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This is to certify that the

dissertation entitled

Type III Protein Secretion in Plant-Bacterial
, Pathogen Interactions

presented by

Wensheng Wei

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1m clowns-p14

Type 111 Protein Secretion in Plant-Bacterial Pathogen Interactions

By

Wensheng Wei

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Genetics Program

1999

ABSTRACT
Type III Protein Secretion in Plant-Bacterial Pathogen Interactions
By

Wensheng Wei

Hypersensitive response and pathogenicity (hrp) genes control the ability of major
groups of plant pathogenic bacteria to elicit the defense response in resistant plants and to
cause disease in susceptible plants. A number of Hrp proteins share significant sequence
similarities with components of type III secretion systems and flagellar assembly
machineries in animal pathogenic bacteria. The hrp gene-encoded type III secretion
system (the Hrp system) is believed to deliver bacterial virulence or avirulence (Avr)
proteins into host cells upon infection. However, the molecular mechanism of type 111
protein secretion is poorly understood in both animal and plant pathogenic bacteria. My
doctoral dissertation research is mainly focused on studying the mechanism of type 111
protein secretion and the function of secreted proteins in a model plant pathogenic
bacterium, Pseudomonas syringae pv. tomato (Pst) DC3000.

I began by examining the function of the hrpA gene, which encodes an
extracellular protein secreted via the Hrp system of Pst DC3000. A non-polar hrpA
mutant was constructed by replacing the hrpA gene open reading frame (ORF) with the
aph ORF. In collaboration with Dr. Martin Romantschuk’s group I demonstrated that
HrpA is a major structural protein of a novel filamentous appendage on the bacterial
surface, called the Hrp pilus. The hrpA mutant could no longer cause disease in

Arabidopsis thaliana or elicit a resistance response in the non-host tobacco or resistant

tomato plants, providing genetic evidence for a critical role of the Hrp pilus in plant-Pst
DC3000 interactions.

In the functional study of the Hrp pilus, I found that the hrpA gene was required
for expression of all examined Pst DC3000 genes (hrc, hrp and avr) that are associated
with the type III secretion. HrpA-mediatcd gene regulation was achieved through
activation of two previously characterized regulatory genes (hrpR and hrpS) at the RNA
level. Ectopic expression of the hrpRS operon restored gene expression in the hrpA
mutant, but did not restore type III protein secretion. These results define a dual role of
the Hrp pilus structural protein in global regulation of the Hrp system and control of type
111 protein secretion in Pst DC3000.

I also explored the possibility of using an Avr protein as a bait to identify host
interacting proteins. Specifically, there is one unique Avr family of proteins, known as
Avr/Pth proteins, that are presumably secreted via the Hrp system of the cotton pathogen
Xanthomonas campestris pv. malvacearum (Xcm). Some members (e. g., Avrb6) of this
family are also required for bacterial virulence in susceptible cotton cultivars. A yeast
two-hybrid system was employed to identify cotton receptors of Avrb6. Afier screening
over 1.8 x 108 cotton cDNA clones, I confirmed that over 152 clones encode proteins that
specifically bind to the bait, the central repeat region of Avrb6. Sequencing analysis
revealed three clones, a serine/threonine kinase homologous to the resistance protein Pto
in tomato, a calcineurin B-like protein, and a putative transcription factor with a C2H2-
type zinc-finger motif. The functional relevance of these cotton proteins to Xcm

avirulence and virulence in plants remains to be determined.

Copyright by
Wensheng Wei

1999

To my parents

ACKNOWLEDGMENTS

I feel fortunate to have Dr. Sheng Yang He as my supervisor and mentor. I thank
him for giving me the opportunity to work on many stimulating'projects. His dedication
and enthusiasm have been a great motivation for me in completing my degree. He has
helped me to become a better scientist and a better person.

I would like to express my gratitude to other members of my advisory committee:
Drs. Johnathan Walton, Pamela Green, Ray Hammerschmidt, and Frans de Bruijn, for
their guidance, encouragement, and valuable advice.

It is always a pleasure to thank my colleagues Anne Plovanich-Jones, Jing Yuan,
Julie Zweisler—Vollick, Paula Lee, Qiaoling Jin, Roger Thilmony, Suresh Gopalan, Sruti
Bandyopadhyay, and Wenqi Hu. It has been most beneficial to work in this joyful,
fiiendly and supportive environment. In my experiments and in writing this dissertation I
have consistently received their advice, support and encouragement. I thank Karen Bird
for critically reading this dissertation, and thank Dr. Tao Sang for help in sequencing.

I want to credit our collaborators Drs. Martin Romantschuk, Elina Roine, Alan
Collmer, Wen-Ling Deng, and Hsiou-Chen Huang.

I wish to acknowledge the role of the Plant Research Labs. It is an outstanding
department that has fostered my development in a supportive atmosphere with colleagues
of the highest standing in the scientific community and support staff that is always
willing to help.

Finally, I would like to thank my friend Tianying Liu for her understanding,

tolerance, and support.

vi

TABLE OF CONTENTS

LIST OF TABLES ............................................................................................................ x

LIST OF FIGURES ..................................................................... . .................................. xi
CHAPTER 1

LITERATURE REVIEW AND INTRODUCTION ........................................................ 1

1.1. Plant Responses to Pathogen Attack ................................................................... 1

1.1.1. Hypersensitive Response (HR) .................................................................. 2

1.1.2. The Gene-for-Gene Hypothesis ................................................................. 4

1.1.3. Disease Resistance (R) Genes .................................................................... 5

1.2. Bacterial Pathogenicity ........................................................................................ 6

1.2.1. Pathogenicity Islands ................................................................................. 7

1.2.2. The Type III Secretion System in Bacteria ................................................ 8

1.2.2.1. The Type 111 Protein Secretion System .......................................... 9

1.2.2.2. hrp Genes ..................................................................................... 11

1.2.2.3. The Regulation Cascade Controlling the Hrp System ................. 15

1.2.2.4. Secreted Proteins and Secretion Signals of the Hrp
Secretion System .......................................................................... 16
1.2.3. Similarity between Type III Secretion System and

F lagellar Assembly ................................................................................ 20

1.2.4. Interkingdom Transport Hypothesis ...................................................... 20

1.3. Project Summary ............................................................................................... 22

1.4. References ......................................................................................................... 25
CHAPTER 2

THE HRPA GENE ENCODES THE STRUCTURAL PROTEIN OF A BACTERIAL
SURFACE APPENDAGE ASSOCIATED WITH TYPE III PROTEIN SECRETION

IN PSEUDOMONAS SYRINGAE PV. TOMA T 0 DC3000 ............................................ 35
2.1. Abstract ............................................................................................................ 36
2.2. Introduction ...................................................................................................... 37
2.3. Materials and Methods ..................................................................................... 39

2.3.1. Culture Conditions ................................................................................. 39
2.3.2. Analysis of Bacterial Extracellular Proteins .......................................... 39
2.3.3. Construction of the hrpA Mutant ........................................................... 40
2.3.4. Partial Purification of Bacterial Surface Structures
(F lagella and Hrp Pili) ........................................................................... 43
2.3.5. Transmission Electron Microscopy (TEM) Analysis ............................ 43
2.3.6. Pathogenesis Assays .............................................................................. 44
2.4. Results .............................................................................................................. 45
2.4.1. Mutational Analysis of the hrpA Gene .................................................. 45
2.4.2. Formation of an hrp Gene-Dependent Pilus by DC3000
on Solid hrp-Inducing Medium ............................................................. 47
2.4.3. Identification of Proteins Associated with the DC3000 Hrp Pilus ........ 49

vii

2.4.4. No Hrp Pilus can be Detected in the hrpA Mutant ................................ 52

2.4.5. Pathogenesis Assays .............................................................................. 54
2.5. Discussion ........................................................................................................ 56
2.6. References ........................................................................................................ 61
CHAPTER 3
Role of the Hrp Pilus in Type III Protein Secretion in PseudomOnas syringae
pv. tomato DC3000 ......................................................................................................... 64
3.1. Abstract ........................................................................................................... 64
3.2. Introduction ..................................................................................................... 65
3.3. Materials and Methods .................................................................................... 67
3.3.1. Bacterial Strains, Plasmids and Growth Conditions ............................. 67
3.3.2. Construction of the hrpS Mutant .......................................................... 68
3.3.3. Protein Immunoblot Analysis ............................................................... 70
3.3.4. RNA Blot Analysis ............................................................................... 71
3.3.5. Mutagenesis of the hrpA Gene .............................................................. 72
3.3.6. Pathogenesis Assays ............................................................................. 73
3.4. Results ............................................................................................................. 74
3.4.1. Effect of a hrpA Deletion Mutation on the Production of HrpW ......... 74
3.4.2. hrpA is Required for Expression of Other Hrc/Hrp Proteins ................ 75
3.4.3. hrpA Controls the Expression of hrp, hrc, and avr Genes at the
RNA Level ............................................................................................ 77
3.4.4. Three Amino Acid Residues Located at the Carboxyl Terminus
Are Essential for HrpA-Mediated Gene Regulation ............................. 79
3.4.5. HrpA-Mediated Gene Regulation is Through Activation of
hrpS and hrpR at the RNA Level .......................................................... 81
3.4.6. hrpA is Required for Secretion of HrpW and AvrPto ........................... 83
3.5. Discussion ....................................................................................................... 86
3.6. References ....................................................................................................... 91
CHAPTER 4
IDENTIFICATION OF COTTON TARGETS OF XANTHOMONAS CAMPESTRIS
PV. MAL VA CEARUM AVR/PTH FAMILY OF PROTEINS ........................................ 96
4.1. Abstract...................... .................................................................................... 96
4.2. Introduction .................................................................................................... 98
4.3. Materials and Methods ................................................................................. 101
4.3. l . Agrobacterium-Mediated Transient Expression ................................ 101
4.3.2. Yeast Two-Hybrid System ................................................................. 102
4.3.3. Total RNA Isolation from Cotton Plant ............................................. 103
4.3.4. cDNA Library Construction .............................................................. 104
4.3.5. Construction of Bait Plasmid ............................................................. 104
4.3.6. DNA Sequencing and Analysis ......................................................... 105
4.3.7. Arabidopsis thaliana Transformation ................................................ 106
4.4. Results .......................................................................................................... 107
4.4.1. Agrobacterium-Mediated Transient Expression of Avrb6
and AvrBlOl in Cotton ...................................................................... 107

viii

4.4.2. Construction and Test of the Bait Plasmid ........................................ 109

4.4.3. Yeast Two-Hybrid Screening ............................................................ 112

4.4.4. Sequencing Analysis of cDNA Clones .............................................. 114

4.4.5. Functional Study ................................................................................ 119

4.5. Discussion .................................................................................................... l 19

4.6. References .................................................................................................... 125
CHAPTER 5

CONCLUSION AND FUTURE PERSPECTIVES ..................................................... 129

References ............................................................................................................ 140

LIST OF TABLES

Table 2.1. Plant reactions to DC3000 and the hrpA mutant ........................................... 55
Table 3.1. Plant reactions to DC3000 and various hrp mutants ................................... 85

Table 4.1. Cotton cDNA clones encoding proteins specifically binding
to Avrb6ANC ............................................................................................... 115

LIST OF FIGURES

Fig. 1.1. The hrp gene cluster of Pseudomonas syringae ............................................... 14
Fig. 2.1. A flow chart of construction of a non-polar hrpA mutant in Pst DC3000 ....... 42

Fig. 2.2. A SDS/PAGE and an immunoblot analysis of protein samples from
surfaces of DC3000, hrpA mutant, and hrpA mutant containing pHRPA ........ 46

Fig. 2.3. Detection of the Hrp pilus on the surface of Pst DC3000 ................................ 48

Fig. 2.4. Purification of Pst DC3000 extracellular appendages
(flagella and Hrp pili) ........................................................................................ 51

Fig. 2.5. Transmission electron microscopic examination of surface appendages
on Pst DC3000 hrpA mutant and hrpA mutant containing pHRPA
grown on solid hrp-inducing medium .............................................................. 53

Fig. 2.6. Models for the function of the Hrp pilus in the transport of Pst DC3000
virulence or avirulence factors from the bacterium to the plant ...................... 60

Fig. 3.1. Immunoblot analysis of HrpW, Hch, and HrcC proteins in Pst DC3000
and hrp mutants in hrp-inducing medium and in planta .................................. 76

Fig. 3.2. RNA blot analysis of hrp W, hrcC and avrPto transcripts in Pst DC3000
and hrp mutants ................................................................................................ 78

Fig. 3.3. A diagram of the P. syringae pv. tomato DC3000 HrpA protein and
immunoblot analysis of the effect of single amino acid mutations of
HrpA on the production of HrpW, Hch, and HrcC in hrp-inducing
medium ............................................................................................................ 80

Fig. 3.4. (A) Effect of the hrpA mutation on the steady-state message abundance
of hrpRS. (B) Effect of ectopic expression of hrpRS on the accumulation
of Hrp/Hrc proteins in the hrpA mutant. (C) Effect of ectopic expression
of hrpRS on the accumulation of Hrp/Hrc proteins in the hrpA mutant

in planta. .......................................................................................................... 82
Fig. 3.5. Effect of ectopic expression of the hrpRS operon in the hrpA mutant

on HrpW and AvrPto secretion ......................................................................... 84
Fig. 4.1. A. tumefaciens-mediated transient expression in cotton leaves ...................... 108

xi

Fig. 4.2. Activation of yeast Gall promoter by different deletion constructs of
Avrb6 ............................................................................................................. 11 1

Fig. 4.3. Summary of yeast two-hybrid screening of cotton cDNA library .................. 113

Fig. 4.4. Amino acid sequence comparison of Cpk with three other protein
kinases from plants ........................................................................................ 116

Fig. 4.5. Amino acid sequence comparison of ch with three calcineurin B-like
proteins from Arabidopsis thaliana ............................................................... 1 17

Fig. 4.6. Amino acid sequence comparison of th with two hypothetical proteins
from Arabidopsis thaliana ............................................................................. 118

Fig. 4.7. Models for the role of Avrb6 in the cotton nucleus ........................................ 124

Fig. 5.1. Regulation of flagellar assembly in S. typhimurium and type 111 protein
secretion in Y. pseudotuberculosis by export of negative regulators ............. 133

Fig. 5.2. Models of the regulatory function of HrpA in the type 111 protein
secretion system in Pseudomonas syringae pv. tomato DC3000 ................... 135

xii

Chapter 1

LITERATURE REVIEW AND INTRODUCTION

Plant disease has had disastrous effects on crop production and the well-being of
human populations. The range of phytopathogenic organisms that attack plants is diverse
and includes viruses, mycoplasmas, bacteria, fungi, nematodes, protozoa, and parasites.
Each has a unique mode of pathogenicity (Staskawicz et al., 1995). Plant disease can
result in enormous economic loss. For example, the worldwide losses of major crops,
including rice, maize, potatoes, soybeans, cotton and coffee, were estimated to be 13.3%
($76.9 billion) from 1988-1990 (Baker et al., 1997). Protection of crops from disease can
substantially improve agricultural production. Thoughtful application of new
technologies based on molecular understanding of plant defense mechanisms, combined
with understanding of the complex ecology of the disease process, can lead to more

effective protection strategies against plant pathogens.

1.1. Plant Responses to Pathogen Attack

Microbial pathogens are responsible for numerous diseases in higher plants,
animals, and humans worldwide, including such common ones as rice leaf blight, apple
fire blight, and plague (He, 1998). Plants are sessile organisms that lack a circulating,
somatically adaptive immune system to protect themselves against pathogens. They
instead have evolved other mechanisms for defense against a spectrum of pathogens.

Plants are, in fact, resistant to most microorganisms by means of constitutive chemical or

physical barriers such as the plant cuticle which consists of layers of wax that a potential
pathogen must penetrate or bypass to incite disease. Disease is therefore the exception
rather than the rule when microbes and plants meet. Yet, yield loss due to plant disease
remains an important component of modern agriculture, as many pathogens are
evolutionarily specialized to overcome preformed defense barriers (Dangl and Holub,

1997).

1.1.1. Hypersensitive Response (HR)

Plant strategies used to inhibit the growth of pathogens include the activation of
localized cell death (classically referred to as the hypersensitive response, HR)
(Greenberg, 1997), the release of reactive oxygen intermediates (Lamb and Dixon, 1997)
and nitric oxide (Delledonne et al., 1998; Dumer et al., 1998), the fortification of plant
cell walls, and the production of numerous antimicrobial secondary metabolites and
defense-related proteins (Hammond-Kosack and Jones, 1997). Physiological features
associated with the HR include a rapid oxidative burst, ion fluxes characterized by K+-H+
exchange, cellular decompartmentalization, cross-linking and strengthening of the plant
cell wall, production of antimicrobial compounds (phytoalexins), and induction of
pathogenesis-related (PR) proteins such as chitinases and glucanases (van Kan et al.,
1992). Although the molecular mechanism is obscure, the HR is likely to be important
for limiting a pathogen’s nutrient supply, since the dying tissue rapidly becomes
dehydrated (Staskawicz et al., 1995).

Programmed cell death (PCD) is the active process of cell death that occurs

during development and in response to environmental cues (Greenberg et al., 1994). The

HR appears to be a form of PCD in plants. The appearance of HR is genetically
controlled and purified HR-inducing factors from bacteria, called harpins, will not induce
the HR unless the plant tissue is transcriptionally active (He at al., 1993). In addition,
HR-inducing bacteria will not cause the HR if protein synthesis 'is blocked in the plant.
In the search for a signal for HR induction, several groups have determined that H202 is
rapidly produced by plant cells in culture during the HR in a phenomenon termed the
oxidative burst. Levine et al. ( 1994) showed that enhancing H202 production during the
HR leads to dramatic increases in the amount of cell death observed in a soybean cell
culture system. Alvarez et al. (1998) showed that inoculation of Arabidopsis leaves with
avirulent Pseudomonas syringae induced secondary oxidative bursts in discrete cells in
distant tissues, leading to low-frequency systemic micro-HRS. The primary oxidative
burst induces these systemic responses, and both the primary burst and the secondary
microbursts are required for systemic immunity. Nitric oxide, which acts as a signal in
the immune, nervous and vascular systems, was found to potentiate the induction of
hypersensitive cell death in soybean cells by reactive oxygen intermediates and function
independently of such intermediates to induce genes for the synthesis of protective
natural products. Moreover, inhibitors of nitric oxide synthesis compromise the
hypersensitive disease-resistance response of Arabidopsis leaves to Pseudomonas
syringae, promoting disease and bacterial growth (Delledonne, 1998).

In addition, the induction of the HR by some pathogens and elicitors may be
mechanistically similar to apoptosis in animals, since apoptotic features such as DNA
breaks with 3’OH ends, blebbing of the plasma membrane, as well as nuclear and

cytoplasmic condensation are present in some cells undergoing the HR. In some cases

the HR is also accompanied by intemucleosomic DNA cleavage, another apoptosis-
associated event (Mittler et al., 1995; Ryerson and Heath, 1996). In addition, some
caspase inhibitors specifically inhibit Pseudomonas syringae pv. tomato DC3000-
induced HR on tobacco (unpublished data from our laboratory). Programmed cell death,

therefore, is likely involved in plant defense against pathogenic attack.

1.1.2. The Gene-for-Gene Hypothesis

Many instances of plant disease resistance follow the gene-for—gene model. It
was first proposed by Flor, who demonstrated that resistance of flax to the fungal
pathogen Melampsora lini was a consequence of the interaction of paired cognate genes
in the host and the pathogen (Flor, 1971). Genetic analyses in many systems have
demonstrated that recognition functions are provided by dominant alleles of genes in the
plant (Resistance, or R-genes) that interact, either directly or indirectly, with either the
direct or indirect product of a pathogen gene (avirulence, or avr gene). The gene-for-
gene hypothesis is a genetic explanation for the interaction between plants and
representatives of all classes of pathogens: fungi, bacteria, viruses, and insects.

The biochemical explanation of gene-for—gene resistance is not known for most
cases. However, evidence suggests that molecular recognition of some bacterial
avirulence determinants occurs inside plant cells by resistance proteins, either directly or
indirectly. This hypothesis is based primarily on research in bacteria showing that
several avirulence proteins function inside the plant to induce resistance-gene-dependent

cell death (Gopalan et al., 1996; Leister et al., 1996; Scofield et al., 1996; Tang et al.,

1996; Van den Ackerveken et al., 1996). More details will be discussed later in this

chapter.

1.1.3. Disease Resistance (R) Genes

Plant disease resistance (R) genes confer an ability to resist infection by pathogens
expressing specific corresponding avirulence genes. Intense effort in the past decade has
led to successes in cloning R genes conferring resistance to a wide spectrum of plant
pathogens including viruses, bacteria, fungi, and nematodes. A number of R genes have
been isolated from several plant species (Hammond-Kosack and Jones, 1997). Many of
them encode transmembrane or cytoplasmic proteins containing leucine-rich repeats
(LRRs), which suggests protein-protein interactions.

R genes can be grouped into five classes (Baker et al., 1997). The structural
feature shared by their products is a leucine-rich repeat (LRR) motif and/or a serine-
threonine kinase domain. The first class encodes cytoplasmic receptor-like proteins that
contain an LRR domain and a nucleotide binding site (NBS). The family of genes
encoding proteins with the LRR-NBS motif includes RPSZ, RPM] and RPS5 from
Arabidopsis (Grant et al., 1995; Mindrinos et al., 1994; Warren et al., 1998); Prf from
tomato (Salmeron et al., 1996); N from tobacco (Whitham et al., 1994); L6 and M from
flax (Lawrence et al., 1995); RPP5 from Arabidopsis (Parker et al., 1997); 12 from tomato
(Ori et al., 1997); Cre3 from wheat (Lagudah et al., 1997); Mi-I from Lycopersicon
peruvianum (Vos et al., 1998); and Dm3 from lettuce (Meyers et al., 1998). The second
class of genes includes Pto, which confers resistance to strains of the bacterial pathogen

Pseudomonas syringae pv. tomato that carry the avrPto gene (Martin et al., 1993). Pto is

the first ‘gene-for-gene’ R gene to be cloned from tomato. Unlike either of the LRR
classes of R genes, Pto is a member of the Raf family of serine-threonine protein kinases.
The third class includes Cf-Z and Cf-9 from tomato and HS]”"’” from sugar beet. Cf-Z
(Dixon et al., 1996) and Cf-9 (Jones et al., 1994) encode putatiVe transmembrane
receptors with large extracytoplasmic LRR domains and confer resistance to different
races of Cladosporiumfulvum. HSIP'O'I (Cai et al., 1997) encodes a transmembrane LRR
protein that confers resistance to the beet cyst nematode Heterodera schachtii. The
fourth class is represented by the rice gene Xa21, which confers resistance to the bacterial
pathogen Xanthomonas oryzae pv. oryzae (Song et al., 1995). Xa21 encodes a putative
transmembrane receptor with an extracellular LRR domain and an intracellular serine-
threonine kinase domain. The Xa21 structure suggests an evolutionary link between
LRR proteins (Ct) and kinases (Pto). The fifth class includes the HM] gene, which
confers resistance to the fungal pathogen Cochliobolus carbonum race 1 (Johal and
Briggs, 1992). HM] encodes a nicotinamide adenine dinucleotide phosphate (NADPH)-
dependent reductase that inactivates the host-selective toxin produced by C. carbonum
race 1. HM] is distinct from other R genes because an Avr component is not involved in
toxin degradation by the HM] product. The common features among different R gene

products suggest that certain signaling events are held in common in plant defense.

