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

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

Savidor A, Chalupowicz L, Teper D, et al. Clavibacter michiganensis subsp michiganensis Vatr1 and Vatr2 Transcriptional Regulators Are Required for Virulence in Tomato. Molecular Plant-Microbe Interactions. 2014;27(10):1035-1047.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 Delta vatr2 mutant showing a more dramatic effect than Delta vatr1. 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 infectionmimicking 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

Characterization of a Clavibacter michiganensis subsp michiganensis population in Israel

Kleitman F, Barash I, Burger A, et al. Characterization of a Clavibacter michiganensis subsp michiganensis population in Israel. EUROPEAN JOURNAL OF PLANT PATHOLOGY. 2008;121(4):463-475.Clavibacter michiganensis subsp. michiganensis (Cmm) strains, collected during the last decade from different locations in Israel, were analyzed by macrorestriction pulsed-field gel electrophoresis (PFGE). Fifty-eight strains from Israel and 18 from other sources were differentiated into 11 haplotypes with either VspI or DraI restriction enzymes. The strains from Israel formed four distinct groups among which groups A (16 strains) and B (32 strains) constituted the major clusters. These two groups originated from the Besor region, which is the main area for growing tomatoes under cover. Rep-PCR, with either ERIC or BOX primers, confirmed results obtained by PFGE. PCR with primers based on three genes - ppaA, chpC and tomA - that spanned the pathogenicity island of the reference strain NCPPB382, produced the expected products with the tested pathogenic strains. Plasmid analysis of representative strains revealed different profiles of one or two plasmids. However all the strains, including five non-pathogenic ones, reacted positively in PCR with primers based on celA gene, which resides on the plasmid pCM1 of NCPPB382. Southern hybridization of total DNA with a 3.2-kb BglII-fragment of pCM1 containing the celA gene was positive when carried out with 31 strains, but the size of the reacting band was not always the same as that of pCM1, suggesting that the plasmids carrying celA may differ in size. Comparison between the colonization rates of strain Cmm42 (group A) and of Cmm32 (group B) did not show any significant differences. The high diversity of the Cmm strains, on the one hand, and the presence of two persistent groups in the Besor region, on the other hand, suggests that the primary inoculum originated each year from residual plants in the soil rather than from infested seeds, in spite of extensive control measures taken by the growers in this area

Tomato Transcriptional Changes in Response to Clavibacter michiganensis subsp. michiganensis Reveal a Role for Ethylene in Disease Development1[W]

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

Characterization of Pectobacterium brasiliense strains from potato and vegetables in Israel

International audiencePectobacterium brasiliense (Pbr) infects a wide range of crops worldwide, causing potato blackleg and soft rot and vegetable soft rots. This study aimed to characterize the genetic diversity and virulence variability among 68 Pbr strains isolated from either symptomless potato progeny tubers, diseased potato plants, ware potatoes wash water, or vegetables grown in Israel, as well as strains isolated from symptomless seed tubers grown in Europe, or diseased potato plants grown in France. The collection was typed using PCR and TaqMan real-time PCR analyses, dnaX sequence analysis, pulsed-field gel electrophoresis (PFGE), and pectolytic activity. dnaX phylogeny grouped almost all strains in a common genetic clade related to Pbr, which was distinct from the other Pectobacterium species. PFGE analysis identified two main clusters, including one major group of 47 strains with 95%-100% similarity. Maceration assays on two potato cultivars showed significant differences between strains but with no correlations with the source of the strains nor the status of the host (with/without symptoms). Molecular (dnaX sequences and PFGE profiles) and phenotypic analyses (tuber maceration tests) showed that the tested Pbr strains are not a homogeneous group. Analysis of the tested Pbr strains isolated from potato and vegetables grown in fields with a history of potato cultivation suggests that seed tubers imported from Europe may be the main source for Pbr in Israel. To the best of our knowledge, this is the first study that describes biodiversity and population structure of P. brasiliense isolated from potato and vegetables under hot climate condition

The Clavibacter michiganensis subsp michiganensis-Tomato Interactome Reveals the Perception of Pathogen by the Host and Suggests Mechanisms of Infection

Savidor A, Teper D, Gartemann K-H, et al. The Clavibacter michiganensis subsp michiganensis-Tomato Interactome Reveals the Perception of Pathogen by the Host and Suggests Mechanisms of Infection. Journal of Proteome Research. 2012;11(2):736-750.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