2 Virulence Strategies of Plant Pathogenic Bacteria - Springer Link

2 Virulence Strategies of Plant Pathogenic Bacteria Maeli Melotto1 . Barbara N. Kunkel2 1 Department of Biology, University of Texas, Arlington, TX, USA 2 Department of Biology, Washington University, St. Louis, MO, USA

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 The Biology of Bacterial Plant Pathogens . . . . . . . . . . . . . . . . . . 62 Virulence Strategies in the Early Stages of Infection . . . . . . . 62 Extracellular Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 Plant Cell Wall Degrading Enzymes . . . . . . . . . . . . . . . . . . . . . . . . 66 Production of Low-Molecular-Weight Phytotoxins . . . . . . . . . 66 Lipodepsipeptide Toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 Modified Peptide Toxins with Antimetabolite Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 Coronatine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 Syringolin Group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 Type III Secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 Structure and Components of TTSS of Bacterial Plant Pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 Identification of Type III Effectors . . . . . . . . . . . . . . . . . . . . . . . 69 Elucidating the Function of Type III Effectors . . . . . . . . . . . 70 Modulation of Plant Hormone Homeostasis and Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 Salicylic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 Jasmonates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 Ethylene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 Abscisic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 Auxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 The Complexities of Hormone Signaling Networks in Plant-Microbe Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 Challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

Abstract Plant pathogenic bacteria have evolved several unique virulence strategies to successfully infect their hosts. One current area of intense research in the field of plant-pathogen interactions is the identification and characterization of pathogen virulence factors and the elucidation of their mode of action within the host. This chapter summarizes recent progress in this area of research, focusing on four Gram-negative bacterial pathogens that grow

on living tissue and cause primarily leaf spotting or wilt diseases of plants: Pseudomonas syringae, Xanthomonas campestris, Ralstonia solanacearum, and Erwinia amylovora, the causal agents of leaf spots, leaf blights, vascular wilts, and fire blights, respectively. The focus is on these pathogens because significant progress has been made in recent years toward elucidating the molecular mechanisms underlying their virulence. The recently available genome sequence data of various strains of several of these pathogens have also begun to provide additional insight into their virulence strategies. Further, because several of these pathogens can infect Arabidopsis thaliana, use of molecular and genetic approaches to investigate the mode of action of pathogen virulence factors within this host has significantly contributed to our understanding of the virulence strategies of these plant pathogenic bacteria.

Introduction Plant pathogenic bacteria, like bacterial pathogens that infect animals, must be able to evade or suppress general antimicrobial defenses and acquire nutrients and water from their hosts to successfully colonize and grow within host tissue. Plant pathogenic bacteria have adapted well to their hosts, which are structurally and physiologically quite different from animals. Since successful infection relies to a great extent on the ability of a pathogen to modulate the physiology of its host, plant pathogenic bacteria have evolved several unique virulence strategies in addition to virulence mechanisms also utilized by bacterial pathogens of animals. One current area of intense research in the field of plantpathogen interactions is the identification and characterization of pathogen virulence factors and the elucidation of their mode of action within the host. This chapter summarizes recent progress in this area of research, focusing on four gram-negative bacterial pathogens that grow on living tissue and cause primarily leaf spotting or wilt diseases of plants: Pseudomonas syringae, Xanthomonas campestris, Ralstonia solanacearum, and Erwinia amylovora, the causal agents of leaf spots, leaf blights, vascular wilts, and fire blights, respectively (Schroth et al. 1981; Chan and Goodwin 1999; Eastgate 2000; Genin and Boucher 2002). The focus is on these pathogens because significant progress has been made toward elucidating the molecular mechanisms underlying their virulence in recent years (Staskawicz et al. 2001; da Silva et al. 2002; Salanoubat et al. 2002; Buell et al. 2003; Buttner and Bonas 2003; Angot et al. 2006; Bocsanczy et al. 2008; Boch and

E. Rosenberg et al. (eds.), The Prokaryotes – Prokaryotic Physiology and Biochemistry, DOI 10.1007/978-3-642-30141-4_62, # Springer-Verlag Berlin Heidelberg 2013

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Virulence Strategies of Plant Pathogenic Bacteria

Bonas 2010). The recent genome sequence data made available for various strains of several of these pathogens have also begun to provide additional insight into their virulence strategies (da Silva et al. 2002; Salanoubat et al. 2002; Buell et al. 2003; Feil et al. 2005; Joardar et al. 2005; Smits et al. 2010; for a complete list of bacterial genomes refer to the National Center for Biotechnology Information database at http://www.ncbi.nlm.nih.gov/). Further, as several of these pathogens can infect Arabidopsis thaliana, a widely studied model plant, use of molecular and genetic approaches to investigate the mode of action of pathogen virulence factors within this host has significantly contributed to our understanding of the virulence strategies of these plant pathogenic bacteria (Quirino and Bent 2003; Koornneef and Meinke 2010; Nishimura and Dangl 2010). For reviews on several other fascinating groups of plant-associated microbes, the tumor-inducing Agrobacterium spp., the soft-rot Erwinia species, and the rootnodulating Rhizobia, refer to several recent articles (Gelvin 2003; Toth et al. 2003; Deakin and Broughton 2009; Mallick et al. 2010; Pitzschke and Hirt 2010; Billing 2011).

The Biology of Bacterial Plant Pathogens Phytopathogenic bacteria colonize all plant tissues. Although bacteria can live in the phyllosphere or rhizosphere without causing any harm to the plant, to become a fully virulent pathogen, they must penetrate plant tissues. In many cases, bacterial penetration is a passive process, in which bacteria take advantage of wounds and openings in the plant body or they can be directly deposited by insect vectors. Once inside the plant tissues, different species can inhabit the dead xylem vessels or live in phloem sieve elements; however, the majority of bacterial pathogens are limited to the intercellular space, that is, apoplast (Beattie and Lindow 1994; Alfano and Collmer 1996; Agrios 2005). Although this view may be changing, in general, the apoplast is considered to be a nutrient-poor, unfavorable environment for most microorganisms, containing several antimicrobial compounds (Dangl and Jones 2001; Dixon 2001; Glazebrook 2001; Pignocchi and Foyer 2003; Rico and Preston 2008). Additionally, plant defenses that are induced upon microbial attack are often targeted to the intercellular space (Wang et al. 2005; Bednarek et al. 2010; Wang and Dong 2011). Given the physiology of plants and the nature of the antimicrobial defense responses they deploy, plant bacterial pathogens have evolved a variety of specialized virulence strategies to facilitate colonization of plant tissue. The achievement of this goal relies to a great extent on the ability of plant pathogens to modulate host physiology. As these pathogens are extracellular, they deploy an arsenal of secreted virulence factors to modulate host cell processes from outside plant cells. These include production of protein virulence factors (or effectors) that are delivered directly into the plant cell cytosol via a specialized, type III secretion system (TTSS; Jin et al. 2003a; Block and Alfano 2011; Lindeberg et al. 2012) illustrated in > Fig. 2.1; cell-cell communication through quorum sensing (Quinõnes et al. 2005; Chatterjee et al. 2007; Barnard and Salmond 2007); production

of low-molecular-weight phytotoxins that are secreted into the apoplast (Bender et al. 1999); exopolysaccharides; and cell wall degrading enzymes. These virulence factors allow the bacteria to evade, overcome, or suppress antimicrobial host defenses and elicit the release of nutrients and water from plant cells to ensure successful colonization of the plant apoplast (> Fig. 2.2). Additionally, some plant pathogens may directly modulate hormone signaling within their hosts through the production of plant hormone analogs (Melotto et al. 2008a; Spoel and Dong 2009; Lee et al. 2009; Grant and Jones 2009; Robert-Seilaniantz et al. 2011; Kazan and Manners 2012). Plant pathogens also express genes believed to help them adapt to the stressful conditions that are constitutively present or that are generated by the host in response to microbial attack. These include the production of proteins and enzymes to counter oxidative stress (e.g., glutathione S-transferase, superoxide dismutase, and catalase), as well as enzymes that may detoxify antimicrobial compounds (Boch et al. 2002; Salanoubat et al. 2002; Buell et al. 2003). Recent studies assessing in planta changes in the transcriptome of X. oryzae pv. oryzicola revealed regulation of genes encoding proteins involved in secretion and transport, tissue adherence, cell wall degradation, and virulence (Soto-Sua´rez et al. 2010). Interestingly, plant-inducible genes in plant pathogens, such as X. oryzae, and plant growth-promoting rhizobacteria, such as P. fluorescens SBW25, fall into similar categories. For instance, P. fluorescens has a functional type III secretion system expressed in the sugar beet rhizosphere (Preston et al. 2001). However, plant-inducible genes in P. fluorescence may be strain-specific indicating that diverse ecological adaptation exists in the pseudomonad group and offering a unique opportunity to address questions about the evolution of plant-microbe interactions (Silby et al. 2009, 2011). In the majority of pathogenic interactions, disease symptoms ensue only after the pathogen has colonized and grown to high levels in the infected tissue. In many cases, disease symptom production is believed to facilitate pathogen release from infected tissue and spread to uninfected tissues and neighboring plants (Agrios 2005). Therefore, the elicitation of disease symptoms is also often considered an important virulence strategy.

