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SUBCELLULAR LOCALIZATION AND FUNCTION OF THE ARABIDOPSIS
THALIANA SMALL GTPASE RABE, A HOST INTERACTING PROTEIN OF THE
PSEUDOMONAS S YRINGAE VIRULENCE EFFECTOR AVRPTO

By

Elena Bray Speth

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Biochemistry and Molecular Biology

2007

ABSTRACT

SUBCELLULAR LOCALIZATION AND FUNCTION OF THE ARABIDOPSIS
THALIANA SMALL GTPASE RABE, A HOST INTERACTING PROTEIN OF THE
PSEUDOMONAS SYRINGAE VIRULENCE EFFECTOR AVRPTO
By

Elena Bray Speth

Pseudomonas syringae pathovar tomato strain DC3000 (Pst DC3000) is a
bacterial pathogen of tomato and of the model plant Arabidopsis thaliana. Like many
Gram-negative bacterial pathogens of animals and plants, Pst DC3000 uses the conserved
type III secretion system (TTSS) to deliver multiple virulence effector proteins directly
into the host cell. Type III effectors collectively participate in causing disease, by
mechanisms that are not well understood. Elucidating the virulence function of individual
effectors is fundamental for understanding bacterial infection of plants.

Transgenic overexpression of AvrPto, one of Pst DC3000 virulence effector
proteins, in Arabidopsis was previously shown to lead to suppression of basal defenses,
thus enabling growth of non-pathogenic "PISS-defective bacteria in the transgenic plants.
AvrPto interacts in the yeast two-hybrid system with the Arabidopsis RabE family of
small GTPases, putative regulators of post-Golgi vesicle trafﬁc to the plasma membrane.
Although the function of RabE homologues in other eukaryotic organisms iS well
understood, the biological role of the Arabidopsis RabE proteins is obscure.

In this study, a live cell imaging approach was applied to investigate the
subcellular localization of one of the ﬁve Arabidopsis RabE proteins, RabEld, and of its

mutant derivatives RabEld-Q74L (predicted to be constitutively active) and RabEld-

SZ9N (predicted to be constitutively inactive), fused to the green ﬂuorescent protein
(GFP). Microscopic analysis and cell fractionation studies revealed that transgenically
expressed GFP-RabEld and endogenous RabE proteins are aSsociated with the Golgi
apparatus and the plasma membrane in Arabidopsis leaves. Strikingly, upon transgenic
expression of AvrPto in planta, the Golgi-localized pool of GFP-RabEld was greatly
reduced and often undetectable. Furthermore, RabEld overexpression could partially
counteract the AvrPto-induced susceptibility to TTSS-defective bacteria. This work
uncovered a novel association between AvrPto virulence function and subcellular
distribution of the RabE protein.

To explore a possible role of RabE in plant growth, development and defense
against Pst DC3000, transgenic Arabidopsis plants overexpressing RabEld or its SZ9N
and Q74L mutant variants were used. Overexpression of wild-type RabEld or of
RabEld-SZ9N resulted in plants that were morphologically and developmentally
indistinguishable from wild-type Arabidopsis and were not altered in disease resistance.
Interestingly, Arabidopsis plants expressing the mutant RabEld-Q74L gained a
signiﬁcant degree of resistance to Pst DC3000, while their growth and development were
Similar to those of wild-type plants. In contrast, RabE Silencing drastically affected
Arabidopsis leaf morphology and rosette Size (suggesting a role for RabE in plant growth
and development) and had a complex effect on host defense.

This study identiﬁed an original case of a virulence effector of a plant-pathogenic
bacterium that alters subcellular localization of a putative regulator of intracellular
trafﬁcking. Additionally, functional study of RabEld laid the basis for further

characterization of the role of the entire RabE family of small GTPases in Arabidopsis.

Copyﬁghtby
Elena Bray Speth

2007

To Soﬁa and Giovanni, who always made me want to be a better person

Acknowledgements

I want to acknowledge ﬁrst of all my advisor, Dr. Sheng Yang He, for giving me
the opportunity to work in his laboratory and for tremendous support throughout the
years. Sheng Yang has been a fantastic mentor, both scientiﬁcally and personally, and I
feel privileged for having had the opportunity to learn from him.

I thank everyone who served on my Graduate Committee, Dr. David Amosti, Dr.
Dean DellaPenna, Dr. Gregg Howe, Dr. Lee MacIntosh, Dr. Steve Triezenberg, for all the
input they gave me through my studies, and Dr. Beronda Montgomery-Kaguri for
agreeing to participate in my Defense. My committee kept me on track when I was
wandering, and as I look back to those times, I truly appreciated them doing so.

The He lab has been a wonderful environment to work, learn and grow in. The
members of this lab have been not only great scientiﬁc collaborators, but people I
enjoyed seeing everyday and Spending time with. For this, I thank the current lab
members Lori Imboden, Wei-ning Huang, Young Nam Lee, Christy Mecey, Maeli
Melotto, Kinya Nomura, Francisco Uribe, J ian Yao and Weiqing Zeng. I also want to
thank all the past lab members whom I had the pleasure to work with: Paula Hauck, Julie
Zwiesler-Vollick, Sruti DebRoy, Roger Thilmony, QiaoLing J in, Anne Plovanich-Jones,
Bill Underwood, Yong Hoon Lee, Ola Kolade, Mingbo Lu, Young Bum Kwack, Sara
Sarkar, Shuo Cheng Zhang and many great undergraduates.

I especially thank Paula Hauck, Lori Imboden and Kinya Nomura for contributing

their data to Chapter 2 of this Dissertation.

vi

The Plant Research Laboratory has been an excellent setting for conducting my
graduate studies: I feel very fortunate for being in such an outstanding scientiﬁc
environment. I am also very thankful to the support staff, especially the oﬁice staff and

Jim Klug for their kindness and helpfulness.

The unconditional love and constant care of my family and ﬁ'iends have given me
the strength and conﬁdence to work through the good and bad times of this journey. I
thank my husband Phil and our son Jonathan, my parents, my brothers and sister and all
my family in Italy and in Reno for believing in me and encouraging me through this. Phil,
especially, could not have been more loving, understanding and supportive.

I have been blessed with God’s presence throughout my life, and with truly
amazing friends: my grandmother Consiglia, Soﬁa Caretto, Giovanni Mita, P. Luigi
Aluisi, Antonio De Benedictis, AnnaMaria Padula, Graziana Giuliani, Silvana Gaetani,
Paula Hauck, Donatella Canella, Giuseppe Verde and all the people who have inspired,
guided, encouraged, nurtured and challenged me. I thank them all and hope they will

continue to enrich my life with their presence.

vii

Table of Contents

 

 

 

List of Tables------ - - -- - - - - - - ............ -- -- - - - .......... x
List of Figures ................ - ....................... -- -- - - - -- ..... ...................... xi
CHAPTER 1 - Literature Review . - - -- ....................... - ---1
INTRODUCTION ................................................................................................................ 2
HOST DEFENSES AGAINST MICROBIAL PATHOGENS ....................................................... 4
Innate immune responses in eukaryotes ...................................................................... 4
Pathogen-speciﬁc and acquired immune responses .................................................... 7
THE BACTERIAL TYPE III SECRETION SYSTEM ............................................................ 10
Host cellular targets of bacterial virulence functions ................................................ 11
Uncovering the function of TTSS effectors of plant pathogens ................................ 12
PSEUDOMONAS SYRINGAE AND ARABIDOPSIS, A MODEL PATHOSYSTEM ...................... 14
Virulence factors of P. syringae pv. tomato (Pst) DC3000 ....................................... 15

The Pst DC3000 effector AvrPto .............................................................................. l6
VESICLE TRAFFICKING IN HOST-PATHOGEN INTERACTIONS ....................................... 19
Protein and membrane trafﬁc in the eukaryotic cell .................................................. 19
Small GTPases: key regulators of vesicle trafﬁcking ............................................... l9
Effectors of animal pathogens target the cell trafﬁcking pathways .......................... 21

The secretory and endocytic pathways in plant innate immunity ............................. 23
RATIONALE ................................................................................................................... 25
REFERENCES ................................................................................................................. 26

CHAPTER 2 - Virulence function of the Pseudomonas syringae effector protein
AvrPto is associated with altered intracellular localization of the small GTPase

 

RabE in Arabidopsis -- -- - - - u - 36
ABSTRACT ..................................................................................................................... 37
INTRODUCTION .............................................................................................................. 38
MATERIALS AND METHODS ........................................................................................... 42

Yeast two-hybrid screen ............................................................................................ 42
Plant growth and bacterial multiplication assay ........................................................ 42
AvrPto and 6xHis-AvrPto transgenic plants ............................................................. 43
RabE cloning and mutagenesis .................................................................................. 43
GFP-RabEld transgenic plants .................................................................................. 45
Protein extraction and immunoblotting ..................................................................... 45
Cell membrane fractionation ..................................................................................... 46
Confocal microscope analysis and imaging .............................................................. 47
Biolistic transformation ............................................................................................. 47
RESULTS ........................................................................................................................ 49
AvrPto membrane localization is required for virulence function in Arabidopsis
plants .......................................................................................................................... 49

viii

AvrPto interacts with the Arabidopsis RabE small GTPases in Y2H assay ............. 52

 

 

Gene expression analysis of the Arabidopsis RabE gene family .............................. 56
AvrPto preferentially interacts with GTP-bound RabE ............................................. 59
RabEld is associated with Golgi apparatus and plasma membrane .......................... 61
AvrPto expression in Arabidopsis alters RabE localization at the Golgi .................. 67
RabE overexpression reduces AvrPto virulence function in transgenic plants ......... 75
DISCUSSION ................................................................................................................... 77
ACKNOWLEDGEMENTS ................................................................................................. 82
REFERENCES ................................................................................................................. 83
CHAPTER 3 - Investigating RabE function in Arabidopsis- - .......... 89
ABSTRACT ..................................................................................................................... 90
INTRODUCTION .............................................................................................................. 91
MATERIALS AND METHODS ........................................................................................... 94
Transgenic plants ....................................................................................................... 94
Plant growth and bacterial multiplication assay ........................................................ 94
Protein extraction and immunoblotting ..................................................................... 95
Confocal microscope analysis and imaging .............................................................. 96
RNA extraction .......................................................................................................... 96
RT-PCR analysis ....................................................................................................... 96
BTH treatment ........................................................................................................... 99
Intercellular Wash Fluid (IWF) collection and analysis ............................................ 99
Callose staining .......................................................................................................... 99
RESULTS ...................................................................................................................... 101
GFP-RabEld-Q74L displays a unique subcellular localization pattern .................. 101
RabEld-Q74L confers resistance against Pst DC3 000 ........................................... 104
RabEld-SZ9N expression does not alter plant growth, development or disease
susceptibility ............................................................................................................ 1 10
Occurrence of RabE Silencing in transgenic plants ................................................. 112
Effect of RabE co-suppression on the expression of individual RabE genes .......... 114
RabE-silenced plants exhibit complex responses to Pst DC3000 infection and
PAMP-induced resistance ........................................................................................ 116
DISCUSSION ................................................................................................................. 1 l8
ACKNOWLEDGEMENTS ............................................................................................... 122
REFERENCES ............................................................................................................... 123
CHAPTER 4 - Conclusions and future perspectives __ - - - -- --127
REFERENCES ............................................................................................................... 133

ix

List of Tables

Table 2 - 1: Primers for RabE cloning and mutagenesis. .................................................. 44

Table 3 - 1: Gene-speciﬁc primers for RT-PCR ................................................ 98

List of Figures

(Figures in this dissertation are presented in color)

Figure 2 - l: Membrane localization is critical for AvrPto virulence function. ................ 51

Figure 2 - 2: ClustalW alignment of the ﬁve Arabidopsis RabE proteins and their closest
homologues in other organisms. ................................................................................ 54

Figure 2 - 3: AvrPto interacts with Arabidopsis RabE in the yeast two-hybrid system. ...55

Figure 2 - 4: RT—PCR Showing RabE gene expression in rosette leaves. ......................... 58

Figure 2 - 5: Wild-type RabE and RabE-Q74L, but not RabE-S29N, interact with AvrPto
in Y2H. ...................................................................................................................... 60

Figure 2 - 6: GFP-RabEld localization in Arabidopsis leaf cells. .................................... 63

Figure 2 - 7: GFP-RabEld iS localized at the plasma membrane and at the Golgi
apparatus in Arabidopsis cells. . ................................................................................. 64

Figure 2 - 8: Detection of subcellular localization of endogenous RabE and transgenically
expressed GFP-RabEld by membrane fractionation technique. ............................... 66

Figure 2 - 9: Western blot indicating expression of GFP-RabEld and of AvrPto in leaves
of double-transgenic plants ........................................................................................ 68

Figure 2 - 10: AvrPto expression alters intracellular distribution of GFP-RabEld. ......... 69

Figure 2 - 11: Subcellular distribution of the GFP-RabEld-SZ9N protein. ...................... 71

Figure 2 - 12: Intracellular localization of RabE-SZ9N is unaffected by AvrPto
expression. ................................................................................................................. 72

Figure 2 - l3: 6xHis-AvrPto does not affect RabE localization at the Golgi. ................... 74

Figure 2 - 14: RabE overexpression limits AvrPto-induced bacterial multiplication. ...... 76

Figure 3 - 1: Localization of GFP-RabEld—Q74L in transgenic Arabidopsis. ................ 102
Figure 3 - 2: GFP-RabE-Q74L is primarily localized in the tonoplast. .......................... 103
Figure 3 - 3: RabE-Q74L overexpression confers resistance to Pst DC3000. ................ 105

xi

Figure 3 - 4: Accumulation of extracellular proteins in plants expressing GFP-RabEld-

Q74L ........................................................................................................................ 107
Figure 3 - 5: Pst DC3000 fails to suppress callose deposition in resistant RabEld-Q74L-

expressing plants ...................................................................................................... 109
Figure 3 - 6: Pst DC3000 grth on plants overexpressing GFP-RabEld-829N ........... 1 11
F igure3 - 7: RabE Silencing severely affects plant morphology. ................................... 113
Figure 3 - 8: Expression of the RabE and RabD genes in RabE-silenced plants. ........... 115

Figure 3 - 9: RabE-silenced plants exhibited complex responses to bacterial infection and
PAMP-induced resistance ........................................................................................ 117

xii

CHAPTER 1

Literature Review

INTRODUCTION

Multicellular organisms live in constant contact with microorganisms. While most
microbes do not pose a threat for plant and animal health, many bacteria, fungi,
protozoans and viruses have the potential of causing disease. Survival of plant and animal
species is dependent on their ability to recognize potential pathogens and to mount
effective defenses. Although higher eukaryotes have evolved elaborate self-protective
mechanisms, many microorganisms can still cause disease, having developed equally
sophisticated strategies to elude or suppress host defenses.

Microbial pathogens of plants are responsible for signiﬁcant crop losses
worldwide (Strange and Scott, 2005). Between 1988 and 1990, 13.3% of the world
agricultural production, equivalent to $76.9 billion, was estimated to be lost due to
pathogens (Baker et al., 1997). In light of the fast-growing human population and relative
limitation of agricultural land, maximizing crop yield and quality and preventing losses
I due to pathogens are among the main concerns of modern society and scientiﬁc
community. Understanding the molecular bases of plant-pathogen interactions is of
fundamental importance for an effective reduction of plant diseases (Strange and Scott,
2005)

Recent research advancements are uncovering the depth and complexity of
interactions between microbial pathogens and their eukaryotic hosts. Fascinating
analogies are emerging in the way animals and plants ward off infectious diseases, and in
the molecular mechanisms by which different microbes achieve pathogenicity on their

hosts (Cao et al., 2001; Buttner and Bonas, 2003).

Gram-negative bacterial pathogens of plants (including Pseudomonas, Ralstom'a,
Erwinia and Xanthomonas spp.) and of animals (including Yersinia, Salmonella, and
Shigella spp.) Share an ancient virulence mechanism, the type III secretion system, that
allows them to deliver disease-promoting proteins directly into the host cells (He et al.,
2004; Galan and Wolf-Watz, 2006). Investigating the biochemical and molecular
function of these secreted bacterial proteins has allowed identiﬁcation of numerous
eukaryotic cellular pathways and processes targeted by pathogens (Mota and Comelis,
2005; Mudgett, 2005). One of the common themes in bacteria-caused diseases is that
pathogens use these type III-secreted proteins to subvert the host cell metabolism and to
create conditions favorable to their own growth; among the most common targets are the
host cell secretory and endocytic pathways. A wealth of studies has explored in depth
how bacterial pathogens of animals subvert the host cell trafﬁcking pathways to their
own beneﬁt (Mota and Comelis, 2005; Schlumberger and Hardt, 2006). It is not yet
understood how phytopathogenic bacteria speciﬁcally interfere with trafﬁcking, although
many studies have indicated that type III effectors target components of the secretory
pathway, or suppress its normal function (Bogdanove and Martin, 2000; Hauck et al.,

2003; Nomura et al., 2005; Soylu et al., 2005).

HOST DEFENSES AGAINST MICROBIAL PATHOGENS

Innate immune responses in eukaryotes

The front line of defense against microbial pathogens in all eukaryotes is innate
(or basal) immunity, an ensemble of non-speciﬁc preformed and inducible defenses that
come into play in the early stage of interaction with microorganisms (Kimbrell and
Beutler, 2001; Nurnberger et al., 2004; Ausubel, 2005). All pathogens need to overcome

basal defenses to successfully colonize their hosts and cause disease.

Preformed defenses

Pre-existing defenses in animals include physical, physiological and chemical
barriers. Physical barriers, such as the skin, mucosae and intestinal epithelium represent
the ﬁrst tier of defense. Temperature and pH of the animal body cavities can be
considered as physiological barriers because they often are non-permissive for non-
adapted microorganisms. Constitutively secreted antimicrobial compounds and peptides
are additional limiting factors for potential pathogens (Schroder, 1999).

Similarly, plants are equipped with preformed structural, anatomical and chemical
defenses. Surface wax, cuticle layers, trichomes, constitutively produced antimicrobial
peptides (Broekaert et al., 1997) and toxic metabolites, along with the plant cell wall
itself are remarkable obstacles against herbivores and microbial pathogens (Thordal-

Christensen, 2003; Field et al., 2006).

Induced innate immune responses

Preformed barriers offer a very effective primary line of protection against
pathogens. Microbes that can breach this layer of defense will come in contact with the
host cells and trigger inducible immune responses. All higher eukaryotes express
receptors that recognize evolutionarily conserved molecules found exclusively in
microorganisms (Nurnberger et al., 2004). These molecules are commonly referred to as
pathogen— or microbe-associated molecular patterns (PAMPS, or MAMPS) and include,
among others, viral and fungal proteins, peptidoglycan of Gram-positive bacteria,
lipOpolysaccharide (LPS) of Gram-negative bacteria and bacterial ﬂagellin (Akira et al.,
2006). Receptors collectively called PRRS (pattern recognition receptors), located in the
plasma membrane or cytoplasm of the animal cell, perceive PAMPS and initiate Signal
transduction cascades leading to innate immune responses (Akira et al., 2006). Certain
PRRS, like Toll-like receptors, are inserted in the plasma membrane and detect microbial
ligands extracellularly; conversely, the cytoplasmic NOD receptors, featuring a
nucleotide binding Site and leucine-rich repeats (N BS-LRR), are responsible for
intracellular sensing (Athman and Philpott, 2004). The ultimate result of these receptors’
stimulation is the host inﬂammatory response, important for controlling infection.
Vertebrates feature an additional layer of surveillance, represented by specialized
phagocytic cells, like macrophages (resident in many tissues throughout the body, such as
the lungs, gut, liver and spleen) and neutrophils, which circulate in the blood. These types
of cells participate in basal immune responses by actively ﬁnding, engulﬁng and killing

microbes (Alberts et al., 2002).

