plasmid which contains several
known avr genes. When cured of this plasmid, strains lose virulence and cause HR in
previously susceptible cultivars of bean. Virulence was restored by complementing these
strains with the region of the cured plasmid encoding vierhA (Jackson et al., 1999).
Thus, VierhA functions as a suppressor of HR. In a subsequent study, another effector
AverhC was found to suppress the HR triggered by AverhF. Both vierhA and
averhC are plasmid-bome and function as avr genes in soybean (Tsiasmis et al., 2000).
Thus, some type III effectors seem to have evolved to mask the presence of endogenous
avr genes that have been previously selected as recognition determinants by the same
plant. Orthologs of vierhA are present in many strains of P. syringae (Jackson et al.,
2002). In Pst DC3000, the ortholog of vierhA is avrPtoB. AvrPtoB has also been shown
to inhibit programmed cell death initiated by Pto and C19 resistance genes (Kim et al.,
2002; Abramovitch et al., 2003). The presence of vierhA-like genes in diverse pathovars
and similarity in their function indicate that it might play a conserved role in suppressing
avr gene-mediated HR.

Another type III effector, HothoD2, suppresses programmed cell death in plants.

The N-terminal domain of HothoD2 shares sequence homology with AverhD, but the

15

C-terminal domain is similar to protein tyrosine phosphatases. Purified HothD2 has
tyrosine phosphatase activity and can suppress HR elicited by an avirulent strain of P.
syringae on Nicotiana benthamaina. The enzymatic activity as well as the HR-
suppressing ability of HothoD2 requires a conserved cysteine residue in the catalytic
site. Using Agrobacterium-mediated gene co-delivery, it was shown that HothoD2 acts
by modulating the MAP kinase pathway in tobacco that involves NtMEKZ. Pst DC3000
mutants lacking HothoD2 were slightly reduced in their ability to multiply in planta and
had enhanced ability to elicit an HR, supporting the role of HothoD2 as a suppressor of
HR (Espinosa et al., 2003; Bretz et al., 2003).

Transgenic expression of effector proteins in susceptible plants has been utilized
to demonstrate the virulence contribution of these proteins. Ectopic expression of aerptZ
in Pst DC3000, results in enhanced growth of Pst DC3000 in Arabidopsis plants lacking
RPSZ, the corresponding R gene (Chen et al., 2003). Interestingly, expression of Aerpt2
in rps2' plants also suppresses the HR elicited by another effector, aerme (Ritter and
Dang], 1996). This interference is apparently mutual, since aerme prevents aerpt2-
elicited host responses (Reuber et al., 1996). Aerpt2 interferes with aerme-mediated
HR by causing the disappearance of RIN4. RIN4 interacts physically with RPMl.
Disappearance of RIN4 prevents RPMl-mediated signal transduction, which normally
leads to the activation of defense responses (Axtell et al., 2003; Mackey et al., 2003;
Mackey et al., 2002).

While type III effectors show no similarity to any proteins of known function,
motif searches reveal the presence of enzymatic motifs in some effectors. AverhB is

similar to a Yersinia type III effector, YopT. Both YopT and AverhB belong to a family

16

of cysteine proteases that are involved in bacterial pathogenesis. YopT cleaves the
posttranslationally modified RhoGTPases leading to the disruption of the actin
cytoskeleton in host cells (Shao and Dixon, 2003; Shao et al., 2003). The enzymatic
activity of AverhB is required for the autoproteolytic activity of the AverhB precursor
in the plant as well as for eliciting the hypersensitive response (Shao et al., 2002). In
Arabidopsis, resistance to P. syringae strains containing AverhB requires RPS5, its
cognate resistance gene and PBSl, a protein kinase. AverhB proteolytically cleaves
PBS] and this activity is required for RPSS-mediated resistance (Shao et al., 2003).

The biochemical function of another Avr protein had been defined previously.
Aer directs the synthesis of low molecular weight elicitors called syringolides, which
elicit an HR on soybean (Midland et al., 1993). Different alleles of aer are involved in
the synthesis of different host-specific syringolides (Yucel et al., 1994a,b). Homologs of
aer are widespread among Pseudomonads (Keith et al., 1997). Strains of Pst PT23 that
lack aer, however, are not impaired in virulence on tomato (Lorang et al., 1994; Murillo
et al., 1994). Ji et al. (1997, 1998), identified a syringolide—binding protein, Rpg4, in
soybean (Ji et al., 1997, 1998). Although aer is co-regulated with the hrp system, it is
still not known whether Aer is translocated into the host cells by the TTSS, or what its
biological function is in P. s. pv tomato (Shen and Keen, 1993).

Testing for the ability of an effector protein to suppress the hypersensitive
response in resistant host plants has provided information about the function of several P.
syringae type III effectors. Previous microscopic studies suggested that in susceptible
plants, the TTSS of plant-pathogenic bacteria also transports suppressors of an HR-

independent cell wall-based plant defense that is activated by the TTSS-defective hrp

17

mutant (Bestwick et.al, 1995; Brown and Bonas, 1995; Brown et al., 1998). However, the
identity of such suppressors remained elusive for many years. It was recently found that
the P. syringae TTSS down-regulates the expression of a set of Arabidopsis genes
encoding putatively secreted cell wall and defense proteins in a salicylic acid-
independent manner. Transgenic expression of AvrPto, a type III effector of Pst DC3000,
represses a similar set of host genes, compromises defense-related callose deposition in
the host cell wall, and permits substantial multiplication of a hrp mutant. AvrPto seems to
be one of the long-postulated suppressors of a salicylic acid-independent, cell wall-based
defense (Hauck etal., 2003).

Another type III effector, HothoAl, contributes to the efficient formation of Pst
DC3000 bacterial colonies in planta. The gene encoding HothoAl is located in the
Conserved Effector Locus and has a functionally redundant partner, hothoAZ, which is
located elsewhere in the genome. Confocal laser-scanning microsocopy of GFP-labelled
bacteria in Arabidopsis leaves show a higher frequency of undeveloped individual
colonies in the hothoAI mutant and an even higher frequency in the

hothoA I/hothoAZ double mutant (Badel et al., 2002).

Type III Chaperones

Studies conducted mainly with mammalian pathogens show that efficient type III
secretion requires chaperones. Type III chaperones are usually co-regulated with the hrp
system and are typically small, acidic proteins (pI<5.5) that often contain an arnphipathic
or-helix near the C-terminus. The first type III chaperone, Sch, discovered in Yersinia, is

required for the secretion of its cognate effector, YopE (Wattiau and Comelis, 1993).

18

Since then, more than 30 chaperones have been found in various plant and animal
pathogenic bacteria and more are predicted from genome-wide analyses (Buell et al.,
2003; van Dijk et al. 2002). In plant pathogens, type III chaperones have been designated
Shc for specific hrp chaperone. ShcA was the first chaperone in P. syringae pv. syringae
that was required for the secretion of HopPsyA and for the ability of HopPsyA to cause
an HR in the non-host tobacco (Van Dijk et al., 2002). Averthto also requires Sth for
efficient secretion into culture media (Shan et al., APS, 2003). Chaperones are thought to
maintain their effectors in a secretion-competent state within the bacterial cytosol and
even prioritize their secretion (Boyd et al., 2000). Chaperones have also been
demonstrated to function to maintain the stability of their cognate effectors within the
bacterium (Cheng and Schneewind, 1999; Frithz-Lindsten et al., 1995, Menard et al.,
1994; Neyt and Comelis, 1999). In the plant pathogen Erwinia amylovora, DspF is

required for the stability and secretion of DspE (Gaudriault et al., 1997; 2002).

Phytotoxins

Toxins have long been known to be virulence factors in P. syringae. For example,
P. s. pv. tomato and P. s. pv. maculicola produce the phytotoxin coronatine (COR). It is
also produced by P. syringae pv. morsprunorum, atropurpurea, and glycinea. Coronatine
is the only known phytotoxin produced by Pst DC3000 (Bender et al., 1999). Coronatine
is a polyketide toxin consisting of coronofacic acid (cfa) and coronarnic acid (cma) joined
by an amide linkage (Bender et al., 1999). The enzymes are required for the biosynthesis
of CFA and CMA are encoded by the cfa and cma genes, respectively, and are

collectively referred to as COR genes. Unlike many P. syringae pathovars where the

19

COR genes are encoded on plasmids, in Pst DC3000 the COR genes are chromosomally
encoded. In Pst DC3000, the corRS operon, which encodes a two-component regulatory
system, regulates the expression of the cfa and cma genes. Two of the cfa genes, cfaI and
cfa6, have been shown to be expressed in a hrp-dependent manner, which implies that the
synthesis of coronatine might be co-ordinately regulated with the hrp system (F outs et al.,
2002). Recently, RpoN, which encodes a sigma 54 factor, was shown to be required for
coronatine synthesis in P. s. pv. glycinea (Alarcon-Chaidez et al., 2003). The primary
symptom associated with coronatine production is chlorosis. Coronatine causes the
development of chlorotic symptoms when applied to tomato leaves (Gnanamanickam et
al., 1982). However, the precise virulence role of coronatine remains unclear. Mutation of
COR genes renders Pst DC3000 less virulent on tomato. There is a reduction in bacterial
multiplication and leaves show smaller chlorotic lesions (Bender et al., 1987). A different
COR mutant of Pst DC3000 (DC3661) was found to grow to lower levels than wildtype
bacteria following surface inoculation of Arabidopsis, but no difference in multiplication
was observed when bacteria were infiltrated into leaf intercellular spaces directly (Mittal
and Davis, 1995)

Other phytotoxins produced by P. syringae pathovars include tabtoxin,
phaseolotoxin, syringomycin, and syringopeptin. Tabtoxin is produced by P. syringae pvs.
tabaci, coronofaciens and garcae. It is a monocyclic B-lactam that elicits chlorosis after
hydrolytic release of tabtoxinine-B-lactam (Levi and Durban,1986). Tabtoxinine-B-
lactam inhibits the plant enzyme glutamine synthetase, causing accumulation of ammonia,

which is thought to cause chlorosis (Thomas et al., 1983). Mutant strains of pathogens

20

that lack tabtoxin remain pathogenic but do not cause chlorosis (Kinscherf et al., 1991;
Willis et al., 1991).

Phaseolotoxin is primarily produced by P. s. phaseolicola. A phaseolotoxin-like
substance has also been characterized from some P. s. tomato strains (Bagdache et al.,
1990). Mutants of P. s. phaseolicola that do not produce phaseolotoxin are reduced in
virulence (Patil, 1974). This toxin consists of ornithine, arginine, homoarginine, and N’-
sulfodiaminophosphinyl (Moore et al., 1984). It inhibits the plant ornithine
carbamoyltransferase resulting in lower levels of arginine, which has been implicated in
the development of chlorosis and inhibition of plant growth (Patil, 1974; Mitchell and
Bielski, 1977).

Syringomycin and syringopeptin are synthesized in P. s. syringae. Mutants of P. s.
syringae that are impaired in the synthesis of both the toxins are reduced in virulence
(Scholz-Schroeder et al., 2001). Syringomycin and syringopeptin are lipopeptide toxins
that trigger necrosis when applied exogenously on plants. They cause pore formation in
plant plasma membranes (Hutchinson and Gross, 1997). Pore formation is thought to

cause electrolyte leakage, cell death and tissue necrosis.

Additional virulence factors

In addition to the TTSS and toxin biosynthesis, several other virulence factors
have been implicated in Pseudomonad pathogenesis.

Several factors have been identified that are linked to epiphytic survival of P.
syringae. These include ice nucleation, protease synthesis, and utilization of citric and

malic acid. Epiphytic growth is a key part of the P. syringae life cycle in nature and could

21

be influenced by a range of traits including chemotaxis, attachment, microcolony
formation, nutrient acquisition, antibiosis and resistance to UV stress (Beattie and
Lindow, 1995; Beattie and Lindow, 1999; Hirano and Upper, 1990). Type IV pili of P. s.
pv. tomato have been implicated in adhesion and UV tolerance (Roine et al., 1998b).
Recent studies from our laboratory have identified a uer mutant of Pst DC3000 that
shows increased sensitivity to UV light. The 11er gene encodes a type II helicase that is
involved in DNA repair and replication. Pst DC3000 strains lacking uer show a 1000-
fold reduction in bacterial multiplication in planta. The rulA mutants also exhibit a
reduced tolerance to UV light and are not as competitive as the wildtype bacteria in the
phyllosphere (Sundin et al., 2000). As the bacteria face an onslaught of UV light during
the epiphytic phase of grth (Sundin and Jacobs, 1999), it is possible that these genes
are required for epiphytic fitness.

Extracellular polysaccharides (BPS) also play an important role in the virulence of
many bacterial phytopathogens including Ralstonia solonacearum, Erwinia amylovora,
Erwinia stewartii, Xanthomonas campestris and Xanthomonas oryzae (Buddenhagen and
Kelrnan, 1964; Denny and Baek, 1991; Kao et al., 1992; Poetter and Coplin, 1991; Geier
and Geider, 1993; Denny, 1995; Rajeshwari and Sonti, 2000). In vascular pathogens,
EPSs cause blockage of the host xylem resulting in wilt. EPSs in R. solanacearum have
been implicated in assisting attachment to the host (Sequira, 1985). Secreted EPS is also
thought to form a layer around the bacteria that provides protection against desiccation,
antimicrobial compounds from the plant, and impedes recognition by preventing contact
with plant cells (Denny, 1995; Braun, 1990). Alginate is an EPS that is an important

virulence factor in P. s. pv. syringae. Alginate has also been linked to epiphytic fitness

22

and production of water-soaking lesions (Jing et al., 1999). P. s. tomato produces alginate
(Gross and Rudolph, 1987), but its contribution to virulence has not been investigated.

Cell wall degrading enzymes (CWDE) are also important virulence factors,
particularly in members of the Erwinia genera of plant bacterial pathogens, which are
collectively referred to as soft-rot bacteria. These bacteria macerate plant tissues via
destruction of pectins and cellulose, which are important components of the plant cell
wall. The enzymes responsible for maceration are pectate lyases, pectin methylesterases
and polygalacturonases, collectively called CWDEs (Barras et al., 1994). In Erwinia
chrysanthemi, there are at least eight pectate lyases. They are encoded by the pel genes,
which contribute to the soft rot ability of E. chrysanthemi on potato tubers (Ried and
Collmer, 1987; Jafra et al., 1999). These enzymes are secreted by the type H secretion
system (Bouley et al., 2001; He et al., 1991). Pectic enzymes and cellulases influence the
development of final symptoms in P. s. pv. lachrymans (Bauer and Colhner, 1997). The
recent completion of the sequence of the Pst DC3000 genome has revealed that Pst
DC3000 contains a pectin lyase, a polygalacturonase and three enzymes with predicted
cellulolytic activity (Buell et al., 2003). At least one pectate lyase gene may be co-
regulated with the hrp system (Fouts et al., 2002).

Pst DC3000 also contains genes required for the synthesis of pyoverdin, a
siderophore that is found in fluorescent Pseudomonads (Buell et al., 2003; Bultreys and
Gheysen, 2000). Siderophores are low molecular weight, high affinity iron chelators that
function as important virulence factors in many bacterial pathogens because of their role
in sustaining growth in iron-limiting host environments. A cluster of genes that are

homologous to those required for biosynthesis of the siderophore pyochelinin in P.

23

aeroginosa was found in Pst DC3000 (Reimmann et al., 2001). Three genes predicted to
encode FHA-like proteins were identified in Pst DC3000 (Buell et al., 2003).
Filamentous haemagglutinin (FHA) has been shown to be an adhesin and a virulence

factor in animal pathogenic bacteria and the plant pathogen Erwinia (Rojas et al., 2002).

Project Summary

Tremendous progress has been made in the last two decades in understanding the
virulence mechanisms utilized by bacterial phytopathogens. This field has witnessed
some exciting discoveries in microbiology and molecular plant pathology. These include
the discovery of the hrp system and the Hrp pilus, demonstration of the action of Avr
proteins within plant cells, finding a large number of effectors in a single bacterial strain
and preliminary evidence for effector function in suppressing host defense responses, and
completion of the Pst DC3000 genome sequence.

In contrast to their mammalian counterparts, plant pathogens have an unusually
large number of effectors. Efforts to understand the virulence role of these effectors face
several challenges, the most obvious being the functional redundancy among type III
effectors. This results in weak loss-of-virulence phenotypes of strains inactivated for
single effectors. This field is also in need of a better understanding of pathogenesis at the
cellular level. A plethora of cellular markers, such as actin, in mammalian cells have
resulted in an avalanche of knowledge about the role of effectors in animal bacterial
pathogens. Identification and development of similar markers in plants would assist in

understanding the role of effector proteins. In planta visualization of infection, functional

24

genomics, and proteomics should also help to elucidate mechanisms by which effectors
contribute to plant pathogenesis.

At the time this project was initiated, only two type III effectors were known to be
present in Pst DC3000. My aim was to identify new type III effectors and to understand
their contribution to virulence of Pst DC3000. I chose to use the Pst DC3000-
Arabidopsis thaliana pathosystem, which is an important model system in molecular
plant pathology for several reasons. It is currently the only pathosystem in which the
genomes of both organisms are completely sequenced. Pst DC3000 is genetically
tractable, and has an extensively studied TTSS. Additionally, it is also a pathogen of the
economically important plant, tomato. This aspect will help in extending the knowledge
acquired through the Arabidopsis-Pst DC3000 pathosystem to design better disease
control techniques for economically important crops. Genomic resources are also
available for the host Arabidopsis, including a large number of mutants, knockouts, and
transgenics that can facilitate the dissection of host pathways that are targeted by the
pathogen during infection.

Chapter 2 describes my research on the Conserved Effector Locus of Pst DC3000
and its contribution to virulence of Pst DC3000 on Arabidopsis. A mutant carrying a
deletion of six genes in the CEL region shows a drastic reduction in grth and symptom
development on Arabidopsis plants. Two ORFs within the deleted region, ORF3 and
ORF4, are sufficient to complement the CEL deletion mutant. ORF3 is a translocated
type III effector which requires ORF4 for secretion and translocation. ORF4, which is a
chaperone of ORF3, is neither secreted nor translocated, but physically interacts with

ORF3 in a yeast two-hybrid system. ORF3 was designated HothoM and ORF4, Sth.

25

Expression of hothoM in Arabidopsis plants causes the development of water soaking,
chlorosis, and necrosis that are very similar to those induced by Pst DC3000 during
infection. Expression of HothoM in planta also complements the growth and symptom
development defects of the ACEL mutant, further confirming that HothoM functions
inside the plant cell. Unlike Pst DC3000, the ACEL mutant is impaired in suppressing
host cell wall-based papilla defenses. HothoM and Sth restores the ability of the
ACEL mutant to suppress papilla formation. Examination of the response of salicylic acid
(SA)-deficient NahG plants and eds5 plants (Delaney et al., 1994; Gaffney et al., 1993;
Nawrath and Metraux, 1999) to the ACEL mutant and a non-pathogenic hrp mutant
showed that activation of papilla formation was partly dependent on SA. Thus, HothoM
is involved in the suppression of SA-dependent activation of plant cell wall-based host
immunity.

Chapter 3 reports my research on the identification of several type 1H effector
genes in Pst DC3000. A set of ten known avr genes from different pathovars of P.
syringae were used to identify homolo gs in Pst DC3000 based on Southern hybridization.
Three orthologues were identified. An Aerpt2 based translocation system was used to
show that all three identified effectors were secreted by the TTSS. Strains lacking
averhEpm or averinm were still able to infect Arabidopsis thaliana and tomato,
whereas an avrPtoB mutant was reduced in virulence. However, I was unable to
complement the avrPtoB mutant. To further investigate the role that AVTPphEpm may
play in virulence, I expressed the averhEpm gene in A. thaliana under the control of a
dexamethasone-(DEX) inducible promoter. Transgenic plants which express AverhEp,o

develop water soaking, chlorosis, and necrosis when maintained under high humidity.

26

These symptoms mirror those seen with Pst DC3000 infection. These plants promoted
the growth of the hrpH mutant. In addition, microarray experiments showed that the
expression profile of the TTSS-dependent host gene cluster of Arabidopsis was 90%
similar between plants expressing AverhEp,o and plants infected with Pst DC3000.
These experiments provided evidence for a virulence role of AverhEpto in disease

promotion.

