Project description:Burkholderia glumae is a causal agent of bacterial grain and seedling rot in rice, and is a threat to stable global food supply. The virulence of B. glumae was suppressed when it was inoculated on budding seed rather than on non-budding seed. To clarify the phenomena, pathogen titer inside the rice plant was measured by serial dilution plating of lysates from budding rice seedlings. Surprisingly, morphologically different types of colonies were observed on the plates. These 'contaminated' rice seed-born bacteria (RSB) were identified by sequencing 16S rRNA genes as three strains of Pseudomonas putida (RSB1, RSB10, RSB15) and Stenotrophomonas maltophilia (RSB2). All bacteria and B. glumae were simultaneously inoculated onto rice seeds, and all three P. putida RSBs suppressed the growth disruption caused by B. glumae, whereas RSB2 had no effect. Thus, the virulence was synergistically suppressed when co-treated with RSBs. The effect could be dependent on the high biofilm formation ability of RSB2. By comprehensive microbiota analysis, endogenous rice flora were changed by RSBs treatment. These results suggest the possibility of novel pathogen control through pre-treatment with endogenous beneficial microorganisms. The method would contribute substantially to the implementation of sustainable agriculture stated in Sustainable Development Goals of United Nations.
Project description:Rice world production is affected due to the growing impact of diseases such as bacterial panicle blight, produced by <i>Burkholderia glumae</i>. The pathogen-induced symptoms include seedling rot, grain rot and leaf-sheath browning in rice plants. It is currently recognized the entrance of this pathogen to the plant, from infected seeds and from environmental sources of the microorganism. However, it is still not fully elucidated the dynamics and permanence of the pathogen in the plant, from its entry until the development of disease symptoms in seedlings or panicles. In this work it was evaluated the infection of <i>B. glumae</i> rice plants, starting from inoculated seeds and substrates, and its subsequent monitoring after infection. Various organs of the plant during the vegetative stage and until the beginning of the reproductive stage, were evaluated. In both inoculation models, the bacteria was maintained in the plant as an endophyte between 1 × 10<sup>1</sup> and 1 × 10<sup>5</sup> cfu of <i>B. glumae</i>.g<sup>-1</sup> of plant throughout the vegetative stage. An increase of bacterial population towards initiation of the panicle was observed, and in the maturity of the grain, an endophyte population was identified in the flag leaf at 1 × 10<sup>6</sup> cfu of <i>B. glumae</i>.g<sup>-1</sup> fresh weight of rice plant, conducting towards the symptoms of bacterial panicle blight. The results found, suggest that <i>B. glumae</i> in rice plants developed from infected seeds or from the substrate, can colonize seedlings, establishing and maintaining a bacterial population over time, using rice plants as habitat to survive endophyticly until formation of bacterial panicle blight symptoms.
Project description:Burkholderia glumae is a motile plant pathogenic bacterium that has multiple polar flagella and one LuxR/LuxI-type quorum sensing (QS) system, TofR/TofI. A QS-dependent transcriptional regulator, QsmR, activates flagellar master regulator flhDC genes. FlhDC subsequently activates flagellar gene expression in B. glumae at 37°C. Here, we confirm that the interplay between QS and temperature is critical for normal polar flagellar morphogenesis in B. glumae. In the wild-type bacterium, flagellar gene expression and flagellar number were greater at 28°C compared to 37°C. The QS-dependent flhC gene was significantly expressed at 28°C in two QS-defective (tofI::? and qsmR::?) mutants. Thus, flagella were present in both tofI::? and qsmR::? mutants at 28°C, but were absent at 37°C. Most tofI::? and qsmR::? mutant cells possessed polar or nonpolar flagella at 28°C. Nonpolarly flagellated cells processing flagella around cell surface of both tofI::? and qsmR::? mutants exhibited tumbling and spinning movements. The flhF gene encoding GTPase involved in regulating the correct placement of flagella in other bacteria was expressed in QS mutants in a FlhDC-dependent manner at 28°C. However, FlhF was mislocalized in QS mutants, and was associated with nonpolar flagellar formation in QS mutants at 28°C. These results indicate that QS-independent expression of flagellar genes at 28°C allows flagellar biogenesis, but is not sufficient for normal polar flagellar morphogenesis in B. glumae. Our findings demonstrate that QS functions together with temperature to control flagellar morphogenesis in B. glumae.
