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Several plant-associated microbes synthesize the auxinic plant growth regulator phenylacetic acid (PAA) in culture; however, the role of PAA in plant-pathogen interactions is not well understood. In this study, we investigated the role of PAA during interactions between the phytopathogenic bacterium Pseudomonas syringae strain Pto DC3000 ( Pto DC3000) and the model plant host, Arabidopsis thaliana . Previous work demonstrated that indole-3-acetaldehyde dehydrogenase A (AldA) of Pto DC3000 converts indole-3-acetaldehyde (IAAld) to the auxin indole-3-acetic acid (IAA). Here, we further demonstrated the biochemical versatility of AldA by conducting substrate screening and steady-state kinetic analyses, and showed that AldA can use both IAAld and phenylacetaldehyde as substrates to produce IAA and PAA, respectively. Quantification of auxin in infected plant tissue showed that AldA-dependent synthesis of either IAA or PAA by Pto DC3000 does not contribute significantly to the increase in auxin levels in infected A. thaliana leaves. Using available arogenate dehydratase ( adt ) mutant lines of A. thaliana compromised for PAA synthesis, we observed that a reduction in PAA-Asp and PAA-Glu is correlated with elevated levels of IAA and increased susceptibility. These results provide evidence that PAA/IAA homeostasis in A. thaliana influences the outcome of plant-microbial interactions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Lee, Harper, Lee, Qi, Clay, Aoi, Jez, Kasahara, Blodgett and Kunkel.)

The plant hormone indole-3-acetic acid (IAA), also known as auxin, plays important roles in plant growth and development, as well as in several plant-microbe interactions. IAA also acts as a microbial signal and in many bacteria regulates metabolism, stress responses, and virulence. In the bacterial plant pathogen Pseudomonas syringae pv. tomato strain DC3000 ( Pto DC3000), exposure to IAA results in large-scale transcriptional reprogramming, including the differential expression of several known virulence genes. However, how Pto DC3000 senses and responds to IAA and what aspects of its biology are regulated by IAA is not understood. To investigate the mechanisms involved in perceiving and responding to IAA, we carried out a genetic screen for mutants with altered responses to IAA. One group of mutants of particular interest carried disruptions in the aefR gene encoding a TetR family transcriptional regulator. Gene expression analysis confirmed that the aefR mutants have altered responses to IAA. Thus, AefR is the first demonstrated auxin response regulator in Pto DC3000. We also investigated several aspects of Pto DC3000 biology that are regulated by both AefR and IAA, including antibiotic resistance, motility, and virulence. The observation that the aefR mutant has altered virulence on Arabidopsis , suggests that the sector of the IAA response regulated by aefR is important during pathogenesis. Our findings also provide evidence that AefR plays a role in coordinating changes in gene expression during the transition from early to late stages of infection. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license., Competing Interests: The author(s) declare no conflict of interest.

Jasmonate (JA) is an important hormone involved in regulating diverse responses to environmental factors as well as growth and development, and its signalling is influenced by other hormones such as ethylene (ET). However, our understanding of the regulatory relationship between the JA and ET signalling pathways is limited. In this study, we isolated an Arabidopsis JA-hypersensitive mutant, jah3 (jasmonate hypersensitive3)-1. Map-based cloning revealed that the JAH3 gene corresponds to At4g16535. JAH3 encodes a protein of unknown function whose amino acid sequence has similarity to leukocyte receptor cluster-like protein. The mutation in jah3-1 is caused by a single nucleotide change from A to T at position 220 of 759 bp. Using CRISPR-Cas9, we generated a second allele, jah3-2, that encodes a truncated protein. Both of these loss-of-function alleles resulted in hypersensitivity to JA, ET-induced root growth inhibition, and accelerated dark-induced senescence. Double mutant analyses employing coronatine insensitive 1 (coi1) and ethylene insensitive 3 (ein3) mutants (jah3 coi1 and jah3 ein3) demonstrated that the hypersensitive phenotypes of the jah3 mutants are mediated by JA and ET signalling components COI1 and EIN3. Therefore, we propose that JAH3 is a negative regulator of both JA and ET signalling., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The auxin indole-3-acetic acid (IAA) is a plant hormone that not only regulates plant growth and development but also plays important roles in plant-microbe interactions. We previously reported that IAA alters expression of several virulence-related genes in the plant pathogen Pseudomonas syringae pv. tomato strain DC3000 ( Pto DC3000). To learn more about the impact of IAA on regulation of Pto DC3000 gene expression, we performed a global transcriptomic analysis of bacteria grown in culture, in the presence or absence of exogenous IAA. We observed that IAA repressed expression of genes involved in the type III secretion (T3S) system and motility and promoted expression of several known and putative transcriptional regulators. Several of these regulators are orthologs of factors known to regulate stress responses and accordingly expression of several stress response-related genes was also upregulated by IAA. Similar trends in expression for several genes were also observed by quantitative reverse transcription PCR. Using an Arabidopsis thaliana auxin receptor mutant that accumulates elevated auxin, we found that many of the P. syringae genes regulated by IAA in vitro were also regulated by auxin in planta . Collectively the data indicate that IAA modulates many aspects of Pto DC3000 biology, presumably to promote both virulence and survival under stressful conditions, including those encountered in or on plant leaves. IMPORTANCE Indole-3-acetic acid (IAA), a form of the plant hormone auxin, is used by many plant-associated bacteria as a cue to sense the plant environment. Previously, we showed that IAA can promote disease in interactions between the plant pathogen Pseudomonas syringae strain Pto DC000 and one of its hosts, Arabidopsis thaliana. However, the mechanisms by which IAA impacts the biology of Pto DC3000 and promotes disease are not well understood. Here, we demonstrate that IAA is a signal molecule that regulates gene expression in Pto DC3000. The presence of exogenous IAA affects expression of over 700 genes in the bacteria, including genes involved in type III secretion and genes involved in stress response. This work offers insight into the roles of auxin-promoting pathogenesis.

The plant hormone auxin governs many aspects of normal plant growth and development. Auxin also plays an important role in plant-microbe interactions, including interactions between plant hosts and pathogenic microorganisms that cause disease. It is now well established that indole-3-acetic acid (IAA), the most well-studied form of auxin, promotes disease in many plant-pathogen interactions. Recent studies have shown that IAA can act both as a plant hormone that modulates host signaling and physiology to increase host susceptibility and as a microbial signal that directly impacts the pathogen to promote virulence, but large gaps in our understanding remain. In this article, we review recent studies on the roles that auxin plays during plant-pathogen interactions and discuss the virulence mechanisms that many plant pathogens have evolved to manipulate host auxin signaling and promote pathogenesis., (Copyright © 2021 Cold Spring Harbor Laboratory Press; all rights reserved.)

