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In JoVE (1)
Other Publications (26)
- Annual Review of Plant Biology
- The Journal of Biological Chemistry
- Molecular Plant-microbe Interactions : MPMI
- Molecular Plant-microbe Interactions : MPMI
- Nature Methods
- Molecular Plant-microbe Interactions : MPMI
- The Plant Cell
- Molecular Microbiology
- Molecular Plant Pathology
- Functional & Integrative Genomics
- Plant Physiology
- Molecular Plant-microbe Interactions : MPMI
- Plant Signaling & Behavior
- Plant Signaling & Behavior
- The Journal of Biological Chemistry
- Molecular Plant-microbe Interactions : MPMI
- The Plant Journal : for Cell and Molecular Biology
- Molecular Plant-microbe Interactions : MPMI
- Phytopathology
- Plant Signaling & Behavior
- Microbiology (Reading, England)
- The Plant Journal : for Cell and Molecular Biology
- Methods in Molecular Biology (Clifton, N.J.)
- PloS One
- Molecular Plant-microbe Interactions : MPMI
- Journal of Proteome Research
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Articles by Guido Sessa in JoVE
JoVE General
Identificação de fenótipos inibição do crescimento induzido pela expressão de Effectors Tipo III bacteriana em Yeast
Department of Plant Sciences, Tel Aviv University
Neste vídeo, descrevemos um procedimento para a expressão de efetores tipo bacteriana III em levedura ea identificação de fenótipos efetoras induzida por inibição do crescimento. Esses fenótipos podem ser posteriormente explorada para elucidar funções efetoras e metas.
Other articles by Guido Sessa on PubMed
Understanding the Functions of Plant Disease Resistance Proteins
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.
LeMPK3 is a Mitogen-activated Protein Kinase with Dual Specificity Induced During Tomato Defense and Wounding Responses
Mitogen-activated protein (MAP) kinase cascades are readily activated during the response of plants to avirulent pathogens or to pathogen-derived elicitors. Here we show that the tomato MAP kinase LeMPK3 is specifically induced at the mRNA level during elicitation of the hypersensitive response in resistant plants infected by avirulent strains of the phytopathogenic bacteria Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. tomato, as well as upon treatment with the fungal elicitor ethylene-inducing xylanase. LeMPK3 gene expression was also induced very rapidly by mechanical stress and wounding much earlier than upon pathogen infection, but not in response to the defense-related plant hormones ethylene and jasmonic acid. Moreover, in resistant tomato plants infected by X. campestris pv. vesicatoria, transcript accumulation was followed by an increase in LeMPK3 kinase activity. Biochemical characterization of a glutathione S-transferase-LeMPK3 fusion protein revealed that the LeMPK3 MAP kinase autophosphorylates in vitro mainly on tyrosine and less so on threonine and serine, whereas it phosphorylates myelin basic protein on serine and threonine. In vitro phosphorylation of a poly-(Glu-Tyr) copolymer by LeMPK3 demonstrated its capability to phosphorylate tyrosine residues on substrates as well. By mutagenesis and phosphoamino acid analysis, Tyr-201 in the kinase activation domain was identified as the main LeMPK3 autophosphorylation site and as critical for kinase activity. Finally, LeMPK3 autophosphorylation showed a preference for Mn(2+) cations and proceeded via an intramolecular mechanism with an estimated K(m) value for ATP of 9.5 microm. These results define LeMPK3 as a MAP kinase with dual specificity and strongly suggest that it represents a convergence point for different signaling pathways inducing the activation of defense responses in tomato.
Identification and Expression Profiling of Tomato Genes Differentially Regulated During a Resistance Response to Xanthomonas Campestris Pv. Vesicatoria
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.