27

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Chapter 2
HothoM is a type III virulence protein of Pseudomonas syringae pv. tomato
DC3000 that suppresses salicylic acid-dependent activation of cell wall-based

extracellular host defenses in Arabidopsis thaliana

I would like to acknowledge Kinya Nomura for contribution of figures 2-5, 2-6 and 2-7.

44

Abstract

Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) is a pathogen of tomato
and Arabidopsis that injects virulence effector proteins into host cells via a type III
secretion system (TTSS). TTSS-deficient mutants have a Hrp' phenotype, that is, they
cannot elicit the hypersensitive response (HR) in nonhost plants or pathogenesis in host
plants. On the other hand, mutations in individual effector genes typically have weak
virulence phenotypes. A very singular case is that of the CEL deletion mutant (ACEL
mutant) of Pst DC3000: deletion of six ORFs in the Pst DC3000 conserved effector locus
(CEL) reduces parasitic growth and abolishes disease symptoms without affecting the
function of the TTSS. The inability of the ACEL mutant to multiply and cause disease
symptoms in Arabidopsis was restored by a clone expressing two of the six deleted
ORFs: CEL ORF3 (HothoM) and ORF4 (Sth). Sth was found to be required for
HothoM to restore virulence to the ACEL mutant. HothoM was secreted in culture and
translocated into Arabidopsis cells by the TTSS during infection. Secretion and
translocation of HothoM were dependent on Sth, which itself, was neither secreted
nor translocated but, like typical TTSS chaperones, could be shown to interact with
HothoM, its cognate effector, in yeast two-hybrid experiments. Expression of HothoM
in Arabidopsis promoted the growth of the ACEL mutant but not of the TTSS-deficient
hrpH mutant. The inability of the ACEL mutant to cause disease was linked to activation
of cell wall-based host defense in wildtype Arabidopsis plants. HothoM and Sth

restored the ability of the ACEL mutant to suppress extracellular cell wall-based host

defenses. The ACEL mutant triggered significantly fewer number of callose deposits and

45

grew better in salicylic acid (SA)-impaired NahG and eds5 plants, than in wildtype plants,

suggesting that HothoM is a suppressor of SA-dependent cell wall-based defenses.

Introduction

Bacterial pathogens cause numerous diseases in animals, plants and humans.
These include members from diverse genera such as Yersinia, Salmonella, Shigella,
Escherichia and Pseudomonas, Xanthomonas, and Ralstonia. All these pathogens
harbour a unique secretion system classified as the type III (Galan and Collmer, 1999).
This secretion apparatus is unique in its ability to deliver proteins from the bacterial
cytoplasm directly into host cells. The TTSS is integral to pathogenicity, which is evident
from an often complete loss of pathogenicity when inactivated. In plant pathogens, the
type III system is encoded by hrp and hrc genes. The hrp genes govern the ability of
bacteria to cause disease in susceptible plants and elicit the hypersensitive response (HR)
in non-host plants (Alfano and Colhner, 1997; Lindgren, 1997; He, 1998). During
pathogenesis, the TTSS secretes numerous virulence effector proteins into the host that
synergistically function to modulate host responses to favour pathogenesis. Recent
completion of the P. syringae pv. tomato DC3000 genome sequence suggests the
presence of at least 31 different type III effectors in this pathogen (Buell et al., 2003).
Historically, most of the type III effectors were identified by their ability to cause an HR
in the presence of their cognate resistance gene in the resistant host genotype and were
named avirulence (avr) genes (Leach and White, 1996; Collmer et al., 2001). Many of the

putative effectors identified from bioinforrnatic analysis of the Pst DC3000 genome have

46

been demonstrated to be secreted by the TTSS and have been renamed Hop for hrp guter
protein.

Another category of proteins that have been shown to be required for efficient
secretion of effectors are the type III chaperones. Chaperones have been found in both
plant and animal pathogens and are typically small, acidic proteins (pI<5.5) with an
amphipathic ct-helix near the C-terminus (Parsot et al., 2003; Feldman and Comelis,
2003; Gaudriault et al., 2002; Van Dijk et al., 2002). In most cases, they bind to the N-
terrninus of their cognate effectors and may stabilize their substrates and maintain them
in a secretion-competent conformation (Stebbins et al., 2001). Chaperones have also been
implicated to prioritize the secretion of their cognate effectors in the presence of other
effectors in the bacterial cytosol (Boyd et al., 2000). Prior to my study, HopPsyA was the
only P. syringae type III effector that had been demonstrated to have a chaperone, ShcA.
ShcA was required for the secretion of HopPsyA (van Dijk et al., 2002). ShcA binds to a
site located within the N-terminal 166 amino acids of HopPsyA (van Dijk et al., 2002).

The most puzzling aspect of type III effectors of plant pathogens is their virulence
function in the host. In comparison, a lot is known about the biochemical activities of
type III effectors injected by mammalian pathogens. Although there has been an ongoing
effort to achieve a better understanding of the functions of type III effectors produced by
plant pathogenic bacteria, these efforts have been thwarted by the typically weak
phenotypes resulting fiom inactivation of single and sometimes multiple effectors,
compared to the strong loss-in-virulence phenotypes observed when hrp genes are
inactivated. This has been attributed to redundancy among the large number of type III

effectors that are present in a single pathogen. Despite these hindrances the functions of

47

several effectors have been identified. Five have been shown to function as suppressors
of cell death (Jackson et al., 1999; Tsiasmis et al., 2000; Abramovitch et al., 2003;
Espinosa et al., 2003; Bretz et al., 2003; Chen et al., 2003), and one was shown to
suppress cell wall-based defense (Hauck et al., 2003).

In P. syringae, genes encoding the TTSS are clustered on a pathogenicity island
(Pai), which also contains two loci that encode putative effector proteins (Alfano et al.,
2000). An exchangeable effector locus (EEL), whose number of ORFs and their
nucleotide sequences vary between closely related strains of the same pathovar, is located
downstream of her. A conserved effector locus (CEL), encoding at least seven ORFs
that are conserved between the divergent strains Pss B728a and Pst DC3000, is located
upstream of hrpR. Deletion of the Pst DC3000 EEL causes a slight reduction of bacterial
growth in tomato, whereas deletion of six ORFs of the CEL drastically reduces bacterial
multiplication in tomato (Alfano et al., 2000). The deleted region contains aer, ORF2,
ORF3, ORF4, hrpW and hothoAI. However, single mutations in aer (Lorang and
Keen, 1995), hrpW (Charkowski et al., 1998), and hothoAI (Badel et al., 2002) do not
cause a reduction in virulence. This is consistent with the idea of functional subtlety
and/or redundancy among effectors in Pst DC3000.

In this study, we found that 1) the ACEL mutant had a drastically reduced
virulence phenotype in Arabidopsis, 2) CEL ORF3 and ORF4 completely restored the
virulence of the ACEL mutant in Arabidopsis, And 3) the CEL ORF3 protein was
secreted in culture and translocated into the plant cell by the TTSS and that translocation
was dependent on a chaperone encoded by CEL ORF4. Accordingly, we designated CEL

ORF3 and CEL ORF4, HothoM and Sth respectively. Transgenic expression of

48

HothoM in planta caused symptoms that are similar to those caused by Pst DC3000 and
allowed the ACEL mutant to grow and cause disease symptoms. Finally, we show that

HothoM is a suppressor of salicylic acid (SA)-dependent host cell wall-based immunity.

Materials and Methods

Bacterial strains and media

E. coli was grown in low salt (5 g/L) Luria Bertani (LB) medium (Sambrook et al.
1989) at 37°C. P. syringae were grown in low salt LB or hrp-inducing minimal medium
(Huynh et al., 1989) at 30°C or 20°C. Antibiotics were used at the following
concentrations unless otherwise specified — rifampicin 100mg/L, kanamycin 50 mg/L,

ampicillin 200mg/L, tetracycline 10mg/L, spectinomycin SOmg/L.

Recombinant DNA techniques

All DNA manipulations including polymerase chain reaction (PCR) were
performed using standard protocols (Sambrook et al., 1989). Oligonucleotide primers for
sequencing or PCR were synthesized at Integrated DNA Technology (Coralville, IA).
PCR was performed using HIFI polymerase (Invitrogen, Carlsbad, CA). DNA
sequencing was done at the Michigan State University Genomic Technology Support
Facility with Automated DNA sequencers model 373A (Applied Biosystems, Foster City,
CA). DNA sequences were analyzed with Assemblylign®, MacVector® and the Sequence

Manipulation Suite (http://www.bioinforrnatics.org/sms/index.htrnl). Database searches

49

were performed using gapped BLASTN, BLASTP, and BLASTX (Altschul et al., 1997)

(http://www.ncbi.n1m.nih. gnv/BLASTD.

 

Construction of complementation plasmids

Various fragments within the CEL were either subcloned from a cosmid clone,
pCEL (for p0RF2-5, p0RF234, p(hrpHRPW-ORF5) and p(0RFf6—10) or amplified by
PCR (for p0RF2, p0RF23, p0RF24, p0RF43, p0RF3, and pORF4) and cloned into
appropriate vectors. The following primers were used to amplify ORF 43 (for construction
of p0RF43): sense primer, 5’-GTGAATTCGCTAAGTGGGCAATTGGAC-3’ and
antisense primer, 5’-CAGGATCCTTTAAGGTTAAAACAGCAT-3’; ORF4 (for
construction of pORF 4): sense primer, 5’-GTGAATTCGCTAAGTGGGCAATTGGAC-
3’ and antisense primer, 5’-CGGGATCCGATCATTGGAATCTCCCAG-3’; and ORF3
(for construction of pORF 3 ): sense primer, 5 ’-
CAGGATCCAAACGCGAGAGCCTTTCGG-3 ’ and antisense primer, 5’-
CTTCTAGATTAAAACAGCATGAAGCATGC-3’. pORF 3 also contains the ORF 4
promoter upstream of ORF 3. The ORF 4 promoter for the above constructs was amplified
using sense primer 5’-GTGAATTCGCTAAGTGGGCAATTGGAC-3’ and antisense
primer 5’-CAGGATCCGTTGATAAGGGTGTGGTAC-3’. The following primers were
used to amplify ORFZ (for construction of pORFZ): sense primer, 5’-
TATCTAGACGCTTTGAATAACATCCGT-3’, and antisense primer, 5’-
GGGGATCCAACTGAAGAGCTAATAACG-3’; and ORF 23 (for construction of
p0RF23); sense primer, 5’-ACTCTAGAGCCTTTCGGCTCCTGGGAG-3’ and

antisense primer, 5’-GGGGATCCAACTGAAGAGCTAATAACG-3’. For constructs

50

p0RF2 and p0RF23, the ORF4 promoter was amplified using sense primer 5’-
GTGAATTCGCTAAGTGGGCAATTGGAC-3’ and antisense primer 5’-
GTTCTAGACATTGTTGGTCATTTCAAG-3’. For construction of pORF 24, ORF 2 was
amplified using sense primer, 5’-TATCTAGACGCTTTGAATAACATCCGT-3’ and
antisense primer, 5’-GGGGATCCAACTGAAGAGCTAATAACG-3’and ORF4 was
amplified with its own promoter using sense primer 5’- GTGAATTCGCTAAGTG
GGCAATTGGAC-3’ and antisense primer 5’-ATTCTAGAACTGATCATTGGAAT

CTCC-3’. Plasmids were introduced into bacteria by electroporation.

Plant growth and bacteria enumeration

Wildtype Arabidopsis accession Columbia glabrous] (Col glI) and transgenic
plants were grown in soil in grth chambers with a day/night cycle of 12h/12h, a light
intensity of 100 pH, and a constant temperature of 20 °C. Four- to five-week-old plants
were used for experiments. Bacteria were grown in low-salt LB to the mid-logarithmic
phase at 30°C. Bacterial cultures were centrifiiged to recover bacterial cells, which were
resuspended in sterile water to a final OD600 of 0.002 (equivalent to 1x106 CFU/ml).
Fully expanded leaves were either vacuum-infiltrated or syringe-infiltrated with bacterial
suspensions, and bacteria were enumerated as described by Katagiri et al. (2002). The
mean values of the bacterial populations were plotted with the standard deviation
displayed as error. Plants analyzed in Figure 2-9 were sprayed daily with a 0.003 uM
dexamethasone (DEX) solution to induce the hothoM transgene. Bacterial suspensions

were infiltrated into leaves one day after the first DEX treatment.

51

Secretion assays

Bacteria were grown in low-salt LB broth until OD600 = 0.6. Bacteria were
collected by centrifugation and resuspended in hrp-inducing minimal medium or hrp-
repressing LB and incubated with shaking at 20°C for 12 h. Cultures were separated into
cell and supernatant fractions by centrifirgation at 15,000g. The cell and supernatant
fractions were concentrated 5 and 50 times, respectively. Proteins were separated on
SDS-PAGE gels and transferred to Immobilin-P membrane (Millipore Corp., Bedford,
MA). Irnmunoblot analyses were performed using rabbit and chicken antibodies raised
against E. coli-expressed HothoM and Sth, respectively, at Cocalico Biologicals, Inc.,

Reamstown, PA.

Type III translocation analysis

The truncated aerpt280-255 gene, which encodes type 1H secretion/translocation-
incompetent, but biologically active, Aerpt2 (Mudgett et al., 2000) was cloned into the
XbaI-HindIII sites of pUCP19 (Schweizer, 1991). Full-length ORF3 or ORF 4 genes were
amplified by PCR and fused to the 5’ end of aerpt23o-255. The recombinant plasmids
were introduced into ACEL mutant by electroporation. The transforrnants were grown in
low-salt LB to OD600 = 0.6. Bacteria were collected by centrifugation and resuspended in
sterile water to OD600 = 0.2. The bacterial suspensions were infiltrated into leaves of 6-
week-old RPS2+ Arabidopsis ecotype Col-O plants or rpsZ mutant plants (Kunkel et al.,
1993). HR was monitored over a 48-h period at room temperature.

The following primers were used in the construction of aerpt230-255 gene fusions.

ORF4::aerpt230_255: sense primer, 5’-GTGAATTCGCTAAGTGGGCAATTGGAC-3’

52

antisense primer, 5 ’-ACTCTAGATTGGAATCTCCCAGGAG—3’;
0RF4+0RF3::aerpt280-255: sense primer, 5’-
GTGAATTCGCTAAGTGGGCAATTGGAC-3 ’ and antisense primer, 5 ’ -
GTTCTAGAAAGCGTCTCGGTACGGTCC-3’, using genomic DNA as a template;
0RF3::aerpt230-255: sense primer, 5’-GTGAATTCGCTAAGTGGGCAATTGGAC-3’
and antisense primer, 5’-GTTCTAGAAAGCGTCTCGGTACGGTCC-3’, using p0RF3

as a template.

Yeast two-hybrid analysis

The LexA-based yeast two-hybrid system (Clontech Laboratories Inc.) was used.
ORF 4 and ORF 3 fragments were amplified by PCR and cloned into pB42AD or pGILDA.
The following primers were used to amplify full-length ORF4: sense primer, 5’-
CGAATTCATGACCAACAATGACCAGTAC-3’ and antisense primer, 5’-
GATCCTCGAGCTGATCATTGGAATCTCC-3’; full-length ORF3: sense primer, 5’-
GGAATTCATGATCAGTTCGCGGATCGGC-3’ and antisense primer, 5’-
CCTGCTCGAGTGACGGATGTTATTCAAAG-3’; sequence corresponding to the first
100 amino acids of ORF3: sense primer, 5’-
GGAATTCATGATCAGTTCGCGGATCGGC-3’ and antisense primer, 5’-
CCTGCTCGAGACTAACCGATCAACAACGC-3’; and sequence corresponding to the
first 200 amino acids of ORF3: sense primer, 5’-
GGAATTCATGATCAGTTCGCGGATCGGC-3’ and antisense primer, 5’-

CTTGCTCGAGCGGCCTATTCGCCAAGGGC-3’. The constructs were transformed

53

into the EGY48 yeast strain carrying the lacZ reporter plasmid. Activation of the LacZ

reporter was determined colorimetrically using X-gal as the substrate.

Generation of transgenic plants

Pst DC3000 genomic DNA was extracted as described by Chen and Kuo (1993).
PCR was used to amplify hothoM using HIFI Taq polymerase and the following
primers :
sense primer 5’GGCTCGAGACCATGGGGCATCATCATCATCATCATATCAGTTC
GCGGATCGGC-3’
antisense primer 5 ’-GCACTAGTTCATAGTCCTTTAAGGTTAAAACAG-3 ’.
The hothoM gene was cloned into the pTA7002 vector (Aoyama and Chua, 1997;
McNellis et al., 1998). pTA7002 allows for inducible expression of transgenes after
application of the animal glucocorticoid hormone, DEX. Electroporation was used to
transform Agrobacterium tumefaciens strain GV3850 with the recombinant plasmid
(Keen et al., 1990). Four pots of Arabidopsis thaliana Col glI plants were transformed
with A. tumefaciens carrying pTA7002-hothoM using the floral dip method (Clough and
Bent, 1998). Seeds collected from each pot were kept separate to ensure that
independently transformed lines could be isolated. T1 seeds were vapor-sterilized in a
dessicator for 4 hours with 80ml of bleach mixed with 3ml of concentrated HCl. Seeds
were placed on Murashige-Skoog (MS) (Gibco BRL, #11117-074) plates supplemented
with 1x vitamins (SIGMA, #M7150) and 40 units/ml hygromycin B (hyg) (Calbiochem
Cat # 400051), kept at 4°C for three days, and then moved to growth chambers (see

previous section). Transforrnants were selected on the basis of hygromycin resistance.

54

Dexamethasone-induction of transgene expression

Dexamethasone (Sigma Aldrich Cat # M6) (DEX) was dissolved in 100%
ethanol to the concentration of 30mM. This stock solution was diluted in water to
required concentrations (see Results section). Plants were sprayed with the DEX solution

to induce the transgene.

Generation of antibody against HothoM and Sth
The hothoM and sth genes were amplified by PCR from Pst DC3000 genomic

DNA using the following primers :
hothoM : sense primer 5’-GGAGATTCATATGATCAGTTCGCGGATC-3’

antisense primer 5 ’-GGAATTCGGATGTTATTCAAAGCGTCTC-3 ’
sth : sense primer 5’-AGGCCTTCATATGACCAACAATGACCAG-3’

antisense primer 5 ’-CGAATTCATCATTGGAATCTCCCAGGAG-3 ’
The genes were individually cloned into the pET28(a) vector (Invitrogen, Carlsbad, CA)
and transformed into E. coli BL21(DE3) cells by electroporation (Sambrook et al., 1989).
Protein was induced by addition of lmM IPTG to a mid-log culture and incubated for 4
hours at 37°C. HothoM and Sth protein was extracted from E. coli cells using
standard protocol (Qiagen, Valencia, CA) and purified using the Ni-NTA column.
Purified protein was analyzed by SDS-PAGE and was used to raise antibodies in rabbit at
Cocalico Biologicals, Inc., Reamstown, PA. Pre and post immune sera was obtained and

checked for the ability to recognize the antigen using immunoblot analysis (see below).

55

Irnmunoblotting

Arabidopsis Col g1] and hothoM plants were sprayed with an appropriate
concentration (see results) of DEX and maintained under high humidity for 24 hours.
Two cm2 tissue was collected using a #5 cork borer (Boekel, #16OIBD 1-10),
homogenized in 100p] 2X treatment buffer (0.125M Tris-HCI pH 6.8, 4% SDS, 20%
glycerol, 10% B-mercaptoethanol) and denatured at 100°C for 10 min. An equal volume
of each sample was separated on a 10% SDS-polyacrylarnide gel (Sambrook et al., 1989)
and proteins were transferred onto Irnmobilin P membrane (Millipore, #IPVH00010)
using a semi-dry apparatus (SEMI PHOR, Hoefer Scientific Instruments).
Irnmunoblotting was carried out using HothoM antiserum and anti-rabbit alkaline
phosphatase conjugate. HothoM protein bands were visualized by a standard colour

reaction using SIGMA FAST (B5655).

