An antibacterial T6SS in Pantoea agglomerans pv. betae delivers a lysozyme-like effector to antagonize competitors.

The type VI secretion system (T6SS) is deployed by numerous Gram-negative bacteria to deliver toxic effectors into neighbouring cells. The genome of Pantoea agglomerans pv. betae (Pab) phytopathogenic bacteria contains a gene cluster (T6SS1) predicted to encode a complete T6SS. Using secretion and competition assays, we found that T6SS1 in Pab is a functional antibacterial system that allows this pathogen to outcompete rival plant-associated bacteria found in its natural environment. Computational analysis of the T6SS1 gene cluster revealed that antibacterial effector and immunity proteins are encoded within three genomic islands that also harbour arrays of orphan immunity genes or toxin and immunity cassettes. Functional analyses indicated that VgrG, a specialized antibacterial effector, contains a C-terminal catalytically active glucosaminidase domain that is used to degrade prey peptidoglycan. Moreover, we confirmed that a bicistronic unit at the end of the T6SS1 cluster encodes a novel antibacterial T6SS effector and immunity pair. Together, these results demonstrate that Pab T6SS1 is an antibacterial system delivering a lysozyme-like effector to eliminate competitors, and indicate that this bacterium contains additional novel T6SS effectors.

Assessing the Ability of Salmonella enterica to Translocate Type III Effectors Into Plant Cells.

Salmonella enterica serovar Typhimurium, a human enteric pathogen, has the ability to multiply and survive endophytically in plants. Genes encoding the type III secretion system (T3SS) or its effectors (T3Es) may contribute to its colonization. Two reporter plasmids for T3E translocation into plant cells that are based on hypersensitive response domains of avirulence proteins from the Pantoea agglomerans-beet and Xanthomonas euvesicatoria-pepper pathosystems were employed in this study to investigate the role of T3Es in the interaction of Salmonella ser. Typhimurium 14028 with plants. The T3Es of Salmonella ser. Typhimurium, SipB and SifA, which are translocated into animal cells, could not be delivered by Salmonella ser. Typhimurium into cells of beet roots or pepper leaves. In contrast, these effectors were translocated into plant cells by the phytopathogenic bacteria P. agglomerans pv. betae, Erwinia amylovora, and X. euvesicatoria. Similarly, HsvG, a T3E of P. agglomerans pv. gypsophilae, and XopAU of X. euvesicatoria could be translocated into beet roots and pepper leaves, respectively, by the plant pathogens but not by Salmonella ser. Typhimurium. Mutations in Salmonella ser. Typhimurium T3SS genes invA, ssaV, sipB, or sifA, did not affect its endophytic colonization of lettuce leaves, supporting the notion that S. enterica cannot translocate T3Es into plant cells.

Clavibacter michiganensis subsp. michiganensis Vatr1 and Vatr2 Transcriptional Regulators Are Required for Virulence in Tomato.

The plant pathogen Clavibacter michiganensis subsp. michiganensis (Cmm) is a Gram-positive bacterium responsible for wilt and canker disease of tomato. While disease development is well characterized and diagnosed, molecular mechanisms of Cmm virulence are poorly understood. Here, we identified and characterized two Cmm transcriptional regulators, Vatr1 and Vatr2, that are involved in pathogenicity of Cmm. Vatr1 and Vatr2 belong to TetR and MocR families of transcriptional regulators, respectively. Mutations in their corresponding genes caused attenuated virulence, with the Δvatr2 mutant showing a more dramatic effect than Δvatr1. While both mutants grew well in vitro and reached a high titer in planta, they caused reduced wilting and canker development in infected plants compared with the wild-type bacterium. They also led to a reduced expression of the ethylene-synthesizing tomato enzyme ACC-oxidase compared with wild-type Cmm and to reduced ethylene production in the plant. Transcriptomic analysis of wild-type Cmm and the two mutants under infection-mimicking conditions revealed that Vatr1 and Vatr2 regulate expression of virulence factors, membrane and secreted proteins, and signal transducing proteins. A 70% overlap between the sets of genes positively regulated by Vatr1 and Vatr2 suggests that these transcriptional regulators are on the same molecular pathway responsible for Cmm virulence.