Virulence Strategies in the Early Stages of Infection The phyllosphere may be a very harsh environment for pathogens. The leaf surface, in particular, is regularly exposed to extreme conditions such as lack of moisture, ultraviolet irradiation, strong winds, and heat. Nonetheless, bacteria, the most abundant organism on the leaf surface (Lindow and Brandl 2003) can reach high population density (106–107 cells/cm2 of leaf; Andrews and Harris 2000). Pathogens arriving to the leaf surface may have to either quickly penetrate the leaf or express traits that ensure survival until environmental conditions are favorable to penetration. To gain entry into the leaf, bacterial pathogens need to overcome the plants’ active defense against penetration, for example, the closure of the stomatal pore as part


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. Fig. 2.1 Activities of bacterial type three secretion effector proteins injected into the host cell. As extracellular pathogens, bacterial pathogens deploy a variety of secreted virulence factors to modulate the biology of their host cells. These factors include protein virulence factors (or ‘‘effectors’’—labeled with an E in the diagram) that are delivered directly into the plant cell cytosol via the specialized type III secretion system (TTSS). Type III-delivered effectors interact with their targets (labeled with a T) in the host cell and modulate host cell physiology by either inhibiting plant immunity components (currently identified components are depicted in the light green box to the right) or altering hormone homeostasis or signaling. Additionally, some effectors enter the nucleus and function as transcription factors (e.g., TAL-like effectors) upregulating the transcription of susceptibility genes. Ultimately, the collective activity of the effectors promotes pathogen growth and disease symptom development. Disease symptoms facilitate pathogen release from infected tissue, and hence pathogen transmission

of their innate immune system (Melotto et al. 2006; Ali et al. 2007; Zhang et al. 2008; Gudesblat et al. 2009). Certain bacterial pathogens such as X. campestris pv. campestris (Gudesblat et al. 2009), P. syringae pv. syringae (Pss) B728a (Schellenberg et al. 2010), and P. syringae pvs. tabaci, tomato, and maculicola (Melotto et al. 2006) produce phytotoxins that promote pathogenesis by overcoming stomatal immunity as discussed below. Survival as epiphytes may also be considered as a virulence strategy. The mechanism by which bacterial pathogens avoid and/or tolerate stress in the phyllosphere is poorly understood. In an attempt to uncover metabolic activities carried out by bacterial cells when they come in contact with the leaf surface,

Marco et al. (2005) assessed the expression of plant-inducible genes using the pathosystem P. syringae pv. syringae B728a and common bean. This study revealed that genes involved in virulence, transcription regulation, transport, and nutrient acquisition are upregulated in epiphytic bacterial populations. In addition, genes of unknown function were also regulated in epiphytes, suggesting new virulence-associated cellular functions yet to be discovered. Recent studies have revealed that bacterial cell-cell communication through diffusible N-acyl homoserine lactones (AHL), which are quorum sensing signals, occurs in epiphytic bacterial aggregates and controls epiphytic fitness, exopolysaccharide

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. Fig. 2.2 Virulence factors secreted by bacterial plant pathogens. Extracellular bacterial phytopathogens colonize the leaf surface and the intercellular space (i.e., apoplast). These organisms deploy an arsenal of secreted virulence factors to modulate host cell processes from outside plant cells. These factors include (1) a type III secretion system (depicted in > Fig. 2.1), (2) extracellular polysaccharides (EPS) and quorum sensing (QS) signals that may increase pathogen fitness and survival as epiphytes, (3) cell wall degrading enzymes (CWDE) that are secreted through a sec-dependent type II secretion system (Alfano and Collmer 1996; Sandkvist 2001) and function to degrade or remodel the plant cell wall, and (4) low molecular weight toxins (labeled with T) that are secreted into the apoplast, many of which presumably enter or are taken up by plant cells (indicated by the questions mark in the diagram). At least two phytotoxins (coronatine and syringolin A) target stomatal immunity (inhibit stomatal closure) and thus facilitate pathogen entry into the apoplast. In addition, coronatine, a mimic of the plant hormone JA-Ile, is perceived by the COI1/JAZ receptor complex (labeled with R) activating jasmonate signaling and leading to suppression of SA-mediated defenses. Some phytopathogenic bacteria produce syringomycins and syringopeptins that form pores (labeled with a P) in the host plasma membrane. Overall, the activities of these virulence factors may promote pathogen growth through suppression of host defenses, modulation of host cell physiology, and release of nutrients and water into the apoplast. Additionally, they may contribute to disease symptom development such as chlorosis and necrosis, thus facilitating pathogen release from infected tissue


production, motility, and virulence of Pseudomonas syringae pv. syringae (Quinõnes et al. 2005; Dulla and Lindow 2009). Quorum sensing signaling also controls virulence determinants in soft-rot Erwinia species including production of extracellular enzymes and exopolysaccharides (EPS; discussed below), tolerance to free oxygen radicals, and subsequent symptom development in the host plant (Molina et al. 2005; Barnard and Salmond 2007). The regulatory system Phc (phenotype conversion) controls virulence factors of Ralstonia solanacearum in a populationdependent manner in the transition from soil to parasitic lifestyle (reviewed by von Bodman et al. 2003). Interestingly, the types of traits that are controlled by QS signaling are different in various bacterial species and seem to be correlated with their lifestyle.

Extracellular Polysaccharides Many plant pathogens produce large amounts of exopolysaccharides (EPSs). EPSs are carbohydrate polymers that are secreted by bacteria and form either a closely attached capsule layer surrounding the bacterial cell or a loosely associated extracellular slime (Denny 1995). The virulence of several phytopathogenic bacteria, including R. solanacearum, E. amylovora, X. campestris, and P. syringae, is associated with their ability to produce various EPS polymers during growth in plant tissue (Denny 1995). EPSs are believed to provide a selective advantage to phytopathogenic bacteria through multiple functions including (1) facilitating absorption of water, minerals and nutrients; (2) providing protection from abiotic stresses encountered during epiphytic or saprophytic growth, as well as from toxic molecules encountered during growth in plant tissue; (3) promoting colonization and spread within host tissue; and (4) contributing to the production of disease symptoms such as watersoaking and wilting (Denny 1995). One of the most important virulence-associated characteristics of the wilt pathogen R. solanacearum is the ability to produce large amounts of a viscous, high-molecular-mass, acidic EPS (EPS1) in planta. Production of large amounts of EPS1 by bacteria colonizing vascular tissue appears to interfere with transduction of water and nutrients within infected plants, resulting in wilting and, in some cases, the ultimate death of aerial portions of the plant (Denny and Baek 1991; Kao et al. 1992). Consistent with these observations, infection with R. solanacearum strains bearing mutations in the EPS1 biosynthetic loci resulted in reduced wilting (Denny and Baek 1991). A study involving detailed microscopic analysis of the infection process revealed that EPS1-deficient mutants of R. solanacearum are less invasive than wild-type strains, suggesting that EPS1 may also be required for efficient colonization and movement within plant roots (Saile et al. 1997; Araud-Razou et al. 1998). Further, the accumulation of electron-dense material in plant tissue infected with eps1 mutants raises the possibility that these mutants elicit nonspecific defenses within the host. Thus, EPS1 may also contribute to pathogen virulence by evading or suppressing host defenses (Araud-Razou et al. 1998).

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Erwinia amylovora, well known as the causal agent of fire blight of pear, produces two major EPSs, levan and amylovoran, that may contribute to this pathogen’s ability to also cause wilting diseases on young plants (Denny 1995). However, only amylovoran, a viscous, acidic heteropolysaccharide containing primarily galactose and glucuronic acid (Eastgate 2000), has been clearly demonstrated to contribute to virulence of E. amylovora, and amylovoran-negative mutants exhibit reduced in planta bacterial growth and symptom development (Bellemann and Geider 1992; Bernhard et al. 1993). Amylovoran is proposed to promote virulence by suppressing pathogen recognition by the host (Metzger et al. 1994), promoting tissue invasion and causing water-soaking and tissue collapse (Eastgate 2000). Xanthomonas campestris strains produce large amounts of the EPS known as xanthan gum that can accumulate to very high levels in infected plant tissues (Denny 1995). Xanthan gum is a high-molecular-weight EPS composed of a cellulose backbone to which trisaccharide side chains are attached. Xanthan exhibits several unique properties in solution that have rendered it useful in industrial applications (Becker et al. 1998). However, despite being one of the best studied polysaccharides produced by phytopathogenic bacteria, the role of xanthan in pathogenesis is not understood. Xanthan clearly contributes to pathogen aggressiveness, as X. campestris strains carrying mutations that specifically disrupt EPS production exhibit reduced virulence (Katzen et al. 1998). It has been proposed that xanthan contributes to X. campestris fitness by providing protection against desiccation and hydrophobic molecules and through facilitating tissue colonization by promoting adhesion of bacteria to biological surfaces (Chan and Goodwin 1999). The recent discovery that xanthan is involved in formation of aggregates of X. campestris pv. campestris in culture suggests that this EPS may be involved in biofilm formation (Dow et al. 2003). Biofilm formation may be important during early stages of tissue colonization, for example, by promoting epiphytic survival or by providing protection against antimicrobial compounds encountered within plant tissues. Interestingly, dispersal of bacteria from such a biofilm at later stages of infection may be required to facilitate colonization of the vascular system (Dow et al. 2003). The major EPS produced by P. syringae pvs. phaseolicola, lachrymans, and tomato growing in planta is alginate, a copolymer of O-acetylated b-1,4-linked D-mannuronic acid and its C-5 epimer, L-glucuronic acid (Osman et al. 1986; Fett and Dunn 1989). Studies have associated P. syringae virulence with the amount of alginate produced in culture (Osman et al. 1986; Denny 1995). A P. syringae pv. syringae alginate lyase (algL) mutant impaired in alginate production exhibited reduced epiphytic fitness, grew to lower levels in plant tissue, and elicited reduced disease symptoms on bean leaves (Yu et al. 1999). However, lack of alginate synthesis by P. syringae pv. glycinea could not be associated with reduction of in planta bacterial multiplication (Schenk et al. 2008). These findings indicate that production of alginate and its role in increased

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epiphytic fitness and pathogen virulence may be pathovar specific. Although the role of alginate in promoting virulence of P. syringae is not fully understood, alginate contributes to the virulence of the human pathogen P. aeruginosa by forming biofilms and providing protection from host defenses and antibiotic treatment (Boyd and Chakrabarty 1995; Franklin et al. 2011).

