Project description:Pectobacterium are Gram-negative rods of the family Pectobacteriaceae. They are the causative agent of soft rot diseases of crops and ornamental plants. However, their virulence mechanisms are not yet fully elucidated. Membrane vesicles (MVs) are universally released by bacteria and are be-lieved to play an important role in pathogenicity, and survival of bacteria in the environment. Our study investigates the role of MVs in the virulence of Pectobacterium. The results indicate that the morphology and yields of MVs depend on medium composition. In polygalacturonic acid (PGA) supplemented media, Pectobacterium produce MVs of a larger size (100-300 nm) apart of vesicles below 100 nm. Proteomic analyses revealed the presence of pectate degrading enzymes in MVs. The pectate plate test and enzymatic assay proved that those enzymes are active and able to de-grade pectates. What is more, pathogenicity test indicated that MVs derived from Pectobacterium were able to induce maceration of Zantedeschia sp. leaves. We also show that MVs of β-lactamase producing strains were able to suppress ampicillin activity and permit the growth of susceptible bacteria. Those findings indicate that MVs of Pectobacterium play an important role in host-pathogen interactions and niche competition with other bacteria. Our research also sheds some light on the mechanism of MVs production. We demonstrate that Pectobacterium strains, which overexpress the green fluorescence protein (GFP), produce more MVs than wild type strains. Moreover, proteomic analysis revealed that GFP was present in MVs. Therefore, we demonstrate that protein sequestration into MVs is not limited strictly to periplasmic proteins and is a common occurrence. Our research highlights the importance of MVs production as a mechanism of cargo delivery in Pectobacterium and an alternative secretion system.
Project description:Pectobacterium atrosepticum (Pba) is a gram-negative bacterium which causes blackleg and tuber soft rot on potato. To investigate the molecular processes and responses involved in Pba-host (potato) and Pba-non-host (radish) interactions, under laboratory conditions, we used total RNA-sequencing to measure the gene expression patterns from all three species. Samples from infected and non-infected plant roots were collected after fourteen days of inoculation with Pba SCRI_1039 and subjected to total RNA-sequencing on an Illumina sequencing platform.
Project description:Importance: Pectobacterium species cause soft rot in potato and other host plants primarily by secreting a battery of plant cell wall degrading enzymes. In addition, several different secretion systems are mobilized during infection. Previous studies of gene expression and regulation thereof primarily focused on the onset of infection. This work investigated transcriptome changes in Pectobacterium during the infection of potato tubers up to 72 hours post inoculation to elucidate biological processes during a longer infection period. Methods: The transcriptomes of aggressive strains of the two species P. carotovorum subsp. carotovorum and P. polaris were investigated during infection of potato minitubers (cv. 'Asterix') at 24, 48 and 72 hours after inoculation by RNA sequencing. The transcriptomes were compared to that of bacteria grown on minimal M9 medium, and transcriptomes from later infection time points (48 and 72 hours after inoculation) were compared to early infection (24 hours after inoculation). Results: Plant cell wall degrading enzymes and secretion system associated genes were largely upregulated during infection compared to in vitro growth, but downregulated in the later phases of infection compared to the early infection phase. The downregulation was not sufficiently explained by the expression of known virulence regulators such as the RsmA/B or the ExpA/S systems.
Project description:The goals of this study are to compare NGS-derived transcriptome profiling (RNA-seq) of Pectobacterium upon treatment of polyphenol compound.
Project description:Small RNAs (sRNAs) have emerged as important regulatory molecules and have been studied in several bacteria. However, to date, there have been no whole-transcriptome studies on sRNAs in any of the Soft Rot Enterobacteriaceae (SRE) group of pathogens. Although the main ecological niches for these pathogens are plants, a significant part of their life cycle is undertaken outside their host within the adverse soil environment. However, the mechanisms of SRE adaptation to this harsh nutrient-deficient environment are poorly understood. In the study reported herein, by using strand-specific RNA-seq analysis and in silico sRNA predictions, we describe the sRNA pool of Pectobacterium atrosepticum and reveal numerous sRNA candidates, including those that are induced during starvation-activated stress response. Consequently, strand-specific RNA-seq enabled detection of 137 sRNAs and sRNA candidates under starvation conditions; 25 of these sRNAs were predicted for this bacterium in silico. Functional annotations were computationally assigned to 68 sRNAs. The expression of sRNAs in P. atrosepticum was compared under growth-promoting and starvation conditions: 63 sRNAs were differentially expressed with 57 sRNAs up-regulated under nutrient-deficient conditions. Conservation analysis using BLAST (e-value < 0.001) showed that most of the identified sRNAs are conserved within the SRE. Subsequently, we identified 9 novel sRNAs within the P. atrosepticum genome. A large number of these sRNAs is starvation-induced, suggesting their role in bacterial adaptive response. Finally, this work provides a basis for future experimental characterization and validation of sRNAs in plant pathogens.