Northern blot analysis

Arabidopsis Col g1] and transgenic plants were sprayed with 0.003 uM DEX and
plants were kept under humidity domes. Tissue was harvested 24 hours later and snap-
frozen in liquid nitrogen. Total RNA was isolated using the Promega RNAgents kit (Cat
# 25110). Ten ug RNA was denatured with an equal volume of loading buffer (500 pl
forrnarnide, 170 pl formaldehyde, 100 pl 10X MOPS buffer, and 10 ul of 1 mg/ml
ethidium bromide) for 15 minutes at 65°C, separated on a 1.2% formaldehyde agarose gel
and transferred to nylon membrane (HybondN; Amersham Pharrnacia Biotech
#RPN303B) (Sambrook et al., 1989). Approximately 100 ng of probe DNA was labeled

with 32P and purified using BIORAD columns (Cat #732-6223) according to the

56

manufacturer’s instructions. Membranes were hybridized overnight at 65°C in
PerfectHyb Plus (SIGMA, #H7033). Membranes were washed to a stringency of 0.1X
SSC (20 mins; 65°C) and exposed to film (Kodak Scientific Imaging Film X-OMAT AR,

#1651454)

Callose assay

Arabidopsis Col g1] leaves were vacuum-infiltrated with a bacterial
suspension of OD600 = 0.2 (1x108 cfu/ml) as described in Katagiri et al. (2002). Leaves
were harvested 7 hours after infiltration, cleared with alcoholic lactophenol (1:1:1:l:2
phenolzglycerolzlactic acidzwaterzethanol), and stained with aniline blue (0.01% aniline
blue, SIGMA #M6900, in 150mM KzHPO4 pH 9.5) for callose as described by Adam and
Somerville (1996). Leaves were examined with a Zeiss Axiophot D-7082
Photomicroscope with an A3 fluorescence cube (ex.: 535 :t 25, DC: 560, EM; 590 long
pass). The number of callose depositions was determined with ImagePro Plus software.
More than 10 adjacent fields of view along the length of the leaf were analyzed and
averaged. The values in Figures 2-12 and 2-13 are the average and standard deviation of

4 or more independent leaves for each treatment.

57

Results

The Pst DC3000 ACEL mutant can be restored by a fragment containing ORF3 and
ORF4

A large deletion mutant was generated in the conserved effector locus (CEL) of
the Pst DC3000 Hrp pathogenicity island (Pai) and was demonstrated to be required for
bacterial virulence on tomato (Alfano et al., 2000). We assessed the ACEL mutant for its
ability to infect Arabidopsis and found that it was severely compromised in its ability to
cause disease symptoms and multiply in the host tissue. The ACEL mutant exhibited 100
to 500 fold reduced growth compared to Pst DC3000 when infiltrated at a concentration
of 106 cfu/ml and elicited no disease symptoms (Figure 2-1). A cosmid, pCEL, was
isolated from a cosmid library of Pst DC3000 genomic DNA by screening for aer, one
of the genes in the deleted region. This cosmid, which carries ORFI through ORFIO,
fully complemented the ACEL mutant (Figure 2-2).

Since the deleted region contained several putative effector genes, we conducted
complementation analysis to identify key players in the ACEL mutant phenotype. We
introduced plasmids carrying different fragments from the deleted region into the ACEL
mutant and tested for restoration of disease symptoms and bacterial multiplication in
Arabidopsis plants. The smallest fragment tested that could complement the ACEL
mutant phenotype contained three genes, ORF2, ORF3 and ORF4 (Figure 2-2). These
three genes constitute an operon and have a hrp box upstream of ORF4 implying
regulation by the Hrp system. Of these genes, ORF 2 is thought to encode a chaperone to

Aer.

58

 

 

 

3 i
7.5 i ‘ +DC3000
+CEL
7 i L;
6.5 i
E |
2 l
.3 5.51‘
.9
sl
1
4.5 l
l
’l
3.5.1
l
3 ‘ . i . -
o 1 2 3 4

days post inoculation

 

1 2

Figure 2-1: Disease symptoms and grth in planta of Pst DC3000 and the ACEL
mutant following vacuum-infiltration with an inoculum of 106 cfu/ml. (A). Bacterial
multiplication in Arabidopsis Col glI of DC3000 (solid diamonds) and ACEL mutant.
Each datum point reflects the average bacterial population recovered from nine 0.5 cm2
leaf discs. Vertical lines indicate standard deviation. (B). Symptoms in Arabidopsis Col
g1] leaves four days after inoculation with 106 cfu/ml DC3000 (leaf 1) and ACEL mutant

(leaf 2).

59

hrpRS 1 aer 2 3 4 hrpWS 6 7 8 91011gstAgafl’

.msl . ‘. _-

 

 
   

 

 

 

 

 

 

 

 

 
   

 

 

 

 

 

 

 

ACEL .............. ILA. ,,,,,,,,,,,,,,,,,,,,,, _
m . %- ~ e ~ - - e- +++
I I
0RF2-5 E L . +++
I I
l I
0RF234 inn—d +++
HrpW-ORF5 i —
0RF6-10 E _

.1

Figure 2-2: Schematic representation of the complementation of the ACEL mutant. Col
glI plants were syringe-infiltrated with 106 cfu/ml of the ACEL mutant carrying different
regions of the conserved effector locus. Plants were scored for symptom development
and bacterial multiplication four days after inoculation. A minus sign indicates no disease
symptom. Three plus signs indicate typical disease symptoms: extensive water-soaking
followed by necrosis and chlorosis. Appearance of disease symptoms was correlated with
high levels of bacterial multiplication.

60

Further complementation studies were conducted within the delineated fragment.
Complementation of the ACEL mutant with ORF 2, ORF23 or 0RF24 did not restore the
virulence defect (Figure 2-3). The adjacent location of the large ORF3 (712 amino acids)
and the small ORF4 (164 amino acids) in the same operon is suggestive of an effector-
chaperone relationship. To determine if ORF4 is required for the ability of ORF3 to
restore virulence to the ACEL mutant, Arabidopsis plants were inoculated with the ACEL
mutant containing either ORF3, ORF4 or ORF43 on a plasmid. Symptom development
and in planta bacterial growth were monitored. We found that ORF4 alone did not
complement the ACEL mutant. The ACEL mutant carrying ORF3 alone was partially
restored in growth, but caused little or no symptom development. However, the presence
of both ORF3 and ORF 4 in the ACEL mutant restored in planta growth and symptom
development completely (Figure 2-4). Based on these results, we concluded that ORF3

and ORF4 were sufficient to restore virulence to the ACEL mutant in Arabidopsis.

61

aer 2 3 4

 

 

 

 

 

 

 

 

 

Symptom
0RF2 1:1 "
ORF3 " "' +*
ORF4 I: —

0RF43 r - ,< . .1 1 +++

 

 

0RF32 :K/I; . , . J _
0RF42 :l\/l::l _

Figure 2-3: Schematic representation of further complementation of the ACEL mutant.
Col g1] plants were syringe-infiltrated with 106 cfu/ml of the ACEL mutant carrying
different ORF3 from the delineated fiagment. Plants were scored for symptom
development and bacterial multiplication (not shown) four days after inoculation. A
minus sign indicates no disease symptom. A plus sign with an asterisk indicates
infrequent yellow spots in only some of the inoculated leaves. No typical water-soaking
or necrosis symptoms were observed. Three plus signs indicate typical disease symptoms:
extensive water-soaking followed by necrosis and chlorosis. Appearance of disease
symptoms was correlated with high levels of bacterial multiplication.

62

log cfu/cm2

 

 

0 1 2 3
days post inoculation

 

1 2 3 4 5

Figure 2-4: Restoration of the ability of the ACEL mutant to multiply and elicit disease
symptoms in Arabidopsis. (A). Bacterial growth curves. Col glI plants were vacuum-
infiltrated with 106 cfu/ml of Pst DC3000 (solid diamonds), ACEL (solid squares), ACEL
mutant containing ORF3 (solid triangles), ORF4 (empty squares) or 0RF43 (empty
triangles). Each time point reflects the mean of nine 0.5 cm2 leaf discs. Error bars indicate
standard deviations. (B). Disease symptoms in C01 gII leaves were scored 4 days after
inoculation with 106cfu/ml of DC3000 (leafl), ACEL mutant (leaf 2), ACEL mutant
containing 0RF43 (leaf 3), ORF 4 (leaf 4) or ORF 3 (leaf 5).

63

ORF3, but not ORF4, is secreted in culture by the Hrp system

The results from the complementation studies prompted us to investigate whether
ORF3 and ORF4 are secreted through the TTSS. For this, ORF3 and ORF4 under the
control of their native promoter (p0RF43), were introduced on a plasmid into the
wildtype Pst DC3000, the ACEL mutant, and the hrcC mutant strain (Yuan and He,
1996). The parental and transformed strains were tested for secretion of ORF3 and ORF4
in both LB and hrp-inducing liquid media. The hrcC mutant is incapable of assembling
the type III apparatus and does not secrete any type III effectors. Both ORF 3 and ORF4
proteins were observed in the cell-bound fractions of all strains tested, except the ACEL
mutant, when grown in hrp-inducing minimal medium but not when grown in LB
medium. This suggests that ORF3 and ORF 4 are induced under conditions that favor hrp
gene expression. As expected, the protein bands observed were stronger in all strains
expressing ORF3 and ORF 4 in trans. However, only ORF3 was detected in the
supernatant fractions of the wild-type Pst DC3000, and ORF43 transformants of Pst
DC3000 and the ACEL mutant, when grown in hrp-inducing minimal medium. This
indicated that only ORF3, but not ORF4, was secreted. ORF3 was not detected in the
supernatant fractions of the hrcC mutant grown in minimal medium, indicating that its

secretion is TTSS-dependent.

64

A

Strain WT WT WT WT hrcC' hrcC' hrcC' hrcC'ACEL
Medium LB MM LB MM LB MM LB MM MM

 

 

 

p0RF43 - - + + - - + +
Sup ----~- _
a-HothoM
_ ...v w wmmt III III.
Sup
a-Sth
C8” —. fl .—
B
0')
C") v v
.1 u. 11. LL
LU c1: 0: 0C
0 O O o
< o. 0. :1.
Sup , _
d-HothoM ”
0°” a... m ...- m '-

Sup
d-Sth

Cell .. ......._._ , ._

Sup
o-B-lacta mase

Cell .— ....,.......... ..-- ._

Figure 2-5: (A). Analysis of type III-dependent secretion of ORF 3 (HothoM) and ORF4
(Sth). Pst DC3000 (WT) or hrcC mutant derivatives carrying (+) or lacking (—)
p0RF43 were grown in rich media (LB) or hrp-inducing media (MM). Cultures were
separated into supernatant (sup) and cell (cell) fractions by centrifugation and the
presence of ORF 3 or ORF4 in each fraction was detected by immunoblot analysis using
an antibody against HothoM (a-HothoM) or Sth (a-Sth). WT: wild-type DC3000.
pORF 43 expresses ORF 3 and ORF 4 under the native promoter to enhance the expression
of both genes in hrp-inducing medium. In the cell fraction of the a-HothoM blot, a
cross-reacting protein present in all lanes migrated slightly faster than ORF3.

(B) Dependence of ORF3 secretion on ORF4. .The ACEL mutant and its derivative
carrying pORF 3, p0RF4, or pORF 43 were grown in hrp-inducing medium (MM). Other
conditions were the same as described in panel A.

65

ORF3 is translocated into plant cells by the TTSS in an ORF4-dependent manner
We decided to further investigate the requirement of ORF4 for the function of
ORF3 during infection by determining whether ORF3 and ORF4 are translocated into
plant cells. We fused full-length ORF3 (712 amino acids) and ORF4 (164 amino acids)
proteins to a truncated Aerpt2 protein (80-255 amino acids, Aerpt230-255). We then
introduced various constructs expressing wildtype Aerpt2, ORF3 + ORF4,
ORF4::Aerpt230-255, ORF3::Aerpt280-255, ORF4 + ORF3::Aerpt280-255 or
ORF4::Aerpt280-255 + ORF3 into the ACEL mutant and inoculated RPS2 and rps2
Arabidopsis plants with these strains. No HR developed in leaves inoculated with
bacteria expressing ORF3 and ORF4. A robust Aerpt2-dependent HR was observed at 9
hours after infiltration for the wildtype Aerpt2 and at 15 hours for the
ORF3::Aerpt280-255 fusion protein expressed together with ORF4 (Figure 2-6). Leaves
inoculated with ORF3::Aerpt2go-255 alone were reduced in turgidity, but no typical HR
occurred. ORF4::Aerpt230-255 alone or when expressed with ORF3 did not give an HR.
Therefore, ORF3 is a translocated type III effector of Pst DC3000. Translocation of

ORF3 requires the presence of ORF4, which itself is not translocated.

66

 

l12/14 I 1/14 I 1/12 I 0/12 l11/14 [0/14 looI-o
l 0/14l 0114 Tom 1 0/12 I 1/141 1/14 ers2

Col-0

rpsz

 

Figure 2-6: Type III translocation analysis of ORF3 and ORF4 in Arabidopsis. Full-
length ORF3 and ORF4 proteins were fused to a truncated Aerpt2 protein (80-255
amino acids, Aerpt230-255). Plasmids were introduced into the ACEL mutant.
Arabidopsis Col 0 (RPS2) or rps2 mutant leaves were infiltrated with bacterial
suspensions at OD600=O.2 and evaluated for HR elicitation. 1: ACEL (pAerpt2), 2:
ACEL (p0RF43), 3: ACEL (pORF3:: Aerpt280_255) , 4: ACEL (p0RF4:: Aerpt230_255),
5: ACEL (p0RF4 + p0RF3: Aerpt230-255), 6: ACEL (p0RF4:: Aerpt2go.255 + p0RF3).
Col-0 leaves (1 and 5) showing HR collapse appear wrinkled. Top: Number of leaves
showing HR/number of leaves infiltrated for Col-O and rps2 plants. Picture was taken 18
h after inoculation. Leaves representing the majority of each treatment are shown.

67

ORF4 physically interacts with ORF3 in the yeast two-hybrid system

The results fiom complementation, virulence, secretion, and translocation
analyses suggested that ORF4 may act as a chaperone for ORF3. To obtain more
conclusive evidence for this possibility, we fused the full-length ORF4 protein (164
amino acids) to the DNA binding domain (BD) and the full-length ORF3 protein (712
amino acids) to the activation domain (AD) of the LexA-based two-hybrid system and
tested for physical interaction of the two proteins. Yeast strains carrying the above
constructs individually, did not turn blue, indicating that these genes were not auto-
activators. A blue color developed in positive control yeast strains, in which BD was
fused to the SV40 activation domain and AD was fused to the p53 binding domain
(Figure 2-6). No color developed when yeast strains carried empty AD or BD, regardless
of the nature of the other partner. A blue color developed in strains carrying AD firsed to
ORF3 and BD fused to ORF4, demonstrating physical interaction of these proteins in
yeast.

To determine the portion of ORF3 that interacts with ORF4, we constructed
fusions of the LexA AD with the N-terminal 100 and 200 amino acids of ORF3 (ORF3100
and ORF3200, respectively) and tested for interaction in yeast as described above. Yeast
strains carrying the N-terminal 200 amino acids of ORF3 turned blue afier two days of
growth in galactose X-gal minimal medium. In contrast, yeast strains carrying the N-
terminal 100 amino acids of ORF3 remained white (Figure2-7). These results indicate

that the first 200 amino acids of ORF3 are required for interaction with ORF4.

68

 

Figure 2-7: Physical interaction between ORF3 and ORF4 in the LexA two-hybrid
system. Full-length ORF4 protein was fused to the DNA binding domain (BD) in
pGILDA and a series of truncated ORF3 proteins were fused to the transcriptional
activation domain (AD) in pB42AD. Yeast strains were grown at 30°C for 2 days on
galactose X-gal complete minimal medium. Blue color indicates interaction, whereas
white color indicates no interaction. 1 = BD::SV40/AD::p53, 2 = BD-empty/AD-empty,
3 = BD-empty/AD::ORF4, 4 = BD::ORF3 (firll—length)/AD-empty, 5 = BD::ORF3
/AD::ORF4, 6 = BD::ORF4/AD::ORF3Hoo (1-100 amino acids), 7 =
BD::ORF4/AD::ORF31-200 (1-200 amino acids), 8 = BD::ORF4/AD::ORF3201-712 (201-
712 amino acids), 9 = BD::ORF4/AD::ORF3301-712 (301-712 arrrino acids), 10 =
BD::ORF4/AD::ORF3401-712 (401-712 amino acids). Yeast colonies containing
AD::ORF31.200 alone, AD::ORF3201-712 alone, and AD::ORF3301-712 alone were white
(data not shown).

69

The results described so far, indicate that DRE 3 is a translocated type III effector
of Pst DC3000 and was therefore renamed HothoM (Hop for hrp outer protein).
Sequence analysis indicated that ORF4 had numerous characteristics of a chaperone
protein. It is a small protein, 18 kDa, with an acidic pI of 5.3 containing an amphipathic
a-helix in the C-terrninal portion. These properties, in combination with the
complementation, secretion, translocation and interaction data, suggest that ORF4 is a
chaperone for HothoM and was therefore renamed Sth (Specific hrp ghaperone of

HothoM).

HothoM transgenic plants exhibit a distinct seedling and growth phenotype

To further examine the function of HothoM, I produced Arabidopsis Col g1]
plants expressing the hothoM gene under the control of a DEX-inducible promoter
system. A 6X His tag was added at the N-terminus of the HothoM protein. Homozygous
lines were chosen on the basis of heritable resistance to hygromycin. Three independent
lines were carried to homozygosity. Western analysis using anti-HothoM and anti His-
tag antibodies confirmed the presence of the protein when plants were induced with DEX.
Data presented here are from two lines, 11 and 44.

These lines were found to have several unique morphological and developmental
features. Transgenic hothoM seeds were delayed in germination compared to wildtype
Col gl1 seeds. When germinated on MS media, hothoM seeds showed a 2-3 day delay.
However, this delay was more marked when seeds were directly sown in soil, being at
least 5-7 days late compared to wildtype seeds. When grown in MS medium and soil,

hothoM seeds also exhibited an inability to achieve synchronous germination, even after

70

stratification for three days at 4°C. Usually, three days of incubation at 4°C causes

wildtype plants to achieve synchronized germination.

Arabidopsis thaliana plants expressing HothoM exhibit chlorosis and necrosis upon
induction

The hothoM plants exhibited a distinct phenotype in response to DEX induction.
When sprayed with a high level of DEX (30uM) and maintained under high humidity,
hothoM plants developed water soaking symptoms after 6 to 12 hours. By 24-36 hours,
severe necrosis and chlorosis developed (Figure 2-8). While this phenotype was similar
to the symptoms caused by Pst DC3000 infection of Arabidopsis, HothoM-caused
symptoms were faster compared to an infection. At lower levels of DEX (0.3 uM), the

HothoM-induced symptoms were comparable to those of an infection.

71

 

Col gl1 hothoM

“.

Col gl1 hothoM HothoM
plants plants E. coli

Figure 2-8: (A) Expression of HothoM in planta causes disease-like symptoms. Six-
week-old Arabidopsis Col g1] and hothoM plants were sprayed with 30 11M DEX and
kept under high humidity. The upper two leaves of Col g]! were inoculated with 106
cfu/ml Pst DC3000 and pictures were taken four days later. Induction of the hothoM
transgene caused development of chlorosis and necrosis that very closely resembled the
disease symptoms caused by Pst DC3000 on Arabidopsis Col glI .

(B) Western blot analysis of HothoM expression in leaves of wild-type Arabidopsis Col
g1] plants and HothoM transgenic plants (hothoM) 24 hours after spraying with 30 11M
DEX. Recombinant HothoM from E. coli was used as control.

72

Expression of HothoM in planta allows the growth of the CEL deletion mutant

We demonstrated that the ACEL mutant is fully complemented by hothoM and
sth. We also showed that Sth is required for the secretion and translocation of
HothoM. Based on these results, we hypothesized that the ACEL mutant may be able to
grow and cause symptoms in plants that express hothoM. In order to investigate this, I
identified a concentration of inducer (0.003 11M DEX), which by itself caused no
macroscopic symptom development in hothoM plants. I was unable to detect the
HothoM protein at this level of DEX by Western blotting. However, Northern blot
analysis confirmed the expression of the transgene at this concentration of DEX (data not
shown).