1.2. Bacterial Pathogenicity

The common Gram-negative phytopathogenic bacteria include Agrobacterium,
Erwim'a, Pseudomonas, Xanthomonas, and Ralstonia. Any given type of symptom can

be caused by bacterial pathogens belonging to several genera, and each genus may

contain pathogens capable of causing different types of diseases, such as leaf spots and
blights; sofi rots of fruits, roots, and storage organs; wilts; overgrowths; scabs; and
cankers. Bacterial pathogens have evolved a variety of strategies to subvert host cellular
functions in order to secure their multiplication and survival. These strategies are often
very complex and fine-tuned, particularly in the case of pathogens that have co-evolved

or have had a long-standing association with their hosts (Galan and Bliska, 1996).

1.2.1. Pathogenicity Islands

Since the early 19808, independent molecular characterization of genes
controlling bacterial pathogenicity of animals and plants have led to the discovery of
large clusters of pathogenicity genes. Such gene clusters are either in extrachromosomal
elements, such as large plasmids in Ralstonia solanacearum (Arlat et al., 1996), Yersinia
(Goguen etal., 1984), and Shigella (Sansonetti et al., 1982), or in specific chromosomal
regions called pathogenicity islands (Hacker et al., 1997), such as those in Salmonella
(Mills et al., 1995), Erwinia amylovora (Barny et al., 1990), Pseudomonas syringae
(Lindgren et al., 1986), Xanthomonas campestris (Bonas et al., 1991), and
enteropathogenic Escherichia coli (EPEC) (Jarvis et al., 1995).

A pathogenicity island can be defined according to the following criteria (Hacker
et al., 1997):

i) Presence of (ofien many) virulence genes
ii) Presence in pathogenic strains and absence or sporadic distribution in less-
pathogenic strains of one species or a related species

iii) Different G-C content in comparison to DNA of host bacterium

iv) Occupation of large chromosomal regions (often 30 kb)
v) Represent compact, distinct genetic units, often flanked by direct repeats
vi) Association with tRNA genes and/or insertion sequence (IS) elements at their
boundaries
vii) Presence of (often cryptic) ‘mobility’ genes (IS elements, integrases, transposases,
origins of plasmid replication)
viii) Instability
The G—C content of pathogenicity islands usually does not match that of other
bacterial DNA. For example, the human intestinal bacterium Escherichia coli has an
average G-C content of 5 1%. But the genes in the diarrhea-causing enteropathogenic
strain of E. coli are on a pathogenicity island with a G-C content of 38% (Barinaga,
1996). It is important to point out that the clustering of genes and the sometimes unusual
G-C content of these gene clusters are often taken as evidence for horizontal transfer of
an entire block of genes with related function. Acquisition of an entire viru1ence system
in a single step would potentially enable an otherwise nonpathogenic or weakly
pathogenic bacterium to become highly virulent on a new host. Acquiring a
pathogenicity island would therefore be a major evolutionary step in the life of a

pathogen.

1.2.2. The Type III Secretion System in Bacteria

In the past decade, research into the molecular basis of bacterial pathogenesis has
led to the conclusion that although different bacteria may use unique mechanisms to

subvert hosts, a few strategies are common. A recent development is the identification of

a highly specialized protein secretion system in several Gram-negative bacteria. This
system, which is termed type III, has evolved to deliver proteins from the bacterial
cytoplasm to the host cytosol (Salmond and Reeves, 1993). These bacterial proteins can
then stimulate or interfere with host cellular processes, thereby dictating the terms of the
bacterial-host cell interaction. Type III secretion systems are present in both animal and
plant pathogenic bacteria, which indicate that they are capable of operating not only

across bacterial genera but also across host kingdoms (Galan and Collmer, 1999).

1.2.2.1. The Type 111 Protein Secretion System

Pathogenicity of Gram-negative bacteria is commonly found to require the type
III secretion system. Type III secretion systems have three distinguishing features: (i) the
absence in the secreted proteins of a cleavable signal peptide that is characteristic of
proteins secreted via the sec-mediated general secretion pathway; (ii) the requirement for
customized accessory proteins (chaperones) for many of the secreted proteins; and (iii) a
widespread requirement of host cell contact for full activation of the secretion pathway
(Galan and Collmer, 1999).

The architecture of the cell envelope of Gram-negative bacteria dictates that
proteins destined to be delivered to the outside must traverse several barriers: the inner
membrane, the periplasmic space, the peptidoglycan layer, and finally the outer
membrane (Agrios, 1997). Consequently, these bacteria have evolved a variety of
mechanisms to transfer proteins from the cytoplasm to the extracellular environment
(Pugsley, 1993). Type III secretion systems are distinct from those of Types I and II

(Salmond, 1994). Type 1 systems are sec-independent, involving secretion via a one-step

mechanism through a channel or gated pore formed between the inner and outer
membranes (Pugsley, 1993; Salmond, 1994). Type II pathways (also known as the sec or
general secretion pathways) involve a two-step mechanism. The first step involves
export from the cytoplasm to the periplasm via a sec-dependent mechanism requiring
cleavage of a NHz-signal sequence, and the second step involves transport across the
outer membrane (Pugsley, 1993; Salmond, 1994). It is believed that all enzymes secreted
via this pathway are synthesized with a classical N-terminal signal sequence and are
processed by signal peptidase en route to the periplasm (Salmond, 1994).

All known type III secretion systems of animal and plant pathogenic bacteria
share a number of core structural components that are highly conserved (Hueck, 1998;
Lee, 1997). These components can be divided into at least two groups. One group
consists of predicted outer membrane proteins, including a protein with sequence
similarity to the secretin family of protein transporters, as well as several less conserved
lipoproteins. The other group consists of several integral membrane proteins with close
similarity to components of the flagellar export apparatus.

Type III secretion systems are used for different purposes in different bacteria,
reflecting the distinct habits of these pathogens (He, 1998). Plant pathogenic bacteria are
extracellular pathogens. The type III secretion system is believed to be used by these
bacteria to secrete virulence proteins that ultimately cause leakage of plant nutrients to
the extracellular space (apoplast) of infected tissues. In contrast, intracellular pathogens
such as Salmonella spp. and Shigella spp. use type III secretion systems for invasion of
host cells (Galan and Bliska, 1996; Menard et al., 1996). Yersim'a spp. also invades host

cells, but the type III secretion system is not used for invasion. Rather, it is used to resist

10

the uptake of bacteria by both phagocytes and non-phagocytic cells in the later stage of
pathogenesis, when bacteria are located extracellularly.

For historical reasons, different names have been assigned in different bacteria to
many apparently functional homologues of the same gene. Recently, efforts have been
made to streamline the nomenclature of type III secretion genes in plant pathogenic
bacteria so that at least nine conserved hrp genes have now been re-named hrc (for [up
conserved) genes and given the last-letter designation of their Yersinia ysc homologues

(Bogdanove et al., 1996).

1.2.2.2. hip Genes

Components of the type III secretion systems of plant pathogenic bacteria are
encoded by hrp (for hypersensitive response and pathogenicity) genes (Lindgren et al.,
1986). hrp genes are so named because they are required for the bacterium to cause
disease in susceptible plants and to elicit the hypersensitive response (HR) in resistant
plants.

hrp mutant bacteria behave very much like saprophytic bacteria that do not have
any apparent interactions with plants. Since the first publications on hrp genes in the
plant pathogenic bacterium P. syringae (Lindgren et al., 1986), three additional bacterial
species have been used as model systems for the study of the organization and fimction of
hrp genes: E. amylovora, Ralstonia (formerly Pseudomonas) solanacearum, and X.
campestris.

Analysis of hrp gene organization, sequence relatedness, and regulatory systems

indicates that hrp genes in P. syringae and E. amylovora belong to one group (group I),

ll

whereas hrp genes in X. campestris and R. solanacearum form another group (group II)
(Alfano et al., 1997). hrp genes in P. syringae pv. syringae (Pss) strain 61 and E.
amylovora Ea321 are clustered in a single 25 to 30-kb chromosomal region to form a
pathogenicity island. A cosmid pHIRl 1, which contains the whole hrp gene cluster of
P53 61, enabled Escherichia coli TBl to elicit an HR-like reaction when infiltrated into
tobacco leaves, thus defining the minimum number of genes required for HR elicitation
by these bacteria (Huang et al., 1988).

There are 27 genes in the P. syringae hrp gene cluster (Fig. 1.1). Three (hrpL,
hrpS, and hrpR) are involved in the up—regulation of many pathogenicity-related genes
(e.g. hrp, avr) in response to the nutrient-poor condition in the plant apoplast, where
bacteria proliferate (Xiao et al., 1994). Three (hrpA, hrpZ, and hrpW) encode proteins
that are secreted outside bacteria in hrp-inducing conditions (Charkowski et al., 1998; He
et al., 1993; Yuan and He, 1996). Most of the other hrp genes are believed to be
involved in protein secretion.

Experiments using reporter genes have demonstrated that hrp genes are actively
transcribed in plants and in minimal media, but are repressed in complex media (Arlat et
al., 1992; Lindgren et al., 1989; Rahme et al., 1992; Schulte and Bonas, 1992; Wei et al.,
1992; Xiao et al., 1992). Expression of hrp genes is affected by various carbon and
nitrogen sources, pH, osmolarity, temperature, and possibly plant signal molecules (Arlat
et al., 1992; Rahme et al., 1992; Schulte and Bonas, 1992; Wei et al., 1992; Xiao et al.,
1992). It is widely believed that the minimal media mimic the apoplastic environment of
plants (Rahme et al., 1992). In contrast to in planta growth, hrp mutants are not impaired

in their ability to grow in culture, since they are able to grow in minimal media (Lindgren

12

et al., 1986). Thus, hrp genes do not encode products required for general growth or
metabolism.

hrp gene clusters were categorized as pathogenicity islands (Alan Collmer,
personal communication). Other genes whose products affect plant-reaction phenotypes
are sometimes physically linked to hrp gene clusters as part of pathogenicity islands. For
example, some avr genes are found linked to hrp clusters in strains of P. syringae
(Lorang and Keen, 1995; Mansfield et al., 1994). The hrmA locus is adjacent to the P. s.
pv. syringae 61 hrp cluster (Heu and Hutcheson, 1993). dsp (for disease-specific) genes
are also linked to hip regions of E. amylovora (Barny et al., 1990) and R. solanacearum
(Arlat et al., 1992); inactivation of dsp genes results in mutants that are nonpathogenic or

reduced in virulence but still retain the ability to elicit the HR.

13

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1.2.2.3. The Regulation Cascade Controlling the Hrp System

In Pseudomonas syringae pv. syringae 61 (Pss 61), hrpR, hrpS, and hrpL have
been shown to be part of a multi-component regulatory system for the expression of hrp
and avr genes. HrpR and HrpS show high sequence similarities to each other and are
related to the NtrC class of response regulators (Grimm et al., 1995; Grimm and
Panopoulos, 1989; Xiao et al., 1994). Proteins of the NtrC class are members of two-
component regulatory systems, consisting of an environmental sensor and a response
regulator, involved with the transcriptional activation of genes required for a wide variety
of metabolic processes (Albright et al., 1989). HrpR and HrpS differ from most members
of the NtrC family in lacking the amino-terminal domain that modulates the regulatory
activity; however, they do contain the conserved carboxy-terminal DNA-binding domain
(Grimm et al., 1995; Grimm and Panopoulos, 1989; Xiao et al., 1994).

Studies have demonstrated that HrpL shares homology with the ECF (extra
cytoplasmic functions) family of response regulators (Lonetto et al., 1994; Xiao et al.,
1994). hrpL contains a conserved (:54 motif in its promoter region, which is consistent
with a requirement for rpoN for its activation (Morett and Segovia, 1993; Xiao et al.,
1994). A consensus sequence has also been found in the promoter region of a number of
Pss hrp genes; this region functions as a HrpL-dependent promoter (Xiao and Hutcheson,
1994). Significantly, this sequence, GGAACCNA-N14-CCACNNA, was originally
identified in the promoter regions of Pss avr genes (Innes et al., 1993) and has been
referred to as the hrp— or avr-box (Innes et al., 1993; Lorang and Keen, 1995).

It is believed that the regulatory cascade for the transcription of P. syringae hrp

genes involves the activation of the hrpL promoter by the HrpS and HrpR proteins in

15

2.

vi

I

conjunction with RpoN (Xiao et al., 1994). The HrpL protein then acts as an alternative
sigma factor for the expression of HrpL-responsive avr and hip genes (Xiao et al., 1994),
which usually have “hrp-box” cis-element in their promoters. A slightly different
regulatory cascade may be operative in P. s. pv. phaseolicola, as genetic analysis
indicated that HrpR functions as a transcriptional activator controlling hrpS expression
(Grimm et al., 1995).

Erwinia amylovora contains genes sharing homology with P. syringae hrpL,
suggesting that these bacteria contain certain common regulatory elements (Wei and

Beer, 1995).

1.2.2.4. Secreted Proteins and Secretion Signals of the Hrp Secretion System

There appear to be two classes of proteins that traverse the Hrp secretion system.
The first class of proteins, exemplified by harpin proteins, is secreted in hrp-inducing
minimal medium. The other class of proteins, typified by avirulence (Avr) proteins,
appear to be secreted through the Hrp system directly into the plant cell (He, 1998).

HrpN of E. amylovora was the first Hrp protein shown to elicit an HR in tobacco
(Wei et al., 1992), and it was subsequently shown to be secreted via the Hrp system (Wei
and Beer, 1993). The properties of HrpN define the characteristics of the class of HR
elicitors known as harpins: They are hydrophilic, rich in glycine, heat stable, lack
cysteine, and elicit HR when injected into the apoplast of certain plants. In P. syringae
pv. syringae, the hrpZ gene product, HrpZ (or harpinpss), was found to exhibit HR-
eliciting activity in the tobacco leaf apoplast. HrpZ shares many properties with HrpN

and therefore is also a member of the Harpin family of proteins. HrpZ was also shown to

16

traverse the Hrp secretion pathway (He at al., 1993). Homologues of HrpZ are present in
all P. syringae pathovars surveyed (He et al., 1993). HrpZ was found to be essential but
not sufficient for HR elicitation by saprophytic bacteria carrying pHIRll (Alfano et al.,
1996).

In addition to secreting HrpZ, P. syringae pv. tomato (Pst) strain DC3000 also
secretes at least three other proteins (EXP-60, EXP-22, and EXP-10) via the Hrp system
in culture (Yuan and He, 1996). The EXP-60 is HrpW, whose gene is immediately
adjacent to the right border of the hrp gene cluster in Pst DC3000 (Charkowski et al.,
1998). hrpW encodes a 42.9-kDa protein with domains resembling harpins and pectate
lyases (Pels). HrpW indeed has HR-eliciting activity, but it does not have measurable
pectate lyase activity. EXP-10 is encoded by the hrpA gene situated adjacent to the hrpZ
gene in the same operon. The primary sequence of HrpA, a small, hydrophilic protein,
shares no homology with any other proteins (Preston et al., 1995). The exact function of
the hrpA gene was unknown.

avr genes mediate the elicitation of hypersensitive disease resistance in plants that
contain the corresponding disease resistance genes (Leach and White, 1996). However,
the phenotypic expression of avr genes in bacteria requires hrp genes (Huynh et al.,
1989). In fact, a 25-kb hip gene cluster of P. syringae pv. syringae strain 61 appears
sufficient to enable E. coli, which otherwise has no apparent interaction with plants, to
deliver Avr proteins to the plant (Gopalan et al., 1996; Pirhonen et al., 1996).

Type III-dependent secretion of some effector proteins can be detected in cultures
of induced mammalian bacterial pathogens in the absence of the host (Hueck, 1998).

However, secretion of avirulence proteins from plant bacterial pathogens has been

17

difficult to detect. Only E. amylovora has been shown to secrete an avirulence protein,
DspE (DspA), into bacterial cultures in a hrp-dependent manner (Bogdanove et al., 1998;
Gaudriault et al., 1997).

The recent development of a heterologous E. coli hrp secretion system capable of
secreting COpious amounts of avirulence proteins into bacterial cultures has provided an
alternative method for studying the hrp secretion pathway (Ham et al., 1998). Using this
system, the secretion of AvrB, AvrPto, and Aerpt2 was efficiently detected (Ham et al.,
1998; Mudgett and Staskawicz, 1999).

It is apparently disadvantageous for bacteria to send their avirulence proteins into
the plant cell because recognition of Avr proteins by the corresponding plant receptors in
resistant plants triggers rapid plant cell death and other defense response, which lead to
plant resistance to bacteria. This puzzle can be explained that bacterial proteins that
traverse the Hrp secretion system originally serve virulence functions. However, during
host-bacterium coevolution, plants have recognized some of these virulenCe proteins as
elicitors of their defense response. The virulence factor therefore becomes avirulence
factor (He, 1998). As a matter of fact, it’s already been found some avr proteins are
actually virulence factors when they are in different plant-bacterial interactions, such as
avrBsZ of X. campestris pv. vesicatoria (Kearney and Staskawicz, 1990), avr/pth genes
of X. campestris pv. malvacearum (Swarup et al., 1992; Yang et al., 1994), avrA and
aer of P. syringae pv. tomato (Lorang et al., 1994), and aerme of P. syringae pv.
maculicola (Ritter and Dangl, 1995).

Proteins traversing the type III pathway carry multiple signals that route them to

the secretion pathway. No common amino acid or peptide sequence has been identified

18

0.4...

It

among the secretion signals. Experiments carried out with YopE and YopN, two type III
secreted proteins of the enteropathogen Yersinia, revealed that the first ~15 amino acids
of these proteins were sufficient to direct the secretion of a heterologous protein (Comelis
et al., 1998; Sory et al., 1995). However, frameshift mutations that completely altered
the peptide sequences of these signals failed to prevent secretion. Thus, the signal that
leads to the type III secretion of Yop proteins appears to be encoded in their messenger
RNA rather than in the peptide sequence (Anderson and Schneewind, 1997). The 5’
mRNA regions of some of the yop messages are predicted to have stem-loop structures
that bury AUG translational signals, which suggests that the translation of these proteins
may be arrested until the 5’ mRNA interacts with a component of the secretion apparatus.
The secretion signals of two proteins from the plant pathogen P. syringae, AvrB and
AvrPto, also appear to reside in their 5’ mRNA, which suggests that this type of substrate
recognition may be a general feature of type III secretion systems (Galan and Collmer,
1999)

Some type III secreted proteins can be secreted posttranslationally through an
alternative secretion signal (Anderson and Schneewind, 1997; Cheng et al., 1997). This
alternative mechanism involves the function of specific chaperones that bind the cognate
secreted proteins on discrete domains located within the first 100 amino acids of many
proteins that travel the type III pathway (Comelis et al., 1998; Sory et al., 1995; Wattiau
et al., 1994; Wattiau and Comelis, 1993). DspF of Erwinia amylovora and Aer of
Pseudomonas syringae are small (16 kDa and 14 kDa) and acidic with predicted

amphipathic alpha helices in their C termini; they resemble chaperones for virulence

l9

factors secreted by type III secretion systems of animal pathogens (Bogdanove et al.,

1998).

1.2.3. Similarity between Type III Secretion System and Flagellar Assembly

At least eight type-III secretion proteins share sequence similarities with flagellar
assembly components (Bogdanove et al., 1996). Intriguingly, type-III secretion
components of plant pathogenic bacteria are more closely related to components of the
flagellar machinery than are their counterparts in animal pathogenic bacteria. Thus, it is
possible that type III secretion systems first emerged in plant pathogenic bacteria as an
evolutionary adaptation of the flagellar export apparatus in order to secrete proteins other
than flagellin, thereby assisting bacteria to form a close association with plant cells
(Galan and Collmer, 1999). Given the similarity of the type III proteins to those that
assemble flagella, it is tempting to hypothesize that they form a flagellum-like structure

that secretes and translocates proteins into the host cells (Barinaga, 1996).

1.2.4. Interkingdom Transport Hypothesis

The avr genes interact genetically with their corresponding disease resistance
genes in plants to trigger resistance responses in a gene-for-gene manner (Leach and
White, 1996). However, purified bacterial Avr proteins elicit no response when added to
the plant apoplast, where bacteria proliferate (Gopalan et al., 1996; Leach and White,
1996), and Avr proteins have not been detected outside bacteria in planta (Gopalan et al.,

1996; Leach and White, 1996).

20

Amino acid sequence analysis predicts that cloned plant disease resistance genes
corresponding to known bacterial avr genes encode intracellular proteins instead of cell
surface receptors (Bent, 1996). With the exception of Aer of Pseudomonas syringae
pv. tomato (Kobayashi et al., 1990), none of the bacterial Avr proteins has been shown to
possess any catalytic activity that could generate diffusible, low-molecular-weight
elicitors of the HR (Leach and White, 1996). Thus, the biochemical basis of the activity
of bacterial avr gene products has remained elusive.

The type III secretion systems in animal pathogenic bacteria translocate effector
proteins from the bacteria to their host cells (Gauthier and Finlay, 1999). The functional
dependence of Avr proteins on hrp genes suggests possible transport of Avr proteins into
the interior of the plant cell via the Hrp secretion system. Indeed, using transgenic and
transient expression of AvrB of P. syringae in leaves of Arabidopsis thaliana, Gopalan et
al. (1996) showed that the putative delivery of AvrB into plant cells required the Hrp
secretion system.