Aldehyde dehydrogenases (ALDHs) catalyze the conversion of various aliphatic and aromatic aldehydes into corresponding carboxylic acids. Traditionally considered as housekeeping enzymes, new biochemical roles are being identified for members of ALDH family. Recent work showed that AldA from the plant pathogen Pseudomonas syringae strain PtoDC3000 (PtoDC3000) functions as an indole-3-acetaldehyde dehydrogenase for the synthesis of indole-3-acetic acid (IAA). IAA produced by AldA allows the pathogen to suppress salicylic acid-mediated defenses in the model plant Arabidopsis thaliana. Here we present a biochemical and structural analysis of the AldA indole-3-acetaldehyde dehydrogenase from PtoDC3000. Site-directed mutants targeting the catalytic residues Cys302 and Glu267 resulted in a loss of enzymatic activity. The X-ray crystal structure of the catalytically inactive AldA C302A mutant in complex with IAA and NAD+ showed the cofactor adopting a conformation that differs from the previously reported structure of AldA. These structures suggest that NAD+ undergoes a conformational change during the AldA reaction mechanism similar to that reported for human ALDH. Site-directed mutagenesis of the IAA binding site indicates that changes in the active site surface reduces AldA activity; however, substitution of Phe169 with a tryptophan altered the substrate selectivity of the mutant to prefer octanal. The present study highlights the inherent biochemical versatility of members of the ALDH enzyme superfamily in P. syringae., (© 2020 The Author(s).)

Aldehyde dehydrogenases are versatile enzymes that serve a range of biochemical functions. Although traditionally considered metabolic housekeeping enzymes because of their ability to detoxify reactive aldehydes, like those generated from lipid peroxidation damage, the contributions of these enzymes to other biological processes are widespread. For example, the plant pathogen Pseudomonas syringae strain Pto DC3000 uses an indole-3-acetaldehyde dehydrogenase to synthesize the phytohormone indole-3-acetic acid to elude host responses. Here we investigate the biochemical function of AldC from Pto DC3000. Analysis of the substrate profile of AldC suggests that this enzyme functions as a long-chain aliphatic aldehyde dehydrogenase. The 2.5 Å resolution X-ray crystal of the AldC C291A mutant in a dead-end complex with octanal and NAD + reveals an apolar binding site primed for aliphatic aldehyde substrate recognition. Functional characterization of site-directed mutants targeting the substrate- and NAD(H)-binding sites identifies key residues in the active site for ligand interactions, including those in the "aromatic box" that define the aldehyde-binding site. Overall, this study provides molecular insight for understanding the evolution of the prokaryotic aldehyde dehydrogenase superfamily and their diversity of function., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Lee et al.)

Modification of host hormone biology is a common strategy used by plant pathogens to promote disease. For example, the bacterial pathogen strain Pseudomonas syringae DC3000 (PtoDC3000) produces the plant hormone auxin (indole-3-acetic acid [IAA]) to promote PtoDC3000 growth in plant tissue. Previous studies suggest that auxin may promote PtoDC3000 pathogenesis through multiple mechanisms, including both suppression of salicylic acid (SA)-mediated host defenses and via an unknown mechanism that appears to be independent of SA. To test if host auxin signaling is important during pathogenesis, we took advantage of Arabidopsis thaliana lines impaired in either auxin signaling or perception. We found that disruption of auxin signaling in plants expressing an inducible dominant axr2-1 mutation resulted in decreased bacterial growth and that this phenotype was suppressed by introducing the sid2-2 mutation, which impairs SA synthesis. Thus, host auxin signaling is required for normal susceptibility to PtoDC3000 and is involved in suppressing SA-mediated defenses. Unexpectedly, tir1 afb1 afb4 afb5 quadruple-mutant plants lacking four of the six known auxin coreceptors that exhibit decreased auxin perception, supported increased levels of bacterial growth. This mutant exhibited elevated IAA levels and reduced SA-mediated defenses, providing additional evidence that auxin promotes disease by suppressing host defense. We also investigated the hypothesis that IAA promotes PtoDC3000 virulence through a direct effect on the pathogen and found that IAA modulates expression of virulence genes, both in culture and in planta. Thus, in addition to suppressing host defenses, IAA acts as a microbial signaling molecule that regulates bacterial virulence gene expression.

Plant pathogens have evolved several strategies to manipulate the biology of their hosts to facilitate colonization, growth to high levels in plant tissue, and production of disease. One of the less well known of these strategies is the synthesis of plant hormones and hormone analogs, and there is growing evidence that modulation of host hormone signaling is important during pathogenesis. Several plant pathogens produce the auxin indole-3-acetic acid (IAA) and/or virulence factors that modulate host auxin signaling. Auxin is well known for being involved in many aspects of plant growth and development, but recent findings have revealed that elevated IAA levels or enhanced auxin signaling can also promote disease development in some plant-pathogen interactions. In addition to stimulating plant cell growth during infection by gall-forming bacteria, auxin and auxin signaling can antagonize plant defense responses. Auxin can also act as a microbial signaling molecule to impact the biology of some pathogens directly. In this review, we summarize recent progress towards elucidating the roles that auxin production, modification of host auxin signaling, and direct effects of auxin on pathogens play during pathogenesis, with emphasis on the impacts of auxin on interactions with bacterial pathogens., (© The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The bacterial pathogen Pseudomonas syringae modulates plant hormone signaling to promote infection and disease development. P. syringae uses several strategies to manipulate auxin physiology in Arabidopsis thaliana to promote pathogenesis, including its synthesis of indole-3-acetic acid (IAA), the predominant form of auxin in plants, and production of virulence factors that alter auxin responses in the host; however, the role of pathogen-derived auxin in P. syringae pathogenesis is not well understood. Here we demonstrate that P. syringae strain DC3000 produces IAA via a previously uncharacterized pathway and identify a novel indole-3-acetaldehyde dehydrogenase, AldA, that functions in IAA biosynthesis by catalyzing the NAD-dependent formation of IAA from indole-3-acetaldehyde (IAAld). Biochemical analysis and solving of the 1.9 Å resolution x-ray crystal structure reveal key features of AldA for IAA synthesis, including the molecular basis of substrate specificity. Disruption of aldA and a close homolog, aldB, lead to reduced IAA production in culture and reduced virulence on A. thaliana. We use these mutants to explore the mechanism by which pathogen-derived auxin contributes to virulence and show that IAA produced by DC3000 suppresses salicylic acid-mediated defenses in A. thaliana. Thus, auxin is a DC3000 virulence factor that promotes pathogenicity by suppressing host defenses.

