Plants and animals possess innate immune systems to prevent infections and are effectively "nonhosts" for most potential pathogens. The molecular mechanisms underlying nonhost immunity in plants remain obscure. In Arabidopsis, nonhost/nonpathogenic Pseudomonas syringae sustains but pathogenic P. syringae suppresses early MAMP (microbe-associated molecular pattern) marker-gene activation. We performed a cell-based genetic screen of virulence factors and identified AvrPto and AvrPtoB as potent and unique suppressors of early-defense gene transcription and MAP kinase (MAPK) signaling. Unlike effectors of mammalian pathogens, AvrPto and AvrPtoB intercept multiple MAMP-mediated signaling upstream of MAPKKK at the plasma membrane linked to the receptor. In transgenic Arabidopsis, AvrPto blocks early MAMP signaling and enables nonhost P. syringae growth. Deletions of avrPto and avrPtoB from pathogenic P. syringae reduce its virulence. The studies reveal a fundamental role of MAMP signaling in nonhost immunity, and a novel action of type III effectors from pathogenic bacteria.

The model pathogen Pseudomonas syringae pv. tomato DC3000 causes bacterial speck in tomato and Arabidopsis, but Nicotiana benthamiana, an important model plant, is considered to be a non-host. Strain DC3000 injects approximately 28 effector proteins into plant cells via the type III secretion system (T3SS). These proteins were individually delivered into N. benthamiana leaf cells via T3SS-proficient Pseudomonas fluorescens, and eight, including HopQ1-1, showed some capacity to cause cell death in this test. Four gene clusters encoding 13 effectors were deleted from DC3000: cluster II (hopH1, hopC1), IV (hopD1, hopQ1-1, hopR1), IX (hopAA1-2, hopV1, hopAO1, hopG1), and native plasmid pDC3000A (hopAM1-2, hopX1, hopO1-1, hopT1-1). DC3000 mutants deleted for cluster IV or just hopQ1-1 acquired the ability to grow to high levels and produce bacterial speck lesions in N. benthamiana. HopQ1-1 showed other hallmarks of an avirulence determinant in N. benthamiana: expression in the tobacco wildfire pathogen P. syringae pv. tabaci 11528 rendered this strain avirulent in N. benthamiana, and elicitation of the hypersensitive response in N. benthamiana by HopQ1-1 was dependent on SGT1. DC3000 polymutants involving other effector gene clusters in a hopQ1-1-deficient background revealed that clusters II and IX contributed to the severity of lesion symptoms in N. benthamiana, as well as in Arabidopsis and tomato. The results support the hypothesis that the host ranges of P. syringae pathovars are limited by the complex interactions of effector repertoires with plant anti-effector surveillance systems, and they demonstrate that N. benthamiana can be a useful model host for DC3000

The ability of Pseudomonas syringae pv. tomato DC3000 to parasitize tomato and Arabidopsis thaliana depends on genes activated by the HrpL alternative sigma factor. To support various functional genomic analyses of DC3000, and specifically, to identify genes involved in pathogenesis, we developed a draft sequence of DC3000 and used an iterative process involving computational and gene expression techniques to identify virulence-implicated genes downstream of HrpLresponsive promoters. Hypersensitive response and pathogenicity (Hrp) promoters are known to control genes encoding the Hrp (type III protein secretion) machinery and a few type III effector proteins in DC3000. This process involved (i) identification of 9 new virulenceimplicated genes in the Hrp regulon by miniTn5gus mutagenesis, (ii) development of a hidden Markov model (HMM) trained with known and transposon-identified Hrp promoter sequences, (iii) HMM identification of promoters upstream of 12 additional virulence-implicated genes, and (iv) microarray and RNA blot analyses of the HrpLdependent expression of a representative subset of these DC3000 genes. We found that the Hrp regulon encodes candidates for 4 additional type III secretion machinery accessory factors, homologs of the effector proteins HopPsyA, AvrPpiB1 (2 copies), AvrPpiC2, AvrPphD (2 copies), AvrPphE, AvrPphF, and AvrXv3, and genes associated with the production or metabolism of virulence factors unrelated to the Hrp type III secretion system, including syringomycin synthetase (SyrE), N -(indole-3-acetyl)-L-lysine synthetase (IaaL), and a subsidiary regulon controlling coronatine production. Additional candidate effector genes, hopPtoA2, hopPtoB2, and an avrRps4 homolog, were preceded by Hrp promoter-like sequences, but these had HMM expectation values of relatively low significance and were not detectably activated by HrpL. P seudomonas syringae pv. tomato DC3000 has emerged as an important model organism in molecular plant pathology because of its genetic tractability, its pathogenicity on both tomato and Arabidopsis thaliana, and its ability to deliver virulence effector proteins into host cells via a type III protein secretion system. To support worldwide functional genomic investigations of P. s. tomato DC3000, we are determining the complete sequence of the DC3000 genome. A draft sequence is now available (http:͞͞www.tigr.org͞tdb͞mdb͞ mdbinprogress.html), and we have initiated a genomewide investigation of potential virulence factors with a search for virulencerelated genes in the same regulon as the type III secretion system.Virulence effector proteins delivered to or into host cells by type III secretion systems are key factors in the pathogenicity of many bacteria, including animal pathogens in the genera Salmonella, Yersinia, Shigella, and Escherichia, and plant pathogens in the genera Pseudomonas, Erwinia, Xanthomonas, Ralstonia, and Pantoea (1). In plant pathogens, the type III secretion machinery is referred to as the hypersensitive response and pathogenicity (Hrp) system ...