Plants do not have mobile defender cells, but express inducible basal immunity
triggered by PAMPS perception at the level of every single cell in contact with a potential
pathogen. The model plant Arabidopsis, for example, can respond to a wide range of
PAMPS including LPS, bacterial elongation factor EF-Tu (Kunze et al., 2004), bacterial
ﬂagellin and its conserved 22-aminoacid peptide ﬂg22 (Gomez-Gomez et al., 1999).
Leucine-rich repeat receptor-like protein kinases (LRR—RLKS), abundant on the plant cell
surface, have been shown to serve as receptors for PAMPS (Nurnberger and Kemmerling,
2006). Some of these receptors have been recently identiﬁed and characterized in
Arabidopsis. F L82, a member of the LRR-RLK protein family, recognizes ﬂagellin and
the ﬂg22 peptide (Gomez-Gomez and Boller, 2000). The EFR receptor kinase perceives
Ef-Tu (Zipfel et al., 2006).

The events following PAMPS recognition include ion ﬂuxes in and out of the
cells, production of reactive oxygen species, activation of MAP kinase signaling
pathways and transcriptional reprogramming. The ﬁnal outputs of induced innate
immunity include cell wall crosslinking, extracellular formation of heterogeneous
appositions called “papillae”, secretion of defense peptides and compounds, and
expression of defense-related proteins (Chisholm et al., 2006). A complete signal
transduction pathway has been described for the plant cell response to ﬂagellin
perception (Asai et al., 2002). The F L82 receptor perceives ﬂg22 in the extracellular
milieu and initiates signal transduction. Downstream of FLSZ, a MAP kinase cascade
(MEKKl , MKK4/MKK5, and MPK3/MPK6) leads to expression and activation of the
WRKY22 and WRKY29 transcription factors, which are key positive regulators of

defense responses (Asai et al., 2002).

In any given plant species, innate immunity seems to be sufﬁcient to confer
broad-Spectrum resistance against most microorganisms, including those capable of
causing disease on other plant Species. This phenomenon is usually referred to as

“nonhost” resistance (Espinosa and Alfano, 2004; Nurnberger and Lipka, 2005).

Pathogen-speciﬁc and acquired immune responses

Vertebrates, in addition to non-speciﬁc basal immunity, express acquired (or
adaptive) immune responses, mediated by specialized white blood cell types (T-cells and
B-cells). Type B lymphocytes, or B cells, recognize non-self antigens and to speciﬁcally
produce antibodies against them (Alberts et al., 2002).

Although plants lack an adaptive immune system Similar to that developed by
animals, they have achieved in a totally different way a signiﬁcant level of speciﬁcity in
their immune response. In the course of co-evolution with their microbial pathogens,
plants have developed a remarkably effective defense mechanism, known as gene-for-
gene resistance, which is pathogen- and cultivar—speciﬁc and can be envisioned as an
additional layer of defense, superimposed on basal immunity (Abramovitch et al., 2006;
Shan et al., 2007). The genetic basis of such resistance lies in the presence of a dominant
allele of a resistance (R) gene in the host, and of the corresponding avirulence (avr) gene
in the pathogen. R genes, with a few exceptions, encode intracellular receptor-like
proteins with a nucleotide-binding Site, leucine-rich repeats (N BS-LRR), and either a
coiled-coil domain or a Toll—interleukin-l -like domain at the N terminus (Martin et al.,
2003). Microbial avr genes typically encode a variety of proteins (effectors) secreted by

the pathogen directly into the cytoplasm of the host cell. While the genetic basis of gene-

for-gene resistance is well understood, the biochemical mechanism is still obscure in the
vast majority of cases. There have been only a few instances in which a direct interaction
was demonstrated between a bacterial Avr and the corresponding host R protein (Scoﬁeld
et al., 1996; Tang et al., 1996; Deslandes et al., 2003). Most Avr-R genetic interactions
are currently interpreted with the “guard” hypothesis, whereby R proteins do not act as
receptors for the Avr factors, but rather guard, or monitor, other host proteins (targets of
effector virulence functions) for effector-induced modiﬁcations (Jones and Dangl, 2006).
The outcome of gene-for-gene resistance is rapid and localized cell death at the
sites of contact with the pathogen (termed hypersensitive response, or HR), which limits
further spread of the infection. The seminal discovery of gene-for-gene resistance in
plants led to the basic distinction between compatible and incompatible interactions,
where “compatible” refers to the interaction between a virulent pathogen and a
susceptible host (the outcome of which is disease), and “incompatible” describes the
interaction between an avirulent pathogen and a resistant host (no disease occurs in this
case). The comparatively more recent understanding of nonhost resistance has brought on
a more complex and multi-layered scenario, in which different forms of resistance
partially overlap and complement each other to increase the immune coverage of plants

(Thordal-Christensen, 2003; da Cunha et al., 2006; Jones and Dangl, 2006).

Systemic Acquired Resistance
Whereas the animal immune system can keep “memory” of encounters with
individual pathogens and establish a speciﬁc long-lasting immune protection of the

exposed individual, plants do not have a similar capability. They do, nonetheless, develop

a broad-range long-term systemic immunity, following localized exposure to pathogens,
called Systemic Acquired Resistance (SAR) (Durrant and Dong, 2004). At the molecular
level, SAR is characterized by systemic expression of defense markers, such as
pathogenesis related (PR) proteins, a group of extracellular proteins with antimicrobial
properties. SAR is strictly dependent on the signaling molecule salicylic acid (SA)
(Gaffney et al., 1993), and can in fact be triggered by exogenous application of SA
(Uknes et al., 1992). Furthermore, SAR is dependent on an intact SA Signal transduction
pathway, and on induction of the cellular secretory pathway (Wang et al., 2005). SAR
expression in tissues far from an infection site implies the existence of long-distance
signals whose identities are still elusive. While it has been conclusively demonstrated that
SA is not the mobile signal that triggers SAR in distal tissues (Vemooij et al., 1994),
several studies indicate that other signaling molecules, possibly lipid-derived, like

jasmonates, may be playing this role (Grant and Lamb, 2006).

THE BACTERIAL TYPE III SECRETION SYSTEM

To successfully colonize their hosts and cause disease, microbial pathogens have
evolved sophisticated strategies that enable them to evade or suppress basal defenses and
to interfere with other cellular processes. Regardless of their lifestyle (intracellular for
most animal pathogens, extracellular for plant pathogens), pathogenic bacteria need to
subvert the host cell metabolism in order to gain access to nutrients, multiply and Spread
further. To achieve these goals, pathogenic bacteria produce and deliver to the host cells a
variety of virulence-mediating proteins that interfere with host defense and housekeeping
cellular pathways.

A powerful virulence mechanism that is conserved among several Gram-negative
bacterial pathogens is the so-called type III secretion system (TTSS), used to deliver
proteinaceous virulence factors directly into the host cells (He et al., 2004; Galan and
Wolf-Watz, 2006). These proteins are referred to as type III effectors, because they are
translocated into the host cell cytoplasm via the TTSS. The molecular machinery
responsible for such translocation is very complex, consisting of more than 20 conserved
proteins, including regulatory and structural components. Among the bacteria that
employ the TTSS there are human pathogens, such as Yersinia, Salmonella, Shigella,
Chlamydia spp. , enteropathogenic and enterohemorragic Escherichia coli, and plant
pathogenic bacteria such as Pseudomonas, Ralstonia, Erwinia and Xanthomonas spp. (He
et al., 2004). Regardless of the high conservation of core components, TTSS structure
and substrates (translocated type III effectors) vary substantially among bacterial species.

The most notable structural distinction is that between the extracellular portion of the

10

TTSS in animal and plant pathogenic bacteria (Buttner and Bonas, 2003; He et al., 2004).
Commonly, each TTSS has a basal apparatus inserted in the inner and outer bacterial
membranes. In animal pathogens, the extracellular component is a stiff needle-like
structure. High resolution electron microscopy, combined with other techniques, allowed
the development of detailed models of the Salmonella typhimurium (Marlovits et al.,
2004) and of the Shigellaﬂexneri (Sani et al., 2007) needle complex, highlighting the
sophistication of this protein-translocation machinery.

In phytopathogenic bacteria, the TTSS basal apparatus is connected to a ﬂexible
ﬁlamentous appendage called a “pilus”, which is sufﬁciently long to potentially cross the
plant cell wall (Brown et al., 2001). The pilus elongates from the tip (Li et al., 2002) and
functions as a conduit for the translocation of secreted proteins into the plant cell cytosol
(J in and He, 2001; Li et al., 2002). Structural and functional components of the type III
secretion system in plant pathogenic bacteria are encoded by the hrp genes (for
hypersensitive response and pathogenicity), so called because they are necessary for the
bacterium to elicit HR in resistant plants and to cause disease in susceptible hosts

(Lindgren, 1997).

Host cellular targets of bacterial virulence functions

Type III effectors of animal pathogens and their functions inside the host cell
have been extensively studied. Many excellent reviews summarize the current knowledge
on bacterial type III effectors and their host targets (Mota and Comelis, 2005; Viboud
and Bliska, 2005; Schlumberger and Hardt, 2006). Investigating the action mechanism of

type III effectors in the eukaryotic cell has revealed how virulent pathogens subvert

11

certain host cellular functions, such as Signal transduction, cytoskeleton dynamics, cell
cycle progression and vesicular trafﬁcking, to their beneﬁt (Galan and Cossart, 2005).
This interference is usually accomplished by physical interaction between the type III
effectors and host cell regulatory proteins. An intriguing aspect of such interactions is
that type III effectors often mimic the action of eukaryotic enzymes and regulators,
acting, for example, as phosphatases, kinases, GTPase activating proteins and so on
(Mota and Comelis, 2005). In particular, several effectors seem to exert their virulence
functions by modulating the activity of host small regulatory GTPases.

Comparatively less is known about virulence functions of type III effectors of
phytopathogens (Grant et al., 2006). Interestingly, common themes can be identiﬁed
across pathogens and host kingdoms, such as suppression of host basal defenses and

modulation of host gene expression (Buttner and Bonas, 2003).

Uncovering the function of TTSS effectors of plant pathogens

It is perhaps not surprising that many type III effectors of plant pathogenic
bacteria were initially identiﬁed based on their ability to trigger a hypersensitive response
in Speciﬁc plant Species or cultivars. These type III effectors were therefore named
“avirulence” (Avr) proteins, because they “betray” the pathogen’s presence to the plant
that has the genetic background to recognize it. In the context of dynamic co-evolution of
plant pathogens and their hosts, the current hypothesis is that all type III effectors have a
virulence function to begin with, but plants have evolved R proteins capable of
recognizing the presence of some of these type III effectors, in a Species- or cultivar-

speciﬁc manner (Alfano and Collmer, 2004; Shan et al., 2007). Validating this

12

hypothesis, several studies have demonstrated how bacterial Avr proteins indeed
contribute to pathogen virulence, in the absence of the cognate R proteins (Shan et al.,
2000; Lim and Kunkel, 2005).

Ascribing virulence functions to the numerous type III effectors secreted by plant
pathogenic bacteria is not an easy task (Chang et al., 2004). One of the critical issues that
hinder studies on effector virulence functions is that of functional subtleness or
redundancy. Deletion or mutation of individual effector genes in a bacterial pathogen,
except in very few cases, does not result in a detectable change in virulence.
Consequently, alternative methods have been adopted for characterizing effectors’
functions. Stable or transient expression of single bacterial effectors in planta has proven
to be one of the most powerful strategies for functional studies. This is also made
possible by the development of model plant-pathogen systems, commonly used to
Simplify and hasten the study of complex interactions. Several agronomically important
diseases, in fact, are difficult to study due to long generation time and poor laboratory
adaptability of the host plants. Hence, the need to develop models that are amenable to

genetic and molecular analyses.

13

PSEUDOMONAS SYRINGAE AND ARABIDOPSIS, A MODEL PATHOSYSTEM

Pseudomonas syringae is a Gram-negative phytopathogenic bacterium that grows
epiphytically (on the leaf surface) on a wide variety of plants and is able to cause disease
in susceptible hosts (Hirano and Upper, 2000). The P. syringae species includes about 40
different pathogenic variants (pathovars) that exhibit different host-speciﬁcities (Hirano
and Upper, 2000).

P. syringae pathovar tomato DC3000 (Pst DC3000) is the causal agent of the
bacterial Speck disease in tomato. Pst DC3000 is also pathogenic on the model plant
Arabidopsis thaliana, and thus is widely used in laboratories to investigate the
mechanisms of plant response to bacterial infection (Preston, 2000; Katagiri et al., 2002).
To initiate pathogenesis, P. syringae enters the intercellular Space of leaves (apoplast)
through stomata or wounds. In the apoplast of susceptible hosts, Pst DC3000 multiplies
to high population levels (in the range of 108 cells/cm2 of leaf). Disease symptoms
develop gradually on infected leaves, starting with the appearance of water-soaking,
followed by the formation of necrotic lesions surrounded by chlorotic halos.

The Arabidopsis-Pst DC3000 interaction represents one of the best studied model
systems in plant pathology. Both the plant and pathogen genomes are fully sequenced
(Buell et al., 2003; The Arabidopsis Genome Initiative, 2000) and they are the subjects of
comprehensive genomic and functional genomics analyses, resulting in resources made
readily accessible to the scientiﬁc community (www.arabidopsis.org;

www.pseudomonas-syringae.org).

l4

Virulence factors of P. syringae pv. tomato (Pst) DC3000

One major known virulence factor of Pst DC3 000 is the phytotoxin coronatine
(Bender et al., 1999). While it is not clear how coronatine reaches the host cell, its
importance for virulence is highlighted by the observation that coronatine-lacking
mutants of Pst DC3000 fail to multiply aggressively and cause disease when surface-
inoculated onto Arabidopsis leaves (Melotto et al., 2006). Coronatine was believed for a
long time to inhibit rapid induction of host defenses in the early stages of pathogen
establishment and disease development (Mittal and Davis, 1995). Only recently, a study
on bacterial entry through stomata has shed light on the key role played by coronatine.
Melotto et al. (2006) showed that, while the plant closes its stomata upon recognizing the
presence of bacteria or bacterial elicitors on the leaf surface, coronatine induces stomatal
re-opening, allowing Pst DC3000 to enter more efﬁciently the apoplastic space, thus
promoting colonization (Melotto et al., 2006).

The other component that is essential for virulence is the type III secretion system
(TTSS). The Pst DC3000 TTSS delivers into the host cell an arsenal of at least thirty
different type III effectors (Lindeberg et al., 2006; Schechter et al., 2006).Their collective
key role in pathogenesis is inferred by the fact that Pst DC3000 hrp' mutants, impaired in
regulation or secretion of type III effectors, lose the ability to multiply and cause disease
in compatible hosts (Yuan and He, 1996; Roine et al., 1997).

The mechanisms by which individual type III effectors of Pst DC3000 affect host
metabolism and promote symptom development are beginning to be elucidated. Targets
being revealed include certain host pathways and processes such as basal defenses, gene

expression, hormone responses and programmed cell death (Grant et al., 2006). At least

15

three effectors of Pst DC3000 (Hole, Aer, AvrPto) and two (Aerpt2, Aerme) of
other P. syringae pathovars were found to be inhibitors of a speciﬁc cell wall-based

immune response, which is callose deposition in papillae (Hauck et al., 2003; DebRoy et

al., 2004; Kim et al., 2005).

The Pst DC3000 effector AvrPto

One of the most thoroughly investigated type III effectors of Pst DC3000 is
AvrPto, a mostly hydrophilic 163 aa polypeptide of 18.3 KDa, expressed in bacteria
during either compatible or incompatible interactions (Salmeron and Staskavvicz, 1993).

AvrPto was initially identiﬁed as an avirulence protein in the Pst-tomato system.
Pseudomonas syringae pv. maculicola transconjugants, expressing the avrPto gene
cloned from an avirulent Pst strain (Race 0), became avirulent on tomato cultivars
encoding the resistance gene Pto (Ronald et al., 1992). Soon aﬁer this discovery, a locus
tightly linked to Pto, named Prf, was found to be also responsible, with Pto, for AvrPto-
triggered resistance to Pst (Salmeron et al., 1994). Tomato Pto encodes a serine-threonine
protein kinase (Martin et al., 1993), whereas Prf encodes a protein with leucine-zipper,
nucleotide-binding, and leucine-rich repeat motifs (Salmeron et al., 1996). AvrPto
interacts with the Pto kinase in the yeast two-hybrid system, and this interaction is the
basis of the gene-for-gene speciﬁcity in this system (Tang et al., 1996). Interestingly, the
AvrPto-Pto interaction has never been observed in planta.

While acting as an avirulence protein in the presence of the cognate R genes,
AvrPto displays a virulence function in their absence. For instance, AvrPto was Shown to

slightly enhance the virulence of Pst strain T1 in tomato plants lacking Pto (Shan et al.,

16

2000). Deletion of AvrPto from the bacterial genome does not result in loss of avirulence,
and this was explained when a second effector was identiﬁed, named AvrPtoB, which
also interacts with the Pto kinase to trigger the hypersensitive response (Kim et al., 2002).
Although AvrPto and AvrPtoB have redundant avirulence activities, they are very
different proteins and have distinct and additive virulence functions: a double
avrPto/avrPtoB bacterial mutant displays a larger decrease in virulence than either of the
single mutants in susceptible tomato plants (Lin and Martin, 2005).

In Arabidopsis (which lacks Pto orthologs), AvrPto contributes to virulence by
suppressing basal defenses. Inducible expression of this effector in planta promotes
susceptibility to TTSS-deﬁcient mutants of Pst DC3000 and suppresses callose
deposition in papillae, a hallmark cell wall-based defense (Hauck et al., 2003). More
recently, AvrPto was Shown to be a potent suppressor of PAMP-induced gene expression
and MPK3/MPK6 activation in Arabidopsis protoplasts, intercepting PAMP-dependent
signaling upstream of MAPKKK (He et al., 2006).

The molecular and cellular mechanism by which AvrPto exerts its virulence
functions is still unknown. Solution of its structure has not provided helpful clues in this
regard (Wulf et al., 2004). A yeast two-hybrid screening for tomato proteins that interact
with AvrPto yielded four interactors: a stress-related protein, a putative N-myristoyl
transferase and two small GTPases, most Similar to mammalian Rab8 (Bogdanove and
Martin, 2000). AvrPto interaction with Rab8-like GTPases represents a particularly
interesting ﬁnding, because Rab8 is a major regulator of polarized secretion in

mammalian cells (Huber et al., 1993; Peranen et al., 1996; Gerges et al., 2004; Hattula et

17

al., 2006). This would indicate a potential interference of AvrPto with the plant cell

secretory pathway.

18

VESICLE TRAFFICKING IN HOST-PATHOGEN INTERACTIONS

Protein and membrane trafﬁc in the eukaryotic cell

Eukaryotic cells are functionally compartmentalized into membrane-bound
organelles with specialized functions. Communication and transport between these
compartments are vital to the cell and are accomplished through complex and tightly
regulated pathways (Harter and Wieland, 1996; Sanderfoot and Raikhel, 2003; van Vliet
et al., 2003; Jurgens, 2004). Some unique features differentiate the plant endomembrane
system from that of animals or yeast. Plant cells, for instance, contain a large number of
Golgi stacks disseminated throughout the cytoplasm, and large distinct vacuoles,
functioning either as Storage or lytic organelles (Jurgens, 2004).

Proteins, metabolites and membrane components to be transported between
compartments are typically “packaged” in membrane-bound vesicles. Movement and
target speciﬁcity of highly diverse vesicles are regulated at the molecular and
biochemical level, through the involvement of sophisticated protein machineries and of
the cytoskeleton. The main players of these pathways are proteins with conserved
functions, Shared between all eukaryotes. The secretory pathway and its components have
been extensively characterized in yeast and mammalian cells, but are still comparatively

less well understood in plants (Sanderfoot and Raikhel, 2003).

