Genomewide Identification of Pseudomonas Syringae Pv. Tomato DC3000 Promoters Controlled by the HrpL Alternative Sigma Factor

Proceedings of the National Academy of Sciences of the United States of America. Feb, 2002  |  Pubmed ID: 11854524

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 HrpL-responsive 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 virulence-implicated 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 HrpL-dependent 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(epsilon)-(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.

Tomato Transcription Factors Pti4, Pti5, and Pti6 Activate Defense Responses when Expressed in Arabidopsis

The Plant Cell. Apr, 2002  |  Pubmed ID: 11971137

The Pti4, Pti5, and Pti6 proteins from tomato were identified based on their interaction with the product of the Pto disease resistance gene, a Ser-Thr protein kinase. They belong to the ethylene-response factor (ERF) family of plant-unique transcription factors and bind specifically to the GCC-box cis element present in the promoters of many pathogenesis-related (PR) genes. Here, we show that these tomato ERFs are localized to the nucleus and function in vivo as transcription activators that regulate the expression of GCC box-containing PR genes. Expression of Pti4, Pti5, or Pti6 in Arabidopsis activated the expression of the salicylic acid-regulated genes PR1 and PR2. Expression of jasmonic acid- and ethylene-regulated genes, such as PR3, PR4, PDF1.2, and Thi2.1, was affected differently by each of the three tomato ERFs, with Arabidopsis-Pti4 plants having very high levels of PDF1.2 transcripts. Exogenous application of salicylic acid to Arabidopsis-Pti4 plants suppressed the increased expression of PDF1.2 but further stimulated PR1 expression. Arabidopsis plants expressing Pti4 displayed increased resistance to the fungal pathogen Erysiphe orontii and increased tolerance to the bacterial pathogen Pseudomonas syringae pv tomato. These results indicate that Pti4, Pti5, and Pti6 activate the expression of a wide array of PR genes and play important and distinct roles in plant defense.

Two Distinct Pseudomonas Effector Proteins Interact with the Pto Kinase and Activate Plant Immunity

Cell. May, 2002  |  Pubmed ID: 12062102

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.

The Tobacco Salicylic Acid-binding Protein 3 (SABP3) is the Chloroplast Carbonic Anhydrase, Which Exhibits Antioxidant Activity and Plays a Role in the Hypersensitive Defense Response

Proceedings of the National Academy of Sciences of the United States of America. Sep, 2002  |  Pubmed ID: 12185253

In plants, salicylic acid (SA) plays an important role in signaling both local and systemic defense responses. Previous efforts to identify SA effector proteins in tobacco have led to the isolation of two soluble cytoplasmic SA-binding proteins (SABPs): catalase, SABP, and an approximately 25-kDa protein, SABP2. Here we describe the identification of an SA-binding protein, SABP3, in the stroma of tobacco chloroplasts. SABP3 bound SA with an apparent dissociation constant (K(d)) of 3.7 microM and exhibited much greater affinity for biologically active than inactive analogs. Purification and partial sequencing of SABP3 indicated that it is the chloroplast carbonic anhydrase (CA). Confirming this finding, recombinant tobacco chloroplast CA exhibited both CA enzymatic and SA-binding activities. Expression of this protein in yeast also demonstrated that CA/SABP3 has antioxidant activity. A second gene encoding CA was also cloned, and its encoded protein was shown to behave similarly to that purified as SABP3. Finally, silencing of CA gene expression in leaves suppressed the Pto:avrPto-mediated hypersensitive response in disease resistance. These results demonstrate that SA may act through multiple effector proteins in plants and shed further light on the function of CA in chloroplasts.

1H, 15N and 13C Chemical Shift Assignments of the Structured Core of the Pseudomonas Effector Protein AvrPto

Journal of Biomolecular NMR. Jul, 2002  |  Pubmed ID: 12238599

Comprehensive Transcript Profiling of Pto- and Prf-mediated Host Defense Responses to Infection by Pseudomonas Syringae Pv. Tomato

The Plant Journal : for Cell and Molecular Biology. Nov, 2002  |  Pubmed ID: 12410809

The disease resistance gene Pto encodes a serine/threonine protein kinase that confers resistance in tomato to Pseudomonas syringae pv. tomato strains that express the effector protein AvrPto. Pto-mediated resistance to bacterial speck disease also requires Prf, a protein with leucine-rich repeats and a putative nucleotide-binding site, although the role of Prf in the defense pathway is not known. We used GeneCalling, an open-architecture, mRNA-profiling technology, to identify genes that are either induced or suppressed in leaves 4 h after bacterial infection in the Pto- and Prf-mediated tomato-Pseudomonas(avrPto) interaction. Over 135 000 individual cDNA fragments representing an estimated 90% of the transcripts expressed in tomato leaves were examined and 432 differentially expressed genes were identified. The genes encode over 25 classes of proteins including 11 types of transcription factors and many signal transduction components. Differential expression of 91% of the genes required both Pto and Prf. Interestingly, differential expression of 32 genes did not require Pto but was dependent on Prf. Thus, our data support a role for Prf early in the Pto pathway and indicate that Prf can also function as an independent host recognition determinant of bacterial infection. Comprehensive expression profiling of the Pto-mediated defense response allows the development of many new hypotheses about the molecular basis of resistance to bacterial speck disease.

Location and Activity of Members of a Family of VirPphA Homologues in Pathovars of Pseudomonas Syringae and P. Savastanoi

Molecular Plant Pathology. Jul, 2002  |  Pubmed ID: 20569328

Summary virPphA is a major determinant of the pathogenicity of Pseudomonas savastanoi pv. phaseolicola to Phaseolus bean. A family of homologues of virPphA was detected in pathovars of P. savastanoi and P. syringae. We examined the structure and activity of alleles designated virPphA, virPphA(Pgy), and virPphA(Psv) from P. savastanoi pathovars phaseolicola, glycinea, and savastanoi, respectively, and avrPtoB from P. syringae pv. tomato. Sequencing showed that the virPphA(Pgy) homologue had a 48-bp central deletion in the open reading frame (ORF) compared with virPphA and virPphA(Psv), but otherwise all three P. savastanoi alleles had > 98% identity at the DNA level. By contrast, AvrPtoB from P. syringae pv. tomato strain DC3000 was predicted to have only 51% amino acid similarity with VirPphA. All ORFs have an upstream hrp-box promoter indicating potential regulation by HrpL. Each cloned homologue was introduced into the P. savastanoi pv. phaseolicola strain RW60, which lacks a native plasmid carrying virPphA as part of a pathogenicity island (PAI), and which is not pathogenic on bean. The homologues all restored virulence, as measured by the development of water-soaked lesions in bean pods, and increased bacterial populations in leaves compared with RW60 alone. RW60 harbouring virPphA or virPphA(Psv) elicited a strong hypersensitive reaction (HR) in soybean cv. Osumi; the presence of avrPtoB caused a weak HR, but virPphA(Pgy) did not affect the null reaction observed in soybean with RW60 alone. A second effector gene, avrPphD, was detected on the genomic clones carrying virPphA(Pgy) and virPphA(Psv). avrPphD was also present in both P. savastanoi pv. phaseolicola and P. syringae pv. tomato, but elsewhere in their genomes. Comparison of the genomic locations of virPphA and other effectors found in the P. savastanoi pv. phaseolicola PAI revealed the greatest divergence of the sequences surrounding virPphA to be in P. syringae pv. tomato.

Pseudomonas Type III Effector AvrPtoB Induces Plant Disease Susceptibility by Inhibition of Host Programmed Cell Death

The EMBO Journal. Jan, 2003  |  Pubmed ID: 12505984

The AvrPtoB type III effector protein is conserved among diverse genera of plant pathogens suggesting it plays an important role in pathogenesis. Here we report that Pseudomonas AvrPtoB acts inside the plant cell to inhibit programmed cell death (PCD) initiated by the Pto and Cf9 disease resistance proteins and, remarkably, the pro-apoptotic mouse protein Bax. AvrPtoB also suppressed PCD in yeast, demonstrating that AvrPtoB functions as a cell death inhibitor across kingdoms. Using truncated AvrPtoB proteins, we identified distinct N- and C-terminal domains of AvrPtoB that are sufficient for host recognition and PCD inhibition, respectively. We also identified a novel resistance phenotype, Rsb, that is triggered by an AvrPtoB truncation disrupted in the anti-PCD domain. A Pseudomonas syringae pv. tomato DC3000 strain with a chromosomal mutation in the AvrPtoB C-terminus elicited Rsb-mediated immunity in previously susceptible tomato plants and disease was restored when full-length AvrPtoB was expressed in trans. Thus, our results indicate that a type III effector can induce plant susceptibility to bacterial infection by inhibiting host PCD.

Overexpression of the Disease Resistance Gene Pto in Tomato Induces Gene Expression Changes Similar to Immune Responses in Human and Fruitfly

Plant Physiology. Aug, 2003  |  Pubmed ID: 12913147

The Pto gene encodes a serine/threonine protein kinase that confers resistance in tomato (Lycopersicon esculentum) to Pseudomonas syringae pv tomato strains that express the type III effector protein AvrPto. Constitutive overexpression of Pto in tomato, in the absence of AvrPto, activates defense responses and confers resistance to several diverse bacterial and fungal plant pathogens. We have used a series of gene discovery and expression profiling methods to examine the effect of Pto overexpression in tomato leaves. Analysis of the tomato expressed sequence tag database and suppression subtractive hybridization identified 600 genes that were potentially differentially expressed in Pto-overexpressing tomato plants compared with a sibling line lacking Pto. By using cDNA microarrays, we verified changes in expression of many of these genes at various time points after inoculation with P. syringae pv tomato (avrPto) of the resistant Pto-overexpressing line and the susceptible sibling line. The combination of these three approaches led to the identification of 223 POR (Pto overexpression responsive) genes. Strikingly, 40% of the genes induced in the Pto-overexpressing plants previously have been shown to be differentially expressed during the human (Homo sapiens) and/or fruitfly (Drosophila melanogaster) immune responses.

The Complete Genome Sequence of the Arabidopsis and Tomato Pathogen Pseudomonas Syringae Pv. Tomato DC3000

Proceedings of the National Academy of Sciences of the United States of America. Sep, 2003  |  Pubmed ID: 12928499

We report the complete genome sequence of the model bacterial pathogen Pseudomonas syringae pathovar tomato DC3000 (DC3000), which is pathogenic on tomato and Arabidopsis thaliana. The DC3000 genome (6.5 megabases) contains a circular chromosome and two plasmids, which collectively encode 5,763 ORFs. We identified 298 established and putative virulence genes, including several clusters of genes encoding 31 confirmed and 19 predicted type III secretion system effector proteins. Many of the virulence genes were members of paralogous families and also were proximal to mobile elements, which collectively comprise 7% of the DC3000 genome. The bacterium possesses a large repertoire of transporters for the acquisition of nutrients, particularly sugars, as well as genes implicated in attachment to plant surfaces. Over 12% of the genes are dedicated to regulation, which may reflect the need for rapid adaptation to the diverse environments encountered during epiphytic growth and pathogenesis. Comparative analyses confirmed a high degree of similarity with two sequenced pseudomonads, Pseudomonas putida and Pseudomonas aeruginosa, yet revealed 1,159 genes unique to DC3000, of which 811 lack a known function.