The plant hormone auxin is perceived by a family of F-box proteins called the TIR1/AFBs. Phylogenetic studies reveal that these proteins fall into four clades in flowering plants called TIR1, AFB2, AFB4, and AFB6. Genetic studies indicate that members of the TIR1 and AFB2 groups act as positive regulators of auxin signaling by promoting the degradation of the Aux/IAA transcriptional repressors. In this report, we demonstrate that both AFB4 and AFB5 also function as auxin receptors based on in vitro assays. We also provide genetic evidence that AFB4 and AFB5 are targets of the picloram family of auxinic herbicides in addition to indole-3-acetic acid. In contrast to previous studies we find that null afb4 alleles do not exhibit obvious defects in seedling morphology or auxin hypersensitivity. We conclude that AFB4 and AFB5 act in a similar fashion to other members of the family but exhibit a distinct auxin specificity., (Copyright © 2016 Prigge et al.)

Posttranslational lipid modifications mediate the membrane attachment of Rab GTPases, facilitating their function in regulating intracellular vesicular trafficking. In Arabidopsis, most Rab GTPases have two C-terminal cysteines and potentially can be double-geranylgeranylated by heterodimeric Rab geranylgeranyltransferases (Rab-GGTs). Genes encoding two putative α subunits and two putative β subunits of Rab-GGTs have been annotated in the Arabidopsis thaliana genome, but little is known about Rab-GGT activity in Arabidopsis. In this study, we demonstrate that four different heterodimers can be formed between putative Arabidopsis Rab-GGT α subunits RGTA1/RGTA2 and β subunits RGTB1/RGTB2, but only RGTA1·RGTB1 and RGTA1·RGTB2 exhibit bona fide Rab-GGT activity, and they are biochemically redundant in vitro. We hypothesize that RGTA2 function might be disrupted by a 12-amino acid insertion in a conserved motif. We present evidence that Arabidopsis Rab-GGTs may have preference for prenylation of C-terminal cysteines in particular positions. We also demonstrate that Arabidopsis Rab-GGTs can not only prenylate a great variety of Rab GTPases in the presence of Rab escort protein but, unlike Rab-GGT in yeast and mammals, can also prenylate certain non-Rab GTPases independently of Rab escort protein. Our findings may help to explain some of the phenotypes of Arabidopsis protein prenyltransferase mutants., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Auxin is a key plant growth regulator that also impacts plant-pathogen interactions. Several lines of evidence suggest that the bacterial plant pathogen Pseudomonas syringae manipulates auxin physiology in Arabidopsis thaliana to promote pathogenesis. Pseudomonas syringae strategies to alter host auxin biology include synthesis of the auxin indole-3-acetic acid (IAA) and production of virulence factors that alter auxin responses in host cells. The application of exogenous auxin enhances disease caused by P. syringae strain DC3000. This is hypothesized to result from antagonism between auxin and salicylic acid (SA), a major regulator of plant defenses, but this hypothesis has not been tested in the context of infected plants. We further investigated the role of auxin during pathogenesis by examining the interaction of auxin and SA in the context of infection in plants with elevated endogenous levels of auxin. We demonstrated that elevated IAA biosynthesis in transgenic plants overexpressing the YUCCA 1 (YUC1) auxin biosynthesis gene led to enhanced susceptibility to DC3000. Elevated IAA levels did not interfere significantly with host defenses, as effector-triggered immunity was active in YUC1-overexpressing plants, and we observed only minor effects on SA levels and SA-mediated responses. Furthermore, a plant line carrying both the YUC1-overexpression transgene and the salicylic acid induction deficient 2 (sid2) mutation, which impairs SA synthesis, exhibited additive effects of enhanced susceptibility from both elevated auxin levels and impaired SA-mediated defenses. Thus, in IAA overproducing plants, the promotion of pathogen growth occurs independently of suppression of SA-mediated defenses., (© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.)

The jasmonates (JAs) comprise a family of plant hormones that regulate several developmental processes and mediate responses to various abiotic and biotic stresses, including pathogens. JA signalling is manipulated by several strains of the bacterial pathogen Pseudomonas syringae, including P. syringae strain DC3000, using the virulence factor coronatine (COR) as a mimic of jasmonyl-L-isoleucine (JA-Ile). To better understand the JA-Ile-mediated processes contributing to P. syringae disease susceptibility, it is important to investigate the regulation of JA signalling during infection. In Arabidopsis thaliana, JASMONATE ZIM-DOMAIN (JAZ) proteins are negative regulators of JA signalling. The transcription factor JASMONATE INSENSITIVE1 (JIN1/ATMYC2) has been implicated in the regulation of JAZ gene expression. To investigate the regulation of JAZ genes during P. syringae pathogenesis, we examined JAZ gene expression during infection of Arabidopsis by DC3000. We found that eight of the 12 JAZ genes are induced during infection in a COR-dependent manner. Unexpectedly, the induction of the majority of JAZ genes during infection was not dependent on JIN1, indicating that JIN1 is not the only transcription factor regulating JAZ genes. A T-DNA insertion mutant and an RNA interference line disrupted for the expression of JAZ10, one of the few JAZ genes regulated by JIN1 during infection, exhibited enhanced JA sensitivity and increased susceptibility to DC3000, with the primary effect being increased disease symptom severity. Thus, JAZ10 is a negative regulator of both JA signalling and disease symptom development., (© 2011 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2011 BSPP AND BLACKWELL PUBLISHING LTD.)