Small GTPases: key regulators of vesicle trafficking

Small monomeric GTPases represent a large superfamily of conserved regulatory

proteins that control several processes in the eukaryotic cell. Based on their sequence,

19

they are subdivided into ﬁve families (RaS, Ran, Rho, Arf and Rab) with distinct
functions (Takai et al., 2001). RaS GTPases regulate cell proliferation in yeast and
animals; they represent the only group of small GTPases that has no known counterparts
in plants (V emoud et al., 2003). The Ran family regulates transport of RNAS and
proteins across the nuclear envelope. The Rab, Arf and Rho families play distinct critical
roles in intracellular vesicle trafﬁcking.

Rab GTPases act as molecular switches to regulate budding of membrane-bound
vesicles ﬁ'om a donor compartment, movement toward a target compartment, tethering
and fusion of the vesicles with the target membrane (Zerial and McBride, 2001). Arf
proteins modulate endocytic and secretory trafficking and organelle structure. Among
their functions are recruitment of vesicle coat proteins and regulation of actin remodeling
near the cell surface (D'Souza-Schorey and Chavrier, 2006). Arfs participate, for
instance, in the biogenesis of clathrin-coated vesicles during endocytosis, and of COPI
/COPII vesicles, which shuttle proteins between the ER and the Golgi apparatus. Rho
GTPases play a critical role in the establishment and maintenance of cell polarization,
mainly by controlling the actin cytoskeleton spatial organization (Park and Bi, 2007).

Most of the current knowledge on polarized vesicle trafficking in plant cells was
gained studying grth in pollen tubes and root hairs, cells that Show a highly polarized
structure and tip growth. Several small GTPases of the Rab, Arf and Rho families were
characterized as necessary regulators of tip growth, controlling vesicular trafﬁcking,

cytoskeleton organization and signaling (Cole and Fowler, 2006; Samaj et al., 2006).

20

The Rab family of small GTPases

Rab proteins represent the largest family of small GTPases, accounting for about
60 members in humans and 57 in Arabidopsis (Vemoud et al., 2003). Like all small
monomeric GTPases, Rabs typically cycle between an “active” (GTP-bound) and an
“inactive” (GDP-bound) state. Many accessory proteins are required for Rabs activation,
inactivation and recycling back from target to donor compartment. Rab escort proteins
(REPS) facilitate delivery of newly synthesized Rabs to the appropriate membrane and
initial loading with GDP. Exchange of GDP for GTP, which activates Rab, is mediated
by guanine nucleotide-exchange factors (GEFs). Hydrolysis of bound GTP is also
mediated by accessory proteins, named GTPase-activating proteins (GAPS), which
accelerate the otherwise very low intrinsic GTPase activity of Rabs. Finally, GDP
dissociation inhibitors (GDIS) extract inactive GDP-Rab from the target membrane and
escort it through the cytoplasm back to the donor membrane, to serve for another round
of transport (Segev, 2001; Stenmark and Olkkonen, 2001).

The so-called “switch” regions of small GTPases adopt very different
conformations in the GDP-bound and in the GTP-bound state; GTP-bound but not GDP-
bound Rabs can interact with downstream components of the intracellular trafﬁcking

machinery, to achieve vesicle tethering and fusion with the target membrane.

Effectors of animal pathogens target the cell trafﬁcking pathways
The cytoskeleton and membrane trafﬁcking system have been known for a long
time to be major targets of TTSS effectors produced by intracellular bacterial pathogens

of animals. To reach their reproductive niche inside the host cell, these pathogens must

21

induce their own phagocytosis ﬁrst, then they must inﬂuence the host endomembrane
system in order to avoid fusion with the lysosome and degradation.

Small GTPases of the Rho subfamily are among the main known host targets of
TTSS effectors of bacteria such as Salmonella and Yersinia, which manipulate host
cytoskeleton dynamics during infection (Juris et al., 2002; Mota and Comelis, 2005;
Viboud and Bliska, 2005; Schlumberger and Hardt, 2006).

Recent studies revealed that human pathogens subvert cell trafﬁcking also by
targeting Rab GTPases. For example, the Legionella pneumophila effector protein
DrrA/SidM acts as a potent GEF on Rabl , to control its intracellular distribution and to
recruit it to the LCV (Legionella-containing vacuole) (Machner and Isberg, 2006; Murata
et al., 2006). An analogous situation was observed in Chlamydia trachomatis, an obligate
intracellular pathogen that multiplies in a non-lysosomal structure called an “inclusion”.
Several host Rab GTPases are recruited to the inclusion membrane, including Rab4A
(Rzomp et al., 2003). A chlamydial inclusion membrane protein, CT229, interacts
speciﬁcally with Rab4A and presumably mediates its recruitment to this highly
specialized host-pathogen interface (Rzomp et al., 2006).

Similarly, during epithelial cells infection with Salmonella enterica serovar
Typhimurium, a large number of Rabs were found to localize to the Salmonella-
containing vacuole (SCV) membrane, including Rab7, which is normally responsible for
late endosome fusion with the lysosome (Smith et al., 2007). Rab7 activity involves
interaction with the microtubule motor complex, mediated by the bridging protein RILP.

SifA, a secreted effector of Salmonella, was able to interact in vitro with Rab7 and was

22

shown to take part in uncoupling Rab7 from RILP (Harrison et al., 2004). This provides

some clue on how the SCV escapes maturation into a lysosome.

The secretory and endocytic pathways in plant innate immunity

In the last few years, the evidence for a major role of the plant secretory pathway
in defense against pathogens has been rapidly growing (Robatzek, 2007).

Among the most common plant defense responses that are associated with
secretory processes is formation of callose-containing papillae. Papillae are
heterogeneous appositions that form between the plasma membrane and the cell wall at
the infection Site and are believed to reinforce the cell wall, acting as a barrier against the
invading microorganisms. Although callose, a [3-1 ,3-glucan, is synthesized at the plasma
membrane (Fink et al., 1987; Kauss, 1987), papillae deposition is coupled with polarized
secretion (Assaad et al., 2004; Soylu et al., 2005). Interestingly, while PAMPS, as well as
TTSS-deﬁcient and nonhost bacteria, induce rapid papilla depoSition, virulent bacteria
suppress this response, via not yet understood mechanisms (Brown et al., 1993; Hauck et
al., 2003; Keshavarzi et al., 2004; Soylu et al., 2005).

SNARE proteins (which include syntaxins) are essential components of the
machinery that drives fusion of secretory vesicles with their appropriate target membrane.
The Arabidopsis plasma membrane-localized syntaxin SYPlZl/PENI was found to be
critical for nonhost resistance against Blumeria graminis, a fungal pathogen of barley
(Collins et al., 2003). Upon fungal infection, PEN] is rapidly recruited at the Sites of

papilla deposition, and seems to favor timely papilla formation; pen] mutant plants,

23

challenged with pathogens, produce papillae with a notable delay compared to wild-type
plants (Assaad et al., 2004).

A key regulator of SAR and SA-dependent defense responses, NPRl , controls
expression of genes encoding secretory pathway proteins in Arabidopsis. Null mutations
in some of these genes cause increased susceptibility to P. syringae and a concomitant
decrease in secretion of the PR1 marker protein (Wang et al., 2005).

One of P. syringae TTSS effectors, Hole, targets several Arabidopsis proteins
for degradation, including AtMIN7 (Nomura et al., 2006). AtMIN7 is a member of the
GEF family of proteins that activate Arf GTPases, which are also involved in regulating
vesicle trafﬁcking.

Furthermore, an important role for the endocytic pathway in PAMP—dependent
signaling has been recently identiﬁed. Upon perception of the ﬂg22 peptide elicitor, the
plasma membrane-localized F L82 ﬂagellin receptor fused to green ﬂuorescent protein
(GFP) rapidly disappears from the plasma membrane and is detected in vesicle-like

structures, indicating internalization by endocytosis (Robatzek et al., 2006).

24

RATIONALE

Previous research conducted in our laboratory addressed the virulence function of
the Pst DC3000 effector AvrPto in Arabidopsis. Transgenic expression of AvrPto in
Arabidopsis plants caused suppression of host defense responses, resulting in
susceptibility to TTSS-deﬁcient mutants of Pst DC3000 (Hauck et al., 2003). AvrPto
interacted in the yeast two-hybrid system with the Arabidopsis RabE proteins, small
GTPases predicted to regulate post-Golgi trafﬁc to the plasma membrane (P. Hauck).

This ﬁnding is particularly interesting because it suggests that AvrPto may exert
at least one of its virulence functions by interfering with the host cell secretory pathway.

In this study, I applied a live cell imaging approach to visualize the Arabidopsis
RabE proteins in the plant cell, and to investigate whether and how AvrPto affects RabE
cell biology. The results of this study are described in Chapter 2 of this dissertation.

The RabE family of GTPases has not been thus far functionally characterized in
Arabidopsis. I, therefore, pursued ﬁmctional analysis of RabE by site-directed
mutagenesis and in planta transgenic expression, with the dual purpose of investigating
RabE role in the plant cell and in defense against pathogens. Chapter 3 of this dissertation

illustrates the progress toward these goals.

25

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35

CHAPTER 2

Virulence function of the Pseudomonas syringae effector protein AvrPto
is associated with altered intracellular localization of the small GTPase

RabE in Arabidopsis

This chapter will be submitted as a manuscript for publication:

Bray Speth, E., Hauck, P., Imboden, L., Nomura, K., and He, S.Y.
Virulence ﬁinction of the Pseudomonas syringae effector protein AvrPto is associated
with altered intracellular localization of the small GTPase RabE in Arabidopsis.

I want to acknowledge the following contributions:

- Paula Hauck performed the yeast two-hybrid analysis and contributed Figure 2 - 3;

- Lori Imboden performed the yeast two-hybrid interaction assay between AvrPto and
mutant variants of RabEld and contributed Figure 2 - 5;

- Kinya Nomura characterized Arabidopsis plants expressing 6xHiS-AvrPto and
contributed Figure 2 - l.

36

ABSTRACT

Many Gram-negative pathogenic bacteria cause disease in their animal or plant
hosts by means of multiple virulence effector proteins, delivered into the host cell via the
type III secretion system (TTSS). Elucidating individual effectors’ ﬁmctions is
fundamental for understanding bacterial infection of plants. We have previously Shown
that transgenic overexpression of the Pseudomonas syringae pv. tomato strain DC3000
effector AvrPto in the host plant Arabidopsis leads to suppression of basal defenses and
enables multiplication of non-pathogenic TTSS-defective bacteria. AvrPto localization at
the host plasma membrane is required for its avirulence and virulence functions in tomato
and in Arabidopsis protoplasts. In this study, we conﬁrmed that membrane localization is
required for AvrPto to exert its virulence function in transgenic Arabidopsis plants as
well. Furthermore, we found that AvrPto interacted in yeast two-hybrid assay with the
Arabidopsis RabE small GTPase proteins, which are homologous to mammalian Rab8
and yeast Sec4p, known regulators of polarized secretion. Microscopic analysis and cell
ﬁ'actionation studies revealed that transgenically expressed GFP-RabEld and endogenous
RabE proteins were associated with the Golgi apparatus and the plasma membrane in
Arabidopsis leaves. Strikingly, transgenic expression of membrane-localized, but not
soluble, AvrPto in Arabidopsis impaired the normal localization of RabEld at the Golgi
apparatus. Such effect on RabEld localization depended on its nucleotide-binding state.
Overexpression of RabEld could partially counteract the AvrPto-induced susceptibility
to TTSS-defective bacteria. Our experiments uncover a novel association between the

AvrPto virulence function and RabE subcellular distribution.

37

INTRODUCTION

A common virulence mechanism, shared by many Gram-negative bacterial
pathogens of plants and animals, is the delivery of bacterial proteins directly into the host
cell via the type III secretion system (TTSS) (Buttner and Bonas, 2003; He et al., 2004;
Mota and Comelis, 2005). These proteins, collectively called TTSS effectors, alter the
host cellular processes to favor pathogen growth. To carry out virulence functions,
effectors interact with (and often biochemically modify) critical regulatory components
of basic host cellular ﬁrnctions. Bacterial pathogens of animals, such as Salmonella and
Yersinia, employ various type III effectors to interfere with host cytoskeleton dynamics,
vesicle trafﬁcking, Signal transduction, apoptosis, and potentially other pathways (Mota
and Comelis, 2005). Only recently, effectors from phytopathogenic bacteria and their
host targets have started to be elucidated. A major virulence activity of TTSS effectors of
Pseudomonas syringae seems to be suppression of host defenses (Alfano and Collmer,
2004; Mudgett, 2005; Nomura et al., 2005; Abramovitch et al., 2006; Desveaux et al.,
2006; Nomura et al., 2006).

Studying the function of effectors delivered by bacterial pathogens of plants is
greatly facilitated by the use of model host-pathogen systems such as the Arabidopsis
thaliana-P. syringae pv. tomato DC3000 (Pst DC3000) interaction. Pst DC3000 is the
causal agent of bacterial Speck disease on Arabidopsis and tomato; its pathogenicity is
dependent on a ﬁinctional TTSS. Pst DC3000 mutant strains that are defective in the
TTSS, such as hrcC (formerly hrpH') and hrpA', are unable to multiply and to cause

disease on host plants, behaving as non-pathogens (Yuan and He, 1996; Roine et al.,

38

1997). Pst DC3000, like several other plant pathogenic bacteria, produces an array of 30
or more type III effectors (Lindeberg et al., 2006; Schechter et al., 2006). The function of
most of these proteins in the host cell has yet to be determined. A common feature of type
III effectors is that bioinfonnatic analysis of protein sequences, in most cases, does not
provide clues to their function. Moreover, mutation or deletion of Single effectors often
does not result in reduced virulence, indicating functional subtlety and/or redundancy,
when delivered by bacteria during infection.

Our group previously Showed that AvrPto, one of Pst DC3000 TTSS effectors,
when transgenically expressed in Arabidopsis, greatly compromises plant basal defenses
and promotes susceptibility to non-pathogenic bacteria, such as TTSS-defective mutants
(Hauck et al., 2003). Other studies have Shown that AvrPto expression promotes
susceptibility to nonhost P. syringae strains (He et al., 2006) and suppresses pathogen-
associated molecular pattern (PAMP)-induced signal transduction, early defense gene
expression and other associated responses (Kang et al., 2004; Li et al., 2005; Oh and
Collmer, 2005; He et al., 2006). However, the molecular mechanism by which AvrPto
exerts its virulence function(s) in the plant cell is still elusive. The best understood
function of AvrPto is that of triggering gene-for-gene resistance in tomato plants carrying
the Prf resistance gene (Mucyn et al., 2006). Such avirulence function depends on
physical interaction, demonstrated in yeast two-hybrid assay (Y2H), between AvrPto and
the Pto kinase (Scoﬁeld et al., 1996; Tang et al., 1996). However, AvrPto contributes to
Pst DC3000 virulence in tomato lacking Prf, and this virulence ﬁmction does not require

Pto (Shan et al., 2000a). Arabidopsis has no known ortholog(s) of Pto or Prf.

39

To gain insight into the virulence effects of AvrPto in Arabidopsis, we conducted
a Y2H screen for Arabidopsis proteins that interact with AvrPto. Among the interactors,
we identiﬁed members of the RabE family of small GTPases, closely related to
mammalian Rab8 (Huber et al., 1993), Saccharomyces pombe th2 (Craighead et al.,
1993), and Saccharomyces cerevisiae Sec4p (Goud et al., 1988), which are well-known
regulators of polarized secretion. Interestingly, a previous Y2H screening of a tomato
cDNA library for AvrPto-interacting proteins also yielded two small GTPase proteins
(named Api2 and Api3) that are Similar to Rab8 (Bogdanove and Martin, 2000). Rab
proteins are key regulators of vesicle formation and transport between membrane-bound
cellular compartments (Stenmark and Olkkonen, 2001; Zerial and McBride, 2001). The
identiﬁcation of Rab GTPases as AvrPto interactors in two different host Species
(Arabidopsis and tomato) raises the intriguing possibility that one of the virulence
frmctions of this effector may be to perturb intracellular vesicle trafﬁcking in the plant.
Interestingly, small GTPases regulating cytoskeleton dynamics and vesicle trafﬁcking are
among the most common host targets of TTSS effectors produced by animal bacterial
pathogens (Harrison et al., 2004; Machner and Isberg, 2006; Murata et al., 2006; Rzomp
et al., 2006; Smith et al., 2007). Importance of vesicle trafﬁc and the secretory pathway
for plant defense and bacterial virulence was recently highlighted by several studies
(Collins et al., 2003; Wang et al., 2005; Nomura et al., 2006) and reviews (Field et al.,
2006; Robatzek, 2007).

Very little is known about the function of RabE GTPases in the Arabidopsis cell.
Rab proteins, in general, are considered as speciﬁc markers of endomembrane

compartments. This Speciﬁcity prompted us to apply a live cell imaging approach to

40

visualize the Arabidopsis RabE proteins in the plant cell, and to investigate whether and
how AvrPto affects RabE cell biology. We found that RabEld is associated with both the
plasma membrane (PM) and the Golgi apparatus in Arabidopsis cells. In planta
expression of AvrPto impaired RabEld localization at the Golgi, without affecting its
localization at the PM. Such effect on RabEld subcellular distribution was dependent on
AvrPto localization at the host membrane and on the RabEld nucleotide binding state.
Furthermore, RabEld overexpression in Arabidopsis transgenic plants reduced the
virulence effect of AvrPto. Our data suggest that one of the mechanisms by which
AvrPto carries out its virulence function in the Arabidopsis cell is by interfering with
subcellular localization of RabE proteins, which likely leads to perturbation of

intracellular vesicle trafﬁcking.

41

MATERIALS AND METHODS

Yeast two-hybrid screen

Arabidopsis proteins that interacted with AvrPto of Pst DC3000 were identiﬁed
by following the Matchmaker LexA-based Y2H system manual (Clontech Laboratories
Inc., Palo Alto, CA). Two Arabidopsis cDNA libraries, made using RNA from pathogen-
infected and uninfected Landsberg erecta plants (kindly provided by Dr. J. Jones,
Sainsbury Laboratory, UK), were screened. The avrPto coding sequence was ampliﬁed
from Pst DC3000 genomic DNA by PCR (sense primer: 5’-
GCGAATTCCGAACCATGGGAAATATATGTGTC-3’; antisense primer: 5’-
GCCTCGAGATTGCCAGTTACGGTA-3’; the EcoRI and XhoI restriction sites, used
for cloning, are underlined) and cloned into pNLexA, to serve as bait in the Y2H

screening.

Plant growth and bacterial multiplication assay

Arabidopsis plants were grown in soil, in grth chambers, under a 12 h dark/12
h light cycle. The light intensity was on average 100 TIE, and the temperature was kept
constant at 20°C. Bacteria were cultured in low-salt LB medium (lOg/l Tryptone, 5g/l
Yeast Extract, Sg/l NaCl), supplemented with the appropriate antibiotics. For
multiplication assays in plants, bacterial liquid cultures were incubated at 30°C to the
mid- to late-logarithmic phase. Bacteria were collected by centrifugation and resuspended
in sterile water with the addition of 0.004% Silwet L-77 (OSI Specialties, Friendship,

WV). Titer of the bacterial inoculurn was 1x106 colony forming units (CFUS)/ml, unless

42

otherwise indicated. Plants were inoculated by vacuum-inﬁltration, and bacteria

enumeration in leaves was conducted as described (Katagiri et al., 2002).

AvrPto and 6xHis-AvrPto transgenic plants

Generation of transgenic Arabidopsis plants that express AvrPto under the control
of the dexamethasone (DEX)-inducible promoter was previously described (Hauck et al.,
2003). To generate 6xHis-AvrPto-expressing plants, a 6xHistidine tag was added to the
N-terminus of AvrPto by PCR, and the PCR product was cloned into the pTA7002 binary

vector (Aoyama and Chua, 1997) for DEX-inducible expression in Arabidopsis.