Project description:Toxoflavin is a major virulence factor of the rice pathogen Burkholderia glumae. The tox operon of B. glumae contains five putative toxoflavin biosynthetic genes toxABCDE. ToxA is a predicted S-adenosylmethionine-dependent methyltransferase, and toxA knockouts of B. glumae are less virulent in plant infection models. In this study, we show that ToxA performs two consecutive methylations to convert the putative azapteridine intermediate, 1,6-didemethyltoxoflavin, to toxoflavin. In addition, we report a series of crystal structures of ToxA complexes that reveals the molecular basis of the dual methyltransferase activity. The results suggest sequential methylations with initial methylation at N6 of 1,6-didemethyltoxoflavin followed by methylation at N1. The two azapteridine orientations that position N6 or N1 for methylation are coplanar with a 140° rotation between them. The structure of ToxA contains a class I methyltransferase fold having an N-terminal extension that either closes over the active site or is largely disordered. The ordered conformation places Tyr7 at a position of a structurally conserved tyrosine site of unknown function in various methyltransferases. Crystal structures of ToxA-Y7F consistently show a closed active site, whereas structures of ToxA-Y7A consistently show an open active site, suggesting that the hydroxyl group of Tyr7 plays a role in opening and closing the active site during the multistep reaction.
Project description:<i>Burkholderia glumae</i> causes rice (<i>Oryza sativa</i>) bacterial panicle blight, which is an increasingly economically important disease worldwide. As the first <i>B. glumae</i> strain isolated from patients with chronic infections, AU6208 has been reported as an opportunistic clinic pathogen. However, our understanding of the molecular mechanism underlying human pathogenesis by <i>B. glumae</i> remains rudimentary. In this study, we report the complete genome sequence of the human-isolated <i>B. glumae</i> strain AU6208 and compare this to the genome of the rice-pathogenic <i>B. glumae</i> type strain LMG 2196<sup>T</sup>. Analysis of the average nucleotide identity demonstrated 99.4% similarity between the human- and plant-pathogenic strains. However, the phenotypic results from this study suggest a history of niche adaptation and divergence. In particular, we found 44 genes were predicted to be horizontally transferred into AU6208, and most of these genes were upregulated in conditions that mimic clinical conditions. In these, the gene pair <i>sbn</i>AB encodes key enzymes in antibiotic biosynthesis. These results suggest that horizontal gene transfer in AU6208 may be responsible for selective advantages in its pathogenicity in humans. Our analysis of the AU6208 genome and comparison with that of LMG 2196<sup>T</sup> reveal the evolutionary signatures of <i>B. glumae</i> in the process of switching niches from plants to humans.
Project description:Burkholderia glumae is a rice pathogenic bacterium that causes bacterial panicle blight. Some strains of this pathogen produce dark brown pigments when grown on casamino-acid peptone glucose (CPG) agar medium. A pigment-positive and highly virulent strain of B. glumae, 411gr-6, was randomly mutagenized with mini-Tn5gus, and the resulting mini-Tn5gus derivatives showing altered pigmentation phenotypes were screened on CPG agar plates to identify the genetic elements governing the pigmentation of B. glumae. In this study, a novel two-component regulatory system (TCRS) composed of the PidS sensor histidine kinase and the PidR response regulator was identified as an essential regulatory factor for pigmentation. Notably, the PidS/PidR TCRS was also required for the elicitation of the hypersensitive response on tobacco leaves, indicating the dependence of the hypersensitive response and pathogenicity (Hrp) type III secretion system of B. glumae on this regulatory factor. In addition, B. glumae mutants defective in the PidS/PidR TCRS showed less production of the phytotoxin, toxoflavin, and less virulence on rice panicles and onion bulbs relative to the parental strain, 411gr-6. The presence of highly homologous PidS and PidR orthologues in other Burkholderia species suggests that PidS/PidR-family TCRSs may exert the same or similar functions in different Burkholderia species, including both plant and animal pathogens.