Molecular Properties of the Xanthomonas AvrRxv Effector and Global Transcriptional Changes Determined by Its Expression in Resistant Tomato Plants
The Xanthomonas campestris pv. vesicatoria avirulence gene AvrRxv specifies resistance on the tomato line Hawaii 7998 by interacting with three nondominant plant resistance genes. AvrRxv molecular properties that impinge on its avirulence activity were characterized and transcriptional changes caused by AvrRxv expression in resistant tomato plants were extensively examined. AvrRxv localized predominantly to the cytoplasm and possibly in association with plasma and nuclear membranes in both resistant and susceptible tomato plants. The AvrRxv cysteine protease catalytic core was found to be essential for host recognition, because introduction of mutations in this domain affected the ability of AvrRxv to elicit a hypersensitive response and the inhibition of bacterial growth in resistant plants. In addition, expression profiles were analyzed for approximately 8,600 tomato genes in resistant plants challenged with X. campestris pv. vesicatoria strains expressing wild-type AvrRxv or a catalytic core AvrRxv mutant. In all, 420 genes were identified as differentially modulated by the expression of a functional AvrRxv, including over 15 functional classes of proteins and a large number of transcription factors and signaling components. Findings of this study allow the development of new hypotheses about the molecular basis of recognition between AvrRxv and the corresponding resistance proteins, and set the stage for the dissection of signaling and cellular responses triggered in tomato plants by this avirulence factor.
A Second-site Suppressor Strategy for Chemical Genetic Analysis of Diverse Protein Kinases
Chemical genetic analysis of protein kinases involves engineering kinases to be uniquely sensitive to inhibitors and ATP analogs that are not recognized by wild-type kinases. Despite the successful application of this approach to over two dozen kinases, several kinases do not tolerate the necessary modification to the ATP binding pocket, as they lose catalytic activity or cellular function upon mutation of the 'gatekeeper' residue that governs inhibitor and nucleotide substrate specificity. Here we describe the identification of second-site suppressor mutations to rescue the activity of 'intolerant' kinases. A bacterial genetic selection for second-site suppressors using an aminoglycoside kinase APH(3')-IIIa revealed several suppressor hotspots in the kinase domain. Informed by results from this selection, we focused on the beta sheet in the N-terminal subdomain and generated a structure-based sequence alignment of protein kinases in this region. From this alignment, we identified second-site suppressors for several divergent kinases including Cdc5, MEKK1, GRK2 and Pto. The ability to identify second-site suppressors to rescue the activity of intolerant kinases should facilitate chemical genetic analysis of the majority of protein kinases in the genome.
Analysis of Promoters Recognized by HrpL, an Alternative Sigma-factor Protein from Pantoea Agglomerans Pv. Gypsophilae
HrpL, an alternative sigma factor, activates the transcription of the Hrp regulon by its binding to a common "hrp box" promoter. Based on computational techniques, the hrp box previously was defined as a consensus bipartite cis element, 5'-GGAACC-N(15-16)-CCACNNA-3'. The present report combines a quantitative in vivo assay for measuring Hrp promoter activity with site-specific mutagenesis to analyze the effect of consensus and nonconsensus nucleotides on promoter activity. The analysis was carried out with Hop effectors of the tumorigenic bacterium Pantoea agglomerans pv. gypsophilae, in which HrpL is indispensable for gall formation. Mutational analysis indicates that the hrp box consensus can be divided into crucial and noncrucial nucleotides. The first 5 nucleotides (nt) of the--35 consensus motif (GGAAC) and the 3 nt of the--10 motif (ACNNA) are crucial, whereas other consensus and adjacent nonconsensus nucleotides exert a significant effect on the promoter's strength. With spacing of 13 or 17 nt between the two motifs, significant activity was still retained. Gel shift assays indicated that deletion of GG from the--35 consensus motif eliminated HrpL binding, whereas mutations in the--10 consensus motif or modification of the spacing, which eliminates promoter activity, did not elicit any effect. The degeneracy in Hrp promoters of four hrp and type III effector genes of P agglomerans pv. gypsophilae indicated significant differences in promoter activity, whereas increasing the promoter strength of the Hop effector, HsvG, resulted in overexpression of gall formation.
Host-mediated Phosphorylation of Type III Effector AvrPto Promotes Pseudomonas Virulence and Avirulence in Tomato
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.