RabE cloning and mutagenesis

The RabE1d(At5g03520) coding sequence was ampliﬁed from Arabidopsis Col-
0 cDNA using the rabE-S’ and rabE-3’ primers (Table 2.1), containing the EcoRI and
BamHI restriction sites, respectively. The PCR product was ligated into a TOPO vector
(Invitrogen), and sequenced. Single nucleotide changes were introduced in the RabE 1 d
sequence by two-step overlapping PCR, to generate the RabEld-S29N and RabEld-
Q74L mutant derivatives. RabEld-SZ9N was obtained through a G—IA substitution; in
the ﬁrst PCR step, two overlapping RabEId fragments were ampliﬁed using the primer
combinations rabE-5’/SZ9N-rev and rabE-3’/SZ9N-for (Table 2 - 1). The products were
puriﬁed from agarose gel, mixed and used as template for a second PCR ampliﬁcation
step, with the rabE-S’ and rabE-3’ primers. The presence of an overlapping region
allowed annealing of the two gene fragments and ampliﬁcation of the full-length coding

sequence. A similar procedure was used for introducing the Q74L mutation, through an

43

A—IT substitution. In this case, the following primer combinations were used in the ﬁrst

PCR step: rabE-5’/Q74L-rev and rabE-3’/Q74L-for (Table 2 - 1). RabEld-S29N and

RabEld-Q74L ampliﬁcation products were introduced in a TOPO vector by TA-cloning

and sequenced.

 

 

Primer Sequence

rabE-5’ 5’thcatggcggﬁgcgccggcaag-3’
rabE-3’ S’Wagcaatcatactcctaaac-S’
Q74L-for 5’-cactgctggtctagaacgtttc-3’
Q74L-rev 5’-gaaacgttctggaccagcagtg-3’
$29N-for 5’-gtggggaagagﬂgtﬂgttac—3’
$29N-rev 5’- gtaacaaacaagtcttccccac-S’

 

Table 2 - 1: Primers for RabE cloning and mutagenesis.

Restriction sites are underlined (EcoRl for rabE-S’, BamHl for rabE-3’).
Start and stop codons are in bold; Single nucleotide changes are in bold and underlined.

44

GFP-RabEld transgenic plants

RabE] d and RabEId-S29N, cloned in the TOPO vector as described above, were
subcloned in the EcoRI and BamHI Sites of the binary expression vector pEGAD (Cutler
et al., 2000), downstream of the enhanced green ﬂuorescent protein (hereafter GFP)
sequence, to create translational fusions. The binary vector was introduced in
Agrobacterium tumefaciens strain GV3 850 via triparental mating, for plant
transformation. Arabidopsis Col-0 glabraus (glI), as well as AvrPto-expressing plants,
were transformed using the ﬂoral dip method (Clough and Bent, 1998). Transgenic plants
were selected based on resistance to the herbicide Basta (glufosinate). A solution
containing 0.012% glufosinate (Finale concentrate, AgrEvo Environmental Health) and
0.025% Silwet L-77 was Sprayed on 2 week-old seedlings growing in soil. Surviving Tl
plants were screened for GF P ﬂuorescence with a Zeiss Axiophot microscope, and

expression of the correct Size GFP-RabE fusion proteins was veriﬁed by western blot.

Protein extraction and immunoblotting

Total proteins were extracted as follows: approximately 20 mg (fresh weight) of
fresh or frozen leaf tissue were ground with a pestle in a microfuge tube in the presence
of 100 pl of 1 X SDS-PAGE loading buffer [90 mM Tris-HCI pH 8.0, 100 mM DTT, 3%
SDS, 22.5% sucrose, 10 ul/ml Protease Inhibitor Cocktail for Plant Cell Extracts (Sigma),
bromophenol blue (to saturation)]. Extracts were immediately heated at 80°C for 10
minutes and then frozen at -20°C. Before loading on gel, extracts were thawed at room
temperature and centrifuged at 20,000 X g for 2 minutes, to pellet debris. An equal

volume of each sample was used for SDS-PAGE electrophoresis. Total proteins were

45

separated on precast gradient gels (4-20%, ISC BioExpress), then transferred onto
Immobilon-P membrane (Millipore, MA) using a semi-dry transfer apparatus (SEMI
PHOR, Hoefer Scientiﬁc Instruments, San Francisco, CA). Protein detection was carried
with the following primary antibodies: anti-AvrPto (raised in rabbit against recombinant
AvrPto protein expressed in E. coli, Cocalico Biological, Inc.), anti-RabE (raised in
chicken against recombinant RabE protein expressed in E. coli, Cocalico Biological,
Inc.), anti-XTl (Faik et al., 2002; Cavalier and Keegstra, 2006), anti-PM ATPase

(Morsomme et al., 1998), and anti-yTIP (gift of Dr. N. Raikhel, unpublished).

Cell membrane fractionation

Leaves were harvested and weighed immediately prior to extraction. Leaf tissue
(2.5 g) was ground with a cold mortar and pestle, in the presence 5 ml of ice-cold
extraction buffer [50 mM HEPES pH 7.5, 100 mM KCl, 10 mM EDTA, 1 mM DTT, and
10 III/ml Protease Inhibitor Cocktail for Plant Cell Extracts (Sigma)] containing 34%
sucrose. The extract was homogenized with a Polytron immersion blender (3 pulses of 10
seconds each), ﬁltered through a single layer of Miracloth and centrifuged for 10 minutes
at 10,000 X g, to remove most unbroken chloroplasts and nuclei. The supernatant was
adjusted to 40% sucrose in about 10 ml ﬁnal volume (concentration was determined with
a refractometer), and layered on top of a 5 ml cushion of 50% sucrose, in clear
ultracentrifugation tubes. The homogenate was subsequently layered with 10 ml of 34%
sucrose, 8 ml of 25% sucrose and 8 mL of 18% sucrose. All sucrose solutions were
prepared in the same buffer used for extraction. Gradients were centrifuged at 100,000 X g

for 3 hours, at 4°C, in a SW28 rotor (Beckman). After centrifugation, the membrane-

46

containing interphases were collected, diluted with sucrose-free extraction buffer, and
membranes were collected by ultracentrifugation (1 hour at 100,000 X g). Membrane
pellets were resuspended in equal volumes of SDS-PAGE loading buffer and heated at
80°C for 10 minutes. Protein electrophoresis and western blot were performed as

described above.

Confocal microscope analysis and imaging

Pieces of leaves were sampled randomly and mounted in water. Imaging was
performed using an LSMSIO META inverted confocal laser scanning microscope (Zeiss),
and either a 20 X or a 40 X oil immersion objective. For GFP-RabE ﬂuorescence
analysis, the 488 nm excitation line of an argon ion laser was used, with a 505 to 530 nm
band-pass ﬁlter, in the Single-track facility of the microscope. Images were processed
with the LSM Image Browser Version 3.1 (Zeiss) and with the Adobe Photoshop
Elements Version 5.0 software (Adobe Systems Inc.). For FM4-64 staining, detached
Arabidopsis leaves were submerged in 8.2 uM FM4-64 (Molecular Probes, Leiden, The
Netherlands) in water for 15 minutes. Leaves were rinsed in distilled water and observed
immediately. For imaging GFP-RabEld and F M4-64 ﬂuorescence, the 488 nm excitation
line was used; GF P ﬂuorescence was collected with a 505 to 530 nm band-pass ﬁlter, and

FM4-64 ﬂuorescence was collected with a 615 nm long-pass ﬁlter.

Biolistic transformation

Transient expression in Arabidopsis leaves was achieved by biolistic

transformation. The binary vector bearing rat Sialyl transferase ﬁrsed to DSRed (ST-RF P)

47

was a kind gift of Dr. F. Brandizzi (Saint-lore et al., 2002). Gold particles (1.0 pm, Bio-
Rad) were coated with the pEGAD::RabE1 d or ST-RFP plasmid DNA as described by
Zhang et al. (Zhang et al., 2001). Arabidopsis leaves were harvested and arranged on MS
agar plates (4.3 g/l MS salts, 0.8% agar, pH 5.7). The DNA-coated particles were
delivered into the lower leaf epidermis with a particle gun (Dupont), using 1100 psi
rupture discs, under a vacuum of 25 in Hg. After bombardment, leaves were incubated in
the sealed plates at room temperature, and ﬂuorescence was observed 24 hours post
transformation.

For co-imaging GFP-RabEld and ST-RF P, the argon ion laser excitation lines of
488 nm (for GFP) and 543 nm (for DSRed) were used. GFP ﬂuorescence was collected
with a 505 to 530 nm band-pass ﬁlter, and DSRed ﬂuorescence was collected with a 615

nm long-pass ﬁlter.

48

RESULTS

AvrPto membrane localization is required for virulence function in
Arabidopsis plants

Hauck et al. (2003) showed that AvrPto protein expression in transgenic
Arabidopsis plants compromised basal defense and allowed increased multiplication of
TTSS-defective mutant bacteria. To characterize this phenomenon ﬁrrther, we wanted to
determine if membrane localization of AvrPto is necessary for these effects. First, we
needed to conﬁrm membrane-association of AvrPto in these transgenic Arabidopsis
plants. Consistent with studies showing that AvrPto is targeted to the host plasma
membrane in transgenic tobacco and tomato plants (Shan et al., 2000b), and in
Arabidopsis protoplasts (He et al., 2006), AvrPto was exclusively detected in the
membrane fraction, but not in the soluble fraction, of the stable transgenic Arabidopsis
plants (Fig. 2 - 1, A).

We also generated Arabidopsis plants that express AvrPto carrying an N-terrninal
6xHis tag, under the control of the DEX-inducible promoter (Aoyama and Chua, 1997).
In contrast to wild-type AvrPto, 6xHis-AvrPto did not localize to the membrane fraction,
but remained in the soluble fraction (Fig 2 - 1, A). This result is consistent with studies
Showing that host-mediated myristoylation of a conserved glycine at the N-terminus of
AvrPto is responsible for AvrPto association with the host membrane. Site-directed
mutagenesis of the myristoylation site disrupts AvrPto plasma membrane localization
(Shan et al., 2000b; He et al., 2006). Our result suggests that addition of the Short 6xHis

tag to the N—terminus of wild-type AvrPto was sufﬁcient to disrupt AvrPto membrane

49

localization, most likely by preventing its myristoylation. Signiﬁcantly, while expression
of membrane-localized AvrPto in Arabidopsis promoted susceptibility to TTSS-deﬁcient
mutants, expression of soluble 6xHis-AvrPto did not (Fig 2 - 1, B). This result
demonstrates that the virulence function of transgenically expressed AvrPto depends not

only on protein production, but on association with the host membrane.

50

T S M
I- u Col/AvrPto

m Col/6xHis-AvrPto

B
107 .
IM

A IAvrPto
NE . I 6xHis-AvrPto
2 106
a:
3
LL
9 105 -
E
3
O
o
.7! 104
a
*5
(U
m 103 .

102 .

 

 

Day 0 Day 3

Figure 2 - 1: Membrane localization is critical for AvrPto virulence function.

(A) Western blot analysis: anti-AvrPto antibody was used to detect AvrPto and 6xHis-
Ath0 in transgenic plants extracts. Transgene expression was induced by spraying the
plants with 30uM DEX, 24h prior to protein extraction. Total extracts were centrifuged
for 1h at 100,000 X g, at 4°C, to separate the membrane fraction (pellet) from the soluble
fraction (supernatant). T = total extract; S = soluble fraction; M = membrane fraction.

(B) AvrPto and 6xHis-AvrPto expression was induced by spraying the plants with 30uM
DEX 24 h prior to bacterial inoculation, and then at 24 h intervals during the course of
the experiment. hrcC TTSS-deﬁcient mutant bacteria were syringe-inﬁltrated in the plant
leaves at a concentration of 106 CFUs/ml.

Figure contributed by Kinya Nomura.

51

AvrPto interacts with the Arabidopsis RabE small GTPases in Y2H assay

To gain insights into the molecular basis of AvrPto-mediated promotion of
Arabidopsis susceptibility to bacterial infection, we conducted Y2H screening of two
Arabidopsis cDNA libraries, using AvrPto as bait. AvrPto was found to interact with
several Arabidopsis proteins, including a member of the RabE family of small GTPases
(At5g59840), a putative cytoplasmic kinase (At4g11890), an auxin Signaling repressor,
IAA7 (At3g23050), two hypothetical proteins (At3g26600, At5g16840) and several
putatively chloroplast- or mitochondria-targeted proteins. Because AvrPto is localized at
the host membrane, we chose to focus on RabE, the only interactor that is also predicted
to be membrane-localized.

There are ﬁve highly similar RabE proteins in Arabidopsis (Rutherford and
Moore, 2002; Vemoud et al., 2003), closely related to several characterized regulators of
the secretory pathway in fungi and animals. Sequence alignment of the ﬁve Arabidopsis
thaliana RabE proteins (AtRabEl a through Ele) with Saccharomyces pombe th2
(Sprt2), human Rab8a (HsRab8a), Drosaphila melanogaster Rab8 (DmRab8) and
Saccharomyces cerevisiae Sec4p (ScSec4p) illustrates this high Similarity (Figure 2 - 2).

Arabidopsis contains a total of 57 Rabs, classiﬁed into eight families (RabA
through H), based on conserved motifs and similarity to the equivalent Rab classes in
yeast and animals (Rutherford and Moore, 2002; Vemoud et al., 2003). We examined the
ability of AvrPto to interact with representative members of the other Rab protein
families. We cloned and expressed RabA Ia, BI b, C1, D2a, F 2a, and G3a in yeast; none
interacted with AvrPto in the Y2H system (Fig. 2 - 3, A). In addition, we investigated

whether AvrPto interacts with other members of the RabE family. Of the ﬁve RabE

52

genes, all but RabElc were successfully cloned and expressed in yeast. All four RabE
proteins tested (RabE! a, b, d, and e) interacted with AvrPto (Figure 2 - 3, B). Thus, it

appears that AvrPto interacts Speciﬁcally with the Arabidopsis RabE family of GTPases.

53

AtRabElb/1-216
AtRabElc/l-le
AtRabEla/l-216
AtRabEld/1-216
AtRabEle/l-ZIB
Sprt2/1-200
HsRabBa/l-ZO?
DmRab8/1-207
SCSec4p/1-215

AtRabElb/1-216
AtRabElC/1-216
AtRabEla/1-216
AtRabEld/l-216
AtRabEle/l-218
Sprt2/1-200
HsRabBa/l-ZO?
DmRab8/1-207
ScSeC4p/1-215

AtRabElb/1-216
AtRabE1C/1-216
AtRabEla/l-le
AtRabEld/l-ZlS
AtRabEle/l-ZIB
Sprt2/l-200
HsRabBa/l-207
DmRab8/1-207
Scsec4p/1-215

AtRabElb/l-216
AtRabE1C/1-2l6
AtRabEla/l-216
AtRabEld/l-216
AtRabEle/1-218
Sprt2/1-200
HsRab8a/1-207
DmRab8/l-207
SCSec4p/1-215

PHI FMZ
----- MAAPPARARADYDYLIKLLLIGDSGVGKSCLLLRFSDGSFTTSFITTIGIDFKIR
----- MAAPPARARADYDYLIKLLLIGDSGVGKSCLLLRFSDGSFTTSFITTIGIDFKIR
----- MAAPPARARADYDYLIKLLLIGDSGVGKSCLLLRFSDGSFTTSFITTIGIDFKIR
----- MAVAPARARSDYDYLIKLLLIGDSGVGKSCLLLRFSDDTFTTSFITTIGIDFKIR
----- MAVAPARARSDYDYLIKLLLIGDSGVGKSCLLLRFSDDTFTTSFITTIGIDFKIR
---------- MST-KSYDYLIKLLLIGDSGVGKSCLLLRFSEDSFTPSFITTIGIDFKIR
------------ MAKTYDYLFKLLLIGDSGVGKTCVLFRFSEDAFNSTFISTIGIDFKIR
------------ MAKTYDYLFKLLLIGDSGVGKTCILFRFSEDAFNTTFISTIGIDFKIR
MSGLRTVSASSGNGKSYDSIMKILLIGDSGVGKSCLLVRFVEDKFNPSFITTIGIDFKIK

PM3
TIELDGKRIKLQIWDTAGQERFRTITTAYYRGAMGILLVYDVTDESSFNNIRNWIRNIEQ
TIELDGKRIKLQIWDTAGQERFRTITTAYYRGAMGILLVYDVTDESSFNNIRNWIRNIEQ
TIELDGKRIKLQIWDTAGQERFRTITTAYYRGAMGILLVYDVTDESSFNNIRNWIRNIEQ
TVELDGKRIKLQIWDTAGQERFRTITTAYYRGAMGILLVYDVTDESSFNNIRNWMKNIEQ
TVELDGKRIKLQIWDTAGQERFRTITTAYYRGAMGILLVYDVTDESSFNNIRNWMKNIEQ
TIELDGKRIKLQIWDTAGQERFRTITTAYYRGAMGILLLYDVTDKKSFDNVRTWFSNVEQ
TIELDGKRIKLQIWDTAGQERFRTITTAYYRGAMGIMLVYDITNEKSFDNIRNWIRNIEE
TIELDNKKIKLQIWDTAGQERFRTITTAYYRGAMGIMLVYDITQEKSFENIKNWIRNIEE
TVDINGKKVKLQLWDTAGQERFRTITTAYYRGAMGIILVYDVTDERTFTNIKQWFKTVNE

G2 G3
HASDNVNKILVGNKADMDESKRAVPKSKGQALADEYGIKFFETSAKTNLNVEEVFFSIAK
HASDNVNKILVGNKADMDESKRAVPTAKGQALADEYGIKFFETSAKTNLNVEEVFFSIGR
HASDSVNKILVGNKADMDESKRAVPKSKGQALADEYGMKFFETSAKTNLNVEEVFFSIAK
HASDNVNKILVGNKADMDESKRAVPTAKGQALADEYGIKFFETSAKTNLNVENVFMSIAK
HASDSVNKILVGNKADMDESKRAVPTSKGQALADEYGIKFFETSAKTNQNVEQVFLSIAK
HASENVYKILIGNKCDCED-QRQVSFEQGQALADELGVKFLEASAKTNVNVDEAFFTLAR
HASADVEKMILGNKCDVND-KRQVSKERGEKLALDYGIKFMETSAKANINVENAFFTLAR
NASADVEKMLLGNKCELTD-KRQVSKERGEQLAIEYGIKFMETSAKASINVEEAFLTLAS
HANDEAQLLLVGNKSDMET-~RVVTADQGEALAKELGIPFIESSAKNDDNVNEIFFTLAK

DIKQRLADTDSRAEPATIKISQTDQA-AGAGQATQKSACCGS-
DIKQRLSDTDSRAEPATIKISQTDQA-AGAGQATQKSACCGT-
DIKQRLADTDARAEPQTIKINQSDQG-AGTSQATQKSACCGT-
DIKQRLTETDTKAEPQGIKITKQDT--AASSSTAEKSACCSYV
DIKQRLTESDTKAEPQGIKITKQDANKASSSSTNEKSACCSYV
EIKKQKIDAENEFSNQANNVDLG-NDRTVKR ------- CC---
DIKAKMDKKLEGNSPQGSNQGVKITPDQQKRSSFFR--CVLL-
DIKAKTEKRMEANNPPKGGHQLKPMDSRTKDSWLSR--CSLL-
LIQEKIDSNKLVGVGNGKEGNISINSGSGNSS---KSNCC---

Figure 2 - 2: ClustalW alignment of the five Arabidopsis RabE proteins and their
closest homologues in other organisms.

Yellow boxes highlight the highly conserved nucleotide-binding domain residues (PM =
phosphate/magnesium-binding domain; G = guanine base-binding domain). Blue boxes
highlight Rab-speciﬁc residues (Stenmark and Olkkonen, 2001). Amino acids in red are
commonly mutated in functional studies, to create Rab variants that have a higher afﬁnity
for GDP than for GTP (S or T in PMl), or that cannot hydrolyze GTP (Q in PM3).
Mutating the N in G2 results in Rabs that cannot bind any nucleotide. The two C-terminal
C residues represent geranylgeranylation Sites.

54

- +
Ata Btb DZa Etd F.2a GBa

D2a E1e E1d 515“”“s1a'

Figure 2 - 3: AvrPto interacts with Arabidopsis RabE in the yeast two-hybrid
system.

