Project description:Some strains of the foliar pathogen Pseudomonas syringae are adapted for growth and survival on leaf surfaces and in the leaf interior. Global transcriptome profiling was used to evaluate if these two habitats offer distinct environments for bacteria and thus present distinct driving forces for adaptation. Further transcriptome profiling was performed to understand the various environmental conditions that P. syringae cells encounter during their association with plants. RNA was collected from P. syringae pv. syringae strain B728a cells that were exposed to seven treatments. The treatments included five in vitro treatments, namely exposing cells to a basal medium, sodium chloride to confer an osmotic stress, hydrogen peroxide to confer an apoplastic growth, iron limitation, nitrogen limitation. They also included two in planta treatments, namely recovering cells from epiphytic sites after surface inoculation and 72 h of growth on bean (Phaseolus vulgaris L.) leaves and recovering cells from apoplastic sites after infiltration and 48 h of growth in bean leaves. The results suggested that B728a cells experience vastly different environments when growing on the surface versus the interior of leaves and identified distinct traits that are likely used for persistence and growth in these environments.
Project description:Some strains of the foliar pathogen Pseudomonas syringae are adapted for growth and survival on leaf surfaces and in the leaf interior. Global transcriptome profiling was used to evaluate if these two habitats offer distinct environments for bacteria and thus present distinct driving forces for adaptation. Further transcriptome profiling was performed to understand the various environmental conditions that P. syringae cells encounter during their association with plants. RNA was collected from P. syringae pv. syringae strain B728a cells that were exposed to seven treatments. The treatments included five in vitro treatments, namely exposing cells to a basal medium, sodium chloride to confer an osmotic stress, hydrogen peroxide to confer an apoplastic growth, iron limitation, nitrogen limitation. They also included two in planta treatments, namely recovering cells from epiphytic sites after surface inoculation and 72 h of growth on bean (Phaseolus vulgaris L.) leaves and recovering cells from apoplastic sites after infiltration and 48 h of growth in bean leaves. The results suggested that B728a cells experience vastly different environments when growing on the surface versus the interior of leaves and identified distinct traits that are likely used for persistence and growth in these environments. RNA was collected from B728a cells that were exposed to seven treatments, each with two biological replicates in each of three laboratories. Experimental methods were standardized across the three laboratories. For the in vitro treatments, exponential cells from two independent cultures were each exposed to the five treatments. For each treatment, the cells originating from the two cultures were pooled and the RNA was extracted; this was repeated in its entirety and the two RNA pools were combined. The resulting RNA representing four independent cultures served as a single biological replicate. Two biological replicates for each treatment were generated in this manner in each of three separate laboratories. Epiphytic B728a populations were established following spray inoculation onto bean leaves and subsequent incubation; the epiphytic cells recovered from 400 to 600 leaves inoculated at one time served as a biological replicate, and the procedure was repeated to provide two biological replicates. Due to the availability of facilities, all of the epiphytic treatments were conducted at as single laboratory, with cultures provided from each of the other laboratories, and the cell pellets were returned to those laboratories for RNA extraction and analysis. Apoplastic B728a populations were established following vacuum infiltration into bean leaves and subsequent incubation; the apoplastic cells recovered from 40 to 80 leaves inoculated at one time served as a biological replicate, and two biological replicates were generated at each of the three laboratories. The RNA from all treatments was submitted to Roche Nimblegen, Inc where it was labeled and hybridized to an ORF-based microarray that included 5,071 ORFs and 61 putative sRNAs, with each ORF represented by 14 60-mer nucleotide probes. The fluorescence intensity for each probe was measured and subjected to robust multiarray averaging, which included adjustment for the background intensity, log2 transformation, quantile normalization and median polishing, and a robust estimated mean value was determined for each ORF and putative sRNA on the array.
Project description:Many genes involve in pathogenicity and virulence are induced only in plant or in the presence of host components. Bean leaf extract was obtained from healthy bean leaves. In this work we investigated the effect of bean leaf extract on the transcriptomic profile of the bacterium, when grown at low temperature in minimal medium with or without extract from healthy bean leaves.