Clavibacter michiganensis subsp. michiganensis Vatr1 and Vatr2 transcriptional regulators are required for virulence in tomato.

The plant pathogen Clavibacter michiganensis subsp. michiganensis is a gram-positive bacterium responsible for wilt and canker disease of tomato. Although disease development is well characterized and diagnosed, molecular mechanisms of C. michiganensis subsp. michiganensis virulence are poorly understood. Here, we identified and characterized two C. michiganensis subsp. michiganensis transcriptional regulators, Vatr1 and Vatr2, that are involved in pathogenicity of C. michiganensis subsp. michiganensis. Vatr1 and Vatr2 belong to TetR and MocR families of transcriptional regulators, respectively. Mutations in their corresponding genes caused attenuated virulence, with the Δvatr2 mutant showing a more dramatic effect than Δvatr1. Although both mutants grew well in vitro and reached a high titer in planta, they caused reduced wilting and canker development in infected plants compared with the wild-type bacterium. They also led to a reduced expression of the ethylene-synthesizing tomato enzyme ACC-oxidase compared with wild-type C. michiganensis subsp. michiganensis and to reduced ethylene production in the plant. Transcriptomic analysis of wild-type C. michiganensis subsp. michiganensis and the two mutants under infection-mimicking conditions revealed that Vatr1 and Vatr2 regulate expression of virulence factors, membrane and secreted proteins, and signal-transducing proteins. A 70% overlap between the sets of genes positively regulated by Vatr1 and Vatr2 suggests that these transcriptional regulators are on the same molecular pathway responsible for C. michiganensis subsp. michiganensis virulence.

Global regulatory networks control the hrp regulon of the gall-forming bacterium Pantoea agglomerans pv. gypsophilae.

Gall formation by Pantoea agglomerans pv. gypsophilae is dependent on the hypersensitive response and pathogenicity (hrp) system. Previous studies demonstrated that PagR and PagI, regulators of the quorum-sensing system, induce expression of the hrp regulatory cascade (i.e., hrpXY, hrpS, and hrpL) that activates the HrpL regulon. Here, we isolated the genes of the Gac/Rsm global regulatory pathway (i.e., gacS, gacA, rsmB, and csrD) and of the post-transcriptional regulator rsmA. Our results demonstrate that PagR and PagI also upregulate expression of the Gac/Rsm pathway. PagR acts as a transcriptional activator of each of the hrp regulatory genes and gacA in a N-butanoyl-L-homoserine lactone-dependent manner as shown by gel shift experiments. Mutants of the Gac/Rsm genes or overexpression of rsmA significantly reduced Pantoea agglomerans virulence and colonization of gypsophila. Overexpression of rsmB sRNA abolished gall formation, colonization, and hypersensitive reaction on nonhost plants and prevented transcription of the hrp regulatory cascade, indicating a lack of functional type III secretion system. Expression of rsmB sRNA in the background of the csrD null mutant suggests that CsrD may act as a safeguard for preventing excessive production of rsmB sRNA. Results presented indicate that the hrp regulatory cascade is controlled directly by PagR and indirectly by RsmA, whereas deficiency in RsmA activity is epistatic to PagR induction.

Tomato fruit and seed colonization by Clavibacter michiganensis subsp. michiganensis through external and internal routes.

The Gram-positive bacterium Clavibacter michiganensis subsp. michiganensis, causal agent of bacterial wilt and canker of tomato, is an economically devastating pathogen that inflicts considerable damage throughout all major tomato-producing regions. Annual outbreaks continue to occur in New York, where C. michiganensis subsp. michiganensis spreads via infected transplants, trellising stakes, tools, and/or soil. Globally, new outbreaks can be accompanied by the introduction of contaminated seed stock; however, the route of seed infection, especially the role of fruit lesions, remains undefined. In order to investigate the modes of seed infection, New York C. michiganensis subsp. michiganensis field strains were stably transformed with a gene encoding enhanced green fluorescent protein (eGFP). A constitutively eGFP-expressing virulent C. michiganensis subsp. michiganensis isolate, GCMM-22, was used to demonstrate that C. michiganensis subsp. michiganensis could not only access seeds systemically through the xylem but also externally through tomato fruit lesions, which harbored high intra- and intercellular populations. Active movement and expansion of bacteria into the fruit mesocarp and nearby xylem vessels followed, once the fruits began to ripen. These results highlight the ability of C. michiganensis subsp. michiganensis to invade tomato fruits and seeds through multiple entry routes.

