Dataset Information

Protein O-linked glycosylation in the plant pathogen Ralstonia solanacearum.

ABSTRACT: Ralstonia solanacearum is one of the most lethal phytopathogens in the world. Due to its broad host range, it can cause wilting disease in many plant species of economic interest. In this work, we identified the O-oligosaccharyltransferase (O-OTase) responsible for protein O-glycosylation in R. solanacearum. An analysis of the glycoproteome revealed that 20 proteins, including type IV pilins are substrates of this general glycosylation system. Although multiple glycan forms were identified, the majority of the glycopeptides were modified with a pentasaccharide composed of HexNAc-(Pen)-dHex(3), similar to the O antigen subunit present in the lipopolysaccharide of multiple R. solanacearum strains. Disruption of the O-OTase led to the total loss of protein glycosylation, together with a defect in biofilm formation and reduced pathogenicity towards tomato plants. Comparative proteomic analysis revealed that the loss of glycosylation is not associated with widespread proteome changes. Only the levels of a single glycoprotein, the type IV pilin, were diminished in the absence of glycosylation. In parallel, disruption of glycosylation triggered an increase in the levels of a surface lectin homologous to Pseudomonas PA-IIL. These results reveal the important role of glycosylation in the pathogenesis of R. solanacearum.

SUBMITTER: Elhenawy W

PROVIDER: S-EPMC4736539 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Protein O-linked glycosylation in the plant pathogen Ralstonia solanacearum.

Elhenawy Wael W Scott Nichollas E NE Tondo M Laura ML Orellano Elena G EG Foster Leonard J LJ Feldman Mario F MF

Glycobiology 20151103 3

Ralstonia solanacearum is one of the most lethal phytopathogens in the world. Due to its broad host range, it can cause wilting disease in many plant species of economic interest. In this work, we identified the O-oligosaccharyltransferase (O-OTase) responsible for protein O-glycosylation in R. solanacearum. An analysis of the glycoproteome revealed that 20 proteins, including type IV pilins are substrates of this general glycosylation system. Although multiple glycan forms were identified, the ...[more]

PMID: 26531228

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Project description:BackgroundPlant pathogens are under significant selective pressure by the plant host. Consequently, they are expected to have adapted to this condition or contribute to evading plant defenses. In order to acquire long-term fitness, plant bacterial pathogens are usually forced to maintain advantageous genetic diversity in populations. This strategy ensures that different alleles in the pathogen's gene pool are maintained in a population at frequencies larger than expected under neutral evolution. This selective process, known as balancing selection, is the subject of this work in the context of a common bacterial phytopathogen. We performed a genome-wide scan of Ralstonia solanacearum species complex, an aggressive plant bacterial pathogen that shows broad host range and causes a devastating disease called 'bacterial wilt'.ResultsUsing a sliding window approach, we analyzed 57 genomes from three phylotypes of the R. solanacearum species complex to detect signatures of balancing selection. A total of 161 windows showed extreme values in three summary statistics of population genetics: Tajima's D, ?w and Fu & Li's D*. We discarded any confounding effects due to demographic events by means of coalescent simulations of genetic data. The prospective windows correspond to 78 genes with known function that map in any of the two main replicons (1.7% of total number of genes). The candidate genes under balancing selection are related to primary metabolism and other basal activities (51.3%) or directly associated to virulence (48.7%), the latter being involved in key functions targeted to dismantle plant defenses or to participate in critical stages in the pathogenic process.ConclusionsWe identified various genes under balancing selection that play a significant role in basic metabolism as well as in virulence of the R. solanacearum species complex. These genes are useful to understand and monitor the evolution of bacterial pathogen populations and emerge as potential candidates for future treatments to induce specific plant immune responses.

Project description:BackgroundThe increased availability of genome sequences has advanced the development of genomic distance methods to describe bacterial diversity. Results of these fast-evolving methods are highly correlated with those of the historically standard DNA-DNA hybridization technique. However, these genomic-based methods can be done more rapidly and less expensively and are less prone to technical and human error. They are thus a technically accessible replacement for species delineation. Here, we use several genomic comparison methods, supported by our own proteomic analyses and metabolic characterization as well as previously published DNA-DNA hybridization analyses, to differentiate members of the Ralstonia solanacearum species complex into three species. This pathogen group consists of diverse and widespread strains that cause bacterial wilt disease on many different plants.ResultsWe used three different methods to compare the complete genomes of 29 strains from the R. solanacearum species complex. In parallel we profiled the proteomes of 73 strains using Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF-MS). Proteomic profiles together with genomic sequence comparisons consistently and comprehensively described the diversity of the R. solanacearum species complex. In addition, genome-driven functional phenotypic assays excitingly supported an old hypothesis (Hayward et al. (J Appl Bacteriol 69:269-80, 1990)), that closely related members of the R. solanacearum could be identified through a simple assay of anaerobic nitrate metabolism. This assay allowed us to clearly and easily differentiate phylotype II and IV strains from phylotype I and III strains. Further, genomic dissection of the pathway distinguished between proposed subspecies within the current phylotype IV. The assay revealed large scale differences in energy production within the R. solanacearum species complex, indicating coarse evolutionary distance and further supporting a repartitioning of this group into separate species.ConclusionsTogether, the results of these studies support the proposed division of the R. solanacearum species complex into three species, consistent with recent literature, and demonstrate the utility of proteomic and genomic approaches to delineate bacterial species.

Project description:Ralstonia solanacearum is a well-known agricultural and ecological threat worldwide. The complexity of the R. solanacearum species complex (Rssc) represents a challenge for the accurate characterization of epidemiological strains by official services and research laboratories. The majority of protocols only focus on a narrow range of strains; however, this species complex includes strains that represent major constraints and are under strict regulation. The main drawback associated with the current methods of detecting and characterizing Rssc strains is their reliance on combining different protocols to properly characterize the strains at the ecotype level, which require time and money. Therefore, we used microarray technology (ArrayTube) to develop a standard protocol, which characterizes 17 major groups of interest in the Rssc, in a single multiplex reaction. These 17 majors groups are linked with a phylogenetic assignation (phylotypes, sequevars), but also with an ecotype assignation associated with a range of hosts (e.g., brown rot, Moko). Probes were designed with a 50-mer length constraint and thoroughly evaluated for any flaws or secondary structures. The strains are characterized based on a DNA extraction from pure culture. Validation data showed strong intra-repeatability, inter-repeatability, and reproducibility as well as good specificity. A hierarchical analysis of the probe groups is suitable for an accurate characterization. Compared with single marker detection tests, the method described in this paper addresses efficiently the issue of combining several tests by testing a large number of phylogenetic markers in a single reaction assay. This custom microarray (RsscAT) represents a significant improvement in the epidemiological monitoring of Rssc strains worldwide, and it has the potential to provide insights for phylogenetic incongruence of Rssc strains based on the host of isolation and may be used to indicate potentially emergent strains.

Dataset Information

Protein O-linked glycosylation in the plant pathogen Ralstonia solanacearum.

Publications

Protein O-linked glycosylation in the plant pathogen Ralstonia solanacearum.

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