Similar evidence supporting interkingdom transport of Avr proteins has been
obtained independently for P. syringae pv. tomato AvrPto (Scofield et al., 1996; Tang et
al., 1996), Aerpt2 (Leister et al., 1996) and X. campestris pv. vesicatoria AvrBs3 (Van
den Ackerveken et al., 1996). In the case of Ath0, the tomato disease resistance gene
Pto product has been shown to bind specifically to AvrPto in the yeast two-hybrid system
(Scofield et al., 1996; Tang et al., 1996).

The AvrBs3 family of Avr proteins was found to contain functional plant nuclear
localization signals (N LSs) that target the reporter protein B-glucuronidase (GUS) to the

plant nucleus (Yang and Gabriel, 1995). It has now been shown that these NLSs are

21

required for the AvrBs3 family of proteins to trigger disease resistance—associated HR,
suggesting that some members of the AvrBs3 family enter not only the plant cell but also

the plant nucleus (Van den Ackerveken et al., 1996).

1.3. Project Summary

Plant pathogenic bacteria are extracellular pathogens, and transport of bacterial
proteins requires passage of the effector proteins across not only the bacterial envelope
but also the plant cell wall (>100 nm thick) and plasmalemma. This is in contrast to
animal-bacterium interactions, in which protein transfer can be accomplished by direct
contact between the membranes of bacteria and host cells (Comelis and Wolf-Watz,
1997). How do bacteria manage to overcome the plant cell wall and send their proteins
into host cells? How do bacteria sense host contact? Are supramolecular appendages
assembled by the Hrp system? Is there a host receptor protein capable of physically
binding to virulence or avirulence proteins? As the key to plant bacterial pathogenicity,
the hrp-encoded type 111 protein secretion system has remained mysterious.

In these thesis projects, I addressed some of these questions. P. syringae pv.
tomato DC3000 (Pst DC3000) was used as a model pathogenic bacterium. The species
P. syringae is divided into pathovars largely on the basis of host specificity, i.e., P.
syringae pathovars syringae (brown spot of beans), tomato (bacterial speck of tomatoes),
and glycinea (bacterial blight of soybeans) (Alfano and Collmer, 1997). Pst DC3000 is a
pathogen of both tomato and Arabidopsis, and it contains a functional hrp gene cluster as
well as several avirulence genes, e.g., avrPto. All these features make it a good

pathogenic bacterium to study the type III secretion system.

22

U)

’TJ

L? 3

At

Chapter 2 describes my efforts to identify and characterize a novel bacterial
surface appendage, called the Hrp pilus. This project was initiated by the finding that the
HrpA protein is secreted outside bacteria in hrp-inducing conditions (Yuan and He,
1996). By constructing a non-polar hrpA mutant, 1 was able to. demonstrate that the hrpA
encodes the major structural protein of a pilus-like structure. I further demonstrated that
hrpA is essential for Pst DC3000 to cause disease in the host plants and to elicit an HR in
a resistant host or nonhost plant. This project was canied out in collaboration with Dr.
Martin Romantschuk’s group at the University of Helsinki, Finland. They independently
made the observation that the HrpA protein is associated with a pilus-like structure.
Results from chapter 2 suggest that the Hrp pilus plays a critical role in the pathogenicity
of Pst DC3000.

Chapter 3 reports my research on determining the function of the Hrp pilus. Two
models were proposed to describe the potential role of Hrp pilus in type 111 protein
secretion: contact model and conduit model. Contact model suggests that the role of Hrp
pilus is to physically attach the bacterium to plant cells. While the conduit model
suggests that the Hrp pilus is directly involved in the protein delivery from bacterium to
plant cell. I was especially interested in knowing whether the Hrp pilus is required for
protein delivery. To test this model, I examined the secretion of HrpW in the hrpA
mutant. I found that HrpW protein secretion was indeed blocked in the hrpA mutant in
which the Hrp pilus is not produced. However, further study revealed that HrpW
production was significantly suppressed in the hrpA mutant. This rather surprising
finding prompted us to consider a possible involvement of the HrpA protein in hrp gene

regulation. Subsequent western and northern blot analyses confirmed that the hrpA

23

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mutation down-regulated expression of all examined hrc/hrp and avr genes at the RNA
level. Furthermore, 1 demonstrated that hrpA acts through two previously known positive
regulatory genes, hrpR and hrpS. Ectopic expression of hrpR/S genes restored gene
expression in the hrpA mutant, but the secretion of HrpW and AvrPto was still blocked.
These results suggest that the Hrp pilus plays a dual role in protein secretion and global
regulation of the Hrp system.

Chapter 4 describes my efforts to identify cotton proteins that interact with Avrb6.

Avrb6 belongs to the Avr/Pth family of proteins in Xanthomonas campestris pv.
malvacearum (Xcm) and is believed to be targeted into the plant nucleus via the Hrp
system. By employing a yeast two-hybrid system, I have isolated several cotton proteins
that specifically interact with the bait protein, Avrb6ANC — the central repeat region of
Avrb6. These proteins include a serine/threonine kinase highly homologous to Pto, a
disease resistance gene product identified from tomato; calcineurin B; and a putative
transcription factor containing a zinc-finger motif. The functional significance of these

proteins in Xcm avirulence and virulence remains to be determined.

24

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Charkowski, A. 0., Alfano, J. R., Preston, G., Yuan, J ., He, S. Y., and Collmer, A.
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Cheng, L. W., Anderson, D. M., and Schneewind, O. (1997). Two independent type III
secretion mechanisms for YopE in Yersinia enterocolitica. Mal Microbial 24, 757-65.

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26

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34

Chapter 2

THE HRPA GENE ENCODES THE STRUCTURAL PROTEIN OF A
BACTERIAL SURFACE APPENDAGE ASSOCIATED WITH TYPE III
PROTEIN SECRETION IN PSEUDOMONAS S YRINGAE PV. TOMA T0 DC3000

Data in this chapter have been previously published in Proc Natl Acad Sci U S A.
REFERENCE: Roine, E., m, Yuan, J ., N urmiaho-Lassila, E. L., Kalkkinen, N.,
Romantschuk, M., and He, S. Y. (1997). Hrp pilus: an hrp-dependent bacterial surface
appendage produced by Pseudomanas syringae pv. tomato DC3 000. Prac Natl Acad Sci

U S A 94, 3459-64. (as co-first author)

35

2.1. ABSTRACT

Hypersensitive response and pathogenicity (hrp) genes control the ability of major
groups of plant pathogenic bacteria to elicit the hypersensitive response (HR) in resistant
plants and to cause disease in susceptible plants. A number of Hrp proteins share
significant similarities with components of the type III secretion system and the flagellar
assembly apparatus in animal pathogenic bacteria. The hrpA gene of Pseudomanas
syringae pv. tomato DC3000 encodes an extracellular protein with unknown function. A
non-polar hrpA mutant was constructed by replacing the hrpA gene open reading frame
(ORF) with the aph (encoding the aminoglycoside phosphotransferase) ORF. The HrpA
protein was not produced in the hrpA mutant. pHRPA, a pUCP18 derivative carrying the
wild type hrpA gene together with its native promoter, restored production and secretion
of the HrpA protein in the hrpA mutant. In collaboration with Dr. Martin Romantschuk’s
group, we identified a filamentous surface appendage, called the Hrp pilus, from DC3000
grown in a solid minimal medium. Using the non-polar hrpA mutant, 1 demonstrated that
the hrpA gene encodes the major structural protein of the Hrp pilus. Transmission
electron microscopy (TEM) showed that hrpA mutant did not form any Hrp pilus,
whereas pHRPA restored the formation of Hrp pili in the hrpA mutant. In addition, the
hrpA mutant was unable to cause disease in Arabidapsis thaliana or to elicit HR in the
non-host tobacco or resistant tomato plants carrying the Pto gene. These results suggest

that the hrpA plays a critical role in plant-Pst DC3000 interactions.

36

2.2. INTRODUCTION

Major groups of Gram-negative plant pathogenic bacteria belonging to the genera
Erwinia, Pseudomanas, Ralstania, and Xanthomonas contain hypersensitive reaction and
pathogenicity (hrp) genes. These genes control the ability of these bacteria to initiate
interactions with plants, including elicitation of the hypersensitive reaction (HR) and
causation of disease (Bonas, 1994; He, 1996).

hrp genes specify the type 111 protein secretion system in pathogenic
phytobacteria, and many of them encode proteins sharing high sequence similarity with
components of the type 111 system of animal pathogenic bacteria, such as ysc genes in
Yersinia spp. (Comelis and Wolf-Watz, 1997). In Yersinia, extracellular proteins that
traverse the type 111 protein secretion system are involved in the translocation of effector
proteins into host cells. For example, it has been suggested that two type III secreted
hydrophobic proteins, YopB and YopD, form a translocation pore in the host cytoplasmic
membrane (Hakansson et al., 1996; Lee and Schneewind, 1999). Other extracellular
proteins, such as YopN, Lch, and TyeA, are involved in sensing of host cells by
Yersinia (Iriarte etal., 1998).

The functional dependence of Avr proteins on hrp genes suggests possible
transport of Avr proteins into the interior of the plant cell via the Hrp secretion system.
Indeed, using transgenic and transient expression of AvrB of P. syringae in leaves of
Arabidapsis thaliana, Gopalan et al. showed that the site of activity of AvrB is inside the
plant cell and that the Hrp system is necessary for the putative delivery of AvrB into plant

cells during infection (Gopalan et al., 1996).

37

rim

'1
r,

The mechanism by which the Hrp secretion system putatively delivers proteins
through the plant cell wall is not known. To send bacterial proteins into plant cells,
bacteria must overcome the thick plant cell wall (>100 nm). Although it is not clear
which proteins are involved in forming the hypothetical transloCation structure, they are
likely secreted outside the bacteria via the Hrp system.

Pst DC3000 produces several major extracellular proteins (EXPs) in a minimal
medium that induces hrp genes. Five EXPs (EXP-60, EXP-45, EXP-43, EXP—22, and
EXP-10) are under the control of the Hip secretion system (Yuan and He, 1996).
Immunoblot analysis with the HrprSS antibody identified EXP-45 as the HrprSS
homolog in DC3000, named the Hrprst protein. The amino-tenninal sequence analysis
revealed that EXP-10 is encoded by the hrpA gene, located upstream of hrpZ in the same
operon (Yuan and He, 1996).

In this study, experiments were carried out to determine the function of the hrpA
gene in plant-Pst DC3000 interactions. For this purpose, a non-polar hrpA mutant was
constructed. Work with the hrpA mutant allowed us to demonstrate that the hrpA gene is
essential for Pst DC3000 to initiate pathogenesis and to elicit the HR in plants, as well as
for the function of AvrB when produced in Pst DC3000. In collaboration with Dr.
Martin Romantschuk’s group, we showed that Pst DC3000 produces a thin, pilus-like
structure on solid hrp-inducing media. HrpA was found associated with the Hrp pilus.
By using the hrpA mutant, I provided evidence that the hrpA gene encodes the major

structural protein of this Hrp pilus.

38

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2.3. MATERIALS AND METHODS

2.3.1. Culture Conditions

For detection of bacterial extracellular proteins (EXPs) in liquid cultures, bacteria
were first grown at 28°C to an 0D600 of 0.8-1.0 in 50 ml King's medium B broth (King et
al., 1954), supplemented with 100 ug/ml rifampicin. Bacteria were then pelleted and
resuspended in 50 ml hrp-inducing broth [NaCl 0.1%, KH2P04 0.17%, MgSO4°7H20
0.07%, potassium citrate 0.324%, (NH4)2804 0.4%, pH5.5; 10 mM fructose added after
autoclaving] (He et al., 1993; Innes et al., 1993) or King's medium B broth, and then
incubated with shaking (250 rpm) at room temperature (21-23°C) for 24 hr. For
preparation of bacterial surface-associated proteins, bacteria were grown on solid hrp-

inducing medium (He et al., 1993) at room temperature (21-23°C) for 2 days.

2.3.2. Analysis of Bacterial Extracellular Proteins

For preparation of bacterial secreted proteins, bacteria were removed from liquid
cultures by centrifugation at 10,000 X g for 10 min. The supernatant was concentrated 50-
fold using centricon concentrators with a molecular weight cutoff of 3,000 daltons
(Amicon) and 10 ul was analyzed by 15% SDS/PAGE followed by staining with 0.025%
Coomassie Brilliant Blue R-250. Immunoblot analysis of HrpA was carried out using
rabbit antibody raised against the HrpA protein. For preparation of bacterial surface-
associated proteins, bacteria from a 76-mm agar plate containing solid hrp-inducing
medium were resuspended in 1 ml of 10 mM sodium phosphate (pH 5.5), pelleted by

centrifugation at 13,000 x g for 10 min to partially remove proteins not associated with

39

cell surface structures, and then resuspended in 0.2 ml of 10 mM sodium phosphate (pH
5.5). The bacterial suspension was forced through a 25 G needle 4 to 5 times to shear
surface structures and proteins (e. g., flagella and pili) from the bacteria, and was then
centrifuged at 13,000 X g for 10 min at 4°C. Twenty microliters of the supernatant was

used for SDS/PAGE analysis.

2.3.3. Construction of the hrpA Mutant

For construction of the nonpolar hrpA mutants, a 5.1-kb EcoRI fragment of
pCPP2201 (Preston et al., 1995) containing hrpRS, hrpAZBCDE, and the 5’ half of hrpF
of Pst DC3000 was subcloned into pBluescript 11 SK(-) (Stratagene) and used as the
template for PCR. A 1.5-kb DNA fragment, 5’ of the hrpA start codon, was amplified by
PCR using the following oligonucleotide set: 5’-GGAAACAGCTATGACCATG-3’
(pBSSK reverse primer) and 5’-GGGGTACCCCTTAAGATTTACCAGCGTGATTGC-
3’ (containing a Kpnl site at the 5’ end). A 3.8-kb fragment 3’ of the hrpA stop codon
was amplified using the following primer set: 5’-CGGGATCCCGTTGCCCCCTCATC-
AGAGG-3’ (containing a BamHI site at the 5’ end) and 5’-GTAAAACGACGGCCAGT-
3’ (M13-20 primer). The 0.8-kb open reading frame (ORF) of the aph gene (encoding
aminoglycoside phosphotransferase) without its own promoter or terminator was
amplified from mini-TnS xylE (de Lorenzo et al., 1990) using the following
oligonucleotides: 5’-GGGGTACCCTGTTATGAGCCATATTCAACG-3 ’ (containing a
KpnI site at the 5’ end) and 5’-CGGGATCCCGTI‘AGAAAAACTCATCGAGCATC-3’
(containing a BamHI site at the 5’ end). Through multiple cloning steps, the three PCR

products were ligated (hmRS-aph-hrpZBCDEF) and cloned into pBluescript 11 SK(-).

40

\2
53

The hrpA ORF was precisely replaced by the aph ORF without changing the promoter or
Shine-Dalgamo sequences upstream of the hrpA ORF, or the sequence and spacing
between hrpA and hrpZ. For marker exchange mutagenesis, the hmRS—aph-hrpZBCDEF
fragment was cloned into pRK415 (Keen et al., 1988), which Was then electroporated
into Pst DC3000. Marker exchange events were selected following a standard procedure
(Huang et al., 1991), except that hrp-inducing medium was used for activating the
expression of the aph gene. The junction sequences of the aph gene in the marker-
exchanged hrpA mutant were cloned, sequenced, and found to be correct. See Fig. 2.1
for details of hrpA non-polar mutant construction. For complementation, the hrpA ORF
and its native promoter were amplified using the following primers: 5’-TTGCAAAGA-
CGCTGGAACCGTATCGC-3 ’ and 5 ’-GGGGTACCTCCTCAAGGTAGCGGCCCCC-
TC-3’. The PCR product was cloned into the SmaI site of pUCPl 8 (Schweizer, 1991),

resulting in pHRPA.

41

BamHI Kpnl KpnI BarnHI

l._.__l
hrpR hrpS aph
BamHI KpnI BamHI BamHI Kpnl
w #
hrpR hrpS aph hrpZBCDEF
BamHI KpnI BamHI Kpnl
hrpR hrpS aph hrpZBCDEF
(ECORI) (EcoRI)

l

 

Pst DC3000 hrpA mutant

Figure 2.1. A flow chart of construction of a non-polar hrpA mutant in Pst DC3000.

42

V“)

«I
.1.

2.3.4. Partial Purification of Bacterial Surface Structures (Flagella and Hrp Pili)

(conducted by Dr. Martin Romantschuk’s group)

All purification steps were performed at 4°C. Bacterial surface structures and
proteins were sheared off as described above and subjected to ultracentrifugation in a
Beckman Ti45 rotor at 100,000 X g for 3 hr. The pellet was resuspended in 10 mM
Tris-HCl (pH 7.5) to which sodium deoxycholate was added to a final concentration of
0.5% (wt/vol). After overnight incubation at 4°C, proteins were subjected to
ultracentrifugation in a 10-60% (wt/wt) sucrose gradient in a Beckman SW27 rotor at
80,000 X g for 20 hr. Ten fractions were taken from the gradient and dialyzed against 10
mM Tris-HCl (pH 7.5) and then against water. The pellet of the gradient was
resuspended in 10 mM Tris-HCl (pH 7.5), repelleted to remove the sucrose (in a
Beckman Ti50 rotor at 125,000 X g for 3 hr), and finally resuspended in 10 mM Tris-HCl
(pH 7.5). Dialyzed gradient fractions and the pellet were then used for transmission

electron microscopy (TEM) and for SDS/PAGE analysis.

2.3.5. Transmission Electron Microscopy (TEM) Analysis

For TEM observation, a drop of bacterium or flagellum plus pilus suspension was
applied to a copper grid coated with pioloforrn and carbon, followed by staining with 1%
potassium phosphotungstic acid adjusted to pH 6.5 with potassium hydroxide. The grids

were then examined with a transmission electron microscope.

43

2.3.6. Pathogenesis Assays

Bacteria were grown in King's B broth to an 0D600 of 0.6-0.8. Bacterial
suspensions in distilled water were infiltrated into leaves of tomato (Lycapersican
esculentum cultivar Rio Grande-PtoR), Arabidapsis thaliana ecotype Columbia (C01),
and tobacco (Nicatiana tabacum cultivar Samsun NN) using needleless syringes. The
concentrations of bacteria used were 2 X 108 colony-forming units (cfu)/ml and 2 X 106
cfu/ml for HR and pathogenesis assays, respectively. Plant responses were recorded at
24 hr (for HR assay) or 4 days (for pathogenesis assay) post-infiltration. HR is indicated
by rapid, localized tissue collapse in the infiltrated area within 24 hr. Disease symptoms
caused by Pst DC3000 and complemented hrpA mutants in tomato and Arabidapsis
leaves were characterized by slowly developing necrosis and spreading tissue chlorosis,
usually observed 3 days afier infiltration. Strain DC3000 and its hrpA and hrpS mutants
contain avrPta in the chromosome (Ronald et al., 1992). Plasmid pAVRB contains P.
syringae pv. glycinea avrB in pDSK609 (kindly provided by N. T. Keen, University of
California). The tobacco and tomato plants were grown in greenhouses. Arabidapsis
plants were grown in grth chambers at 20°C with 70% relative humidity and a 12-hr
photoperiod. HR assays with tobacco and tomato plants were performed at room
temperature in the laboratory. Pathogenesis assays with Arabidapsis plants were
performed at 20°C in growth chambers with 70% relative humidity and a 12-hr

photoperiod.

44

2.4. RESULTS

2.4.1. Mutational Analysis of the hrpA Gene

To study the function of HrpA protein, a nonpolar hrpA mutation was constructed
by precisely replacing the hrpA open reading frame (ORF) with the aph (conferring
kanamycin resistance) ORF, which confers kanamycin resistance (de Lorenzo et al.,
1990) (Fig. 2.1). For complementation, pHRPA (containing the hrpA gene with its native
promoter; see MATERIALS AND METHODS) was introduced into the nonpolar hrpA
mutant. SDS-PAGE was used to examine bacterial surface-associated proteins. A 10-
kDa protein was secreted in both DC3000 and the hrpA mutant carrying pHRPA;
however, it was absent in the hrpA mutant (Fig. 2.2A). Immunoblot analysis using an
HrpA antibody showed that HrpA was not detectable at the surface of the hrpA mutant,
while pHRPA restored the production of HrpA at the bacterial surface (Fig. 2.23). These

results confirmed that I had obtained a non-polar hrpA mutant.

45

107

 

 

 

 

b. q
u v. - . _
.‘ni.
~ .w
‘M'. . ' '_‘
" -* ' r.
. . 1,;- “-

 

 

Figure 2.2. (A) A 15% SDS/PAGE and (B) an immunoblot analysis of protein samples
prepared from surfaces of DC3000 (lane 1), hrpA mutant (lane 2), and hrpA mutant
containing pHRPA (lane 3) grown on solid hrp-inducing medium. Lane M, molecular
weight markers (Bio-Rad) in kDa. The gel (A) was stained with 0.025% Coomassie
Brilliant Blue R-250. The immunoblot analysis was carried out using an anti-HrpA
antibody.

46

2.4.2. Formation of an hrp Gene-Dependent Pilus by DC3000 on Solid hrp-Inducing

Medium

Examination of bacteria grown on solid hrp-inducing medium by TEM revealed
that DC3000 produced two or three polar flagella (15-18 nm in diameter) on both King's
medium B (Fig. 2.3A) and hrp-inducing agar plates (Fig. 2.33). In addition, DC3000
also produces many pilus-like appendages (6-8 nm in diameter) on solid hrp-inducing
medium (Fig. 2.3B), but not on King's medium B plates (Fig. 2.3A). These pilus-like
appendages were found both on the bacterial surface with no consistent distribution
pattern, and as detached pilus clusters. The pilus-like appendages were easily fragmented
during sample preparation, as evidenced by the presence of many short pieces of pili (Fig.
2.38); therefore, the length of these pilus-like appendages could not be determined. The
effect of temperature (from 16-28°C) on the formation of the pilus-like appendages was
examined. The number of pili observed decreased dramatically when incubation
temperature exceeded 25°C. This result suggests that the pilus-like structure is unstable

in high temperature.