Understanding the Functions of Plant Disease Resistance Proteins

Annual Review of Plant Biology. 2003  |  Pubmed ID: 14502984

Many disease resistance (R) proteins of plants detect the presence of disease-causing bacteria, viruses, or fungi by recognizing specific pathogen effector molecules that are produced during the infection process. Effectors are often pathogen proteins that probably evolved to subvert various host processes for promotion of the pathogen life cycle. Five classes of effector-specific R proteins are known, and their sequences suggest roles in both effector recognition and signal transduction. Although some R proteins may act as primary receptors of pathogen effector proteins, most appear to play indirect roles in this process. The functions of various R proteins require phosphorylation, protein degradation, or specific localization within the host cell. Some signaling components are shared by many R gene pathways whereas others appear to be pathway specific. New technologies arising from the genomics and proteomics revolution will greatly expand our ability to investigate the role of R proteins in plant disease resistance.

Molecular Basis of Pto-mediated Resistance to Bacterial Speck Disease in Tomato

Annual Review of Phytopathology. 2003  |  Pubmed ID: 14527329

The Pto gene in tomato confers gene-for-gene resistance to Pseudomonas syringae pv. tomato, the causative agent of bacterial speck disease. Pto was first introgressed from a wild species of tomato into cultivated tomato varieties over 60 years ago and is now widely used to control speck disease. Cloning of the Pto gene revealed that it encodes a cytoplasmically localized serine-threonine protein kinase. The molecular basis of gene-for-gene recognition in this pathosystem is the direct physical interaction of the Pto kinase with either of two Pseudomonas effector proteins, AvrPto and AvrPtoB. Upon recognition of AvrPto or AvrPtoB, the Pto kinase acts in concert with Prf, a leucine-rich repeat-containing protein, to activate multiple signal transduction pathways. There has been much progress in understanding the evolutionary origin of the Pto gene, structural details about how the Pto kinase interacts with AvrPto and AvrPtoB, signaling steps downstream of Pto, and defense responses activated by the Pto pathway. Future work on this model system will focus on how the interaction of the Pto kinase with bacterial effector proteins activates signal transduction, defining the specific role of signaling components, and ultimately, determining which host defense responses are most responsible for inhibiting growth of the pathogen and suppressing symptoms of bacterial speck disease.

The Tomato Transcription Factor Pti4 Regulates Defense-related Gene Expression Via GCC Box and Non-GCC Box Cis Elements

The Plant Cell. Dec, 2003  |  Pubmed ID: 14630974

The tomato transcription factor Pti4, an ethylene-responsive factor (ERF), interacts physically with the disease resistance protein Pto and binds the GCC box cis element that is present in the promoters of many pathogenesis-related (PR) genes. We reported previously that Arabidopsis plants expressing Pti4 constitutively express several GCC box-containing PR genes and show reduced disease symptoms compared with wild-type plants after inoculation with Pseudomonas syringae pv tomato or Erysiphe orontii. To gain insight into how genome-wide gene expression is affected by Pti4, we used serial analysis of gene expression (SAGE) to compare transcripts in wild-type and Pti4-expressing Arabidopsis plants. SAGE provided quantitative measurements of >20,000 transcripts and identified the 50 most highly expressed genes in Arabidopsis vegetative tissues. Comparison of the profiles from wild-type and Pti4-expressing Arabidopsis plants revealed 78 differentially abundant transcripts encoding defense-related proteins, protein kinases, ribosomal proteins, transporters, and two transcription factors (TFs). Many of the genes identified were expressed differentially in wild-type Arabidopsis during infection by Pseudomonas syringae pv tomato, supporting a role for them in defense-related processes. Unexpectedly, the promoters of most Pti4-regulated genes did not have a GCC box. Chromatin immunoprecipitation experiments confirmed that Pti4 binds in vivo to promoters lacking this cis element. Potential binding sites for ERF, MYB, and GBF TFs were present in statistically significantly increased numbers in promoters regulated by Pti4. Thus, Pti4 appears to regulate gene expression directly by binding the GCC box and possibly a non-GCC box element and indirectly by either activating the expression of TF genes or interacting physically with other TFs.

Two MAPK Cascades, NPR1, and TGA Transcription Factors Play a Role in Pto-mediated Disease Resistance in Tomato

The Plant Journal : for Cell and Molecular Biology. Dec, 2003  |  Pubmed ID: 14675454

The tomato Pto kinase confers resistance to the causative agent of bacterial speck disease, Pseudomonas syringae pv. tomato, by recognizing the pathogen effector proteins AvrPto or AvrPtoB. Pto-mediated resistance requires multiple signal transduction pathways and has been shown to activate many defense responses including an oxidative burst, rapid changes in the expression of over 400 genes, and localized cell death. We have tested the role in Pto-mediated resistance in tomato of a set of 21 genes from other species known to be involved in defense-related signaling. Expression of each gene was suppressed by virus-induced gene silencing (VIGS) and the effect on disease symptoms and bacterial growth during the tomato-Pseudomonas incompatible interaction was determined. We found that Pto-mediated resistance was compromised by silencing of genes encoding two mitogen-activated protein (MAP) kinase kinases, MEK1 and MEK2, two MAP kinases, NTF6 and wound-induced protein kinase (WIPK), a key regulator of systemic acquired resistance (SAR), NPR1, and two transcription factors, TGA1a and TGA2.2. A lesser impact on Pto-mediated resistance was observed in plants silenced for RAR1 and COI1. The identification of nine genes that play a role in resistance to bacterial speck disease both advances our knowledge of Pto signal transduction and demonstrates the conservation of many defense signaling components among diverse plant species.

Silencing of Subfamily I of Protein Phosphatase 2A Catalytic Subunits Results in Activation of Plant Defense Responses and Localized Cell Death

The Plant Journal : for Cell and Molecular Biology. May, 2004  |  Pubmed ID: 15125764

The central importance of protein phosphorylation in plant defense responses has been demonstrated by the isolation of several disease-resistance genes that encode protein kinases. In addition, there are many reports of changes in protein phosphorylation accompanying plant responses to pathogens. In contrast, little is known about the role of protein dephosphorylation in regulating plant defenses. We report that expression of the LePP2Ac1 gene, which encodes a catalytic subunit of the heterotrimeric protein phosphatase 2A (PP2Ac), is rapidly induced in resistant tomato leaves upon inoculation with an avirulent strain of Pseudomonas syringae pv. tomato. By analysis of PP2Ac gene sequences from several plant species, we found that PP2Ac genes cluster into two subfamilies, with LePP2Ac1 belonging to subfamily I. Virus-induced gene silencing (VIGS) in Nicotiana benthamiana was used to suppress expression of genes from subfamily I and not from subfamily II. The PP2Ac-silenced plants had greatly decreased PP2A activity, constitutively expressed pathogenesis-related (PR) genes, and developed localized cell death in stems and leaves. In addition, the plants were more resistant to a virulent strain of P. syringae pv. tabaci and showed an accelerated hypersensitive response (HR) to effector proteins from both P. syringae and the fungal pathogen, Cladosporium fulvum. Thus, catalytic subunits of PP2Ac subfamily I act as negative regulators of plant defense responses likely by de-sensitizing protein phosphorylation cascades.

Suppression of Pathogen-inducible NO Synthase (iNOS) Activity in Tomato Increases Susceptibility to Pseudomonas Syringae

Proceedings of the National Academy of Sciences of the United States of America. May, 2004  |  Pubmed ID: 15146069

Inducible NO synthase (iNOS) activity is induced upon pathogen inoculation in resistant, but not susceptible, tobacco and Arabidopsis plants. It was shown recently that a variant form of the Arabidopsis P protein (AtvarP) has iNOS activity. P protein is part of the glycine decarboxylase complex (GDC). It is unclear whether P protein also has iNOS activity and, if so, whether AtvarP, P, or both, play a role in plant defense. Here, we show that iNOS activity is induced in both resistant and susceptible tomato leaves upon inoculation with the Pseudomonas syringae pv. tomato strain DC3000. Virus-induced gene-silencing targeting LevarP, a putative tomato ortholog of AtvarP, led to complete suppression of DC3000-induced iNOS activation and an approximately 80% reduction in GDC activity; it also increased disease-symptom severity and DC3000 growth in both resistant and susceptible tomato. To determine whether enhanced susceptibility exhibited by LevarP-silenced, susceptible tomato was due to loss of (i) iNOS activity, (ii) GDC activity, or (iii) both, GDC activity was inhibited with or without concurrent suppression of iNOS. Treatment with methotrexate inhibited both iNOS and GDC activities and resulted in increased susceptibility, comparable with that observed in LevarP-silenced plants. When normal iNOS activity was maintained in the presence of methotrexate by the addition of tetrahydrobiopterin, there was no change in susceptibility, despite a dramatic reduction in GDC activity. Together, these results indicate that iNOS contributes to host defense response against DC3000.

PeerGAD: a Peer-review-based and Community-centric Web Application for Viewing and Annotating Prokaryotic Genome Sequences

Nucleic Acids Research. 2004  |  Pubmed ID: 15184545

PeerGAD is a web-based database-driven application that allows community-wide peer-reviewed annotation of prokaryotic genome sequences. The application was developed to support the annotation of the Pseudomonas syringae pv. tomato strain DC3000 genome sequence and is easily portable to other genome sequence annotation projects. PeerGAD incorporates several innovative design and operation features and accepts annotations pertaining to gene naming, role classification, gene translation and annotation derivation. The annotator tool in PeerGAD is built around a genome browser that offers users the ability to search and navigate the genome sequence. Because the application encourages annotation of the genome sequence directly by researchers and relies on peer review, it circumvents the need for an annotation curator while providing added value to the annotation data. Support for the Gene Ontology vocabulary, a structured and controlled vocabulary used in classification of gene roles, is emphasized throughout the system. Here we present the underlying concepts integral to the functionality of PeerGAD.

Strategies Used by Bacterial Pathogens to Suppress Plant Defenses

Current Opinion in Plant Biology. Aug, 2004  |  Pubmed ID: 15231256

Plant immune systems effectively prevent infections caused by the majority of microbial pathogens that are encountered by plants. However, successful pathogens have evolved specialized strategies to suppress plant defense responses and induce disease susceptibility in otherwise resistant hosts. Recent advances reveal that phytopathogenic bacteria use type III effector proteins, toxins, and other factors to inhibit host defenses. Host processes that are targeted by bacteria include programmed cell death, cell wall-based defense, hormone signaling, the expression of defense genes, and other basal defenses. The discovery of plant defenses that are vulnerable to pathogen attack has provided new insights into mechanisms that are essential for both bacterial pathogenesis and plant disease resistance.