(A) Y2H assay demonstrating that AvrPto (in the bait vector pNLexA) interacts with
Arabidopsis RabEld, but not with other members of the Rab superfarnily (in the prey
vector pB42AD). Interaction is visualized by development of the blue color on media
containing X-Gal.

(-) empty vectors, negative control; (+) pLexA-A53 + pB42AD-T, positive control.

(B) Y2H assay demonstrating AvrPto (in pNLexA) interaction with four of the ﬁve
Arabidopsis RabE proteins (in pB42AD). Interaction is visualized by development of the
blue color on media containing X-Gal. Yeast expressing AvrPto in pNLexA and RabDZa
in pB42AD is shown as negative control.

(+) pLexA-A53 + pB42AD-T, positive control.

Figure contributed by Paula Hauck.

55

Gene expression analysis of the Arabidopsis RabE gene family

Some Arabidopsis Rab families are uncommonly large. Careful analysis of
genome duplications Showed that 44 out of 57 Rob genes reside in duplicated regions. Of
the ﬁve RabE genes, for instance, RabE] d and E] e appear to be deriving from a major
duplication event between chromosomes III and V, and the same holds true for RabEIb
and E] c (Rutherford and Moore, 2002).

The high degree of sequence identity among RabE proteins (equal or higher than
86%) suggests functional redundancy. Members of gene families are, in some cases,
preferentially expressed in different tissues, or at Speciﬁc developmental stages, or in
response to stresses. We, therefore, investigated whether this is the case with the ﬁve
RabE genes, by gathering information on their expression through the TAIR website
(www.arabidopsis.org). In silica analysis revealed that all ﬁve genes are expressed in all
Arabidopsis tissues and developmental stages. RabE] d and E I e (encoding 94% identical
proteins) were the only two family members whose expression was much lower in pollen
than in all other tissues. According to the TAIR database, RabEId was the most highly
expressed in rosette leaves, immediately followed by RabE1c. The RabE] a, E] e and E I b
genes had the lowest expression levels. RT-PCR analysis on rosette leaves conﬁrmed
these data (Figure 2 - 4).

To gain insight on potential up- or down-regulation of members of the RabE
family in responses to pathogens or other stresses, the expression pattern of the RabE
genes was analyzed with the AtGenExpreSS Visualization Tool
(http://jSp.weigelworld.org/expviz/expviz.iSp) (Schmid et al., 2005), across several

publicly available microarray datasets. None of the ﬁve genes appeared to be

56

Signiﬁcantly (more than 2.5-fold) up- or down- regulated in response to pathogens or
elicitors. In the absence of any expression-based indication on whether the ﬁve RabE
proteins have redundant or distinct functions, we chose to utilize RabEld (At5 g03 520),

which has the highest mRNA expression level in rosette leaves.

 

 

57

E18 E1b E1c E1d E16

  

awn &¢.i-~i W m Act8

RabE

 

Figure 2 - 4: RT-PCR showing RabE gene expression in rosette leaves.

RT-PCR reaction products representing the ﬁve RabE genes, reverse-transcribed and
ampliﬁed from Arabidopsis Col-0 rosette leaf total RNA. A single reverse transcription
reaction was performed, and equal amounts of the resulting cDNA were used as template
in PCR reactions. Each PCR reaction contained primers for the Actin8 gene, in addition
to primers for one of the RabE genes (procedure described in detail in Chapter 3; primers
are listed in Table 3 - 1). Five out of 25 ul of PCR product were loaded on 1% agarose
gel; 21 cycles of ampliﬁcation were used for Act8, 25 for the RabE genes.

58

AvrPto preferentially interacts with GTP-bound RabE

In the cell, Rab GTPases act as molecular switches that cycle between an “active”
GTP-bound and an “inactive” GDP-bound state. Interconversion between the two forms
is stimulated by accessory proteins. A guanosine nucleotide exchange factor (GEF)
converts a GDP-bound Rab into the GTP-bound form, whereas a GTPase-activating
protein (GAP) stimulates the Rab low intrinsic GTPase activity, causing hydrolysis of
GTP into GDP (Novick and Brennwald, 1993). Mutation of conserved residues is widely
used to alter the nucleotide-exchange and the GTP-hydrolysis activities of Rab proteins
for functional analysis (Stenmark et al., 1994). Substitution of a conserved serine or
threonine with asparagine in the PMl nucleotide-binding domain results in RabS that
have a stronger preference for GDP than for GTP, whereas substitution of a conserved
glutamine with leucine in the PM3 catalytic domain inhibits both intrinsic and GAP-
stimulated GTP hydrolysis (Figure 2 - 2) (Stenmark et al., 1994). We engineered the
mutant forms RabEld—SZ9N and RabE1d-Q74L by site-directed mutagenesis.

In the Y2H system, AvrPto interacted with only wild-type RabEld or RabEld-Q74L
(predicted to be mostly in the GTP-bound form), but not with RabEld-SZ9N (predicted to
be mostly GDP-bound) (Fig 2 - 5). Nucleotide-binding state is known to affect the
conformation of Rab proteins (Stenmark and Olkkonen, 2001). Our results indicate that
the active GTP-bound form of RabE, but not the inactive GDP-bound, is in the

appropriate conformation for interacting with AvrPto.

59

pGiIda

 

RabE1d RabE1d- RabE1d-
Q74L $29N

.. I ‘: AvrPto
- ' ‘ pB42AD

‘ ' no insert

 

 

Figure 2 - 5: Wild-type RabE and RabE-Q74L, but not RabE-S29N, interact with
AvrPto in Y2H.

Yeast two-hybrid assay demonstrating interaction between AvrPto (in the prey vector
pB42AD) and RabEld or RabE1d-Q74L, but not RabE1d-SZ9N (all in the bait vector
pGILDA). For this experiment only, the mutant RabEld proteins, as well as wild type
RabEld as a control, were modiﬁed by substituting the two C-terrninal conserved
cysteine residues, which are sites of geranylgeranylation (Figure 2 - 2), with glycine and
serine, to prevent prenylation and membrane association.

As a negative control, yeast expressing the three RabEld versions in pGILDA and empty
pB42AD vector is shown. Interaction in visualized by development of blue color on X-
Gal-containing media.

(+) pLexA-A53 + pB42AD-T, positive control.

Figure contributed by Lori Imboden.

60

RabEld is associated with Golgi apparatus and plasma membrane

Each Rab protein is normally present in cells in two pools, one of which is
cytoplasmic, the other is membrane-associated (Novick and Brennwald, 1993).
Nucleotide-binding state and interaction with accessory proteins determine whether a Rab
is in the cytosol or the membrane, at any given time. A hallmark feature of Rab proteins
is that they localize to the speciﬁc membrane compartments in which they function. It
was previously reported that Arabidopsis RabEl d, when transiently and heterologously
expressed in tobacco epidermal cells as a ﬁrsion with yellow ﬂuorescent protein (YFP),
was detected in the Golgi apparatus and in the cytoplasm (Zheng et al., 2005). However,
AvrPto was reported to be localized at the host plasma membrane (Shan et al., 2000; He
et al., 2006). To determine RabE localization in Arabidopsis cells, we created stable
transgenic plants that express RabEld fused with enhanced green ﬂuorescent protein
(GF P), under the control of the CaMV 358 promoter. GF P was fused to the N-terminus
of RabEld, to preserve the C-terminal CAAX geranylgeranylation site, critical for
membrane association and function (Calero et al., 2003). Several independent transgenic
lines were analyzed by confocal laser scanning microscopy. GF P ﬂuorescence was
observed in intracellular punctate structures consistent with the Golgi apparatus, as
detected in tobacco cells (Zheng et al., 2006), but also at the cell periphery (Fig 2 - 6).

Leaf epidermal cells typically contain a very large vacuole that accounts for most
of the cell volume. Fluorescence detected at the cell periphery may represent the PM, or
the vacuolar membrane (tonoplast), or even the thin layer of cytoplasm that is between
the PM and the tonoplast. To more precisely determine whether GFP-RabE was also

localized at the PM, we stained live leaf tissue with the lipophylic dye FM4-64 (Fischer-

61

Parton et al., 2000; Bolte et al., 2004). FM4-64, which produces a bright red ﬂuorescence
when it is in membranes but not in aqueous solutions, is rapidly incorporated in the PM.
It is often used in microscopy as an endocytic tracker, because it is retained in the
portions of PM that are internalized by endocytosis (Ueda et al., 2001). Within the ﬁrst
15-30 minutes after incorporation (depending on the system used), FM4-64 will
selectively stain the PM. Confocal microscope analysis revealed overlap of GFP-RabEld
ﬂuorescence with FM4-64 ﬂuorescence, immediately after staining, suggesting RabE
association with the plasma membrane (Fig 2 - 7, A).

To investigate whether the punctate structures labeled by GFP-RabEld
corresponded to the Golgi apparatus, we examined co-localization with rat Sialyl
transferase, a Golgi marker protein (Wee et al., 1998) fused to DSRed (ST-RFP). ST-RFP
was transiently expressed in the GFP-RabE transgenic leaves, via particle bombardment.
Observation of cells co-expressing GFP-RabEld and ST-RFP revealed overlapping
ﬂuorescence Signals, conﬁrming RabE association with the Golgi apparatus (Fig 2 - 7,
B).

Taken together, microscopic analysis of GFP-RabEld localization suggested that,
in native Arabidopsis leaf cells, membrane-associated RabEld is found not only in the

Golgi apparatus, as previously reported, but also in the PM.

62

 

Figure 2 - 6: GFP-RabEld localization in Arabidopsis leaf cells.

Confocal microscope image of the leaf epidermis of a transgenic Arabidopsis plant
expressing GFP-RabEld. This image represents a projection along the Z-axiS of several
optical planes intersecting the leaf epidermal cell layer. GFP-RabEld is visible in
intracellular punctate structures and at the cell periphery.

Scale bar = 50pm.

63

 

Figure 2 - 7: GFP-RabEld is localized at the plasma membrane and at the Golgi
apparatus in Arabidopsis cells.

(A) Confocal microscope image Showing overlapping ﬂuorescence of GFP-RabEld and
FM4-64, at the plasma membrane. The image is a Single focal plane crossing adjacent
cells (40x oil immersion objective, 4x zoom). Left panel: GFP-RabEld ﬂuorescence,
green; center panel: FM4-64 ﬂuorescence, red; right panel: merged image, in which the
yellow color results from the overlap of red and green.

Scale bar = 10 um.

(B) Confocal microscope image showing overlapping ﬂuorescence of GFP-RabE and
RFP fused to Sialyl transferase (ST), a marker of the Golgi apparatus. The image is a
single focal plane crossing the cytoplasm of a cell (40x oil immersion objective, 4x
zoom). Left panel: GFP-RabEld ﬂuorescence, green; center panel: ST-RFP ﬂuorescence,
red; right panel: merged image, in which the yellow color results from the overlap of red
and green. The arrowheads point at some of the co—labeled Golgi stacks.

Scale bar = 10 um.

64

Endogenous RabE co-fractionates with PM and Golgi markers

GFP-RabEld expression in the transgenic plants was driven by the strong 3SS
constitutive promoter. To exclude the possibility that the observed localization of RabE
reﬂects patterns of only the overexpressed protein, we analyzed the localization of
endogenous RabE in transgenic as well as wild-type Arabidopsis plants. We performed
subcellular fractionation by centriﬁrgating clariﬁed plant extracts on sucrose step
gradients. Our ﬂotation method allowed separation of the PM from a fraction containing
lighter membranes (Golgi, tonoplast). The endogenous RabE proteins, as well as the
transgenically expressed GFP-RabEld, were detected in both fractions, with the bulk of
membrane-associated RabE in the same fraction as the PM marker H+-ATPase
(Morsomme et al., 1998). A lower amount of endogenous RabE as well as GFP-RabEld
co-fractionated with XTl , a Golgi apparatus resident protein (Faik et al., 2002; Cavalier
and Keegstra, 2006) (Fig. 2 - 8) . The tonoplast marker, yTIP, was found predominantly
in the same fraction as the Golgi marker. Overall, the membrane fractionation
experiments complemented the microscope analysis; together, they indicate that
endogenous and ectopically expressed RabE proteins are not only localized at the Golgi

apparatus, but a signiﬁcant pool is associated with the PM in Arabidopsis leaf cells.

65

/\ I3
Col/GFP-RabEtd Col-O 911

1 2 3 4 1 2 3 4
n: PM-ATPase ...L.. PM-ATPase
”I ’ x“ Q xr1
y—TIP -2
.' g’ * y-TIP
GFP-RabE1d .’
. RabE . " ' - RabE
. “

 

Figure 2 - 8: Detection of subcellular localization of endogenous RabE and
transgenically expressed GFP-RabEld by membrane fractionation technique.

Western blot of membrane fractions from (A) transgenic plants overexpressing GFP-
RabEld and (B) wild-type Arabidopsis plant extract.

Total membranes were separated by ﬂotation on a sucrose step gradient. Lanes 1 through
4 represent the 4 membrane fractions collected at the interfaces between layers of
different sucrose concentration, after ultracentrifugation: 18-25% (1), 25-34% (2), 34-
40% (3) and 40-50% (4). Equal volumes of each fraction were loaded on SDS-PAGE gel.
I’M-ATPase is a PM marker, XT l iS a trans-Golgi resident protein, and y—TIP is a marker
for the tonoplast. GFP-RabEld and endogenous RabE proteins were detected with a
P01yclonal chicken anti-RabE antibody developed for this study (K. Nomura).

66

AvrPto expression in Arabidopsis alters RabE localization at the Golgi

To investigate a possible effect of AvrPto on RabE in vivo, we produced double
transgenic plants by transforming pEGAD::RabEId into AvrPto-expressing plants
(Hauck et al., 2003). In these double transgenic plants, expression of GFP-RabEld is
under the control of CaMV 358 promoter, whereas AvrPto expression is under the
control of the DEX-inducible promoter. Several independent transgenic lines were
obtained, which produced the ﬁrsion protein, as conﬁrmed by microscope analysis and by
western blot with anti-RabE antibodies (Figure 2 - 9).

Microscopic examination revealed a striking effect of AvrPto on the subcellular
distribution of GFP-RabEld. After AvrPto induction, the level of GFP-RabEld in the
Golgi apparatus was greatly reduced and, in some experiments, was undetectable (Figure
2 - 10). This effect seems speciﬁc to the Golgi-associated pool of GFP-RabEld, as

localization of GFP-RabEld at the plasma membrane appeared unperturbed.

67

Col-0 AvrPto AV'PIO’
GFP-RabEtd

V 1%“ VII-

“ GFP-RabEtd

H

 

--'.--"-~-a.¢- ....- ..-\ H " ' . RabE

_ a AvrPto

Figure 2 - 9: Western blot indicating expression of GFP-RabEld and of AvrPto in
leaves of double-transgenic plants.

GFP-RabEld was constitutively expressed (under the CaMV 3SS promoter); AvrPto
expression was induced by Spraying the leaves with 30uM DEX, 24 hours prior to protein

extraction. Equal volumes of protein extracts, containing roughly equal amounts of
proteins, were loaded in each lane. At least three independent transgenic lines were

analyzed (not shown), Showing Similar protein expression levels.

68

 

h—g I—-I

Figure 2 - 10: AvrPto expression alters intracellular distribution of GFP-RabEld.

 

Confocal microscopic images of Arabidopsis plants co-expressing GFP-RabEld and
AvrPto. AvrPto expression was induced by Spraying plants with 30uM DEX; water was
sprayed on a different set of plants, aS a control.

(A) and (B) represent water- and DEX- treated samples, respectively. Each image is a
projection along the Z—axis of several focal planes intersecting the upper epidermis and
the palisade mesophyll cell layers. Scale bar = 50 um.

(C) and (D) represent water- and DEX- treated samples, respectively, at higher
magniﬁcation. Scale bar = 10 um.

69

To further characterize this phenomenon, we generated Arabidopsis Col-0 plants
expressing GFP-RabEld-SZ9N (under the control of CaMV 358 promoter) and AvrPto
(under the control of DEX-inducible promoter). RabEld-SZ9N failed to interact with
AvrPto in the Y2H system, as shown in Fig. 2 - 5. In the absence of inducer, the
subcellular distribution of GFP-RabE1d-SZ9N closely mirrored that of wild-type
RabEld. The GFP-RabE-SZ9N protein was partly cytosolic, as demonstrated by diffuse
ﬂuorescence and abundant cytoplasmic strands. A pool of GFP-RabE1d-SZ9N was
observed in association with membranes; the distribution was reminiscent of that of wild-
type RabE, at the cell periphery and in intracellular punctate structures (Figure 2 - 11, A).
A closer look at the peripheral localization, using FM4-64 as a PM marker, revealed that
the RabE-SZ9N mutant was also localized at the PM (Figure 2 - 11, B).

Interestingly, DEX-induction of AvrPto expression in the transgenic plant did not

affect GFP-RabE1d-SZ9N localization at the Golgi (Figure 2 - 12).

70

 

 

 

Figure 2 - 11: Subcellular distribution of the GFP-RabE1d-SZ9N protein.

(A) Confocal microscope image of a representative Arabidopsis leaf expressing GFP-
RabEld-SZ9N. Projection along the Z-axis of several focal planes crossing the epidermal
cell layer. Arrowheads point at cytoplasmic Strands, and asterisks mark ﬂuorescence in
the perinuclear region. Scale bar = 50pm.

(B) Overlapping ﬂuorescence of GFP—RabE-SZ9N and FM4-64 at the plasma membrane.
From left to right: GFP-RabE1d-SZ9N, green (left); FM4-64, red (center); merged
image, in which yellow results from the overlap of green and red (right). The image
represents a single focal plane. Scale bar = 20pm.

71

 

Figure 2 - 12: Intracellular localization of RabE-SZ9N is unaffected by AvrPto
expression.

Confocal microscope images of double transgenic plants expressing either GFP-RabEld
and AvrPto (left panels), or GFP-RabE1d-SZ9N and AvrPto (right panels). Expression of
AvrPto was induced with DEX. A 30uM DEX solution in water, or water alone (as a
control), were inﬁltrated in leaves with a needle-less syringe. Control and treated leaves
were detached and observed 24 hours after inﬁltration.

(A) and (B) represent, respectively, RabEld and RabE1d-SZ9N localization upon water
inﬁltration.

(C) and (D) represent, respectively, RabEld and RabEld-SZ9N localization upon AvrPto
induction.

All images are projections along the Z-axis of several focal planes. Scale bar = 20 um.

72

We extended our analysis of AvrPto-induced RabE relocalization to the
transgenic Arabidopsis plants expressing 6xHis-AvrPto. As shown in Figure 2 — 1, 6xHis-
AvrPto was not membrane-associated and did not exert its virulence function in
Arabidopsis. We investigated whether soluble 6xHis-AvrPto can affect RabE subcellular
localization. GFP-RabEld was transiently expressed in Arabidopsis leaves that also
expressed 6xHis-AvrPto or AvrPto (as a control). Remarkably, GFP-RabEld
ﬂuorescence at the Golgi apparatus was unaltered in the presence of 6xHis-AvrPto,
whereas the Golgi-associated ﬂuorescence was greatly reduced in the presence of
untagged AvrPto (Fig. 2 - 13). This result indicates that reduction of the Golgi-localized

RabEld pool requires AvrPto localization at the host membrane.

73

 

 

Figure 2 - 13: 6xHis-AvrPto does not affect RabE localization at the Golgi.

Confocal microscope images of Arabidopsis epidermal leaf cells transiently expressing
GFP-RabEld. A 30 1.1M DEX solution was inﬁltrated with a needle-less syringe in leaves
of Col-0 glI (A) and of transgenic Arabidopsis expressing 6xHis-AvrPto (B) or AvrPto
(C), respectively. Two to three hours after DEX inﬁltration, leaves were detached and the
pEGAD: :RabEId plasmid was delivered by particle bombardment. Transformed leaves
were observed 24 hours after bombardment.