Comparative sequence analysis of pPATH pathogenicity plasmids in Pantoea agglomerans gall-forming bacteria.

Acquisition of the pathogenicity plasmid pPATH that encodes a type III secretion system (T3SS) and effectors (T3Es) has likely led to the transition of a non-pathogenic bacterium into the tumorigenic pathogen Pantoea agglomerans. P. agglomerans pv. gypsophilae (Pag) forms galls on gypsophila (Gypsophila paniculata) and triggers immunity on sugar beet (Beta vulgaris), while P. agglomerans pv. betae (Pab) causes galls on both gypsophila and sugar beet. Draft sequences of the Pag and Pab genomes were previously generated using the MiSeq Illumina technology and used to determine partial T3E inventories of Pab and Pag. Here, we fully assembled the Pab and Pag genomes following sequencing with PacBio technology and carried out a comparative sequence analysis of the Pab and Pag pathogenicity plasmids pPATHpag and pPATHpab. Assembly of Pab and Pag genomes revealed a ~4 Mbp chromosome with a 55% GC content, and three and four plasmids in Pab and Pag, respectively. pPATHpag and pPATHpab share 97% identity within a 74% coverage, and a similar GC content (51%); they are ~156 kb and ~131 kb in size and consist of 198 and 155 coding sequences (CDSs), respectively. In both plasmids, we confirmed the presence of highly similar gene clusters encoding a T3SS, as well as auxin and cytokinins biosynthetic enzymes. Three putative novel T3Es were identified in Pab and one in Pag. Among T3SS-associated proteins encoded by Pag and Pab, we identified two novel chaperons of the ShcV and CesT families that are present in both pathovars with high similarity. We also identified insertion sequences (ISs) and transposons (Tns) that may have contributed to the evolution of the two pathovars. These include seven shared IS elements, and three ISs and two transposons unique to Pab. Finally, comparative sequence analysis revealed plasmid regions and CDSs that are present only in pPATHpab or in pPATHpag. The high similarity and common features of the pPATH plasmids support the hypothesis that the two strains recently evolved into host-specific pathogens.

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.

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.

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.

Recent evolution of bacterial pathogens: the gall-forming Pantoea agglomerans case.

Pantoea agglomerans, a widespread epiphyte and commensal bacterium, has evolved into an Hrp-dependent and host-specific tumorigenic pathogen by acquiring a plasmid containing a pathogenicity island (PAI). The PAI was evolved on an iteron plasmid of the IncN family, which is distributed among genetically diverse populations of P. agglomerans. The structure of the PAI supports the premise of a recently evolved pathogen. This review offers insight into a unique model for emergence of new bacterial pathogens. It illustrates how horizontal gene transfer was the major driving force in the creation of the PAI, although a pathoadaptive mechanism might also be involved. It describes the crucial function of plant-produced indole-3-acetic acid (IAA) and cytokinines (CK) in gall initiation as opposed to the significant but secondary role of pathogen-secreted phytohormones. It also unveils the role of type III effectors in determination of host specificity and evolution of the pathogen into pathovars. Finally, it describes how interactions between the quorum sensing system, hrp regulatory genes, and bacterially secreted IAA or CKs affect gall formation and epiphytic fitness.

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.

Two Pantoea agglomerans type III effectors can transform nonpathogenic and phytopathogenic bacteria into host-specific gall-forming pathogens.