Project description:Many genes involve in pathogenicity and virulence are induced only in plant or in the presence of host components. Plant apoplast is the primary site of infection for P. syringae, which obtain nutrients directly from apoplastic fluid of host plants. In this work we investigated the effect of apoplastic fluid on the transcriptomic profile of the bacterium, when grown at low temperature in minimal medium with or without apoplastic fluid extracted from healthy bean leaves.
Project description:P. syringae pv. phaseolicola is the causal agent of the halo blight disease of beans (Phaseolus vulgaris L). The disease attacks both foliage and pods of plant host. Many genes involve in pathogenicity and virulence are induced only in plant or in the presence of host components. In this work we investigated the effect of bean pod extract on the transcriptomic profile of the bacterium, when grown at low temperature in minimal medium with or without bean pod extract.
Project description:Many bacteria can transition from a planktonic lifestyle to life attached to a surface. Changes in gene expression have been documented in bacteria in mature biofilms, but few studies have looked at gene expression during the initial stages of surface attachment. To investigate this, we performed RNA-Seq using the model organism Pseudomonas syringae B728a which has been found in rivers and lakes but is known for living on the leaf surface. We compared gene expression of wild-type P. syringae B728a cells attached to a filter for 2 hours to the gene expression of wild-type P. syringae B728a cells in King's medium B broth. We found that certain gene catergories were quickly induced when cells were on a surface such as flagellar synthesis and motility while other gene categories were quickly repressed such as phytotoxin synthesis and transport. These fast changes in gene expression suggest that P. syringae B728a uses surface attachment as a potential cue to better adapt to life on a surface.
Project description:P. syringae pv. phaseolicola is the causal agent of the halo blight disease of beans (Phaseolus vulgaris L). The disease attacks both foliage and pods of plant host. Many genes involve in pathogenicity and virulence are induced only in plant or in the presence of host components. In this work we investigated the effect of bean pod extract on the transcriptomic profile of the bacterium, when grown at low temperature in minimal medium with or without bean pod extract. Two RNA samples were compared, one prepared from cells grown in minimal medium M9 and the other from cells grown in minimal medium supplemented with bean pod extract.To control de biological variation that might interfere with data interpretation, a minimum of three biological replicates and two technical replicates (swap) were prepared.
Project description:Pseudomonas syringae pv. syringae 9644 (Pss9644) is a causal agent of bacterial cherry canker causing necrotic symptoms on leaves, fruits, gummosis and canker in woody tissues of sweet cherry (Prunus avium). To understand which virulent factor genes were expressed in vitro, Pss9644 was grown in rich media (King's B Broth) and minimum media (hrp-inducing minimum media). The latter mimics the in planta environment.
Project description:The complete genomic sequence of Pseudomonas syringae pv. syringae B728a (Pss B728a) has been determined and is compared with that of P. syringae pv. tomato DC3000 (Pst DC3000). The two pathovars of this economically important species of plant pathogenic bacteria differ in host range and other interactions with plants, with Pss having a more pronounced epiphytic stage of growth and higher abiotic stress tolerance and Pst DC3000 having a more pronounced apoplastic growth habitat. The Pss B728a genome (6.1 Mb) contains a circular chromosome and no plasmid, whereas the Pst DC3000 genome is 6.5 mbp in size, composed of a circular chromosome and two plasmids. Although a high degree of similarity exists between the two sequenced Pseudomonads, 976 protein-encoding genes are unique to Pss B728a when compared with Pst DC3000, including large genomic islands likely to contribute to virulence and host specificity. Over 375 repetitive extragenic palindromic sequences unique to Pss B728a when compared with Pst DC3000 are widely distributed throughout the chromosome except in 14 genomic islands, which generally had lower GC content than the genome as a whole. Content of the genomic islands varies, with one containing a prophage and another the plasmid pKLC102 of Pseudomonas aeruginosa PAO1. Among the 976 genes of Pss B728a with no counterpart in Pst DC3000 are those encoding for syringopeptin, syringomycin, indole acetic acid biosynthesis, arginine degradation, and production of ice nuclei. The genomic comparison suggests that several unique genes for Pss B728a such as ectoine synthase, DNA repair, and antibiotic production may contribute to the epiphytic fitness and stress tolerance of this organism.