The arrowheads indicate autoﬂuorescent chloroplasts in the mesophyll layer (beneath the
epidermal layer); the asterisks indicate autoﬂuorescence from adjacent stomatal guard
cells. Scale bar = 10m.

74

RabE overexpression reduces AvrPto virulence function in transgenic plants

In Arabidopsis protoplasts, expression of the tomato Pto protein partially relieved
the AvrPto-mediated suppression of ﬂg22 marker gene induction, likely due to AvrPto
sequestration by Pto (He at al., 2006). We investigated whether RabEld overexpression
could similarly reduce the virulence effect of AvrPto in transgenic plants. To address this
question, we examined the ability of the TTSS-defective hrpA' mutant to multiply in
AvrPto/GFP-RabEld double transgenic plants. We found that GFP-RabEld
overexpression partially restricted the multiplication of hrpA' mutant bacteria to a level
that was intermediate between those achieved on wild-type Arabidopsis and on AvrPto

single transgenic plants (Fig. 2 - 14, A).

Importantly, the increased resistance from overexpression of GFP-RabEld was
not caused by a nonspeciﬁc, broad-spectrum resistance mechanism because, when
challenged with Pst DC3000, Arabidopsis expressing GFP-RabEld remained as
susceptible as wild-type Arabidopsis (Fig. 2 - 14, B). These results therefore suggest that
RabEld overexpression speciﬁcally counteracts the virulence function of AvrPto. Pst
DC3000 secretes many effectors; therefore the unaltered susceptibility of EGFP-RabEld

transgenic plants to this bacterium reinforces the notion that one or more other effectors

produced by Pst DC3000 may be functionally redundant to AvrPto.

75

 

l Col wt
\‘ I AvrPto
. El AvrPto/RabE1 d

Bacterial count (CFUs/cmz)

 

Day 0 Day 3
109* lColwt
I RabE1d
108-1

Bacterial count (CFUs/cmz)

 

 

Day 0 Day 3

Figure 2 - 14: RabE overexpression limits AvrPto-induced bacterial multiplication.

(A) Multiplication of the TTSS-deﬁcient hrpA- mutant on wild-type Arabidopsis,
AvrPto-expressing plants and AvrPto/RabEld-expressing plants. A 0.3uM DEX solution
was sprayed on the plants 24 hours before bacterial inoculation, and then every 24 h
throughout the entire course of the experiment. Bacteria were vacuum-inﬁltrated in the
leaves at a titer of 106 CFUS/ml.

(B) Multiplication of Pst DC3000 on wild-type Arabidopsis and on RabE1d-expressing
plants. Bacteria were vacuum-inﬁltrated in the leaves at a titer of 106 CFUS/ml.

76

DISCUSSION

To successfully colonize their hosts and cause disease, bacterial pathogens of
plants and animals have evolved virulence mechanisms, such as the TTSS, that enable
them to evade or suppress host defenses and to interfere with host cellular functions.
AvrPto of Pst DC3000 is one of several bacterial TTSS effectors shown to act inside the
plant cell to suppress basal defenses (Hauck et al., 2003), nonhost-resistance-associated
cell death (Kang et al., 2004) and PAMP Signaling (He et al., 2006; Harm and Rathjen,
2007). However, the exact mechanism by which AvrPto exerts its virulence ﬁmction(s)
is not yet understood.

Bogdanove and Martin (2000) ﬁrst identiﬁed small GTPases as AvrPto
interactors in tomato, which, together with our identiﬁcation of the Arabidopsis RabE
family of small GTPases as AvrPto-interacting proteins, prompted us to conduct in-depth
cell biology experiments, described in this study. These experiments revealed a striking
AvrPto-dependent effect on the Golgi-localization of RabE1d, and showed that this effect
requires both the AvrPto localization to the host membrane and the appropriate
nucleotide-binding state of RabE1d. Furthermore, we found that overexpression of
RabE1d alone was sufficient to partially counteract the AvrPto-induced susceptibility to
nonpathogenic hrpA' mutant bacteria, without activating a nonspeciﬁc resistance to Pst
DC3000 (Figure 2 - 8). This result implicates the involvement of RabE1d in AvrPto-
induced susceptibility in Arabidopsis and Shows a ﬁrst example of a plant pathogen
effector altering subcellular localization of a host small GTPase implicated in vesicle

trafﬁc.

77

The inability of AvrPto expression to affect Golgi-localization of the RabE1d-
S29N mutant, which does not interact with AvrPto in Y2H assay (Figure 2 - 2), suggests
that AvrPto-dependent alteration of RabE1d localization requires physical interaction
between the two proteins. AvrPto was Shown to be located exclusively at the host PM in
tomato and Arabidopsis (Shan et al., 2000; He et al., 2006). In this study, we found that a
substantial pool of RabE1d is also localized at the PM. Despite the apparent co-
localization of AvrPto and RabE1d to the same host membrane, we were unable to detect
stable in planta interaction using co-irrununoprecipitation methods. It was previously
reported that in viva co-immunoprecipitation could not be accomplished in interaction
studies involving some Rab proteins (Monier and Goud, 2005). Co-immunoprecipitation
of yeast Sec4p and its effector SeclSp was only achieved by using chemical crosslinkers
to stabilize the interaction (Guo et al., 1999). In addition, AvrPto may represent a
particularly difﬁcult bait for co-immunoprecipitation experiments: even the interaction
between AvrPto and its best-known host target, the tomato Pto kinase, was demonstrated
in Y2H (Scoﬁeld et al., 1996; Tang et al., 1996) but has not yet been documented in
planta. It is possible that AvrPto interaction with its host target proteins may be
inherently transient and unstable or require special conditions. The lack of demonstrable
Stable interaction in planta, however, does not affect our conclusion that AvrPto
speciﬁcally alters Golgi-localization of wild-type RabE1d, but not of the RabE1d-SZ9N
mutant, which does not interact with AvrPto in yeast.
Previous studies showed that localization to the host PM is crucial for the

virulence and avirulence activities of AvrPto in transgenic tobacco and tomato plants

(Shan et al., 2000b), and in Arabidopsis protoplasts (He et al., 2006). It is therefore very

78

interesting to ﬁnd that N-terrninally tagged 6xHis-AvrPto, which was not targeted to the
host membrane and had no virulence function in Arabidopsis, failed to affect RabE
subcellular localization (Fig. 2 - 8). Rab proteins undergo cyclical interconversion
between the GDP- and the GTP-bound form, accompanied by shuttling between donor
and target membranes. Given AvrPto localization and its preference for GTP-RabE, our
results can be best explained by a model in which PM-localized AvrPto encounters GTP-
bound RabE1d and traps it, directly or indirectly, so that RabE1d can no longer be
recycled into the cytoplasm and back to the Golgi/ trans-Golgi network (TGN).
Alternatively, PM-localized AvrPto mediates a RabE1d modiﬁcation so that, either the
modiﬁed RabE1d cannot be extracted by host proteins to be recycled back to the
Golgi/TGN, or the modiﬁed RabE1d disrupts Golgi integrity when it reaches the Golgi
apparatus.

Rab proteins are often considered as molecular markers of speciﬁc cellular
compartments in which they function. Arabidopsis RabE1d localization at the Golgi
apparatus had been previously described (Zheng et al., 2005). In this study, we further
characterized RabE subcellular distribution and discovered that a substantial amount of
protein is associated with the PM, in addition to the Golgi, in Arabidopsis cells. Based on
its localization in the plant cell, and on the role of its yeast and mammalian homologs,
RabE is likely to ﬁmction in mediating trafﬁc of secretory vesicles between the Golgi or
TGN and the PM. Although the identity of RabE-dependent trafﬁcking cargo remains
unknown, an attractive possibility is that the RabE family of small GTPases controls
polarized secretion of some type of antimicrobial peptides/compounds or papilla

components. Interestingly, callose deposition in papillae, a hallmark response to non-

79

pathogenic bacteria, is suppressed in plants expressing AvrPto (Hauck et al., 2003).
Although callose is synthesized at the plasma membrane (Scheible and Pauly, 2004),
papillae contain enzymes and numerous other structural compounds, and their deposition
is associated with polarized vesicle trafﬁcking (Huckelhoven et al., 1999; Assaad et al.,
2004). RabE proteins may also participate in a vesicle trafﬁcking pathway that targets
PAMP receptors to the PM to establish plant basal defense. Future research is needed to
test these hypotheses and to determine whether AvrPto interferes with the RabE-mediated
vesicle trafﬁcking route to impair delivery of antimicrobials and/or PAMP receptors
during infection. It also remains to be determined whether AvrPto affects the subcellular
localization of the other four RabE family members (i.e., RabEla, b, c, and e). As shown
in Fig. 2 - 2, AvrPto interacts with four RabE GTPases, suggesting potential redundancy
among RabE family members.

The availability of sophisticated techniques for imaging live cells, such as
confocal laser scanning microscopy with ﬂuorescent tags, has opened up a whole new
realm of possibilities for investigating plant-pathogen interactions (Koh and Somerville,
2006). One of the obvious advantages is the ability to examine such interactions at the
single cell level, rather than at the global tissue scale. Cellular responses such as
movement of the nucleus, focal accumulation of secretory vesicles and other organelles at
the Site of pathogen attack, papillae deposition and reorganization of actin
microﬁlaments, have been extensively documented (Koh and Somerville, 2006). While
the overall reorganization of plant cells upon microbe infection has been known for a
long time, only recently individual plant proteins were identiﬁed whose subcellular

localization is altered in response to pathogens or pathogen-derived elicitors (Lipka and

80

Panstruga, 2005). For instance, the Arabidopsis PM syntaxin PENl focally accumulates
at the Sites of attempted penetration by Blumeria graminis, deﬁning microdomains
reminiscent of lipid rafts in animal cells (Assaad et al., 2004; Bhat et al., 2005). The PM-
localized FLSZ ﬂagellin receptor, upon perception of the ﬂ g22 peptide elicitor, rapidly
disappears from the PM and is detected in vesicle-like structures, indicating endocytosis
(Robatzek et al., 2006).

Our study provides an example in which a virulence-promoting bacterial TTSS
effector alters the subcellular localization of a host vesicle trafﬁcking regulatory protein.
Further analysis of RabE function in vesicle trafﬁc may shed light not only on P.
syringae pathogenesis and host immunity, but importantly also on the plant cellular

vesicle trafﬁc system.

81

ACKNOWLEDGEMENTS

In the ﬁrst place, I want to thank Dr. Paula Hauck and my advisor, Dr. Sheng
Yang He, for starting the study of RabE proteins. Paula identiﬁed RabE as an interactor
of AvrPto in Y2H, and Sheng Yang worked on RabE1d mutagenesis and subcloning

during his sabbatical in North Carolina.

I would also like to thank: Dr. Federica Brandizzi, for her helpful suggestions and
discussion of the microscopy data presented in this Chapter, and for providing the ST-
RFP construct; Dr. Shirley Owens and Dr. Melinda Frame, for assistance with the
confocal microscope; Dr. Jonathan Jones, for sharing the Arabidopsis cDNA libraries
used in the yeast two-hybrid analysis; Dr. Mingbo Lu, for contributing to construction of
the 6xHis-tagged version of AvrPto and generation of transgenic plants; Ms. Beth
Rzendzian for invaluable laboratory assistance and plant care. Many thanks also to Dr.
Marc Boutry, Dr. Ken Keegstra and Dr. Natasha Raikhel, for kindly sharing the

antibodies I used in this study.

82

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CHAPTER 3

Investigating RabE function in Arabidopsis

89

ABSTRACT

The virulence effector protein AvrPto, produced by the plant pathogen
Pseudomonas syringae pv. tomato strain DC3000 (Pst DC3000), interacts in the yeast
two-hybrid system with the Arabidopsis thaliana RabE family of small GTPases, putative
regulators of post-Golgi vesicle trafﬁc to the plasma membrane. When expressed in the
plant cell, AvrPto altered the subcellular distribution of RabE, while RabE
overexpression was sufﬁcient to partially counteract the virulence function of AvrPto.
These ﬁndings suggest that Pst DC3000 may use AvrPto (and possibly other effectors) to
interfere with the host vesicle trafficking system. Although the ﬁrnction of RabE
homologs in other eukaryotic organisms is well understood, a speciﬁc role of the RabE
proteins in Arabidopsis growth, development, or defense has not yet been established. To
gain insights into the biological ftmction of RabE proteins in Arabidopsis, we produced
and studied transgenic Arabidopsis plants that overexpressed either the wild-type RabE1d
protein or its mutant derivatives RabE1d-SZ9N and RabE1d-Q74L, which are expected to
be in the GTP-bound (active) or GDP-bound (inactive) state, respectively. We found that
Arabidopsis plants expressing the GTP-bound mutant RabE1d-Q74L gained a Signiﬁcant
degree of resistance to Pst DC3 000, while their growth and development are similar to
those of wild-type plants. In contrast, partial Silencing of RabE genes drastically affected
Arabidopsis leaf morphology and rosette size (suggesting a role of RabE proteins in plant

growth and development) and had a complex effect on host defense.

90

INTRODUCTION

Rab proteins are conserved regulators of vesicle trafﬁcking between membrane-
bound compartments in eukaryotic cells. They participate in vesicle formation, transport
along the cytoskeleton, tethering and fusion to the target membranes. Their functional
speciﬁcity is determined, in part, by their unique subcellular distribution (Stenmark and
Olkkonen, 2001; Zerial and McBride, 2001). The Arabidopsis thaliana genome encodes
57 Rab proteins, divided in eight subfamilies (RabA to RabH) based on sequence
Similarity (Rutherford and Moore, 2002; Vemoud et al., 2003). The RabE clade includes
ﬁve highly similar proteins, whose biological function in Arabidopsis is not well-
understood. RabE GTPases are highly Similar to a class of eukaryotic Rabs, including
yeast Sec4p and animal Rab8, extensively characterized regulators of vesicle transport
from the trans—Golgi network (TGN) to Speciﬁc regions of the plasma membrane (PM).

S. cerevisiae Sec4p is associated with the cytoplasmic side of the PM and of
secretory vesicles directed to speciﬁc regions of the PM, such as the budding Sites, and is
essential for exocytosis in yeast (Goud et al., 1988). During bud formation, Sec4p is
localized at the tip of the daughter cell (Novick and Brennwald, 1993; Walch-Solimena et
al., 1997). In addition to Sec4p, several other fungal Sec4-like proteins have been
identiﬁed and studied. CLPT] of the plant-pathogenic fungus Calletotrichum
lindemuthianum is a Sec4-like GTPase that is required for protein secretion and
pathogenesis. CLPT] can complement the S. cerevisiae sec4-8 mutant (Dumas et al.,
2001). Expression of the nucleotide-binding-deﬁcient CLPTl-N123I form in C.

lindemuthianum results in a dominant-negative phenotype, blocking secretion and leading

91

to accumulation of intracellular vesicular aggregates (Siriputthaiwan et al., 2005). CLPT]
is thus believed to contribute to fungal pathogenesis by regulating the transport of
secretory vesicles that may deliver extracellular enzymes potentially involved in
pathogenesis (Siriputthaiwan et al., 2005). Rab8 directs vesicle trafficking to the
basolateral membrane in polarized Madin-Darby Canine Kidney (MDCK) epithelial cells
(Huber et al., 1993). In addition, Rab8 promotes the polarized transport of newly
synthesized membrane proteins in ﬁbroblasts (Peranen et al., 1996) and iS involved in cell
morphogenesis (Hattula et al., 2006). In rat brain, Rab8 is a critical component of the
cellular machinery that controls both constitutive cycling and regulated delivery of a
speciﬁc type of receptors (AMPA-type glutamatergic receptors, AMPARS) into synapses
(Gerges et al., 2004).

Unlike its fungal and animal counterparts, plant RabE proteins have only recently
begun to be characterized. The RabE1d subcellular localization results presented in
Chapter 2 of this dissertation, together with previously published data (Zheng et al.,
2005), support the hypothesis that the Arabidopsis RabE proteins function in trafﬁcking
between the Golgi apparatus and the PM.

Fungal and bacterial infections, in plants, are often associated with activation (or
suppression) of extracellular defense responses, including secretion of antimicrobial
phytoalexins and formation of cell wall appositions known as papillae (Snyder and
Nicholson, 1990; Snyder et al., 1991; Brown et al., 1995; Soylu et al., 2005; Field et al.,
2006). An important role for an intact secretory pathway in plant defense against
pathogens has been demonstrated in several studies (Snyder and Nicholson, 1990; Snyder

et al., 1991; Collins et al., 2003; Assaad et al., 2004; Soylu et al., 2005; Wang et al.,

92

2005; Field et al., 2006). However, the molecular mechanisms underlying vesicle
trafﬁcking leading to defense, and the Speciﬁc cargos transported by these vesicles have
yet to be elucidated.

The physical interaction between RabE proteins and AvrPto detected in the yeast
two-hybrid system, the AvrPto-induced alteration of RabE1d subcellular localization, and
the ability of transgenically overexpressed RabE1d to partially counteract the virulence
effect of AvrPto altogether suggest that RabE may mediate intracellular transport events
important for establishing defenses against bacterial pathogens. Virulent Pst DC3000
may use AvrPto, and potentially other effectors, to interfere with RabE-dependent
trafﬁcking, therefore weakening plant defenses. In addition, RabE proteins may have a
function in basic cellular trafﬁc necessary for plant growth and development. As a ﬁrst
step toward understanding a possible role of the RabE family of proteins in plant defense,
growth, and/or development at the whole plant level, we generated transgenic plants
overexpressing wild-type RabE1d , RabE1d-Q74L (predicted to be active and
preferentially GTP-bound) or RabE1d-S29N (predicted to be inactive and preferentially
GDP-bound form), and examined these plants for morphological and developmental

phenotypes and response to pathogen infection.

93

MATERIALS AND METHODS

Transgenic plants

Generation of Arabidopsis plants expressing RabE1d and RabE1d-829N was
described in the Materials and Methods section of Chapter 2 of this dissertation. Plants
expressing GFP-RabE-Q74L were produced by subcloning the RabE1d-Q74L sequence
into the EcoRI and BamHI Sites of the binary expression vector pEGAD (Cutler et al.,
2000), in frame with the GFP sequence. The binary vector was introduced in A.
tumefaciens strain GV3 850 via triparental mating, for plant transformation. Arabidopsis
Col-0 glabraus (gll) plants were transformed using the ﬂoral dip method (Clough and
Bent, 1998). Transgenic plants were selected based on resistance to the herbicide Basta
(glufosinate). A solution containing 0.012% glufosinate (Finale concentrate, AgrEvo
Environmental Health) and 0.025% Silwet L-77 was Sprayed on 2 week-old seedlings
growing in soil. Surviving Tl plants were screened for GFP ﬂuorescence with a Zeiss
Axiophot microscope, and expression of the correct Size GFP-RabE fusion proteins was

veriﬁed by western blot.

Plant growth and bacterial multiplication assay

Arabidopsis plants were grown in soil, in grth chambers, under a 12 h dark/12
h light cycle. The light intensity was on average 100 uE m'2 sec", and the temperature
was kept constant at 20°C. Pst DC3000 bacteria were cultured in low-salt LB medium
(lOg/l Tryptone, 5g/l Yeast Extract, 5g/l NaCl), supplemented with lOOug/ml

Rifarnpicin. For plant surface inoculation, bacterial liquid cultures were incubated at

94

30°C to the mid- to late-logarithrnic phase. Bacteria were collected by centrifugation and
resuspended in sterile water with the addition of 0.05% Silwet L-77 (OSI Specialties,
Friendship, WV). Titer of the bacterial inoculum was 5x107 colony forming units
(CFUs)/ml. Arabidopsis plants growing on mesh-covered pots were submerged for a few
seconds in the bacterial suspension, to completely coat the leaves, and incubated for three
days under a tight-ﬁtting dome, to maintain high humidity. Bacteria enumeration in
leaves was conducted on day 3 post-inoculation, as previously described (Katagiri et al.,

2002)

Protein extraction and immunoblotting

Total proteins were extracted as follows: approximately 20 mg (fresh weight) of
fresh or frozen leaf tissue were ground with a pestle in a microfuge tube in the presence
of 100 pl of l X SDS-PAGE loading buffer [90 mM Tris-HCI pH 8.0, 100 mM DTT, 3%
SDS, 22.5% sucrose, l0 III/ml Protease Inhibitor Cocktail for Plant Cell Extracts (Sigma),
bromophenol blue (to saturation)]. Extracts were immediately heated at 80°C for 10
minutes and then frozen at -20°C. Before loading on gel, extracts were thawed at room
temperature and centrifuged at 20,000 X g for 2 minutes, to pellet debris. An equal
volume of each sample was used for SDS-PAGE electrophoresis. Total proteins were
separated on precast gradient gels (4-20%, ISC BioExpress), then transferred onto
Immobilon-P membrane (Millipore, MA) using a semi-dry transfer apparatus (SEMI
PHOR, Hoefer Scientiﬁc Instruments, San Francisco, CA). Protein detection was carried

with the following anti-AvrPto and anti-RabE antibodies (developed by Dr. K. Nomura).