The Type III Effectors HsvG and HsvB of Gall-forming Pantoea Agglomerans Determine Host Specificity and Function As Transcriptional Activators
Pantoea agglomerans pv. gypsophilae (Pag) elicits galls on gypsophila and a hypersensitive response on beet, whereas P. agglomerans pv. betae (Pab) induces galls on both beet and gypsophila. The pathogenicity of both pathovars is dependent on the presence of a plasmid harbouring type III secretion system (TTSS) components and effectors. The HsvG TTSS effectors of Pag (HsvG-Pag) and Pab (HsvG-Pab) determine the host specificity of both pathovars on gypsophila. Here we describe a novel HsvG homologue, HsvB, which determines the host specificity of Pag and Pab on beet. HsvG requires two direct amino acid repeats for pathogenicity on gypsophila, whereas one repeat in HsvB is sufficient for pathogenicity on beet. Exchanging repeats between HsvG-Pag and HsvB-Pab resulted in a switch of host specificities. Transient expression of GFP-HsvG or GFP-HsvB fusions in gypsophila, beet or melon leaves showed that HsvG and HsvB were localized to the nuclei of host and non-host plants. A yeast one-hybrid assay revealed that a single repeat of HsvG or HsvB was sufficient to activate transcription. By employing random binding-site selection and gel-shift assay HsvG was demonstrated to be a double-stranded DNA-binding protein with an ACACC/aAA consensus binding site. These results suggest that HsvG and HsvB are host-specificity determinants and bear the potential to affect the host transcriptional machinery.
A Novel Link Between Tomato GRAS Genes, Plant Disease Resistance and Mechanical Stress Response
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.
Transcriptional Analysis of the Tomato Resistance Response Triggered by Recognition of the Xanthomonas Type III Effector AvrXv3
The type III effector AvrXv3 from Xanthomonas campestris pv. vesicatoria (Xcv) elicits a resistance response in the tomato line Hawaii 7981. To test whether similar genes participate in responses triggered by recognition of different avirulence proteins, we examined the effect of AvrXv3 expression on the plant transcriptome as compared to that of other avirulence proteins. By microarray analysis we monitored expression of approximately 8,600 tomato genes upon inoculation with isogenic Xcv strains differing only by the avrXv3 gene. Changes in transcript levels of 139 genes were observed within 8 h, and a massive shift in expression of 1,294 genes was detected at 12 h. Recognition of AvrXv3 modulated a large number of genes encoding transcription factors and signaling components. In addition, genes involved in defense and stress responses, lipid metabolism, protein degradation, and secondary metabolism were mainly up-regulated. Conversely, genes related to photosynthesis and protein synthesis were generally down-regulated. Many novel genes encoding proteins of unknown function were also identified. A comparison between AvrXv3-modulated genes and those differentially expressed in tomato plants recognizing other bacterial effectors revealed partial overlap and similar distribution in functional classes. The identification of tomato genes modulated by AvrXv3 expression paves the way for dissecting defense networks activated by recognition of this effector in resistant plants.
Tomato Transcriptional Changes in Response to Clavibacter Michiganensis Subsp. Michiganensis Reveal a Role for Ethylene in Disease Development
Clavibacter michiganensis subsp. michiganensis (Cmm) is a gram-positive actinomycete, causing bacterial wilt and canker disease in tomato (Solanum lycopersicum). Host responses to gram-positive bacteria and molecular mechanisms associated with the development of disease symptoms caused by Cmm in tomato are largely unexplored. To investigate plant responses activated during this compatible interaction, we used microarray analysis to monitor changes in host gene expression during disease development. This analysis was performed at 4 d postinoculation, when bacteria were actively multiplying and no wilt symptoms were yet visible; and at 8 d postinoculation, when bacterial growth approached saturation and typical wilt symptoms were observed. Of the 9,254 tomato genes represented on the array, 122 were differentially expressed in Cmm-infected plants, compared with mock-inoculated plants. Functional classification of Cmm-responsive genes revealed that Cmm activated typical basal defense responses in the host, including induction of defense-related genes, production and scavenging of free oxygen radicals, enhanced protein turnover, and hormone synthesis. Cmm infection also induced a subset of host genes involved in ethylene biosynthesis and response. After inoculation with Cmm, Never ripe (Nr) mutant plants, impaired in ethylene perception, and transgenic plants with reduced ethylene synthesis showed significant delay in the appearance of wilt symptoms, compared with wild-type plants. The retarded wilting in Nr plants was a specific effect of ethylene insensitivity, and was not due to altered expression of defense-related genes, reduced bacterial populations, or decreased ethylene synthesis. Taken together, our results indicate that host-derived ethylene plays an important role in regulation of the tomato susceptible response to Cmm.