Plant Cell Wall Degrading Enzymes Plant cell walls also play an important role in plant-pathogen interactions. Unlike animal cells, plant cells are surrounded by a semirigid cell wall that provides structural support, maintains cell shape, cements adjacent plant cells together, and serves as a barrier to pathogen invasion and spread within infected tissue. Plant cell walls are composed of several complex carbohydrate polymers, the most abundant of which are cellulose, hemicelluloses, and pectin (Carpita and McCann 2000; Pauly and Keegstra 2010). As extracellular pathogens, phytopathogenic bacteria encounter plant cell walls as barriers preventing access to the cytoplasmic contents of host cells, as deterrents to pathogen spread within infected tissue, as physical substrates on which to grow, and as a potentially rich source of carbon (Agrios 2005). Thus, not surprisingly, many plant pathogens include a battery of cell wall degrading enzymes in their repertoire of virulence factors. These enzymes include pectinases (e.g., polygalacturonases, pectate lyases, and pectin methyl esterases), cellulases, and proteases that work collectively to soften or break down plant cell walls, thereby facilitating pathogen entry and the release of nutrients for pathogen growth (Barras et al. 1994). The secretion of these exoenzymes may also result in the loosening of the middle lamellae that hold together adjacent plant cell walls, thus promoting the spread of pathogens between host cells and beyond the initial infection site. The soft-rot pathogens, such as E. chrysanthemi and E. carotovora, which make their living by macerating the plants’ tissue, secrete multiple cell wall degrading enzymes. The importance of these enzymes in the virulence of these pathogens is well established (Toth et al. 2003). X. campestris pv. campestris also has an extensive collection of genes encoding putative cell wall degrading enzymes, including several pectic enzymes and cellulases (da Silva et al. 2002). Presumably, these enzymes contribute to the massive degeneration of plant tissue that occurs during development of black rot disease in plants infected with X. campestris pv. campestris (Agrios 2005). However, as Erwinia and X. campestris pathogens secrete complex mixtures of degradative enzymes and possess multiple genes encoding functionally redundant isoenzymes, the precise role of any one of these enzymes in pathogenesis has been difficult to determine (Chan and Goodwin 1999; Toth et al. 2003). The roles of plant cell wall degrading enzymes during pathogenesis of vascular wilt and leaf spotting pathogens such as R. solanacearum and P. syringae are less clear. Ralstonia solanacearum encodes multiple known or predicted pectolytic

enzymes, including endoglucanases, polygalacturonases, and a pectin methyl esterase (Genin and Boucher 2002; Salanoubat et al. 2002). Genetic studies have revealed that several of these pectolytic enzymes contribute quantitatively to bacterial wilt disease development by facilitating invasion, colonization, and systemic spread of the pathogen within host tissue (Schell et al. 1988; Huang and Allen 1997, 2000). Recent sequence analysis has revealed that P. syringae pv. tomato strain DC3000 also encodes several potential cell wall degrading enzymes, including a polygalacturonase, a pectin lyase, and three enzymes predicted to have cellulolytic activity (Buell et al. 2003). The role of these enzymes in DC3000 virulence is not known, and no cell wall degrading activity has been reported for this strain. However, pectolytic enzymes have been reported to contribute to symptom development during infection by P. syringae pv. lachrymans (Bauer and Collmer 1997). Interestingly, three TTSS effector proteins (HopPmaHPto, HrpW, and HopPtoP), classified as ‘‘helper proteins’’ that may assist in delivery of TTSS-secreted proteins, possess carboxyterminal domains with similarity to pectolytic enzymes (Charkowski et al. 1998; Boch et al. 2002; Collmer et al. 2002). The secretion of these potential pectolytic enzymes (either through sec-dependent or TTSS-dependent processes) could possibly facilitate the assembly of functional type III secretion complexes at the bacteria-plant cell wall interface.

Production of Low-Molecular-Weight Phytotoxins Many plant pathogens produce low-molecular-weight, nonhost specific phytotoxins that are not essential for pathogenicity but yet contribute to bacterial virulence and increase disease symptoms such as chlorosis and necrosis. These toxins act either by directly damaging plant cells or by modulating host cellular metabolism to promote symptom development (Alfano and Collmer 1996; Bender et al. 1999; Birch 2001). The best characterized of these phytotoxins are those produced by P. syringae species and include lipodepsipeptide toxins (e.g., syringomycins and syringopeptins), modified peptides (e.g., tabtoxin and phaseolotoxin), polyketides (e.g., coronatine), and a combination of peptides and polyketides (e.g., syringolin group).

Lipodepsipeptide Toxins Syringomycins and syringopeptins are examples of the two classes of lipodepsipeptide toxins produced by P. syringae pv. syringae during infection. They are synthesized by nonribosomal peptide synthetases which are encoded by the syr and syp genomic islands of P. syringae pv. syringae. The genomic structure of these genomic islands including the regulatory network controlling toxin production has been determined (Scholz-Schroeder et al. 2003; Lu et al. 2005). The syringomycins are cyclic lipodepsinonapeptide phytotoxins that consist of a polar cyclic


peptide head containing nine amino acids attached to a hydrophobic 3-hydroxy carboxylic acid tail (Bender and Scholz-Schroeder 2004). Several structurally similar syringomycins are produced by different P. syringae pv. syringae strains that contain different amino acid residues in the nine-peptide ring. Syringopeptins are larger than the syringomycins and contain a peptide moiety of 22 or 25 amino acids attached to either a 3-hydroxydecanoic or a 3-hydroxydodecanoic acid (Bender and Scholz-Schroeder 2004). As in syringomycins, the amino acid chain is cyclized to form a nine-peptide ring. Many of the amino acids present in the syringopeptins are hydrophobic and thus contribute to the amphipathic nature of these toxins. The syringomycins and syringopeptins induce necrosis in plant tissues by forming pores in the plant cell plasma membrane possibly through a mechanism involving initial insertion of toxin monomers into the membrane, followed by aggregation of multiple monomers to form a pore (Bender and ScholzSchroeder 2004). The formation of pores in lipid membranes increases transmembrane ion flux, causing disruption of membrane electrical potential and eventual plant cell death (Hutchison and Gross 1997). The amphipathic nature of these toxins is likely to facilitate their insertion into plant cell membranes. Lipodepsipeptide phytotoxins are likely to play an important role in interactions between P. syringae pv. syringae and its hosts, as all strains of P. syringae pv. syringae analyzed to date produce both syringomycins and syringopeptins. Genes encoding these toxins are induced by plant-derived phenolic compounds (Wang et al. 2006), and they contribute quantitatively to P. syringae pv. syringae virulence; however, the relative importance of these toxins vary among different pathogen-host interactions (Bender et al. 1999; Scholz-Schroeder et al. 2001). Much progress has been made toward understanding the biosynthesis and pore-forming activities of the lipodepsipeptide phytotoxins (Bender and Scholz-Schroeder 2004), and they contribute to pathogen virulence by stimulating plant cell necrosis and disease lesion development. Additionally, as both syringomycins and syringopeptins exhibit biosurfactant activities, they could potentially contribute to virulence by reducing the surface tension of water and thus facilitate the spread of bacteria across plant surfaces (Bender et al. 1999), thereby promoting tissue colonization and spread of the pathogen within infected plant tissue. Interestingly, the recent completion of the genomic sequence of R. solanacearum has revealed two large open reading frames predicted to encode proteins with high similarity to syringomycin synthase (Salanoubat et al. 2002), suggesting that this pathogen may also produce syringomycin.

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phaseolotoxin produced by P. syringae pvs. phaseolicola and actinidae (Mitchell 1976; Bender et al. 1999), tabtoxin produced by P. syringae pvs. tabaci, coronafaciens, and garcae (Uchytil and Durbin 1980; Moore et al. 1984), and mangotoxin produced by P. syringae pvs. syringae and avellanae (Gasson 1980; Arrebola et al. 2003). These toxins can interfere with host nitrogen metabolism by inhibiting specific enzymes involved in the biosynthesis of essential amino acids, leading to amino acid deficiencies and concomitant accumulation of nitrogen-containing intermediates that can be utilized by the pathogens (Snoeijers et al. 2000; Arrebola et al. 2011). Tabtoxin is a dipeptide toxin that contains tabtoxinine-blactam (TbL), linked by a peptide bond to threonine (Bender et al. 1999). TbL is the toxic moiety of tabtoxin and is released from the intact toxin upon hydrolysis of the peptide bond by the action of aminopeptidases within the plant (Levi and Durbin 1986). TbL, which induces the degradation of chlorophyll in plant cells (thus causing yellowing or ‘‘chlorosis’’), irreversibly inhibits the enzyme glutamine synthetase (Thomas et al. 1983). As glutamine synthetase is required for efficient detoxification of ammonia in plant cells, inactivation of this enzyme results in accumulation of high levels of ammonia and the disruption of thylakoid membranes within the chloroplast. Phaseolotoxin is a tripeptide consisting of ornithine, alanine, and a homoarginine linked to a sulfo-diaminophosphinyl moiety (Moore et al. 1984). When taken up by plant cells, phaseolotoxin is hydrolyzed to produce octicidine, an irreversible inhibitor of ornithine carbamoyl transferase (OCTase; Mitchell and Bieleski 1977). OCTase is a key enzyme in the urea cycle that converts ornithine and carbamoyl phosphate to citrulline. Inhibition of OCTase by phaseolotoxin results in accumulation of ornithine and reduction in arginine levels, leading to the production of severe chlorosis within plant tissue (Bender et al. 1999). Both tabtoxin and phaseolotoxin contribute significantly to pathogen virulence, presumably by inhibiting photosynthesis and thus limiting available resources within the plant for mounting a successful defense response and by contributing to the severe yellowing of plant tissues associated with disease (Agrios 2005). The structure of mangotoxin has not been conclusively determined yet; however, it is most likely to consist of two amino acids linked by a sugar residue (Arrebola et al. 2003). It inhibits the ornithine N-acetyltransferase (OAT) that converts a-N-acetyl-ornithine to L-ornithine during arginine biosynthesis. The gene cluster encoding enzymes for mangotoxin biosynthesis has been identified (Arrebola et al. 2011). Studies with the mgoA mutant of P. syringae pv. syringae reveal that it is incapable of producing mangotoxin and shows reduced virulence on tomato leaves (Arrebola et al. 2007).