Arabidopsis Col g1] and hothoM plants were sprayed with 0.003 uM DEX,
6 hours prior to bacterial inoculation and daily thereafter. Both plant genotypes were
inoculated with Pst DC3000, ACEL mutant, and the hrpH mutant. The hrpH mutant is
unable to assemble a functional type III secretion apparatus and is thus incapable of
secreting any type III effector proteins (Yuan and He, 1996). Bacterial population was
estimated 3 days later. Pst DC3000 showed similar grth in both wildtype and
transgenic plants. The hrpH mutant grew 5-fold in hothoM plants as compared to C01
g1] plants. While the ACEL mutant grew a lOO-fold in C01 g1] plants, it multiplied 5000-
fold in the hothoM plants, representing a 50-fold increase. In fact, the ACEL mutant
multiplied only 5-fold less than Pst DC3000 in hothoM plants. We concluded that the
ACEL mutant can be complemented by in planta expression of hothoM. The hothoM
plants inoculated with the ACEL mutant also developed chlorosis and necrosis.

Uninoculated leaves remained asymptomatic during the experiment.

73

  
   
  
  
   
 

I Col/DC3000
7'5 } n hothoM/DC3000
7 l I Coll hrpH
t I hothoM/hrpH
6'5 l n Col/CEL
s i I h—othoMlCEL

 

log cfulcm 2
U!
"I

 

 

days post inoculation

Figure 2-9: Complementation of the ACEL mutant multiplication in plants expressing
HothoM. Arabidopsis Col gl1 (Col) plants and hothoM transgenic plants (hothoM)
were sprayed with 0.003 uM DEX, 6 hours prior to bacterial infiltration and dail during
the course of the experiment. Bacteria were syringe-infiltrated into plants at 10 cfu/ml.
Bacterial grth was monitored after three days. Bacterial numbers are the average of 12
leaf discs from three individual leaves. Error bars indicate standard deviation. DC3000
represents Pst DC3000. hrpH represents the hrpH mutant and CEL represents the ACEL
mutant. The hothoM transgenic plants support a 50-fold higher growth of the ACEL
mutant compared to wildtype Col glI .

74

DC3000 uninoculated

  

Col gl1 0.003 uM DEX

hothoM 0.003 uM DEX

   

Figure 2-10: Growth of the ACEL mutant in hothoM transgenic plants is accompanied
by the development of symptoms similar to those of Pst DC3000 infection. Wildtype Col
g1] and hothoM transgenic plants were induced with 0.003 uM DEX, 6 hours prior to
inoculation and daily during the course of the experiment. Bacterial inoculum of 106
cfu/ml was syringe-infiltrated. Symptoms were recorded three days later. DC3000
represents Pst DC3000, hrpH represents the hrpH mutant and ACEL represents the ACEL
mutant. Leaves labeled “uninoculated” were treated with DEX but were not infiltrated
with bacteria.

75

on
J

 

 

 

 

J I hothoM/DCIBOOO
7 I hothoM/hrpH

J El hothoM/CEL
6

 

log cfulcm 2
01

A

0)
1. 4._1_ __._ _L___ _1.

 

 

 

 

 

N

days post inoculation

Figure 2-11: The multiplication of the ACEL mutant is not complemented in uninduced
hothoM transgenic plants. Bacteria were syringe-infiltrated into plants at 106 cfu/ml.
Bacterial growth was monitored after three days. Each bar represents the mean titer of
12 leaf discs from three individual leaves. Error bars indicate standard deviation.
hothoM represents hothoM transgenic plants, DC3000 represents Pst DC3000, hrpH
represents the hrpH mutant, and CEL represents the ACEL mutant.

76

The ACEL mutant is impaired in suppressing cell wall-based defenses

As a successful pathogen of Arabidopsis, Pst DC3000 is capable of suppressing
defense responses mounted by the host in response to bacterial attack. One type of
defense elicited by non-pathogenic bacteria, such as hrp mutants, are extracellular cell
wall-based defense including the formation of papillae. Papillae are characterized by
highly localized depositions of callose, phenolic compounds, and hydroxyproline-rich
glycoproteins into paramural deposits directly beneath sites of bacterial interaction with
plant cells (Bestwick et.al, 1995; Brown and Bonas, 1995; Brown et al., 1998). Staining
for callose, as a marker for papilla, has revealed that wildtype Pst DC3000 has the ability
to suppress papilla deposition through the action of type III effector proteins.

To investigate if the ACEL mutant was impaired in suppressing extracellular host
defenses, I analyzed callose deposition in C01 glI plants in response to Pst DC3000, the
ACEL mutant, and the non-pathogenic hrpA mutant. The hrpA mutant is unable to
assemble the Hrp pilus and does not secrete any type III effectors (Roine et al., 1997).
The hrpA mutant induced a high number of callose deposits. In contrast, leaves infected
with Pst DC3000 showed very low numbers of callose deposits, clearly demonstrating
that the TTSS of Pst DC3000 is involved in the suppression of callose-associated cell
wall modifications in Arabidopsis. The ACEL mutant induced a large number of callose
deposits in wildtype leaves as compared to Pst DC3000. The number of callose deposits
were about 50% of that caused by the hrpA mutant. Because HothoM and Sth are
sufficient to restore virulence to the ACEL mutant, I analyzed the callose response to the

ACEL mutant containing hothoM and sth, to see if these genes are sufficient to

suppress callose deposition. I found that hothoM and sth restored the ability of ACEL

77

mutant to suppress callose accumulation to a level comparable to that of Pst DC3000

(Figure 2-12).

The ACEL mutant is compromised in eliciting callose formation in NahG and eds5
plants

I also analyzed the callose response of Pst DC3000, ACEL mutant and hrpA
mutant in NahG plants. NahG plants express the salicylate hydroxylase gene from
Pseudomonas putida. Salicylate hydroxylase converts SA to catechol and these plants are
therefore incapable of accumulating SA and are compromised in the induction of SA-
dependent defenses (Delaney et al., 1994; Gaffney et al., 1993). The hrpA mutant induced
similar numbers of callose deposits in NahG leaves and wildtype Col glI leaves (Figure
2-13). This result also confirmed the earlier study which showed largely SA-independent
regulation of Arabidopsis genes by the Pst DC3000 hrpH mutant. In contrast, the ACEL
mutant failed to trigger a high level of callose deposits in NahG leaves. The number of
callose deposits in NahG leaves infected with the ACEL mutant was very similar to those
induced by Pst DC3000 in wildtype leaves. As expected, Pst DC3000 did not induce a
significant number of callose deposits in NahG leaves. This result indicated that the
ACEL mutant activated SA-dependent host cell wall-based defense. To further assess the
SA-dependency of the ACEL mutant-activated cell wall defenses, we analyzed the ability
of the ACEL mutant to induce callose deposition in eds5 plants. The eds5 mutant of
Arabidopsis, accumulate little or no SA after pathogen inoculation and are
hypersusceptible to pathogens (Nawrath and Metraux, 1999). Leaves of eds5 plants

inoculated with the ACEL mutant exhibited a similar number of callose deposits as that in

78

NahG leaves. The response of eds5 plants to Pst DC3000 and hrpA mutant was similar to
that of wildtype Col g1] i.e. very few callose deposits in response to Pst DC3000 and a
high number of callose deposits to the hrpA mutant. These results confirmed that the

ACEL mutant activates SA-dependent papilla formation in Arabidopsis.

NahG plants support growth of the ACEL mutant

The near absence of papillae formation in NahG leaves in response to the ACEL
mutant suggested that the ACEL mutant might be able to multiply better in NahG plants
compared to wildtype plants. Arabidopsis Col gl] and NahG plants were infiltrated with
Pst DC3000, the hrpH mutant, and the ACEL mutant. Growth and symptom development
were monitored over a period of 4 days. We found that the growth of the ACEL mutant
and symptom development on the NahG plants were partially restored to those of Pst
DC3000 on Col g1]. Pst DC3000 and the hrpH mutant grew slightly more in NahG plants
(about 5-fold) compared to that in C01 g1] plants. The ACEL mutant multiplied 200-fold
more at day 3, and 400-fold more at day 4 in NahG plants than in C01 gl1 plants. These
results further demonstrated that the effector genes in the CEL region interfere with SA-

mediated defenses in the host.

79

Pst DC3000

 

C EL CEL(pORF43)

   

Pst DC3000 hrpA mutant ACEL mutant ACEL(porf43)

Col gl1 26 d: 8 438 :t 48 202 i 36 23 :t 2

Figure 2-12: The ACEL mutant triggers the callose response in Arabidopsis. (A) Portions
of wildtype Arabidopsis Col g1] leaves stained with Aniline blue for callose (white dots
in these images) after inoculation with Pst DC3000, the hrpA mutant, the ACEL mutant,
and the ACEL mutant containing p0RF43. The ability of the ACEL mutant to suppress
callose deposition was restored by ORF3 and ORF4. Scale bar, 100 um. (B) Average
number of callose depositions per field of view (0.9 m2) with standard deviation
displayed as error.

80

Pst DC3000

    

I.

Col g1]

 

NahG

 

B
Pst DC3000 hrpA mutant ACEL mutant
C01 g1] 30 :l: 7 491 :l: 49 223 :1: 29
NahG 29i5 473i32 31:1:3
C
Pst DC3000 ACEL mutant hrpA mutant
Col g1] 31 :h 6 232 :t 37 353 :1: 47
eds5 21 :E 7 26 :l: 4 378 :l: 45

Figure 2-13: The ACEL mutant is compromised in the ability to activate callose response
in leaves of the SA-deficient NahG plant. (A) Portions of wild-type Arabidopsis Col g1]
and NahG leaves stained with aniline blue for callose (white dots in these images) after
inoculation with Pst DC3000, the hrpA mutant, and the ACEL mutant. Scale bar, 100 um.
(B) Average number of callose depositions per field of view (0.9 m2) with standard
deviation displayed as error in C01 gl1 and NahG plants. (C) Average number of callose
depositions per field of view (0.9 m2) with standard deviation displayed as error in C01
g1] and eds5 plants.

81

   
 

1.E+09
+NahG DC3000

 
 
 

-D- Col DC3000

1.E+08
+NahG CEL

 
 
   
 

'- -Col CEL

1.E+07
+Nahc hrpH

  
   
 

 

" _._,_ Col hrpH
E 1.E+06
1'
_ f

1.E+05 -
1.E+04 “/3.
1.E+03 El . . . .
0 1 2 3 4

days post inoculation

 

DC/NahG DC/Col CEL/NahG CEL/Col

Figure 2-14: The ACEL mutant proliferates and causes symptoms more aggressively in
NahG plants than in C01 glI plants. (A) Bacterial growth curves: Col g1] and NahG
plants were vacuum-infiltrated with 106 cfu/ml of Pst DC3000, the ACEL,mutant and the
hrpH mutant. Each time point reflects the mean of nine 0.5 cm2 leaf discs. Error bars
indicate standard deviations. (B) Disease symptoms in C01 g1] and NahG leaves were

scored 4 days after inoculation.

82

Discussion

During infection, Pst DC3000 utilizes the TTSS to inject at least 31 different
effector proteins into the host. These effectors are believed to collectively promote the
development of disease. Attempts to understand the function of individual effectors by
gene inactivation in bacteria has proven to be challenging because of the typically weak
contributions that individual effectors make to virulence. This is in sharp contrast to the
strong loss-of-virulence phenotype observed when hrp genes are inactivated. Deletion of
six ORFs in the CEL of Pst DC3000 created a mutant that was exceptional, in that it
possessed a strong reduced-virulence phenotype in tomato (Alfano et al., 2000). This
deletion however, did not affect its ability to cause HR in tobacco plants indicating that
the TTSS was not aberrant in this mutant.

In this chapter I have demonstrated that the ACEL mutant also has a strong
reduced-virulence phenotype in Arabidopsis thaliana and that this loss in virulence can
be fully complemented by two genes from the deleted region, CELORF 3 (hothoM) and
CEL ORF 4 (sth). Moreover, we have found that HothoM is secreted in culture and is
translocated into plant cells in a T TSS-dependent manner. hothoM is conserved and
linked to the Hrp TTSS genes in divergent P. syringae pathovars, suggesting that this
effector has played a significant function in the evolution of the species P. syringae as an
aggressive pathogen of diverse plants.

In this study, we demonstrated that Sth is a chaperone for HothoM. The
demonstration of the requirement of Sth for the efficient translocation and function of
HothoM in the plant cell is consistent with the presence of customized chaperones in

plant pathogenic bacteria and supports recent findings with Pss ShcA and E. amylovora

83

DspB/F (Gaudriault et al., 2002; van Dijk et al., 2002). Bacterial chaperones have been
suggested to maintain their cognate effectors in a state that is competent for secretion,
thereby conferring a competitive advantage over other non-chaperoned effectors for
traffic through the TTSS.

Although HothoM can be translocated efficiently in the presence of Sth, our
results suggest that some HothoM may still be injected into the plant cell in the absence
of its chaperone. This conclusion is supported by two observations. First, the ACEL
mutant carrying the HothoMzzAerpt230-255 fusion without simultaneous expression of
Sth is able to cause loss of turgidity in Arabidopsis leaves. Second, the ACEL mutant
complemented with only hothoM shows partial restoration of bacterial growth even
though symptom development is not augmented substantially. Both complementation and
translocation analyses suggest that secretion and/or translocation of HothoM in the
absence of its chaperone is probably an inefficient process. Consistent with our
interpretation, an E. amylovora dspB/F mutant retains some virulence to pear seedlings,
suggesting that some DspA/E still travels the TTSS in the absence of the chaperone
(Gaudriault et al., 2002). In addition, it has been shown that chaperones are not
absolutely required for translocation of some effectors of animal pathogenic bacteria. For
instance, deletion of the binding site for the chaperone Sch in the Yersinia enterocolitica
YopE effector does not prevent its translocation into the eukaryotic cell owing to the
presence of the N-terminal secretion signal that is present in all type III effectors (Boyd et
al,2000)

Based on a study of the Yersinia type III effector YopE, and its chaperone, Sch,

Boyd et al. (2000) suggested that chaperoned effectors may be secreted by the bacterium

84

either more efficiently or at an early stage during the interaction with the eukaryotic cell
(Boyd et al., 2000). HothoM is the only effector of the P. syringae CEL locus explored
thus far whose virulence function is chaperone-dependent. Thus, it is possible that
HothoM is translocated early into the plant cell. Being probably one of the first effectors
to encounter the host cytoplasm, HothoM could be expected to target early host
responses that may promote the establishment of an infection.

1 demonstrated the ability of HothoM expressed in planta to complement the
ACEL mutant to multiply and cause symptoms. This is especially noteworthy since such
complementation has not been demonstrated for any P. syringae type III effectors. It is
also worth noting that this complementation was observed at very low levels of the DEX.
It is known that during infection, the pathogen injects minute quantities of effectors into
the host. I speculate that the very low level of DEX that allows hothoM plants to
support growth of the ACEL mutant must have resulted in a HothoM concentration that
is comparable to that delivered by Pst DC3000. These results also suggest that HothoM
can correctly fold into its functional conformation in the host cytoplasm without
assistance from its cognate chaperone or other bacterial factors.

Immunoblotting analyses sometimes revealed the presence of a second band
below that of HothoM in hothoM transgenic plants. This band was not present in
uninduced transgenic plants or in C01 g1] plants treated with DEX. Thus, this band is
specific to expression of the transgene. This band could result from a non-specific
degradation of HothoM. Alternatively, this band was generated from specific processing

of the HothoM protein within the plant cytoplasm. Because this extra band was not

85

observed consistently, it might be the result of non-specific protein degradation rather
than specific host-dependent processing.

Several years ago, electron microscopic studies revealed that pepper and lettuce
leaf cells deposit papilla on the inner side of the cell wall at the site of attempted infection
by non-pathogenic bacteria including hrp mutants (Bestwick et al., 1995; Brown et al.,
1995, 1998). This highly localized cell wall thickening response could be a critical part of
the poorly defined basal resistance that prevents multiplication of the vast majority of
non-pathogenic bacteria to which plants are exposed in nature. Since the cell wall is the
first host barrier that pathogens encounter, strengthening the cell by forming papilla
would exert a local inhibitory effect on bacterial infection. Such fortification could
theoretically reduce the penetration capacity of the type III pilus as well as reduce the
movement of nutrients into the apoplastic space that would otherwise promote bacterial
multiplication. In a previous study, another effector, AvrPto, was demonstrated to
function in reducing the deposition of callose in the Arabidopsis cell walls during
infection by the non-pathogenic hrpH mutant. It was also noticed that the transcriptional
profile of plants expressing AvrPto showed repression of a large percentage of genes that
are putatively predicted to be localized to the cell wall. It was therefore suggested that
AvrPto is involved in overcoming the cell wall-based defense of the host (Hauck et al.,
2003).

I tested the ability of the ACEL mutant to suppress the deposition of callose in the
host cell wall. I found that unlike the wildtype Pst DC3000 bacteria, the ACEL mutant

was severely compromised in suppressing callose deposition in C01 glI plants.

86

Furthermore, HothoM and Sth were able to completely restore the papilla-
suppressing ability of the ACEL mutant.

We have therefore identified a second type III effector in Pst DC3000 that thwarts
plant cell wall-based defense. The role of HothoM in the suppression of one of the first
lines of host defense further strengthens the concept that this effector is one of the first
type III effectors to be translocated into the host during infection.

I also showed that while the hrpA mutant elicited callose deposition in a SA-
independent manner, the ACEL mutant activated the cell wall response in a SA-
dependent manner. The elicitation of callose response by the ACEL mutant was not as
dramatic as the non-pathogenic hrp mutant, suggesting that effectors (e. g. AvrPto) other
than HothoM function in the ACEL mutant to suppress callose deposition. Specifically,
the ACEL mutant does not activate high levels of papilla response in NahG or eds5 plants.
In contrast, the non-pathogenic hrpA mutant triggered callose deposition in wildtype
Arabidopsis, NahG, and eds5 leaves. Thus, there appears to be two pathways leading to
callose response. While AvrPto seems to suppress the SA-independent callose response,
HothoM inactivates the SA-independent pathway. One of the major inducers of the SA-
independent pathway seems to be the flagellin protein that is the major constituent of the
flagella ( Underwood and He, unpublished data).

A previous study had also reported a SA-dependent reduction in the amount of
callose deposits formed around the fungal haustoria during infection of Arabidopsis
thaliana by Pernospora parasitica. Callose encasements formed in response to fungal
penetration have been suggested to function as defense structures that decrease the

efficiency of the haustorium to obtain nutrients from host cells (Allen and Friend, 1983 ).

87

NahG plants, which permit higher growth of the pathogen, had a smaller number of
haustoria surrounded by thick callose encasements, and in most haustoria callose was
limited to thin collars around haustoria] necks (Donofrio and Delaney 2001).

We have demonstrated that the ACEL mutant can multiply in NahG plants to
levels close to those achieved by the wildtype Pst DC3000. This result, coupled with the
papilla suppression data, supports a role for papilla to function as a defense structure that
reduces the efficiency of bacterial multiplication in the apoplast perhaps by affecting type
III secretion and/or nutrient movement across the host cell wall.

At this point, we do not know which bacterial factors trigger the SA-dependent
papilla deposition. However, this factor must not be present in the hrp mutant, therefore
must either be the components of type III secretion system or effectors injected into the
host cell (Figure 2-15).

Overall, this study has led to the identification of an effector-chaperone system in
Pst DC3000 that is a key player in pathogenesis. We show here that HothoM
contributes to Pst DC3000 virulence by suppression of SA-dependent host cell wall-

based immunity.

88

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Chapter 3

Homology-based identification of type III effectors in Pseudomonas syringae pv.

tomato DC3000 and characterization of transgenic Arabidopsis thaliana plants

expressing averhEpw.

