Chemical or Genetic Alteration of Proton Motive Force Results in Loss of Virulence of Burkholderia glumae, the Cause of Rice Bacterial Panicle Blight.

Rice is an important source of food for more than half of the world's population. Bacterial panicle blight (BPB) is a disease of rice characterized by grain discoloration or sheath rot caused mainly by Burkholderia glumae. B. glumae synthesizes toxoflavin, an essential virulence factor that is required for symptoms of the disease. The products of the tox operons, ToxABCDE and ToxFGHI, are responsible for the synthesis and the proton motive force (PMF)-dependent secretion of toxoflavin, respectively. The DedA family is a highly conserved membrane protein family found in most bacterial genomes that likely function as membrane transporters. Our previous work has demonstrated that absence of certain DedA family members results in pleiotropic effects, impacting multiple pathways that are energized by PMF. We have demonstrated that a member of the DedA family from Burkholderia thailandensis, named DbcA, is required for the extreme polymyxin resistance observed in this organism. B. glumae encodes a homolog of DbcA with 73% amino acid identity to Burkholderia thailandensis DbcA. Here, we created and characterized a B. glumae ΔdbcA strain. In addition to polymyxin sensitivity, the B. glumae ΔdbcA strain is compromised for virulence in several BPB infection models and secretes only low amounts of toxoflavin (∼15% of wild-type levels). Changes in membrane potential in the B. glumae ΔdbcA strain were reproduced in the wild-type strain by the addition of subinhibitory concentrations of sodium bicarbonate, previously demonstrated to cause disruption of PMF. Sodium bicarbonate inhibited B. glumae virulence in rice, suggesting a possible non-toxic chemical intervention for bacterial panicle blight. IMPORTANCE Bacterial panicle blight (BPB) is a disease of rice characterized by grain discoloration or sheath rot caused mainly by Burkholderia glumae. The DedA family is a highly conserved membrane protein family found in most bacterial genomes that likely function as membrane transporters. Here, we constructed a B. glumae mutant with a deletion in a DedA family member named dbcA and report a loss of virulence in models of BPB. Physiological analysis of the mutant shows that the proton motive force is disrupted, leading to reduction of secretion of the essential virulence factor toxoflavin. The mutant phenotypes are reproduced in the virulent wild-type strain without an effect on growth using sodium bicarbonate, a nontoxic buffer that has been reported to disrupt the PMF. The results presented here suggest that bicarbonate may be an effective antivirulence agent capable of controlling BPB without imposing an undue burden on the environment.

Identification of potential genetic components involved in the deviant quorum-sensing signaling pathways of Burkholderia glumae through a functional genomics approach.

Burkholderia glumae is the chief causal agent for bacterial panicle blight of rice. The acyl-homoserine lactone (AHL)-mediated quorum-sensing (QS) system dependent on a pair of luxI and luxR homologs, tofI and tofR, is the primary cell-to-cell signaling mechanism determining the virulence of this bacterium. Production of toxoflavin, a major virulence factor of B. glumae, is known to be dependent on the tofI/tofR QS system. In our previous study, however, it was observed that B. glumae mutants defective in tofI or tofR produced toxoflavin if they grew on the surface of a solid medium, suggesting that alternative signaling pathways independent of tofI or tofR are activated in that growth condition for the production of toxoflavin. In this study, potential genetic components involved in the tofI- and tofR-independent signaling pathways for toxoflavin production were sought through screening random mini-Tn5 mutants of B. glumae to better understand the intercellular signaling pathways of this pathogen. Fifteen and three genes were initially identified as the potential genetic elements of the tofI- and tofR-independent pathways, respectively. Especially, the ORF (bglu_2g06320) divergently transcribed from toxJ, which encodes an orphan LuxR protein and controls toxoflavin biosynthesis, was newly identified in this study as a gene required for the tofR-independent toxoflavin production and named as toxK. Among those genes, flhD, dgcB, and wzyB were further studied to validate their functions in the tofI-independent toxoflavin production, and similar studies were also conducted with qsmR and toxK for their functions in the tofR-independent toxoflavin production. This work provides a foundation for future comprehensive studies of the intercellular signaling systems of B. glumae and other related pathogenic bacteria.

The roles of the shikimate pathway genes, aroA and aroB, in virulence, growth and UV tolerance of Burkholderia glumae strain 411gr-6.

Burkholderia glumae is the major causal agent of bacterial panicle blight of rice, which is a growing disease problem for rice growers worldwide. In our previous study, some B. glumae strains showed pigmentation phenotypes producing at least two (yellow-green and purple) pigment compounds in casein-peptone-glucose agar medium. The B. glumae strains LSUPB114 and LSUPB116 are pigment-deficient mutant derivatives of the virulent and pigment-proficient strain 411gr-6, having mini-Tn5gus insertions in aroA encoding 3-phosphoshikimate 1-carboxyvinyltransferase and aroB encoding 3-dehydroquinate synthase, respectively. Both enzymes are known to be involved in the shikimate pathway, which leads to the synthesis of aromatic amino acids. Here, we demonstrate that aroA and aroB are required for normal virulence in rice and onion, growth in M9 minimal medium and tolerance to UV light, but are dispensable for the production of the phytotoxin toxoflavin. These results suggest that the shikimate pathway is involved in bacterial pathogenesis by B. glumae without a significant role in the production of toxoflavin, a major virulence factor of this pathogen.