Modified Peptide Toxins with Antimetabolite Activity

Coronatine

Antimetabolite toxins are oligopeptides produced by several pathovars of P. syringae that can inhibit host metabolite pathways. The most well-studied antimetabolite phytotoxins include

Coronatine is produced by several P. syringae strains (Bender et al. 1999) and contributes to the virulence of P. syringae via several mechanisms. It plays important roles in early and late

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stages of disease development including suppression of stomatal immunity and thus enhancing entry into host tissue (Melotto et al. 2006; Zeng et al. 2010); promoting pathogen growth in the apoplast subsequent to entry (Zeng et al. 2010); and enhancing disease symptom development (Brooks et al. 2005; Melotto et al. 2008a). Results from genetic studies, utilizing both P. syringae mutants impaired in coronatine biosynthesis and Arabidopsis and tomato mutants impaired in JA signaling, indicate that coronatine promotes pathogen virulence by stimulating JA signaling within the plant (Feys et al. 1994; Kloek et al. 2001; Zhao et al. 2003; Brooks et al. 2005; Laurie-Berry et al. 2006). These studies revealed that SA- and JA-dependent defense signaling pathways are mutually antagonistic; coronatine-induced activation of JA signaling results in inhibition of SA-dependent defense responses, which are effective in limiting P. syringae infection and disease (Brooks et al. 2005; Laurie-Berry et al. 2006; Uppalapati et al. 2007). In support of this antagonist interaction between JA and SA signaling, reduced susceptibility to P. syringae in Arabidopsis coronatine insensitive1 (coi1) and JA insensitive1 (jin1/myc2) mutant plants is associated with increased signaling through the SA-dependent defense pathway (Kloek et al. 2001; Nickstadt et al. 2004; Laurie-Berry et al. 2006), and coronatine suppresses induction of several SA-dependent defense-related genes in tomato (Zhao et al. 2003; Uppalapati et al. 2005). Thus, coronatine suppresses SA-mediated host defenses, thereby providing P. syringae with a window of opportunity during which it can colonize and grow within host tissue. Coronatine may also be directly involved in disease symptom development via multiple SA-independent processes. The in planta growth defect of P. syringae coronatine biosynthetic mutants is suppressed in Arabidopsis mutants in which SA-dependent defenses are compromised (Brooks et al. 2005; Zeng et al. 2010). However, although the coronatine mutants grow to wild-type levels in SA-deficient plants, disease symptom development is not fully restored (Brooks et al. 2005). Recent studies suggest that coronatine may contribute to disease symptom production via two mechanisms: (1) by stimulating production of reactive oxygen species that lead to disease-associated necrotic cell death (Ishiga et al. 2009) and (2) by stimulating expression of the STAYGREEN (SGR) gene, which results in enhanced chlorophyll degradation and chlorosis of the infected tissue (Mecey et al. 2011). At the molecular level, the polyketide phytotoxin coronatine is of interest to both plant biologists and plant pathologists, as it is a structural and functional mimic of the endogenous plant hormone jasmonyl-L-isoleucine (JA-Ile) (Staswick and Tiryaki 2004; Fonseca et al. 2009). Coronatine is comprised of two distinct chemical moieties, coronafacic acid (CFA; a polyketide) that shares structural and functional relatedness with jasmonic acid (JA) and coronamic acid (CMA; an ethylcyclopropyl amino acid) that is a cyclized form of isoleucine. CFA and CMA are joined by an amide linkage forming the intact coronatine molecule (Bender et al. 1999), which is required for full virulence of P. syringae (Brooks et al. 2005; Uppalapati et al. 2007). Specifically, the intact coronatine

molecule closely resembles the active form of the plant jasmonate (+)-7-iso-JA–L-Ile (Fonseca et al. 2009). JA-Ile is a member of a family of fatty acid-derived signaling molecules, the jasmonates, which play an important role in many aspects of plant growth, development, and defense (Wasternack 2007; Browse 2009). The biological effects of coronatine closely resemble those induced by jasmonates and include induction of chlorosis, production of the protective pigment anthocyanin, inhibition of root growth, promotion of plant cell growth, and the induction of several JA-responsive genes (Feys et al. 1994; Bender et al. 1999; Zhao et al. 2003; Uppalapati et al. 2005). The most compelling evidence that coronatine is a functional analog of JA-Ile is the observation that it competitively binds to the JA-Ile receptor complex. This receptor complex is formed by the CORONATINE INSENSITIVE1 (COI1) and JASMONATE ZIM-DOMAIN (JAZ) proteins (Katsir et al. 2008; Yan et al. 2009; Melotto et al. 2008b; Sheard et al. 2010). COI1 is the F-box protein in the SCFCOI1 ubiquitin E3 ligase complex that targets JAZ proteins for degradation (Chini et al. 2007; Thines et al. 2007). Binding of JAZ proteins to the SCFCOI1 complex is stabilized in the presence of coronatine (or JA-Ile), leading to the degradation of JAZ proteins, which are transcriptional repressors of JA signaling. JAZ protein degradation, therefore, results in the expression of JA-responsive genes and the activation of JA-mediated responses leading to disease progression through a yet-to-bediscovered mechanism(s).

Syringolin Group Phytotoxins in the syringolin group contain a 12-membered ring structure consisting of 5-methyl-4-amino-2-hexenoic acid, 3, 4-dehydrolysine, and a dipeptide tail. The substitution of one of more of these amino acids forms syringolin A–F (Wa¨spi et al. 1998). Syringolin A is the best characterized among this group of small cyclic tripeptide phytotoxins produced by P. syringae pv. syringae. Recently, it has been shown that syringolin A negatively acts on the catalytic activity of eukaryotic proteasomes (Groll et al. 2008) and functions in disease development. Surface inoculation of a syringolin A-deficient strain of P. syringae pv. syringae B728a shows reduced bacterial multiplication and symptoms on the host plant Phaseolus vulgaris (Groll et al. 2008; Schellenberg et al. 2010). Similar to coronatine, syringolin A also stimulates opening of the stomatal pore and belongs to a growing group of antistomate defense factors (Melotto et al. 2006; Gudesblat et al. 2009; Schellenberg et al. 2010). Syringolin A-deficient bacteria are unable to open stomata of bean leaves; however, this effect can be reverted by exogenous application of syringolin A or the proteasome inhibitor MG132, suggesting that syringolin A acts through protein turnover affecting guard cell movement (Schellenberg et al. 2010). Interestingly, the defense hormone SA and the SA signaling component NPR1 (non-expressor of pathogenesis-related genes) are required for stomatal immunity (Melotto et al. 2006; Zeng and He 2010).


NPR1 is a transcription activator of systemic acquired resistance that is regulated in part through protein turnover, and it is degraded through the proteasome (Spoel et al. 2009; Trujillo and Shirasu 2010) suggesting that several pathogen-produced phytotoxins act as antistomate defense factors, at least in part, by blocking NPR1-dependent SA signaling.

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presumably long enough to span the plant cell wall (Brown et al. 2001; Jin and He 2001). Several studies suggest (but do not directly demonstrate) that the Hrp pilus serves as the conduit through which bacterial proteins are secreted (Brown et al. 2001; Jin and He 2001).

Identification of Type III Effectors

Type III Secretion Although bacterial pathogens of plants such as P. syringae and Xanthomonas have multiple secretion pathways as revealed by genome analysis (Buell et al. 2003; Cunnac et al. 2009; Ryan et al. 2011), like most gram-negative bacterial pathogens of animals, the bacterial plant pathogens discussed in this chapter require a functional type III secretion system (TTSS) for pathogenesis (Galan and Collmer 1999; Cornelis and Van Gijsegem 2000; Staskawicz et al. 2001; Buttner and Bonas 2003; Jin et al. 2003a; Cunnac et al. 2009; Ryan et al. 2011; Lindeberg et al. 2012; Buttner and He 2009). Type III secretion systems mediate the transfer of bacterial proteins (also referred to as ‘‘effectors’’) directly into the cytosol of the host cell, where they interfere with or modulate normal host cell processes to facilitate bacterial invasion, growth, and disease production (> Fig. 2.1). Plant pathogenic bacterial mutants defective in TTSS are usually unable to grow or cause disease on normally susceptible hosts, indicating that the integrity of the TTSS is essential for pathogenesis (Mudgett 2005; Boch and Bonas 2010; Lindeberg et al. 2012). Much progress has been made toward elucidating the structure and components of the TTSS and in identifying the effector proteins secreted through this apparatus. Insights into the function of several type III effectors and how they contribute to the virulence of plant pathogens have also been recently obtained.