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Abstract

Pseudomonas syringae pv. tomato strain DC3000 (Pst DC3000) is a virulent
pathogen of Arabidopsis thaliana and tomato. It utilizes the type IH secretion system
(TTSS) to deliver into host cells, effector proteins that function to promote pathogenesis.
These effector proteins were initially identified as mediators of gene-for-gene resistance
responses and thereby named Avr (avirulence) proteins. Increasing evidence supports
dual functions for Avr proteins as recognition determinants in resistant hosts and
virulence determinants in susceptible hosts. Screening a Pst DC3000 cosmid library using
ten known P. syringae avr genes as probes, led to the identification of three new effector
genes. These are avrPtoB, averhEpm, and averinm and are homologous to vierhA
and averhE from P. syringae pv. phaseolicola, and averiB from P. syringae pv. pisi,
respectively. All three genes encode bona fide effectors that are translocated in a type III
dependent manner. Insertional inactivation of avrPtoB caused a dramatic loss of
virulence on tomato but not on Arabidopsis. The lack of averinm or averhEpm or both,
did not result in a loss of virulence on Arabidopsis or tomato. However, expression of
averhEpm in Arabidopsis caused development of symptoms that closely resembled those
caused by Pst DC3000 during infection. Microarray analysis revealed that transgenic
expression of averhEpw in Arabidopsis was capable of regulating host genes in a
manner that closely resembled that of the Pst DC3000 TTSS. These transgenic plants
allowed the non-pathogenic hrpH mutant to grow like Pst DC3000, supporting the role

for averhEpm in promoting pathogenesis.

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Introduction

Pseudomonas syringae infects numerous plant species and can be classified into
more than 40 pathovars on the basis of host specificity (Gardan et al., 1999). Pst DC3000
is a virulent pathogen of both tomato and the model plant Arabidopsis thaliana. Like
most plant pathogenic bacteria, Pst DC3000 is a noninvasive, extracellular pathogen that
colonizes the host intercellular space outside the plant cell wall. During infection in
Arabidopsis, the pathogen multiplies vigorously for two days before the onset of disease
symptoms, which is characterized by water-soaking in the apoplast, followed by tissue
necrosis and chlorosis (Whalen et al., 1991; Katagiri et al., 2002 ). The TTSS is encoded
by hrp (for hypersensitive reaction and pathogenicity) and hrc (h_rp gene gonserved)
genes (Van Gijsegem et al., 1993; Bonas, 1994; Alfano and Collmer, 1997; Lindgren,
1997; He, 1998; Hutcheson, 1999; Mudgett and Staskawicz, 1998). The TTSS is crucial
to pathogenesis because hrp mutants (e.g., hrpS and hrcC [formerly hrpH] mutants) of
Pst DC3000 are unable to multiply or cause disease in susceptible hosts (Yuan and He,
1996; Roine et al., 1997). This system, which is also utilized by pathogens of mammalian
hosts , encodes a protein secretion machinery that is believed to translocate proteins from
the bacteria directly into the eukaryotic host cell. Pst DC3000 secretes and translocates
numerous effector proteins into the plant host cell, which collectively function to
modulate host cellular processes and cause disease development. Although the primary
function of type III effectors is to promote plant susceptibility, in the event that the
pathogen attempts to infect a resistant cultivar or a non-host plant, some of these effectors
may be recognized by the corresponding plant disease resistance proteins and trigger the

hypersensitive response (HR) (Keen, 1990). HR is a defense-associated programmed cell

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death at the site of attempted infection. In such situations, these virulence effector
proteins have been named avirulence (Avr) proteins (Leach and White, 1996; Colhner et
al., 2001). In fact, most of the avr genes known so far were identified by screening for
altered host response to transformants of a virulent race of P. syringae carrying a
genomic library of an avirulent strain (Staskawicz et. al., 1984; Ronald et al., 1992;
Lorang and Keen, 1995). The presence of an avr gene would result in HR instead of
pathogenesis. Although avr genes were traditionally considered to be negative
determinants of host specificity at the race-cultivar level, there is evidence that many of
these avr genes actually contribute to the virulence of the pathogen in susceptible hosts.
Among these are avrBs2 of Xanthomonas campestris pv. vesicatoria, pthA of X. citri,
aerme from P. syringae pv. maculicola, avrA and aer from P. syringae pv. tomato
strain PT23, avrBs6 from X. campestris pv. maculicola and avaa7 from X. oryzae pv.
oryzae (Kearney and Staskawicz, 1990; Swarup et al., 1991; Swarup et al., 1992; Dangl,
1994; Lorang et al., 1994; Ritter and Dangl, 1995; Yang et al., 1996; Choi, 1993).

Several effectors have also been identified based on approaches that do not
depend on plant reactions. Surveys of secreted proteins found in culture led to the
identification of HrpA, HrpZ and HrpW Wuan and He, 1996). In strains where the
pathogenicity island (PAI) has been sequenced (Alfano et al., 2000), some putative
effectors have been identified by their inclusion in the Conserved Effector Locus (CEL)
or Exchangeable Effector Locus (EEL) associated with the hrp PAI (Badel et al., 2002).
Potential effectors have also been identified either by their location downstream of a
putative hrpL-dependent promoter or by the presence of a potential type 1H secretion

signal (Fouts et al., 1999; Petnicki-chieja et al., 2002; Zwiesler-Vollick et al., 2002).

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Another group used a modified in vivo expression technology (IVET) to identify genes
from the plant pathogen that are induced upon infection of Arabidopsis thaliana (Boch et
al., 2002). Type III effectors have also been predicted based on a genetic screen that
identified effectors based on their ability to be translocated into the host (Guttman et al.,
2002). Combined, these studies, estimated Pst DC3000 to have at least 31 type III
effectors.

Despite the identification of a number of new effectors, demonstration of a
virulence function/contribution of single effectors remains a major challenge in the field.
While P. syringae mutants blocked in the hrp secretion pathway are deficient in their
interactions with plants, phenotypes of mutants deficient in individual effector genes are
too subtle to be observed by traditional methods of virulence assessment such as
determining bacterial populations isolated from infected plant tissues (Kjemtrup et al.
2000). The lack of an effect on virulence has been attributed primarily to functional
redundancy amongst effectors harbored in a single bacterium.

At the time this study was initiated, Pst DC3000 was known to harbor only two
type III effector genes — aer and avrPto. To identify unknown effector genes in Pst
DC3000, I screened for the presence of orthologs of known avr genes from various P.
syringae pathovars. Three type III effectors were identified and were demonstrated to be
translocated by the hrp secretion system. To understand the virulence contribution of
effectors, I expressed averhEpm in susceptible Arabidopsis using a dexamethasone-
inducible promoter (Aoyoma and Chua, 1997). averhEpm transgenic Arabidopsis
exhibited symptoms that mimic those caused by wildtype Pst DC3000 during infection.

Global host gene profiling analysis showed that AverhEp,0 modulated host gene

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expression in a fashion similar to that caused by the Pst DC3000 TTSS. The averhEpm
transgenic plants also supported significant growth of the non-pathogenic hrpH mutant.

These results strongly support that AverhEpto contributes to Pst DC3000 virulence.

Materials and Methods

Bacterial strains and media

Bacterial cultures of Escherichia coli (E. coli) were grown in low salt (5g/L NaCl)
Luria Bertani (LB) medium (Sambrook et a1. 1989) at 37°C. P. syringae cultures were
grown in low salt LB or hrp-inducing minimal medium (Huynh et al., 1989) at 30°C or
20°C. Antibiotics were used at the following concentrations unless otherwise specified —
rifampicin lOOmg/L, kanamycin 50 mg/L, ampicillin 200mg/L, tetracycline lOmg/L,

spectinomycin 50mg/L.

Recombinant DNA techniques

DNA manipulations and polymerase chain reaction (PCR) were performed using
standard protocols (Sambrook et al., 1989). Oligonucleotide primers for sequencing or
PCR were synthesized at Integrated DNA Technology (Coralville, IA). PCR was
performed using HIFI DNA polymerase (Stratagene, Madison, WI). DNA sequencing
was done at the Michigan State University Genomic Technology Support Facility with
Automated DNA sequencers, model 373A (Applied Biosystems, Foster City, CA). DNA
sequences were analyzed with the Assemblylign®, MacVector®, and the Sequence

Manipulation Suite (http://www.bioinformatics.org/sms/index.html). Database searches

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were performed using gapped BLASTN, BLASTP, and BLASTX (Altschul et al., 1997)

( http://www.ncbi.nlm.nih.gov/BLASTO.

DNA gel blots

Pst DC300 genomic DNA was extracted (Chen and Kuo, 1993) and digested
overnight at 37°C with appropriate restriction enzymes (New England Biolabs, Beverly,
MA). The digested DNA was electrophoretically separated on a 0.7 % agarose gel. The
gel was treated with 0.25 M HCl for 5 min and rinsed with distilled water. DNA was
transferred onto nylon membranes (HybondN; Amersham Pharrnacia Biotech
#RPN303B) in 0.4M sodium hydroxide (Sambrook et al., 1989). Approximately 100 ng
of probe DNA was labeled with 32F ATP and purified using BIORAD columns (Cat
#732-6223) according to the manufacturer’s instructions. Membranes were hybridized
overnight at 65°C in hybridization buffer (6X SSC, 1% SDS, 5X Denhardt’s solution,
0.1mg/ml ssDNA, 0.05M Tris pH 8.0, 0.0125M EDTA). Membranes were washed to a
stringency of 0.1X SSC (20 mins; 65°C) and exposed to X-ray film (Kodak Scientific

Imaging Film X-OMAT AR, #1651454).

Cosmid library screening for detection of effector orthologs

A Pst DC3000 cosmid library was plated on LB plates containing tetracycline.
After overnight growth at 37°C, plates were chilled for 30 min at 4°C. Colonies were
lifted onto nylon membranes (MagnaLift; Micron Separation Inc., #NL4HY 08250) and
both the plates and the membrane were marked with ink for orientation. The membranes

were treated twice with 0.5N sodium hydroxide for 2 min each, followed by two

101

treatments with 1M Tris for 5 min each. Membranes were finally treated with 1.5M
sodium chloride, 0.5 M Tris for 5 min and then air dried. The membranes were UV-
crosslinked in UV Stratalinker 1800 (Stratagene, Madison, WI). Bacterial debris sticking
to the membranes were removed by washing the membranes with hybridization buffer,
prior to hybridization. Hybridization was performed as described in the previous section.
Positive colonies for each probe were identified by comparing the films and plates from
which they had been lifted. These colonies were subjected to a second hybridization
screen.
The following primer pairs were used in PCR reactions to amplify full-length avr genes
for use as probes :
averaI : sense 5 ’CCGCTCGAGATGGGCTGTGTATCGAGCACTTCA3 ’
antisense 5 ’ GGACTAGTTTAAAAGTCATCTTCTGAGTCAGAC 3 ’
averhB : sense 5’CCGCTCGAGATGAAAATAGGTACGCAGGCCACC3’
antisense 5 ’ GGACTAGTTTACGAAACTCTAAACTCGTTTACGCA3 ’
aerpt2: sense 5 ’CCGCTCGAGATGAAAATTGCTCCAGTTGCCATAAAT3 ’
antisense 5 ’ GGACTAGTTTAGCGGTAGAGCATTGCGTG3 ’
averhE: sense 5 ’CCGCTCGAGATGAGAATTCACAGTGCTGGTCACAGC3 ’
antisense 5 ’ GGACTAGTTTATCTTCGTGGAGGCATGCCTTT3 ’
averiB: sense 5 ’CCGCTCGAGATGCACGCAAATCCTTTAAGCTCTTTCAAC3 ’
antisense 5 ’ GGACTAGTTTAGTCGCCTAGGAAATTATTTAGTTC3 ’
ORF 4 : sense 5’AGCATATGGGTATCAACAGAGCAGGGTCA3’
antisense 5 ’ C CACTAGTTCACTCCACACGCTGAATAACCATGCTCTC 3 ’

vierhA: sense 5 ’ CTATATGCCGGGTATCAACGGAGCAGGACCA3 ’

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antisense 5 ’ CCACTAGTTCATGGAACAATTTTAAAAGCGTACTT3 ’
averiA: sense 5 ’ C CGCTCGAGATGGGCTGTGTATCGAGCACTTCAAGA3 ’
antisense 5 ’ GGACTAGTTTAAAAGTCATCTTCTGAGTCAGACTG3 ’
averhF
ORFI : sense 5’CCGCTCGAGATGAAGAATTCGTTCGACCGCTTAATCGAT3’
antisense 5 ’GGACTAGTGACCGAACTCTCAGACAAATCCAA3 ’
averhF
ORF 2: sense 5’CCGCTCGAGATGGGTAATATCTGCAATTGCGGGGGCGTC3’

antisense 5 ’GGACTAGTATCGAGGATATTGACCGGTACTTT 3 ’

Cloning and sequencing full-length effector genes

Cosmid DNA was isolated from the positive colonies by a modified alkaline lysis
method (Sambrook et al., 1989), digested with different restriction enzymes, and
separated on 0.7% agarose gels. Southern hybridization was used to identify the single,
smallest fragments that hybridized to their respective probes. Appropriate DNA bands
were excised from agarose gels and cloned into pBS SK (Invitrogen, Carlsbad, CA).
These constructs were sequenced with the M13 forward and reverse primers. The DNA
sequence obtained was identified by BLASTN. Using the initial sequence, further

primers were designed to obtain the complete sequence of the ORF.

Type III translocation analysis using an AerptZ fusion

The truncated aerpt28o-255 gene, which encodes type III secretion/translocation-

incompetent, but biologically active Aerpt2 (Mudgett et al., 2000), was cloned into the

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XbaI-HindIII sites of pUCP19 (Schweizer, 1991). Candidate type III effector genes were
amplified using apprOpriate primers (see below) and cloned into the EcoRI- XbaI sites in
pUCPl9::aerpt2go-255 to create in-frame fusions (5’-effector gene-aerpt280-255-3’). All
gene fusions contained the full-length putative effector genes. The recombinant plasmids
were then electroporated into Pst DC3000. The transformants were grown in LB to OD600
= 0.6. Bacteria were collected by centrifugation and resuspended in sterile water to an
OD600 = 0.2. The bacterial suspensions were infiltrated into leaves of six-week-old RPS2+
Arabidopsis accession Columbia glaborous 1 (Col glI) or rps2 mutant (Kunkel et al.,
1993) plants. Tissue collapse was monitored over a 48 hour period at room temperature.
The following primers were used for making aerpt2 fiisions:
avrPtoB: sense 5’GCGAATTCGGGCATGGAAAAATCCTCTTC3’

antisense 5 ’ GCTCTAGAGCTGTCAGCGCGGTCAGGGGA3 ’
averinw: sense 5’GCG GAT CCG GCT CGA ATC GAT CTT CCC AA3’

antisense 5’GCT CTA GAG TCG CCT AGG AAA TTA TTT AGT TC3’
averhEpm: sense 5’ GCG AAT TCG CAA CTG CTG GAG GTG AGA GC3’

antisense 5’GCT CTA GAG ACA TCT CGT CTC GCC AAG CC3’

Transposon mutagenesis and marker exchange

Cosmid clones containing the full-length ORFs of the identified genes and their
flanking sequences were cloned into the broad host range vector pRK415 (Keen et al.,
1988) and transformed into E. coli DHSa. Two transposons were used for generating the
mutants — mini-T n5 Sp and mini-T n5 Km (Lorenzo et al., 1990). E. coli strains containing

the target cosmids and the transposon were grown to log phase in LB medium. Bacteria

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were then centrifuged and the pellet resuspended in lOmM MgClz. The donor and
recipient strains were mixed and plated onto LB plates and incubated overnight at 30°C.
Colonies were scraped off the LB plate, resuspended in sterile water and plated onto
selective plates containing nalidixic acid (lOmg/L), tetracycline and spectinomycin or
kanamycin. Plasmid DNA was extracted from the transconjugants and the presence of
transposon insertions was confirmed by restriction enzyme digestion and Southern
blotting. Plasmids were then introduced into Pst DC3000 by electroporation. The
inactivated copy of the gene was introduced into the Pst DC3000 genomic DNA by
allelic exchange. Genomic DNA was isolated from the mutant strain and was used to

confirm a successful marker exchange by Southern hybridization.

Plant growth and bacteria enumeration

Arabidopsis Col g1] and transgenic plants were grown in soil or in growth
chambers with a day/night cycle of 12h/12h, a light intensity of 100 uE, and a constant
temperature of 20 °C. Four- to five-week-old plants were used for experiments. Tomato
(Lycopersicon esculentum) cv Castlemart was used for all tomato experiments. Tomato
seedlings were grown in Jiffy peat pots (Hummert International, Earth City, MO) in a
grth chamber maintained under 17 h of light (200 uE-m'z's'l) at 28°C and 7 h of dark
at 18°C. Six- to eight-week-old plants were used for experiments.

Bacteria were grown in low-salt LB to the mid-log phase at 30°C. Bacterial
cultures were centrifuged to recover bacteria, which were resuspended in sterile water to
the required level of inoculum (see Results section). Fully expanded leaves were either

vacuum-infiltrated or syringe-infiltrated with bacterial suspensions, and bacteria were

105

enumerated as described by Katagiri et al. (2002). The mean values of the bacterial
populations were plotted with the standard deviation displayed as error. Plants analyzed
in Figure 3-9 and 3-11 were sprayed daily with 30 uM and 0.1 11M dexamethasone
(DEX) solution respectively, to induce the transgene. Bacterial suspensions were

infiltrated into leaves one day after the first DEX treatment

Generation of transgenic plants

Pst DC3000 genomic DNA was extracted as described by Chen and Kuo (1993).
PCR was used to amplify averhEpm using HIFI Taq polymerase and the following
primers :
averhEpm sense primer 5’-GCACTAGTACCATGAAAATACATAACGCTGGC-3’
averhEpm antisense primer 5’-GGACTAGTTTATCTTCGTGGAGGCATGCCTTT-3’

The averhEpm gene was cloned into the pTA7002 vector (Aoyama and Chua,
1997; McNellis et al., 1998). pTA7002 allows for inducible expression of transgenes
after application of the animal glucocorticoid hormone, dexamethasone (DEX).
Electroporation was used to transform Agrobacterium tumefaciens strain GV3850 with
the recombinant plasmid (Keen et al., 1990). Four pots of Arabidopsis thaliana Col glI
plants were transformed with A. tumefaciens carrying pTA7002-averhEpm using the
floral dip method (Clough and Bent, 1998). Seeds collected from each pot were kept
separate to ensure that independently transformed lines could be isolated. T1 seeds were
vapor-sterilized in a dessicator for 4 hours with 80ml of bleach mixed with 3ml of
concentrated HCl. Seeds were placed on Murashige-Skoog (MS) (Gibco BRL, #11117-

074) plates supplemented with 1x vitamins (SIGMA, #M7150) and 40 units/ml

106

hygromycin B (hyg) (Calbiochem Cat # 400051), kept at 4°C for three days, and then
moved to growth chambers (see previous section).Transformants were selected on the

basis of hygromycin resistance.

Production of antibody

The averhEpm gene was amplified by PCR from Pst DC3000 genomic DNA
using the following primers :
sense primer 5’-GCCATATGAAAATACATAACGCTGGCC -3’
antisense primer 5’- GCCTCGAGGACATCTCGTCTCGCCAAGCC -3’.
The gene was cloned into the pET28(a) vector (Invitrogen, Carlsbad, CA) and
transformed into E. coli BL21(DE3) cells by electroporation (Sambrook et al., 1989).
Protein was induced by addition of lmM IPTG to a mid-log culture and incubated for 4
hours at 37°C. AverhEp,0 protein was extracted from E. coli cells using a standard
protocol (Qiagen, Valencia, CA) and purified using the Ni-NTA column. Purified protein
was analyzed by SDS-PAGE and used to raise antibodies in rabbit at Cocalico
Biologicals, Inc., Reamstown, PA. Pre and post immune sera were obtained and checked

for the ability to recognize the antigen using immunoblot analysis (see below).

Irnmunoblotting

Arabidopsis Col gll and averhEPm plants were sprayed with the appropriate
concentration of DEX and maintained under high humidity for 24 hours. Leaf disks of
2cm2 tissue were collected using a #5 cork borer ( Boekel, #1601BD 1-10), homogenized

in 100111 2X treatment buffer (0.125M Tris Cl pH 6.8, 4% SDS, 20% glycerol, 10% B-

107

mercaptoethanol)and denatured at 100°C for 10 mins. Protein samples were separated on
a 10% SDS-polyacrylamide gel (Sambrook et al., 1989) and transferred onto Irnmobilin—
P membrane (Millipore, #lPVH00010) using a semi-dry transfer apparatus (SEMI PHOR,
Hoefer Scientific Instruments). AverhEp,0 was visualized using the AverhEptO

antiserum, followed by anti-rabbit alkaline phosphatase conjugate and colour reaction

using SIGMA FAST (B5655) .