Diversities in virulence, antifungal activity, pigmentation and DNA fingerprint among strains of Burkholderia glumae.

Burkholderia glumae is the primary causal agent of bacterial panicle blight of rice. In this study, 11 naturally avirulent and nine virulent strains of B. glumae native to the southern United States were characterized in terms of virulence in rice and onion, toxofalvin production, antifungal activity, pigmentation and genomic structure. Virulence of B. glumae strains on rice panicles was highly correlated to virulence on onion bulb scales, suggesting that onion bulb can be a convenient alternative host system to efficiently determine the virulence of B. glumae strains. Production of toxoflavin, the phytotoxin that functions as a major virulence factor, was closely associated with the virulence phenotypes of B. glumae strains in rice. Some strains of B. glumae showed various levels of antifungal activity against Rhizoctonia solani, the causal agent of sheath blight, and pigmentation phenotypes on casamino acid-peptone-glucose (CPG) agar plates regardless of their virulence traits. Purple and yellow-green pigments were partially purified from a pigmenting strain of B. glumae, 411gr-6, and the purple pigment fraction showed a strong antifungal activity against Collectotrichum orbiculare. Genetic variations were detected among the B. glumae strains from DNA fingerprinting analyses by repetitive element sequence-based PCR (rep-PCR) for BOX-A1R-based repetitive extragenic palindromic (BOX) or enterobacterial repetitive intergenic consensus (ERIC) sequences of bacteria; and close genetic relatedness among virulent but pigment-deficient strains were revealed by clustering analyses of DNA fingerprints from BOX-and ERIC-PCR.

WtsE, an AvrE-family type III effector protein of Pantoea stewartii subsp. stewartii, causes cell death in non-host plants.

Pantoea stewartii subsp. stewartii (Pnss) causes Stewart's bacterial wilt of sweet corn and leaf blight of maize. The pathogenicity of Pnss depends on synthesis of extracellular polysaccharide and an Hrp type III secretion system. WtsE, a type III secreted effector protein, is essential for the virulence of Pnss on corn. It belongs to the AvrE family of effectors, which includes DspA/E from Erwinia amylovora and AvrE1 from Pseudomonas syringae. Previously, WtsE was shown to cause disease-associated cell death in its host plant, sweet corn. Here, we examine the biological activity of WtsE in several non-host plants. WtsE induced cell death in Nicotiana benthamiana, tobacco, beet and Arabidopsis thaliana when it was transiently produced in plant cells following agroinfiltration or translocated into plant cells from Pnss, Escherichia coli or Pseudomonas syringae pv. phaseolicola (Pph). WtsE-induced cell death in N. benthamiana, tobacco and beet resembled a hypersensitive response and in N. benthamiana it was delayed by cycloheximide. Interestingly, WtsE strongly promoted the growth of Pnss in N. benthamiana prior to the onset of cell death. Deletion derivatives of WtsE that failed to induce cell death in N. benthamiana and tobacco also did not complement wtsE mutants of Pnss for virulence in sweet corn, indicating a correlation between the two activities. WtsE also induced cell death in A. thaliana, where it suppressed basal defences induced by Pph. Thus, WtsE has growth-promoting, defence-suppressing and cell death-inducing activities in non-host plants. Expression of WtsE also prevented the growth of yeast, possibly due to an innate toxicity to eukaryotic cells.

Expression of peroxidase-like genes, H2O2 production, and peroxidase activity during the hypersensitive response to Xanthomonas campestris pv. vesicatoria in Capsicum annuum.

Pepper ascorbate peroxidase-like (CAPOA1), thioredoxin peroxidase-like (CAPOT1), and peroxidase-like (CAPO1) clones were isolated from pepper leaves inoculated with avirulent strain Bv5-4a of Xanthomonas campestris pv. vesicatoria. CAPOA1, CAPOT1, and CAPO1 mRNA disappeared 18 to 30 h after the bacterial infection when the hypersensitive response (HR) was visible. In contrast, peroxidase activity reached a peak at 18 h after infection and then declined at 24 and 30 h when H2O2 accumulation level was maximal. These results suggest that the striking accumulation of H2O2 and strong decrease in peroxidase activity during the programmed cell death may be due to the strong suppression of CAPOA1, CAPOT1, and CAPO1 gene expression. Infection by Phytophthora capsici or Colletotricum gloeosporioides also induced the expression of the three putative peroxidase genes in pepper tissues. CAPOA1 mRNAs were in situ localized in phloem areas of vascular bundles in pepper tissues infected by Colletotricum. coccodes, P. capsici, or C. gloeosporioides. Exogenous treatment with H2O2 strongly induced the CAPOA1 and CAPOT1 transcription 1 h after treatment, while the CAPO1 transcripts accumulated 12 h after H2O2 treatment. We suggest that pepper ascorbate peroxidase and thioredoxin peroxidase genes may function as regulators of H2O2 level and total peroxidase activity in the oxidative burst during the HR to incompatible pathogen interaction in pepper plant.