Structure and Components of TTSS of Bacterial Plant Pathogens The structural components of the TTSS of gram-negative bacterial pathogens of animal and plants are highly conserved. These systems have been extensively described elsewhere (Galan and Collmer 1999; Collmer et al. 2000; Cornelis and Van Gijsegem 2000; Buttner and Bonas 2002; Jin et al. 2003a; Buttner and He 2009); therefore, the TTSS apparatus will not be discussed in detail. However, note that structurally the TTSSs of plant pathogenic bacteria are slightly different from those described for the animal pathogens. For example, the TTSS of several mammalian pathogens, such as Salmonella enterica serovar Typhimurium and Shigella flexneri, are associated with protruding, needle-like surface structures that are approximately 80 nm in length (Kubori et al. 1998; Blocker et al. 1999). The TTSS of several plant pathogenic bacteria are associated with relatively longer, pilus-like structures referred to as ‘‘Hrp pili’’ (He and Jin 2003). The Hrp pilus of P. syringae pv. tomato strain DC3000 is approximately 8 nm in diameter and has been observed to be up to 200 nm in length, which is

The importance of TTSS for pathogenesis has prompted many research groups to direct a significant amount of effort toward identifying and characterizing proteins that are secreted through the TTSS. An inventory of effector proteins secreted by plant pathogenic bacteria has been compiled in several excellent reviews (Collmer et al. 2002; Buttner and Bonas 2003; Buttner et al. 2003; Greenberg and Vinatzer 2003; Jin et al. 2003a, Cunnac et al. 2009; Lindeberg et al. 2012). A variety of approaches have been used to identify these effectors. The first type III-secreted proteins studied were those identified on the basis of their ability to elicit TTSS-dependent host defense responses on resistant plant genotypes (Staskawicz 2001). This may not be surprising, given the eagerness of plant pathologists to elucidate the mechanisms underlying pathogen recognition and disease resistance. Further, given that type III effectors are secreted directly into host cells, and thus may serve as ‘‘easy targets’’ for recognition during the evolution of host surveillance systems, it may not be surprising that many effector molecules serve as elicitors of plant defense. Recently, more comprehensive approaches for identifying genes encoding type III effectors have been employed. These approaches include (1) utilizing information regarding gene location and gene regulation, (2) direct functional assays to screen for secreted proteins, and (3) taking advantage of common structural features of known TTSS-secreted proteins to carry out ‘‘genomic mining’’ experiments. For instance, in P. syringae and X. campestris, several genes encoding effector proteins are located within or adjacent to the gene clusters encoding the structural components of the TTSS (Alfano et al. 2000; Noel et al. 2002; Charity et al. 2003). Further, the expression of many P. syringae genes encoding either structural TTSS components or TTSS effector proteins depends on HrpL, an alternative RNA polymerase s factor required for pathogenesis (Xiao et al. 1994). The HrpL s factor directs transcription of TTSS-associated genes by recognizing a consensus ‘‘hrp box’’ in the promoter regions of these genes (Innes et al. 1993; Xiao and Hutcheson 1994). This information has been used as the basis of genetic screens to identify genes potentially encoding TTSS effector proteins (Fouts et al. 2002; Zwiesler-Vollick et al. 2002; Bretz et al. 2003). Likewise, a molecular genetic strategy has been used to identify X. campestris genes whose expression is dependent on HrpX, an AraC-like transcriptional activator that is essential for induction of TTSS-related genes in this organism (Noel et al. 2001). Although type III effectors do not have an obvious signal sequence targeting them for secretion, the proteins are modular

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in structure, with the amino terminal region carrying information required for secretion (Guttman et al. 2002). Analyses of the primary amino acid sequences of several known P. syringae type III effectors has revealed a strikingly well-conserved pattern of amino acid biases within the first 50 residues that is essential for secretion (Guttman et al. 2002; Petnicki-Ocwieja et al. 2002). As the genomes of several plant pathogenic bacteria have been sequenced, the above features have facilitated genomic mining experiments to identify the entire repertoire of type III effectors secreted by these pathogens (Collmer et al. 2002; Greenberg and Vinatzer 2003; Ryan et al. 2011; Potnis et al. 2011; Lindeberg et al. 2012). Interestingly, the available genomes of plant pathogenic bacteria revealed large inventories of type III effectors. For instance, P. syringae pv. tomato strain DC3000 and pv. phaseolicola race 6 have 29 and 19 effector proteins, respectively, that are translocated to and elicit hypersensitive response in plant cells as detected by a variety of functional screens (Chang et al. 2005; Cunnac et al. 2009) The fact that plant pathogenic bacteria secrete many type III effectors could be an adaptive feature of plant pathogens and suggests that functional redundancy may exist among these effectors (Buttner and Bonas 2003). Consistent with this hypothesis, mutations in single effector genes usually do not dramatically alter bacterial virulence, at least when assayed under laboratory conditions (Ponciano et al. 2003). In contrast to TTSS structural components, which are highly conserved between various plants and animals, the sequences and inventories of type III effectors vary considerably among different plant pathogens and even among different strains of the same species (Collmer et al. 2002; Greenberg and Vinatzer 2003). This variation suggests that different strains have evolved different repertoires of virulence factors to infect and cause disease on specific host plants. Thus, characterizing type III effectors and elucidating the mechanisms through which they contribute to pathogenesis is of great interest and may eventually provide insight into the molecular basis of host specificity.

Elucidating the Function of Type III Effectors The majority of TTSS effectors secreted by bacterial plant pathogens are predicted to function inside plant cells, and secretion into the host cell has been experimentally demonstrated for many effector proteins (Casper-Lindley et al. 2002; Szurek et al. 2002; Hotson et al. 2003; Chang et al. 2005; Cunnac et al. 2009). Several type III effectors have been directly demonstrated to have elicitor and virulence activities inside plant cells (Leister et al. 1996; Chen et al. 2000; Marois et al. 2002; Hauck et al. 2003; Jamir et al. 2004; Block and Alfano 2011), and are predicted to modulate various aspects of host cell biology and physiology to support bacterial virulence, proliferation, dissemination, and promote disease (Alfano and Collmer 1996; Greenberg and Vinatzer 2003; Jin et al. 2003a; Ponciano et al. 2003; Boch and Bonas 2010; Block and Alfano 2011). Various strategies to elucidate the activities of these effectors have been employed, including protein sequence and structural

analyses, biochemical approaches to identify interacting proteins, and the analysis of transgenic plants expressing effector proteins. Such studies have revealed that, despite their prokaryotic origin, many type III effectors have features typical of eukaryotic proteins, consistent with their activity within plant cells. For instance, the P. syringae effector proteins AvrRpm1, AvrB, AvrPto, and AvrPphB have consensus N-terminal myristoylation sites that are myristoylated inside host cells (Nimchuk et al. 2000). This modification is required for the proper localization of these type III effectors at the host plasma membrane (Nimchuk et al. 2000; Shan et al. 2000). Another effector from P. syringae, HopI1, is targeted to the chloroplast and suppresses the accumulation of the defense hormone SA (Jelenska et al. 2007). Furthermore, all members of the AvrBs3/ PthA family of effectors and XopD, found in the genus Xanthomonas, carry a functional nuclear localization signal (NLS), share structural features of eukaryotic transcription factors, and are classified as the ‘‘transcription activator-like’’ (TAL) family (Schornack et al. 2006; Kim et al. 2008). An NLS at the carboxy-terminus of AvrBs3 from X. campestris is required for interaction with importin a, which is part of the host nuclear import machinery (Szurek et al. 2001). The function of these Xanthomonas effectors, several of which activate host gene expression, either to promote virulence or to trigger defense responses, have recently been comprehensively reviewed (Boch and Bonas 2010; Rivas and Genin 2011; Ryan et al. 2011). These studies illustrate that type III effectors of prokaryotic origin have evolved to take advantage of eukaryote-specific post-translational modification and targeting mechanisms to access specific subcellular compartments within the host cell. As is described in more detail below, the molecular activities identified to date for TSS effectors include facilitating type III secretion; suppressing plant immune defense by altering host cell biology at various levels; causing proteolysis of host proteins; regulating host transcription; modulating plant RNA metabolism and vesicle trafficking; and altering plant hormone synthesis, homeostasis, and signaling. Facilitators of Type III Secretion. Several type III effector proteins, known as hairpins, are secreted into the apoplast and function as ‘‘helper proteins’’ to facilitate translocation of type III secretion effectors through the host plasma membrane during pathogenesis. Some examples are HrpZ and HrpW of P. syringae (Lee et al. 2001), HrpF of X. campestris pv. vesicatoria (Buttner et al. 2002), and HrpN of E. amylovora (Bocsanczy et al. 2008). The P. syringae HrpZ protein forms oligomers and has strong affinity for the plasma membrane lipid phosphatidic acid (Haapalainen et al. 2011). Likewise, HrpF from X. campestris contains two putative transmembrane regions, suggesting its association with membranes (Buttner et al. 2002), and is required to translocate type III effectors essential for pathogenicity in the host plant (Jiang et al. 2009). Both proteins have lipid-binding activity and form ion-conducting pores in vitro when associated with lipid bilayers (Lee et al. 2001; Buttner et al. 2002). The pore-forming activity of these proteins suggests that they function in assisting delivery of effectors into the plant cell cytoplasm and/or by mediating nutrient and water release from