95

Confocal microscope analysis and imaging

Pieces of leaves were sampled randomly and mounted in water. Imaging was
performed using an LSM510 META inverted confocal laser scanning microscope (Zeiss),
and either a 20 X or a 40 X oil immersion objective. For GFP-RabE ﬂuorescence
analysis, the 488 nm excitation line of an argon ion laser was used, with a 505 to 530 nm
band-pass ﬁlter, in the single-track facility of the microscope. Images were processed
with the LSM Image Browser Version 3.1 (Zeiss) and with the Adobe Photoshop
Elements Version 5.0 software (Adobe Systems Inc.). For FM4-64 staining, detached
Arabidopsis leaves were submerged in 8.2 uM FM4-64 (Molecular Probes, Leiden, The
Netherlands) in water for 15 minutes. Leaves were rinsed in distilled water and observed
immediately. For imaging GFP-RabE1d and FM4-64 ﬂuorescence, the 488 nm excitation
line was used; GFP ﬂuorescence was collected with a 505 to 530 nm band-pass ﬁlter, and

FM4-64 ﬂuorescence was collected with a 615 nm long-pass ﬁlter.

RNA extraction

Total RNA was extracted from 100 mg of Arabidopsis leaf tissue with the
RNeasy Plant Mini Kit (QUIAGEN), according to the manufacturer’s speciﬁcations.
RNA concentration in samples was determined with a NanoDrop ND-1000

Spectrophotometer (N anoDrop, Wilmington, DE).

lRT-PCR analysis

RNA reverse transcription and target gene ampliﬁcation (RT-PCR) were

performed using the RNA LA PCR Kit (AMV), Ver. 1.] (TaKaRa, Japan). Reverse

96

transcription reaction mixture was prepared according to the manufacturer’s protocol
(5mM MgClz, l X RNA PCR Buffer, 1 mM dNTP mixture, 1 unit/u] RNase Inhibitor,
0.25 units/u] AMV Reverse Transcriptase, 0.125 M Oligo-dT Adaptor Primer, RNase-
free water and 1 ug total RNA). For ampliﬁcation of the RabE and RabD transcripts, a
single RT reaction was carried in a total volume of 50 u], and incubated in a thermal
cycler for 30 minutes at 45°C, followed by 5 minutes at 99°C and 5 minutes at 5°C. Five
microliters of reverse transcribed cDNA were used as template in each of ten PCR
reactions with gene-speciﬁc primer pairs designed to amplify the ﬁve RabE gene family
members, the four RabD genes, and the Actin8 gene as a control. Primer sequences are
listed in Table 1. Each PCR reaction contained 2.5 mM MgC12, 1 X LA PCR Buffer II,
0.2 uM Forward Primer and 0.2 uM Reverse Primer, sterilized distilled water and 5 u] of
the RT reaction described above, in a ﬁnal volume of 25 u]. The reactions were placed in
a thermal cycler and ampliﬁcation was performed under the following conditions: 94°C,
2 minutes (1 cycle), 94°C, 30 seconds, 54°C, 30 seconds, 72°C, 1 minute (22 or 25
cycles), 72°C, 1 minute (1 cycle), 4°C. Ten microliters of the PCR reactions were loaded
on a 1% agarose gel. Gels were photographed with a Bio-Rad Gel Documentation

System, and band intensity was analyzed with the Quantity One software (Bio-Rad).

97

 

 

Gene Locus Forward and Reverse Primers
Actin8 (ACT8) At1g49240 F: 5'-GCTTCATCGGCCGTI’GCA1TI'C-3'

R: 5'-GATCCCGTCATGGAAACGATGTCTC-3'
AtRabD1 At391 1730 F: 5'-CTCGGAAACGCAGTCTTCAGC-3’

R: 5'-GCT1'ATTCAAGACACAGCGACATGG-3'
AtRabDZa At19021 30 F: 5'-GATCTCTGGCTCTGTATCGCTCG-B'

R: 5'-GGATATTGCTAGGCTGGTCACGTC—3'
AtRabDZb At5g47200 F: 5'-CTGAATTGACTGCCGGAGATTCC-S'

R: 5'-GATGATCGAAAGAGGAGTGGTGAC-3'
AtRabDZc At4g17530 F: 5'-CATCACCGACGAAGATCACGG-B‘

R: 5'-GCGAATTAAGAGGAGCAGCAGC-3'
AtRabE1a At3953610 F: 5'-CCGACGATCTATCTTCCCCGAGTAG-B'

R: 5'-GACAGGCGTCGTGGACCC-3'
AtRabE 1b At5g59840 F: 5'-CCAACAAGGTCTCTI'CTCITCTC-S'

R: 5'-CAACTTTGGAGCCTTT1 GGGAC—3’
AtRabE1c At3g46060 F: 5'-GTCGTCCGCCATAACC1TC-3'

R: 5'-CACTTCACCCCCAAACTTTTTTCG-3’
AtRabE1d At5903520 F: 5'-G'lTl’CTGACGATGGCGGTTGC-3'

R: 5'-CAGCAAGCTGACTTCTCGGCTG-S’
AtRabE 1e At3909900 F: 5'-GGCTGTCTCCGGCGAGAAG-3'

R: 5'-CATAGGACGATCCCTI'GAATGATGC-3'

 

Table 3 - l: Gene-speciﬁc primers for RT-PCR.

98

BTH treatment

Benzothiadiazole (BTH; Actigard) was prepared at a ﬁnal concentration of 300
M in water and Sprayed onto potted GFP-RabEld-Q74L and Col-O glI Arabidopsis
plants. A separate set of plants was sprayed with water, as a control. Plants were covered

with a tight-ﬁtting dome and assayed for responses 3 days after BTH application.

Intercellular Wash Fluid (IWF) collection and analysis

BTH-treated and control plants were harvested three days after treatment. Whole
plants were vacuum-inﬁltrated for 2 minutes with distilled water containing 0.002%
Silwet L-77 (08] Specialties, Friendship, WV). The plants were placed in conical
centrifuge tubes (Nalgene) containing a mesh septum placed about 2 cm above the
bottom. IWF was collected by centrifuging the inﬁltrated plants at 400 X g for 20
minutes, at 4°C. The IWF volume was measured with a micropipette, and the appropriate
volume of 5 X SDS-PAGE loading buffer was immediately added. Samples were heated

at 85°C for 5 minutes, then frozen or loaded on acrylarnide gel.

Callose staining

Pst DC3000 from a fresh culture plate was inoculated into liquid LB and grown to
mid-logarithmic phase. Bacteria were pelleted by centrifugation (10 minutes at 2,000 X g)
and resuspended in distilled water. A bacterial suspension of O.D.6oo= 0.4 (equivalent to
2 x 108 CFUS/ml), or water, were inﬁltrated with a needle-less syringe in leaves of
Arabidopsis Col-0 g1] and of EGFP-RabEld-Q74L plants. Six hours after inﬁltration,

leaves were collected and vacuum-inﬁltrated with 95% ethanol, followed by incubation at

99

50°C for 30 minutes. The 95% ethanol was replaced with 75% ethanol and the leaves
were incubated overnight at room temperature. Cleared leaves were rinsed in 50%
ethanol, then in water, and placed in staining solution [150 mM KzHPO4, pH 9.5, and
0.01% aniline blue (Sigma)] for about 15 minutes at room temperature. Stained leaves
were mounted in 50% glycerol on microscope slides and observed with a Zeiss Axiophot
D-7082 ﬂuorescence microscope with an excitation ﬁlter of 365 nm, a 400 nm dichroic

mirror and a 450 nm long-pass emission ﬁlter.

100

RESULTS

GFP-RabEld-Q74L displays a unique subcellular localization pattern

Rab proteins engineered by substitution of a conserved glutamine with leucine in
the PM3 catalytic domain (Figure 2 - 2) are unable of catalyzing both intrinsic and GAP-
stimulated GTP hydrolysis, but are not affected in their nucleotide-binding properties
(Stenmark et al., 1994). Rabs carrying this mutation were demonstrated to be mostly in
the GTP-bound (active) state, and often have a constitutive-active phenotype.

Transgenic expression of GFP-RabE-Q74L had no signiﬁcant effects on
Arabidopsis grth and development, other than appearance of minute sparse
indentations in mature rosette leaves, one or two weeks prior to bolting. Interestingly,
unlike wild-type GFP-RabE1d (see Chapter 2), GFP-RabEld-Q74L was not observed in
association with any intracellular punctate structures, but was exclusively found at the
cell periphery (Figure 3 - 1, A). Initial observation of the fusion protein peripheral
distribution was suggestive of a tonoplast, rather than PM localization. To conﬁrm this
suspicion, we obtained seeds of Arabidopsis transgenic lines expressing GFP ﬁrsions to a
PM-localized channel protein (line Q8) and to a tonoplast marker (line Q5) (Cutler et al.,
2000) from ABRC (Arabidopsis Biological Resource Center, Ohio State University).
Comparison of our GFP-RabE1d-Q74L localization with that of the PM and tonoplast
markers further suggested RabE1d-Q74L was not located at the PM (Figure 3 - l, B).
Staining with FM4-64 conﬁrmed that the bulk of GFP-RabE1d-Q74L ﬂuorescence was

indeed not overlapping with the PM membrane, but labelled the tonoplast (Figure 3 - 2).

101

 

 

B

Figure 3 - 1: Localization of GFP-RabE1d-Q74L in transgenic Arabidopsis.

(A) Confocal microscope image of a representative Arabidopsis leaf expressing GFP-
RabEld-Q74L. Projection along the Z-axis of several focal planes crossing the epidermal
cell layer.

 

(B) Localization pattern of GFP—RabEld-Q74L compared to a PM marker and a tonoplast
marker. From left to right: line Q8, expressing a GFP fusion to the Plasma membrane
Integral Protein PIP2A (left); line Q5, expressing a fusion to delta-TIP (Tonoplast
Integral Protein), a vacuolar membrane channel protein (center); Arabidopsis expressing
GFP-RabE1d-Q74L (right). Scale bar = 50pm.

102

II—l

CI] p m

:0 LI r n

 

Figure 3 - 2: GFP-RabE-Q74L is primarily localized in the tonoplast.

Confocal microscope image of epidermal leaf cells of Arabidopsis expressing GFP-
RabE-Q74L, indicating that GFP-RabEld—Q74L accumulates mostly in the tonoplast.
Leaves were stained with FM4-64, to visualize the PM, and immediately observed. The
image represents a single focal plane (40x oil-immersion objective).

(A) GFP ﬂuorescence;

(B) FM4-64 ﬂuorescence (the asterisks indicate autoﬂuorescence of chloroplasts in the
mesophyll layer, below the epidermis);

(C) Merged image: arrowheads point at places where the tonoplast is most clearly distinct
Item the PM. Invaginations and formation of membranous structures are typical of the
highly dynamic vacuolar membrane. Even in the areas where the PM and tonoplast are
closest, still green and red ﬂuorescence are visibly distinct.

103

RabE1d-Q74L confers resistance against Pst DC3000

Although overexpression of constitutively active GFP-RabE1d-Q74L did not
affect plant growth or development, it had a remarkable effect on plant responses to P.
syringae infection. Upon challenge with Pst DC3000, the GFP-RabE]d-Q74L-expressing
plants displayed a considerable degree of resistance, reﬂected by bacterial multiplication
being restricted 10- to 100-fold, compared to multiplication on wild-type Arabidopsis
(Figure 3 - 3, A). Visible disease symptoms, namely chlorosis and necrosis, were also

markedly reduced (Figure 3 - 3, B).

104

10’ . um
I: Q74L

105 —,

104-

Bacterial count (CFUs/cmz)

103—

1024 E

Day 0 Day 1 Day 3

 

 

Figure 3 - 3: RabE-Q74L overexpression confers resistance to Pst DC3000.

(A) Bacterial multiplication in GFP-RabE]d-Q74L-expressing plants (Q74L), compared
to that in wild-type Arabidopsis (Col). Pst DC3000 was vacuum-inﬁltrated at a density of
105 CFUs/ml.

(B) Disease symptoms 3 days after inoculation with Pst DC3000 at a density of 105

CFUS/ml. On the left, Arabidopsis Col-O glI (wild-type); on the right, Arabidopsis
expressing GFP-RabE-Q74L.

105

Up-regulation of the secretory pathway was recently demonstrated in Systemic Acquired
Resistance (SAR) (Wang et al., 2005), and it was known for a long time that SAR-
expressing plants accumulate in the apoplast secreted proteins, which include
antimicrobial polypeptides (U knes et al., 1992). Because RabE proteins are predicted to
be involved in regulating secretory vesicle trafﬁcking, the enhanced resistance to Pst
DC3000 in GFP-RabE]d-Q74L-expressing plants could be caused by constitutive
stimulation of defense-associated secretion. To test this possibility, Arabidopsis wild-type
and GFP-RabE]d-Q74L-expressing plants were sprayed with benzothiadiazole (BTH), a
synthetic activator known to trigger SAR in plants (Lawton et al., 1996), or with water, as
a control. Three days later, protein secretion in the apoplast and secretion of the
extracellular marker of plant defenses PR] (Pathogenesis Related protein 1) were
monitored. Intercellular wash ﬂuid (IWF) collected from water-treated GFP-RabE] d-
Q74L-expressing plants contained PR] and several unknown proteins that were absent
from the water-treated Arabidopsis wild-type IWF, indicating a constitutive activation of
secretary and defense pathways. BTH application resulted in Similar levels of secreted
PR] and other proteins in the apoplast, in both wild-type and transgenic plants (Figure 3 -
4). Interestingly, some protein bands were exclusively detected in the IWF of water- and
BTH-treated RabE1d-Q74L-expressing plants, but not in the BTH-treated wild-type
plants IWF. These unique extracellular proteins, associated with expression of RabE1d-
Q74L suggest that additional secretory pathways are activated in these plants, in addition

to the SAR pathway.

106

H2O BTH
Col Q74L

 

   

_ ' :«4 20kDa
_ 7 :4. i; a"

 

”—r -& PR1

Figure 3 - 4: Accumulation of extracellular proteins in plants expressing GFP-
RabE1d-Q74L

Proteins in the intercellular wash ﬂuid from wild type (Cal) and RabE1d-Q74L-
expressing plants (Q74L) were separated by SDS-PAGE. In the top panel, Coomassie
Blue-stained gel, representing total proteins; the arrowheads indicate bands which seem
to be exclusive to the Q74L plants. In the bottom panel, western blot with the anti-PR1
antibody (gift of Dr. X. Dong, Duke Univ.).

107

Another cellular event that is associated with defense against microbes and involves the
secretory pathway is deposition of callose-containing papillae. Virulent bacteria are able
to suppress papilla formation in a TTSS-dependent manner (Brown et al., 1995; Hauck et
al., 2003). Interestingly, AvrPto expression in Arabidopsis is sufﬁcient to suppress
bacteria-induced callose deposition, and this correlates with elevated susceptibility to
non-pathogenic TTSS-deﬁcient hrp' mutants (Hauck et al., 2003). When inoculated at
high density (2x108 CFUs/ml) on Arabidopsis expressing GFP-RabEld-Q74L, virulent
Pst DC3000 failed to suppress callose deposition (Figure 3 - 5) suggesting that the
transgenically expressed RabE1d-Q74L mutant could counteract the ability of Pst
DC3000 to suppress cell wall-associated defense. Absence of callose deposits in the
water-inﬁltrated leaves demonstrates that GFP-RabE1d-Q74L expression is not
promoting constitutive callose deposition. This experiment, rather, indicates that the
transgene expression is counteracting speciﬁcally Pst DC3000-mediated suppression of

callose production.

108

Col + water Col + Pst DC3000

 

Q74L + water Q74L + Pst DC3000

Figure 3 - 5: Pst DC3000 fails to suppress callose deposition in resistant RabE1d-
Q74L-expressing plants.

Callose staining results on Co] and Q74L leaves inﬁltrated with either water or Pst
DC3000. Callose deposits are visible as bright spots against the dark background. Six to
eight leaves per treatment were analyzed; the pictures represent the average callose
distribution observed.

This experiment was done twice, with comparable results.

109

RabE1d-SZ9N expression does not alter plant growth, development or disease
susceptibility

The serine/threonine to asparagine mutation in the PM] domain of Rabs (Fig. 2 -
2), which greatly increases Rab afﬁnity for GDP over that for GTP, was found to confer,
in most cases, a dominant-negative phenotype. The dominant-negative effect is often
associated with an impaired ability of the mutant Rab to be delivered to the apropriate
membrane compartment. This is often interpreted as the result of highly increased afﬁnity
of the mutant Rabs for guanine exchange factor (GEF) (Burstein et al., 1992), and
consequent sequestration of this important activating protein (Peranen et al., 1996).

As described in Chapter 2, however, the intracellular distribution of the GFP-
RabE-S29N protein was Similar to that of wild-type RabE, at both the cell periphery
(plasma membrane) and in intracellular punctate structures. GFP-RabE1d-SZ9N-
overexpressing plants were phenotypically and developmentally indistinguishable from
wild-type Arabidopsis. When surface-inoculated with Pst DC3000, GFP-RabE1d-SZ9N-
overexpressing plants exhibited a Similar degree of susceptibility as wild-type plants
(Figure 3 - 6). Taken together, these results suggest that transgenic expression of

RabE1d-S29N does not affect growth, development or disease resistance.

110

transgenic lines
Col-0 gI1 #1-3 #2-1 #24
i H " ‘ ' m» GFP-RabE1d-329N

 

   

...._'- mar-g «1 RabE

GFP-RabE1 d-829N
transgenic plants

 

 

Bacterial count (CFUs/cmz)

 

Col wt Line # 1-3 Line # 2-1 Line # 2-4

Figure 3 - 6: Pst DC3000 growth on plants overexpressing GFP-RabEld-S29N

(A) Western blot analysis, with anti-RabE primary antibody, indicating expression of the
GFP-RabEld-SZ9N fusion protein in different transgenic lines. Endogenous RabE
proteins are also detected (lower band).

(B) Bacterial population in Arabidopsis leaves 3 days after surface-inoculation with Pst
DC3000 at a density of 5x107 CFUs/ml.

111

Occurrence of RabE-silencing in transgenic plants

When creating transgenic plants, it is common to generate, in some individuals,
transgene Silencing; if gene silencing spreads from the transgene to the endogenous copy,
it is generally a post-transcriptional event, termed co-suppression (V aucheret et al.,
1998). A large percentage of GFP-RabE1d, GFP-RabEld-Q74L, and GFP-RabE1d-SZ9N
transgenic Arabidopsis plants generated in this study showed co-suppression of the
transgene and of endogenous RabE, as demonstrated by western blot analysis using a
polyclonal antibody that reacts with all RabE proteins (Figure 3 - 7). Severe reduction of
the overall endogenous RabE protein level in transgenic plants invariably correlated with
a distinct morphological phenotype. Rosette leaves developed normally for the ﬁrst 3-4
weeks (when Arabidopsis development is usually slower), the plants being
indistinguishable from wild-type. In the following two weeks, when Arabidopsis size
increases rapidly, the leaves of RabE-silenced plants did not fully elongate; midribs
remained short, while the leaf lamina continued to expand, producing a characteristic
wavy phenotype. Mature (5-6 week-old) RabE-silenced plants were signiﬁcantly smaller
than wild-type and had Short midribs and stems.

