SummaryThe bacterial pathogen Pseudomonas syringae pv tomato DC3000 suppresses plant innate immunity with effector proteins injected by a type III secretion system (T3SS). The cysteine protease effector HopN1, which reduces the ability of DC3000 to elicit programmed cell death in non-host tobacco, was found to also suppress the production of defence-associated reactive oxygen species (ROS) and callose when delivered by Pseudomonas fluorescens heterologously expressing a P. syringae T3SS. Purified His 6-tagged HopN1 was used to identify tomato PsbQ, a member of the oxygen evolving complex of photosystem II (PSII), as an interacting protein. HopN1 localized to chloroplasts and both degraded PsbQ and inhibited PSII activity in chloroplast preparations, whereas a HopN1D299A non-catalytic mutant lost these abilities. Gene silencing of NtPsbQ in tobacco compromised ROS production and programmed cell death by DC3000. Our data reveal PsbQ as a contributor to plant immunity responses and a target for pathogen suppression.

Pseudomonas savastanoi pv. savastanoi NCPPB 3335 causes olive knot disease and is a model pathogen for exploring bacterial infection of woody hosts. The type III secretion system (T3SS) effector repertoire of this strain includes 31 effector candidates plus two novel candidates identified in this study which have not been reported to translocate into plant cells. In this work, we demonstrate the delivery of seven NCPPB 3335 effectors into Nicotiana tabacum leaves, including three proteins from two novel families of the P. syringae complex effector super-repertoire (HopBK and HopBL), one of which comprises two proteins (HopBL1 and HopBL2) that harbor a SUMO protease domain. When delivered by P. fluorescens heterologously expressing a P. syringae T3SS, all seven effectors were found to suppress the production of defense-associated reactive oxygen species. Moreover, six of these effectors, including the truncated versions of HopAA1 and HopAZ1 encoded by NCPPB 3335, suppressed callose deposition. The expression of HopAZ1 and HopBL1 by functionally effectorless P. syringae pv. tomato DC3000D28E inhibited the hypersensitive response in tobacco and, additionally, expression of HopBL2 by this strain significantly increased its competitiveness in N. benthamiana. DNA sequences encoding HopBL1 and HopBL2 were uniquely detected in a collection of 31 P. savastanoi pv. savastanoi strains and other P. syringae strains isolated from woody hosts, suggesting a relevant role of these two effectors in bacterial interactions with olive and other woody plants.

Dickeya dadantii 3937 (ex Erwinia chrysanthemi), a member of the Enterobacteriaceae, causes soft rot in many economically important crops. A successful pathogen has to reach the interior of the plant in order to cause disease. To study the role of motility and chemotaxis in the pathogenicity of D. dadantii 3937, genes involved in the chemotactic signal transduction system (cheW, cheB, cheY and cheZ) and in the structure of the flagellar motor (motA) were mutagenized. All the mutant strains grew like the wild-type in culture media, and the production and secretion of pectolytic enzymes was not affected. As expected, the swimming ability of the mutant strains was reduced with respect to the wild-type: motA (94 %), cheY (80 %), cheW (74 %), cheB (54 %) and cheZ (48 %). The virulence of the mutant strains was analysed in chicory, Saintpaulia and potato. The mutant strains were also tested for their capability to enter into Arabidopsis leaves. All the mutants showed a significant decrease of virulence in certain hosts; however, the degree of virulence reduction varied depending on the virulence assay. The ability to penetrate Arabidopsis leaves was impaired in all the mutants, whereas the capacity to colonize potato tubers after artificial inoculation was affected in only two mutant strains. In general, the virulence of the mutants could be ranked as motA,cheY,cheB5cheW,cheZ, which correlated with the degree to which swimming was affected. These results clearly indicate that motility plays an important role in the pathogenicity of this bacterium.