Plant pathogenic bacteria, such as Pseudomonas syringae pv. tomato strain DC3000, the causative agent of tomato bacterial speck disease, grow to high levels in the apoplastic space between plant cells. Colonization of plant tissue requires expression of virulence factors that modify the apoplast to make it more suitable for pathogen growth or facilitate adaptation of the bacteria to the apoplastic environment. To identify new virulence factors involved in these processes, DC3000 Tn5 transposon insertion mutants with reduced virulence on Arabidopsis thaliana were identified. In one of these mutants, the Tn5 insertion disrupted the malate:quinone oxidoreductase gene (mqo), which encodes an enzyme of the tricarboxylic acid cycle. mqo mutants do not grow to wild-type levels in plant tissue at early time points during infection. Further, plants infected with mqo mutants develop significantly reduced disease symptoms, even when the growth of the mqo mutant reaches wild-type levels at late stages of infection. Mutants lacking mqo function grow more slowly in culture than wild-type bacteria when dicarboxylates are the only available carbon source. To explore whether dicarboxylates are important for growth of DC3000 in the apoplast, we disrupted the dctA1 dicarboxylate transporter gene. DC3000 mutants lacking dctA1 do not grow to wild-type levels in planta, indicating that transport and utilization of dicarboxylates are important for virulence of DC3000. Thus, mqo may be required by DC3000 to meet nutritional requirements in the apoplast and may provide insight into the mechanisms underlying the important, but poorly understood process of adaptation to the host environment.

The Pseudomonas syringae type III effector AvrRpt2 promotes bacterial virulence on Arabidopsis thaliana plants lacking a functional RPS2 gene (rps2 mutant plants). To investigate the mechanisms underlying the virulence activity of AvrRpt2, we examined the phenotypes of transgenic A. thaliana rps2 seedlings constitutively expressing AvrRpt2. These seedlings exhibited phenotypes reminiscent of A. thaliana mutants with altered auxin physiology, including longer primary roots, increased number of lateral roots, and increased sensitivity to exogenous auxin. They also had increased levels of free indole acetic acid (IAA). The presence of AvrRpt2 also was correlated with a further increase in free IAA levels during infection with P. syringae pv. tomato strain DC3000 (PstDC3000). These results indicate that AvrRpt2 alters A. thaliana auxin physiology. Application of the auxin analog 1-naphthaleneacetic acid promoted disease symptom development in PstDC3000-infected plants, suggesting that elevated auxin levels within host tissue promote PstDC3000 virulence. Thus, AvrRpt2 may be among the virulence factors of P. syringae that modulate host auxin physiology to promote disease.

The roles of the phytotoxin coronatine (COR) and salicylic acid (SA)-mediated defenses in the interaction of Pseudomonas syringae pv. tomato DC3000 and tomato (Solanum lycopersicum) were investigated. Unlike findings reported for Arabidopsis thaliana, DC3000 mutants impaired for production of COR or one of its components, coronafacic acid (CFA) or coronamic acid (CMA), induced distinctly different disease lesion phenotypes in tomato. Tomato plants inoculated with the CFA- CMA- mutant DB29 showed elevated transcript levels of SlICS, which encodes isochorismate synthase, an enzyme involved in SA biosynthesis in S. lycopersicum. Furthermore, expression of genes encoding SA-mediated defense proteins were elevated in DB29-inoculated plants compared with plants inoculated with DC3000, suggesting that COR suppresses SlICS-mediated SA responses. Sequence analysis of SlICS revealed that it encodes a protein that is 55 and 59.6% identical to the A. thaliana ICS-encoded proteins AtICS1 and AtICS2, respectively. Tomato plants silenced for SlICS were hypersusceptible to DC3000 and accumulated lower levels of SA after infection with DC3000 compared with inoculated wild-type tomato plants. Unlike what has been shown for A. thaliana, the COR- mutant DB29 was impaired for persistence in SlICS-silenced tomato plants; thus, COR has additional roles in virulence that are SA independent and important in the latter stages of disease development. In summary, the infection assays, metabolic profiling, and gene expression results described in this study indicate that the intact COR molecule is required for both suppression of SA-mediated defense responses and full disease symptom development in tomato.

The phytotoxin coronatine (COR) is produced by various pathovars of Pseudomonas syringae, including P. syringae pv. tomato DC3000, which is pathogenic on crucifers and tomato, and P. syringae pv. glycinea PG4180, a soybean pathogen. The COR molecule contains two distinct components, coronafacic acid (CFA) and coronamic acid (CMA), which are intermediates in the COR biosynthetic pathway. In P. syringae pv. tomato DC3000, it is not clear whether corR, which encodes a response regulator, positively regulates CFA and CMA synthesis as it does in P. syringae pv. glycinea PG4180. In this study, a corR mutant of P. syringae pv. tomato DC3000 was constructed and was shown to be defective in the production of COR, CFA, and CMA. Furthermore, disease severity was greatly reduced in tomato plants inoculated with the corR mutant compared with wild-type P. syringae pv. tomato DC3000. We also showed that a mutation in hrpL, which encodes an alternate RNA polymerase sigma factor (sigmaL) required for the expression of genes encoding components of the type III secretion system, abrogated production of COR in P. syringae pv. tomato DC3000. The presence of a potential hrp box, the recognition site for sigmaL, upstream of corR suggested that corR might be regulated by hrpL. This was confirmed in reverse-transcription polymerase chain reaction experiments showing that the upstream effector gene holPtoAA, which was associated with the hrp box, was cotranscribed with corR. Furthermore, studies also were conducted to investigate whether mutations in corR had effects on the expression of hrpL. The corR mutant of P. syringae pv. tomato DC3000 showed both a reduction and delay in the expression of hrpL and was impaired in its ability to elicit a hypersensitive response on Nicotiana benthamiana. A putative CorR-binding site was identified upstream of hrpL, and gel shift studies confirmed the binding of CorR to this region. These results indicate that corR directly impacts the expression of the hrp regulon in P. syringae.

Many plant pathogens suppress antimicrobial defenses using virulence factors that modulate endogenous host defenses. The Pseudomonas syringae phytotoxin coronatine (COR) is believed to promote virulence by acting as a jasmonate analog, because COR-insensitive 1 (coil) Arabidopsis thaliana and tomato mutants are impaired in jasmonate signaling and exhibit reduced susceptibility to P. syringae. To further investigate the role of jasmonate signaling in disease development, we analyzed several jasmonate-insensitive A. thaliana mutants for susceptibility to P. syringae pv. tomato strain DC3000 and sensitivity to COR. Jasmonate-insensitive 1 (jin1) mutants exhibit both reduced susceptibility to P. syringae pv. tomato DC3000 and reduced sensitivity to COR, whereas jasmonate-resistant 1 (jar1) plants exhibit wild-type responses to both COR and P. syringae pv. tomato DC3000. A jin1 jar1 double mutant does not exhibit enhanced jasmonate insensitivity, suggesting that JIN1 functions downstream of jasmonic acid-amino acid conjugates synthesized by JAR1. Reduced disease susceptibility in jin1 mutants is correlated with elevated expression of pathogenesis-related 1 (PR-1) and is dependent on accumulation of salicylic acid (SA). We also show that JIN1 is required for normal P. syringae pv. tomato DC3000 symptom development through an SA-independent mechanism. Thus, P. syringae pv. tomato DC3000 appears to utilize COR to manipulate JIN1-dependent jasmonate signaling both to suppress SA-mediated defenses and to promote symptom development.