Quorum-sensing System Affects Gall Development Incited by Pantoea Agglomerans Pv. Gypsophilae
The quorum-sensing (QS) regulatory system of the gall-forming Pantoea agglomerans pv. gypsophilae was identified. Mass spectral analysis, together with signal-specific biosensors, demonstrated that P. agglomerans pv. gypsophilae produced N-butanoyl-l-homoserine lactone (C4-HSL) as a major and N-hexanoyl-l-homoserine lactone (C6-HSL) as a minor QS signal. Homologs of luxI and luxR regulatory genes, pagI and pagR, were characterized in strain P. agglomerans pv. gypsophilae Pag824-1 and shown to be convergently transcribed and separated by 14 bp. The deduced PagI (23.8 kDa) and PagR (26.9 kDa) show high similarity with SmaI (41% identity) and SmaR (43% identity), respectively, of Serratia sp. American Type Culture Collection 39006. PagR possesses characteristic autoinducer binding and a helix-turn-helix DNA-binding domain. Gall formation by P. agglomerans pv. gypsophilae depends on a plasmid-borne hrp/hrc gene cluster, type III effectors, and phytohormones. Disruption of pagI, pagR, or both genes simultaneously in Pag824-1 reduced gall size in gypsophila cuttings by 50 to 55% when plants were inoculated with 10(6) CFU/ml. Higher reductions in gall size (70 to 90%) were achieved by overexpression of pagI or addition of exogenous C4-HSL. Expression of the hrp/hrc regulatory gene hrpL and the type III effector pthG in the pagI mutant, as measured with quantitative reverse-transcriptase polymerase chain reaction, was reduced by 5.8 and 6.6, respectively, compared with the wild type, suggesting an effect of the QS system on the Hrp regulon.
Activation and Manipulation of Host Responses by a Gram-positive Bacterium
The interaction between tomato plants and Clavibacter michiganensis subsp. michiganensis (Cmm) represents a model pathosystem to study the interplay between the virulence determinants of a Gram-positive bacterium and the attempt of a crop plant to counteract pathogen invasion. To investigate plant responses activated during this compatible interaction, we recently analyzed gene expression profiles of tomato stems infected with Cmm. This analysis revealed activation of basal defense responses that are typically observed upon plant perception of pathogen-associated molecular patterns. In addition, Cmm infection upregulated the expression of host genes related to ethylene synthesis and response. Further analysis of tomato plants impaired in ethylene perception and production demonstrated an important role for ethylene in the development of disease symptoms. Here we discuss possible molecular strategies used by the plant to recognize Cmm infection and possible mechanisms employed by the pathogen to interfere with the activation of plant defense responses and promote disease.
A Chemical-genetic Approach for Functional Analysis of Plant Protein Kinases
Plant genomes encode hundreds of protein kinases, yet only for a small fraction of them precise functions and phosphorylation targets have been identified. Recently, we applied a chemical-genetic approach to sensitize the tomato serine/threonine kinase Pto to analogs of PP1, an ATP-competitive and cell-permeable small-molecule inhibitor. The Pto kinase confers resistance to Pst bacteria by activating immune responses upon specific recognition of bacterial effectors. By using PP1 analogs in combination with the analog-sensitive Pto, we shed new light on the role of Pto kinase activity in effector recognition and signal transduction. Here we broaden the use of this chemical-genetic approach to another defense-related plant protein kinase, the MAP kinase LeMPK3. In addition, we show that analog-sensitive but not wild-type kinases are able to use unnatural N(6)-modified ATP analogs as phosphodonors that can be exploited for tagging direct phosphorylation targets of the kinase of interest. Thus, sensitization of kinases to analogs of the small-molecule inhibitor PP1 and ATP can be an effective tool for the discovery of cellular functions and phosphorylation substrates of plant protein kinases.