The Solution Structure of Type III Effector Protein AvrPto Reveals Conformational and Dynamic Features Important for Plant Pathogenesis

Structure (London, England : 1993). Jul, 2004  |  Pubmed ID: 15242602

Pseudomonas syringae pv. tomato, the causative agent of bacterial speck disease of tomato, uses a type III secretion system (TTSS) to deliver effector proteins into the host cell. In resistant plants, the bacterial effector protein AvrPto physically interacts with the host Pto kinase and elicits antibacterial defense responses. In susceptible plants, which lack the Pto kinase, AvrPto acts as a virulence factor to promote bacterial growth. The solution structure of AvrPto reveals a functional core consisting of a three-helix bundle motif flanked by disordered N- and C-terminal tails. Residues required for Pto binding lie in a 19 residue Omega loop. Modeling suggests a hydrophobic patch involving the activation loop of Pto forms a contact surface with the AvrPto Omega loop and that helix packing mediates interactions between AvrPto and putative virulence targets Api2 and Api3. The AvrPto structure has a low stability that may facilitate chaperone-independent secretion by the TTSS.

MAPKKKalpha is a Positive Regulator of Cell Death Associated with Both Plant Immunity and Disease

The EMBO Journal. Aug, 2004  |  Pubmed ID: 15272302

Many plant pathogens cause disease symptoms that manifest over days as regions of localized cell death. Localized cell death (the hypersensitive response; HR) also occurs in disease-resistant plants, but this response appears within hours of attempted infection and may restrict further pathogen growth. We identified a MAP kinase kinase kinase gene (MAPKKKalpha) that is required for the HR and resistance against Pseudomonas syringae. Significantly, we found that MAPKKKalpha also regulates cell death in susceptible leaves undergoing P. syringae infection. Overexpression of MAPKKKalpha in leaves activated MAPKs and caused pathogen-independent cell death. By overexpressing MAPKKKalpha in leaves and suppressing expression of various MAPKK and MAPK genes by virus-induced gene silencing, we identified two distinct MAPK cascades that act downstream of MAPKKKalpha. These results demonstrate that signal transduction pathways associated with both plant immunity and disease susceptibility share a common molecular switch.

Applications and Advantages of Virus-induced Gene Silencing for Gene Function Studies in Plants

The Plant Journal : for Cell and Molecular Biology. Sep, 2004  |  Pubmed ID: 15315635

Virus-induced gene silencing (VIGS) is a recently developed gene transcript suppression technique for characterizing the function of plant genes. The approach involves cloning a short sequence of a targeted plant gene into a viral delivery vector. The vector is used to infect a young plant, and in a few weeks natural defense mechanisms of the plant directed at suppressing virus replication also result in specific degradation of mRNAs from the endogenous plant gene that is targeted for silencing. VIGS is rapid (3-4 weeks from infection to silencing), does not require development of stable transformants, allows characterization of phenotypes that might be lethal in stable lines, and offers the potential to silence either individual or multiple members of a gene family. Here we briefly review the discoveries that led to the development of VIGS and what is known about the experimental requirements for effective silencing. We describe the methodology of VIGS and how it can be optimized and used for both forward and reverse genetics studies. Advantages and disadvantages of VIGS compared with other loss-of-function approaches available for plants are discussed, along with how the limitations of VIGS might be overcome. Examples are reviewed where VIGS has been used to provide important new insights into the roles of specific genes in plant development and plant defense responses. Finally, we examine the future prospects for VIGS as a powerful tool for assessing and characterizing the function of plant genes.

Comprehensive EST Analysis of Tomato and Comparative Genomics of Fruit Ripening

The Plant Journal : for Cell and Molecular Biology. Oct, 2004  |  Pubmed ID: 15361140

A large tomato expressed sequence tag (EST) dataset (152 635 total) was analyzed to gain insights into differential gene expression among diverse plant tissues representing a range of developmental programs and biological responses. These ESTs were clustered and assembled to a total of 31 012 unique gene sequences. To better understand tomato gene expression at a plant system level and to identify differentially expressed and tissue-specific genes, we developed and implemented a digital expression analysis protocol. By clustering genes according to their relative abundance in the various EST libraries, expression patterns of genes across various tissues were generated and genes with similar patterns were grouped. In addition, tissues themselves were clustered for relatedness based on relative gene expression as a means of validating the integrity of the EST data as representative of relative gene expression. Arabidopsis and grape EST collections were also characterized to facilitate cross-species comparisons where possible. Tomato fruit digital expression data was specifically compared with publicly available grape EST data to gain insight into molecular manifestation of ripening processes across diverse taxa and resulted in identification of common transcription factors not previously associated with ripening.

Identification of MAPKs and Their Possible MAPK Kinase Activators Involved in the Pto-mediated Defense Response of Tomato

The Journal of Biological Chemistry. Nov, 2004  |  Pubmed ID: 15371431

The Pto kinase mediates resistance to bacterial speck disease in tomato by activating host defenses upon recognition of Pseudomonas syringae pv. tomato strains expressing the AvrPto or AvrPtoB proteins. Previous gene-silencing experiments have indicated that mitogen activated protein kinase (MAPK) cascades play a key role downstream of the Pto kinase to activate host defense responses. Here we use biochemical methods to demonstrate that two tomato MAPKs, LeMPK2 and LeMPK3, are activated in leaves in a Pto-specific manner upon expression of AvrPto and AvrPtoB. We show that these same MAPKs are activated upon overexpression of LeMAPKKKalpha, a protein previously demonstrated to be involved in Pto-mediated immunity. We identified two phylogenetically unrelated MAPK kinases (LeMKK2 and LeMKK4) that when overexpressed in leaves elicit cell death and activate LeMPK2 and LeMPK3. In vitro analysis demonstrated that LeMKK2 and LeMKK4 each phosphorylate the same subset of three MAPKs. Together these data provide biochemical evidence for the involvement of MAPK cascades in Pto-mediated resistance.

Suppression of Pathogen-inducible NO Synthase (iNOS) Activity in Tomato Increases Susceptibility to Pseudomonas Syringae

Proceedings of the National Academy of Sciences of the United States of America. Nov, 2004  |  Pubmed ID: 15516371

Identification and Expression Profiling of Tomato Genes Differentially Regulated During a Resistance Response to Xanthomonas Campestris Pv. Vesicatoria

Molecular Plant-microbe Interactions : MPMI. Nov, 2004  |  Pubmed ID: 15553246

The gram-negative bacterium Xanthomonas campestris pv. vesicatoria is the causal agent of spot disease in tomato and pepper. Plants of the tomato line Hawaii 7981 are resistant to race T3 of X. campestris pv. vesicatoria expressing the type III effector protein AvrXv3 and develop a typical hypersensitive response upon bacterial challenge. A combination of suppression subtractive hybridization and microarray analysis identified a large set of cDNAs that are induced or repressed during the resistance response of Hawaii 7981 plants to X. campestris pv. vesicatoria T3 bacteria. Sequence analysis of the isolated cDNAs revealed that they correspond to 426 nonredundant genes, which were designated as XRE (Xanthomonas-regulated) genes and were classified into more than 20 functional classes. The largest functional groups contain genes involved in defense, stress responses, protein synthesis, signaling, and photosynthesis. Analysis of XRE expression kinetics during the tomato resistance response to X. campestris pv. vesicatoria T3 revealed six clusters of genes with coordinate expression. In addition, by using isogenic X. campestris pv. vesicatoria T2 strains differing only by the avrXv3 avirulence gene, we found that 77% of the identified XRE genes were directly modulated by expression of the AvrXv3 effector protein. Interestingly, 64% of the XRE genes were also induced in tomato during an incompatible interaction with an avirulent strain of Pseudomonas syringae pv. tomato. The identification and expression analysis of X. campestris pv. vesicatoria T3-modulated genes, which may be involved in the control or in the execution of plant defense responses, set the stage for the dissection of signaling and cellular responses activated in tomato plants during the onset of spot disease resistance.

An AvrPto/avrPtoB Mutant of Pseudomonas Syringae Pv. Tomato DC3000 Does Not Elicit Pto-mediated Resistance and is Less Virulent on Tomato

Molecular Plant-microbe Interactions : MPMI. Jan, 2005  |  Pubmed ID: 15672817

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: a Bacterial Type III Effector That Both Elicits and Suppresses Programmed Cell Death Associated with Plant Immunity

FEMS Microbiology Letters. Apr, 2005  |  Pubmed ID: 15796972

Pseudomonas syringae pv. tomato DC3000 is a model pathogen for studying the molecular basis of plant immunity and disease susceptibility in tomato and Arabidopsis. DC3000 uses a type III secretion system to inject effector proteins into the plant cell. Type III effectors are thought to promote bacterial virulence by suppressing plant defenses and enhancing access to nutrients trapped in the plant cell. The AvrPtoB type III effector elicits immunity-associated programmed cell death (PCD) when expressed in tomato plants carrying the Pto resistance protein. However, in the absence of Pto, AvrPtoB functions to suppress PCD and immunity in tomato. Here, we review current research examining the molecular basis of AvrPtoB-mediated elicitation and suppression of plant PCD. In addition, the "trump model" is proposed to explain how resistance proteins successfully elicit immunity-associated PCD in response to effectors that suppress PCD.

Role of Mitogen-activated Protein Kinases in Plant Immunity

Current Opinion in Plant Biology. Oct, 2005  |  Pubmed ID: 16043387

Studies of the early events that follow pathogen recognition have established the importance of mitogen-activated protein kinase (MAPK) cascades in plant defense signaling. Specific MAPK cascade components have now been identified in several species, and so it has become possible to compare defense responses to diverse pathogens and to elucidate downstream events that are initiated by these key signaling molecules. Recent work utilizing post-transcriptional gene silencing techniques and the development of stable transgenic lines for both gain- and loss-of-function analysis have further advanced our understanding of the role of MAPK cascades in plant immunity.

Pseudomonas Syringae Pv. Tomato Type III Effectors AvrPto and AvrPtoB Promote Ethylene-dependent Cell Death in Tomato

The Plant Journal : for Cell and Molecular Biology. Oct, 2005  |  Pubmed ID: 16167902

The type III secretion system (TTSS) of Pseudomonas syringae pv. tomato (Pst) injects into the plant cell effector proteins that play an essential role in the formation of bacterial speck disease. To investigate the molecular roles of TTSS effectors in disease formation, we used a cDNA microarray to analyze the expression of approximately 8600 random tomato genes in response to wild-type Pst strain DC3000 and a mutant lacking a functional TTSS. Many of the differentially expressed genes identified encode proteins associated with hormone response or hormone biosynthesis pathways. Using isogenic mutant strains of DC3000, we monitored host transcriptional changes in response to the TTSS effector proteins AvrPto and AvrPtoB, both of which are important virulence factors on susceptible tomato lines. We found that AvrPto and AvrPtoB induce a set of host genes involved in ethylene biosynthesis and signaling, and in particular they regulate the expression of two genes, LeACO1 and LeACO2, encoding the ethylene-forming enzyme ACC oxidase. Analysis of transgenic tomato lines with diminished ACC oxidase activity revealed that ethylene production by the host is required for the full virulence activity of both AvrPto and AvrPtoB. AvrPto and AvrPtoB therefore appear to promote enhanced disease in tomato leaves, in part, by upregulating genes involved in ethylene production.