host cells. Since HrpF is dispensable for protein secretion in vitro, but is required for the recognition of an effector with elicitor activity in vivo, it has been proposed that HrpF may facilitate translocation of one or more effector proteins into the host cell (Rossier et al. 2000). Suppression of Host Immunity. Plants possess a complex immune system that efficiently detects potential microbial invaders and fends off dangerous microbes. Plant innate immunity is associated with perception of pathogen- or microbeassociated molecular patterns (PAMPs or MAMPs) by pattern recognition receptors (PRRs) at the plant’s cell surface. For example, two well-characterized MAMPs, the flagellin peptide flg22 and elongation factor Tu (EF-Tu), are detected by the PRRs FLS2 and EFR1, respectively (Zipfel et al. 2004, 2006). This branch of innate immunity is also known as PTI (PAMPtriggered immunity; Jones and Dangl 2006; Boller and Felix 2009). Several type III effectors can effectively suppress PTI, and this is hypothesized to be fundamental in the evolution of pathogen virulence (Jones and Dangl 2006). However, pathogens do not always cause disease, as plants have evolved another level of innate immunity known as ETI (effector-triggered immunity), formerly referred to as ‘‘gene-for-gene resistance’’ (Jones and Dangl 2006; Thomma et al. 2011). ETI is established by either direct binding of host resistance proteins to bacterial effectors or by host resistance proteins monitoring the modification of (or ‘‘guarding’’) effector targets in the host cell. Although the latter mechanism appears to be more common than the former, in both cases, a rapid, localized host cell death, known as the hypersensitive responses (HR), is typical of ETI (Lewis et al. 2009). Plant host immunity and the molecular activities of bacterial effectors suppressing both PTI and ETI are the subject of several excellent, recent reviews (Chisholm et al. 2006; Jones and Dangl 2006; Block et al. 2008; Boller and He 2009; Lewis et al. 2009; Block and Alfano 2011; Chen and Ronalds 2011; Spoel and Dong 2012). Proteolysis of Host Proteins. Several type III effectors have proteolytic activity that interferes with normal host physiology and actively suppresses plant immunity. For instance, two effectors from P. syringae pv. tomato strain DC3000, HopM1 and AvrPtoB, promote degradation of host proteins through the 26S proteasome pathway. HopM1 interferes with vesicle trafficking of possible defense cargoes to the plasma membrane by triggering the degradation of the ARF guanine exchange factor AtMIN7 through the proteasome pathway. Plants lacking AtMIN7 are exceedingly susceptible to bacteria (Nomura et al. 2006). The specificity of ubiquitinylation of proteins targeted for degradation in eukaryotes is controlled by a variety of E3 ligase complexes. Of relevance to this discussion is the SCF-type E3 ubiquitin ligase complex, containing Skp1, Cullin1, and an F-box protein subunit. Interestingly, some bacterial effectors seem to highjack the host SCF-type E3 ubiquitin ligase mechanism to promote disease. For instance, AvrPtoB has an E3 ligase activity that targets the PAMP receptors FLS2, ERF1, CERK1, and the receptor partner BAK1 thus suppressing plant immunity in vivo (Gohre et al. 2008; Shan et al. 2008; Gimenez-Ibanez et al. 2009). The GALA effectors from R. solanacearum contain an

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F-box domain that is required for several stages of disease development on plants (Angot et al. 2006; Turner et al. 2009). The host targets of GALA effectors remain to be identified. The AvrPphB cysteine protease from P. syringae pv. phaseolicola cleaves both itself and PBS1, an Arabidopsis protein kinase required for ETI (Shao et al. 2003). Interestingly, AvrPphB can inhibit PTI by cleaving PBS1-like kinases (Zhang et al. 2010) that interact with FLS2, demonstrating that PTI and ETI are connected by the activities of pathogen effector proteins. A model for the action of AvrPphB is still under development and has been recently reviewed by Day and He (2010). AvrRpt2 from P. syringae pv. tomato is a cysteine protease that cleaves itself to form a mature protease inside the host plant (Jin et al. 2003b) as well as several host proteins, including the Arabidopsis RIN4 protein, a negative regulator of PTI and ETI (Axtell and Staskawicz 2003; Axtell et al. 2003; Chisholm et al. 2005; Day et al. 2005; Kim et al. 2005; Dodds and Rathjen 2010). As will be discussed further below, AvrRpt2 also promotes pathogen virulence and modulates auxin sensitivity in the host. However, it is not clear whether the cysteine protease activity of AvrRpt2 is also required for both of these activities. RIN4 appears to be an important virulence target or defense decoy, as it is also targeted by several other type III effectors (AvrB, AvrRpm1, and HopF2). However, it is not clear how alteration of RIN4 protein enhances bacterial virulence (Dodds and Rathjen 2010). The C-terminal portion of the XopD protein from X. campestris pv. vesicatoria has a high degree of similarity with the C-terminal catalytic domain of the Ulp1 ubiquitin-like protease protein family and has a cysteine protease activity specific for small ubiquitin-like modifier (SUMO)-lated substrates found specifically in plants (Hotson et al. 2003). The XopD SUMO protease effector promotes pathogen growth and delays the onset of symptoms in the host plant tomato (Kim et al. 2008). On the basis of amino acid sequence similarity, three additional effectors from X. campestris pv. vesicatoria, AvrRxv, AvrBsT, and AvrXv4, as well as PopP1 from R. solanacearum appear to belong to the YopJ family of ubiquitin-like protein proteases. Interestingly, like XopD, YopJ exhibits specificity for SUMO-lated proteins (Orth et al. 2000; Lavie et al. 2002). Although the importance of the above demonstrated or predicted proteolytic activities in pathogen virulence in some cases is not clear, these findings suggest that the use of type III effectors to cleave specific host signaling molecules is a common strategy of bacterial pathogens. Analysis of Type III Effector Function. The findings that the primary amino acid sequences of most effector proteins do not provide much insight regarding function and that, in most cases, mutation of the effector genes does not result in pronounced virulence phenotypes have not helped to hasten our understanding of these proteins. One of the major challenges in understanding TTSS effector function is to identify the targets of these virulence factors within the host and to elucidate the roles of these molecules in pathogenesis. Despite these challenges, the scientific community has recently made tremendous progress in this area and has gained much knowledge about the function of

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TTSS effectors. Importantly, new components of the plant innate immune system, such as vesicle trafficking (Nomura et al. 2006, 2011) and RNA metabolism (Fu et al. 2007), have been identified in the search for host targets of bacterial effectors (Speth et al. 2007). Although a tremendous amount of progress toward identifying and characterizing bacterial type III effectors has been recently made, the mode of action of most of these effectors is still unknown. Future studies involving host gene expression profiling and proteomic approaches to reveal the potential effects of these pathogen molecules on plant defense and other aspects of host physiology may help elucidate the function of some of these effectors. Likewise, localization of effector proteins within host cells, as well as identification of plant proteins that interact with the effectors, will also continue to contribute to our understanding of effector function within plant cells. One experimental approach that has provided important new insight into the function of several effector proteins has been to express them ectopically in transgenic plants. Several such studies have revealed that some effectors modulate host hormone biology. For example, transgenic Arabidopsis expressing the P. syringae effector AvrRpt2 exhibit increased sensitivity to the plant hormone auxin, suggesting that AvrRpt2 modulates host auxin physiology to promote pathogen virulence (Chen et al. 2007). Likewise, expression of AvrPtoB and HopAM1 in planta results in increased levels of abscisic acid (ABA; Goel et al. 2008) and hypersensitivity to ABA, respectively (Goel et al. 2008; de Torres-Zabala et al. 2007). Thus, one role of type III effectors appears to be modulation of hormone production and/or signaling in the host. Further discussion as to how altering host hormone physiology might contribute to pathogenesis is provided below.

Modulation of Plant Hormone Homeostasis and Signaling Plant hormones, which are also often referred to as ‘‘plant growth regulators,’’ are endogenous signaling molecules important for many aspects of plant growth and development. The best known phytohormones are auxin, ethylene, cytokinins, abscisic acid (ABA), and gibberellins. More recently, several additional chemicals, including jasmonates (JA), salicylic acid (SA), brassinosteroids, nitric oxide, and strigolactones, have been recognized as phytohormones (Santner and Estelle 2009). Three of these hormones in particular, SA, JA, and ethylene, are important in mediating plant defenses in response to pathogen or herbivore attack (Hammond-Kosack and Jones 2000; Kunkel and Brooks 2002; Kazan and Manners 2009, 2012; Spoel and Dong 2009; Robert-Seilaniantz et al. 2011). Other hormones that have been implicated in plant-pathogen interactions are ABA, auxins, gibberellins, cytokinins, and brassinosteroids; their complex signaling network is nicely reviewed by Pieterse et al. (2009) and Choi et al. (2011). It is not surprising that many plant pathogens have evolved mechanisms for modulating endogenous hormone signaling

and homeostasis in their hosts, presumably as a mechanism for promoting pathogenesis. However, the specific hormone signaling pathway(s) targeted by a given pathogen, and the manner in which they are modulated seems to depend on the virulence strategy employed by the pathogen, that is, whether it is a necrotroph that rapidly kills plant cells to obtain nutrients or a biotroph that colonizes living plant tissue. Several excellent reviews on this subject are available (Spoel and Dong 2009; Grant and Jones 2009; Bari and Jones 2009; Choi et al. 2011; Robert-Seilaniantz et al. 2011; Kazan and Manners 2012). In general, there appear to be three basic strategies utilized by pathogens to manipulate the hormone physiology of their plants hosts: (1) synthesis of the hormone, or a hormone mimic; (2) perturbing hormone homeostasis (e.g., metabolism or conversion into inactive forms); and (3) modulating hormone signaling pathways. As this newly emerging paradigm has been recently discussed in several excellent reviews (Kazan and Manners 2009, 2012; Spoel and Dong 2009; Robert-Seilaniantz et al. 2011), we briefly discuss examples of these processes below.