RabE-silenced plants ﬂowered at the same time as wild-type Arabidopsis, and
produced fertile seeds. The progeny of a selected Silenced line (B11) also manifested
silencing and had the same phenotype as the parental plant. Interestingly, RabE-silenced
plants Spontaneously arose also among the progeny of established GFP-RabE1d

OVCI’CXPI’CSSOTS.

112

 

Col oe sil
h-gv GFP-RabE1d

~~ -- RabE

 

Figure 3 - 7: RabE silencing severely affects plant morphology.
(A) Size and morphology of RabE1d-overexpressing plants (bottom left) and RabE-
Silenced plants (top and bottom right), compared to Arabidopsis wild type (top left).

(B) Enlarged picture of the RabE-silenced plant in (A) top right; the arrowheads point at
the wavy leafs.

(C) Western blot (with anti-RabE antibody) illustrating how RabE-silenced plants (sil)

have a considerably lower amount of endogenous RabE, compared to both wild-type
(C01) and GFP-RabEld-overexpressors (06).

113

Effect of RabE co-suppression on the expression of individual RabE genes

The RabE gene family expression proﬁle was analyzed by RT-PCR in the co-
suppressed lines, in comparison to wild type Arabidopsis. The RT—PCR demonstrated
that not all RabE gene family members are equally affected by co-suppression. RabE1d
and RabEI e were the most severely knocked-down, followed, to a lesser extent, by
RabE] b. RabEIa and RabElc showed only mild down-regulation (Figure 3 - 8). Given
the high degree of sequence similarity among small GTPases of the Arabidopsis Rab
superfarnily, we tested whether other closely related RabS were affected by Silencing. The
closest relatives of the RabE clade, in Arabidopsis, are the four RabD proteins (RabDl,
D2a, D2b and D2c). RabD was previously characterized as a regulator of the early
secretory pathway, being involved in transport from the endoplasmic reticulum to the
Golgi (Batoko et al., 2000; Zheng et al., 2005). RT—PCR revealed that the transcripts of
all four RabD genes were present at similar levels in RabE-Silenced plants and in wild-
type Arabidopsis (Figure 3 - 8). Silencing, in the transgenic plants, is therefore

speciﬁcally limited to the RabE genes, primarily RabE1d and E 1 e.

114

 

DColwt
lsilB11

 

 

 

100.00 4-. -------- - ------

.................

75.00 I“

........

50.00 «H

 

25.00 4* '*

Band intensity (% of wild-type)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0.00 . - .f .
'"s " Ira-‘- um - ...— m... m d!!! '

E1a E1 1) BIG E1d E19 01 02a D2b DZc ACT8

 

  

Figure 3 - 8: Expression of the RabE and RabD genes in RabE-silenced plants.

RT-PCR analysis of expression of the ﬁve RabE and four RabD genes in the RabE-
silenced Arabidopsis plants. Equal volumes of the PCR reactions were loaded on 1%
agarose gel. The gel was photographed with a Bio-Rad imager and the Quantity One
software was used to quantify the bands. Intensity values, normalized to those of Actin8,
are represented in the chart as percent of wild-type value.

115

RabE-silenced plants exhibit complex responses to Pst DC3000 infection and

PAMP-induced resistance

The RabE-silenced plants, although morphologically abnormal, represent an
opportunity for exploring the effect of partially down-regulating the production of more
than one RabE member on plant defense against pathogens. An interesting observation
revealed that RabE may play a positive role in establishment of PAMP-induced basal
resistance. It was previously Shown that pre-treatrnent of Arabidopsis leaves with the
conserved ﬂagellin peptide ﬂg22 could induce resistance against P. syringae, restricting
bacterial multiplication up to 100-fold (Zipfel et al., 2004). In my experiments, I
conﬁrmed that ﬂg22-induced basal resistance was associated with 100-fold reduction in
Pst DC3000 population in wild-type plants (Figure 3 - 9). However, ﬂg22 pretreatment
of RabE-Silenced plants caused a signiﬁcantly lower degree of resistance to Pst DC3000,
only about l0-fold reduction in Pst DC3000 multiplication (Figure 3 - 9). However, Pst
DC3000 multiplied on RabE-silenced plants to levels that were consistently 0- to 10-fold
lower than on wild-type Arabidopsis (across several experiments). This result suggests a
low level of basal resistance in RabE-silenced plants, possibly due to general stress.
Therefore, RabE-silenced plants seem to have two opposing phenotypes with regard to
pathogen responses: they have a reduced ability to exhibit ﬂg22-induced resistance, but,
at the same time, they seem to have a slightly elevated constitutive basal resistance

against Pst DC3000, perhaps through a distinct mechanism.

116

 

109 7

  

lwater
108 .11922
‘E
2
(0
EB 107
9,
g 106
E
“E, 105
8
m
104
1O3

Col-0 sil-RabE

Figure 3 - 9: RabE-silenced plants exhibited complex responses to bacterial infection
and PAMP-induced resistance.

The ﬂg22 peptide was inﬁltrated in the leaves with a needle-less syringe, at a
concentration of 2 pM. Water was inﬁltrated in a separate set of leaves, as a control.
Plants were inoculated with Pst DC3000 24 hours after ﬂg22 treatment. Bacteria (105
CFUs/ml) were inoculated by vacuum-inﬁltration. Bacteria in leaves were enumerated on
Day 3 post-inoculation.

117

DISCUSSION

The presence of ﬁve highly Similar RabE genes in Arabidopsis presents a
signiﬁcant challenge to investigating the function of this family of small GTPases in
plant growth, development and pathogen defense. This study focused on functional
analysis of one member of the RabE family, RabE1d. We implemented stable transgenic
expression of RabE1d, as well as its Q74L and SZ9N mutant derivatives, hoping to alter
the normal RabE-mediated vesicle trafﬁc in Arabidopsis and to observe any effects from
such perturbation on Arabidopsis growth, development and defense. Many studies have
shown that the presence of GF P (or other tags) at the N terminus of Rab proteins does not
affect their subcellular localization or function, in both plant (Ueda et al., 200]; Ueda et
al., 2004) and animal systems (Bucci et al., 2000; Mesa et al., 2001; Galperin and Sorkin,
2003), but allows Simultaneous analysis of protein function and subcellular localization.
We therefore used GFP-fused RabE1d, as well as its Q74L and SZ9N mutant derivatives,
for functional study.

We found that transgenic expression of the RabE1d-Q74L variant (expected to be
in active conformation) conferred on Arabidopsis a signiﬁcant degree of resistance to Pst
DC3 000. Based on the data gathered in this study, it is not yet possible to discern whether
this resistance is a direct effect of the mutated protein, due to enhancement of defense-
related vesicle trafﬁc, or rather an indirect effect, due to overall perturbation of cellular
vesicle trafﬁc, not necessarily associated with defense. There are numerous examples of
constitutively resistant Arabidopsis mutants, such as dnd (disease, no death), cim

(constitutive immunity) and cpr (constitutive PR-expression), which display upregulated

118

defenses against P. syringae and other pathogens. Common characteristics of these
mutants include accumulating high levels of salicylic acid and PR proteins, and being
signiﬁcantly dwarfed, or otherwise morphologically altered, compared to wild type plants
(Bowling et al., 1994; Clarke et al., 1998; Yu et al., 1998; Li et al., 200]; Maleck et al.,
2002). In this respect, the RabE1d-Q74L-expressing plants may be particularly
interesting and unique, because they exhibit a high level of resistance to P. syringae
without being adversely affected in overall plant Size or leaf shape. However, more
experiments will be needed to further characterize growth, development and pathogen
resistance in these transgenic plants.

In addition to enhanced resistance, the GFP-RabE1d-Q74L transgenic plants
displayed unexpected localization of the fusion protein at the vacuolar membrane
(tonoplast). The peculiar GFP-RabE1d-Q74L localization pattern could be interpreted as
passive ﬂow of the protein from the PM to the tonoplast via endocytosis. According to
the general Rab recycling model, after its synthesis, RabE is supposed to be delivered to
the donor compartment (e.g., the Golgi apparatus) in which it ﬁrnctions, and loaded with
GDP. GDP exchange with GTP activates the protein, promoting interaction with the
downstream machinery needed for vesicle targeting and fusion with the target membrane
(e. g., the PM). Once vesicle delivery is completed, Rabs are inactivated by GTP
hydrolysis. A GDP dissociation inhibitor (GDI) extracts GDP-bound (but not GTP-
bound) Rab from the membrane and escorts it through the cytosol back to the donor
compartment, for a new transport cycle. It is possible that RabE-Q74L, upon reaching the
PM, cannot be extracted by GDI because it remains in its GTP-bound state. A default

non-speciﬁc endocytosis pathway such as that observed for the FM4-64 dye (Ueda et al.,

119

2001) could carry the RabE-Q74L protein to the tonoplast, where it accumulates. If this is
the case, a more accurate microscopic analysis may reveal a low transient pool of GFP-
RabEld-Q74L at the PM and Golgi apparatus, which was not detectable during my
routine observation. Alternatively, co-expression with AvrPto may be useful: as
described in Chapter 2, AvrPto interacts with wild-type RabE or RabE-Q74L in Y2H.
Transgenic expression of AvrPto in planta results in GFP-RabE mislocalization, which
we interpreted as AvrPto “trapping” active RabE proteins at at the PM. Based on this
model, AvrPto may similarly “trap” the GFP-RabEld-Q74L protein at the PM, resulting
in increased PM and reduced tonoplast level of the ﬂuorescent protein. Further
experiments are needed to examine this possibility.

In contrast to the GFP-RabEld-Q74L transgenic plants, Arabidopsis plants
expressing RabE1d-$29N (expected to be in inactive conformation) were Similar to wild-
type plants in their morphology and pathogen response. Furthermore, the subcellular
localization pattern of GFP-RabEld-SZ9N was Similar to that of GFP-RabE1d (as shown
in Chapter 2). These results could be explained in at least two ways. The ﬁrst possibility
is that GFP-RabEld-SZ9N, in Arabidopsis, has a dominant-negative effect an
endogenous RabE1d protein (and possibly other RabE members) but this RabE protein is
not necessary for plant development or defense. The second possibility is that GFP-
RabEld-SZ9N is not exerting a dominant-negative effect. We could not distinguish
between these two possibilities, although the remarkable effect of RabE downregulation
on plant size and morphology (seen in RabE-Silenced plants) seems to argue against the

ﬁrst hypothesis.

120

Transgene-mediated RabE co-suppression correlated with Signiﬁcant changes in
plant Size and leaf morphology, and with complex defense phenotypes. Co-suppression
affected not only RabE1d, but also other RabE genes (Figure 3 - 8). Therefore, the
multiple phenotypes of RabE-silenced plants are likely caused by simultaneous silencing
of more than one RabE gene. Consistent with this speculation, individual knock-out
mutants of RabE1d and RabE] b, two of the most severely silenced genes, did not exhibit
any defect in grth and development, nor in disease susceptibility to Pst DC3000 (data
not shown). Unfortunately, a T-DNA insertion upstream of the RabE I e gene did not
affect gene expression, leaving thus open the possibility that RabE] e downregulation
may alone be responsible for the RabE1d-silenced plants phenotypes. AS loss-of-
function mutants provide a powerful tool in the study of gene functions, additional
analysis of RabE] e, RabE] a, and RabEIC single mutants and various combinations of
multiple RabE gene mutations is needed to further assess the biological function(s) of the
RabE family of GTPases in Arabidopsis.

Pathogenesis assays with and without ﬂg22 pre-treatment unveiled a complex
overlap of two apparently opposing defense responses in the RabE-silenced plants. The
origin of the low but reproducible constitutive resistance exhibited by RabE-silenced
plants is unclear; resistance could be caused by an indirect effect of long-term
perturbation of the RabE1d-mediated traffic, resulting in nonspeciﬁc general stress on
plants. On the other hand, ﬂg22-induced resistance was reduced in the RabE-Silenced
plants, suggesting that RabE may be involved in regulating trafﬁcking events that
mediate PAMP-triggered cellular responses. In the future, inducible RNAi-mediated

RabE gene silencing may circumvent the low constitutive resistance observed in stable

121

RabE-silenced transgenic plants, enabling a more deﬁnitive evaluation of the apparently

positive role of RabE proteins in PAMP-triggered plant resistance.

ACKNOWLEDGEMENTS

Dr. Federica Brandizzi and Marika Rossi gave me invaluable help with the
acquisition and interpretation of microscopic data. I also want to thank Dr. Melinda
F rame for assistance with the confocal microscope, Dr. Xinnian Dong for sharing the

anti-PR1 antibody, and MS. Beth Rzendzian for helping me with plant care.

122

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CHAPTER 4

Conclusions and future perspectives

127

A very signiﬁcant amount of crops are lost every year to pathogens worldwide
(Baker et al., 1997; Strange and Scott, 2005). In order to devise effective strategies for
preventing such losses, it is critical that we deepen our understanding of the molecular
and cellular bases of plant-pathogen interactions. Gram-negative plant pathogenic
bacteria use the highly conserved type III secretion system (TTSS) to deliver virulence-
mediating proteins inside the plant cell. Bacterial virulence effectors are apparently not
generic bullets aimlessly shot in the host cell cytoplasm through the TTSS. Rather, they
are precisely targeted at those host cellular processes the pathogen needs to manipulate to
its own advantage (Grant et al., 2006). One of the major current endeavors in the ﬁeld of
plant pathology is that of understanding individual effector functions. Importantly,
identifying the host targets of pathogen effectors and studying their role in the host cell is
often a way to gain insight on previously uncharacterized plant cell functions.

In the absence of a circulatory system and of Specialized cell types, such as those
found in animals, plants ﬁght their battle against microbial invaders at the level of each
single cell in contact with a potential pathogen. Every plant cell can be envisioned as a
battleground, Where pathogens deploy their virulence factors to interfere with Speciﬁc
host targets. It was known for a long time that the plant cell responds to pathogen attack
with cytoskeleton and organelle rearrangements, secretion of antimicrobial compounds
and peptides, papilla deposition and more (Lipka and Panstruga, 2005; Field et al., 2006).
Most of the knowledge in this ﬁeld was obtained by studying plant cell defense responses
against fungal pathogens. The recent application of sophisticated microscopy techniques
to the study of plant pathology allows us to take a closer look at the fascinating

interaction between plants and pathogens at the cellular and subcellular level (Koh and

128

Somerville, 2006). The use of live cell imaging techniques for investigating the effect of
disease-promoting virulence factors of bacterial pathogens on the plant cell is a relatively

new and unexplored ﬁeld.

My dissertation work focused on investigating intracellular localization and
function of the poorly characterized Arabidopsis RabE small GTPases, which were
previously found to interact in yeast two-hybrid with the Pseudomonas syringae pv.
tomato (Pst) DC3000 effector AvrPto. I speciﬁcally pursued analysis of RabE1d, one of
the ﬁve highly similar Arabidopsis RabE GTPases, and therefore, the following
conclusions apply to RabEld, and more investigation will be necessary to ﬁnd out
whether they can be extended to other members of the family.

The ﬁrst part of this study demonstrated that transgenically expressed GFP-
RabEld and endogenous RabE proteins are localized at the Golgi apparatus and at the
plasma membrane (PM) in Arabidopsis cells. Analysis of GFP-RabE1d subcellular
localization in the presence of AvrPto revealed a novel and interesting phenomenon.
AvrPto expression in planta induced a remarkable change in GFP-RabE1d intracellular
distribution, which was dependent on AvrPto membrane-localization and on RabE1d
nucleotide-binding state. The Golgi-localized pool of RabEld, but not of GDP-bound
RabE1d-S29N, was greatly reduced in the presence of membrane-associated, but not
soluble, AvrPto. Furthermore, overexpression of RabE1d proved to be sufﬁcient to
speciﬁcally counteract AvrPto virulence function. These results, altogether, revealed a
novel connection between the virulence function of the bacterial effector AvrPto and the

subcellular distribution of RabE, a putative regulator of plant intracellular vesicle

129

trafﬁcking. In the future, it will be interesting to perform more in—depth microscopic
analysis, to understand whether AvrPto is speciﬁcally impairing RabE localization at the
Golgi, without affecting overall Golgi integrity, or whether the Golgi itself (or part of it)
is disassembled as a consequence of AvrPto virulence function.

The second part of my dissertation focused on investigating the biological
function of RabE in Arabidopsis growth, development and response to bacterial
pathogens. A major obstacle toward this functional analysis was represented by the
presence of ﬁve highly similar RabE genes in Arabidopsis. I chose to begin the analysis
by exploring the effect of constitutively overexpressing RabE1d and its mutant
derivatives Q74L and S29N in stable transgenic plants. Overexpression of RabE1d-Q74L
resulted in particular interesting phenotypes. The mutant protein targeted GFP to the
tonoplast, a novel and unexpected subcellular localization and, most importantly, the
transgenic plants manifested a notable resistance to P. syringae infection, which
correlated with constitutive activation of defense and secretion pathways and with callose
deposition that was not suppressed by the pathogen. Remarkably, these transgenic plants
were not negatively affected in their growth and development, Whereas the vast majority
of all known constitutively resistant Arabidopsis mutants are dwarfed or otherwise
morphologically altered.

Future work will be necessary to further characterize the actual mechanism
underlying RabE] d-Q74L-mediated defense responses in these plants. It is known that
activation of SAR is accompanied by accelerated secretion of certain PR proteins (Wang
et al., 2005). However, I detected the presence of several unique extracellular protein

bands in the intercellular wash ﬂuid (IWF) of BTH-treated RabEld-Q74L-expressing

130

plants, but not in the IWF of BTH-treated wild-type plants (Figure 3 - 4), suggesting that
additional secretory pathways (perhaps Speciﬁc to RabE) are activated in these plants, in
addition to the SAR-associated pathway. To unequivocally assign RabE a role in
trafﬁcking and in defense, identiﬁcation of a marker (protein or compound) that is
transported or secreted in a RabE-dependent fashion is absolutely critical. One possible
approach toward this goal would be that of determining the identity of those proteins,
recovered in the IWF of plants expressing RabE1d-Q74L, which appeared to be unique to
the transgenic plants (Figure 3 - 4). Also, my current results were obtained from
constitutive expression of RabE1d-Q74L. AS long-term overexpression may be more
likely to activate SAR than short-terrn gene expression, it Will be of interest in the future
to produce transgenic plants that conditionally express the RabE1d-Q74L transgene (e. g.,
dexamethasone-inducible) to separate SAR-dependent protein secretion from a possible
RabE-speciﬁc secretory process.

In the process of selecting transgenic plants overexpressing RabE1d or its mutant
derivatives, I noticed a Signiﬁcant number of primary transforrnants with short, curly
leaves, smaller than their sibling plants. Molecular characterization of these individuals
revealed that they were RabE-cosuppressed plants. The overall level of RabE proteins in
these plants is considerably lower than in wild-type Arabidopsis, due to partial silencing
of the RabE1d, E1 e and E1b genes, primarily. The pathogenesis assays performed on
these plants revealed a complex overlay of distinct defense responses, including a low
basal level of constitutive resistance, and an apparent impairment in PAMP-induced
defenses. More work is needed to dissect these responses. Speciﬁcally, in-depth analysis

of individual RabE gene knock-out mutants, and of different combinations of individual

13]

 

mutations (obtained by crosses), may shed light on the contribution of different RabE
genes to the plant’s response to pathogens. Moreover, the low basal level of constitutive
resistance could be caused by long-term Silencing of multiple RabE genes. If so,
chemically inducible RNAi-mediated silencing of RabE genes could be used to attempt
uncoupling the basal resistance observed in silenced-RabE plants from the possible defect

in PAMP-triggered defense responses.

132

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133

   

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