47

 

Figure 2.3. Detection of the Hrp pilus on the surface of Pst DC3000. (A) DC3000
grown on King's medium B agar plates, and (B) DC3000 mutants grown on solid
hrp-inducing medium were examined with a transmission electron microscope
after staining with 1% potassium phosphotungstic acid (pH 6.5). One to three
polar flagella of 15-18 nm in diameter are present on most rod-shaped bacteria
(surrounded by dark shadows) in samples (A and B); in B, many Hrp pili of 6—8
nm in diameter are also present (indicated by arrows). (Scale bars = 200 nm).

48

2.4.3. Identification of Proteins Associated with the DC3000 Hrp Pilus

(carried out mainly by Dr. M. Romantschuk’s group; independently confirmed by SY He)

The presence of the Hrp pilus is specifically correlated with the appearance of a
10-kDa extracellular protein. Only DC3000 grown on solid hrp-inducing medium
produced the lO-kDa protein (Fig. 2.2A & B, lane 1). Neither DC3000 grown on King's
B agar medium, nor the hrcC mutant bacteria grown on solid hrp-inducing medium,
produced the protein (Roine et al., 1997).

To determine the identity of the Hrp pilus, a preparation of cell surface structures
(flagella and Hrp pili) from DC3000 grown on solid M9 minimal medium was subjected
to ultracentrifugation in a 10-60% sucrose gradient. Hrp pili together with flagella were
found in the pellet (Fig. 2.4A). SDS/PAGE analysis of the pellet fraction revealed three
major proteins of 50, 36, and 10 kDa in size, respectively, associated with these
filamentous structures (Fig. 2.4C, lane 1). A minor, contaminating, 100-kDa protein was
also present in some, but not all, preparations (Fig. 2.4C, lane 1). Another fraction from
the middle of the sucrose gradient contained only flagella (Fig. 2.43). When this fraction
was analyzed by SDS/PAGE, only the 36-kDa protein, but not the 10- or 50-kDa protein,
was found. This result suggests that the 36-kDa protein is flagellin that makes up
flagella, and that the 10- and 50-kDa proteins are associated with the Hrp pilus.

The N-tenninal sequences of the 36-, 10-, and 50-kDa proteins were determined.
The N-terrninal sequence (ALTVNTNVASLNVQKNLGRASDALST) of the 36-kDa
protein, from both the pellet fraction containing flagella and Hrp pili (Fig. 2.4C, lane 1)
and the flagella fraction (Fig. 2.4C, lane 2), is almost identical to those of flagellins of

Pseudomanas aeruginosa and Pseudomanas putida (Totten and Lory, 1990; Winstanley

49

et al., 1994), confirming that the 36-kDa protein is the DC3000 flagellin. The first 35
amino acids (VAFAGLTSKLTNLGNSAVGGVGGALQGVNTVASNA) of the 10-kDa
protein matched exactly that of HrpA encoded by hrpA (Preston et al., 1995). The
sequence of the first 16 amino acids (ASPITSTTGLGSGLAI) of the N terminus of the
50-kDa protein does not show any significant similarity to any proteins in the current

gene/protein databases.

50

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51

2.4.4. No Hrp Pilus can be Detected in the hrpA Mutant

Since the HrpA protein is associated with the Hrp pilus, it is important to know
whether HrpA is required for the assembly of the Hrp pilus. Examination of bacteria
grown on solid hrp-inducing medium by TEM revealed that the hrpA mutant produces
only two to three polar flagella (15-18 nm in diameter), and no pilus-like structure could
be detected (Fig. 2.5A). pHRPA (containing the hrpA gene with its native promoter)
enabled the hrpA mutant to produce Hrp pili on the bacterial surface (Fig. 2.58).

Therefore we conclude that HrpA is likely the major structural protein of Hrp pili.

52

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53

2.4.5. Pathogenesis Assays

To determine the biological role of the Hrp pilus in interactions of plant and Pst
DC3000, the effect of the hrpA mutation on bacterial pathogenesis and HR elicitation was
examined. The results are summarized in Table 2.1. The hrpA mutant was unable to
elicit the HR in leaves of the nonhost tobacco or to cause disease symptoms (tissue
chlorosis and necrosis) in leaves of the host Arabidapsis thaliana. The responses of
tobacco and A. thaliana leaves to the hrpA mutant were very similar to the responses to a
hrpS mutant, which is a typical hrp mutant unable to initiate any hrp-mediated plant
responses. Plasmid pHRPA restored the ability of the hrpA mutant to elicit HR necrosis
in tobacco leaves and to cause disease symptoms in A. thaliana leaves. HrpA was also
required for two well-characterized P. syringae avr genes, avrB and avrPta, to trigger
genotype-specific HR (Table 2.1). DC3000 contains avrPto, which mediates the
elicitation of an HR on tomato cv. Rio Grande-PtoR containing the Pto resistance gene
(Martin etal., 1993; Ronald et al., 1992). avrB was originally cloned from P. syringae
pv. glycinea and later was found to mediate the elicitation of an RPM] -dependent HR
and resistance in A. thaliana (Bisgrove et al., 1994). Therefore, the Hrp pilus structure
gene, hrpA, is critical for DC3000 in both pathogenesis and HR elicitation in non-host

plants or in host plants carrying the proper avr gene(s).

54

Table 2.1. Plant reactions to DC3000 and the hrpA mutant.

 

 

Bacteria Arabidapsis Tobacco Tomato
(Col) (Samsun NN) (Rio Grande—PtoR)
DC3000, avrPta+ D HR HR
hrpA, avrPta+ NR NR NR
hrpA/pHRPA, avrPto+ D HR HR
DC3000/pAVRB HR HR HR
hrpA/pAVRB NR NR NA

 

HR: rapid, localized tissue collapse in the infiltrated area; D: tissue chlorosis and necrosis
typical of disease symptoms caused by DC3000; NR: no visible reaction; NA: not
assayed. DC3000 and its derivative, hrpA mutant, contain avrPto in the chromosome

(Ronald et al., 1992).

55

2.5. DISCUSSION

In this study, I (in collaboration with Dr. Martin Romantschuk’s group) showed
that Pst DC3000 produced a novel pilus-like structure on the cell surface when bacteria
are grown on solid hrp-inducing medium at low temperature. Furthermore, I have

provided genetic evidence that HrpA is a structural protein of this Hrp pilus. Finally, I

have shown that a nonpolar hrpA mutant strain cannot initiate pathogenesis or elicit the l _
HR in plants.
The nucleic acid sequence of the Pst DC3000 hrpA gene, the first gene of the a

hrpZ operon, was previously determined by Preston et al. (1995). hrpA codes for a
hydrophilic protein with a predicted molecular weight of 11 kDa (Preston et al., 1995).
The primary amino acid sequence of the HrpA protein does not show any significant
homology to those of characterized pilin proteins. However, our computer analysis using
the PROPSEARCH program (Hobohm and Sander, 1995), which identifies structural
similarities between proteins without looking for primary amino acid sequence
homology, indicates that HrpA is structurally similar to several pilin proteins, especially
to the AF/Rl pilus chain A precursor of E. coli (Wolf and Boedeker, 1990). This
analysis is in agreement with our results showing that HrpA is the structural protein of
the Hrp pilus.

We do not know the role, if any, of the 50-kDa protein in the assembly of the Hrp
pilus. The 50-kDa protein was produced by Pst DC3000 growing in King's B medium,
which represses hrp gene expression (Yuan and He, 1996). Furthermore, the hrpA
mutant, which does not produce the Hrp pilus, still produces the 50-kDa protein (Fig.

2.2A). These two observations suggest that the production and secretion of this protein is

56

 

independent of the Hrp secretion system. It is possible that the 50-kDa protein is
involved in the formation of some surface structure independent of the Hrp pilus.

To our knowledge, this is the first study showing that the Hrp pathway is involved
in the formation of a pilus structure. Our finding is consistent with the observation that
many Hrp proteins are structurally related to those that participate in the construction of
bacterial flagella (Kihara et al., 1989), suggesting an involvement of Hrp proteins in the
assembly of an extracellular macromolecular structure. Salmonella, Shigella, and
Yersinia, all of which contain type III secretion systems, secrete proteins required for
pathogenesis (Hueck et al., 1995; Michiels et al., 1990; Parsot et al., 1995; Rosqvist et
al., 1994), (Persson et al., 1995). Both Salmonella typhimurium and Shigellaflexneri are
enteroinvasive pathogens. S. typhimurium transiently produces filamentous surface
appendages of 60 nm in diameter upon contact with epithelial cells during its invasion of
host cells (Ginocchio et al., 1994). The structural components of these appendages have
not been identified. Yersinia spp. are not intracellular pathogens, but during infection
they secrete virulence proteins through contact zones between bacteria and host cells
(Rosqvist et al., 1994). The secreted proteins of S. flexneri and Yersinia spp. form
various aggregates and protein complexes in liquid, stationary-phase cultures (Michiels et
al., 1990; Parsot et al., 1995). However, the relationship between these protein
aggregates and possible formation of surface appendages in these bacteria remains to be
determined.

Agrabacterium tumefaciens has been shown to produce pili involved in T-DNA
transfer (Fullner et al., 1996). Pili of 3.5 nm in diameter were formed under vir gene-

inducing conditions. These pili were proposed to function as conjugation pili in T-DNA

57

transfer between bacteria and plant cells. The protein components of the T-DNA
secretion pathway encoded by virB genes share sequence similarities with proteins
involved in the assembly of conjugative pili, but not with protein components of the type
III secretion system. The structural proteins of the A. tumefaciens pilus have yet to be
identified.

Morphologically, the Hrp pilus of Pst DC3000 characterized in this study
resembles most closely the pilus produced by A. tumefaciens. Both pili are much thinner
than the surface appendages of S. typhimurium. This similarity may reflect an adaptation
of the two bacteria in the infection of wall-bound plant cells. Conditions for pilus
production by the two bacteria are also very similar. Like the Hrp pilus, far fewer A.
tumefaciens pili are produced at higher temperatures (e. g., 28°C) than at lower
temperatures (e.g., 19°C). Furthermore, formation of the Hrp pilus requires solid growth
medium, the condition used also for growing A. tumefaciens for pilus production (Fullner
et al., 1996). This may reflect the instability of the pilus under mechanical shaking
condition.

Recent results strongly suggest that the action sites of P. syringae pv. glycinea
AvrB and possibly X. campestris pv. malvacearum Avr/Pth proteins are inside the plant
cell. A previous study showed that close bacterial contact is required for bacterial
elicitation of HR (Stall and Cook, 1979). The requirement of the Hrp pilus structural
gene hrpA in the phenotypic expression of avrB, as demonstrated in this study, suggests
that the Hrp pilus may be involved in the delivery of AvrB and possibly other virulence
and avirulence proteins into the plant cell (conduit model, Fig. 2.6). Alternatively, it may

be involved in mediating contact between bacteria and plant cells in the plant intercellular

58

space (contact model, Fig. 2.6). The exact function of the Hrp pilus in protein transfer or

cell-cell contact remains to be determined.

59

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2.6. REFERENCES

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A disease resistance gene in Arabidapsis with specificity for two different pathogen
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Bonas, U. (1994). Bacterial pathogenesis of plants and animals-molecular and cellular
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Comelis, G. R., and Wolf-Watz, H. (1997). The Yersinia Yop virulon: a bacterial
system for subverting eukaryotic cells. Mol Microbiol 23, 861-7.

de Lorenzo, V., Herrero, M., J akubzik, U., and Timmis, K. N. (1990). Mini-TnS
transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal
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Fullner, K. J., Lara, J. C., and Nester, E. W. (1996). Pilus assembly by Agrabacterium
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Ginocchio, C. C., Olmsted, S. B., Wells, C. L., and Galan, J. E. (1994). Contact with
epithelial cells induces the formation of surface appendages on Salmonella typhimurium.
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Gopalan, S., Bauer, D. W., Alfano, J. R., Loniello, A. 0., He, S. Y., and Collmer, A.
(1996). Expression of the Pseudomanas syringae avirulence protein AvrB in plant cells
alleviates its dependence on the hypersensitive response and pathogenicity (Hrp)
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Hakansson, S., Schesser, K., Persson, C., Galyov, E. E., Rosqvist, R., Homble, F.,
and Wolf-Watz, H. (1996). The YopB protein of Yersinia pseudotuberculosis is
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He, S. Y. (1996). Elicitation of plant hypersensitive response by bacteria. Plant Physiol
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He, S. Y., Huang, H. C., and Collmer, A. (1993). Pseudomanas syringae pv. syringae
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61

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cluster from Pseudomanas syringae pv. syringae 61 and TnphoA tagging of genes

encoding exported or membrane-spanning Hrp proteins. Mol Plant Microbe Interact 4,
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Hueck, C. J., Hantman, M. J., Bajaj, V., Johnston, C., Lee, C. A., and Miller, S. I.
(1995). Salmonella typhimurium secreted invasion determinants are homologous to
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Innes, R. W., Bent, A. F., Kunkel, B. N., Bisgrove, S. R., and Staskawicz, B. J.
(1993). Molecular analysis of avirulence gene aerptZ and identification of a putative

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Iriarte, M., Sory, M. P., Boland, A., Boyd, A. P., Mills, S. D., Lambermont, I., and
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Keen, N. T., Tamaki, S., Kobayashi, D., and Trollinger, D. (1988). Improved broad-
host-range plasmids for DNA cloning in gram-negative bacteria. Gene 70, 191-7.

Kihara, M., Homma, M., Kutsukake, K., and Macnab, R. M. (1989). Flagellar switch
of Salmonella typhimurium: gene sequences and deduced protein sequences. J Bacteriol
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King, E. 0., Ward, M. K., and Raney, D. E. (1954). J Lab Med 22, 301-307.

Lee, V. T., and Schneewind, O. (1999). Type 111 machines of pathogenic Yersiniae
secrete virulence factors into the extracellular milieu. Mal Microbial 31, 1619-29.

Martin, G. B., Brommonschenkel, S. H., Chunwongse, J ., Frary, A., Ganal, M. W.,
Spivey, R., Wu, T., Earle, E. D., and Tanksley, S. D. (1993). Map-based cloning of a
protein kinase gene conferring disease resistance in tomato. Science 262, 1432-6.

Michiels, T., Wattiau, P., Brasseur, R., Ruysschaert, J. M., and Comelis, G. (1990).
Secretion of Yop proteins by Yersiniae. Infect Immun 58, 2840-9.

Parsot, C., Menard, R., Gounon, P., and Sansonetti, P. J. (1995). Enhanced secretion
through the Shigellafiexneri Mxi-Spa translocon leads to assembly of extracellular
proteins into macromolecular structures. Mol Microbiol 16, 291-300.

Persson, C., Nordfelth, R., Holmstrom, A., Hakansson, S., Rosqvist, R., and Wolf-
Watz, H. (1995). Cell-surface-bound Yersinia translocate the protein tyrosine
phosphatase YopH by a polarized mechanism into the target cell. Mal Microbial 18, 135-
50.

62

'T1—a

Preston, G., Huang, H. C., He, S. Y., and Collmer, A. (1995). The HrpZ proteins of
Pseudomanas syringae pvs. syringae, glycinea, and tomato are encoded by an operon
containing Yersinia ysc homologs and elicit the hypersensitive response in tomato but not
soybean. Mol Plant Microbe Interact 8, 717-32.

Raine, E., Wei, W., Yuan, J ., Nurmiaho-Lassila, E. L., Kalkkinen, N., Romantschuk,
M., and He, S. Y. (1997). Hrp pilus: an hrp-dependent bacterial surface appendage
produced by Pseudomanas syringae pv. tomato DC3000. Proc Natl Acad Sci U S A 94,
3459-64.

Ronald, P. C., Salmeron, J. M., Carland, F. M., and Staskawicz, B. J. (1992). The
cloned avirulence gene avrPto induces disease resistance in tomato cultivars containing
the Pto resistance gene. J Bacteriol 174, 1604-11.

Rosqvist, R., Magnusson, K. E., and Wolf-Watz, H. (1994). Target cell contact triggers
expression and polarized transfer of Yersinia YopE cytotoxin into mammalian cells.
EMBOJ 13, 964-72.

Schweizer, H. P. (1991). Escherichia-Pseudomanas shuttle vectors derived from
pUC18/19. Gene 97, 109-21.

Stall, R. E., and Cook, A. A. (1979). Evidence that bacterial contact with the plant cell is
necessary for the hypersensitive reaction but not the susceptible reaction Xanthomonas
vesicatoria, pepper. Physiol Plant Pathol 14, 77-84.

Totten, P. A., and Lory, S. (1990). Characterization of the type a flagellin gene from
Pseudomanas aeruginosa PAK. J Bacteriol 172, 7188-99.

Winstanley, C., Morgan, J. A., Pickup, R. W., and Saunders, J. R. (1994). Molecular
cloning of two Pseudomanas flagellin genes and basal body structural genes.
Microbiology 140, 2019-3 1.

Wolf, M. K., and Boedeker, E. C. (1990). Cloning of the genes for AF/Rl pili from
rabbit enteroadherent Escherichia coli RDEC-l and DNA sequence of the major
structural subunit. Infect Immun 58, 1124-8.

Yuan, J ., and He, S. Y. (1996). The Pseudomanas syringae Hrp regulation and secretion

system controls the production and secretion of multiple extracellular proteins. J
Bacterial 178, 6399-402.

63

Chapter 3

ROLE OF THE HRP PILUS IN TYPE III PROTEIN SECRETION IN
PSEUDOMONAS S YRINGAE PV. TOMA T0 DC3000

3.1. ABSTRACT

Bacterial surface appendages called pili are often required for DNA and/or protein
transfer between cells. The exact function of pili in the transfer process is not understood
and is a matter of considerable debate. The Hip pilus is assembled by the Hrp type III
protein secretion system of Pseudomanas syringae pv. tomato (Pst) strain DC3000. In
this study, we show that the hrpA gene, which encodes the major subunit of the Hrp pilus,
is required for expression of all examined genes that are associated with type III
secretion. HrpA-mediated gene regulation occurs through activation of two previously
characterized regulatory genes (hrpR and hrpS) at the RNA level. Ectopic expression of
the hrpRS gene operon restored gene expression in the hrpA mutant. Three single amino
acid mutations at the HrpA carboxyl terminus were identified that affect the regulatory
function of the HrpA protein. In addition to being required for gene regulation, a
functional HrpA protein was found to be necessary for extracellular secretion of putative
virulence proteins, such as HrpW and AvrPto. These results define an essential role of
the Hrp pilus structural protein in protein secretion and global regulation of a major

protein secretion system in Gram-negative bacteria.

64

3.2. INTRODUCTION

Many Gram-negative bacterial pathogens, including Pst DC3000, possess a
unique protein secretion system called the type III protein secretion system that is
necessary for transfer of virulence proteins directly into the host cell (Galan and Collmer,
1999; He, 1998; Hueck, 1998). This system has been shown to play a critical role in
bacterial infection of plants, and animals. Genes involved in type 111 protein secretion
have been characterized extensively in several model human, animal and plant bacteria
(Galan and Collmer, 1999; He, 1998; Hueck, 1998). Several general features of type III
protein secretion have been revealed: i) type 111 protein secretion appears to be activated
fully upon contact with the host cells in viva (Pettersson et al., 1996); ii) extracellular
filamentous appendages are often associated with type 111 protein secretion (Ginocchio et
al., 1994; Knutton et al., 1998; Roine et al., 1997); iii) a secretion signal is localized in
the 5’ region of the mRNA of the secreted protein (Anderson and Schneewind, 1997;
Anderson and Schneewind, 1999); and iv) the secretion apparatus is genetically and
morphologically similar to the bacterial flagellum (Kubori et al., 1998).

In plant pathogenic bacteria, the type 111 protein secretion system is encoded by
hrp (for hypersensitive reaction and pathogenicity) genes (He, 1998; Lindgren, 1997;
Lindgren et al., 1986). Nine hrp genes have been renamed hrc (for hrp conserved) genes
because of their broad conservation among all bacteria that harbor type 111 protein
secretion systems (Bogdanove et al., 1996). The Hrp secretion system of Pseudomanas
syringae has been shown to secrete two families of proteins that elicit host responses:
Harpins, such as HrpZ and HrpW (Charkowski et al., 1998; He et al., 1993; Yuan and

He, 1996), and Avr proteins (Alfano et al., 1997; Gopalan et al., 1996; Scofield et al.,

65

1996; Tang et al., 1996); (Mudgett and Staskawicz, 1999; Stevens et al., 1998; van Dijk,
1999). The expression of P. syringae hrc/hip genes is tightly controlled. Most hrp genes
are expressed at a very low level in standard nutrient-rich media. The expression of
hrc/hip genes is induced in infected plant tissues or in artificial hrp-inducing minimal
media that presumably mimic the in planta conditions (Huynh et al., 1989; Rahme et al.,
1992; Xiao et al., 1992). The mechanism by which host and other signals induce hrc/hrp
genes is not clear. Three intracellular positive regulatory proteins are required: HrpR and
HrpS, which belong to the NtrC family of two-component regulatory proteins (Grimm et
al., 1995; Grimm and Panopoulos, 1989; Xiao et al., 1994), and HrpL, a member of the
ECF family of alternate sigma factors (Xiao and Hutcheson, 1994). The HrpS, HrpR, and
HrpL proteins appear to function as a regulatory cascade in which HrpS and HrpR
activate the expression of HrpL in response to a signal in host tissue or in hip-inducing
minimal medium (Grimm et al., 1995; Xiao et al., 1994). HrpL is presumed to activate
all hip and avr genes by recognizing a consensus sequence motif (“harp box”) present in
the upstream regions of many hrp and avr genes (Xiao et al., 1994; Xiao and Hutcheson,
1994). Recently, a putative negative regulator encoded by the hrpV gene has been
identified in P. syringae (Preston et al., 1998). In hip-inducing minimal medium,
overexpression of the htpV gene down-regulates hrp/hrc gene expression, whereas a
hrmeutant is elevated in hip/hrc gene expression (Preston et al., 1998).