Characteristics of forty strains from macerated potato tubers and water-soaked lesions of some ornamental plants were studied in north parts of Iran. The causal organisms isolated from infected tissues were identified as Pectobacterium spp. based on their physiological and biochemical assays and confirmed by species and subspecies specific PCR and RFLP analysis of 16S-23S intergenic transcribed spacer region. Artificial inoculation of isolates to their related hosts generated the same symptoms on potato and ornamental plants, from which the same bacteria were isolated and identified. We detected two groups of atypical isolates in this study. The first group from potato classified as Pectobacterium carotovorum subsp. carotovorum by phenotypic tests but was unable to elicit HR on tobacco leaves, to grow at 37°C and to amplify the pel gene relevant to this subspecies. The second one from ornamental plants which was again characterized as Pectobacterium carotovorum subsp. carotovorum in biochemical assays, produced a unique ITS-RFLP profile different from all of known Pectobacterium species and subspecies. Our findings based on phylogenetic analysis using concatenated partial sequences of housekeeping genes mdh and gapA, indicated the occurrence of P. wasabiae as a novel species in potato storage in Iran. Furthermore we detected a distinct clade of Pectobacterium spp. from some ornamental plants including Schlumbergera bridgesii, Syngonium podophyllum and Iris spp.

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SUMMARYChemotaxis enables bacteria to move towards an optimal environment in response to chemical signals. In the case of plantpathogenic bacteria, chemotaxis allows pathogens to explore the plant surface for potential entry sites with the ultimate aim to prosper inside plant tissues and to cause disease. Chemoreceptors, which constitute the sensory core of the chemotaxis system, are usually transmembrane proteins which change their conformation when sensing chemicals in the periplasm and transduce the signal through a kinase pathway to the flagellar motor. In the particular case of the soft-rot pathogen Dickeya dadantii 3937, jasmonic acid released in a plant wound has been found to be a strong chemoattractant which drives pathogen entry into the plant apoplast. In order to identify candidate chemoreceptors sensing wound-derived plant compounds, we carried out a bioinformatics search of candidate chemoreceptors in the genome of Dickeya dadantii 3937. The study of the chemotactic response to several compounds and the analysis of the entry process to Arabidopsis leaves of 10 selected mutants in chemoreceptors allowed us to determine the implications of at least two of them (ABF-0020167 and ABF-0046680) in the chemotaxis-driven entry process through plant wounds. Our data suggest that ABF-0020167 and ABF-0046680 may be candidate receptors of jasmonic acid and xylose, respectively.

SummaryJasmonate is a key signalling compound in plant defence that is synthesized in wounded tissues. In this work, we have found that this molecule is also a strong chemoattractant for the phythopathogenic bacteria Dickeya dadantii (ex-Erwinia chysanthemi ). Jasmonic acid induced the expression of a subset of bacterial genes possibly involved in virulence/ survival in the plant apoplast and bacterial cells pretreated with jasmonate showed increased virulence in chicory and Saintpaulia leaves. We also showed that tissue wounding induced bacterial spread through the leaf surface. Moreover, the jasmonate-deficient aos1 Arabidopsis thaliana mutant was more resistant to bacterial invasion by D. dadantii than wild-type plants. These results are consistent with the hypothesis that sensing jasmonic acid by this bacterium helps the pathogen to ingress inside plant tissues.

Protein secretion plays a very important role in the virulence of the bacterium Dickeya dadantii, the causative agent of soft rot disease, in a wide range of plant species. We studied the contribution of the twin-arginine translocation (Tat) protein system to the adaptation of D. dadantii 3937 to different growth conditions and to the interaction with the plant host. First, a list of 44 putative Tat substrates was obtained using bioinformatic programs taking advantage of the availability of the complete sequence of this bacterium. Second, a tatC mutant strain was constructed and analysed. The mutant displayed a pleiotropic phenotype, showing limited growth in an iron-depleted medium, higher sensitivity to copper, reduced motility on soft agar plates and attenuated virulence in witloof chicory leaves. Our results indicate the Tat system as an important determinant of the virulence and fitness of D. dadantii 3937. Potential Tat substrates related to the tatC mutant phenotype are discussed.