DEX-induction of transgene expression
A 30mM stock of dexamethasone (Sigma Aldrich Cat # D1756) (DEX) was made
in 100% ethanol. This stock solution was diluted in water to required concentrations (see

Results section) and sprayed onto the plants to induce the transgene.

Microarray analysis

Four- to five-week-old Arabidopsis thaliana Col g1] leaves were vacuum-
infiltrated with bacterial suspensions containing 1x106 cfu/ml bacteria (Katagiri et al.,
2002). Infiltrated leaves were collected at 12, 24, and 36 h post-inoculation, before the
appearance of water-soaking symptoms (at about 48 h) and necrosis and chlorosis (at
about 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 previously (Schaffer et al., 2001). The microarray experiments were
performed using a 864-element subarray enriched for Pst DC3000-regulated genes (R.
Thilmony and S. Y. He, unpublished). The subarray was derived from the Arabidopsis

Functional Genomic Consortium’s microarray slides, each containing about 7,200 unique

108

genes (Schaffer et al., 2001). For microarray analysis of transgenic plants, five to six
week-old averhEPm transgenic plants and Col gl] plants were sprayed with appropriate
concentration of DEX (see Results section) and kept under humidity domes. Tissue was
collected at 24 h post induction and total RNA was isolated from each sample for
microarray analysis.

Supporting Information Table B-1 and B-2 (Appendix B) lists genes that are
regulated in a TTSS-dependent manner in at least two of the three biological replicates
for the DC3000/hrpS mutant comparison in C01 gl1 plants and in both biological
replicates for the averhEpm/Col gl1 comparison. Gene clustering analysis was

performed using the Cluster and TreeView programs (Eisen et al., 1998).

109

Results

Identification of putative effectors genes in Pst DC3000 that are homologous to
known avr genes

At the time this study was initiated, there were only two effector genes known to
be present in Pst DC3000 — aer and avrPto. To discover additional effector genes in Pst
DC3000, ten known avr genes from four different pathovars were chosen (Table 3-1).
The genes were amplified from genomic DNA by PCR using gene-specific primers (see
Materials and Methods). Pst DC3000 genomic DNA was digested with restriction
enzymes, electrophoretically separated, transferred to nylon membranes and hybridized
with the amplified avr gene probes. This analysis revealed the presence of three genes in
Pst DC3000 that were putative orthologs of averhE (Stevens et al., 1998; Mansfield et
al., 1994) and vierhA (Jackson et al., 1999) from P. s. pv. phaseolicola and averiB
(Arnold et al., 2001; Coumoyer et al., 1995) from P. s. pv. pisi respectively. These genes

were named averhEpm, avrPtoB, and averinm.

110

Table 3-1: List of avr genes used for identification of orthologs in Pst DC3000

 

 

 

 

 

 

 

 

 

 

 

Avirulence/virulence gene Pseudomodnas syringae pathovar
averaI maculicola
averh3 phaseolicola
averhE phaseolicola
averhF—ORF l phaseolicola
averhF-ORFZ phaseolicola
vierhA phaseolicola
ORF4 phaseolicola
averiA pisi
averiB pisi
aerptZ tomato

 

 

 

 

111

averhEm averiBm avrPtoB

 

averhE averiB vierhA

Figure 3-1: Southern blot analysis of genomic DNA from Pst DC3000 for the presence
of putative orthologs of known avr genes. Ten ug of genomic DNA was digested with
restriction enzymes (B: BamHI; E: EcoRI; P: PstI) and hybridized with 32P-labelled probe
of the avr gene listed below each set of lanes. The ortholog of averiB has two copies in
Pst DC3000, while orthologs of averhE and vierhA are single-copy genes. The name
designated to each ortholog is indicated on top.

112

Isolation of full-length clones of the three putative effector genes

In order to isolate full-length genes of the orthologs identified in Pst DC3000 by
Southern hybridization, averhE, averiB and vierhA were used as probes to screen a
genomic library of Pst DC3000 constructed in the cosmid pCPP47 (Bauer and Collmer,
1997) using colony hybridization. Three independent cosmids were isolated for avrPtoB,
fourteen cosmids for averinm, and three cosmids for averhEpw. These cosmid clones
were subjected to digestion with three different restriction enzymes and the single,
smallest fragments that hybridized to their respective probes, were identified by Southern
blotting. The identified fragment could contain the full length gene The respective
fragments were subcloned into pBluescript SKII (Strategene, Madison, WI).

Sequencing analysis revealed that all three genes encode hydrophilic proteins with
no similarity to any proteins of known biological functions. A conserved hrp box was
found in the putative promoter region of avrPtoB and averinm, implying the
requirement of the hrp regulatory circuit for expression and secretion. averhEpm
however, had a modified hrp box in its promoter region. The ORF of avrPtoB was 1,652
nt long and encoded a protein of 551 amino acids. AvrPtoB shares 58% similarity with
VierhA at the amino acid level. The averinm ORF was 828 nt long and encoded a
protein of 276 amino acids, identical to that of AveriB. The averhEpm ORF was 1,156
nt long and shared 83% identity with averhE at the nucleotide level and 67% identity at
the amino acid level. Recent analysis of the Pst DC3000 genome reveals that averinm
and averhEpm are located on one of the two plasmids, pDC3000A. A second copy of
averinw and avrPtoB are located on the single circular chromosome (Buell et al., 2003).

The G+C contents of avrPtoB (59%), averhEpm (54%) and averinm (39%) are lower

113

than the G+C content of the Pst DC3000 genome (about 60%; Alfano et al., 2000). No
specific plant organelle-targeting sequences are present in these proteins. None of the

identified effectors have any known functional domains or structural motifs.

Translocation analysis of identified orthologs

The presence of the hrp box motif in the promoter region of all three orthologs
implies that they are possibly secreted and translocated into the host cytoplasm in a hrp-
dependent fashion. To investigate this, we utilized a translocation assay that is based on
the properties of another avr gene, aerpt2, from P. s. pv. tomato JL1065. It has been
established that Aerpt2 has a modular structure and the N-terminally localized secretion
signal can be physically separated from the C-terminal HR-inducing domain. When type
III secretion/translocation-incompetent, but biologically active, Aerpt230-255 (80-255
amino acids; C-terrninal portion) is fused to a protein carrying a type III secretion signal,
the fusion protein can be translocated into the plant cell and elicits a hypersensitive
response (HR) in Arabidopsis plants carrying a functional copy of the corresponding
resistance gene, RPS2 (Mudgett et al., 2000; Guttman and Greenberg, 2001).

We fused full-length AverhEpto, AvrPtoB, and Averinto proteins to the HR
inducing domain of Aerpt2. These expression constructs were electroporated into Pst
DC3000. The ability of Pst DC3000 carrying the recombinant fusions to elicit an HR in
leaves of RPS2 and rpsZ Arabidopsis plants (ecotype Col-0) was examined. The negative
control, wildtype Pst DC3000 carrying the empty vector, caused RPS2-independent, slow
disease necrosis visible only after 24 h post-inoculation. Pst DC3000 expressing the wild-

type Aerpt2 elicited an RPSZ-dependent HR at about 9 hours post-inoculation.

114

Arabidopsis plants exhibited a RPS2-dependent HR for all three effectors at about 14 to
16 hrs after inoculation. These results confirmed that all three identified orthologs were

bona fide type III effectors that travel the hrp pathway and enter the host cell.

115

 

0/14 14/14 14/14 13 /14 11/14 Col gl1 (RPSZ)
0/14 0/ 14 0/14 1/ 14 1/14 rps2

 

 

 

 

 

 

 

 

Col gl1
(RPSZ)

rpsZ

 

Figure 3-2: Type III translocation analysis of identified effectors in Arabidopsis. Full-
length proteins were fused to a truncated Aerpt2 protein (80-255 arrrino acids,
Aerpt230-255). Plasmids were introduced into Pst DC3000. Arabidopsis Col glI (RPS2+)
or rps2 mutant leaves were infiltrated with bacterial suspensions at OD600=0.2 and
evaluated for HR elicitation. 1: DC3000, 2: DC3000 (pAVRRPTZ), 3: DC3000
(pAvrPtoBzzAerpt280_255), 4: DC3000 (pAverinto::Aerpt280-255), 5:
DC3000(pAverhEpto::Aerpt230-255). Col g1] leaves (2 - 5) showing HR collapse appear
wrinkled. Top: Number of leaves showing PER/number of leaves infiltrated for Col g1]
and rps2 plants. Picture was taken 18 h after inoculation. Leaves representing the
majority of each treatment are shown.

116

Generation of mutants by transposon mutagenesis

All three effector genes were cloned into the broad host range vector pRK415
(Keen et al., 1988) and subjected to transposon mutagenesis. avrPtoB and averinm
were mutagenized using the mini-T n5 Sp transposon while the mini-T n5 Km (Lorenzo et
al., 1990) was used to create insertion mutants of averhEpm. Transposons conferring
resistance to different antibiotics were used in order to facilitate the generation of double
mutants of the effector genes.

The insertion events were confirmed by restriction digestion and Southern blot
analyses. The inactivated genes were then introduced into the Pst DC3000 genome by
marker exchange. Genomic DNA isolated from the mutants was analyzed by Southern
blotting to confirm successful marker exchange. Double mutants were generated by
sequential marker exchanges with two effector genes and were also confirmed by

Southern blot analysis.

Analysis of the mutants on Arabidopsis and tomato plants

To determine if the inactivation of one or more of these three genes affects Pst
DC3000 virulence in Arabidopsis, mutant strains were infiltrated into Arabidopsis Col
gll and the plants were scored for both symptom development and bacterial
multiplication for a period of 4 days. Using an inoculum of 105 cfu/ml, a small, but
insignificant reduction in bacterial growth (about 1-fold) was observed for the avrPtoB
mutant. The double mutant of avrPtoB and averhEpw had a more significant growth
reduction, about lO-fold, as compared to that of the wild-type strain. However, symptom

development was not dramatically affected for either the single or the double mutant. The

117

other effectors, Averinto and AverhEp,O did not have a demonstrable virulence
function in this pathosystem, although a double mutant of averinm and averhEpm did
exhibit a slight reduction of about 1-2 fold in bacterial multiplication.

The virulence function of these three effectors was also assayed in tomato plants.
Eight-week-old Castlemart II plants were vacuum infiltrated at 5 X 105 cfu/ml and
disease symptoms and bacterial multiplication were scored 5 days post inoculation. While
averinw and averhEpm mutants showed no significant changes in either symptom
development or bacterial multiplication, the avrPtoB mutant exhibited a significant
reduction in virulence. Tomato plants infiltrated with the avrPtoB mutant showed
reduced chlorosis with a near absence of necrotic specks as compared to plants infiltrated
with Pst DC3000. There was a slight, further reduction in both chlorosis and necrosis in
plants infiltrated with the avrPtoB/averhEpm double mutant. The reduction in disease
symptoms was also accompanied by a 50-fold reduction in bacterial multiplication in this
host.

Although the avrPtoB mutant showed a reduction in virulence on tomato, I was
unable to complement this mutant with a plasmid-bome copy of the gene. It is possible,
that the observed phenotype may be an artifact of secondary mutations that might have
occurred during allelic exchange.

To investigate the possibility of ecotype-specific virulence functions of the three
effector, I also analyzed the response of seventeen Arabidopsis ecotypes to all single and
double effector mutants. None of the ecotypes tested showed any significant alteration in

symptom development for any of the mutants.

118

E. coli (pavr) E. coli (paw-Tn5)

Mini-Tn5
E> %
d

   

Pst DC3000 (pavr-Tn5)

Infiltration Allelic exchange

 

A. thaliana Col gl1
Pst D03000 avr mutant

Figure 3-3: Schematic representation of the strategy employed to obtain insertion
mutants of the identified Pst DC3000 effectors genes. E. coli strains carrying full-length
clones of a given effector gene was mutagenized with mini-Tn5 Sp or mini-Tn5 Km
which confers Sp and Km resistance respectively. The insertion event was confirmed by
restriction analysis and Southern blotting. Plasmids were then introduced into Pst
DC3000 by conjugation and the inactivated copy of the gene was introduced into the Pst
DC3000 genomic DNA by allelic exchange. Genomic DNA from the mutant strain was
used to confirm successful marker exchange by Southern hybridization.

119

Pst pcaooo avrPPhEpi.

avrPtoB

avrPtoB averhEm

averiBMo

averiBm averhEm

 

Figure 3-4: Symptoms of Pst DC3000 mutants in A. thaliana Col glI plants. Bacteria
were vacuum-infiltrated into Col glI leaves at 105 cfu/ml. Pictures were taken four days
after infection.

120

(D

    
  
 
 
   

8 I
I DC3000
7 l
a. I avrPtoB-
E 6
g El averiB-
7’ 5
3 El averhE-
4 I avrPtoB-
3 averhE-
I averiB-

 

averhE-

 

 

days post inoculation

Figure 3-5: Bacterial proliferation in A. thaliana Col glI plants. Bacteria were vacuum-
infiltrated into Col gl1 plants at 105 cfu/ml. Bacterial growth was monitored after four
days. Each bar represents the mean titer of 12 leaf discs from three individual leaves.

Error bars represent standard deviation. DC3000 represents Pst DC3000, averiB
represents averinm, and averhE represents averhEpw.

121

9° so
0 O
71 I #4

7‘
O
1

 

F”
O
1

9‘
o

11

log cfulcm 2

Figure 3- 6. Bacterial proliferation in tomato Castlemart 11 plants. Bacteria were
syringe- -infiltrated into tomato plants at 5 X 105 cfu/ml. Bacterial growth was monitored
after four days. Each bar represents the mean titer of 9 leaf discs fi'om three individual
leaves. Error bars represent standard deviation. DC3000 represents Pst DC3000,

  
 
   

 

I DC3000

El avrPtoB-
averhE-

El averiB-
I averhE-

I avrPtoB-

lavrPtoB-
l
i averhE-

averiB represents averinm and averhE represents averhEpm.

122

 

In planta expression of AverhEpto

Because of the presumed redundancy among type III effectors, inactivation of one
or more genes often does not result in a measurable difference in the ability of the mutant
strain to cause disease symptom development or reduce bacterial multiplication, as
indicated in my work with averiBPm and averhEpw. This redundancy is probably
caused by the presence of a large number of effector genes in a single strain. For example,
analysis of the recently sequenced Pst DC3000 genome suggests the presence of at least
31 different effectors in Pst DC3000. In order to study the effect of a single effector, I
have adopted a new approach that involves the expression of a single effector in
Arabidopsis plants. Of the three genes that were identified, averhEPm was chosen for
further study.

Transgenic Arabidopsis thaliana Col g11 plants that express averhEp“, under the
control of the DEX-inducible promoter were generated. Transgenic lines were selected
based on their resistance to hygromycin. Homozygous lines were used for all experiments.
Four independent averhEpm lines, (141, 222, 342 and 422) were chosen for initial study.
None of these lines showed any gross morphological or developmental abnormalities in
comparison to wildtype Col g1] plants. All four lines were sprayed with 3011M DEX to
induce the transgene. Leaf tissue samples collected 24 hours later were analyzed for
protein expression by immunoblot analysis. Two of these lines, 141 and 222, expressed
high levels of AverhEpt0 upon induction, whereas lines 342 and 422 expressed lower
levels of the protein (Figure 3-7).

Induction of the averhEpm transgene led to the development of a distinct

phenotype. When the averhEpm plants were maintained under high humidity, they

123

showed water-soaking 24 hours after DEX exposure. Chlorosis and necrotic spots started
developing 36 to 48 hours post induction. The induced phenotype in these lines mirrored
the phenotype of Pst DC3000 infection in Arabidopsis. There was a close correlation
between the severity of the induced phenotype and the amount of protein produced upon
induction. The lines that expressed higher amounts of AverhEpto, 141 and 222,
developed extensive necrosis and chlorosis, rendering these lines unfit for further
characterization using bacterial multiplication and gene expression profiling. The 342 and
422 lines showed low and moderate levels of AverhEpto induction and developed
relatively less chlorosis and necrosis. These two lines were used for further
characterization. Protein expression was also analyzed at 24 hours post induction using
different levels of DEX. Decreasing levels of the inducer was found to cause decreasing
amounts of protein production in these lines and decreasing disease-like symptoms (data

not shown).

124

No DEX

3011M DEX

 

Col gl 342 422
B
-
Col gl1 342 422 AverhEm
plants plants plants E.coli

Figure 3-7: (A) Phenotype of averhEpm transgenic plants, lines 342 and 422. Six-week-
old plants were used. Uninduced plants, labeled “No DEX” looked similar to wildtype A.
thaliana Col glI plants. averhEpm transgenic plants labeled “30 11M DEX” were
sprayed with 30 11M DEX and kept under high humidity. Pictures were taken four days
later. Induction of the averhEpm transgene caused the development of chlorosis and
necrosis.

(B) Western blot analysis of AverhEp,o expression in leaves of wild—type A. thaliana Col
g1] plants and AverhEp,o transgenic plants 24 hours after spraying with 30 uM DEX.

125

Growth of non-pathogenic hrpH mutant in averhEm Plants

The striking similarity between the induced phenotype of averhEpw plants and
that of Pst DC3000 infection on Arabidopsis plants prompted us to examine the virulence
contribution of AverhEpm by assessing the susceptibility of the averhEpm transgenic
plants to the hrpH mutant. The hrpH mutant is unable to assemble a functional type III
secretion apparatus and is thus incapable of secreting any type III effector proteins (Yuan
and He, 1996). Wildtype Col g1] and transgenic averhEpm plants were sprayed with
30uM DEX, 24 hours prior to inoculation with bacteria and daily thereafter during the
course of the experiment. While the hrpH mutant was unable to grow beyond the
inoculation level in DEX treated Col gl1 plants, DEX treated averhEPm plants allowed
the hrpH mutant to grow to levels very similar to that of Pst DC3000. Transgenic
expression of averhEPw did not significantly affect Pst DC3000 multiplication because
Pst DC3000 multiplied similarly in averhEpm plants and wild-type Col glI plants. In the
absence of DEX induction, the transgenic averhEpm plants did not allow hrpH to grow
beyond the inoculation level, indicating that the increased susceptibility observed was
due to the presence of the effector protein. The ability of averhEpm plants to support the
growth of the hrpH mutant bacteria could be due to extensive cellular damage caused by
overexpression of averhEpm, resulting in non-specific nutrient leakage. To determine if
the multiplication of the hrpH mutant bacteria can occur in the absence of tissue damage,
I identified a level of induction which did not promote any visible macroscopic
symptoms during the course of the experiment. When induced with 0.1uM DEX,
averhEpm plants did not exhibit any chlorosis or necrosis in response to DEX.

Irnmunoblotting confirmed the presence of AverhEptO in these plants after induction

126

with luM DEX (data not shown). However, I was unable to detect protein in plants
treated with 0.1 11M DEX. Transgenic averhEpm plants and Col gl] plants were sprayed
with 0.1 uM DEX, 24 hours prior to bacterial inoculation and daily during the experiment.
I found that expression of averhEpm allowed 10 to 50-fold increase in the hrpH mutant
population in line 342 and lOO-fold in line 422. At this DEX concentration, leaves
inoculated with the hrpI-I mutant showed some chlorosis in line 342. In line 422
multiplication of the hrpH mutant led to the development of chlorosis and few necrotic
spots. Thus, AverhEpt0 could promote multiplication of and symptom production by the

hrpH mutant in the absence of extensive tissue damages.

127

a ‘ I 342 ocaooo

I 342 hrpH

7 1' [3422 ocaooo

c1422 hrpH

 

log cfulcm2

 

 

 

 

days post lnoculatlon

Figure 3-8: Bacterial proliferation in uninduced averhEpw transgenic plants. Bacteria
were syringe-infiltrated into plants at 106 cfu/ml. Bacterial grth was monitored after
four days. Each bar represents the mean titer of 12 leaf discs from three individual
leaves. Error bars indicate standard deviation. “342" and “422” represent two
independent lines of averhEpm transgenic plants. “DC3000” represents Pst DC3000.
“hrpH” represents the hrpH mutant.