Calmodulin-like Proteins from Arabidopsis and Tomato Are Involved in Host Defense Against Pseudomonas Syringae Pv. Tomato

Plant Molecular Biology. Aug, 2005  |  Pubmed ID: 16240180

Complex signal transduction pathways underlie the myriad plant responses to attack by pathogens. Ca(2+) is a universal second messenger in eukaryotes that modulates various signal transduction pathways through stimulus-specific changes in its intracellular concentration. Ca(2+)-binding proteins such as calmodulin (CaM) detect Ca(2+) signals and regulate downstream targets as part of a coordinated cellular response to a given stimulus. Here we report the characterization of a tomato gene (APR134) encoding a CaM-related protein that is induced in disease-resistant leaves in response to attack by Pseudomonas syringae pv. tomato. We show that suppression of APR134 gene expression in tomato (Solanum lycopersicum), using virus-induced gene silencing (VIGS), compromises the plant's immune response. We isolated APR134-like genes from Arabidopsis, termed CML42 and CML43, to investigate whether they serve a functionally similar role. Gene expression analysis revealed that CML43 is rapidly induced in disease-resistant Arabidopsis leaves following inoculation with Pseudomonas syringae pv. tomato. Overexpression of CML43 in Arabidopsis accelerated the hypersensitive response. Recombinant APR134, CML42, and CML43 proteins all bind Ca(2+ )in vitro. Collectively, our data support a role for CML43, and APR134 as important mediators of Ca(2+)-dependent signals during the plant immune response to bacterial pathogens.

Transcriptome and Selected Metabolite Analyses Reveal Multiple Points of Ethylene Control During Tomato Fruit Development

The Plant Cell. Nov, 2005  |  Pubmed ID: 16243903

Transcriptome profiling via cDNA microarray analysis identified 869 genes that are differentially expressed in developing tomato (Solanum lycopersicum) pericarp. Parallel phenotypic and targeted metabolite comparisons were employed to inform the expression analysis. Transcript accumulation in tomato fruit was observed to be extensively coordinated and often completely dependent on ethylene. Mutation of an ethylene receptor (Never-ripe [Nr]), which reduces ethylene sensitivity and inhibits ripening, alters the expression of 37% of these 869 genes. Nr also influences fruit morphology, seed number, ascorbate accumulation, carotenoid biosynthesis, ethylene evolution, and the expression of many genes during fruit maturation, indicating that ethylene governs multiple aspects of development both prior to and during fruit ripening in tomato. Of the 869 genes identified, 628 share homology (E-value < or = 1 x 10(-10)) with known gene products or known protein domains. Of these 628 loci, 72 share homology with previously described signal transduction or transcription factors, suggesting complex regulatory control. These results demonstrate multiple points of ethylene regulatory control during tomato fruit development and provide new insights into the molecular basis of ethylene-mediated ripening.

Adi3 is a Pdk1-interacting AGC Kinase That Negatively Regulates Plant Cell Death

The EMBO Journal. Jan, 2006  |  Pubmed ID: 16362044

Bacterial speck disease in tomato is caused by Pseudomonas syringae pv. tomato. Resistance to this disease is conferred by the host Pto kinase, which recognizes P. s. pv. tomato strains that express the effector AvrPto. We report here that an AvrPto-dependent Pto-interacting protein 3 (Adi3) is a member of the AGC family of protein kinases. In mammals, AGC kinases are regulated by 3-phosphoinositide-dependent protein kinase-1 (Pdk1). We characterized tomato Pdk1 and showed that Pdk1 and Pto phosphorylate Adi3. Gene silencing of Adi3 in tomato causes MAPKKKalpha-dependent formation of necrotic lesions. Use of a chemical inhibitor of Pdk1, OSU-03012, also implicates Pdk1 and Adi3 in plant cell death regulation. Adi3 thus appears to function analogously to the mammalian AGC kinase protein kinase B/Akt by negatively regulating cell death via Pdk1 phosphorylation. We speculate that the negative regulatory function of Adi3 might be subverted by interaction with Pto/AvrPto, leading to host cell death that is associated with pathogen attack.

A Bacterial Inhibitor of Host Programmed Cell Death Defenses is an E3 Ubiquitin Ligase

Science (New York, N.Y.). Jan, 2006  |  Pubmed ID: 16373536

The Pseudomonas syringae protein AvrPtoB is translocated into plant cells, where it inhibits immunity-associated programmed cell death (PCD). The structure of a C-terminal domain of AvrPtoB that is essential for anti-PCD activity reveals an unexpected homology to the U-box and RING-finger components of eukaryotic E3 ubiquitin ligases, and we show that AvrPtoB has ubiquitin ligase activity. Mutation of conserved residues involved in the binding of E2 ubiquitin-conjugating enzymes abolishes this activity in vitro, as well as anti-PCD activity in tomato leaves, which dramatically decreases virulence. These results show that Pseudomonas syringae uses a mimic of host E3 ubiquitin ligases to inactivate plant defenses.

Diverse AvrPtoB Homologs from Several Pseudomonas Syringae Pathovars Elicit Pto-dependent Resistance and Have Similar Virulence Activities

Applied and Environmental Microbiology. Jan, 2006  |  Pubmed ID: 16391110

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 DC3000DeltaavrPtoDeltaavrPtoB, a P. syringae pv. tomato strain with a deletion of both avrPto and avrPtoB. Like AvrPtoB, all of the homologs enhanced the ability of DC3000DeltaavrPtoDeltaavrPtoB 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.

Host-mediated Phosphorylation of Type III Effector AvrPto Promotes Pseudomonas Virulence and Avirulence in Tomato

The Plant Cell. Feb, 2006  |  Pubmed ID: 16399801

The AvrPto protein from Pseudomonas syringae pv tomato is delivered into plant cells by the bacterial type III secretion system, where it either promotes host susceptibility or, in tomato plants expressing the Pto kinase, elicits disease resistance. Using two-dimensional gel electrophoresis, we obtained evidence that AvrPto is phosphorylated when expressed in plant leaves. In vitro phosphorylation of AvrPto by plant extracts occurs independently of Pto and is due to a kinase activity that is conserved in tomato (Solanum lycopersicum), tobacco (Nicotiana tabacum), and Arabidopsis thaliana. Three Ser residues clustered in the C-terminal 18 amino acids of AvrPto were identified in vitro as putative phosphorylation sites, and one site at S149 was directly confirmed as an in vivo phosphorylation site by mass spectrometry. Substitution of Ala for S149 significantly decreased the ability of AvrPto to enhance disease symptoms and promote growth of P. s. tomato in susceptible tomato leaves. In addition, S149A significantly decreased the avirulence activity of AvrPto in resistant tomato plants. Our observations support a model in which AvrPto has evolved to mimic a substrate of a highly conserved plant kinase to enhance its virulence activity. Furthermore, residues of AvrPto that promote virulence are also monitored by plant defenses.

Type III Effector AvrPtoB Requires Intrinsic E3 Ubiquitin Ligase Activity to Suppress Plant Cell Death and Immunity

Proceedings of the National Academy of Sciences of the United States of America. Feb, 2006  |  Pubmed ID: 16477026

Microbial pathogens of both plants and animals employ virulence factors that suppress the host immune response. The tomato pathogen Pseudomonas syringae injects the AvrPtoB type III effector protein into the plant cell to suppress programmed cell death (PCD) associated with plant immunity. AvrPtoB also inhibits PCD in yeast, indicating that AvrPtoB manipulates a conserved component of eukaryotic PCD. To identify host targets of AvrPtoB, we performed a yeast two-hybrid screen and identified tomato ubiquitin (Ub) as a strong AvrPtoB interactor. AvrPtoB is ubiquitinated in vitro and exhibits E3 Ub ligase activity in the presence of recombinant E1 activating enzyme and specific E2 Ub-conjugating enzymes. The C terminus of AvrPtoB is sufficient for both anti-PCD and E3 Ub ligase activities, suggesting the two functions are associated. Indeed, mutation of AvrPtoB lysine residues in the C terminus, between K512 and K529, disrupts AvrPtoB-Ub interactions, decreases AvrPtoB-mediated anti-PCD activity, and abrogates P. syringae pathogenesis of susceptible tomato plants. Remarkably, quantitative decreases in AvrPtoB anti-PCD activity are correlated with decreases in AvrPtoB ubiquitination and E3 Ub ligase activity. Overall, these data reveal a unique bacterial pathogenesis strategy, where AvrPtoB manipulates the host Ub system and requires intrinsic E3 Ub ligase activity to suppress plant immunity.

Specific Bacterial Suppressors of MAMP Signaling Upstream of MAPKKK in Arabidopsis Innate Immunity

Cell. May, 2006  |  Pubmed ID: 16678099

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.

Bacterial Elicitation and Evasion of Plant Innate Immunity

Nature Reviews. Molecular Cell Biology. Aug, 2006  |  Pubmed ID: 16936700

Recent research on plant responses to bacterial attack has identified extracellular and intracellular host receptors that recognize conserved pathogen-associated molecular patterns and more specialized virulence proteins, respectively. These findings have shed light on our understanding of the molecular mechanisms by which bacteria elicit host defences and how pathogens have evolved to evade or suppress these defences.

Comparative Genomics of Host-specific Virulence in Pseudomonas Syringae

Genetics. Oct, 2006  |  Pubmed ID: 16951068

While much study has gone into characterizing virulence factors that play a general role in disease, less work has been directed at identifying pathogen factors that act in a host-specific manner. Understanding these factors will help reveal the variety of mechanisms used by pathogens to suppress or avoid host defenses. We identified candidate Pseudomonas syringae host-specific virulence genes by searching for genes whose distribution among natural P. syringae isolates was statistically associated with hosts of isolation. We analyzed 91 strains isolated from 39 plant hosts by DNA microarray-based comparative genomic hybridization against an array containing 353 virulence-associated (VA) genes, including 53 type III secretion system effectors (T3SEs). We identified individual genes and gene profiles that were significantly associated with strains isolated from cauliflower, Chinese cabbage, soybean, rice, and tomato. We also identified specific horizontal gene acquisition events associated with host shifts by mapping the array data onto the core genome phylogeny of the species. This study provides the largest suite of candidate host-specificity factors from any pathogen, suggests that there are multiple ways in which P. syringae isolates can adapt to the same host, and provides insight into the evolutionary mechanisms underlying host adaptation.

Whole-genome Expression Profiling Defines the HrpL Regulon of Pseudomonas Syringae Pv. Tomato DC3000, Allows De Novo Reconstruction of the Hrp Cis Clement, and Identifies Novel Coregulated Genes

Molecular Plant-microbe Interactions : MPMI. Nov, 2006  |  Pubmed ID: 17073300

Pseudomonas syringae pv. tomato DC3000 is a model pathogen of tomato and Arabidopsis that uses a hypersensitive response and pathogenicity (Hrp) type III secretion system (T3SS) to deliver virulence effector proteins into host cells. Expression of the Hrp system and many effector genes is activated by the HrpL alternative sigma factor. Here, an open reading frame-specific whole-genome microarray was constructed for DC3000 and used to comprehensively identify genes that are differentially expressed in wild-type and deltahrpL strains. Among the genes whose differential regulation was statistically significant, 119 were upregulated and 76 were downregulated in the wild-type compared with the deltahrpL strain. Hierarchical clustering revealed a subset of eight genes that were upregulated particularly rapidly. Gibbs sampling of regions upstream of HrpL-activated operons revealed the Hrp promoter as the only identifiable regulatory motif and supported an iterative refinement involving real-time polymerase chain reaction testing of additional HrpL-activated genes and refinements in a hidden Markov model that can be used to predict Hrp promoters in P. syringae strains. This iterative bioinformatic-experimental approach to a comprehensive analysis of the HrpL regulon revealed a mix of genes controlled by HrpL, including those encoding most type III effectors, twin-arginine transport (TAT) substrates, other regulatory proteins, and proteins involved in the synthesis or metabolism of phytohormones, phytotoxins, and myo-inositol. This analysis provides an extensively verified, robust method for predicting Hrp promoters in P. syringae genomes, and it supports subsequent identification of effectors and other factors that likely are important to the host-specific virulence of P. syringae.