The Pto serine/threonine kinase of tomato confers resistance to speck disease by recognizing strains of Pseudomonas syringae that express the protein AvrPto. Pto and AvrPto physically interact, and this interaction is required for activation of host resistance. We identified a second Pseudomonas protein, AvrPtoB, that interacts specifically with Pto and is widely distributed among plant pathogens. AvrPtoB is delivered into the plant cell by the bacterial type III secretion system, and it elicits Pto-specific defenses. AvrPtoB has little overall sequence similarity with AvrPto. However, AvrPto amino acids, which are required for interaction with Pto, are present in AvrPtoB and required for its interaction with Pto. Thus, two distinct bacterial effectors activate plant immunity by interacting with the same host protein kinase through a similar structural mechanism.

AvrPto and AvrPtoB are type III effector proteins expressed by Pseudomonas syringae pv. tomato strain DC3000, a pathogen of both tomato and Arabidopsis spp. Each effector physically interacts with the tomato Pto kinase and elicits a hypersensitive response when expressed in tomato leaves containing Pto. An avrPto deletion mutant of DC3000 previously was shown to retain avirulence activity on Pto-expressing tomato plants. We developed an avrPtoB deletion mutant of DC3000 and found that it also retains Pto-specific avirulence on tomato. These observations suggested that avrPto and avrPtoB both contribute to avirulence. To test this hypothesis, we developed an deltaavrPtodeltaavrPtoB double mutant in DC3000. This double mutant was able to cause disease on a Pto-expressing tomato line. Thus, avrPto and avrPtoB are the only avirulence genes in DC3000 that elicit Pto-mediated defense responses in tomato. When inoculated onto susceptible tomato leaves and compared with wild-type DC3000, the mutants DC3000deltaavrPto and DC3000deltaavrPtoB each caused slightly less severe disease symptoms, although their growth rate was unaffected. However, DC3000deltaavr PtodeltaavrPtoB caused even less severe disease symptoms than the single mutants and grew more slowly than them on susceptible leaves. Our results indicate that AvrPto and AvrPtoB have phenotypically redundant avirulence activity on Pto-expressing tomato and additive virulence activities on susceptible tomato plants.