128

    
 
  

 

 

l
ma
4 +Col DC3000
7'5 j-i-Col hrpH j
‘ +342 pcaooo
. 342 hrpH
6.5 4 +422 DC3000 ‘
422 hrpH
E 5.5 ‘
3
s l
8’ l
...l
...i
2.5 4— - ~ . .

0 2 4
days post inoculation

Figure 3-9: Multiplication of the hrpH mutant in averhEpw transgenic plants. Two
independent lines of averhEPw plants were analysed — 342 and 422. Plants were sprayed
with 30 11M DEX, 24 hours prior to bacterial infiltration and daily during the course of
the experiment. Bacteria were syringe-infiltrated into plants at 106 cfu/ml. Bacterial
growth was monitored over four days. Bacterial numbers are the average of 12 leaf discs
from three individual leaves. Error bars indicate standard deviation. Col represents Col
glI plants. “DC3000” represents Pst DC3000. “hrpH” represents the hrpH mutant.

129

7 5 i l IcoI ocsooo
' l I Col hrpH
l l :1 342 DC3000
6.5 g E] 342 hrpH ‘

1 ‘ I422 Dcaooo
l I422 hrpH

   
  

log cfulcmz
9'
0|

9
u
1

 

 

 

days post Inoculation

Figure 3-10: Growth of the hrpH in A. thaliana Col gll and averhEpm plants. Plants
were sprayed with 0.1 uM DEX, 24 hours prior to bacterial infiltration and dail during
the course of the experiment. Bacteria were syringe-infiltrated into plants at 10 cfu/ml.
Bacterial growth was monitored after four days. Bacterial numbers are the average of 12
leaf discs from three individual leaves. Error bars indicate standard deviations. Col
represents Col gll plants. “342” and “422” represent the two lines of averhEpm
transgenic plants. “DC3000” represents Pst DC3000. “hrpH” represents the hrpH mutant.

130

DC3000 hrpH none

 

.’ mxa
l1
3‘ .' I?)
“I
aE 342
f is; A n
it ’.
i 9‘4 4 5‘1.
. *1 .
\
aE 422

Figure 3-11: Growth of the hrpH mutant in averhE p10 transgenic plants is accompanied
by the development of symptoms similar to those of Pst DC3000 infection. Wildtype Col
gll and averhEpw transgenic plants were induced with 0.1 uM DEX, 24 hours prior to
inoculation and daily during the course of the experiment. Bacterial inoculum of 106
cfu/ml was syringe-infiltrated. Symptoms were recorded four days later. “aE 342” and
“aE 422” represent two independent lines of averhEpw transgenic plants, “DC3000”
represents Pst DC3000 and “hrpH” represents the hrpH mutant. Leaves labeled “none”
were treated with DEX but were not infiltrated with bacteria.

131

Expression profiling of plants expressing AverhEpto

The ability of transgenic averhEpm plants to support high levels of the non-
pathogenic hrpH mutant, prompted us to examine the host gene expression profile in
response to the expression of averhEpm in Arabidopsis plants. For this, we used a cDNA
microarray that had been developed in our laboratory and essentially constitutes a gene
expression signature for Pst DC3000 infection of Arabidopsis — PAGES (Pseudomonas
Arabidopsis Gene Expression Signature). The PAGES array was developed by examining
the expression of about 7,200 randomly chosen Arabidopsis genes in pre-symptomatic
leaves inoculated with Pst DC3000 or hrp mutants. A set of 864 genes that were
identified as being reproducibly regulated during the infection of Arabidopsis thaliana
Col gll plants with Pst DC3000 constitutes the PAGES array. The expression of a subset
of 117 genes on the array was found to be associated with the functions of the Pst
DC3000 TTSS. Of the 117 genes, 53 were repressed and 64 were induced in a TTSS-
dependent manner.

In order to determine the virulence contribution of averhEpm, transgenic plants
were sprayed with 30 uM DEX and kept under high humidity conditions. Leaf tissue was
harvested 24 hours later. At this time, the plants develop small amounts of water soaking,
but do not develop any chlorosis or necrosis. Total RNA from this tissue sample was used
to hybridize the microarray. Wildtype Col gll plants sprayed with DEX was used as
control. Both averhEpm lines were assessed in a similar manner and two independent
biological replicates were performed for each line. In planta expression of AverhEptO

was found to regulate approximately 50% of the Arabidopsis genes known to be

132

regulated by Pst DC3000 infection. We also found that 90% of the TTSS-specific gene
cluster was regulated similarly by Pst DC3000 and AverhEpto (Table B-l).

Similar microarray analysis was also performed with averhEpm plants that had
been sprayed with 0.1uM DEX. At this lower level of induction, which causes neither
chlorosis nor necrosis, the transcriptional profile resembled that observed at the higher
level of DEX but appeared to be dampened (Table B-2). The striking similarity between
the Pst DC3000 TTSS- and AverhEpto-regulated host gene expression profiles
demonstrates that averhEPm expression in transgenic Arabidopsis globally mimicked

the Pst DC3000 TTSS functions at the molecular level.

133

few... _..-»:. » :4- I .
K -3 5:17

DC3000 Bi
ocaooo cj I

DC3000A1 u _ _ I
342 j I I E j

I;,1_.,.-.'., W .P.'.. 2;: , _>,,‘_ 1

 

 

DC3000 B
DC3000 C
DC3000 A
342
422

 
    

ItL‘

 

0.2 0.3 0.4 0.5 0.6 1.0 1.5 2.0 2.5 3.0 5.0

 

Fold expression

Figure 3-12: Cluster analysis of the expression profiles of 117 TTSS-regulated genes
(colored bars) after Pst DC3000 infection and transgenic expression of AverhEp,o with
30 1.1M DEX. Rows DC3000 A, DC3000 B and DC3000 C represent Pst DC3000 TTSS-
regulated genes from three independent biological replicates (Table B-l). Rows 342 and
422 represent two independent biological replicates in two independent averhEpm
transgenic lines 342 and 422, respectively, 24 hours after induction with 30 11M DEX

(Table B-l).

134

DC3000 C
DC3000 A t
422
342

i h- a - .l ‘5' rear...» - - .-
DC3000 B i

 

 

DC3000 B
DC3000 C
DC3000 A
422
342

 

0.2 0.3 0.4 0.5 0.6 1.0 1.5 2.0 2.5 3.0 5.0

Fold expression

Figure 3-13: Cluster analysis of the expression profiles of 117 TTSS-regulated genes
(colored bars) afier Pst DC3000 infection and transgenic expression of AverhEpto with
0.1 pM DEX Rows DC3000 A, DC3000 B and DC3000 C represent Pst DC3000 TTSS-
regulated genes from three independent biological replicates (Table B-2). Rows 342 and
422 represent two independent biological replicates in two independent averhEpm
transgenic lines 342 and 422, respectively, 24 hours afier induction with 0.1 pM DEX
(Table B-2).

135

Discussion

The aim of this study was to identify type III effectors of Pst DC3000 and to
investigate their contribution towards the development of disease. At the time this project
was initiated, the Pst DC3000 genome had not been sequenced and Pst DC3000 was
known to harbour only two effector genes — aer and avrPto. While aer is required for
full virulence of Pst strain PT23 on tomato (Lorang et al., 1995; Lorang et al., 1994), and
the homolog of aer in Erwinia amylovora, dspE, is required for pathogenicity on apple
and pear (Bogdanove et al., 1998; Gaudriault et al., 1997; Tharaud et al., 1994), the role
of aer in Pst DC3000 virulence is not very clear. Characterization of transgenic plants
conditionally expressing ssaer (the aer gene tagged with the PR-l signal sequence)
suggested that aer has a role in virulence in the Pst DC3000-Arabidopsis pathosystem
(Zwiesler-Vollick et al., unpublished data). Bacterial mutants lacking aer, however, are
not affected in their ability to infect Arabidopsis (S. Y. He, unpublished results).
Inactivation of avrPto also does not affect virulence of Pst DC3000 on Arabidopsis (Q. L.
Jin, unpublished results), but the expression of avrPto in a less virulent strain, T1, that
lacks the gene, gives it a grth advantage on tomato, indicating that avrPto contributes
to virulence (Shan et al., 2000; Chang et al., 2000). In Pst DC3000, avrPto has recently
been shown to function as a suppressor of the extracellular cell wall-based defenses of its
host, Arabidopsis (Hauck et al., 2003)

In order to identify other putative type III effectors in Pst DC3000, I searched for
putative orthologs of ten known avr genes that had been identified in other pathovars of P.
syringae (Table 3-1). I was able to detect the presence of orthologs of three of the genes

examined using Southern hybridization. These were putative orthologs of vierhA and

136

averhE from P. s. pv. phaseolicola and averiB from P. s. pv. pisi. Of these, only
VierhA has previously been demonstrated to function as a virulence factor. Pph race 7
strain 1449B harbors vierhA on a 154 Kb plasmid. When cured of this plasmid, strains
lose virulence in bean and instead cause an HR defense response in previously
susceptible cultivars. Virulence was restored when vierhA was re-introduced on a
plasmid. Although vierhA has a HR-suppressing function in bean, it was shown to
function like an avr gene in soybean (Jackson et al., 1999). The ortholog of vierhA in
Pst DC3000, avrPtoB, has been shown to be widely conserved among different genera of
plant pathogens including Xanthomonas, Erwinia and many strains of Pseudomonas
(Jackson et al., 1999; Kim et al., 2002; Guttman et al., 2002). AvrPtoB has been shown to
play a role in suppressing programmed cell death and HR-based immunity in tomato
plants (Abramovich et. al, 2003).

averhE was initially cloned from Pph race 4 strain 1302A and confers resistance
on bean cultivars harboring the R2 resistance gene. averhE, like all other avr genes, is
hrp-regulated and shares no similarity with any protein of known function. It is near the
left border of the Pph hrp cluster and is linked to her. Orthologs have been identified in
all eight races of Pph and P. s. tabaci. Disruption of averhE by transposon mutagenesis
blocks induction of HR but does not seem to affect virulence. An interesting aspect of
this gene, however, is that orthologs have also been found in races that are virulent on
cultivars with the R2 resistance locus (Stevens et al., 1998; Mansfield et al., 1994).

averiB confers race-specific resistance to pea cultivars harboring the R3
resistance locus. It contains the hrp box and has homologs in three other races of P. s.

pisi and several other P. syringae pathovars. PCR-RFLP patterns of averiB and its

137

homologs are identical, suggesting conservation of gene structure among P. syringae
strains (Arnold et al., 2001; Coumoyer et al., 1995).

Analysis of the recently sequenced Pst DC3000 genome revealed that our
approach had failed to identify the putative orthologs of two avr genes that were used for
the screen. These were orthologs of averhF ORFl and ORF2. Pst DC3000 has a single
copy of averhF ORF] and ORF2 (Buell et al., 2003; Fouts et al., 2002). These two
genes constitute an operon and have been named averthm. Sequence analysis shows
that averhF p10 is only 59% identical at the nucleotide level to averhF from P. s. pv.
phaseolicola (Tsiasmis et al., 2000), probably explaining our inability to identify this
gene. Consistent with this reasoning, all the orthologs that I identified had high levels of
identity at the nucleotide level to their respective probes: avrPtoB (79%), averhEPm
(72%), and averinm (99%).

The fact that averhEpm and averinm mutants had no detectable reduction in
virulence is not surprising. Bacterial mutagenesis approaches have often proved to be
unsuccessful in detecting subtle virulence phenotypes because of presumed functional
redundancy among the set of effectors harbored by a single pathogen. As a result,
inactivation of one or more genes often does not have a measurable impact on virulence
as assessed by symptom development and bacterial multiplication. The relatively few
effectors that have been shown to have virulence phenotypes were largely demonstrated
to do so by heterologous expression in weakly virulent strains or pathovars (Chang et al.,
2000; Shan et al., 2000; Chen et al., 2000; Guttman and Greenberg, 2001). To date, in P.
s. pv. tomato, only avrA and aer in P. syringae pv. tomato PT23 (Lorang et al., 1994)

and aerme in P. syringae pv. maculicola Psm M2 (Ritter and Dangl, 1995) have been

138

shown to contribute visibly to the symptom development and growth of native strains on
their susceptible hosts.

The avrPtoB mutant was found to be reduced in its ability to multiply in both
Arabidopsis and tomato with a significant reduction in symptom development in tomato
(data not shown). Although this mutation was confirmed by Southern analysis, I was
unable to complement the mutant. I provided the avrPtoB gene in trans on several
different plasmids that range from high to single copy number, as well as the original
cosmid that had been isolated from the Pst DC3000 genomic library. I cloned avrPtoB
both with its own promoter as well as the promoter of the plasmid. None of these
attempts were successful in complementing the mutant. It is possible that the mutant
phenotype observed is not due to inactivation of avrPtoB, but due to secondary mutations
that occurred during the allelic exchange. Plasmid instability could also be an explanation.
Perhaps a wildtype copy integrated into the genome would function better. Interestingly,
an independent study in Dr. Greg Martin’s lab (Cornell University) also showed a
reduced virulence phenotype for avrPtoB. Complementation of the mutant with avrPtoB
was found to be only partial in that case as well (Abramovitch et al., 2003).

Given the limitation of bacterial genetics, we adopted the approach of generating
transgenic Arabidopsis plants that express the desired effector in an effort to study the
virulence function of a single effector in susceptible plants In this chapter, I have
demonstrated that expression of averhEpm in plants caused the development of
macroscopic symptoms such as water soaking, chlorosis and necrosis, that closely
resemble those caused by Pst DC3000 during infection. Arabidopsis plants expressing

averhEPw support growth of the non-pathogenic hrpH mutant.

139

The macroscopic necrosis caused by the high level of AverhEpto protein
complicates the interpretation of the grth promotion by AverhEpto. The aggressive
growth of the hrpH mutant could be due to massive cellular damage in host cells. To
address this issue, I analyzed averhEpm plants at a level of DEX that does not cause the
development of any symptoms during the experiment. It was found that even at this low
level of induction, the transgenic lines still allowed the hrpH mutant to multiply
substantially. Surprisingly, line 342, which allowed 10-fold increase in hrpH mutant
growth, also developed patchy chlorosis. In line 422, the hrpH mutant attained population
levels close to 5.5 log cfu/cm2 leaf tissue. These leaves developed even more significant
symptoms including chlorosis and necrotic spots. Generally, when Pst DC3000 is present
in infected tissue at levels near 5.5 log cfu/cm2 leaf tissue, macroscopic disease
symptoms are not observed. For example, plants infected with the CEL deletion mutant
(Chapter 2), which multiplied to ~ 5.5 log cfii/cm2 leaf tissue, are completely symptom-
free. Therefore, even though low levels of DEX did not elicit macroscopic symptoms in
averhE p10 plants, these plants are potentiated to cellular damages in response to bacterial
inoculation.

A low level of expression of AverhEpto appears to cause microscopic changes in
the host physiology, making the plant more fit for supporting bacterial growth. Such a
function of averhEpm is further supported by the microarray results. At 30uM DEX,
expression of AverhEpto generated a host gene expression profile that is very similar to
that seen in wildtype plants infected with Pst DC3000, with more than 90% of the TTSS-
dependent gene cluster being regulated in a manner similar to that of the Pst DC3000. At

the lower level of DEX induction, the gene expression profile of the transgenic lines is

140

qualitatively similar to that observed at the high level of the inducer. However, the
overall expression is dampened. Therefore, expression of the effector at this low level
brings about molecular changes in the host that occur during disease progression and
renders the host susceptible to the non-pathogenic hrpH mutant, which is not able to
inject any type III effectors into the host. Apparently, the expression of AverhEp,0
compensated for the lack of the effector translocated by the hrp mutant. Our results
strongly support a role for AverhEpto in Pst DC3000 virulence since it renders the host a
better niche for the hrp mutant despite its inability to inject type III effectors into the host
cell.

While we have evidence that supports a role for AverhEpto in Pst DC3000
virulence, we do not know how this effector brings about changes that promote the
diseased state. Pst DC3000 is an extracellular pathogen and colonizes the intercellular
space in the leaf tissue. It is thought that the apoplast maybe limited in nutrients. As a
major component of the Pst DC3000 arsenal, the type III effectors are believed to cause
leakage of water and nutrients into the apoplastic space to allow for high levels of
bacterial multiplication. Water soaking that occurs during the early stage of infection is
believed to reflect the leakage of water/nutrients into the apoplast. Since we observed
water-soaking after DEX-induced expression of AverhEpto in transgenic plants, it is
possible that this effector is involved in nutrient/water release. However, the fact that we
did not detect microscopic water soaking at the low level of DEX treatment, but still
observed significant growth of the hrpH mutant suggests that this is not the primary or

the only function of AverhEpto.

141

Overall, this study identified three type III effectors that were not previously
known to be present in Pst DC3000. The mutagenesis approach failed to demonstrate a
virulence contribution of two effectors in Pst DC3000. Although we did observe a
reduced-virulence phenotype for the avrPtoB mutant, we were unable to draw any
conclusions due to the inability to achieve successful complementation. However, the
transgenic approach coupled with microarray analysis has been helpful in showing that
averhEpm contributes to Pst DC3000 virulence. Given the difficulty in uncovering
functions of individual effectors due to redundancy, this approach might provide a way of

determining the functions of type III effectors in disease development

142

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149

Chapter 4

Conclusions and Future Perspectives

150

The long term objective of this study is to elucidate mechanisms of bacterial
pathogenesis in plants, which should help in the development of improved methods of
pathogen control and reduction of losses in crop production. Bacterial plant pathogens
infect a wide range of crop plants and cause severe losses in crop production worldwide.
Current practices of pathogen control involve the use of large quantities of pesticides and
antibiotics. These methods are costly, non-specific, and a major source of environmental
toxicity. An understanding of the process by which a pathogen triggers disease in a host
plant will enable us to design strategies that target key steps in the process of
pathogenesis, leading to the development of specific, environment-friendly and durable
methods to combat pathogens.

Pseudomonas syringae pathovars cause diseases in important crop plants such as
beans, peas, cucumber, tomatoes and tobacco. Although much research has been
conducted on crop plants, many of them are not very amenable to genetic manipulations.
The model plant Arabidopsis thaliana has many advantages in this respect. These include
a rapid generation time, small genome size, established methods for transformation,
completely sequenced genome, large collection of tagged mutants, and availability of
microarray and other genomic resources. A tomato pathogen, Pseudomonas syringae pv.
tomato DC3000 (PstDC3000), was found to be pathogenic on most tested ecotypes of
Arabidopsis thaliana in the early 19905 (Whalen et al., 1991). Since then, the
Arabidopsis thaliana-PstDC3000 pathosystem has proven to be a useful model system to
study plant-pathogen interactions and results are generally correlated with observations
from other systems (Baker et al., 1997). Pst DC3000 utilizes two known virulence

systems. One is the phytotoxin coronatine. Although coronatine is thought to be primarily

151

responsible for causing chlorosis (Bender, 1987), the mechanism by which this toxin
promotes disease is not yet known. Pst DC3000 also utilizes a type 111 protein secretion
system (TTSS) during pathogenesis. The TTSS secretes bacterial proteins, collectively
referred to as effectors, directly into the plant host cytoplasm. Once they are translocated
into the plant host cell, the effectors promote disease presumably by suppressing host
defense responses and promoting the release of nutrients for utilization by bacteria. My
thesis research focused on understanding the virulence function of type III effectors of
Pst DC3000.

When this study was initiated, only two effectors, Aer and AvrPto, were known
to be present in Pst DC3000. However, bacterial mutants of aer and avrPto do not show
any reduction in virulence (Lorang and Keen, 1995). The lack of phenotype of individual
effector mutations has been attributed to functional redundancy. The ACEL mutant was
generated by the deletion of six ORFs in the Conserved Effector Locus of Pst DC3000
(Alfano et al., 2000). This mutant is unique in that it has a dramatic reduced-virulence
phenotype. Typically, mutation of single and even multiple type III effectors results in
weak virulence phenotypes.