Previously, we conducted a mutant screen of Pseudomonas syringae pv. tomato strain DC3000 to identify genes that contribute to virulence on Arabidopsis thaliana plants. Here we describe the characterization of one mutant strain, DB4H2, which contains a single Tn5 insertion in PSPTO3576, an open reading frame that is predicted to encode a protein belonging to the TetR family of transcriptional regulators. We demonstrate that PSPTO3576 is necessary for virulence in DC3000 and designate the encoded protein TvrR (TetR-like virulence regulator). TvrR, like many other TetR-like transcriptional regulators, negatively regulates its own expression. Despite the presence of a putative HrpL binding site in the tvrR promoter region, tvrR is not regulated by HrpL, an alternative sigma factor that regulates the expression of many known DC3000 virulence genes. tvrR mutant strains grow comparably to wild-type DC3000 in culture and possess an intact type III secretion system. However, tvrR mutants do not cause disease symptoms on inoculated A. thaliana and tomato plants, and their growth within plant tissue is significantly impaired. We demonstrate that tvrR mutant strains are able to synthesize coronatine (COR), a phytotoxin required for virulence of DC3000 on A. thaliana. Given that tvrR mutant strains are not defective for type III secretion or COR production, tvrR appears to be a novel virulence factor required for a previously unexplored process that is necessary for pathogenesis.

SUMMARY Successful pathogen infection likely involves the suppression of general antimicrobial host defences. One Pseudomonas syringae virulence factor proposed to act in this manner is coronatine (COR), a phytotoxin believed to function as an analogue of one or more jasmonates, a family of plant growth regulators. COR biosynthetic (COR(-)) mutants of P. syringae pv. tomato strain DC3000 exhibit reduced virulence on Arabidopsis thaliana and tomato. In the present study, three genetically and biochemically defined COR(-) mutants of DC3000 were used to explore potential effects of COR and its precursors, coronafacic acid (CFA) and coronamic acid (CMA), on defence signalling pathways in A. thaliana. Inoculation with wild-type DC3000 resulted in the accumulation of several jasmonate-responsive transcripts, whereas infection with a mutant strain that accumulates CFA, which is structurally similar to methyl jasmonate (MeJA), did not. Thus, COR, but not CFA, stimulates jasmonate signalling during P. syringae infection of A. thaliana. The ability of the COR(-) mutants to grow to high levels in planta was fully restored in A. thaliana lines deficient for salicylic acid (SA) accumulation. Although the COR(-) mutants grew to high levels in SA-deficient plants, disease symptoms were reduced in these plants. Collectively, these results indicate that COR is required both for overcoming or suppressing SA-dependent defences during growth in plant tissue and for normal disease symptom development in A. thaliana.

In order to cause disease on plants, gram-negative phytopathogenic bacteria introduce numerous virulence factors into the host cell in order to render host tissue more hospitable for pathogen proliferation. The mode of action of such bacterial virulence factors and their interaction with host defense pathways remain poorly understood. avrRpt2, a gene from Pseudomonas syringae pv. tomato JL1065, has been shown to promote the virulence of heterologous P. syringae strains on Arabidopsis thaliana. However, the contribution of avrRpt2 to the virulence of JL1065 has not been examined previously. We show that a mutant derivative of JL1065 that carries a disruption in avrRpt2 is impaired in its ability to cause disease on tomato (Lycopersicon esculentum), indicating that avrRpt2 also acts as a virulence gene in its native strain on a natural host. The virulence activity of avrRpt2 was detectable on tomato lines that are defective in either ethylene perception or the accumulation of salicylic acid, but could not be detected on a tomato mutant insensitive to jasmonic acid. The enhanced virulence conferred by the expression of avrRpt2 in JL1065 was not associated with the suppression of several defense-related genes induced during the infection of tomato.

Plant pathogens can severely reduce host yield and fitness. Thus, investigating the genetic basis of plant response to pathogens is important to further understand plant-pathogen coevolution and to improve crop production. The interaction between Arabidopsis thaliana and Pseudomonas syringae is an important model for studying the genetic basis of plant-pathogen interactions. Studies in this model have led to the discovery of many genes that differentiate a resistant from a susceptible plant. However, little is known about the genetic basis of quantitative variation in response to P. syringae. In this study, we investigate the genetic basis of three aspects of A. thaliana's response to P. syringae: symptom severity, bacterial population size and fruit production using a quantitative trait loci (QTL) analysis. We found two QTL for symptom severity and two for fruit production (possible candidate genes for observed QTL are discussed). We also found significant two-locus epistatic effect on symptom severity and fruit production. Although bacterial population size and symptom severity were strongly phenotypically correlated, we did not detect any QTL for bacterial population size. Despite the detected genetic variation observed for susceptibility, we found only a weak overall relationship between susceptibility traits and fitness, suggesting that these traits may not respond to selection.

AvrRpt2, an effector protein from Pseudomonas syringae pv. tomato (Pst), behaves as an avirulence factor that activates resistance in Arabidopsis thaliana lines expressing the resistance gene RPS2. AvrRpt2 can also enhance pathogen fitness by promoting the ability of the bacteria to grow and to cause disease on susceptible lines of A. thaliana that lack functional RPS2. The activation of RPS2 is coupled to the AvrRpt2-induced disappearance of the A. thaliana RIN4 protein. However, the significance of this RIN4 elimination to AvrRpt2 virulence function is unresolved. To clarify our understanding of the contribution of RIN4 disappearance to AvrRpt2 virulence function, we generated new avrRpt2 alleles by random mutagenesis. We show that the ability of six novel AvrRpt2 mutants to induce RIN4 disappearance correlated well with their avirulence activities but not with their virulence activities. Moreover, the virulence activity of wild-type AvrRpt2 was detectable in an A. thaliana line lacking RIN4. Collectively, these results indicate that the virulence activity of AvrRpt2 in A. thaliana is likely to rely on the modification of host susceptibility factors other than, or in addition to, RIN4.