Salicylic Acid Salicylic acid (SA) plays a central role in defense against pathogen attack. During infection, plants often accumulate SA, and exogenous application of SA or SA analogs results in enhanced resistance to a wide variety of pathogens (Durrant and Dong 2004; Bari and Jones 2009; Spoel and Dong 2009). Plant mutants that are impaired in their ability to accumulate SA exhibit enhanced susceptibility to many pathogens (Nawrath and Metraux 1999; Wildermuth et al. 2001). Thus, to successfully colonize host tissue, virulent bacterial pathogens presumably have evolved mechanisms for interfering with SA-mediated defense responses, for instance, by delaying or preventing the accumulation of high levels of SA within host tissue or by suppressing SA-dependent signaling downstream of SA accumulation. For example, the deployment of several P. syringae type III effectors, including AvrRpt2, AvrPphC, VirPphA, and AvrPphF, delays the accumulation of SA within the infected plant by inhibiting host recognition of bacteria expressing specific avirulence factors (Ritter and Dangl 1996; Jackson et al. 1999; Chen et al. 2000; Tsiamis et al. 2000; Cunnac et al. 2009; Block and Alfano 2011). Another potential strategy for interfering with induction of SA-dependent defenses is degradation of SA. This mechanism may be deployed by R. solanacearum, whose genome includes several genes encoding putative SAdegrading enzymes (Salanoubat et al. 2002). Pseudomonas syringae, and presumably other pathogens, may also facilitate colonization of host tissue by suppressing SA synthesis and signaling. As discussed above, coronatine appears to be utilized by P. syringae to downregulate SAdependent defense responses (Brooks et al. 2005; Laurie-Berry et al. 2006). Likewise, the P. syringae type III effectors AvrRpt2 and HopPtoD2 suppress the expression of SA-regulated defenserelated genes during infection on susceptible plants (Bretz et al. 2003; Chen et al. 2004). In the case of AvrRpt2, this appears to


occur without altering SA levels (Chen et al. 2004). The HopI1 type III effector acts directly at the site of SA synthesis, the chloroplast, disrupting thylakoid structure and suppressing SA accumulation (Jelenska et al. 2007). Therefore, plant pathogenic bacteria appear to deploy several different strategies to interfere with various aspects of SA-dependent defenses within the host.

Jasmonates A group of biochemically related plant growth regulators collectively referred to as ‘‘jasmonates’’ (‘‘JAs’’) are involved in defense against both herbivorous insect pests and necrotrophic bacterial and fungal pathogens that colonize dead plant tissues. Thus, intact JA signaling processes are required for resistance to attack by these organisms (Browse 2009). In contrast, JA signaling is required for disease susceptibility of Arabidopsis and tomato plants to the biotrophic bacterial pathogen P. syringae (Feys et al. 1994; Kloek et al. 2001; Zhao et al. 2003; Nickstadt et al. 2004; Laurie-Berry et al. 2006). This may not be surprising, as discussed earlier, coronatine, an important virulence factor for P. syringae, is a molecular mimic of JA-Ile (Melotto et al. 2008a; Staswick 2008; Browse 2009). Coronatine stimulates JA signaling within the plant, by binding to the receptor complex COI1/ JAZ (Katsir et al. 2008; Sheard et al. 2010). COI1-dependent activation of JA signaling is required to promote pathogenesis and disease development (Feys et al. 1994; Weiler et al. 1994; Kloek et al. 2001; Zhao et al. 2003). However, the molecular mechanism(s) underlying this process downstream of coronatine perception by the COI1/JAZ receptors is not well understood. Mounting evidence suggests that stimulation of JA signaling results in antagonism of SA-dependent defenses thereby promoting pathogen growth (Kloek et al. 2001; Zhao et al. 2003; Brooks et al. 2005; Laurie-Berry et al. 2006; Uppalapati et al. 2007; Zheng et al. 2012). Additionally, stimulation of JA appears to result in enhanced disease production, via a mechanism that is independent of SA (Brooks, et al. 2005; Laurie-Berry et al. 2006).

Ethylene The role of the gaseous plant hormone ethylene in plantmicrobe interactions is complex, as it is required for resistance against some pathogens and for disease susceptibility in others (Kunkel and Brooks 2002; Van Loon et al. 2006; RobertSeilaniantz et al. 2011). Like JA, in general, ethylene contributes to defense against necrotrophic pathogens but promotes disease in plants infected by biotrophic pathogens. Ralstonia solanacearum and P. syringae, two pathogens for which normal ethylene responsiveness in the host is important for disease development (Bent et al. 1992; Lund et al. 1998; Hoffman et al. 1999; Weingart et al. 2001; Hirsch et al. 2002), have been reported to produce ethylene, both in culture and in planta (Freebairn and Buddenhagen 1964; Weingart and Volksch 1997; Valls et al. 2006). These findings suggest that ethylene

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production by R. solanacearum and P. syringae plays an important role in pathogenesis. Consistent with this hypothesis, ethylene synthesis mutants of some strains of P. syringae pv. glycinea grow to significantly reduced levels in bean and soybean plants (Weingart et al. 2001). Interestingly, in addition to encoding the ethylene biosynthetic gene ACC oxidase, the R. solanacearum genome contains a gene encoding ACC deaminase (Salanoubat et al. 2002), an enzyme involved in ethylene degradation. This suggests that R. solanacearum may carefully modulate ethylene levels within the plant for a maximal virulence effect. The role of ethylene in interactions with bacterial plant pathogens is somewhat confusing, as it is reported to both suppress and stimulate host defenses responses, depending upon the nature of the interaction under study. For example, ethylene signaling is required for normal susceptible responses to P. syringae; ethylene-insensitive mutants (e.g., ein2 and ein3 eil1) are less susceptible to infection (Bent et al. 1992; Chen et al. 2009; Boutrot et al. 2010), and overexpression of ERF1, an ethylene-responsive transcription factor, results in increased susceptibility to P. syringae (Berrocal-Lobo et al. 2002). These observations are consistent with studies indicating that ethylene antagonizes SA signaling in these interactions (Chen et al. 2009). However, ethylene is also required for normal induction of PTI by the MAMP flg22. Challenge of Arabidopsis plants with flg22 results in increased ethylene production, and ethylene perception and signaling mutants are impaired for flg22-mediated responses (Boutrot et al. 2010). This effect appears to be mediated through the expression of the flg22 receptor, FLS2. Although ethylene plays multiple roles in plant-pathogen interactions, in the case of virulent pathogens such as P. syringae, it seems that the primary role of ethylene is to promote disease, as the basal defenses induced by MAMP perception are normally suppressed by pathogen virulence factors (van Loon et al. 2006).

Abscisic Acid Abscisic acid (ABA) is well known for the roles it plays in regulating seed development and germination and in mediating responses to abiotic stress induced by drought, salt, and cold (Hirayama and Shinozaki 2007). ABA and its downstream signaling components induce stomatal closure in response to drought and epiphytic bacteria, suggesting a common mechanism of guard cell response to both biotic and abiotic stresses (Melotto et al. 2006). However, the role of ABA in modulating post-invasion plant-bacterium interactions is not always clear. For instance, how ABA impacts the outcome of a pathogenic interaction appears to depend not only upon the lifestyle of the pathogen (i.e., whether it is a biotroph or a necrotroph) but also on the specific interaction (Fan et al. 2009; Robert-Seilaniantz et al. 2011). This may be due, in part, to the fact that ABA is involved in regulatory cross talk with the ethylene, JA, and SA defense signaling pathways (Bari and Jones 2009; Pieterse et al. 2009; Robert-Seilaniantz et al. 2011). More recent evidence suggests that ABA signaling may also impact pathogen growth by influencing physiological conditions in infected tissue.

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Several studies indicate that ABA promotes susceptibility to biotrophic pathogens, such as the bacterium P. syringae and the oomycete Hyaloperonospora arabidopsidis. Upon infection with virulent P. syringae, the ABA level increases in the host tissue (Fan et al. 2009; de Torres-Zabala et al. 2007). Additionally, overexpression of ABA biosynthetic genes (e.g., NCED5) and consequent overproduction of ABA render plants hypersusceptible to P. syringae and H. arabidopsidis, while mutant plants impaired in ABA biosynthesis or responsiveness exhibit reduced susceptibility (de Torres-Zabala et al. 2007; Fan et al. 2009). There is growing evidence that P. syringae modulates ABA signaling as a virulence strategy. The increase in ABA levels observed upon infection with P. syringae is correlated with the induction of ABA biosynthetic genes, and this appears to be dependent upon type three secretion effectors, such as AvrPtoB (de Torres-Zabala et al. 2007; Fan et al. 2009). Furthermore, at least one P. syringae effector, HopAM1, enhances both ABA sensitivity and pathogen virulence within the host. The effect of HopAM1 on pathogen virulence is most pronounced on drought-stressed plants, suggesting that one role for ABA in contributing to host susceptibility is by modulating water availability in infected tissue in a manner that favors pathogen growth (Goel et al. 2008; Beattie 2011). Fan et al. (2009) also demonstrated that the increase in ABA in infected tissue stimulated pathogen-induced JA accumulation. Thus, one mechanism by which ABA enhances pathogen growth appears to be through promoting the antagonistic cross talk between JA and SA signaling pathways, inhibiting defenses against biotrophic pathogens. However, the overall role of ABA in impacting the outcome of plant-pathogen interactions is clearly not that simple, as in some situations, ABA and JA appear to act antagonistically in modulating defense responses (de Torres-Zabala et al. 2009). Although some necrotrophic fungi, such as Botrytis cinerea and Fusarium oxysporum, produce ABA, we are not aware of any reports that bacterial pathogens can synthesize ABA. However, it would not be necessary for them to do so in order to modulate ABA signaling in the host, as this can be accomplished by stimulating ABA synthesis and/or by promoting relocalization within the plant or release from internal stores (RobertSeilaniantz et al. 2011).