A Novel Link Between Tomato GRAS Genes, Plant Disease Resistance and Mechanical Stress Response

Molecular Plant Pathology. Nov, 2006  |  Pubmed ID: 20507472

SUMMARY Members of the GRAS family of transcriptional regulators have been implicated in the control of plant growth and development, and in the interaction of plants with symbiotic bacteria. Here we examine the complexity of the GRAS gene family in tomato (Solanum lycopersicum) and investigate its role in disease resistance and mechanical stress. A large number of tomato ESTs corresponding to GRAS transcripts were retrieved from the public database and assembled in 17 contigs of putative genes. Expression analysis of these genes by real-time RT-PCR revealed that six SlGRAS transcripts accumulate during the onset of disease resistance to Pseudomonas syringae pv. tomato. Further analysis of two selected family members showed that their transcripts preferentially accumulate in tomato plants in response to different avirulent bacteria or to the fungal elicitor EIX, and their expression kinetics correlate with the appearance of the hypersensitive response. In addition, transcript levels of eight SlGRAS genes, including all the Pseudomonas-inducible family members, increased in response to mechanical stress much earlier than upon pathogen attack. Accumulation of SlGRAS transcripts following mechanical stress was in part dependent on the signalling molecule jasmonic acid. Remarkably, suppression of SlGRAS6 gene expression by virus-induced gene silencing impaired tomato resistance to P. syringae pv. tomato. These results support a function for GRAS transcriptional regulators in the plant response to biotic and abiotic stress.

DspA/E, a Type III Effector of Erwinia Amylovora, is Required for Early Rapid Growth in Nicotiana Benthamiana and Causes NbSGT1-dependent Cell Death

Molecular Plant Pathology. May, 2007  |  Pubmed ID: 20507497

SUMMARY DspA/E is a pathogenicity factor of Erwinia amylovora that is translocated into the plant cell cytoplasm through an Hrp type III secretion system. Transient expression of dspA/E in Nicotiana benthamiana or yeast induced cell death, as it does in N. tabacum and apple as described previously. DspA/E-induced cell death in N. benthamiana was not inhibited by coexpression of AvrPtoB of Pseudomonas syringae pv. tomato, which inhibits programmed cell death (PCD) induced by several other elicitors in plants. Silencing of NbSGT1, the expression of which is required for PCD mediated by several resistance proteins of plants, prevented DspA/E-induced cell death in N. benthamiana. However, silencing of NbRAR1, or two MAP kinase kinase genes, which are required for PCD associated with many resistance genes in plants, did not prevent cell death induced by DspA/E. Silencing of NbSGT1 also compromised non-host resistance against E. amylovora. E. amylovora grew rapidly within the first 24 h after infiltration in N. benthamiana, and DspA/E was required for this early rapid growth. However, bacterial cell numbers decreased after 24 h in TRV-vector-transformed plants, whereas a dspA/E mutant strain grew to high populations in NbSGT1-silenced plants. Our results indicate that DspA/E enhances virulence of E. amylovora in N. benthamiana, but the bacteria are then recognized by the plant, resulting in PCD and death of bacterial cells or restriction of bacterial cell growth.

Manipulation of Plant Programmed Cell Death Pathways During Plant-pathogen Interactions

Plant Signaling & Behavior. May, 2007  |  Pubmed ID: 19704693

The interaction of plants with bacterial pathogens involves the manipulation of programmed cell death (PCD) pathways. During a resistance interaction PCD is induced in a process termed the hypersensitive response (HR) which may function to limit pathogen spread. In a susceptible plant-pathogen interactions, the pathogen both inhibits and/or induces host PCD depending on the infection stage and lifestyle of the pathogen. Genes/pathways regulating PCD in plants have been difficult to identify due to a lack of homologous sequences in plants for mammalian genes that control apoptosis and possibly due to functional redundancy. Our labs study plant PCD pathways and bacterial speck disease in tomato which is caused by Pseudomonas syringae pv. tomato (Pst). We recently identified the tomato protein kinases Pdk1 and Adi3 as negative regulators of plant PCD. The plant Pdk1/Adi3 pathway appears to function similarly to the Pdk1/PKB (Akt) pathway in mammals which functions as a major apoptosis negative regulation pathway. Here we discuss regulation of Pdk1/Adi3 and targeting of this pathway during the tomato-Pst interaction for modulation of host PCD.

Identification and Characterization of Plant Genes Involved in Agrobacterium-mediated Plant Transformation by Virus-induced Gene Silencing

Molecular Plant-microbe Interactions : MPMI. Jan, 2007  |  Pubmed ID: 17249421

Genetic transformation of plant cells by Agrobacterium tumefaciens represents a unique case of trans-kingdom sex requiring the involvement of both bacterial virulence proteins and plant-encoded proteins. We have developed in planta and leaf-disk assays in Nicotiana benthamiana for identifying plant genes involved in Agrobacterium-mediated plant transformation using virus-induced gene silencing (VIGS) as a genomics tool. VIGS was used to validate the role of several genes that are either known or speculated to be involved in Agrobacterium-mediated plant transformation. We showed the involvement of a nodulin-like protein and an alpha-expansin protein (alpha-Exp) during Agrobacterium infection. Our data suggest that alpha-Exp is involved during early events of Agrobacterium-mediated transformation but not required for attaching A. tumefaciens. By employing the combination of the VIGS-mediated forward genetics approach and an in planta tumorigenesis assay, we identified 21 ACG (altered crown gall) genes that, when silenced, produced altered crown gall phenotypes upon infection with a tumorigenic strain of A. tumefaciens. One of the plant genes identified from the screening, Histone H3 (H3), was further characterized for its biological role in Agrobacterium-mediated plant transformation. We provide evidence for the role of H3 in transfer DNA integration. The data presented here suggest that the VIGS-based approach to identify and characterize plant genes involved in genetic transformation of plant cells by A. tumefaciens is simple, rapid, and robust and complements other currently used approaches.

An NB-LRR Protein Required for HR Signalling Mediated by Both Extra- and Intracellular Resistance Proteins

The Plant Journal : for Cell and Molecular Biology. Apr, 2007  |  Pubmed ID: 17346268

Tomato (Solanum lycopersicum) Cf resistance genes confer hypersensitive response (HR)-associated resistance to strains of the pathogenic fungus Cladosporium fulvum that express the matching avirulence (Avr) gene. Previously, we identified an Avr4-responsive tomato (ART) gene that is required for Cf-4/Avr4-induced HR in Nicotiana benthamiana as demonstrated by virus-induced gene silencing (VIGS). The gene encodes a CC-NB-LRR type resistance (R) protein analogue that we have designated NRC1 (NB-LRR protein required for HR-associated cell death 1). Here we describe that knock-down of NRC1 in tomato not only affects the Cf-4/Avr4-induced HR but also compromises Cf-4-mediated resistance to C. fulvum. In addition, VIGS using NRC1 in N. benthamiana revealed that this protein is also required for the HR induced by the R proteins Cf-9, LeEix, Pto, Rx and Mi. Transient expression of NRC1(D481V), which encodes a constitutively active NRC1 mutant protein, triggers an elicitor-independent HR. Subsequently, we transiently expressed this auto-activating protein in N. benthamiana silenced for genes known to be involved in HR signalling, thereby allowing NRC1 to be positioned in an HR signalling pathway. We found that NRC1 requires RAR1 and SGT1 to be functional, whereas it does not require NDR1 and EDS1. As the Cf-4 protein requires EDS1 for its function, we hypothesize that NRC1 functions downstream of EDS1. We also found that NRC1 acts upstream of a MAP kinase pathway. We conclude that Cf-mediated resistance signalling requires a downstream NB-LRR protein that also functions in cell death signalling pathways triggered by other R proteins.

A Pseudomonas Syringae Pv. Tomato DC3000 Mutant Lacking the Type III Effector HopQ1-1 is Able to Cause Disease in the Model Plant Nicotiana Benthamiana

The Plant Journal : for Cell and Molecular Biology. Jul, 2007  |  Pubmed ID: 17559511

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.

Pto- and Prf-mediated Recognition of AvrPto and AvrPtoB Restricts the Ability of Diverse Pseudomonas Syringae Pathovars to Infect Tomato

Molecular Plant-microbe Interactions : MPMI. Jul, 2007  |  Pubmed ID: 17601168

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.

A Bacterial E3 Ubiquitin Ligase Targets a Host Protein Kinase to Disrupt Plant Immunity

Nature. Jul, 2007  |  Pubmed ID: 17637671

Many bacterial pathogens of plants and animals use a type III secretion system to deliver diverse virulence-associated 'effector' proteins into the host cell. The mechanisms by which these effectors act are mostly unknown; however, they often promote disease by suppressing host immunity. One type III effector, AvrPtoB, expressed by the plant pathogen Pseudomonas syringae pv. tomato, has a carboxy-terminal domain that is an E3 ubiquitin ligase. Deletion of this domain allows an amino-terminal region of AvrPtoB (AvrPtoB(1-387)) to be detected by certain tomato varieties leading to immunity-associated programmed cell death. Here we show that a host kinase, Fen, physically interacts with AvrPtoB(1-387 )and is responsible for activating the plant immune response. The AvrPtoB E3 ligase specifically ubiquitinates Fen and promotes its degradation in a proteasome-dependent manner. This degradation leads to disease susceptibility in Fen-expressing tomato lines. Various wild species of tomato were found to exhibit immunity in response to AvrPtoB(1-387 )and not to full-length AvrPtoB. Thus, by acquiring an E3 ligase domain, AvrPtoB has thwarted a highly conserved host resistance mechanism.

Pseudomonas Syringae Type III Effector AvrPtoB is Phosphorylated in Plant Cells on Serine 258, Promoting Its Virulence Activity

The Journal of Biological Chemistry. Oct, 2007  |  Pubmed ID: 17711844

The Pseudomonas syringae pv. tomato protein AvrPtoB is translocated into plant cells via the bacterial type III secretion system. In resistant tomato leaves, AvrPtoB acts as an avirulence protein by interacting with the host Pto kinase and eliciting the host immune response. Pto-mediated immunity requires Prf, a Pto-interacting protein with a putative nucleotide-binding site and a region of leucine-rich repeats. In susceptible tomato plants, which lack either Pto or Prf, AvrPtoB acts as a virulence protein by promoting P. syringae pv. tomato growth and enhancing symptoms associated with bacterial speck disease. The N-terminal 307 amino acids of AvrPtoB (designated AvrPtoB(1-307)) are sufficient for these virulence activities and for Pto-mediated avirulence. We report that AvrPtoB is phosphorylated by a Pto- and Prf-independent kinase activity that is conserved in several plant species, including tomato (Solanum lycopersicum), Nicotiana benthamiana, and Arabidopsis thaliana. AvrPtoB(1-307) was phosphorylated in tomato protoplasts, and mass spectrometry identified serine 258 as the major in vivo phosphorylation site of this protein. An alanine substitution of Ser(258) resulted in the loss of virulence and the diminution of avirulence activity of AvrPtoB(1-307), whereas a phosphomimetic S258D mutant had activities similar to wild type AvrPtoB(1-307). These observations suggest that AvrPtoB has evolved to mimic a substrate of a conserved plant kinase, leading to enhancement of its virulence and avirulence activities in the host cell.