Project description:We analyzed B. glumae transcriptome from infected rice tissue using an RNAseq technique. To identify unique expression of B. glumae genes within rice tissues, we compared in vivo transcriptome data of B. glumae to in vitro data. To accomplish this, we analyzed differentially expressed genes (DEGs) and identified 2,906 transcripts that were significantly altered. Three in vitro (culture) and three in vivo (in plant) RNAseqs in B. glumae
Project description:The lipopolysaccharide (LPS) composed of lipid A, core, and O-antigen is the fundamental constituent of the outer membrane in gram-negative bacteria. This study was conducted to investigate the roles of LPS in Burkholderia glumae, the phytopathogen causing bacterial panicle blight and seedling rot in rice. To study the roles of the core oligosaccharide (OS) and the O-antigen region, mutant strains targeting the waaC and the wbiFGHI genes were generated. The LPS profile was greatly affected by disruption of the waaC gene and slight reductions were observed in the O-antigen region following wbiFGHI deletions. The results indicated that disruption in the core OS biosynthesis-related gene, waaC, was associated with increased sensitivity to environmental stress conditions including acidic, osmotic, saline, and detergent stress, and to polymyxin B. Moreover, significant impairment in the swimming and swarming motility and attenuation of bacterial virulence to rice were also observed in the waaC-defective mutant. The motility and virulence of O-antigen mutants defective in any gene of the wbiFGHI operon, were not significantly different from the wild-type except in slight decrease in swimming and swarming motility with wbiH deletion. Altogether, the results of present study indicated that the LPS, particularly the core OS region, is required for tolerance to environmental stress and full virulence in B. glumae. To our knowledge, this is the first functional study of LPS in a plant pathogenic Burkholderia sp. and presents a step forward toward full understanding of B. glumae pathogenesis.
Project description:Burkholderia glumae is the causative agent of grain and seedling rot in rice and of bacterial wilt in many field crops. Here, we report the complete genome sequence of B. glumae BGR1 isolated from a diseased rice panicle in Korea.
Project description:Panicle blight and seed rot disease caused mainly by Burkholderia glumae and Burkholderia gladioli is threatening rice cultivation worldwide. The bacteria have been reported as seed-borne pathogens from rice. Accurate detection of both pathogens on the seeds is very important for limiting the disease dissemination. Novel primer pairs targeting specific molecular markers were developed for the robust detection of B. glumae and B. gladioli. The designed primers were specific in detecting the target species with no apparent crossreactions with other related Burkholderia species at the expected product size. Both primer pairs displayed a high degree of sensitivity for detection of B. glumae and B. gladioli separately in monoplex PCR or simultaneously in duplex PCR from both extracted gDNA and directly preheated bacterial cell suspensions. Limit of detection was as low as 0.1 ng of gDNA of both species and 3.86 × 102 cells for B. glumae and 5.85 × 102 cells for B. gladioli. On inoculated rice seeds, the designed primers could separately or simultaneously detect B. glumae and B. gladioli with a detection limit as low as 1.86 × 103 cells per rice seed for B. glumae and 1.04 × 104 cells per rice seed of B. gladioli. The novel primers maybe valuable as a more sensitive, specific, and robust tool for the efficient simultaneous detection of B. glumae and B. gladioli on rice seeds, which is important in combating rice panicle blight and seed rot by early detection and confirmation of the dissemination of pathogen-free rice seeds.