Bypassing Kinase Activity of the Tomato Pto Resistance Protein with Small Molecule Ligands
The tomato (Solanum lycopersicum) protein kinase Pto confers resistance to Pseudomonas syringae pv. tomato bacteria expressing the AvrPto and AvrPtoB effector proteins. Pto specifically recognizes both effectors by direct physical interactions triggering activation of immune responses. Here, we used a chemical-genetic approach to sensitize Pto to analogs of PP1, an ATP-competitive small molecule inhibitor. By using PP1 analogs in combination with the sensitized Pto (Pto(as)), we examined the role of Pto kinase activity in effector recognition and signal transduction. Strikingly, while PP1 analogs efficiently inhibited kinase activity of Pto(as) in vitro, they enhanced interactions of Pto(as) with AvrPto and AvrPtoB in a yeast two-hybrid system. In addition, in the presence of PP1 analogs, Pto(as) bypassed mutations either at an autophosphorylation site critical for the Pto-AvrPto interaction or at catalytically essential residues and interacted with both effectors. Moreover, in the presence of the PP1 analog 3MB-PP1, a kinase-deficient form of Pto(as) triggered an AvrPto-dependent hypersensitive response in planta. These findings suggest that, rather than phosphorylation per se, a conformational change likely triggered by autophosphorylation in Pto and mimicked by ligand binding in Pto(as) is a prerequisite for recognition of bacterial effectors. Following recognition, kinase activity appears to be dispensable for Pto signaling in planta. The chemical-genetic strategy applied here to develop specific small molecule inhibitors of Pto represents an invaluable tool for the study of biological functions of other plant protein kinases in vivo.
Regulatory Interactions Between Quorum-sensing, Auxin, Cytokinin, and the Hrp Regulon in Relation to Gall Formation and Epiphytic Fitness of Pantoea Agglomerans Pv. Gypsophilae
Gall formation by Pantoea agglomerans pv. gypsophilae is controlled by hrp/hrc genes, phytohormones, and the quorum-sensing (QS) regulatory system. The interactions between these three components were investigated. Disruption of the QS genes pagI and pagR and deletion of both substantially reduced the transcription levels of the hrp regulatory genes hrpXY, hrpS, and hrpL, as determined by quantitative reverse-transcriptase polymerase chain reaction. Expression of hrpL in planta was inhibited by addition of 20 microM or higher concentrations of the QS signal C(4)-HSL. The pagR and hrpL mutants caused an equivalent reduction of 1.3 orders in bacterial multiplication on bean leaves, suggesting possible mediation of the QS effect on epiphytic fitness of P. agglomerans pv. gypsophilae by the hrp regulatory system. indole-3-acetic acid (IAA) and cytokinin significantly affected the expression of the QS and hrp regulatory genes. Transcription of pagI, pagR, hrpL, and hrpS in planta was substantially reduced in iaaH mutant (disrupted in IAA biosynthesis via the indole-3-acetamide pathway) and etz mutant (disrupted in cytokinin biosynthesis). In contrast, the ipdC mutant (disrupted in IAA biosynthesis via the indole-3-pyruvate pathway) substantially increased expression of pagI, pagR, hrpL, and hrpS. Results presented suggest the involvement of IAA and cytokinins in regulation of the QS system and hrp regulatory genes.
Tomato MAPKKKε is a Positive Regulator of Cell-death Signaling Networks Associated with Plant Immunity
Mitogen-activated protein (MAP) kinase cascades are fundamental components of the signaling pathways associated with plant immunity. Despite the large number of MAP kinase kinase kinases (MAPKKK) encoded in the plant genome, only very few of them have an assigned function. Here, we identified MAPKKK gene of tomato (Solanum lycopersicum), SIMAPKKKε, which is required for hypersensitive response cell death and disease resistance against Gram-negative bacterial pathogens. Silencing of SIMAPKKKε compromised tomato resistance to Xanthomonas campestris and Pseudomonas syringae strains, resulting in the appearance of disease symptoms and enhanced bacterial growth. In addition, silencing of NbMAPKKKε in Nicotiana benthamiana plants significantly inhibited the cell death triggered by expression of different R gene/effector gene pairs. Conversely, overexpression of either the full-length SIMAPKKKε gene or its kinase domain in N. benthamiana leaves caused pathogen-independent activation of cell death that required an intact kinase catalytic domain. Moreover, by suppressing the expression of various MAPKK and MAPK genes and overexpressing the SIMAPKKKε kinase domain, we identified a signaling cascade acting downstream of SIMAPKKKε that includes MEK2, WIPK and SIPK. Additional epistasis experiments revealed that SIPKK functions as a negative regulator of SIMAPKKKε-mediated cell death. Our results provide evidence that SIMAPKKKε is a signaling molecule that positively regulates cell death networks associated with plant immunity.