The N-terminal Region of Pseudomonas Type III Effector AvrPtoB Elicits Pto-dependent Immunity and Has Two Distinct Virulence Determinants

The Plant Journal : for Cell and Molecular Biology. Nov, 2007  |  Pubmed ID: 17764515

Resistance to bacterial speck disease in tomato is activated by the physical interaction of the host Pto kinase with either of the sequence-dissimilar type III effector proteins AvrPto or AvrPtoB (HopAB2) from Pseudomonas syringae pv. tomato. Pto-mediated immunity requires Prf, a protein with a nucleotide-binding site and leucine-rich repeats. The N-terminal 307 amino acids of AvrPtoB were previously reported to interact with the Pto kinase, and we show here that this region (AvrPtoB(1-307)) is sufficient for eliciting Pto/Prf-dependent immunity against P. s. pv. tomato. AvrPtoB(1-307) was also found to be sufficient for a virulence activity that enhances ethylene production and increases growth of P. s. pv. tomato and severity of speck disease on susceptible tomato lines lacking either Pto or Prf. Moreover, we found that residues 308-387 of AvrPtoB are required for the previously reported ability of AvrPtoB to suppress pathogen-associated molecular patterns-induced basal defenses in Arabidopsis. Thus, the N-terminal region of AvrPtoB has two structurally distinct domains involved in different virulence-promoting mechanisms. Random and targeted mutagenesis identified five tightly clustered residues in AvrPtoB(1-307) that are required for interaction with Pto and for elicitation of immunity to P. s. pv. tomato. Mutation of one of the five clustered residues abolished the ethylene-associated virulence activity of AvrPtoB(1-307). However, individual mutations of the other four residues, despite abolishing interaction with Pto and avirulence activity, had no effect on AvrPtoB(1-307) virulence activity. None of these mutations affected the basal defense-suppressing activity of AvrPtoB(1-387). Based on sequence alignments, estimates of helical propensity, and the previously reported structure of AvrPto, we hypothesize that the Pto-interacting domains of AvrPto and AvrPtoB(1-307) have structural similarity. Together, these data support a model in which AvrPtoB(1-307) promotes ethylene-associated virulence by interaction not with Pto but with another unknown host protein.

Aconitase Plays a Role in Regulating Resistance to Oxidative Stress and Cell Death in Arabidopsis and Nicotiana Benthamiana

Plant Molecular Biology. Jan, 2007  |  Pubmed ID: 17013749

In animals, aconitase is a bifunctional protein. When an iron-sulfur cluster is present in its catalytic center, aconitase displays enzymatic activity; when this cluster is lost, it switches to an RNA-binding protein that regulates the translatability or stability of certain transcripts. To investigate the role of aconitase in plants, we assessed its ability to bind mRNA. Recombinant aconitase failed to bind an iron responsive element (IRE) from the human ferritin gene. However, it bound the 5' UTR of the Arabidopsis chloroplastic CuZn superoxide dismutase 2 (CSD2) mRNA, and this binding was specific. Arabidopsis aconitase knockout (KO) plants were found to have significantly less chlorosis after treatment with the superoxide-generating compound, paraquat. This phenotype correlated with delayed induction of the antioxidant gene GST1, suggesting that these KO lines are more tolerant to oxidative stress. Increased levels of CSD2 mRNAs were observed in the KO lines, although the level of CSD2 protein was not affected. Virus-induced gene silencing (VIGS) of aconitase in Nicotiana benthamiana caused a 90% reduction in aconitase activity, stunting, spontaneous necrotic lesions, and increased resistance to paraquat. The silenced plants also had less cell death after transient co-expression of the AvrPto and Pto proteins or the pro-apoptotic protein Bax. Following inoculation with Pseudomonas syringae pv. tabaci carrying avrPto, aconitase-silenced N. benthamiana plants expressing the Pto transgene displayed a delayed hypersensitive response (HR) and supported higher levels of bacterial growth. Disease-associated cell death in N. benthamiana inoculated with P. s. pv. tabaci was also reduced. Taken together, these results suggest that aconitase plays a role in mediating oxidative stress and regulating cell death.

Bacterial Effectors Target the Common Signaling Partner BAK1 to Disrupt Multiple MAMP Receptor-signaling Complexes and Impede Plant Immunity

Cell Host & Microbe. Jul, 2008  |  Pubmed ID: 18621007

Successful pathogens have evolved strategies to interfere with host immune systems. For example, the ubiquitous plant pathogen Pseudomonas syringae injects two sequence-distinct effectors, AvrPto and AvrPtoB, to intercept convergent innate immune responses stimulated by multiple microbe-associated molecular patterns (MAMPs). However, the direct host targets and precise molecular mechanisms of bacterial effectors remain largely obscure. We show that AvrPto and AvrPtoB bind the Arabidopsis receptor-like kinase BAK1, a shared signaling partner of both the flagellin receptor FLS2 and the brassinosteroid receptor BRI1. This targeting interferes with ligand-dependent association of FLS2 with BAK1 during infection. It also impedes BAK1-dependent host immune responses to diverse other MAMPs and brassinosteroid signaling. Significantly, the structural basis of AvrPto-BAK1 interaction appears to be distinct from AvrPto-Pto association required for effector-triggered immunity. These findings uncover a unique strategy of bacterial pathogenesis where virulence effectors block signal transmission through a key common component of multiple MAMP-receptor complexes.

A Draft Genome Sequence of Pseudomonas Syringae Pv. Tomato T1 Reveals a Type III Effector Repertoire Significantly Divergent from That of Pseudomonas Syringae Pv. Tomato DC3000

Molecular Plant-microbe Interactions : MPMI. Jan, 2009  |  Pubmed ID: 19061402

Diverse gene products including phytotoxins, pathogen-associated molecular patterns, and type III secreted effectors influence interactions between Pseudomonas syringae strains and plants, with additional yet uncharacterized factors likely contributing as well. Of particular interest are those interactions governing pathogen-host specificity. Comparative genomics of closely related pathogens with different host specificity represents an excellent approach for identification of genes contributing to host-range determination. A draft genome sequence of Pseudomonas syringae pv. tomato T1, which is pathogenic on tomato but nonpathogenic on Arabidopsis thaliana, was obtained for this purpose and compared with the genome of the closely related A. thaliana and tomato model pathogen P. syringae pv. tomato DC3000. Although the overall genetic content of each of the two genomes appears to be highly similar, the repertoire of effectors was found to diverge significantly. Several P. syringae pv. tomato T1 effectors absent from strain DC3000 were confirmed to be translocated into plants, with the well-studied effector AvrRpt2 representing a likely candidate for host-range determination. However, the presence of avrRpt2 was not found sufficient to explain A. thaliana resistance to P. syringae pv. tomato T1, suggesting that other effectors and possibly type III secretion system-independent factors also play a role in this interaction.

Xanthomonas T3S Effector XopN Suppresses PAMP-Triggered Immunity and Interacts with a Tomato Atypical Receptor-Like Kinase and TFT1

The Plant Cell. Apr, 2009  |  Pubmed ID: 19366901

XopN is a virulence factor from Xanthomonas campestris pathovar vesicatoria (Xcv) that is translocated into tomato (Solanum lycopersicum) leaf cells by the pathogen's type III secretion system. Xcv DeltaxopN mutants are impaired in growth and have reduced ability to elicit disease symptoms in susceptible tomato leaves. We show that XopN action in planta reduced pathogen-associated molecular pattern (PAMP)-induced gene expression and callose deposition in host tissue, indicating that XopN suppresses PAMP-triggered immune responses during Xcv infection. XopN is predicted to have irregular, alpha-helical repeats, suggesting multiple protein-protein interactions in planta. Consistent with this prediction, XopN interacted with the cytosolic domain of a Tomato Atypical Receptor-Like Kinase1 (TARK1) and four Tomato Fourteen-Three-Three isoforms (TFT1, TFT3, TFT5, and TFT6) in yeast. XopN/TARK1 and XopN/TFT1 interactions were confirmed in planta by bimolecular fluorescence complementation and pull-down analysis. Xcv DeltaxopN virulence defects were partially suppressed in transgenic tomato leaves with reduced TARK1 mRNA levels, indicating that TARK1 plays an important role in the outcome of Xcv-tomato interactions. These data provide the basis for a model in which XopN binds to TARK1 to interfere with TARK1-dependent signaling events triggered in response to Xcv infection.

Deletions in the Repertoire of Pseudomonas Syringae Pv. Tomato DC3000 Type III Secretion Effector Genes Reveal Functional Overlap Among Effectors

PLoS Pathogens. Apr, 2009  |  Pubmed ID: 19381254

The gamma-proteobacterial plant pathogen Pseudomonas syringae pv. tomato DC3000 uses the type III secretion system to inject ca. 28 Avr/Hop effector proteins into plants, which enables the bacterium to grow from low inoculum levels to produce bacterial speck symptoms in tomato, Arabidopsis thaliana, and (when lacking hopQ1-1) Nicotiana benthamiana. The effectors are collectively essential but individually dispensable for the ability of the bacteria to defeat defenses, grow, and produce symptoms in plants. Eighteen of the effector genes are clustered in six genomic islands/islets. Combinatorial deletions involving these clusters and two of the remaining effector genes revealed a redundancy-based structure in the effector repertoire, such that some deletions diminished growth in N. benthamiana only in combination with other deletions. Much of the ability of DC3000 to grow in N. benthamiana was found to be due to five effectors in two redundant-effector groups (REGs), which appear to separately target two high-level processes in plant defense: perception of external pathogen signals (AvrPto and AvrPtoB) and deployment of antimicrobial factors (AvrE, HopM1, HopR1). Further support for the membership of HopR1 in the same REG as AvrE was gained through bioinformatic analysis, revealing the existence of an AvrE/DspA/E/HopR effector superfamily, which has representatives in virtually all groups of proteobacterial plant pathogens that deploy type III effectors.

Crystal Structure of the Complex Between Pseudomonas Effector AvrPtoB and the Tomato Pto Kinase Reveals Both a Shared and a Unique Interface Compared with AvrPto-Pto

The Plant Cell. Jun, 2009  |  Pubmed ID: 19509331

Resistance to bacterial speck disease in tomato (Solanum lycopersicum) is activated upon recognition by the host Pto kinase of either one of two sequence-unrelated effector proteins, AvrPto or AvrPtoB, from Pseudomonas syringae pv tomato (Pst). Pto induces Pst immunity by acting in concert with the Prf protein. The recently reported structure of the AvrPto-Pto complex revealed that interaction of AvrPto with Pto appears to relieve an inhibitory effect of Pto, allowing Pto to activate Prf. Here, we present the crystal structure of the Pto binding domain of AvrPtoB (residues 121 to 205) at a resolution of 1.9A and of the AvrPtoB(121-205)-Pto complex at a resolution of 3.3 A. AvrPtoB(121-205) exhibits a tertiary fold that is completely different from that of AvrPto, and its conformation remains largely unchanged upon binding to Pto. In common with AvrPto-Pto, the AvrPtoB-Pto complex relies on two interfaces. One of these interfaces is similar in both complexes, although the primary amino acid sequences from the two effector proteins are very different. Amino acid substitutions in Pto at the other interface disrupt the interaction of AvrPtoB-Pto but not that of AvrPto-Pto. Interestingly, substitutions in Pto affecting this unique interface also cause Pto to induce Prf-dependent host cell death independently of either effector protein.