In a previous study, we found that P. syringae pv. tomato strain DC3000
assembles a hrp-dependent pilus (the Hrp pilus) (Roine et al., 1997). We showed that the
structural protein of the Hrp pilus, HrpA, is required for P. syringae pv. tomato (Pst)

DC3000 to cause disease in Arabidapsis and to elicit the hypersensitive response (HR) in

66

tobacco and tomato (Roine et al., 1997). The exact function of the Hrp pilus in plant-Pst
DC3000 interactions, however, is not clear. Pili have also been shown to be required for
bacterial conjugation (Willetts and Skurray, 1980) and for transfer of T-DNA to plant
cells by Agrobacterium tumefaciens (Fullner et al., 1996). The conjugative F pilus plays
a major role in mediating contact between donor and recipient bacteria during mating
(Willetts and Skurray, 1980). However, whether pili have other functions in protein
and/or DNA transfer is not understood and is a matter of considerable debate.
Specifically, it is not known whether these pili are involved in the actual transfer of DNA
or protein or only in mediating cell-cell contact. In this study, we show that the Hrp pilus
structural protein HrpA plays a key role in secretion of putative virulence proteins and

global regulation of the Pst DC3000 Hrp system.

3.3. MATERIALS AND METHODS

3.3.1. Bacterial Strains, Plasmids and Growth Conditions

Pst DC3000 and four hrp mutant derivatives (hrpA, hrpS, hrcC, and hrcChrpT V)
were used in this study. The hrpA and hrcChrpT V (formerly hrpH) mutants were
described before (Roine et al., 1997; Yuan and He, 1996). The hrcC mutant was
constructed by replacing the hrcC gene with the terrninatorless nptII gene (kindly
provided by Dr. Alan Collmer’s laboratory). The hrpS mutant construction is described
below. Pst strains were grown at 28°C in Luria-Bertani (LB) broth (Sambrook et al.,
1989). For induction of hrp genes, bacteria were grown at 20°C in hrp-inducing minimal
medium (NaCl 0.1%, KH2P04 0.17%, MgSO4-7H20 0.07%, potassium citrate 0.324%,

(NH4)2SO4 0.4%, pH 5.5, and 10 mM fructose added after autoclaving) (Innes et al.,

67

1993). E. coli DHSa, grown in LB medium at 37°C, was used for all cloning
experiments. pHRPA contains the Pst DC3000 hrpA gene under the control of its native
promoter (Roine et al., 1997). pHRPRSl carries the P. syringae pv. syringae 61 hrpRS
ORFs under the control of the salicylic acid-inducible PG promoter in pKMY299 (Yen,
1991). pHRPRSZ carries the Pst DC3000 hrpRS ORFs under the control of Plac in
pUCP18 (Schweizer, 1991). pHRPCT contains the hrcC and hrpT genes downstream of
the lac promoter of pUCPl 8 (Schweizer, 1991) and it was able to restore the ability of
hrcChrpTV mutant to elicit an HR in tobacco and to cause disease in Arabidapsis (data
not shown). All plasmids were introduced into P. syringae by electroporation. The
antibiotics used were: rifampicin, 100 mg/l; ampicillin, 100 mg/l; chloramphenicol, 34

mg/l; and kanamycin, 50 mg/l.

3.3.2. Construction of the hrpS Mutant

An hrpS mutant was constructed through transposon-mediated mutagenesis by
using TnSSp/Sm carried on the suicide plasmid pUT (Herrero et al., 1990) in E. coli strain
SMlO ltpir (Taylor et al., 1989). Previously, the Pst DC3000 hrp gene cluster was
cloned in two plasmids (pDC3000-l and pDC3000-2) by Sheng Yang He, using pUCPl9
as the cloning vector (Schweizer, 1991). Hybridization with individual 32P-labeled Pss
61 hm gene probes allowed me to determine that the 10.9-kb insert in pDC3000-1
contains the hrcC, hrpZ, and hrpRS operons. Ms. J. Yuan (in our laboratory) subcloned
the 10.9-kb insert in pDC3000-1 into the pRK415 vector (Keen et al., 1988), and the
resulting plasmid was designated pRK3000-1. In order to mutagenize the hrpS gene,

donor (pUT-TnSSp/Sm in SMlO kpir) and recipient cells (pRK3000-l in DHSa) were

68

plated together on LB agar plates and incubated at 37°C for 8—10 hours. The bacterial
mixture was subsequently plated on LB plates containing 10 ug/ml nalidixic acid

(selecting for DHSa), 10 ug/ml tetracycline (selecting for pRK3000-1), and 50 ug/ml
spectinomycin (selecting for TnSSp/Sm). The pRK3000-1 plasmid DNA was isolated
from the surviving transconjugants, transformed en masse into DHSa, and plated on LB
plates containing tetracycline and spectinomycin (selecting for pRK3000-1 carrying the
sTnSSp/Sm insertion). Individual transformant was examined by polymerase chain
reaction (PCR) analysis to identify TnSSp/Sm insertions in the hrpRS operon. The
following two oligonucleotide primers were used to amplify the hrpR and hrpS ORFs: 5’-
GGGGTACCTGCTCTTGTCCCGGATCACAGC-3’ (annealing to the promoter
sequence of the hrpR/S gene) and 5’-GCTCTAGATCAGATCTGCAATTCTTTGATG-
CG-3’ (annealing to the hrpS 3’ end). One pRK3000-l carrying a TnSSp/Sm insertion in
the hrpRS operon was isolated and electroporated into Pst DC3000. The hrpRS gene
operon in the DC3000 chromosome was replaced with the mutated hrpRS copy by
standard marker-exchange mutagenesis (de Lorenzo et al., 1990). The hrpS mutant was
verified by PCR analysis using the above two primers and confirmed by its failure to
elicit HR on tobacco and to cause disease on Arabidapsis thaliana (data not shown).
Furthermore, the hrpS mutant could be complemented by both pHRPRSl and pHRPRS2

(data not shown).

69

3.3.3. Protein Immunoblot Analysis

For immunoblot analysis of Hrp/Hrc proteins in culture, bacteria were first grown
at 28°C to an OD600 of 0.8 to 1.0 in 5 ml of LB broth supplemented with appropriate
antibiotics. Bacteria were then pelleted and resuspended in 51ml of hrp-inducing broth or
LB broth and incubated with shaking (250 rpm) at 20°C for 6 hrs. Cultures were then
separated into cell (C) and supernatant (S) fractions by centrifugation. The cell fraction
was resuspended in 0.5 ml of sterile water, whereas the supernatant fraction was
concentrated lO-fold with Microcon 10 microconcentrators (Amicon) or the 30,000
NMWL filter unit of Ultrafree-MC (Millipore Corporation). Hrp/Hrc proteins in these
fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) followed by immunoblotting with appropriate antibodies. For immunoblot
analysis of Hrp/Hrc proteins expressed in planta, bacteria were grown in LB broth at
28°C to an OD600 of 0.8. Bacterial suspensions (OD600 of 1.5) prepared in distilled water
were infiltrated into leaves of tobacco using needleless syringes. Four hours after
infiltration, the infiltrated leaf tissue was excised and bacteria were expelled from the
infiltrated leaf tissue by re-infiltrating the leaf tissue with an excess amount of distilled
water. Bacteria were collected by centrifugation and the levels of Hrp/Hrc proteins in the
expelled bacteria were analyzed by immunoblotting with appropriate antibodies. The
protocol of immunoblot analysis of AvrPto secretion is as follows (conducted by Wen-
Ling Deng in Alan Collmer’s lab): Bacteria were first grown at 30°C to an OD600 of 0.8-
1.0 in 40 ml of LM broth supplemented with appropriate antibiotics. The bacteria were
then pelleted, washed with 10- to 15-ml hrp-inducing minimal medium (MM) twice, and

resuspended in lOO-ml hrp-inducing MM to an OD600 of 0.22-0.23. Cultures were

70

incubated at 20°C with shaking (180 rpm) until the Optical density reached 0.4 (Pst
DC3000 took about 24 hr to reach 0.4; different strains took different lengths of time).
Bacterial cultures were then separated into cell and supernatant fractions by
centrifugation (in a 45-ml Oakridge tube) (12,000 x g, 10 min, to pellet the bacteria first;
then the supernatant was transferred to a clean tube for another spin at the same speed for
another 10 min). Afier the second centrifugation, the upper lS-ml of the supernatant was
carefully withdrawn to a clean tube, and proteins were precipitated with 20% (w/w) TCA
at 4°C for at least 1 hr. The precipitated proteins were washed once with cold acetone
once, and resuspended in 60 ml SDS sample buffer. In all immunoblotting experiments,
gel staining with Coomassie Brilliant Blue R-250 was used to ensure equal loading of the

protein samples.

3.3.4. RNA Blot Analysis

For determination of steady-state levels of hrp W, hrcC, avrPta, and hrpRS
transcripts, total RNA was first isolated from bacteria according to the following
protocol: Bacteria were first grown at 28°C to an OD600 of 03-06 in 10 ml of LB broth
supplemented with appropriate antibiotics. The bacteria were then pelleted, washed with
10 ml of 10 mM MgC12, and resuspended into 5 ml of LB or hip-inducing MM, and
incubated with shaking for another 3 hr. 1.5 ml of bacterial culture was pelleted in a
microtube. The cell pellet was resuspended in 0.5 m1 lysis buffer (SOmM Tris-HCl, pH
9.0, 50 mM EDTA, 300 mM NaOAc, 0.625% [w/v] sodium dodecyl sulfate), incubated
in boiling water for 30 sec, extracted with phenol twice at 65°C, and extracted with

chloroform twice. The supernatant was added to 0.5 ml of isopropyl alcohol, and the

71

visible clumps of DNA were taken out. The rest of the solution was then centrifuged for
5 min. The pellet was resuspended in 0.5 ml DEPC-treated water, incubated with 0.5 ml
4M lithium acetate for 60 min on ice, and then centrifuged for 5 min. The pellet was
resuspended in 0.5 ml DEPC-treated water, incubated with 0.5 ml 4M lithium acetate for
60 min on ice, and then centrifuged for 5 min. The pellet was resuspended in 0.5 ml of
DEPC-treated water and precipitated with 1 ml cold (0°C) 100% ethanol. The pellet was
washed with 70% ethanol, dried, and dissolved in 25 ml of DEPC-treated water. Five pg
of total RNA from each bacterial strain were analyzed by northern blotting (Sambrook et

al., 1989).

3.3.5. Mutagenesis of the hrpA Gene (by Anne Plovanich-J ones and Sheng Yang He)

For site-directed mutagenesis, the hrpA gene from pHRPA (Roine etal., 1997)
was cloned into pAlterl (Promega). Six amino acid residues (G23, A54, K93’ D95, 11°], and
1'”) that are conserved between the HrpA proteins of P. syringae pv. tomato and Erwinia
amylovora and/or among the HrpA proteins of P. syringae pvs. tomato, syringae, and
glycinea (Kim et al., 1997; Preston et al., 1995) were individually replaced by residues
with different properties (i.e., A23, E54, 193, 895, T1°', and Prol ‘ l). The mutagenized hrpA
inserts were then recloned into pUCP18 (Schweizer, 1991). For random mutagenesis of
the hrpA gene, pHRPA was transformed into the E. coli mutator strain XLl-Red
(Stratagene). After being subcultured four times, each for 12 hr at 37°C, pHRPA was
isolated from XLl—Red and introduced into the Pst DC3000 hrpA nonpolar mutant en

masse by electroporation. Transforrnants were individually grown in LB broth in

microtiter plates overnight at 28°C (OD600 > 1.0). The bacterial cultures were then

72

diluted ten-fold in distilled water to an OD600 of 0.1 to 0.2 and infiltrated into tobacco and
Arabidapsis leaves for HR and pathogenesis tests (see (Roine et al., 1997), for

procedures).

3.3.6. Pathogenesis Assays

Bacteria were grown in LB broth to an OD600 of 0.6-0.8. Bacterial suspensions in
distilled water were infiltrated into leaves of Arabidapsis thaliana ecotype Columbia
(C01), and tobacco (Nicotiana tabacum cultivar Samsun NN) using needleless syringes.
The concentrations of bacteria used were 2 X 108 colony-forming units (cfu)/ml and 2 X
10° cfu/ml for HR and pathogenesis assays, respectively. Plant responses were recorded
at 24 hr (for HR assay) or 4 days (for pathogenesis assay). The HR is characterized by
rapid, localized tissue collapse in the infiltrated area within 24 hr. Disease symptoms
were characterized by slowly developing necrosis and spreading tissue chlorosis, usually
observed 3 days afler infiltration. Tobacco plants were grown in greenhouses.
Arabidapsis plants were grown in a growth chamber at 20°C with 70% relative humidity
and a 12-hr photoperiod. HR assays with tobacco plants were performed at room
temperature in the laboratory. Pathogenesis assays with Arabidapsis plants were
performed at 20°C in growth chambers with 70% relative humidity and a 12-hr

photoperiod.

73

3.4. RESULTS

3.4.1. Effect of a hrpA Deletion Mutation on the Production of HrpW

To examine the role of the Hrp pilus in protein secretion, I investigated the effect
of a hrpA deletion mutation on secretion of HrpW in culture, in which a possible
involvement of the Hrp pilus in mediating bacterial attachment to host cells is excluded.
The cellular distribution of HrpW in Pst DC3000 and hrpA, hrcC, hrcChrpTV, and hrpS
mutants was determined. As shown in Fig. 3.1A, HrpW was produced and secreted in
DC3000 and in the hrpA mutant complemented by pHRPA, which carries the wild-type
hrpA gene (Roine et al., 1997). HrpW was produced but not secreted in the hrcC and
hrcChrpTV secretion mutants, and was not detectable in the hrpS regulatory mutant. In
the hrpA deletion mutant, however, HrpW was not detectable in the medium and was
only barely detectable in the cells (Fig. 3.1A). The higher level of HrpW (and other Hrp,
Hrc, and Avr proteins; see below) in the hrcChrpTV mutant, compared with that in the
wild-type DC3000, may be due to the polar effect of the Tn5Cm-induced mutation in the
hrcC gene on the downstream hrpV gene, which encodes a putative negative regulator
(Preston etal., 1998). HrpW was not overproduced in the nonpolar hrcC mutant, but it
was overproduced in the hrcChrpTV mutant carrying pHRPCT, which contains hrcC and

hrpT genes driven by P100 promoter (Fig. 3. 1 A).

74

3.4.2. hrpA is Required for Expression of Other Hrc/Hrp Proteins

The unexpected down-regulation of the secreted protein, HrpW, in the hrpA
mutant prompted me to examine a possible effect of the hrpA mutation on the expression
of other hrc/hrp genes. Specifically, I examined expression of two membrane-bound
components (HrcC and Hch) of the Hrp secretion machinery (Deng and Huang, 1999;
Huang et al., 1992). In hrp-inducing medium Hch was highly expressed in DC3000, the
hrcC and hrcChipT V mutants, and the hrpA mutant complemented by pHRPA, but not in
the hrpS mutant. However, the expression of Hch was significantly suppressed in the
hrpA mutant (Fig. 3.1A). The same expression pattern was observed for HrcC, except
that HrcC was not detected in the hrcC or hrcChrpTV mutant (Fig. 3.1A). To rule out the
possibility that the effect of the hrpA mutation on expression of hrc/hrp genes is due to
the difference between hrp-inducing culture and the actual environment in planta, I
determined the expression patterns of HrpW, Hch, and HrcC in DC3000 and the hrp
mutants in planta. I found an expression pattern similar to that in hrp-inducing culture
(Fig. 3.1B). Specifically, HrpW, Hch, and HrcC were produced in large amounts in
DC3000, but not in the hrpS mutant. The amounts of these proteins were very low in the
hrpA mutant. pHRPA restored the expression of these proteins in the hrpA mutant (Fig.
3.1B). Thus, I conclude that the pilus structural gene hrpA is required for expression of
secreted proteins (HrpW) and components of the Hrp secretion machinery (HrcC and

Hch) in both hrp-inducing minimal medium and in planta.

75

 

 

 

 

 

 

 

 

 

 

 

_u-_
W.-

NJ 0..

MC

 

 

 

 

 

 

 

Figure 3.1. Immunoblot analysis of HrpW, Hch, and HrcC proteins in Pst DC3000
and hrp mutants in hrp-inducing medium (A) and in planta (B). For analysis of HrpW
expression in hrp-inducing cultures, the levels of HrpW in cell-associated (c) and
supernatant (5) fractions were examined. (C) A diagram of Hrp/Hrc protein
production and secretion in Pst DC3000, hrcC mutant, and hrpA mutant.

76

3.4.3. hrpA Controls the Expression of hrp, hrc, and avr Genes at the RNA Level

To determine whether the hrpA gene controls the expression of hrc/hrp genes at
the RNA or protein level, I directly measured the steady-state RNA level of the hrpW
operon in DC3000 and various mutants. Consistent with the immunoblot results, this
operon was significantly down-regulated in the hrpA and hrpS mutants, compared with
that in DC3000, in the hrpA deletion mutant complemented by pHRPA, and in the
hrcChrpTV mutant (Fig. 3.2). I also determined the effect of the hrpA mutation on the
expression of avrPto, which encodes a protein that is probably delivered into the plant
cell via the Hrp secretion system (Scofield et al., 1996; Tang et al., 1996). As shown in
Fig. 3.2, the steady-state RNA level of avrPto was also significantly suppressed in the
hrpA and hrpS mutants. The higher level of steady-state RNA in the hrcChrpTV mutant is
likely due to the absence of the negative regulator HrpV. The same result was obtained
for the hrcC operon (Fig. 3.2). Thus, both the negative effect of the hrpA mutation and
the positive effect of the himeutation on the expression of hrc, hrp, and avr genes in

hrp-inducing medium are at the RNA level.

77

 

hrpW

 

 

 

hrcC

 

a WHO

 

 

 

 

 

23$rRNA ~~~~~~

 

 

 

Figure 3.2. RNA blot analysis of hip W, hrcC and avrPto transcripts in Pst DC3000

and hip mutants. The genes indicated on the left were used as probes. The 23S rRNA
visualized after ethidium bromide staining was used as a loading control. RNA was
isolated from bacteria grown in hrp-repressing Luria-Bertani (LB) broth or hip-inducing
minimal medium (MM).

78

3.4.4. Three Amino Acid Residues Located at the Carboxyl Terminus Are Essential

for HrpA-Mediated Gene Regulation

We next attempted to define amino acid substitution mutations that affect the
regulatory function of the HrpA protein. An HR assay was used to screen for these
mutations. As reported previously, the wild-type hrpA gene cloned in pHRPA restored
the ability of an hrpA deletion mutant to elicit an HR in tobacco (Roine etal., 1997). We
reasoned that a mutation that affects the regulatory function of the HrpA protein would
eliminate the ability of the hrpA gene (pHRPA) to complement the genomic hrpA
deletion mutation. We used both site-directed and random mutagenesis procedures in the
screen. Site-directed mutagenesis was facilitated by the relatively few residues (six were
substituted in this study) conserved among the hypervariable HrpA proteins of P.
syringae pathovars and E. amylovora (Kim et al., 1997; Preston et al., 1995). Residue
substitutions in two positions (D95 to 895 and Il 1' to P1 1 1) were found to eliminate the
ability of the hrpA gene to complement the hrpA mutation for the elicitation of HR in
tobacco (Fig. 3.3A). Random mutagenesis enabled us to identify a third mutant hrpA
gene that fails to complement the hrpA mutation. Sequence analysis revealed an amino
acid substitution at position 94 (from E94 to K94) in the HrpA protein (Fig. 3.3A). All
three mutant hrpA genes were found to be defective in positive regulation of hrc/hrp

expression (Fig. 3.3B).

79

1 113
0 ”at 0'

l

 

 

m.

 

 

 

G45 A“ Koo E94 095 I101 I111
045 554 [93 K“ 395 T‘°‘ pm
HrpA function 4» + + +

 

 

 

 

 

 

 

 

 

Figure 3.3. (A) A diagram of the P. syringae pv. tomato DC3000 HrpA protein (113
amino acids in length). Six amino acid residues (*) conserved among HrpA proteins of
P. syringae pvs. tomato, syringae, and glycinea and Erwinia amylovora were mutated by
site-directed mutagenesis. The E94 to K94 mutation, indicated by an arrowhead, was
obtained by random mutagenesis. Amino acid residue substitutions that did not affect
the HrpA function are indicated with hatched bars. Those substitutions that eliminated
the ability of pHRPA to complement the genomic hrpA mutation are indicated with filled
bars. The HrpA function was assayed by the ability (+) or inability (-) of the
corresponding pHRPA derivatives to complement the genomic hrpA deletion mutation
for HR elicitation in tobacco leaves and disease causation in Arabidapsis thaliana leaves.
(B) Immunoblot analysis of the effect of single amino acid mutations of HrpA on the
production of HrpW, Hch, and HrcC in hrp-inducing medium. Both cell-associated

(C) and supernatant (S) fractions were analyzed.

80

3.4.5. HrpA-Mediated Gene Regulation is Through Activation of hrpS and hrpR at

the RNA Level

To examine the relationship between hrpA gene and other regulatory genes, I first
determined the steady-state message levels of the hrpRS positive regulatory operon in
DC3000 and the hrpA mutant. As expected, the expression of the hrpRS operon was
induced when DC3000 and the hrcChtpT V mutant were grown in hrp-inducing minimal
medium, whereas this operon was barely expressed in DC3000 grown in LB or in the
hrpS mutant grown in the hrp-inducing medium. I found significant down-regulation of
the hrpRS operon in the hrpA mutant, whereas overexpression of hrpA from pHRPA
elevated the expression of the hrpRS operon (Fig. 3.4A). Furthermore, when the hrpRS
operon was expressed ectopically from a plasmid, hrc/hrp gene expression in the hrpA
mutant was completely restored. Specifically, HrpW and HrcC were produced in both
DC3000 and the hrpA mutant when hrpRS genes were ectopically expressed in the
otherwise hrp-repressing LB broth (Fig. 3.4B). Similarly, ectopic expression of the
hrpRS operon allowed expression of HrpW and HrcC proteins in the hrpA mutant in
planta (Fig. 3.4C). The same results were obtained for Hch protein (Fig. 3.4C). These
experiments suggest that hrpR and hrpS genes act downstream of hrpA in regulation of
hrc, hip, and avr genes. A previous study, however, has clearly shown that the
expression of the hrpA gene is under the control of hrpR and hrpS (Xiao et al., 1994).
Therefore, the activation of the type 111 protein secretion system in P. syringae involves a
closed regulatory network consisting of the extracellular pilus protein HrpA and at least

three intracellular positive regulators, HrpS, HrpR, and HrpL.