Expression of Xanthomonas Campestris Pv. Vesicatoria Type III Effectors in Yeast Affects Cell Growth and Viability
The gram-negative bacterium Xanthomonas campestris pv. vesicatoria is the causal agent of spot disease in tomato and pepper. X. campestris pv. vesicatoria pathogenicity depends on a type III secretion system delivering effector proteins into the host cells. We hypothesized that some X. campestris pv. vesicatoria effectors target conserved eukaryotic cellular processes and examined phenotypes induced by their expression in yeast. Out of 21 effectors tested, 14 inhibited yeast growth in normal or stress conditions. Viability assay revealed that XopB and XopF2 attenuated cell proliferation, while AvrRxo1, XopX, and XopE1 were cytotoxic. Inspection of morphological features and DNA content of yeast cells indicated that cytotoxicity caused by XopX and AvrRxo1 was associated with cell-cycle arrest at G0/1. Interestingly, XopB, XopE1, XopF2, XopX, and AvrRxo1 that inhibited growth in yeast also caused phenotypes, such as chlorosis and cell death, when expressed in either host or nonhost plants. Finally, the ability of several effectors to cause phenotypes in yeast and plants was dependent on their putative catalytic residues or localization motifs. This study supports the use of yeast as a heterologous system for functional analysis of X. campestris pv. vesicatoria type III effectors, and sets the stage for identification of their eukaryotic molecular targets and modes of action.
Silencing of Host Basal Defense Response-related Gene Expression Increases Susceptibility of Nicotiana Benthamiana to Clavibacter Michiganensis Subsp. Michiganensis
Clavibacter michiganensis subsp. michiganensis is an actinomycete, causing bacterial wilt and canker disease of tomato (Solanum lycopersicum). We used virus-induced gene silencing (VIGS) to identify genes playing a role in host basal defense response to C. michiganensis subsp. michiganensis infection using Nicotiana benthamiana as a model plant. A preliminary VIGS screen comprising 160 genes from tomato known to be involved in defense-related signaling identified a set of 14 genes whose suppression led to altered host-pathogen interactions. Expression of each of these genes and three additional targets was then suppressed in larger-scale VIGS experiments and the effect of silencing on development of wilt disease symptoms and bacterial growth during an N. benthamiana-C. michiganensis subsp. michiganensis compatible interaction was determined. Disease susceptibility and in planta bacterial population size were enhanced by silencing genes encoding N. benthamiana homologs of ubiquitin activating enzyme, snakin-2, extensin-like protein, divinyl ether synthase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase 2, and Pto-like kinase. The identification of genes having a role in the host basal defense-response to C. michiganensis subsp. michiganensis advances our understanding of the plant responses activated by C. michiganensis subsp. michiganensis and raises possibilities for devising novel and effective molecular strategies to control bacterial canker and wilt in tomato.
The SlMKK2 and SlMPK2 Genes Play a Role in Tomato Disease Resistance to Xanthomonas Campestris Pv. Vesicatoria
Xanthomonas campestris pv. vesicatoria (Xcv) is the causal agent of bacterial spot disease in tomato (Solanum lycopersicum) plants. We recently identified a MAPKKK gene, SlMAPKKKε, which is required for tomato resistance to Xcv strains and encodes a positive regulator of cell death. We also provided evidence that the MEK2 MAPKK, and the WIPK and SIPK MAPKs act downstream to MAPKKKε in Nicotiana benthamiana plants. Here, we used the virus-induced gene silencing technique to assess whether tomato homologs of MEK2 (SlMKK2), SIPK (SlMPK1 and SlMPK2), WIPK (SlMPK3), and other components of MAP kinase cascades (SlNPK1, SlMEK1 and SlNTF6), which were previously implicated in plant immunity, are involved in disease resistance to Xcv. Silencing of none of the tested genes caused the appearance of disease symptoms in tomato leaves challenged with an avirulent Xcv strain. However, bacterial populations were significantly higher in leaves of plants silenced for SlMKK2 and SlMPK2, as compared to control plants, suggesting that these two genes contribute to disease resistance to Xcv. It remains to be established whether SlMKK2 and SlMPK2 are activated by SlMAPKKKε directly or through a distinct MAPKKK.