Advances in Experimental Methods for the Elucidation of Pseudomonas Syringae Effector Function with a Focus on AvrPtoB

Molecular Plant Pathology. Nov, 2009  |  Pubmed ID: 19849784

Pseudomonas syringae infects a wide range of plant species through the use of a type III secretion system. The effector proteins injected into the plant cell through this molecular syringe serve as promoters of disease by subverting the plant immune response to the benefit of the bacteria in the intercellular space. The targets and activities of a subset of effectors have been elucidated recently. In this article, we focus on the experimental approaches that have proved most successful in probing the molecular basis of effectors, ranging from loss-of-function to gain-of-function analyses utilizing several techniques for effector delivery into plants. In particular, we highlight how these diverse approaches have been applied to the study of one effector--AvrPtoB--a multifunctional protein with the ability to suppress both effector-triggered immunity and pathogen (or microbe)-associated molecular pattern-triggered immunity. Taken together, advances in this field illustrate the need for multiple experimental approaches when elucidating the function of a single effector.

Tomato 14-3-3 Protein 7 Positively Regulates Immunity-associated Programmed Cell Death by Enhancing Protein Abundance and Signaling Ability of MAPKKK {alpha}

The Plant Cell. Jan, 2010  |  Pubmed ID: 20061552

Programmed cell death (PCD) is triggered when Pto, a Ser-Thr protein kinase, recognizes either the AvrPto or AvrPtoB effector from Pseudomonas syringae pv tomato. This PCD requires mitogen-activated protein kinase kinase kinase (MAPKKK alpha ) as a positive regulator in tomato (Solanum lycopersicum) and Nicotiana benthamiana. To examine how PCD-eliciting activity of the tomato MAPKKK alpha protein is regulated, we screened for MAPKKK alpha -interacting proteins in tomato and identified a 14-3-3 protein, TFT7. Virus-induced gene silencing using the TFT7 gene in N. benthamiana compromised both Pto- and MAPKKK alpha -mediated PCD, and coexpression of TFT7 with tomato MAPKKK alpha enhanced MAPKKK alpha -mediated PCD. TFT7 was also required for PCD associated with several other disease resistance proteins and contributed to resistance against P. syringae pv tomato. Coexpression of TFT7 with MAPKKK alpha in vivo caused increased accumulation of the kinase and enhanced phosphorylation of two MAP kinases. TFT7 protein contains a phosphopeptide binding motif that is present in human 14-3-3 epsilon, and substitutions in this motif abolished interaction with MAPKKK alpha in vivo and also the PCD-enhancing activity of TFT7. A 14-3-3 binding motif, including a putative phosphorylated Ser-535, is present in the C-terminal region of MAPKKK alpha. An S535A substitution in MAPKKK alpha reduced interaction with TFT7 and both PCD-eliciting ability and stability of MAPKKK alpha. Our results provide new insights into a role for 14-3-3 proteins in regulating immunity-associated PCD pathways in plants.

Two Virulence Determinants of Type III Effector AvrPto Are Functionally Conserved in Diverse Pseudomonas Syringae Pathovars

The New Phytologist. Sep, 2010  |  Pubmed ID: 20122130

*The Pseudomonas syringae pv. tomato type III effector protein AvrPto has two functional domains that contribute additively to its ability to promote pathogen virulence in susceptible tomato plants and also defense responses in resistant tomato and tobacco genotypes. Here, we test the hypothesis that key amino acid residues in these two domains will be conserved even in sequence-divergent AvrPto proteins expressed by diverse P. syringae pathovars. *We cloned avrPto homologs from diverse P. syringae pathovars and characterized the four most diverse homologs from P. syringae pathovars mori, lachrymans, myricae and oryzae for their virulence activity and ability to elicit resistance in tomato and tobacco. *Key residues within the two AvrPto domains are conserved in three of the four homologs and are required for virulence activity and defense elicitation. AvrPto(oryzae), lacks conserved residues in each domain, but was found to be recognized by a previously unknown resistance gene in both tomato and tobacco. *Our results indicate that the two virulence domains of AvrPto are conserved in diverse pathovars despite the fact these domains are recognized by certain plant species. AvrPto may therefore function in pathovars infecting diverse plant species by targeting conserved host processes.

A Secreted Effector Protein (SNE1) from Phytophthora Infestans is a Broadly Acting Suppressor of Programmed Cell Death

The Plant Journal : for Cell and Molecular Biology. May, 2010  |  Pubmed ID: 20128886

Evasion or active suppression of host defenses are critical strategies employed by biotrophic phytopathogens and hemibiotrophs whose infection mechanism includes sequential biotrophic and necrotrophic stages. Although defense suppression by secreted effector proteins has been well studied in bacteria, equivalent systems in fungi and oomycetes are poorly understood. We report the characterization of SNE1 (suppressor of necrosis 1), a gene encoding a secreted protein from the hemibiotrophic oomycete Phytophthora infestans that is specifically expressed at the transcriptional level during biotrophic growth within the host plant tomato (Solanum lycopersicum). Using transient expression assays, we show that SNE1 suppresses the action of secreted cell death-inducing effectors from Phytophthora that are expressed during the necrotrophic growth phase, as well as programmed cell death mediated by a range of Avr-R protein interactions. We also report that SNE1 contains predicted NLS motifs and translocates to the plant nucleus in transient expression studies. A conceptual model is presented in which the sequential coordinated secretion of antagonistic effectors by P. infestans first suppresses, but then induces, host cell death, thereby providing a highly regulated means to control the transition from biotrophy to necrotrophy.

Endosome-associated CRT1 Functions Early in Resistance Gene-mediated Defense Signaling in Arabidopsis and Tobacco

The Plant Cell. Mar, 2010  |  Pubmed ID: 20332379

Resistance gene-mediated immunity confers protection against pathogen infection in a wide range of plants. A genetic screen for Arabidopsis thaliana mutants compromised for recognition of turnip crinkle virus previously identified CRT1, a member of the GHKL ATPase/kinase superfamily. Here, we demonstrate that CRT1 interacts with various resistance proteins from different structural classes, and this interaction is disrupted when these resistance proteins are activated. The Arabidopsis mutant crt1-2 crh1-1, which lacks CRT1 and its closest homolog, displayed compromised resistance to avirulent Pseudomonas syringae and Hyaloperonospora arabidopsidis. Additionally, resistance-associated hypersensitive cell death was suppressed in Nicotiana benthamiana silenced for expression of CRT1 homolog(s). Thus, CRT1 appears to be a general factor for resistance gene-mediated immunity. Since elevation of cytosolic calcium triggered by avirulent P. syringae was compromised in crt1-2 crh1-1 plants, but cell death triggered by Nt MEK2(DD) was unaffected in CRT1-silenced N. benthamiana, CRT1 likely functions at an early step in this pathway. Genome-wide transcriptome analysis led to identification of CRT1-Associated genes, many of which are associated with transport processes, responses to (a)biotic stress, and the endomembrane system. Confocal microscopy and subcellular fractionation revealed that CRT1 localizes to endosome-like vesicles, suggesting a key process in resistance protein activation/signaling occurs in this subcellular compartment.

The T-loop Extension of the Tomato Protein Kinase AvrPto-dependent Pto-interacting Protein 3 (Adi3) Directs Nuclear Localization for Suppression of Plant Cell Death

The Journal of Biological Chemistry. Jun, 2010  |  Pubmed ID: 20371603

In tomato (Solanum lycopersicum), resistance to Pseudomonas syringae pv. tomato is elicited by the interaction of the host Pto kinase with the pathogen effector protein AvrPto, which leads to various immune responses including localized cell death termed the hypersensitive response. The AGC kinase Adi3 functions to suppress host cell death and interacts with Pto only in the presence of AvrPto. The cell death suppression (CDS) activity of Adi3 requires phosphorylation by 3-phosphoinositide-dependent protein kinase 1 (Pdk1) and loss of Adi3 function is associated with the hypersensitive response cell death initiated by the Pto/AvrPto interaction. Here we studied the relationship between Adi3 cellular localization and its CDS activity. Adi3 is a nuclear-localized protein, and this localization is dictated by a nuclear localization signal found in the Adi3 T-loop extension, an approximately 80 amino acid insertion into the T-loop, or activation loop, which is phosphorylated for kinase activation. Nuclear localization of Adi3 is required for its CDS activity and loss of nuclear localization causes elimination of Adi3 CDS activity and induction of cell death. This nuclear localization of Adi3 is dependent on Ser-539 phosphorylation by Pdk1 and non-nuclear Adi3 is found in punctate structures throughout the cell. Our data support a model in which Pdk1 phosphorylation of Adi3 directs nuclear localization for CDS and that disruption of Adi3 nuclear localization may be a mechanism for induction of cell death such as that during the Pto/AvrPto interaction.

Identification of Nicotiana Benthamiana Genes Involved in Pathogen-associated Molecular Pattern-triggered Immunity

Molecular Plant-microbe Interactions : MPMI. Jun, 2010  |  Pubmed ID: 20459311

In order to identify components of pathogen-associated molecular pattern-triggered immunity (PTI) pathways in Nicotiana benthamiana, we conducted a large-scale forward-genetics screen using virus-induced gene silencing and a cell-death-based assay for assessing PTI. The assay relied on four combinations of PTI-inducing nonpathogens and cell-death-causing challenger pathogens and was first validated in plants silenced for FLS2 or BAK1. Over 3,200 genes were screened and 14 genes were identified that, when silenced, compromised PTI as judged by the cell-death-based assay. Further analysis indicated that the 14 genes were not involved in a general cell death response. A subset of the genes was found to act downstream of FLS2-mediated PTI induction, and silencing of three genes compromised production of reactive oxygen species in leaves exposed to flg22. The 14 genes encode proteins with potential functions in defense and hormone signaling, protein stability and degradation, energy and secondary metabolism, and cell wall biosynthesis and provide a new resource to explore the molecular basis for the involvement of these processes in PTI.