Pantoea agglomerans (Pa), a widespread commensal bacterium, has evolved into a host-specific gall-forming pathogen on gypsophila and beet by acquiring a plasmid harbouring a type III secretion system (T3SS) and effectors (T3Es). Pantoea agglomerans pv. gypsophilae (Pag) elicits galls on gypsophila and a hypersensitive response on beet, whereas P. agglomerans pv. betae (Pab) elicits galls on beet and gypsophila. HsvG and HsvB are two paralogous T3Es present in both pathovars and act as host-specific transcription activators on gypsophila and beet, respectively. PthG and PseB are major T3Es that contribute to gall development of Pag and Pab, respectively. To establish the minimal combinations of T3Es that are sufficient to elicit gall symptoms, strains of the nonpathogenic bacteria Pseudomonas fluorescens 55, Pa 3-1, Pa 98 and Escherichia coli, transformed with pHIR11 harbouring a T3SS, and the phytopathogenic bacteria Erwinia amylovora, Dickeya solani and Xanthomonas campestris pv. campestris were transformed with the T3Es hsvG, hsvB, pthG and pseB, either individually or in pairs, and used to infect gypsophila and beet. Strikingly, all the tested nonpathogenic and phytopathogenic bacterial strains harbouring hsvG and pthG incited galls on gypsophila, whereas strains harbouring hsvB and pseB, with the exception of E. coli, incited galls on beet.

Virulence mechanisms and host specificity of gall-forming Pantoea agglomerans.

Pantoea agglomerans has been transformed from a commensal bacterium associated with many plants into a host-specific gall-forming pathogen by acquiring a plasmid-borne pathogenicity island. This pathogenicity island harbors the hrp/hrc gene cluster, in addition to genes encoding type III effector proteins, biosynthesis of the phytohormones indole-3-acetic acid and cytokinin, multiple diverse insertion sequences and pseudogenes. This review describes a unique model for understanding the emergence of new pathogens or new pathogenic variants, offering an insight into the function of type III effectors in host specificity and the evolution of a pathogen into pathovars. It also addresses the primary role of type III effectors in gall initiation as compared with a secondary role of phytohormones secreted by the pathogen.

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.

Revealing the inventory of type III effectors in Pantoea agglomerans gall-forming pathovars using draft genome sequences and a machine-learning approach.

Pantoea agglomerans, a widespread epiphytic bacterium, has evolved into a hypersensitive response and pathogenicity (hrp)-dependent and host-specific gall-forming pathogen by the acquisition of a pathogenicity plasmid containing a type III secretion system (T3SS) and its effectors (T3Es). Pantoea agglomerans pv. betae (Pab) elicits galls on beet (Beta vulgaris) and gypsophila (Gypsophila paniculata), whereas P. agglomerans pv. gypsophilae (Pag) incites galls on gypsophila and a hypersensitive response (HR) on beet. Draft genome sequences were generated and employed in combination with a machine-learning approach and a translocation assay into beet roots to identify the pools of T3Es in the two pathovars. The genomes of the sequenced Pab4188 and Pag824-1 strains have a similar size (∼5 MB) and GC content (∼55%). Mutational analysis revealed that, in Pab4188, eight T3Es (HsvB, HsvG, PseB, DspA/E, HopAY1, HopX2, HopAF1 and HrpK) contribute to pathogenicity on beet and gypsophila. In Pag824-1, nine T3Es (HsvG, HsvB, PthG, DspA/E, HopAY1, HopD1, HopX2, HopAF1 and HrpK) contribute to pathogenicity on gypsophila, whereas the PthG effector triggers HR on beet. HsvB, HsvG, PthG and PseB appear to endow pathovar specificities to Pab and Pag, and no homologous T3Es were identified for these proteins in other phytopathogenic bacteria. Conversely, the remaining T3Es contribute to the virulence of both pathovars, and homologous T3Es were found in other phytopathogenic bacteria. Remarkably, HsvG and HsvB, which act as host-specific transcription factors, displayed the largest contribution to disease development.

Distribution and replication of the pathogenicity plasmid pPATH in diverse populations of the gall-forming bacterium Pantoea agglomerans.