Characterization of Nuclear Localization Signals in the Type III Effectors HsvG and HsvB of the Gall-forming Bacterium Pantoea Agglomerans
HsvG and HsvB, two paralogous type III effectors of the gall-forming bacteria Pantoea agglomerans pv. gypsophilae and P. agglomerans pv. betae, determine host specificity on gypsophila and beet, respectively. They were previously shown to be DNA-binding proteins imported into host and non-host nuclei and might act as transcriptional activators. Sequence analysis of these effectors did not detect canonical nuclear localization signals (NLSs), but two basic amino acid clusters designated putative NLS1 and NLS2 were detected in their N-terminal and C-terminal regions, respectively. pNIA assay for nuclear import in yeast and bombardment of melon leaves with each of the NLSs fused to a 2xYFP reporter indicated that putative NLS1 and NLS2 were functional in transport of HsvG into the nucleus. A yeast two-hybrid assay showed that HsvB, HsvG, putative NLS1, putative NLS2, HsvG converted into HsvB, or HsvB converted into HsvG by exchanging the repeat domain, all interacted with AtKAP-α and importin-α3 of Arabidopsis thaliana. Deletion analysis of the NLS domains in HsvG suggested that putative NLS1 or NLS2 were required for pathogenicity on gypsophila cuttings and presumably for import of HsvG into the nucleus. This study demonstrates the presence of two functional NLSs in the type III effectors HsvG and HsvB.
Endosomal Signaling of the Tomato Leucine-rich Repeat Receptor-like Protein LeEix2
Extracellular leucine-rich repeat (LRR) receptor-like proteins (RLPs) represent a unique class of cell-surface receptors, as they lack a functional cytoplasmic domain. Our knowledge of how RLPs that do not contain a kinase or Toll domain function is very limited. The tomato RLP receptor LeEix2 signals to induce defense responses mediated by the fungal protein ethylene-inducing xylanase (EIX). The movement of FYVE-positive endosomes before and after EIX application was examined using spinning disc confocal microscopy. We found that while FYVE-positive endosomes generally observe a random movement pattern, following EIX application a subpopulation of FYVE-positive endosomes follow a directional movement pattern. Further, cellular endosomes travel greater distances at higher speeds following EIX application. Time-course experiments conducted with specific inhibitors demonstrate the involvement of endosomal signaling in EIX-triggered defense responses. Abolishing the existence of endosomes or the endocytic event prevented EIX-induced signaling. Endocytosis/endosome inhibitors, such as Dynasore or 1-butanol, inhibit EIX-induced signaling. Moreover, treatment with Endosidin1, which inhibits an early step in plasma membrane/endosome trafficking, enhances the induction of defense responses by EIX. Our data indicate a distinct endosomal signaling mechanism for induction of defense responses in this RLP system.
Sensitizing Plant Protein Kinases to Specific Inhibition by ATP-competitive Molecules
The highly conserved nature of the protein kinase catalytic domain and the low permeability of plant cell membranes pose a challenge to the development of specific inhibitors that target individual protein kinases in vivo. Here, we describe a chemical-genetic approach to specifically sensitize individual plant kinases to cell-permeable small molecules that do not inhibit wild-type kinases. In this approach, a single amino-acid substitution is introduced in the ATP-binding site of the enzyme enabling specific binding of ATP-competitive molecules. Cell-permeable molecules can then be used to specifically target the sensitized allele in transgenic Arabidopsis thaliana plants that do not express the wild-type form of the kinase. This strategy provides a useful tool for the functional characterization of protein kinases in planta and for the dissection of the signaling pathways in which they are involved.