81

 

hrpRS

 

 

 

23$ rRNA _—~~~—

 

 

 

 

 

B ocsooo hrpA-
(pHRPRS1)(pHRPRS1) DC3000
SA - + - + - +

 

HrpW - _.— fi’ ‘

 

 

 

 

 

HrcC ' ..-~- --
C
.56
9%
00 ‘69.
'59 '
Y‘

 

 

 

Figure 3.4. (A) Effect of the hrpA mutation on the steady-state message abundance of
hrpRS. The procedures for bacterial growth and RNA preparation and blotting are the
same as in the legend to Fig. 2, except that the RNA gel blot was hybridized to the hrpRS
gene probe. (B) Effect of ectopic expression of hrpRS on the accumulation of Hrp/Hrc
proteins in the hrpA mutant. Bacteria were grown in LB broth supplemented with (+) or
without (-) 35 uM salicylic acid (SA). The original culture was directly used for analysis
by SDS-PAGE followed by immunoblot analysis with antibodies against HrpW and
HrcC, respectively, without further fractionation. pHRPRSl carries the P. syringae pv.
syringae 61 hrpRS open reading frames under the control of the SA-inducible PG promoter
in pKMY299 (Yen, 1991). (C) Effect of ectopic expression of hrpRS on the
accumulation of Hrp/Hrc proteins in the hrpA mutant in planta. Bacteria were
supplemented with 35 11M SA before infiltration. Ectopic expression of Pst DC3000
hrpRS genes also restored the expression of HrpW and HrcC in the hrpA mutant

(see Fig. 3.5).

82

3.4.6. hrpA is Required for Secretion of HrpW and AvrPto

The restoration of gene expression in the hrpA mutant by the ectopically
expressed hrpRS operon enabled the testing of a direct role of HrpA in protein secretion.
As shown in Fig. 3.5A, when grown in the hip-inducing medium, DC3000 produced and
secreted HrpW to the medium with or without ectopic expression of the hrpRS gene
operon, whereas HrpW was not detected in the culture medium of the hrpA mutants even
when the hrpRS gene operon was constitutively expressed. Instead, the expressed HrpW
was recovered exclusively in the cell fraction. Similarly, secretion of AvrPto was
completely blocked by both mutation of hrpA and hrp/19955 (Fig. 3.5B). Consistent with
a defect in protein secretion, the hrpA mutant ectopically expressing the hrpRS genes did
not elicit HR in tobacco or cause disease in Arabidapsis (Table 3.1). Thus, in addition to
its critical role in regulation of the Hrp secretion system, the Hrp pilus structural protein

is required for secretion of HrpW and AvrPto.

83

 

 

 

 

 

.23?
(V o}- ~2‘
0’ \ \ 9
<2~ n, Q or q)
0. 0) 0) 5» Ca
Q~ QQ‘ Q~ v- Q~
i Q Q <1 <1
Q 08 ‘2‘ {it I? I?
go , go .8 00° 3 >3 >8
0‘” <1” (3’ <1 6° 3 Q" q"
o e o e o a a c
l ' ‘ l
s ~. M l s . .
l _'_,. .
l 7 ’ 77' 77 '7 T7 ’77 7"
VA A, _ w c 4m
L ‘ ‘ ' J

 

 

C .\\'rl’1n
a

R
Hrp“ . O

 

 

 

DC3000 hag/1' hmA'lpHRPRS

 

Figure 3.5. Effect of ectopic expression of the hrpRS operon in the hrpA mutant

on HrpW and AvrPto secretion. Bacteria were grown in hrp- inducing minimal
medium. Bacterial supernatant (s) and cell (c) fractions were analyzed by SDS-
PAGE followed by immunoblotting with HrpW (A) or AvrPto antibody (B).
pHRPRSZ carries the Pst DC3000 hrpRS open reading frames under the control of

PM in pUCP18. (C) Diagram of protein production and secretion in Pst DC3000. hrpA
mutant. and hrpA mutant carrying pHRPRSl or pHRPRSZ.

Table 3.1. Plant reactions to DC3000 and various hrp mutants.

 

 

Bacteria Arabidapsis thaliana Tobacco
(ecotype Columbia) . (cv. Samsun NN)
DC3000 i SA D HR
hrpS" NR NR
hipA‘ NR NR
hrpA‘/pHRPA D HR
hrpS” /pHRPRS1 + SA D HR
hrpS/pHRPRSZ D HR
hrpA‘/pHRPRSl + SA NR NR
hrpA'/pHRPRSZ NR NR

%

hrpA"/pHRPAD955, PHRPRSl NR
+SA

 

HR, rapid, localized tissue collapse in the infiltrated area within 24 hours. D, disease
symptoms (slowly developing necrosis and spreading tissue chlorosis) observed 3 days

after infiltration; NR, no visible reaction; SA, 0.35 mM salicylic acid.

85

3.5. DISCUSSION

In this study, I have attempted to define the function of the Hrp pilus structural
protein HrpA in type III protein secretion in P. syringae pv. tomato DC3000. I found that
the HrpA protein is required for expression of all examined genes that are associated with
type 111 protein secretion. I showed that the positive regulatory function of HrpA on all
hrc, hip, and avr genes is achieved through activation of the hrpR and hrpS regulatory
genes at the RNA level. Ectopic expression of the hrpRS gene operon can completely
restore gene expression in the hrpA mutant. We identified three amino acid residues at
the carboxyl terminus that affect the regulatory function of the HrpA protein. Finally, we
showed that a functional HrpA protein is required not only for gene expression, but also
for secretion of HrpW and AvrPto in culture. To our knowledge, this is the first
demonstration of participation of a pilus structural protein in global regulation of a major
protein secretion system and control of protein secretion in bacteria. We suggest that the
Hrp pilus is an integral component of a supramolecular protein secretion structure that
enables Pst DC3000 to turn on virulence genes and inject virulence proteins at the right
place and time during bacterial infection of plants.

Pathogenic bacteria devote a large number of genes to type 111 protein secretion.
In the P. syringae hrp gene cluster alone, for example, about 27 genes are co-regulated
and they encode either regulatory, secretion, or effector proteins (Huang et al., 1995). In
addition, several hrp-regulated genes that encode effector proteins have been shown to be
unlinked to the hrp gene cluster (Collmer, 1998; Leach and White, 1996). Thus, turning
on the type 111 protein secretion system is an energy-consuming process. Because the

final outcome of activating the type III secretion system is delivery of some virulence

86

proteins into the host cell, it would be beneficial for bacteria to prevent full induction of
type-III-secretion-associated genes until host cells are available for protein injection.
This prediction is consistent with the observed “contact-dependent activation”
(transcriptional or posttranscriptional) of type 111 protein secretion in Yersinia
pseudotuberculosis, Salmonella typhimurium, Shigellaflexneri, and E. coli (Ginocchio et
al., 1994; Knutton et al., 1998; Menard et al., 1994; Pettersson et al., 1996; Rosqvist et
al., 1994). Expression of hip genes has also been shown to be induced in infected plant
tissues (Huynh et al., 1989; Rahme et al., 1992; Xiao et al., 1992). Host-bacterium
contact appears to be important for bacterial elicitation of an HR (Stall and Cook, 1979),
although direct evidence for contact-dependent activation of the Hrp system has yet to be
obtained. The molecular basis of bacterial sensing of host-bacterium contact is not
understood in any bacterium. In Yersinia, the current hypothesis for contact-dependent
activation assumes that host-bacterium contact removes bacterial surface sensors, such as
YopN (F orsberg et al., 1991), Lch (Sarker et al., 1998; Skryzpek and Straley, 1993),
and TyeA (Iriarte et al., 1998), that normally prevent secretion (Cornelis, 1998).
However, an involvement of secreted proteins (e. g. Lch) as part of an extracellular
secretion appendage in positive regulation of Yop secretion is a possibility (Cornelis,
1998).

The significant down-regulation of hrp, hrc, and avr genes in the hrpA mutants in
viva makes the Hrp pilus a strong candidate for mediating the presumed contact-
dependent regulation of the Hrp system in Pst DC3000. Although bacterial sensors in the
inner membrane or within the cytoplasm are often used to detect diffusible signals, they

would not be suitable for detection of extracellular physical contact. Pili, on the other

87

hand, because of their extracellular location and supramolecular nature, appear to be
suitable for sensing extracellular physical contact. In support of a role of pili in contact-
based gene regulation, the adhesive P pilus of E. coli has been shown to sense the host-
bacterium contact signal, resulting in the induction of bacterial iron starvation response
genes (Zhang and Norrnark, 1996). The growing Hrp pilus may therefore be used by Pst
DC3000 to sense the availability of host cells in viva. In Yersinia spp., an artificial
minimal medium has been shown to mimic the host-bacterium contact signal to induce
the Ysc type III secretion system (Comelis, 1998; Rosqvist et al., 1994). It is therefore
likely that the composition of the hrp-inducing minimal medium and growth conditions
in vitra (usually with constant shaking, resulting in frequent pilus breakage) also mimic
the plant-bacterium contact signal in viva.

Whether pili are involved directly in the transfer of DNA or protein or indirectly
in mediating cell-cell contact has been a long-standing and unresolved question in any
system. We show here that in addition to its involvement in gene regulation, the HrpA
protein is necessary for secretion of HrpW and AvrPto in culture, in which the possible
function of pilus-mediated bacterial attachment to the host cell is excluded. The
demonstrated role of HrpA in secretion of AvrPto and HrpW is therefore consistent with
a hypothesis that the Hrp pilus is an integral component of the Hrp secretion structure.
The Hrp pilus could be the functional equivalent of the extracellular part (called the short
needle extension) of the S. typhimurium type III secretion supramolecular structure
(Kubori et al., 1998), providing a conduit for protein secretion and/or maintenance of the
integrity of the Hrp secretion structure. Assembly of the longer pilus in Pst DC3000 may

reflect a need for this bacterium to deliver proteins through the thick plant cell wall,

88

which is lacking in animal cells. Alternatively, the HrpA protein may function as a
chaperone protein that pilots HrpW, AvrPto, and other secreted proteins through the Hrp
secretion machinery (and along the Hrp pilus).

The observed dual function of HrpA in gene regulation and protein secretion
strongly suggests that HrpA-mediated gene regulation is linked to the HrpA function in
protein secretion. One explanation would be that HrpA (and hence the Hrp pilus) is also
required for secretion of a negative regulator. Gene regulation based on export of a
negative regulator (i.e., Lch) via the type III protein secretion system, but not involving
a pilus, has been shown in Yersinia pseudotuberculosis (Pettersson et al., 1996).
According to this model, a negative regulator would accumulate in the cytoplasm of any
type III secretion mutant (e.g., the hrpA mutant), thus repressing the expression of hrc,
hip, and avr genes. In support of this mechanism, a putative negative regulatory gene,
hrp V, has recently been identified in P. syringae pv. syringae strain 61 (Preston et al.,
1998). However, this simple model does not appear to be consistent with the observed
normal gene expression in the hrcC (Figs. 1 and 2) and other secretion mutants in P.
syringae (Preston et al., 1998). In fact, the apparently different effects of secretion
mutations on gene expression are the key differences in the regulation of the type 111
protein secretion systems of Y. pseudotuberculosis and P. syringae. The molecular basis
for this difference remains to be determined. However, the fact that HrpW, HrcC, and
Hch proteins are expressed in much higher levels in the hrcC mutant than in the hrpA
mutant suggests that HrpA accumulated inside the hrcC mutant causes a high level of
gene expression. The HrpA-mediated positive regulatory function in the hrcC mutant

may reflect a normal function of HrpA in the wild-type bacteria, assuming that there is a

89

pool of intracellular HrpA protein in Pst DC3000. Alternatively, the aberrant,
intracellular localization of HrpA in the hrcC mutant may directly or indirectly interfere
with the action of a negative regulator, such as HrpV.

The requirement of the hrpA gene for expression of the hrpRS operon argues that
transcriptional activation of the Hrp secretion system in P. syringae involves a ‘closed’
regulatory circuit consisting of the extracellular pilus structural protein HrpA and three
intracellular positive regulators, HrpS, HrpR and HrpL. Where is the start point of this
regulatory circuit? The experimental results shown in Figs. 1, 3, and 5 suggest a two-
phase activation model. Specifically, we found that in both hrp-inducing medium and in
vivo, the levels of HrpW, Hch, and HrcC expression were slightly higher in the hrpA
mutant than in the hrpS mutant. In most experiments we observed detectable levels of
HrpW, HrcC, and Hch in the hrpA mutant (Figs. 3.1, 3.3, and 3.5), whereas we have
never been able to detect any HrpW, HrcC, or Hch protein in the hrpS mutant. The HrpS
and HrpR positive regulators must therefore be involved in detection of both a hrpA-
dependent signal and a hrpA-independent signal in the plant apoplast. Perhaps the hrpA-
independent signal triggers the first phase, low-level activation of hrp gene operons in the
plant apoplast, as revealed in the hrpA mutant. The low-level expression of hrp genes
initiates the growth of Hrp pili, which are then required for the second-phase, hrpA (and
presumably contact)-dependent induction of hrc, hrp, and avr genes. The increased
hrc/hrp/avr gene expression would enable further assembly of the Hrp secretion structure
on top of or along with the existing Hrp pilus to penetrate the plant cell wall and host
membrane, which eventually leads to injection of expressed virulence proteins into the

host cytoplasm.

90

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95

Chapter 4

IDENTIFICATION OF COTTON TARGETS OF XANTHOMONAS CAMPES TRIS

PV. MAL VA CEAR UM AVR/PTH FAMILY OF PROTEINS

4.1. ABSTRACT

The function of avr genes is dependent on hrp genes, which are involved in the
assembly of a type III secretion system. Recent studies have shown that Avr proteins are
probably translocated into plant cells to trigger host responses. One unique Avr family of
proteins, Avr/Pth, from the cotton pathogen Xanthomonas campestris, shares very high
sequence similarity among each other. Members of this family differ primarily in the
multiplicity of a tandemly repeated 102-base pair motif within the central portions of the
genes. The repeated regions have been shown to confer specificity in avr-R (Resistance)
gene interactions. Their C-terrninal regions contain eukaryotic nuclear localization
signals (NLSs), which have been demonstrated to be capable of delivering reporter fission
proteins into the plant nucleus, and are critical for their avirulence functions. Some
members (e.g. Avrb6) of this family are also responsible for the virulence of X.
campestris pv. malvacearum (Xcm) in susceptible cotton. This chapter describes
experiments aimed at identifying cotton proteins that interact with Avrb6, a member of
the Avr/Pth family of proteins. The yeast two-hybrid system was employed for this
purpose. After screening over 1.8 x 108 clones, 1 confirmed that more than 152 of them

encode proteins that specifically bind to the bait 6ANC, the central repeat region of

96

_..r m-‘w ______

Avrb6. Sequencing analysis revealed three interesting clones: a serine/threonine kinase
homologous to Pto in tomato, a calcineurin B-like protein, and a putative transcription
factor with a zinc-finger motif. The functional significance of these putative target
proteins in Xcm avirulence and virulence and their interaction with the entire length

Avrb6 protein remain to be determined.

97

4.2. INTRODUCTION

Bacterial pathogens are responsible for numerous diseases in higher plants,
animals and humans. Plants have evolved a variety of mechanisms to defend themselves
from pathogens (Lamb et al., 1989; Lamb, 1994). Among them, the gene-for-gene type
resistance response is being studied intensively. According to the gene-for-gene
hypothesis (Flor, 1971), disease resistance is conditioned by the presence of a single
dominant or semidominant disease resistance (R) gene in the host and a corresponding
avirulence (avr) gene in the pathogen. The absence of an avr gene in the pathogen or its
matching R gene in the plant, or both, results in a compatible interaction and leads to
plant disease.

A type 111 protein secretion system found in many bacteria transports virulence or
avirulence proteins to host cells. In animal pathogens, the type III secreted proteins affect
host cell signaling. For example, Yersinia species secrete several Yops (Xersinia Quter
Proteins), including a tyrosine phosphatase (YopH), an actin cytotoxin (YopE), and a
Ser/Thr kinase (kaA), into the host cell following host cell contact (Galyov et al., 1993;
Sory et al., 1995).

Indirect evidence suggests that bacterial virulence and avirulence proteins are also
transported into plant cell via type 111 protein secretion system. The first line of evidence
was provided by Gopalan et al. (1996) from our laboratory. By using transgenic and
transient expression of AvrB of P. syringae in leaves of Arabidapsis thaliana, they
showed that the site of activity of AvrB is inside the plant cell and that the Hip secretion
system is necessary for the delivery of AvrB into plant cells during infection. Similar

indirect evidence supporting transport of Avr proteins has independently been obtained

98

for P. syringae pv. tomato AvrPto (Scofield et al., 1996; Tang et al., 1996) and Aerpt2
(Leister et al., 1996). In the case of the avrPto/Pto system, the Pto protein appears to be
a cytoplasmic protein. It has been shown that AvrPto and Pto physically bind to each
other when expressed in yeast (Tang et al., 1996).

Additionally, Yang and Gabriel (Yang and Gabriel, 1995) reported that members
of the Xanthomonas Avr/Pth family of proteins might be recognized inside the plant cell.
PthA contains functional nuclear localization signals (NLSs). Subsequently, Van den
Ackerveken et al. (1996) showed that the NLSs in AvrBs3, another member of the
Avr/Pth family of proteins, were able to target the reporter protein B-glucuronidase
(GUS) to the plant nucleus. They also showed that these NLSs are required for AvrBs3
to trigger disease resistance-associated HR, suggesting that some members of the Avr/Pth
family not only enter the plant cell, but also enter the plant nucleus. Expression of
avrBs3 in pepper cells, using A grabacterium tumefaciens for gene delivery, resulted in
induction of hypersensitive cell death specifically on plants carrying the avrBs3-
corresponding R gene, BS3. This response resembled phenotypically the HR induced by
the pathogen Xanthomonas campestris pv. vesicatoria expressing avrBs3.

The research described in this chapter was aimed at identifying cotton proteins
that interact with Avrb6. Xanthomonas campestris pv. malvacearum (Xcm) causes
bacterial blight of cotton (Gossypium hirsutum L.). The disease symptoms include severe
water soaking and eventual necrosis. Xcm also elicits HR in resistant cotton cultivars. At
least 16 genetically characterized resistance genes against Xcm have been identified in
cotton. Many of these R genes have been introduced into a common (Acala-44) genetic

background by repeated backcrossing (Brinkerhoff et al., 1984; De Feyter and Gabriel,

99

1991), resulting in the resistant line named IM216. A large avirulence
(avr)/pathogenicity (pth) gene family has been found in Xcm (Yang et al., 1996). The
published members of the avr/pth gene family include avrB4, avrb6, avrBIOZ, avrB] 01,
avrBIn, and avrb7 of X. campestris pv. malvacearum (De F eyter and Gabriel, 1991; De
Feyter et al., 1993). All family members sequenced to date are 95 to 98% identical to
each other and to the prototype of this Avr protein family, AvrBs3 (Bonas et al., 1993;
De Feyter et al., 1993; Hopkins et al., 1992; Yang and Gabriel, 1995). They differ
primarily in the multiplicity of a tandemly repeated 102-base pair motif within the central
portions of the genes, repeated from 14 to 23 times in members of this gene family. The
changes in the repeat region can give rise to different specificity. This strongly suggests
that the Avr/Pth protein is the actual elicitor molecule that is being recognized by the
plant, rather than functioning in the bacterium as an enzyme required for the production
of elicitor molecules (De Feyter et al., 1993).
7 By constructing chimeric genes among pthA, avrB4, avrb6, avrb 7, avrBIn,

avrB] 01, and avrB102, the 102-bp tandem repeats of the genes were found to determine
gene-for-gene specificity (Yang et al., 1994). avrb6 was also found to be required for
Xcm to cause severe water-soaking and subsequent necrosis in susceptible Acala-44
cotton lines (Yang et al., 1994). Using Avrb6, it should therefore be possible to find the
receptors responsible for both plant resistance and susceptibility.

The yeast two-hybrid assay approach was used for identifying cotton proteins that
interact with Avrb6. The yeast two-hybrid system provides a sensitive method for
detecting relatively weak and transient protein-protein interactions. Such interactions

may not be biochemically detectable, but may be critical for the proper functioning of

100

complex biological systems (Estojak et al., 1995). In addition, because the two-hybrid
assay is performed in vivo, the proteins are most likely to be in their native
conformations, which may lead to increased sensitivity and accuracy of detection. For
the purpose of identifying cotton receptors of Avrb6, I chose the GAL4
MATCHMAKER Two-hybrid System 2 from CLONTECH Company. The GAL4
systems utilize two reporter genes (HIS3 and lacZ) under the control of a GAL4-
responsive UAS. All reporter cassettes are integrated into the genome of the host strain.
Screening His+ clones for the expression of the lacZ reporter, which has a promoter other
than the HIS3 reporter, eliminates many of the false positives that arise in a typical two-

hybrid library screening.

4.3. MATERIALS AND METHODS

4.3.1. Agrabacterium-Mediated Transient Expression

A. tumefaciens strain GM3 850 containing the Ti-plasmid pKYLX7l (Maiti et al.,
1993) or individual transconjugants were grown in 2 ml LB medium containing
appropriate antibiotics. Following overnight grth at 28°C, bacteria were pelleted by
centrifugation and resuspended to an optical density at 600 nm (OD600) equal to 0.25 in
induction medium according to Bundock et al. (1995), with minor modifications. The
medium contained 10.5 g/l KzHPO4, 4.5 g/l KH2P04, 1.0 g/l (NI-I4)ZSO4, 0.5 g/l
NaCitrate.2H20, 1 mM MgSO4.H20, 0.2% glucose, 0.5% glycerol, 50 uM
acetosyringone (Aldrich), and 10 mM N-morpholino-ethanesulfonic acid (MES) (pH

5.6). After overnight incubation at 28°C, at OD600 = 0.5-0.6, bacteria were pelleted by

101

centrifugation, washed with Murashige and Skoog’s medium (MS, Sigma) with 10 mM
MES QrH 5.6), and resuspended to OD600 = 0.5 in MS-MES medium with 150 uM
acetosyringone. Bacterial suspensions were infiltrated into young but fully expanded
leaves of 6- to 8-week-old cotton plants using a needleless syringae. Alter infiltration,
plants were immediately covered with transparent plastic bags and placed at 20°C
(reported optimal for T-DNA transfer, (Fullner and Nester, 1996)). After 40-48 hr,
plastic bags were removed and plants placed at 28°C, 16 hr light/8hr dark, and 80%

humidity to allow symptoms to develop (Van den Ackerveken et al., 1996).