Auxin The role of auxin in promoting plant cell division and growth in diseases caused by tumorigenic plant pathogens such as A. tumefaciens and P. savastanoi is well established (Pitzschke and Hirt 2010; Rodrı´guez-Palenzuela et al. 2010). Although, a number of non-gall-forming plant pathogens, including R. solanacearum, X. oryzae pv. oryzae, and several P. syringae strains, have been reported to produce indole acetic acid (IAA), the predominant naturally occurring active form of auxin, when grown in culture (Phelps and Sequeira 1968; Fett et al. 1987; Glickmann et al. 1998; Ansari and Sridhar 2000; Valls et al. 2006; Spaepen and Vanderleyden 2011), the involvement of this plant

growth regulator in disease caused by nontumorigenic bacterial pathogens is less well understood. Several P. syringae strains harbor an iaaL gene that encodes an enzyme believed to catalyze the conversion of IAA to IAA-lysine, a conjugated form of IAA that is believed to be biologically less active than free IAA (Glickmann et al. 1998). Thus, presumably these bacteria are not only able to produce auxin but are also able to adjust free IAA levels within the plant. IAA levels increase in Arabidopsis plants infected with virulent X. campestris or P. syringae strains (O’Donnell et al. 2003; Chen et al. 2007; Spaepen and Vanderleyden 2011). Although the source (i.e., host or pathogen) of this increase in free IAA has not been established, the fact that several Arabidopsis genes encoding enzymes involved either in IAA biosynthesis or in hydrolysis of IAA-amino acid conjugates are upregulated upon infection with P. syringae (Niyogi et al. 1993; Bartel and Fink 1994; Zhao and Last 1996; Hull et al. 2000; Tao et al. 2003; Thilmony et al. 2006; Kazan and Manners 2009) suggests that the increase in free IAA in infected plants is produced, at least in part, by the plant. There is growing evidence that this increase in free IAA levels is of benefit to the pathogen. For example, exogenous application of the auxin analogs 1-naphthaleneacetic acid (NAA) or 2,4-D to Arabidopsis plants resulted in increased disease susceptibility to P. syringae, while plant mutants or transgenic lines with impaired auxin signaling exhibit reduced susceptibility to P. syringae (Chen et al. 2007; Wang et al. 2007; Navarro et al. 2006). Thus, it is reasonable to propose that some plant pathogens may modulate free auxin levels within the plant as a strategy to promote pathogen growth and disease development. Three virulence factors that appear to contribute to this process have been identified: the P. syringae phytotoxin coronatine and two TTSS effector proteins, AvrRpt2 from P. syringae and AvrBs3 from X. campestris. For instance, the expression of several genes involved in either producing IAA or releasing free IAA from conjugated pools within the plant (e.g., IAR3) is induced by infection with wild-type DC3000 but not with coronatinedefective mutants (Thilmony et al. 2006; Uppalapati et al. 2005). Transgenic Arabidopsis plants constitutively expressing the P. syringae type III effector AvrRpt2 (and lacking the corresponding resistance gene, RPS2) exhibit enhanced sensitivity to auxin and accumulate elevated levels of free IAA (Chen et al. 2007), suggesting that AvrRpt2 may promote pathogen virulence by modulating host auxin physiology. Finally, AvrBs3 from X. campestris stimulates host cell enlargement, a process associated with auxin, and induces expression of a group of auxin-regulated genes (Marois et al. 2002). In susceptible pepper plants, delivery of AvrBs3 specifically induces the expression of a group of auxin-induced SAUR genes (Marois et al. 2002). However, unlike AvrRpt2, the presence of AvrBs3 does not appear to affect free IAA levels in infected plants (Marois et al. 2002). Thus, AvrBs3 may alter host auxin physiology by altering IAA responses downstream of free IAA production and release from internal pools. Several studies indicate that auxin promotes susceptibility by suppressing host defenses. For example, auxin downregulates


expression of defense-related genes in cultured tobacco cells and plant tissues (Shinshi et al. 1987; Rezzonico et al. 1998), and injection of auxin-producing A. tumefaciens or P. savastanoi bacteria into tobacco leaves prior to injection of an avirulent P. syringae strain inhibits the development of visible tissue collapse (i.e., the hypersensitive response or HR) associated with the host defense response. The ability of these strains to suppress the P. syringae-induced HR was dependent on the presence of functional auxin biosynthetic genes, suggesting that auxin is directly involved in suppressing the HR (Robinette and Matthysse 1990). Exogenous application of auxin represses induction of pathogenesis-related (PR) genes upon treatment with SA (Wang et al. 2007). Although not directly demonstrated, it is possible that IAA promotes susceptibility by suppressing SA-mediated defenses. Additional, although somewhat indirect, evidence that auxin promotes pathogenesis comes from studies of basal defenses responses. Navarro et al. (2006) demonstrated that flg22 treatment of Arabidopsis induces a microRNA (miR393) that targets several TIR1 family auxin receptor genes and thus inhibits auxin signaling. Likewise, Wang et al. (2007) demonstrated that SA inhibits auxin signaling and that this appears to be mediated through the repression of the TIR1 family of auxin receptors. Thus, one component of MAMP-mediated basal defense appears to involve suppression of auxin responses. Auxin may also promote pathogen virulence via SAindependent mechanism(s). For example, auxin may alter the physiology of host cells at the site of infection in a manner that could render host tissue more suitable for pathogen growth. Alternatively, or additionally, IAA or IAA-amino acid conjugates could impact the pathogen directly, for example, by regulating virulence gene expression (Yang et al. 2007). A recent study by Gonza´lez-Lamothea et al. (2012) shows that Botrytis cinerea and P. syringae infection promote the accumulation of an IAAamino acid conjugate, IAA-Asp, and that this compound directly impacts the pathogen by stimulating transcription of virulence genes.

The Complexities of Hormone Signaling Networks in Plant-Microbe Interactions Note that the amount of crosstalk between the hormone signaling pathways discussed above is significant (Gazzarrini and McCourt 2003; Pieterse et al. 2009; Grant and Jones 2009). For instance, after reading this chapter, it should be clear that a large amount of interplay exists between the auxin, ABA, SA, and JA signaling pathways, and pathogens such as P. syringae may take advantage of the mutually antagonistic crosstalk between these pathways to manipulate signaling within the plant (Kazan and Manners 2009, 2012; Spoel and Dong 2009; Grant and Jones 2009; Robert-Seilaniantz et al. 2011). Likewise, auxin appears to upregulate the expression of ACC synthase 4 (ACS4; Abel et al. 1995), an enzyme that catalyzes a rate-limiting step in ethylene biosynthesis. Thus, auxin may also induce ethylene biosynthesis. Moreover, increasing evidence suggests that auxin and JA

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signaling pathways are interconnected. Therefore, the involvement of one plant hormone in a plant-pathogen interaction could be mediated, at least in part, through the action of one or more other plant hormones. Future studies aimed at untangling these complicated signaling networks will undoubtedly provide valuable insight into the virulence mechanisms used by bacterial plant pathogens.

Challenges The recent use of a combination of genetic, molecular, and genomic approaches has led to major advances in the identification of numerous potential new virulence factors. Studies involving plant genetic, genomic, and biochemical approaches, as well as physiological and gene expression analyses of transgenic plants expressing specific pathogen virulence factors (e.g., type III effector proteins), have provided insights on the action of virulence factors in the host cell. However, despite many recent advances, little is known about the plant processes modulated by pathogens. The challenge that lies ahead is to continue to develop experimental strategies that will facilitate the investigation of the mode of action of these factors and how they function collectively within the plant to promote pathogen virulence and disease. Given the potential functional redundancy of these factors, and the fact that their mode of action may not always be accurately predicted, it would be wise to utilize a variety of approaches in these future studies. In certain situations, advantage can also be taken of the observations that certain bacterial virulence factors are active in yeast (for examples, see Abramovitch et al. (2003) and Jamir et al. (2004)). The power of yeast genetics and the use of heterologous systems will likely facilitate the identification of host components that are important in mediating the activity of these virulence factors. It is unclear how plants prioritize their response to multiple stresses they face in nature. Additional studies addressing the uniqueness and commonalities of regulatory networks controlling responses to abiotic and biotic stressors will shed some light on response output in the field and provide new tools for crop improvement. Collectively, these studies are likely to provide valuable information regarding the molecular mechanisms underlying pathogen virulence, the host processes that are modulated during pathogenesis, as well as how environmental inputs impact host-pathogen interactions. The insights gained from these experiments may also lead to the development of new approaches for controlling virulence and disease development in agronomically important plant-pathogen interactions.

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