A Simple Yeast-based Strategy to Identify Host Cellular Processes Targeted by Bacterial Effector Proteins
Bacterial effector proteins, which are delivered into the host cell via the type III secretion system, play a key role in the pathogenicity of gram-negative bacteria by modulating various host cellular processes to the benefit of the pathogen. To identify cellular processes targeted by bacterial effectors, we developed a simple strategy that uses an array of yeast deletion strains fitted into a single 96-well plate. The array is unique in that it was optimized computationally such that despite the small number of deletion strains, it covers the majority of genes in the yeast synthetic lethal interaction network. The deletion strains in the array are screened for hypersensitivity to the expression of a bacterial effector of interest. The hypersensitive deletion strains are then analyzed for their synthetic lethal interactions to identify potential targets of the bacterial effector. We describe the identification, using this approach, of a cellular process targeted by the Xanthomonas campestris type III effector XopE2. Interestingly, we discover that XopE2 affects the yeast cell wall and the endoplasmic reticulum stress response. More generally, the use of a single 96-well plate makes the screening process accessible to any laboratory and facilitates the analysis of a large number of bacterial effectors in a short period of time. It therefore provides a promising platform for studying the functions and cellular targets of bacterial effectors and other virulence proteins.
The Type III Effector HsvG of the Gall-forming Pantoea Agglomerans Mediates Expression of the Host Gene HSVGT
The type III effector HsvG of the gall-forming Pantoea agglomerans pv. gypsophilae is a DNA-binding protein that is imported to the host nucleus and involved in host specificity. The DNA-binding region of HsvG was delineated to 266 amino acids located within a secondary structure region near the N-terminus of the protein but did not display any homology to canonical DNA-binding motifs. A binding site selection procedure was used to isolate a target gene of HsvG, named HSVGT, in Gypsophila paniculata. HSVGT is a predicted acidic protein of the DnaJ family with 244 amino acids. It harbors characteristic conserved motifs of a eukaryotic transcription factor, including a bipartite nuclear localization signal, zinc finger, and leucine zipper DNA-binding motifs. Quantitative real-time polymerase chain reaction analysis demonstrated that HSVGT transcription is specifically induced in planta within 2 h after inoculation with the wild-type P. agglomerans pv. gypsophilae compared with the hsvG mutant. Induction of HSVGT reached a peak of sixfold at 4 h after inoculation and progressively declined thereafter. Gel-shift assay demonstrated that HsvG binds to the HSVGT promoter, indicating that HSVGT is a direct target of HsvG. Our results support the hypothesis that HsvG functions as a transcription factor in gypsophila.
The Clavibacter Michiganensis Subsp. Michiganensis-Tomato Interactome Reveals the Perception of Pathogen by the Host and Suggests Mechanisms of Infection
The Gram-positive bacterium Clavibacter michiganensis subsp. michiganensis (Cmm) causes wilt and canker disease of tomato (Solanum lycopersicum). Mechanisms of Cmm pathogenicity and tomato response to Cmm infection are not well understood. To explore the interaction between Cmm and tomato, multidimensional protein identification technology (MudPIT) and tandem mass spectrometry were used to analyze in vitro and in planta generated samples. The results show that during infection Cmm senses the plant environment, transmits signals, induces, and then secretes multiple hydrolytic enzymes, including serine proteases of the Pat-1, Ppa, and Sbt familes, the CelA, XysA, and NagA glycosyl hydrolases, and other cell wall-degrading enzymes. Tomato induction of pathogenesis-related (PR) proteins, LOX1, and other defense-related proteins during infection indicates that the plant senses the invading bacterium and mounts a basal defense response, although partial with some suppressed components including class III peroxidases and a secreted serine peptidase. The tomato ethylene-synthesizing enzyme ACC-oxidase was induced during infection with the wild-type Cmm but not during infection with an endophytic Cmm strain, identifying Cmm-triggered host synthesis of ethylene as an important factor in disease symptom development. The proteomic data were also used to improve Cmm genome annotation, and thousands of Cmm gene models were confirmed or expanded.