4.3.2. Yeast Two-Hybrid System

Yeast two-hybrid screening was conducted according to instructions with the
CLONTECH MATCHMAKER GAL4 Two-hybrid system 2 (K1604-1) and Yeast
Protocols Handbook (PT3024-l). The pAS2-1 plasmid was used to construct the bait
plasmid. Yeast strain CG-1945 was used as the host for the library screening. An
alternative HIS3 “jump-start” procedure (CLONTECH) was also used in which the yeast
strain Y190 was used as the host. The “jump-start” procedure permits detection of some
interactions in which the affinity is too low to allow the HIS3 reporter to produce enough
HIS3 protein for survival if plated directly on the SD (synthetic dropout)/-His selection

medium.

102

4.3.3. Total RNA Isolation from Cotton Plant

Acala-44 is a susceptible cotton line lacking most of resistance genes. IM216 is a
resistant cotton line which has many R genes (Brinkerhoff et al., 1984; De F eyter and
Gabriel, 1991). Avrb6 proteins are responsible not only for triggering HR in line IM216,
but also for causing water-soaking disease in Acala-44. The total cotton RNA was
isolated from both IM216 and Acala-44 lines by using a modified hot borate method in
order to cope with cotton’s high levels of phenolics, terpenes, secondary metabolites, and,
in some tissues, polysaccharides (Wan and Wilkins, 1994). Preheated (80°C) RNA
extraction buffer (200 mM sodium borate decahydrate (Borax), pH 9.0, 30 mM ethylene
glycol bis(B-aminoethyl ether)-N,N ’-tetraacetic acid, 1% (w/v) sodium dodecyl sulfate
(SDS), 10 mM dithiothreitol, 2% (w/v) polyvinylpyrrolidone (PVP)-40,000) was added
to frozen ground tissue at a ratio of 5 ml of buffer per gram of fresh weight and
homogenized for 2 min. The homogenate was transferred to a 50-ml Oak Ridge tube
containing 0.5 mg of proteinase K per milliliter of extraction buffer and incubated with
mild agitation on a rotary shaker at 100 rpm for 1.5 hr at 42°C. The homogenate was
adjusted to 160 mM potassium chloride and chilled on ice for 1 hr. After centrifugation
at 12,000 x g for 20 min, the supernatant was filtered through miracloth and the RNA was
precipitated overnight in 2 M LiCl on ice. Following centrifugation as above, the RNA
pellet was washed two to three times with 5 ml of cold 2 M LiCl until the supernatant
was relatively colorless. The LiCl-RNA pellet was suspended in 2 ml of 10 mM Tris-
HCl, pH 7.5, and clarified by centrifugation for 10 min. 200 mM potassium acetate (pH
5.5) was added and following an incubation of 15 min on ice, salt-insoluble material was

removed from the supernatant containing the RNA by centrifugation. The RNA was

103

precipitated overnight with 2.5 x volume of 100% ethanol at —20°C. The RNA was
pelleted, washed with 70% cold ethanol, dried briefly under vacuum, and suspended in
DEPC (diethylpyrocarbonate)-treated deionized water. Agarose gel electrophoresis was

used to examine the quality of the isolated cotton leaf total RNA.

4.3.4. cDNA Library Construction

In order to have the chance to identify both resistance and susceptibility factors, I
combined both IM216 and Acala-44 total RNA in 50:50 ratio to make the cDNA library.
F or construction of an AD library, 3.6 mg of Acala-44 and 5.4 mg of IM216 total RNA
was sent to CLONTECH.

Both oligo(dT) and random primers were used for cDNA synthesis. EcoRI
adapters were ligated to the cDNA before being cloned into the pGADlO vector. The
amplified library had a titer of 2108 cfu/ml (in DHSa), 75% of colonies were estimated
to have inserts, and the number of independent clones was 2.5 x 10° with an average
insert size of 1.0 kb (ranging from 0.2 - 4.0 kb). Fifteen colonies were randomly picked
and subjected to polymerase chain reaction (PCR) analysis; 14 of these contained inserts

(data not shown).

4.3.5. Construction of Bait Plasmid

The avrb6 ORF was amplified by PCR from plasmid pAVRB6-1 (avrb6 gene
clone in pBSSKII(-) from pUFR106 (DeFeyter et al., 1990) using the NdeI-tagged

upstream primer (5’-CTAGACCACAIAIGGATCCCATTCGTTCGCGCACGCCAAG-

104

”TC-3’), and EcoRI-tagged downstream primer (5’-CQAAIICCCTCACTGAGGCAAT-
AGCTCCATCAAC-3’). The PCR product was digested with NdeI and EcoRI, and
cloned into the corresponding sites in vector pAS2- l. The avrb6 sequence was inserted
in the correct reading frame and orientation to make a fusion of Avrb6 and the GAL4
binding domain (BD). This construct was then transformed into yeast strain CG-l945 or
Y190 using a small-scale yeast transformation protocol (CLONTECH K1604-1).
Transformants were assayed for lacZ reporter gene expression.

Sequential deletion of avrb6 was accomplished by PCR cloning using primers
matching the sequences of different regions of the avrb6 open reading frame. PCR
products were cloned into a pASZ-l and transformed into yeast strains CG-l945 or Y190,
as described above. The primers used to amplify the central repeat region of avrb6 for
construction of the bait plasmid 6ANC were: NdeI-tagged upstream primer (5’-
CCCAAGCTTCAIAIQGTGCAATCGGGTCTGCGGGC-3 ’) and EcoRI-tagged
downstream primer (5’-GCTCTAQAAII_QAAAATGCTTGCGCTGGGTGGGAGT-

GGC-3’).

4.3.6. DNA Sequencing and Analysis

For sequence analysis of putative clones, the cDNA inserts were amplified by
using pAD10 vector primers: S’AD (5’-TACCACTACAATGGATG-3’) and 3’AD (5’-
TGCACGATGCACAGTTGAAGTAAC-3’). To obtain full-length sequences, primers
were designed according to the internal sequence information. In combination with
either the S’AD or the 3’AD primer, the 5’ and 3’ end sequences of each cDNA insert

were amplified from the AD-cDNA library by using gene-specific primers and 5’AD or

105

3’AD primer. DNA sequence data were analyzed using the MacVector software (Oxford
Molecular Group) and BLAST search program (http://www.ncbi.nlm.nih.gov/blast/).

The deduced amino acid sequences were compared with sequences in the current
databases (GENBANK, SWISS PROTEIN, and EMBL) using the BLAST, MOTIFS, and
PROPSEARCH programs. Sequences were aligned using the BOXSHADE 3.21
program (http://www.ch.embnet.org/software/box_form.html). Protein motifs were
examined by using the PROSITE PATTERN search program
(http://www.motif.genome.ad.jp/). The protein localization prediction was analyzed by

using the PSORT program (http://psort.nibb.ac.jp:8800/form.html).

4.3.7. Arabidapsis thaliana Transformation

The full length the avrb6 gene was amplified from plasmid pAVRB6-1 using the
HindIII-tagged upstream primer (5’-CCCAAG£I[ACCATGGATCCCATTCGTTCGC-
GC-3’) and XbaI-tagged downstream primer (5’-GCIE_IAGATCACTGAGGCAATAG-
CTCCATCAACC-3’), digested with HindIII and XbaI, and cloned into the corresponding
sites in pKYLX7l ::3582, a plant transformation vector (Maiti et al., 1993). The resulting
construct was electroporated into Agrabacterium tumefaciens GV3 850 and transformed
into Arabidapsis thaliana (Col) by vacuum infiltration (Bent et al., 1994). Following the
same strategy, the avrB] 01 gene was also transformed into Arabidapsis thaliana (C01).
The same avrb6 and avrB] 01 clones in pKYLX71 were also used in an Agrabacterium-

mediated transient assay in cotton.

106

4.4. RESULTS

4.4.1. Agrabacterium-Mediated Transient Expression of Avrb6 and AvrBlOl in

Cotton

AvrBs3 of Xanthomonas campestris pv. vesicatoria has been shown to induce
hypersensitive cell death when transiently expressed in pepper leaves that contains BS3
resistance gene (Van den Ackerveken et al., 1996). This suggests that AvrBs3 fimctions
inside the plant cell. To determine whether the Avrb6 protein also acts inside plant cells,
transient expression of avrb6 in cotton leaves was carried out. The PCR-amplified avrb6
gene was cloned into the HindIII /XbaI sites of pKYLX71 ::35S2 (Maiti et al., 1993).
Using A. tumefaciens for gene delivery, avrb6 was expressed transiently in cotton leaves.
As shown in Fig. 4.1, leaves of the cotton resistant line IM216 responded to avrb6
expression with an HR, whereas those of the susceptible line Acala-44 did not. In the
absence of the avrb6 gene, the HR was not induced by A. tumefaciens. Deletion of the
central repeat region also abolished ability of Avrb6 to elicit HR in IM216 (Fig. 4.1).
These results indicate that Avrb6 by itself is sufficient to trigger HR in the resistant
cotton line IM216 when expressed inside plant leaves. The same results were obtained
for AvrBlOl (Fig. 4.1). Therefore, Avrb6 and AvrBlOl appear to function inside cotton

cells to trigger HR.

107

   

IM216 AC44

1. Control 1. Control

2. avrb6 2. avrb6

3.avrB101 3.avrB101

4. avrb6Arepeats 4. avrb6Arepeats

5. avrB10mrepeats 5. avrBiOMrepeats

Figure 4.1. A. tumefaciens-mediated transient expression in cotton leaves.

A. tumefaciens transconjugants carrying different constructs were grown under vir gene-
inducing conditions and inoculated into the intercellular space of cotton leaves. Symptoms
were photographed 48 hours post-infiltration. avrb6, avrB] 01 , avrb6Arepeats and

avrBI 01 Arepeats (Mscl complete digestion to delete the central repeat region) were cloned
into Hind III / XbaI sites of pKYLX71::35S2. Transient expression of avrb6 and avrB] 01 ,
but not the control (pKYLX alone), avrb6Arepeats, or avrB] 01 Arepeats led to hypersensitive
cell death in leaves of the resistant cotton Im 216, but not the susceptible line Acala-44,
within 48 hours.

108

4.4.2. Construction and Test of the Bait Plasmid

The full length of the avrb6 ORF was cloned into the bait vector pAS2-1. The
resulting construct was transformed into the yeast strain CG-l945 and assayed for the
expression of the lacZ reporter gene. To our surprise, thisbait construct was able to
activate the expression of both reporter genes to a high level even in the absence of any
AD protein. Although this result indicates that Avrb6 may firnction as a transcription
activator in plants, the full length of the avrb6 could therefore not be used as a bait to
screen the AD library.

Activation domains of many eukaryotic transcription factors share a common
feature, i.e., presence of a net negative charge (Ma and Ptashne, 1988; Remacle et al.,
1997; Saha et al., 1993). In fact, E. coli genomic DNA fragments that bear an excess
number of acidic amino acids can function as yeast transcriptional activators (Ma and
Ptashne, 1987; Ruden et al., 1991). By using the DNASTAR Protean program
(DNASTAR Inc., Madison, Wisconsin), I defined several acidic domains in Avrb6 (data
not shown). There is a small acidic domain in the N-terminus (102-108 residues). At the
C-terminus, starting from residue 782, there are several acidic regions (Fig. 4.2). I
sequentially deleted different numbers of acidic domain(s) from Avrb6 and tested for the
transcription activation activity of the remaining polypeptides. The C-terminal acidic
domain alone was capable of activating reporter gene transcription. To my surprise, the
N-tenninal small acidic sequence also had the ability to turn on reporter gene
transcription. When the N-terrninal (1-128 amino acid) acidic region was deleted, the
rest of Avrb6 had no transcription activation activity despite the presence of the C-

terminal acidic domain. Deletion of both the N-terminal and C-terrninal acidic domains

109

also abolished the transcription activation activity (Fig. 4.2). I therefore had two bait
choices: the central repeated region plus C-terrninal (385-3291 bp), designated 6AN, and
the central region only (385-2498 bp), designated 6ANC. 6ANC was chosen as the bait
for the library screening. The reason I did not choose to use 6AN as the bait will be

discussed in the DISCUSSION (4.5).

110

NLS ACTIVITY

 

 

 

 

 

 

 

 

 

 

34-AA REPEATS ——
irvri
E] IIIIIIIIIIIIIIIIIII E] +
A All 41 1
AD AD
or I." “Iii +

+

+

+
mm» :mnmrmumn: -
mm -

fll-llllllllIIIIIIIIIIIIIIII‘ +

 

Figure 4.2. Activation of the yeast Gall promoter by different deletion constructs of
Avrb6 (AD: acidic domain; NLSs: nuclear localization signals).

Avrb6, when fused with Gal4 BD, activates the transcription of reporter genes (HIS3

and lacZ) in yeast, unless the acidic domain in the N-terminus or those in both N- and C—
terrninus are deleted.

11]

4.4.3. Yeast Two-Hybrid Screening

Yeast two-hybrid screening was carried out using a standard protocol with CG-
1945 as the yeast host strain, and using a “jump-start” protocol with Y190 as the host
(CLONTECH K1604-l). To increase the yeast transformation efficiency, I employed a
sequential transformation method instead of simultaneous transformation because my bait
protein was not toxic to the yeast and sequential transformation was 10 times more
efficient (data not shown).

The number of independent clones of the cotton cDNA library was about 2.5 x
10°. To identify rare clones, at least 10 times the number of independent clones in the
library should be screened according to the “jump-start” protocol. Alter screening 1.8 x
108 clones, I obtained over one thousand His+ clones, among which 372 clones were both
His+ and LacZ+. After eliminating false positive clones (see CLONTECH Manual

K1604-l), at least 152 clones were confirmed to specifically bind to 6ANC (Fig. 4.3).

112

 

mluufli‘lfififi —-|-C-t‘- u. - Ii! - -—-.. m—u—h .- _,. II“

1.8 _x 108 yeast transformants screened“
31.096319th

i

372 LacZ"/His+

i

> 152 6ANC-dependent LacZ+/Hrs

 

i

2 81 groups

Figure 4.3. Summary of yeast two-hybrid screening of cotton cDNA library.

113

4.4.4. Sequencing Analysis of cDNA Clones

Putative clones were further sorted to eliminate duplicates. For this purpose, AD
plasmids from the His’L/LacZ+ yeast transformants were isolated. The AD library inserts
were characterized by restriction digestion with the frequent-cutting restriction enzyme,
AluI. Clones sharing the same digestion pattern were sorted into the same group. The
152 putative clones were found to form 81 groups. .1

One or two clones from each group were sequenced. The sequencing data are I

summarized in Table 4.1. Most of the sequences turned out to be clearly uninteresting,

 

such as Rubisco gene, rRNA genes, etc. However, proteins encoded by three genes were 3i
found to be of interest: Cpk (for Cotton Pto-like Kinase), a serine/threonine kinase highly

homologous to Pto (a disease resistance gene product identified from tomato); ch (for

Cotton Calcineurin B-like protein), a calcineurin B homologous protein; and th (for

Cotton Transcription Factor), a putative transcription factor containing a zinc-finger

motif. The full-length sequences were obtained for all three genes. Their deduced amino

acid sequences and the alignment with the homologous gene products are shown in Figs.

4.4-4.6.

114

Table 4.1. Cotton cDNA clones encoding proteins specifically binding to Avrb6ANC.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Clones (Na.) Encoded Protein Notes
18005 (1) novel named th, with NLSs and Csz-type Zinc finger motif
JSl75 (l) serine/threonine named Cpk, homologous to Pto kinase
kinase
YBBZ4 (l) calcineurin B named ch, homologous to calcineurin B-like protein from
A rabidopsis thaliana
13013 (1) eIF-Z [3 subunit homologous to eukaryotic translation initiation factor [3 subunit
JSOSO ( l) K“ channel protein glutathione-regulated potassium-efflux system protein
IS 160 (l) putative
phosphatase
18192 (1) tenascin precursor
JS094 ( l) cyclophilin
IS 157 (l) heat-shock protein
18279 (1) EF—la translation elongation factor a
15200 (1) K+ transporter similar to high affinity potassium transporter
18297 (1) metallothionein- from Gossypium hirsutum
like protein
Y6-72 (l) homologous to bcr/C-abl oncogene protein
YBB140 (1) ATP synthase delta chain, mitochondrial precursor
(19) Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase, rbcL or rch
gene product
(23) PS M1 or CAB photosystem I/II reaction centre subunit, PS 11 10 KD polypeptide
precursor, or chlorophyll A/B-binding protein

JS084 (1) F BPASE fructose-1,6-bisphosphatase, cytosolic

(46) tRNA 165, 185, 23s, 26s

(58) misc. short peptide

 

 

115

 

H} w t ,_ ; '_‘_ { fI“u* gm
R1}: 5 14 if”? , 5 ; GHGRVYIGEL GTKVAVKRG
sck 476 ' GFGQVYK'I GTKfi

Pto 24 u‘ EA-RJFD H :GEGHFGKHYKGELRDGTKVAK

 

   

 
  

  
  
 

  
  

 
 
 
 

 

 
 
 

Cpk 61
le S74 _
Stk 536 RHRHLYSLEGYC'ENSEMILVYEFMEOGTLI‘ L.
Pto 84 ':' r EMBLIYIIMEHGNI\SH

I i
Cpk 121 fl~fifi
le 626 -ICIGIARGLHYLHI uVAFGQEHv-
Stk 588 ICIGAARGLBYLH; GIIIHRDVKSTNILLDB
Pto 136 i71GAARGLHY1HT- N---G'iHRDVKSlNIIID;E *’ ---REELYQu
Cpk 179 5 KI FGYLUPEIIHC p 1.1RgggiNLp_J . E; a
le 684 r thLTIKSDVYsFGVVLLEflLCARpAIIpfiLpREflVNLAEH- “,4
Stk 647 IN 'K FGYLDPEYLQTH«LTEKSDVYIHGVVLLEVLEARparupuLpflEEVNLIEHfiMF

Pto 189 ID K FGY'DPEY23IEG'ITEKSDVYSFGVVL s:vrcaps IoGLPRHMVNLAEIA E

Cpk 239 n_fi MEVIKIIDISMIS“IlrI\L I‘SIIUBK LIP G‘SFP MLJ, ”LLLIHLXIC

le 744 -KG LEKIIDHELAGEINHES aEnAEKCLEDY DRP GDVLHILEYALQLQE
Stk 707 I ,oEIaDHs IGQIET s HIEIAEKCLK Y " RP 'DVIWDLEYULQLQ
Pto 249 SIHNGoLEoI HIVL Dtl'PESL KHGH 'ATrch-LSSIDHH CUVIWQIL’AIU pr

 

Cpk 2 99 - -mDASFN- - - -MSSRPFQRLPSNNCPIEKVDVPMEID
le 804
Stk 767

Pto 309

  
  

I aDSmNSGGSLVAPRLMVSDSF- - - STNSIFQNGDESKIR

Figure 4.4. Amino acid sequence comparison of Cpk with three other protein kinases
from plants.

The kinases include one receptor-like protein kinase from Arabidapsis thaliana (le,
accession AL050351), one putative serine/threonine protein kinase from A. thaliana (Stk,
accession AC002391), and serine/threonine kinase Pto from tomato (accession U593 16).
Amino acids identical with Cpk are highlighted in black, and conservative sequences are
highlighted in gray. The invariant residues conserved in all protein kinases are
underlined, and residues that indicate serine-threonine specificity are double-underlined
(in Cpk sequence).

116

 

ch

Cnb-3
Cub-2
Cub-1

ch

Cub—3
Cub—2
Cnb-l

ch

Cub-3
Cnb-Z
Cnb—l

 

121
121
121
106

181

181:
181’

166

HLUGIGILK :II.oc GILL;KgsHGKuDHELLAKETVHsstIEALKELIKKIHGK
HSQCIDGEKHIC SEEECFDIDIYKQS GLGDPELLARETVFSVSEIEALYELFKKISSR
G

Hsocfin

 

HLCrsVLGCFDIDLYI KQs CLCDPELLARITVFSVSEIEALYELFKKISC}

IIL:lLlillflifilflle\i&?K11dKrJiSl.FPMUR17FIHJFLfFKlHJCiILK3FEHSKEXRXKLSVJl1iPPLAE'
”lDDGLINKEEFQLALFKTNKKESLFADRVFDLFDTKHNGILGFEEFARALSVFHPNDPI
IDDGLINKEEFQLALFKTNKKESLFADRVFDLFDTKHNGILGFEEFARALSVFHPN

  
  
 
  

  
  
 
  

.DKIDFSFQLYDLKQQGFIERQEVKQMVVATLAESGMNLSDE IESIIDKTFEEADTKHI
WDKIMFSFQLYDLKQQGFIERQEVKQMVVATLAESGMNLED- IEDIIDKTFEEADTKHLI

 

 

IDKEEWRILVLRHPSLLKNMTLQYLKDITTTFPSFVFHSQ
IDKEEWRSLVLRHPSLLKNMTLQYLKDITTTFPSFVFHSQ

 

Figure 4.5. Amino acid sequence comparison of ch with three calcineurin B-like
proteins from Arabidapsis thaliana.

The calcineurin B-like proteins include Cnb-3 (accession AF 076253), Cub-2 (accession
AFO76252), and Cnb-l (accession AF 07625 1). Amino acids identical with ch are
highlighted in black, and conservative sequences are highlighted in gray.

ll7

 

 

 
 
 

th 1 MALLTFLPEAAAEPMKQPEKREEKQVIRQKQPT MP KN
Hyp-l 32 ---------------------------------- Rs: KS -u SK

cm: 61 pHHI‘T-‘HG ss ------ cs sm-
Hyp-1 57 VGGRSGCS L l
th 108 ------- TLGoE GFJSSRSLSGSTRS SSRHIoFR ----

Hyp-l 117 NPITHDVIFsfis G’EEFVGNLRPGTP 81'sz IfiREAss REG F

th 157 YECF'ITI SRYPIR
Hyp-l 177 Hos: RE EAAINGD :ISNSGEEC E
Hyp-z 120

  
  

   

  

a ~$LCS IGSSEFL

all

th 217
Hyp-l 237
Hyp~2 167

 

  
 
 

th 269 “-3 ill: JIMENR If" :7? I ‘ QGFQKKGC‘A‘AME

Hyp-1 289 rxAHINELIRIHfiir r‘ —---RNGFS KREMNNGI

Hyp-z 225 "UNI IIIIILL RI" " " :.;: II". V --------- EggpspKL
s
G
D

 

$11 LlLl vyi .
¥N§R LPC‘I‘JLIC P
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Figure 4.6. Amino acid sequence comparison of th with two hypothetical proteins from
Arabidapsis thaliana.

