SummaryBurkholderia glumae BGR1 produces a broad-host range phytotoxin, called toxoflavin, which is a key pathogenicity factor in rice grain rot and wilt in many field crops. Our molecular and genetic analyses of toxoflavin-deficient mutants demonstrated that gene clusters for toxoflavin production consist of four transcriptional units. The toxoflavin biosynthesis genes were composed of five genes, toxA to toxE , as Suzuki et al. (2004) reported previously. Genes toxF to toxI , which are responsible for toxoflavin transport, were polycistronic and similar to the genes for resistancenodulation-division (RND) efflux systems. Using Tn 3 -gusA reporter fusions, we found that ToxR, a LysRtype regulator, regulates both the toxABCDE and toxFGHI operons in the presence of toxoflavin as a coinducer. In addition, the expression of both operons required a transcriptional activator, ToxJ, whose expression is regulated by quorum sensing. TofI, a LuxI homologue, was responsible for the biosynthesis of both N -hexanoyl homoserine lactone and Noctanoyl homoserine lactone (C8-HSL). C8-HSL and its cognate receptor TofR, a LuxR homologue, activated toxJ expression. This is the first report that quorum sensing is involved in pathogenicity by the regulation of phytotoxin biosynthesis and its transport in plant pathogenic bacteria.
SummaryThe bacterium Burkholderia glumae causes rice grain rot by producing toxoflavin, whose expression is regulated by quorum sensing (QS). We report a major deviation from the current paradigm for the regulation of bacterial polar flagellum genes. The N-octanoyl homoserine lactone (C8-HSL)-deficient mutant of B. glumae is aflagellate and has lost the ability to swim and swarm at 37°C. Mutagenesis of the bacterium with the mini-Tn5rescue identified an IclR-type transcriptional regulator, called QsmR, which is important for flagellum formation. TofR, which is a cognate C8-HSL receptor, activated qsmR expression by binding directly to the qsmR promoter region. From the flagellum gene cluster, we identified flhDC homologues that are directly activated by QsmR. FlhDC in turn activates the expression of genes involved in flagellum biosynthesis, motor functions and chemotaxis in B. glumae. Non-motile qsmR, fliA and flhDC mutants produced toxoflavin, but lost pathogenicity for rice. The unexpected discovery of FlhDC in a polarly flagellate bacterium suggests that exceptions to the typical regulatory mechanisms of flagellum genes exist in Gram-negative bacteria. The finding that functional flagella play critical roles in the pathogenicity of B. glumae suggests that either QS or flagellum formation constitutes a good target for the control of rice grain rot.
Severe wilt symptoms similar to bacterial wilt caused by Ralstonia solanacearum were observed in tomato, hot pepper, eggplant, potato, perilla, sesame, and sunflower in 2000 and 2001 in Korea. From diseased crops at 65 different locations, we obtained 106 isolates that produced green pigment on CPG medium; 36 were isolated from discolored rice panicles. The causal pathogen was identified as Burkholderia glumae based on its biochemical characteristics, fatty acid methyl ester analysis, and 16S rRNA gene sequence. Nine representative isolates produced toxoflavin, as determined by electrospray ionization mass spectrometry using a direct inlet system and TLC analyses, and caused bacterial wilt on tomato, sesame, perilla, eggplant, and hot pepper. However, BGR12, a wild-type isolate lacking toxoflavin production and toxoflavin-deficient mutants generated by Tn5lacZ failed to cause bacterial wilt on those five field crops. Cells of B. glumae and synthetic toxoflavin caused wilt symptoms on field crops, demonstrating a lack of host specificity. Synthetic toxoflavin caused wilt symptoms on tomato, sesame, perilla, eggplant, and hot pepper at 10 μg/ml concentration 1 day after treatment. This is the first report of bacterial wilt on various crops caused by B. glumae, and our results clearly demonstrate that toxoflavin is a key factor in wilt symptom development.
A central mechanism of virulence of extracellular bacterial pathogens is the injection into host cells of effector proteins that modify host cellular functions. HopW1 is an effector injected by the type III secretion system that increases the growth of the plant pathogen Pseudomonas syringae on the Columbia accession of Arabidopsis. When delivered by P. syringae into plant cells, HopW1 causes a reduction in the filamentous actin (F-actin) network and the inhibition of endocytosis, a known actin-dependent process. When directly produced in plants, HopW1 forms complexes with actin, disrupts the actin cytoskeleton and inhibits endocytosis as well as the trafficking of certain proteins to vacuoles. The C-terminal region of HopW1 can reduce the length of actin filaments and therefore solubilize F-actin in vitro. Thus, HopW1 acts by disrupting the actin cytoskeleton and the cell biological processes that depend on actin, which in turn are needed for restricting P. syringae growth in Arabidopsis.
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