The avrRpt2 gene from Pseudomonas syringae pv. tomato exhibits avirulence activity on Arabidopsis expressing the resistance gene RPS2 but promotes bacterial virulence on susceptible rps2 Arabidopsis. To understand the functional relationship between the avirulence and virulence activities of avrRpt2, we analyzed a series of six avrRpt2 mutants deficient in eliciting the RPS2-dependent hypersensitive response. We show that the mutants are also severely impaired in triggering RSP2-dependent resistance. Four of these mutants are severely impaired in their virulence activity, whereas two alleles, encoding C-terminal deletions of AvrRpt2, retain significant but slightly reduced virulence activity. Thus, the avirulence and virulence activities of avrRpt2 can be genetically uncoupled. We tested the ability of the two C-terminal deletion mutants to trigger AvrRpt2-induced elimination of the Arabidopsis RIN4 protein and show that they retain this activity but are less efficient than wild-type AvrRpt2. Thus, reduced AvrRpt2 virulence activity is correlated with reduced efficiency in the induction of RIN4 disappearance. This suggests that an alteration in kinetics of RIN4 disappearance triggered by the C-terminal deletion mutants may provide the mechanistic basis for the uncoupling of the avirulence and virulence activities of avrRpt2.

AvrRpt2, a Pseudomonas syringae type III effector protein, functions from inside plant cells to promote the virulence of P. syringae pv. tomato strain DC3000 (PstDC3000) on Arabidopsis thaliana plants lacking a functional copy of the corresponding RPS2 resistance gene. In this study, we extended our understanding of AvrRpt2 virulence activity by exploring the hypothesis that AvrRpt2 promotes PstDC3000 virulence by suppressing plant defenses. When delivered by PstDC3000, AvrRpt2 suppresses pathogen-related (PR) gene expression during infection, suggesting that AvrRpt2 suppresses defenses mediated by salicylic acid (SA). However, AvrRpt2 promotes PstDC3000 growth on transgenic plants expressing the SA-degrading enzyme NahG, indicating that AvrRpt2 does not promote bacterial virulence by modulating SA levels during infection. AvrRpt2 general virulence activity does not depend on the RPM1 resistance gene, as mutations in RPM1 had no effect on AvrRpt2-induced phenotypes. Transgenic plants expressing AvrRpt2 displayed enhanced susceptibility to PstDC3000 strains defective in type III secretion, indicating that enhanced susceptibility of these plants is not because of suppression of defense responses elicited by other type III effectors. Additionally, avrRpt2 transgenic plants did not exhibit increased susceptibility to Peronospora parasitica and Erysiphe cichoracearum, suggesting that AvrRpt2 virulence activity is specific to P. syringae.

To identify Pseudomonas syringae pv. tomato genes involved in pathogenesis, we carried out a screen for Tn5 mutants of P. syringae pv. tomato DC3000 with reduced virulence on Arabidopsis thaliana. Several mutants defining both known and novel virulence loci were identified. Six mutants contained insertions in biosynthetic genes for the phytotoxin coronatine (COR). The P. syringae pv. tomato DC3000 COR genes are chromosomally encoded and are arranged in two separate clusters, which encode enzymes responsible for the synthesis of coronafacic acid (CFA) or coronamic acid (CMA), the two defined intermediates in COR biosynthesis. High-performance liquid chromatography fractionation and exogenous feeding studies confirmed that Tn5 insertions in the cfa and cma genes disrupt CFA and CMA biosynthesis, respectively. All six COR biosynthetic mutants were significantly impaired in their ability to multiply to high levels and to elicit disease symptoms on A. thaliana plants. To assess the relative contributions of CFA, CMA, and COR in virulence, we constructed and characterized cfa6 cmaA double mutant strains. These exhibited virulence phenotypes on A. thalliana identical to those observed for the cmaA or cfa6 single mutants, suggesting that reduced virulence of these mutants on A. thaliana is caused by the absence of the intact COR toxin. This is the first study to use biochemically and genetically defined COR mutants to address the role of COR in pathogenesis.

Gram-negative bacteria use a variety of virulence factors including phytotoxins, exopolysaccharides, effectors secreted by the type III secretion system, and cell-wall-degrading enzymes to promote parasitism in plants. However, little is known about how these virulence factors alter plant cellular responses to promote disease. In this study, we show that virulent Pseudomonas syringae strains activate the transcription of an Arabidopsis ethylene response factor (ERF) gene, RAP2.6, in a coronatine insensitive 1 (COI1)-dependent manner. A highly sensitive RAP2.6 promoter-firefly luciferase (RAP2.6-LUC) reporter line was developed to monitor activities of various bacterial virulence genes. Analyses of P. syringae pv. tomato DC3000 mutants indicated that both type III secretion system and the phytotoxin coronatine are required for RAP2.6 induction. We show that at least five individual type III effectors, avirulence B (AvrB), AvrRpt2, AvrPphB, HopPtoK, and AvrPphEPto, contributed to RAP2.6 induction. Gene-for-gene recognition was not involved in RAP2.6 induction because plants lacking RPM1 and RPS2 responded normally to AvrB and AvrRpt2 in RAP2.6 expression. Interestingly, the role of coronatine in RAP2.6 induction can be partially substituted by the addition of avrB in DC3000, suggesting that AvrB may mimic coronatine. These results suggest that P. syringae type III effectors and coronatine act by augmenting a COI1-dependent pathway to promote parasitism.

Pseudomonas syringae pv. tomato (Pst) strain DC3000 infects the model plants Arabidopsis thaliana and tomato, causing disease symptoms characterized by necrotic lesions surrounded by chlorosis. One mechanism used by Pst DC3000 to infect host plants is the type III protein secretion system, which is thought to deliver multiple effector proteins to the plant cell. The exact number of type III effectors in Pst DC3000 or any other plant pathogenic bacterium is not known. All known type III effector genes of P. syringae are regulated by HrpS, an NtrC family protein, and the HrpL alternative sigma factor, which presumably binds to a conserved cis element (called the "hrp box") in the promoters of type III secretion-associated genes. In this study, we designed a search motif based on the promoter sequences conserved in 12 published hrp operons and putative effector genes in Pst DC3000. Seventy-three predicted genes were retrieved from the January 2001 release of the Pst DC3000 genome sequence, which had 95% genome coverage. The expression of the 73 genes was analysed by microarray and Northern blotting, revealing 24 genes/operons (including eight novel genes), the expression of which was consistently higher in hrp-inducing minimal medium than in nutrient-rich Luria-Bertani broth. Expression of all eight genes was dependent on the hrpS gene. Most were also dependent on the hrpL gene, but at least one was dependent on the hrpS gene, but not on the hrpL gene. An AvrRpt2-based type III translocation assay provides evidence that some of the hrpS-regulated novel genes encode putative effector proteins.