Chapter 2 summarized my research to identify the effector gene(s) that restores
the virulence of the ACEL mutant, and elucidating the mechanism that it plays in
promoting pathogenesis. HothoM (ORF3) and Sth (ORF4), two of the deleted ORFs,
were found to be sufficient to restore the ACEL mutant phenotype to wildtype.
Translocation of HothoM is dependent on Sth, and the first 200 amino acids of
HothoM are required for interaction with Sth. The ACEL mutant was found to be

compromised in its ability to suppress basal host immunity that is characterized by the

152

deposition of callose-containing papillae in the cell wall in response to pathogen attack.
Wildtype Pst DC3000 suppresses this defense response. Furthermore, I found that the
ACEL mutant-activated papillae formation was dependent on salicylic acid, whereas the
hrp mutant-activated papilla formation was independent of salicylic acid. The inability of
the ACEL mutant to suppress papilla formation can also be restored by providing
HothoM and Sth in trans, suggesting that HothoM is a suppressor of an SA-
dependent cell wall defense in Arabidopsis.

Clearly, the next step is to identify Arabidopsis proteins that are targeted by
HothoM to suppress the SA-dependent cell wall based defense. In a yeast two hybrid
screen, I identified a putative aminocyclopropane carboxylic acid (ACC) synthase,
ACSlO, as the only Arabidopsis protein that interacts with HothoM. The first 100 amino
acids of HothoM were found to be sufficient to interact with ACSlO. However,
HothoM with a lOO-amino acid C-terrninal truncation was unable to interact with
ACSlO (Figure A-l). ACC synthase catalyzes the first committed step in ethylene
biosynthesis, and converts S-adenosyl-L-methionine to ACC (Bleeker and Kende, 2000).
Ethylene has been implicated in symptom development during the compatible interaction.
Pst DC3000 causes fewer symptoms in the ethylene insensitive ein2 mutant, with no

reduction of bacterial growth (Bent et al., 1992).

It is possible that the interaction of ACSlO with HothoM alters ACSlO in a
positive or negative fashion, modulating ethylene levels in the plant. In other words,
inability of the ACEL mutant to grow in planta could be associated with its inability to
elevate or repress ethylene levels in the host. To test this hypothesis, I sprayed

Arabidopsis plants with either ACC, which is the substrate for ethylene, or with AVG,

153

which is an inhibitor of ACC synthase, followed by bacterial inoculation. Neither
treatment affected the virulence of ACEL mutant or Pst DC3000. I also monitored the
ability of ACEL to infect ein2 mutant plants, which had been pre-treated with either ACC
or AVG or untreated. However, I did not observe any alterations in ACEL mutant or Pst
DC3000 virulence under any of the experimental conditions (Figure A-2). I also adopted
a genetic approach to examine whether ACSlO is involved in Pst DC3000 pathogenesis. I
obtained two knockout lines for ACSIO from the SALK collection
(http://signal.salk.edu/tdna) and assessed them for differences in their ability to
support/enhance the growth of the ACEL mutant and/or the wildtype pathogen. Although
I confirmed the T DNA insertion by PCR, the mutant plants behaved like wildtype plants
in their responses to both the wildtype pathogen and the ACEL mutant (Figure A-3 and
A-4). The T-DNA insertions in both SALK lines are upstream of the start codon of the
ACSIO gene. It is possible that insertion of the T-DNA in the 5’ UTR did not eliminate
the transcription and translation of this gene. To further investigate whether ACSlO is
involved in HothoM action and Pst DC3000 pathogenesis, it would be helpful to
generate transgenic plants that overexpress the ACSIO gene, and to produce antisense
lines, or new T-DNA insertion lines to provide us new clues about the function of

HothoM.

Because high level DEX-induced expession of HothoM in Arabidopsis results in
a necrosis phenotype, I also initiated a genetic approach to find putative host proteins that
are targeted/required by HothoM function in plants. I have generated a population of
EMS—mutagenized hothoM transgenic seed. Screening for suppressor mutants from this

population is currently underway. We hope to obtain at least two groups of mutants from

154

this screen: i) those that carry lesions in an Arabidopsis protein that is required by
HothoM to cause necrosis and ii) mutants carrying lesions within HothoM itself that
affect its function. This screen can yield mutants from both of the above mentioned
categories. Western blotting will be used to determine if the mutant still synthesizes
HothoM. Only those that synthesize HothoM, but do not develop any symptoms upon
30 uM DEX induction, will be characterized on the basis of their ability to support the
growth of the ACEL mutant when the transgene is induced with low dose of DEX (3 nM)
and the ability to suppress callose deposition activated by the ACEL mutant. We can
determine if the phenotype results from an alteration in the hothoM gene or a host gene
by testing for the ability of ACEL mutant containing HothoM and Sth in trans to
cause disease and suppress callose deposits in the mutant plants. Mutation in a host gene
may block the action of HothoM delivered from bacteria and therefore make bacteria
less virulent, whereas mutation within the hothoM transgene should not affect the
function of bacteria-delivered HothoM, and therefore these mutant plants should remain
susceptible to the bacteria. In summary, we may get mutants that will now enable us to

dissect symptom development, grth promotion and callose suppression by HothoM.

Chapter 3 summarized my research to identify more effectors in Pst DC30000 by
using a gene homology-based method. I was successfirl in identifying three new effectors
in Pst DC3000. These were orthologs of vierhA and averhE from P. s. pv.
phaseolicola and averiB from P. s. pv. pisi, respectively (Stevens et al., 1998;
Mansfield et al., 1994; Jackson et al., 1999; Arnold et al., 2001; Coumoyer et al., 1995).
Using the traditional bacterial mutagenesis method, I did not detect a virulence effect for

either averhEpm or averinm. On the other hand, even though I detected a significant

155

virulence effect for avrPtoB in both tomato and Arabidopsis, I was unable to complement
this mutant and hence the virulence function of avrPtoB was not conclusive. Transgenic
expression of AverhEpto in Arabidopsis revealed its ability to support grth of the non-
pathogenic hrpH mutant. Microarray analysis of transgenic averhEpm plants show
mimicry of Pst DC3000 infection in terms of host gene expression profile by AverhEpto.

Although, my study suggests that transgenic expression of type HI effectors in
Arabidopsis can be used to assess the virulence contribution of a single effector, such
results should be interpreted carefully. During an infection, Pst DC3000 injects effectors
into its host in minute quantities. These quantities are so small, that they are not
detectable using any currently available detection techniques. Thus, we have no estimate
of the quantity of effector that the host experiences during pathogenesis. In a transgenic
plant, even low doses of the inducer probably results in effector quantities that are larger
compared to that during an infection. Hence, our observations could be to an extent, non-
physiological. Therefore, great care should be taken in interpreting data and drawing
conclusions when transgenic plants are used.

How AverhEptO promotes disease remains to be determined. I have performed
experiments that provide clues for future elucidation of the function of AverhEpm. I
conducted a yeast two-hybrid screen to identify Arabidopsis proteins that interact with
AVTPphEth I identified three interactors (Table B-3). Of these, one was a receptor
protein kinase-like protein (At5g20480). Sequence analysis revealed the presence of 13
leucine rich repeats (LRR). It is predicted to be a transmembrane protein with two short
membrane-spanning domains. The LRRs and kinase domain are probably

extracytoplasmic (Figure B-l). The other interactors were a putative phosphoprotein

156

phosphatase (At2g27210) and a putative protein (At3g51650). Interestingly, the putative
phosphoprotein phosphatase transcript accumulated in Arabidopsis plants in response to
infection with the hrpRS mutant. The hrpRS mutant does not assemble the TTSS and
therefore does not secrete any effectors. The At2g27210 transcript was strongly induced
by the hrpRS mutant, only mildly induced by Pst DC3000, and almost not induced by the
ACEL mutant (Figure B-2). It would be interesting to detect whether AverhEptO or other
effectors mediate suppression of this transcript.

SALK lines that contain T-DNA insertions in the receptor-like protein and the
putative phosphoprotein phosphatase are available (Table B-3). These SALK lines can be
used to assess if the presence or absence of the identified interactors is required for Pst
DC3000 to establish a successful infection. This could be done by infecting these plants
with Pst DC3000 as well as averhEpm mutants and observing them for possible
alterations in bacterial multiplication and symptom development. Transgenic plants
overexpressing these interactors can also be useful in studying the impact these host
proteins have on the ability of Pst DC3000 to cause infection. Transgenic plants that
overexpress these interactors can also be crossed with the averhEpm plants. Analysis of
progeny could determine whether overexpression of these host proteins has an inhibitory
effect on the necrosis phenotype induced by transgene expression of AverhEpto.

The field of molecular plant pathology was initially focused on deciphering gene-
for-gene resistance that restricts an avirulent pathogen from infecting its host. Clearly, a
new trend is to understand how compatible interactions proceed in the absence of gene-
for-gene resistance. A better understanding of the compatible interaction can also help us

better understand resistance because type III effectors have been shown to target defense

157

 

 

pathways. The recent completion of the Pst DC3000 genome and its subsequent analysis
by several independent studies has estimated the presence of at least 30 different type III
effectors in Pst DC3000. An understanding of how these effectors modulate the host can
yield a lot of information about the molecular basis of pathogenesis.

My research has provided two important pieces of information in bacterial
pathogenesis. First, I have shown that the CEL of Pst DC3000 encodes effectors that
suppress extracellular cell wall-based defense. The presence of papilla during interaction
of wildtype Arabidopsis plants with non-pathogenic bacteria, including the hrp mutant,
and the ACEL mutant is correlated with their inability to multiply in plants. The cell wall-
based defense could therefore be a critical part of basal plant immunity that keeps
myriads of non-pathogens from establishing themselves in the plants. Second, I have
demonstrated that this basal immunity is partly dependent on salicylic acid. The ACEL
mutant is impaired in suppressing this SA-dependent triggering of callose deposition in
wildtype leaves. SA has been demonstrated to play a major role in mediating defenses,
especially those related to gene-for-gene related resistance (Hunt et al., 1996; Shirasu et
al., 1996). This study demonstrates that SA is also involved in mediating basal defenses
that function even against the successful pathogen. This study provides new insight into
the evolution of a successful pathogen. Acquiring the CEL might have been an important
step in the evolution of the successful pathogen and that is why it is so highly conserved
among the strains in which it has been studied so far.

Finally, I also identified three type III effectors that were not known to be present

in Pst DC3000 at the time this research was initiated. My research suggests that

158

 

transgenic expression is a useful tool to study the function of type III effectors and to

demonstrate their contribution to disease development.

159

References

Alfano, J. R., Charkowski, A. O., Deng, W. L., Badel, J. L., Petnicki-chieja, T., van
Dijk, K., and Collmer, A. (2000) The Pseudomonas syringae Hrp pathogenicity island
has a tripartite mosaic structure composed of a cluster of type III secretion genes bounded
by exchangeable effector and conserved effector loci that contribute to parasitic fitness
and pathogenicity in plants. Proceedings of the National Academy of Sciences of the
United States of America 97, 4856-4861.

Arnold, D. L., Jackson, R. W., Fillingham, A. J ., Goss, S. C., Taylor, J. D., Mansfield, J.
W., and Vivian, A. (2001) Highly conserved sequences flank avirulence genes: isolation
of novel avirulence genes from Pseudomonas syringae pv. pisi. Microbiology 147, 1171-
1 182.

Baker, B., Zambryski, P., Staskawicz, B. and Dinesh-Kumar, S. P. (1997) Signaling in
plant-microbe interactions. Science 276, 726-733.

Bender, C. L., Stone, H. E., Sims, J. J., and Cooksey, D. A. (1987) Reduced pathogen
fitness of Pseudomonas syringae pv tomato Tn5 mutants defective in coronatine
production. Physiological and Molecular Plant Pathology 30, 273-283.

Bent, A. F ., Innes, R. W., Ecker, J. R. and Staskawicz, B. J. (1992) Disease development
in ethylene-insensitive Arabidopsis thaliana infected with virulent and avirulent
Pseudomonas and Xanthomonas pathogens. Molecular Plant Microbe Interactions 5, 372-
378.

Bleeker, A. B. and Kende, H. (2000) Ethylene: a gaseous signal molecule in plants.
Annual Review of Cell and Developmental Biology 16, 1-18.

Coumoyer, B., Sharp, J. D., Astuto, A., Gibbon, M. J ., Taylor, J. D., and Vivian, A.
(1995) Molecular characterization of the Pseudomonas syringae pv. pisi plasmid-bome
avirulence gene averiB which matches the R3 resistance locus in pea. Molecular Plant
Microbe Interactions 8, 700-708.

Jackson, R. W., Athanassopoulos, E., Tsiamis, G., Mansfield, J. W., Sesma, A., Arnold,
D. L., Gibbon, M. J ., Murillo, J ., Taylor, J. D., and Vivian, A. (1999) Identification of a
pathogenicity island, which contains genes for virulence and avirulence, on a large native

plasmid in the bean pathogen Pseudomonas syringae pathovar phaseolicola. Proceedings
of the National Academy of Sciences of the United States of America 96, 10875-10880.

Lorang, J. M., and Keen, N. T. (1995) Characterization of aer from Pseudomonas

syringae pv. tomato: A hrp-linked avirulence locus consisting of at least two
transcriptional units. Molecular Plant Microbe Interactions 8, 49-57.

160

Mansfield, J ., Jenner, C., Hockenhull, R., Bennett, M. A., and Stewart, R. (1994)
Characterization of averhE, a gene for cultivar-specific avirulence from Pseudomonas
syringae pv. phaseolicola which is physically linked to hrp Y, a new hrp gene identified in
the halo-blight bacterium. Molecular Plant Microbe Interactions 7, 726-739.

Stevens, C., Bennett, M. A., Athanassopoulos, E., Tsiamis, G., Taylor, J. D., and
Mansfield, J. W. ( 1998) Sequence variations in alleles of the avirulence gene averhE.R2
from Pseudomonas syringae pv. phaseolicola lead to loss of recognition of the AverhE
protein within bean cells and a gain in cultivar-specific virulence. Molecular
Microbiology 29, 165-177.

Whalen, M. C., Innes, R. W., Bent, A. F. and Staskawicz, B. J. (1991) Identification of

Pseudomonas syringae pathogens of Arabidopsis and a bacterial locus determining
avirulence on both Arabidopsis and soybean. Plant Cell 3, 49-59.

161

 

 

 

Appendix A

This Appendix contains supplementary information for Chapter 2

162

 

Figure A-l: Physical interaction between ACSlO and HothoM in the LexA two-hybrid
system. ACSlO was identified as the only Arabidopsis host protein that interacts with
HothoM. The isolated ACSlO interactor was fused to the DNA binding domain (BD) in
pGILDA and a series of truncated HothoM proteins were fused to the transcriptional
activation domain (AD) in pB42AD. Yeast strains were grown at 30°C for 5 days on
galactose X—gal complete minimal medium. Blue color indicates interaction, whereas
white color indicates no interaction. ACSlO interacted with all fragments of HothoM,
but there was a significant decrease in the strength of interaction between ACSlO and
HothoM lacking 124 amino acids at the C-terminal. 1: BD::HothoM/ADzzempty;
2: BD::HothoM/ADzzACSlO; 3:BD::HothoM100 (first 100 aa)/AD::ACSlO;
4:BD::HothoM200/AD::ACS10;5:BD::HothoM300/AD::ACSlO;6:BD::HothoM400/
AD::ACSlO;7: BD::HothoMSOO/AD22ACSIO; 8: BD::HothoM600/ADzzACSIO.

163

8.5

(I)

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log cfulcm 2

0')

 

5"
01

 

I Col/DC300 I ein2/DC3000 D ColICEL I ein2/CEL
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I ein2/DC3000IAVG El CollDCSOOO/AVG I Col/CELIAVG I ein2/CELIAVG

5

 

 

 

 

Figure A-2 : Multiplication of Pst DC3000, and the ACEL mutant in wildtype
Arabidopsis and ein2 mutant plants after treatment with ACC or AVG. The interaction
of ACSlO with HothoM raised the possibility that alteration of ethylene levels in
plants might alter the interaction with wild-type pathogen or the ACEL mutant. Plants
were sprayed with ACC (ethylene precursor) or AVG (ACS inhibitor) and vacuurn-
infiltrated with 106 cfu/ml bacteria. Each bar represents the mean titer of 12 leaf discs
from three individual leaves. Error bars represent standard deviation. “DC3000”
represents Pst DC3000, “CEL” represents the ACEL mutant, “Col” represents wild-
type Arabidopsis, and “ein2” represents the ethylene insensitive ein2 mutant plant.

164

 

+CoI/Dcaodbw I
+CollCEL
8 . +SALKO48483/DC3000

-)(-SALK048483/CEL __ E

 

 

 

 

a:

 

log cfulcm 2

(’I
1

 

 

l
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days post inoculation

Figure A-3: Growth of Pst DC3000 and the ACEL mutant in Arabidopsis Col gll and
ACSIO knockout lines. Arabidopsis plants containing T-DNA insertions in ACSIO were
obtained from the SALK collection. Wild-type and homozygous mutant plants were
vacuum-infiltrated with 106 cfu/ml bacteria. Bacterial grth was monitored over four
days. Bacterial numbers are the average of 12 leaf discs from three individual leaves.
Error bars indicate standard deviation. “Col” represents Col gll plants. “SALK048483”
represents the T-DNA insertion mutant of ACSlO. “DC3000” represents Pst DC3000.

“CEL” represents the ACEL mutant.

165

Pst DC3000

SALK_048483

 

Figure A-4: Symptom development on wild-type Arabidopsis and ACS10 knockout
plants. Plants were inoculated with 106 cfu/ml bacteria. Pictures were taken four days
later. The knockout line was not altered in response to either bacterium.

166

Appendix B

This appendix contains supplementary information for Chapter 3

167

 

 

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Table B-3: Arabidopsis proteins that interact with AverhEpto in yeast

 

 

 

 

 

Interactor ID At locus Description SALK knockout lines
EIP 1 At5g20480 Receptor SALK_O44334, SALK_O44305,
(3,096 bp) protein kinase- SALK_O44338, SALK_O68675;
like
EIP 2 At3g51650 Putative protein SALK_O3 7943, SALK_O37935,
(2,480 bp) SALK_O79737, SALK_O85968
EIP 3 At2g27210 Putative SALK_O71689, SALK_O72431,
(2,148 bp) phosphoprotein SALK_O72437
phosphatase

 

 

 

 

Interactors were identified in a yeast two-hybrid screen using a LexA-based system
(Clontech, Palo Alto, CA). Two independent libraries (kindly provided by Jonathan
Jones) made using infected and uninfected Landsberg erecta plants were screened. None

of these proteins autoactivated reporters in the absence of AverhEpto.

178

 

MKLsmyrxALruiox'rriierA RFSNETDMQALLEFKSQVSENNKREVLAS
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GSLSKLAII.DLSKNNLTGNFPASLGNLTSLQKLDFAYNQMRGEIPDEVARLTQM
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NQFSSAGVRGTIGYAAPEYGMGGQPSIQGDVYSFGILLLEMFSGKEPTDESFAGD
YNLHSYTKSILSGCTSSGGSNAHDEGLRLVLQVGIKCSEEYPRDRMRTDEAVRELI
SIRSKFFSSKTTITESPRDAPQSSPQEWMLNTDMHTM

Figure B-l: Predicted structural domains of At5g20480, the receptor-like protein kinase
that interacts with AverhEpto. Sequence colored in red denote transmembrane regions;
sequence in blue denote LRR repeats; sequence highlighted in green indicates the protein
kinase ATP binding region signature, and sequence highlighted in yellow indicates —the
Serine/Threonine protein kinases active-site signature. Predictions for the kinase domain
and signatures were obtained using ScanProsite (http://us.expasv.org/cgi-bin/scanprosite).

 

179

hrpRS ' Pst DC3000 ACEL
O 3 6 9 3 6 9 3 6 9

 

 

 

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hrpRS ' Pst DC3000 ACEL

0369 6369

 

 

   

Figure B-2: Northern blot analysis of total RNA isolated from Arabidopsis plants
infected with Pst DC3000, the non-pathogenic hrpRS mutant, and the ACEL mutant with
(A) At3g51650 and (B) At2g27210 as probes. The transcript for At3g51650, encoding a
hypothetical protein, showed early and transient induction during infection by hrpRS and
ACEL mutants but retained elevated transcript levels during Pst DC3000 infection. The
putative phosphoprotein phosphatase transcript was strongly induced by the hrpRS
mutant, mildly induced by Pst DC3000, and almost not induced by the ACEL mutant.

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