AvrPtoB is a type III effector protein from Pseudomonas syringae pv. tomato that physically interacts with the tomato Pto kinase and, depending on the host genotype, either elicits or suppresses programmed cell death associated with plant immunity. We reported previously that avrPtoB-related sequences are present in diverse gram-negative phytopathogenic bacteria. Here we describe characterization of avrPtoB homologs from P. syringae pv. tomato T1, PT23, and JL1065, P. syringae pv. syringae B728a, and P. syringae pv. maculicola ES4326. The avrPtoB homolog from P. syringae pv. maculicola, hopPmaL, was identified previously. The four new genes identified in this study are designated avrPtoB T1 , avrPtoB PT23 , avrPtoB JL1065 , and avrPtoB B728a . The AvrPtoB homologs exhibit 52 to 66% amino acid identity with AvrPtoB. Transcripts of each of the avrPtoB homologs were detected in the Pseudomonas strains from which they were isolated. Proteins encoded by the homologs were detected in all strains except P. syringae pv. tomato T1, suggesting that T1 suppresses accumulation of AvrPtoB T1 . All of the homologs interacted with the Pto kinase in a yeast two-hybrid system and elicited a Pto-dependent defense response when they were delivered into leaf cells by DC3000⌬avrPto⌬avrPtoB, a P. syringae pv. tomato strain with a deletion of both avrPto and avrPtoB. Like AvrPtoB, all of the homologs enhanced the ability of DC3000⌬avrPto⌬avrPtoB to form lesions on leaves of two susceptible tomato lines. With the exception of HopPmaL which lacks the C-terminal domain, all AvrPtoB homologs suppressed programmed cell death elicited by the AvrPto-Pto interaction in an Agrobacterium-mediated transient assay. Thus, despite their divergent sequences, AvrPtoB homologs from diverse P. syringae pathovars have conserved avirulence and virulence activities similar to AvrPtoB activity.

dWhen analyzing the secretome of the plant pathogen Pseudomonas syringae pv. tomato DC3000, we identified hemolysin-coregulated protein (Hcp) as one of the secreted proteins. Hcp is assumed to be an extracellular component of the type VI secretion system (T6SS). Two copies of hcp genes are present in the P. syringae pv. tomato DC3000 genome, hcp1 (PSPTO_2539) and hcp2 (PSPTO_5435). We studied the expression patterns of the hcp genes and tested the fitness of hcp knockout mutants in host plant colonization and in intermicrobial competition. We found that the hcp2 gene is expressed most actively at the stationary growth phase and that the Hcp2 protein is secreted via the T6SS and appears in the culture medium as covalently linked dimers. Expression of hcp2 is not induced in planta and does not contribute to virulence in or colonization of tomato or Arabidopsis plants. Instead, hcp2 is required for survival in competition with enterobacteria and yeasts, and its function is associated with the suppression of the growth of these competitors. This is the first report on bacterial T6SS-associated genes functioning in competition with yeast. Our results suggest that the T6SS of P. syringae may play an important role in bacterial fitness, allowing this plant pathogen to survive under conditions where it has to compete with other microorganisms for resources.

The molecular basis underlying the ability of pathogens to infect certain plant species and not others is largely unknown. Pseudomonas syringae is a useful model species for investigating this phenomenon because it comprises more than 50 pathovars which have narrow host range specificities. Tomato (Solanum lycopersicum) is a host for P. syringae pv. tomato, the causative agent of bacterial speck disease, but is considered a nonhost for other P. syringae pathovars. Host resistance in tomato to bacterial speck disease is conferred by the Pto protein kinase which acts in concert with the Prf nucleotide-binding lucine-rich repeat protein to recognize P. syringae pv. tomato strains expressing the type III effectors AvrPto or AvrPtoB (HopAB2). The Pto and Prf genes were isolated from the wild tomato species S. pimpinellifolium and functional alleles of both of these genes now are known to exist in many species of tomato and in other Solanaceous species. Here, we extend earlier reports that avrPto and avrPtoB genes are widely distributed among pathovars of P. syringae which are considered nonhost pathogens of tomato. This observation prompted us to examine the possibility that recognition of these type III effectors by Pto or Prf might contribute to the inability of many P. syringae pathovars to infect tomato species. We show that 10 strains from presumed nonhost P. syringae pathovars are able to grow and cause pathovar-unique disease symptoms in tomato leaves lacking Pto or Prf, although they did not reach the population levels or cause symptoms as severe as a control P. syringae pv. tomato strain. Seven of these strains were found to express avrPto or avrPtoB. The AvrPto- and AvrPtoB-expressing strains elicited disease resistance on tomato leaves expressing Pto and Prf. Thus, a gene-for-gene recognition event may contribute to host range restriction of many P. syringae pathovars on tomato species. Furthermore, we conclude that the diverse disease symptoms caused by different Pseudomonas pathogens on their normal plant hosts are due largely to the array of virulence factors expressed by each pathovar and not to specific molecular or morphological attributes of the plant host.