Plant defense in response to microbial attack is regulated through a complex network of signaling pathways that involve three signaling molecules: salicylic acid (SA), jasmonic acid (JA) and ethylene. The SA and JA signaling pathways are mutually antagonistic. This regulatory cross talk may have evolved to allow plants to fine-tune the induction of their defenses in response to different plant pathogens.

Phytopathogenic bacteria possess a large number of genes that allow them to grow and cause disease on plants. Many of these genes should be induced when the bacteria come in contact with plant tissue. We used a modified in vivo expression technology (IVET) approach to identify genes from the plant pathogen Pseudomonas syringae pv. tomato that are induced upon infection of Arabidopsis thaliana and isolated over 500 in planta-expressed (ipx) promoter fusions. Sequence analysis of 79 fusions revealed several known and potential virulence genes, including hrp/hrc, avr and coronatine biosynthetic genes. In addition, we identified metabolic genes presumably important for adaptation to growth in plant tissue, as well as several genes with unknown function that may encode novel virulence factors. Many ipx fusions, including several corresponding to novel genes, are dependent on HrpL, an alternative RNA polymerase sigma factor that regulates the expression of virulence genes. Expression analysis indicated that several ipx fusions are strongly induced upon inoculation into plant tissue. Disruption of one ipx gene, conserved effector locus (CEL) orf1, encoding a putative lytic murein transglycosylase, resulted in decreased virulence of P. syringae. Our results demonstrate that this screen can be used successfully to isolate genes that are induced in planta, including many novel genes potentially involved in pathogenesis.

Plant pathogen resistance is mediated by a large repertoire of resistance (R) genes, which are often clustered in the genome and show a high degree of genetic variation. Here, we show that an Arabidopsis thaliana R-gene cluster is also subject to epigenetic variation. We describe a heritable but metastable epigenetic variant bal that overexpresses the R-like gene At4g16890 from a gene cluster on Chromosome 4. The bal variant and Arabidopsis transgenics overexpressing the At4g16890 gene are dwarfed and constitutively activate the salicylic acid (SA)-dependent defense response pathway. Overexpression of a related R-like gene also occurs in the ssi1 (suppressor of SA insensitivity 1) background, suggesting that ssi1 is mechanistically related to bal.

A new allele of the coronatine-insensitive locus (COI1) was isolated in a screen for Arabidopsis thaliana mutants with enhanced resistance to the bacterial pathogen Pseudomonas syringae. This mutant, designated coi1-20, exhibits robust resistance to several P. syringae isolates but remains susceptible to the virulent pathogens Erisyphe and cauliflower mosaic virus. Resistance to P. syringae strain PstDC3000 in coi1-20 plants is correlated with hyperactivation of PR-1 expression and accumulation of elevated levels of salicylic acid (SA) following infection, suggesting that the SA-mediated defense response pathway is sensitized in this mutant. Restriction of growth of PstDC3000 in coi1-20 leaves is partially dependent on NPR1 and fully dependent on SA, indicating that SA-mediated defenses are required for restriction of PstDC3000 growth in coi1-20 plants. Surprisingly, despite high levels of PstDC3000 growth in coi1-20 plants carrying the salicylate hydroxylase (nahG) transgene, these plants do not exhibit disease symptoms. Thus resistance to P. syringae in coi1-20 plants is conferred by two different mechanisms: (i) restriction of pathogen growth via activation of the SA-dependent defense pathway; and (ii) an SA-independent inability to develop disease symptoms. These findings are consistent with the hypotheses that the P. syringae phytotoxin coronatine acts to promote virulence by inhibiting host defense responses and by promoting lesion formation.

Several bacterial avr genes have been shown to contribute to virulence on susceptible plants lacking the corresponding resistance (R) gene. The mechanisms by which avr genes promote parasitism and disease, however, are not well understood. We investigated the role of the Pseudomonas syringae pv. tomato avrRpt2 gene in pathogenesis by studying the interaction of P. syringae pv. tomato strain PstDC3000 expressing avrRpt2 with several Arabidopsis thaliana lines lacking the corresponding R gene, RPS2. We found that PstDC3000 expressing avrRpt2 grew to significantly higher levels and often resulted in the formation of more severe disease symptoms in ecotype No-0 plants carrying a mutant RPS2 allele, as well as in two Col-0 mutant lines, cpr5 rps2 and coil rps2, that exhibit enhanced resistance. We also generated transgenic A. thaliana lines expressing avrRpt2 and demonstrated, by using several different assays, that expression of avrRpt2 within the plant also promotes virulence of PstDC3000. Thus, AvrRpt2 appears to promote pathogen virulence from within the plant cell.

Abstract To identify virulence genes of P. syringae pv. tomato strain DC3000 we screened for mutants with reduced virulence on its plant hosts, Arabidopsis thaliana and tomato. We isolated a Tn5-insertion mutant that exhibited reduced virulence on both hosts. Further characterization showed that this mutant carried a single Tn5 insertion in the dsbA gene, which encodes a periplasmic disulphide bond-forming protein. In addition to reduced virulence, the dsbA mutant exhibits mucoid colony morphology, loss of fluorescence, decreased motility, and a reduced growth rate in culture. The dsbA mutant is able to multiply in A. thaliana and tomato plants, trigger the hypersensitive response on tobacco and elicit Pto-mediated resistance in tomato, indicating that type III secretion occurs in this background. However, type III secretion appears to function with reduced efficiency in the dsbA mutant, as type III-dependent secretion of HrpZ and AvrRpt2 is impaired. These findings indicate that while the dsbA gene is required for multiple cellular functions in P. syringae, type III secretion in P. syringae is only partially dependent on dsbA.