The hypothetical proteins are from the Arabidapsis genome sequencing project, including
Hyp-l (accession ALO30978.1) and Hyp-2 (accession AC005496.1). Amino acids
identical with th are highlighted in black, and conservative sequences are highlighted in
gray. The putative zinc finger motifs (Csz type) are double-underlined. The putative
nuclear localization signals are underlined.

118

 

4.4.5. Functional Study

The cotton RNA preparation for cDNA library construction included RNA from
both the resistant line IM216 and susceptible line Acala-44 for the purpose of having the
opportunity to fish out both resistance and susceptibility factors. The putative clones
therefore could be proteins involved in either resistance or susceptibility. To distinguish
these two possibilities, transient assays were conducted by co-expressing the avrb6 gene
with the putative target genes in IM216 and Ac44 cotton leaves. The Cpk and ch genes
were cloned into pKYLX7] 33582. Transient co-expression of avrb6 with cpk or ccb did
not result in an HR in cotton Acala-44 (susceptible line) leaves (data not shown).
However, this experiment was very preliminary, and needs to be repeated with
appropriate modifications. Thus, no conclusions can be drawn at this point.

I also transformed the avrb6 gene into Arabidapsis thaliana (C01). The T2 seeds
were collected. Once the homozygous transgenic plants are obtained, the putative target
gene will be introduced into this transgenic plant to examine the phenotypic symptoms

resulting from the interactions between Avrb6 and the putative cotton target protein.

4.5. DISCUSSION

Two constructs were used to screen the yeast two-hybrid library. I first tried the
longer bait (6AN) in library screening because I thought that the longer protein might
exhibit a closer conformation to the native protein. By using yeast strain CG-1945, a
total of 3.35 x 106 clones were screened. Among them, about 700 clones were both His+

and LacZ+. However, in the subsequent verification analysis (Chien et al., 1991), none of

119

 

the AD clones was able to bind to the original bait protein. One possible explanation
could be that the “positive” clones were due to spontaneous mutations in the bait (6AN).
In fact, it was reported by De Feyter et al. (1993) that spontaneous race-change mutations
in X. c. pv. malvacearum involved avr genes and were readily obtained at unusually high
frequencies of 10'3 to 10“. To confirm if this indeed was the cause, I designed the
following experiments.

“Positive” clones were grown in SD/-Trp+Leu plates that select only the BD
plasmid so that the AD plasmids would be randomly lost. After obtaining the yeast

colonies carrying only the bait (BD) plasmid, I tested whether the yeast retained the

 

ability to turn on reporter gene transcription in the absence of AD protein. All of the
clones tested retained the transcription activation activity. Therefore, those “positive”
clones were due to spontaneous mutations in the bait. The mutation frequency was about
2.3 x 10”, which is consistent with De Feyter’s report that the central repeat regions
mutate at a high rate (De Feyter et al., 1993). The C-terminal region of Avrb6 alone, as
we tested before, had transcriptional activation activity. This activity was curiously
absent in 6AN. One explanation is that the acidic-rich activation domains were buried
inside the original fusion protein, but became exposed outside when spontaneous
mutations occurred in the repeat regions.

For this reason, the central repeat region of Avrb6 (6ANC) was used as bait in the
subsequent screening. Following the “jump-start” protocol, I screened over 1.8 x 108
clones, from which about 152 clones were shown to specifically bind to the bait protein.

Further verification and sequencing analysis revealed three clones of interest, i.e., Cpk, (a

120

serine-threonine kinase homologous to Pto), ch (a calcineurin B-like protein), and Of (a
putative transcription factor with zinc-finger motif).

Pto is a disease resistance gene product identified from tomato, which confers
resistance to bacterial pathogens carrying avrPto (Martin et al., 1993). It has been shown
that AvrPto and Pto physically bind to each other in the yeast nucleus (Tang et al., 1996).
Like Pto, Cpk is predicted to be cytoplasmic based on its sequencing information. Avrb6
contains functional NLSs and is predicted to act inside the plant nucleus. It is possible,
however, that Avrb6 binds to Cpk first in the cytoplasm, and then Avrb6 carries Cpk into
the nucleus. Alternatively, Cpk could phosphorylate Avrb6 first to expose its NLSs to
aid the nuclear-importing process. The function of Cpk in Avrb6-mediated host response
remains to be determined.

Calcineurin is a ubiquitously expressed serine/threonine phosphatase that exists as
a heterodimer comprised of a 59-kDa calmodulin-binding catalytic A subunit and a 19-
kDa Ca2+-binding regulatory B subunit (Stemmer and Klee, 1994). ch shares
significant homology with an Arabidapsis thaliana calcineurin B protein (about 92%
identity and 95% similarity). Activation of calcineurin is mediated by binding of Ca2+
and calmodulin to the regulatory and catalytic subunits, respectively. It has been found
that calcineurin is involved in many eukaryotic cell signal transduction pathways
(Crabtree, 1999), programmed cell death (Wang et al., 1999), and pathogenesis
(Alspaugh et al., 1998; Odom et al., 1997). For example, the calcium-dependent
phosphatase calcineurin has been found to induce cardiac hypertrophy in response to
numerous pathologic stimuli (Molkentin et al., 1998). It will be interesting to determine

if ch plays any role in cotton/Xcm interactions, especially in pathogenesis.

121

 

th is a nuclear protein with a Csz type zinc-finger motif. Since Avrb6 contains
functional NLSs, and is believed to be targeted into the plant nucleus, it was predicted to
bind to a nuclear protein. th contains typical NLSs that could target the protein to the
nucleus. Again, a functional study is required to determine if th plays any role in cotton
disease resistance and/or response to pathogens.

Since the bait I used for library screening is the central repeated portion of Avrb6,
the question remains whether the truncated sequence retained the native protein
conformation so that the interaction between 6ANC and its putative receptor(s) are
biological significant. It is important to determine whether Cpk, ch, and th also bind
to the full length Avrb6 protein. One way to test this is to use these proteins as bait and
to express avrb6 in the AD vector. This assumes that AD-Avrb6 by itself does not turn
on reporter gene transcription.

Alternatively, biochemical approaches, e.g., affinity capillary electrophoresis
(Chu et al., 1994) and affinity chromatography (Olson et al., 1991 ),- could be used to
examine interactions between Avrb6 and these three proteins.

One way to test the biological function of the identified genes is to make
transgenic cotton plants (cultivars IM216 or Acala-44) that express the genes either in
sense or antisense. Cotton plants are transformable (Estruch et al., 1997; John, 1996;
John and Crow, 1992; Millar and Dennis, 1996; Rinehart et al., 1996). However, it takes
a relatively long time to get transgenic plants. Two alternative methods may be
considered for the functional test: one is the transient expression assay as described
above; and the second is to sequentially introduce avrb6 and a cotton target gene into

Arabidapsis thaliana to see if the interaction between the two gene products resulted in

122

 

any phenotypic change in the transgenic plant. The advantage of using Arabidapsis is
that it is much easier to transform. However, the genetic differences between cotton and
Arabidapsis may make this approach unrealistic.

It is worth mentioning that the transcriptional activation activity of Avrb6 in yeast
nucleus might reflect its role in plants. Recently, Zhu et al. (1998) showed that the C-
terminal coding region of AvaalO, another member of the Avr/Pth family, has r-]
transcriptional activation activity in both yeast and Arabidapsis thaliana, and the .
transcription activation activity was critical for its avirulence activity. Therefore, Avrb6

and other members of the Avr/Pth family may function as transcription factors during

 

pathogenesis. Avrb6 may bind to other transcription factor(s) to activate target gene
transcription (Figure 4.7, model 2), or it may work alone by binding to the promoter

region of target genes in the host (Figure 4.7, model 1).

123

 

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.‘I‘-' ’I-

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128

 

 

I1:

Chapter 5

CONCLUSION AND FUTURE PERSPECTIVES

The research for my doctoral dissertation has mainly been focused on studying the
mechanism of type 111 protein secretion in Pseudomanas syringae pv. tomato DC3000.
Type III secretion is controlled by hrp genes in DC3000 and other phytopathogenic
bacteria. The bacterial hrp system is believed to be responsible for secretion and
translocation of virulence and avirulence factors into the plant cell. The actual
mechanisms of this interkingdom transfer was previously poorly understood.

By constructing a non-polar hrpA mutant of DC3000, I demonstrated that the
hrpA gene encodes an essential protein for bacterial pathogenesis and HR elicitation. In
collaboration with Martin Romantschuk’s group, I have characterized a novel pilus-like
structure (named the Hrp pilus), assembled by DC3000. The Hrp pili are about 6-8 nm in
diameter, and can be easily distinguished from polar flagella, which are about 15-18 mm
in diameter. The pilus may function as a bridge that connects the bacterium to the plant
cell. The HrpA protein was found to be associated with this structure. By mutant
analysis and transmission electron microscopic examination, I demonstrated that HrpA is
a structural protein of the Hrp pilus.

A supramolecular structure controlled by the type 111 system has recently been
isolated from Salmonella typhimurium (Kubori et al., 1998). This structure, termed the
needle complex, spans both the inner and outer membranes of the bacterial envelope.

The basal structure resembles the flagellar basal body (Aizawa, 1996) because it contains

two upper (or outer) and two lower (or inner) rings. The needle structure itself is a stiff,

129

straight tube, 80 nm long and 13 nm wide. It was suggested that S. typhimurium uses this
needle structure to send its effector proteins into host cells. The Hrp pilus is much longer
than the needle structure. This may reflect bacterial adaptation to the different surface
barriers of the host, because, unlike animal bacteria, phytobacteria have to overcome a
thick cell wall (~100 nm). Consistent with the view that the Hrp pilus is involved in
protein transfer, another pilus-like structure has also been identified from Agrabacterium “‘"xl
tumefaciens. The pili varied in length, but pili longer than the length of bacterium were

observed. Agrabacterium pili, 3.8 nm in diameter, are required for transfer of DNA to

 

plant cells in a process similar to that of bacterial conjugation (Fullner et al., 1996). In E j
contrast, no pilus-like structures are associated with type III secretion in animal
pathogenic bacteria.

Since we first reported the finding of the Hrp pilus, similar cell surface
appendages have been found in all major Gram-negative bacteria carrying hrp genes,
including Erwinia amylovora (W. Hu and S.Y. He, unpublished results), Xanthomonas
campestris (U. Bonas, personal communication), and Ralstania solanacearum (C.
Boucher, personal communication). Thus, formation of the Hrp pilus appears to be a
common function of hrp genes.

To define the function of the Hrp pilus, I first examined the effect of the hrpA
mutation on protein secretion through the Hrp system. Unexpectedly, the hrpA mutation
was found not only to block type III protein secretion, but also to suppress the expression
of other hrc/hrp and avr genes. Northern analysis showed that hrpA-mediated gene
activation of hrpS and hrpR was at the RNA level. Although ectopic expression of

hrpR/S restored hrc/hrp and avr gene expression in the hrpA mutant, Hrp and Avr protein

130

secretion was still blocked. However, ectopic expression of hrpR/S genes was unable to
restore the hrpA mutant’s ability to cause disease in susceptible Arabidapsis thaliana
leaves or to elicit an HR in resistant tobacco leaves. These data suggest a dual function
of the Hrp pilus in protein secretion and coordinate regulation of the Hrp system. My
experimental results are therefore in favor of the conduit model, in which the Hrp pilus
functions as an integral part of the Hrp secretion structure.

At least eight type III secretion proteins share sequence similarities with the
components of flagellar assembly machinery (Bogdanove et al., 1996). In fact, these
eight Hrp proteins are more closely related to components of the flagella machinery than
to their counterparts in animal pathogenic bacteria. Thus, it is possible that type III
secretion systems first evolved in plant pathogenic bacteria from the flagellar export
apparatus in order to secrete proteins other than flagellin (Galan and Collmer, 1999).
The Salmonella typhimurium flagellar assembly senses structural integrity by export of a
negative regulator, F lgM (Hughes et al., 1993). In cells with a functional hook-basal
body complex, the flagellin genes are transcribed normally and the negative regulator
F lgM is expelled into the grth medium. In strains with a defective hook-basal body
structure, the anti-o28 F lgM protein accumulates inside the cell and inhibits flagellin
transcription (Fig. 5.1A).

A similar mechanism has been found in Yersinia pseudotuberculosis (Pettersson
et al., 1996). Microbe-host contact triggers export of Lch, a negative regulator of Yop
expression, via the Yop-type III secretion system. The intracellular concentration of

Lch is thereby lowered upon host-bacterial contact, resulting in increased expression of

131

 

Yops (Fig. 5.1B). However, the actual molecule or structure that senses host-bacterial
contact has not been identified.

Recently, a putative negative regulator, called HrpV, has been identified from
Pseudomanas syringae pv. syringae (Preston et al., 1998). hrpV is the last gene in the
hrcC operon (Fig. 1.1). A hrpV mutant produced higher levels of proteins encoded by all
three examined hrp gene operons—Hch (hrpZ operon), HrcC (hrcC operon), and HchB
(her operon)—-and constitutive expression of hrpV in trans abolished the production of
each of these proteins. It was further demonstrated that HrpV acts upstream of HrpR and

HrpS in the regulation cascade (Preston et al., 1998).

132

 

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In my own experiments, the hrcChrpTV triple mutant of DC3000 overproduced
HrpW, Hch, and AvrPto. pHRPCT, which contains the hrcChrpT genes driven by the
Plac promoter, complemented the hrcChrpTV triple mutant in HrcC production and
pathogenicity in planta. However, the protein production was still significantly higher in
hrcChrpTV/pHRPCT than in DC3000. These data confirmed that the HrpV protein in
DC3000 also negatively regulates hrc/hrp and avr gene expression. Figure 5.2 provides a
model of how the Hrp pilus might control gene expression in Pst DC3000. We believe
that there are two phases of induction for the Hrp system. The HrpS and HrpR positive
regulators first detect a signal (nutritional signal?) in the plant apoplast, leading to the
first-phase, low-level activation of hrc/hrp and avr genes, including the hrpA gene. The
low-level expression of hrc/hrp genes initiates the assembly of Hrp pili, which are
required for the second-phase, much higher (contact-dependent?) induction of hrc/hrp
and avr genes. ‘The extracellular location of the Hrp pilus appears to be especially
suitable for sensing host-bacterial contact. For second phase induction, there are three
possible mechanisms: 1) HrpV proteins are normally secreted through the Hrp pilus,
either into the plant apoplast or into the plant cell; in the hrpA mutant, the secretion of
this negative regulator is blocked, thereby repressing hrp gene expression (Fig. 5.2.
Model 1). 2) HrpA regulates other genes by directly inducing hrpRS transcription. The
negative regulatory function of HrpV is independent of the HrpA pathway (Fig. 5.2.
Model 2). 3) HrpA protein suppresses the negative regulation of HrpV, possibly by
physically binding to and inactivating the repression function of HrpV (Fig. 5.2. Model

3).

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135

The first model is consistent with what is known about flagellar assembly in S.
typhimurium and the negative regulation of type III secretion in Y. pseudotuberculosis.
To study the possible secretion of HrpV, HrpV has been fused to the 6xHis epitope tag at
the C-terminus. I have been unsuccessful so far in identifying the secretion of HrpV
using the His-tagged antibody. It is possible that the amount of HrpV is too low to be
detected. Alternatively, the 6xHis epitope might have interfered with the function or
secretion of HrpV. An antibody raised against the native HrpV may have a better chance
of localizing this protein. Another alternative would be to create a hrpA/hrpV double
mutant. If hrpA regulates gene expression exclusively through hrp V, I would expect
normal gene expression in the hrpA hrpV double mutant. An effort has been made to
create this double mutant.

According to the negative regulation model, gene expression in any secretion
mutant should be suppressed by the retention of HrpV in the cytoplasm. I found,
however, that hrc/hm and avr gene expression is normal, instead of being suppressed, in
the hrcC mutant. These results suggest that HrpA has a positive regulation function, by
either directly binding to HrpV to prevent it from suppressing hrpR/S gene transcription,
or indirectly competing with HrpV to bind to the promoter of the hrpR/S operon. Based
on these results I decided to determine whether the HrpA protein physically binds to
HrpV in the yeast two-hybrid system. I have cloned the hrpV ORF into both the pAS-Z
and pACT-2 vectors to create fusion proteins BD-HrpV and AD-HrpV. The HrpA
protein can be fused with the Gal4 activation or DNA binding domain. Using these

constructs, a direct interaction between HrpV and HrpA will be examined in yeast.

136

I am personally in favor of Model 1 and 3. The real mechanism is likely to be the
combination of these two mechanisms. Under hrp-inducing conditions, HrpV negative
regulator is secreted through the Hrp pilus in Pst DC3000, and most of HrpA proteins are
also secreted to outside the bacterium to form the Pilus. In the hrcC mutant, both HrpA
and HrpV proteins are accumulated inside bacterium, however, HrpA protein physically
binds to HrpV and thus inactivates its repression function. The hrp gene expression is
therefore normal in the secretion mutant. Only in the absence of functional hrpA gene,
HrpV is able to suppress hrp gene expression. The ratio of HrpA and HrpV seems to be
critical because, at least in Pss 61, over-expression of hreresulted in down-regulation
of hrp gene expression (Preston et al., 1998). This hypothesis remains to be determined.

If the HrpV protein is not directly involved in the HrpA regulatory pathway, it is
still possible that there is some other negative regulator. Several of the 28 hrp/hrc genes
have not been assigned a function. These genes, such as her and her, may be
candidates for mutant analysis.

To study a possible interaction of type III effector proteins with plant proteins, I
carried out the yeast two-hybrid screening experiment. This work is described in Chapter
4. After screening over 1.8 x 108 clones from a cotton AD library, I obtained over a
hundred clones that specifically bind to the bait, 6ANC, which is the central repeat region
of the Avrb6 protein of Xanthomonas campestris pv. malvacearum. Sequence analysis
revealed three clones: Cpk, (a serine-threonine kinase homologous to Pto), ch (a
calcineurin B-like protein), and th (a putative transcription factor with a zinc-finger
motif). A fimctional assay is still required to determine whether any of these proteins

plays a role in avrb6-mediated Xcm-cotton interactions.

137

Cotton is transformable, but it is a time-consuming and labor-intensive process
(Estruch et al., 1997; John, 1996; John and Crow, 1992; Millar and Dennis, 1996;
Rinehart et al., 1996). I have explored the possibility of transforming the avrb6 and
avrB] 01 genes into Arabidapsis thaliana. The function of the putative target genes could
be tested in the homozygous avrb6 (or avrB] 01) transgenic Arabidapsis plants.

Attempts have also been made to transiently express both the avrb6 and the Cpk
or ch genes in cotton leaves using the Agrabacterium gene delivery system. Neither
Cpk nor ch elicited HR in susceptible cotton plant Acala-44 when co-expressed with
avrb6. However, transient expression is affected by many environmental factors. Future
work is needed to optimize the transient expression method.

In addition, it is necessary to verify further binding between Avrb6 and the
proteins encoded by the cotton clones. For example, all the putative clones were
demonstrated to bind specifically to the central repeat region of Avrb6. It is important to
show that such an interaction also occurs between the target proteins and the full-length
Avrb6. Additionally, northern analysis should be conducted to examine whether genes of
the interacting clones are inducible during Xcm infection of cotton plant.

In an effort to identify the interacting protein of Avrb6, I discovered that Avrb6
had transcription activation activity in the yeast nucleus, perhaps due to the acidic
domains at both the C- and N-terminal regions. This result suggests that Avrb6 might be
a transcription factor in cotton plant cells. The Avrb6 protein may bind to the target gene
promoter or dimerize with other transcription factors within the cotton nucleus to induce

gene expression. It is attempting to identify those genes whose expression could be

138

induced by Avrb6. Many powerful techniques could be used to find out these genes,

such as differential display and DNA Microarray.

139

REFERENCES

Aizawa, S. I. (1996). F lagellar assembly in Salmonella typhimurium. Mol Microbiol 19,
1-5.

Bogdanove, A. J., Beer, S. V., Bonas, U., Boucher, C. A., Collmer, A., Coplin, D. L.,
Comelis, G. R., Huang, H. C., Hutcheson, S. W., Panopoulos, N. J., and Van
Gijsegem, F. (1996). Unified nomenclature for broadly conserved hrp genes of
phytopathogenic bacteria. Mol Microbiol 20, 681-3.

Estruch, J. J., Carozzi, N. B., Desai, N., Duck, N. B., Warren, G. W., and Koziel, M.
G. (1997). Transgenic plants: an emerging approach to pest control. Nat Biotechnol 15,
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Fullner, K. J., Lara, J. C., and Nester, E. W. (1996). Pilus assembly by Agrobacterium
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Galan, J. E., and Collmer, A. (1999). Type III secretion machines: bacterial devices for
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Hughes, K. T., Gillen, K. L., Semon, M. J., and Karlinsey, J. E. (1993). Sensing
structural intermediates in bacterial flagellar assembly by export of a negative regulator.
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John, M. E. (1996). Structural characterization of genes corresponding to cotton fiber
mRNA, E6: reduced E6 protein in transgenic plants by antisense gene. Plant Mol Biol 30,
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John, M. E., and Crow, L. J. (1992). Gene expression in cotton (Gossypium hirsutum
L.) fiber: cloning of the mRNAs. Proc Natl Acad Sci U S A 89, 5769-73.

Kubori, T., Matsushima, Y., Nakamura, D., Uralil, J., Lara-Tejero, M., Sukhan, A.,
Galan, J. E., and Aizawa, S. I. (1998). Supramolecular structure of the Salmonella
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Millar, A. A., and Dennis, E. S. (1996). The alcohol dehydrogenase genes of cotton.
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Pettersson, J ., Nordfelth, R., Dubinina, E., Bergman, T., Gustafsson, M.,
Magnusson, K. E., and Wolf-Watz, H. (1996). Modulation of virulence factor
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Preston, G., Deng, W. L., Huang, H. C., and Collmer, A. (1998). Negative regulation
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140

Rinehart, J. A., Petersen, M. W., and John, M. E. (1996). Tissue-specific and
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