Pantoea agglomerans has been transformed from a commensal bacterium into two related gall-forming pathovars by acquisition of pPATH plasmids containing a pathogenicity island (PAI). This PAI harbors an hrp/hrc gene cluster, type III effectors, and phytohormone biosynthetic genes. DNA typing by pulsed-field gel electrophoresis revealed two major groups of P. agglomerans pv. gypsophilae and one group of P. agglomerans pv. betae. The pPATH plasmids of the different groups had nearly identical replicons (98% identity), and the RepA protein showed the highest level of similarity with IncN plasmid proteins. A series of plasmids, designated pRAs, in which the whole replicon region (2,170 bp) or deleted derivatives of it were ligated with nptI were generated for replicon analysis. A basic 929-bp replicon (pRA6) was sufficient for replication in Escherichia coli and in nonpathogenic P. agglomerans. However, the whole replicon region (pRA1) was necessary for expulsion of the pPATH plasmid, which resulted in the loss of pathogenicity. The presence of direct repeats in the replicon region suggests that the pPATH plasmid is an iteron plasmid and that the repeats may regulate its replication. The pPATH plasmids are nonconjugative but exhibit a broad host range, as shown by replication of pRA1 in Erwinia, Pseudomonas, and Xanthomonas. Restriction fragment length polymorphism analyses indicated that the PAIs in the two groups of P. agglomerans pv. gypsophilae are similar but different from those in P. agglomerans pv. betae. The results could indicate that the pPATH plasmids evolved from a common ancestral mobilizable plasmid that was transferred into different strains of P. agglomerans.

Differential contribution of Clavibacter michiganensis ssp. michiganensis virulence factors to systemic and local infection in tomato.

Clavibacter michiganensis ssp. michiganensis (Cmm) causes substantial economic losses in tomato production worldwide. The disease symptoms observed in plants infected systemically by Cmm are wilting and canker on the stem, whereas blister-like spots develop in locally infected leaves. A wide repertoire of serine proteases and cell wall-degrading enzymes has been implicated in the development of wilt and canker symptoms. However, virulence factors involved in the formation of blister-like spots, which play an important role in Cmm secondary spread in tomato nurseries, are largely unknown. Here, we demonstrate that Cmm virulence factors play different roles during blister formation relative to wilting. Inoculation with a green fluorescent protein (GFP)-labelled Cmm382 indicates that penetration occurs mainly through trichomes. When spray inoculated on tomato leaves, the wild-type Cmm382 and Cmm100 (lacking plasmids pCM1 and pCM2) strains form blister-like spots on leaves, whereas Cmm27 (lacking the chp/tomA pathogenicity island) is non-pathogenic, indicating that plasmid-borne genes, which have a crucial role in wilting, are not required for blister formation. Conversely, mutations in chromosomal genes encoding serine proteases (chpC and sbtA), cell wall-degrading enzymes (pgaA and endX/Y), a transcriptional regulator (vatr2), a putative perforin (perF) and a putative sortase (srtA) significantly affect disease incidence and the severity of blister formation. The transcript levels of these genes, as measured by quantitative reverse transcription-polymerase chain reaction, showed that, during blister formation, they are expressed early at 8-16 h after inoculation, whereas, during wilting, they are expressed after 24-72 h or expressed at low levels. Plant gene expression studies suggest that chpC is involved in the suppression of host defence.

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.

Virulence and in planta movement of Xanthomonas hortorum pv. pelargonii are affected by the diffusible signal factor (DSF)-dependent quorum sensing system.

Xanthomonas hortorum pv. pelargonii (Xhp), the causal agent of bacterial blight in pelargonium, is the most threatening bacterial disease of this ornamental worldwide. To gain an insight into the regulation of virulence in Xhp, we have disrupted the quorum sensing (QS) genes, which mediate the biosynthesis and sensing of the diffusible signal factor (DSF). Mutations in rpfF (encoding the DSF synthase) and rpfC (encoding the histidine sensor kinase of the two-component system RfpC/RpfG) and overexpression of rpfF showed a significant reduction in incidence and severity of the disease on pelargonium. Confocal laser scanning microscopy images of inoculated plants with a green fluorescent protein (GFP)-labelled wild-type strain showed that the pathogen is homogeneously dispersed in the lumen of xylem vessels, reaching the apex and invading the intercellular spaces of the leaf mesophyll tissue within 21 days. In contrast, the rpfF and rpfC knockout mutants, as well as the rpfF-overexpressing strain, remained confined to the vicinity of the inoculation site. The rpfF and rpfC mutants formed large incoherent aggregates in the xylem vessels that might interfere with upward movement of the bacterium within the plant. Both mutants also formed extended aggregates under in vitro conditions, whereas the wild-type strain formed microcolonies. Expression levels of putative virulence genes in planta were substantially reduced within 48 h after inoculation with the QS mutants when compared with the wild-type. The results presented indicate that an optimal DSF concentration is crucial for successful colonization and virulence of Xhp in pelargonium.