Phosphorylation of the Pseudomonas Syringae Effector AvrPto is Required for FLS2/BAK1-independent Virulence Activity and Recognition by Tobacco

The Plant Journal : for Cell and Molecular Biology. Jan, 2010  |  Pubmed ID: 19793077

The type III effector protein AvrPto from Pseudomonas syringae pv. tomato is secreted into plant cells where it promotes bacterial growth and enhances symptoms of speck disease on susceptible tomato plants. The virulence activity of AvrPto is due, in part, to its interaction with components of host pattern recognition receptor complexes, which disrupts pathogen-associated molecular pattern-triggered immunity. This disruption mechanism requires a structural element of the AvrPto protein, the CD loop, which is also required for triggering Pto/Prf-mediated resistance in tomato. We have shown previously that the carboxyl-terminal domain (CTD) of AvrPto is phosphorylated and also contributes to bacterial virulence. Here we report that phosphorylation of the CTD on S147 and S149 promotes bacterial virulence in an FLS2/BAK1-independent manner, which is mechanistically distinct from the CD loop. In a striking corollary with Pto recognition of the CD loop in tomato, the tobacco species Nicotiana sylvestris and Nicotiana tabacum have a recognition mechanism that specifically detects the phosphorylation status of the CTD. Thus different species in the Solanaceae family have evolved distinct recognition mechanisms to monitor the same type III effector.

Methods to Study PAMP-triggered Immunity Using Tomato and Nicotiana Benthamiana

Molecular Plant-microbe Interactions : MPMI. Aug, 2010  |  Pubmed ID: 20615110

Understanding the molecular basis of plant responses to pathogen-associated molecular patterns (PAMPs) is an active area of research in the field of plant-microbe interactions. A growing number of plant genes involved in various steps of PAMP-triggered immunity (PTI) pathways and microbial factors involved in the elicitation or suppression of PTI have been identified. These studies have largely relied on Arabidopsis thaliana and, therefore, most of the PTI assays have been developed and optimized for that model plant system. Although PTI is a conserved feature among plants, the response spectra vary across different species. Thus, there is a need for robust PTI assays in other pathosystems, such as those involving Solanaceae plant-pathogen interactions, which include many economically important plants and their diseases. We have optimized molecular, cellular, and whole-plant methods to measure PTI responses in two widely studied solanaceous species, tomato (Solanum lycopersicum) and Nicotiana benthamiana. Here, we provide detailed protocols for measuring various PTI-associated phenotypes, including bacterial populations after pretreatment of leaves with PAMPs, induction of reporter genes, callose deposition, activation of mitogen-activated protein kinases, and a luciferase-based reporter system. These methods will facilitate limited genetic screens and detailed characterization of potential PTI-related genes in model and economically important Solanaceae spp.-pathogen interactions.

Effector-triggered Immunity Mediated by the Pto Kinase

Trends in Plant Science. Mar, 2011  |  Pubmed ID: 21112235

Pto was the first disease-resistance gene cloned from a plant that confers recognition of a specific pathogen. The intracellular protein kinase that it encodes activates an immune response in tomato (Solanum lycopersicum) to bacterial speck disease by interacting with either the AvrPto or AvrPtoB type III effector proteins that are delivered into the plant cell by Pseudomonas syringae pathovar tomato. This recognition event triggers signaling pathways leading to effector-triggered immunity (ETI), which inhibits pathogen growth. During the past 15 years, ~25 genes have been identified by loss-of-function studies to have a role in Pto-mediated ETI. Here, we review the experimental approaches that have been used in these studies, discuss the proteins that have been identified and characterized, and present a current model of Pto-mediated ETI.

Genetic Disassembly and Combinatorial Reassembly Identify a Minimal Functional Repertoire of Type III Effectors in Pseudomonas Syringae

Proceedings of the National Academy of Sciences of the United States of America. Feb, 2011  |  Pubmed ID: 21282655

The virulence of Pseudomonas syringae and many other proteobacterial pathogens is dependent on complex repertoires of effector proteins injected into host cells by type III secretion systems. The 28 well-expressed effector genes in the repertoire of the model pathogen P. syringae pv. tomato DC3000 were deleted to produce polymutant DC3000D28E. Growth of DC3000D28E in Nicotiana benthamiana was symptomless and 4 logs lower than that of DC3000ΔhopQ1-1, which causes disease in this model plant. DC3000D28E seemed functionally effectorless but otherwise WT in diagnostic phenotypes relevant to plant interactions (for example, ability to inject the AvrPto-Cya reporter into N. benthamiana). Various effector genes were integrated by homologous recombination into native loci or by a programmable or random in vivo assembly shuttle (PRIVAS) system into the exchangeable effector locus in the Hrp pathogenicity island of DC3000D28E. The latter method exploited dual adapters and recombination in yeast for efficient assembly of PCR products into programmed or random combinations of multiple effector genes. Native and PRIVAS-mediated integrations were combined to identify a minimal functional repertoire of eight effector genes that restored much of the virulence of DC3000ΔhopQ1-1 in N. benthamiana, revealing a hierarchy in effector function: AvrPtoB acts with priority in suppressing immunity, enabling other effectors to promote further growth (HopM1 and HopE1), chlorosis (HopG1), lesion formation (HopAM1-1), and near full growth and symptom production (AvrE, HopAA1-1, and/or HopN1 functioning synergistically with the previous effectors). DC3000D28E, the PRIVAS method, and minimal functional repertoires provide new resources for probing the plant immune system.

Tomato 14-3-3 Protein TFT7 Interacts with a MAP Kinase Kinase to Regulate Immunity-associated Programmed Cell Death Mediated by Diverse Disease Resistance Proteins

The Journal of Biological Chemistry. Apr, 2011  |  Pubmed ID: 21378171

Programmed cell death (PCD) associated with immunity is triggered when a plant disease resistance (R) protein recognizes a corresponding pathogen virulence protein. In tomato, detection by the host Pto kinase of the Pseudomonas syringae proteins AvrPto or AvrPtoB causes localized PCD. Previously, we reported that both MAPKKKα (mitogen-activated protein kinase kinase kinase) and the tomato 14-3-3 protein 7 (TFT7) positively regulate Pto-mediated PCD in tomato and Nicotiana benthamiana. In addition, in contrast to MAPKKKα, TFT7 is required for PCD mediated by four other R proteins. Here we investigate why TFT7 is required for PCD induced by diverse R proteins in plants. We discovered that a MAPKK, SlMKK2, which acts downstream of SlMAPKKKα, also interacts with TFT7 in plant cells. Gene silencing experiments revealed that the orthologous genes of both SlMKK2 and TFT7 in N. benthamiana are required for PCD mediated by the same set of R proteins. SlMKK2 and its orthologs contain a 14-3-3 binding site in their N terminus, and Thr(33) in this site is required for interaction with TFT7 in vivo. Like the structurally similar human 14-3-3ε protein, TFT7 forms a homodimer in vivo. Because TFT7 interacts with both SlMAPKKKα and SlMKK2 and also forms a homodimer, we propose that TFT7 may coordinately recruit these client proteins for efficient signal transfer, leading to PCD induction.

Structural Analysis of Pseudomonas Syringae AvrPtoB Bound to Host BAK1 Reveals Two Similar Kinase-interacting Domains in a Type III Effector

Cell Host & Microbe. Dec, 2011  |  Pubmed ID: 22169508

To infect plants, Pseudomonas syringae pv. tomato delivers ~30 type III effector proteins into host cells, many of which interfere with PAMP-triggered immunity (PTI). One effector, AvrPtoB, suppresses PTI using a central domain to bind host BAK1, a kinase that acts with several pattern recognition receptors to activate defense signaling. A second AvrPtoB domain binds and suppresses the PTI-associated kinase Bti9 but is conversely recognized by the protein kinase Pto to activate effector-triggered immunity. We report the crystal structure of the AvrPtoB-BAK1 complex, which revealed structural similarity between these two AvrPtoB domains, suggesting that they arose by intragenic duplication. The BAK1 kinase domain is structurally similar to Pto, and a conserved region within both BAK1 and Pto interacts with AvrPtoB. BAK1 kinase activity is inhibited by AvrPtoB, and mutations at the interaction interface disrupt AvrPtoB virulence activity. These results shed light on a structural mechanism underlying host-pathogen coevolution.

Type III Secretion and Effectors Shape the Survival and Growth Pattern of Pseudomonas Syringae on Leaf Surfaces

Plant Physiology. Feb, 2012  |  Pubmed ID: 22319072

The bacterium Pseudomonas syringae pv syringae B728a (PsyB728a) uses a type III secretion system (T3SS) to inject effector proteins into plant cells, a process that modulates the susceptibility of different plants to infection. Analysis of GFP-expressing PsyB728a after spray inoculation without additives under moderate relative humidity conditions permitted (1) a detailed analysis of this strain's survival and growth pattern on host (Nicotiana benthamiana) and non-host (tomato) leaf surfaces, (2) an assessment of the role of plant defenses in affecting PsyB728a leaf surface (epiphytic) growth and (3) the contribution of the T3SS and specific effectors to PsyB728a epiphytic survival and growth. On host leaf surfaces, PsyB728a cells initially persist without growing, and show an increased population only after 48 h, unless plants are pretreated with the defense-inducing chemical benzothiazole. During the persistence period, some PsyB728a cells induce a T3SS reporter, whereas a T3SS-deficient mutant shows reduced survival. By 72 h, rare invasion by PsyB728a to the mesophyll region of host leaves occurs, but endophytic and epiphytic bacterial growths are not correlated. The effectors HopZ3 and HopAA1 delay the onset of epiphytic growth of PsyB728a on N. benthamiana, whereas they promote epiphytic survival/growth on tomato. These effectors localize to distinct sites in plant cells and likely have different mechanisms of action. HopZ3 may enzymatically modify host targets, as it requires residues important for the catalytic activity of other proteins in its family of proteases. Thus, the T3SS, HopAA1, HopZ3 and plant defenses strongly influence epiphytic survival and/or growth of PsyB728a.

A Tomato LysM Receptor-like Kinase Promotes Immunity and Its Kinase Activity is Inhibited by AvrPtoB

The Plant Journal : for Cell and Molecular Biology. Jan, 2012  |  Pubmed ID: 21880077

Resistance in tomato (Solanum lycopersicum) to infection by Pseudomonas syringae involves both detection of pathogen-associated molecular patterns (PAMPs) and recognition by the host Pto kinase of pathogen effector AvrPtoB which is translocated into the host cell and interferes with PAMP-triggered immunity (PTI). The N-terminal portion of AvrPtoB is sufficient for its virulence activity and for recognition by Pto. An amino acid substitution in AvrPtoB, F173A, abolishes these activities. To investigate the mechanisms of AvrPtoB virulence, we screened for tomato proteins that interact with AvrPtoB and identified Bti9, a LysM receptor-like kinase. Bti9 has the highest amino acid similarity to Arabidopsis CERK1 among the tomato LysM receptor-like kinases (RLKs) and belongs to a clade containing three other tomato proteins, SlLyk11, SlLyk12, and SlLyk13, all of which interact with AvrPtoB. The F173A substitution disrupts the interaction of AvrPtoB with Bti9 and SlLyk13, suggesting that these LysM-RLKs are its virulence targets. Two independent tomato lines with RNAi-mediated reduced expression of Bti9 and SlLyk13 were more susceptible to P. syringae. Bti9 kinase activity was inhibited in vitro by the N-terminal domain of AvrPtoB in an F173-dependent manner. These results indicate Bti9 and/or SlLyk13 play a role in plant immunity and the N-terminal domain of AvrPtoB may have evolved to interfere with their kinase activity. Finally, we found that Bti9 and Pto interact with AvrPtoB in a structurally similar although not identical fashion, suggesting that Pto may have evolved as a molecular mimic of LysM-RLK kinase domains.