Phenylalanine, tyrosine, and tryptophan have a dual biosynthetic role in plants; they are required for protein synthesis and are also precursors to a number of aromatic secondary metabolites critical to normal development and stress responses. Whereas much has been learned in recent years about the genetic control of tryptophan biosynthesis in Arabidopsis and other plants, relatively little is known about the genetic regulation of phenylalanine and tyrosine synthesis. We have isolated, characterized and determined the expression of Arabidopsis thaliana genes encoding chorismate mutase, the enzyme catalyzing the first committed step in phenylalanine and tyrosine synthesis. Three independent Arabidopsis chorismate mutase cDNAs were isolated by functional complementation of a Saccharomyces cerevisiae mutation. Two of these cDNAs have been reported independently (Eberhard et al., 1993. FEBS 334, 233-236; Eberhard et al., 1996. Plant J. 10, 815-821), but the third (designated CM-3) represents a novel gene. The different organ-specific expression patterns of these cDNAs, their regulation in response to pathogen infiltration, as well as the different enzymatic characteristics of the proteins they encode are also described. Together, these data suggest that each isoform may play a distinct physiological role in coordinating chorismate mutase activity with developmental and environmental signals.

The RPS2 gene in Arabidopsis thaliana governs resistance to strains of the bacterial pathogen, Pseudomonas syringae pv. tomato, that express the avrRpt2 gene. The two loci are involved in a gene-for-gene interaction. Seventeen accessions of A. thaliana were sequenced to explore the diversity present in the coding region of the RPS2 locus. An unusually high level of nucleotide polymorphisms was found (1.26%), with nearly half of the observed polymorphisms resulting in amino acid changes in the RPS2 protein. Seven haplotypes (alleles) were identified and their evolutionary relationships deduced. Several of the alleles conferring resistance were found to be closely related, whereas susceptibility to disease was conferred by widely divergent alleles. The possibility of selection at the RPS2 locus is discussed.

We report the existence of a small open reading frame (usd) that is located between the promoter and coding sequence for the sporulation gene spoIIID in Bacillus subtilis. The mRNA from the usd-spoIIID operon contains an inverted repeat sequence that is predicted to form a stem-loop structure that would sequester the ribosome binding site for spoIIID. A mutation eliminating the ribosome binding site for the upstream open reading frame caused an oligosporogenous phenotype and interfered with the translation, but not the transcription, of the downstream gene spoIIID. We propose that efficient synthesis of SpoIIID requires that the putative stem-loop structure be disrupted by translation through the upstream open reading frame.

Plant resistance to disease caused by phytopathogenic organisms is often triggered by the ability of the plant to specifically recognize the invading pathogen. One of the most fascinating areas in plant biology research focusses on understanding the mechanisms governing this process. Several recent breakthroughs in this area have come from the genetic analyses of disease resistance in Arabidopsis thaliana.

Plant disease resistance genes function is highly specific pathogen recognition pathways. PRS2 is a resistance gene of Arabidopsis thaliana that confers resistance against Pseudomonas syringae bacteria that express avirulence gene avrRpt2. RPS2 was isolated by the use of a positional cloning strategy. The derived amino acid sequence of RPS2 contains leucine-rich repeat, membrane-spanning, leucine zipper, and P loop domains. The function of the RPS2 gene product in defense signal transduction is postulated to involve nucleotide triphosphate binding and protein-protein interactions and may also involve the reception of an elicitor produced by the avirulent pathogen.

The interaction between Arabidopsis thaliana and the bacterium Pseudomonas syringae is being developed as a model experimental system for plant pathology research. Race-specific ("gene-for-gene") resistance has been demonstrated for this interaction, and pathogen genes that determine avirulence have been isolated and characterized. Because certain lines of both Arabidopsis and soybean are resistant to bacteria carrying the avirulence genes avrRpt2 and avrB, extremely similar pathogen recognition mechanisms are apparently present in these two plant species. Isogenic bacterial strains that differ by the presence of single avirulence genes are being used to analyze plant resistance. Plant resistance genes have been identified in crosses between resistant and susceptible lines. The extensive map-based cloning tools available in Arabidopsis are being used to isolate these resistance genes. In a related project, ethylene-insensitive Arabidopsis mutants are being used to examine the role of ethylene in disease development. Ethylene apparently mediates symptom formation in susceptible plants and is not required for resistance, suggesting possible strategies for enhancement of disease tolerance in crops.

A molecular genetic approach was used to identify and characterize plant genes that control bacterial disease resistance in Arabidopsis. A screen for mutants with altered resistance to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) expressing the avirulence gene avrRpt2 resulted in the isolation of four susceptible rps (resistance to P. syringae) mutants. The rps mutants lost resistance specifically to bacterial strains expressing avrRpt2 as they retained resistance to Pst strains expressing the avirulence genes avrB or avrRpm1. Genetic analysis indicated that in each of the four rps mutants, susceptibility was due to a single mutation mapping to the same locus on chromosome 4. Identification of a resistance locus with specificity for a single bacterial avirulence gene suggests that this locus, designated RPS2, controls specific recognition of bacteria expressing the avirulence gene avrRpt2. Ecotype Wü-0, a naturally occurring line that is susceptible to Pst strains expressing avrRpt2, appears to lack a functional allele at RPS2, demonstrating that there is natural variation at the RPS2 locus among wild populations of Arabidopsis.

The avrRpt2 locus from Pseudomonas syringae pv. tomato causes virulent strains of P. syringae to be avirulent on some, but not all, lines of Arabidopsis thaliana and Glycine max (soybean). We determined the DNA sequence of the avrRpt2 locus and identified the avrRpt2 gene as a 768-bp open reading frame encoding a putative 28.2-kDa protein. Deletion analysis and transcription studies provided further evidence that this open reading frame encodes AvrRpt2. We found that the avrRpt2 gene also has avirulence activity in P. syringae pathogens of Phaseolus vulgaris (common bean), suggesting that disease resistance genes specific to avrRpt2 are functionally conserved among diverse plant species. The predicted AvrRpt2 protein is hydrophilic and contains no obvious membrane-spanning domains or export signal sequences, and there was no significant similarity of AvrRpt2 to sequences in the GenBank, EMBL, or Swiss PIR data bases. A comparison of the avrRpt2 DNA sequence to nine other P. syringae avirulence genes revealed a highly conserved sequence, GGAACCNA-N14-CCACNNA, upstream of the translation initiation codon. This motif is located 6 to 8 nucleotides upstream of the transcription start site in all four P. syringae avirulence genes for which a transcription start site has been determined, suggesting a role as a binding site for a novel form of RNA polymerase. Regulation of avrRpt2 was similar to other P. syringae avirulence genes; expression was high in minimal medium and low in rich medium and depended on the hrpRS locus and an additional locus at the opposite end of the hrp cluster of P. syringae pv. tomato.