Ingyu Hwang

Quorum sensing and the LysR-type transcriptional activator ToxR regulate toxoflavin biosynthesis and transport in Burkholderia glumae.

Burkholderia 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 resistance‐nodulation‐division (RND) efflux systems. Using Tn3‐gusA reporter fusions, we found that ToxR, a LysR‐type 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 N‐octanoyl 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.

Characterization of the Xanthomonas axonopodis pv. glycines Hrp Pathogenicity Island

We sequenced an approximately 29-kb region from Xanthomonas axonopodis pv. glycines that contained the Hrp type III secretion system, and we characterized the genes in this region by Tn3-gus mutagenesis and gene expression analyses. From the region, hrp (hypersensitive response and pathogenicity) and hrc (hrp and conserved) genes, which encode type III secretion systems, and hpa (hrp-associated) genes were identified. The characteristics of the region, such as the presence of many virulence genes, low G+C content, and bordering tRNA genes, satisfied the criteria for a pathogenicity island (PAI) in a bacterium. The PAI was composed of nine hrp, nine hrc, and eight hpa genes with seven plant-inducible promoter boxes. The hrp and hrc mutants failed to elicit hypersensitive responses in pepper plants but induced hypersensitive responses in all tomato plants tested. The Hrp PAI of X. axonopodis pv. glycines resembled the Hrp PAIs of other Xanthomonas species, and the Hrp PAI core region was highly conserved. However, in contrast to the PAI of Pseudomonas syringae, the regions upstream and downstream from the Hrp PAI core region showed variability in the xanthomonads. In addition, we demonstrate that HpaG, which is located in the Hrp PAI region of X. axonopodis pv. glycines, is a response elicitor. Purified HpaG elicited hypersensitive responses at a concentration of 1.0 micro M in pepper, tobacco, and Arabidopsis thaliana ecotype Cvi-0 by acting as a type III secreted effector protein. However, HpaG failed to elicit hypersensitive responses in tomato, Chinese cabbage, and A. thaliana ecotypes Col-0 and Ler. This is the first report to show that the harpin-like effector protein of Xanthomonas species exhibits elicitor activity.

Pyrroloquinoline Quinone Is a Plant Growth Promotion Factor Produced by Pseudomonas fluorescens B16

Pseudomonas fluorescens B16 is a plant growth-promoting rhizobacterium. To determine the factors involved in plant growth promotion by this organism, we mutagenized wild-type strain B16 using ΩKm elements and isolated one mutant, K818, which is defective in plant growth promotion, in a rockwool culture system. A cosmid clone, pOK40, which complements the mutant K818, was isolated from a genomic library of the parent strain. Tn3-gusA mutagenesis of pOK40 revealed that the genes responsible for plant growth promotion reside in a 13.3-kb BamHI fragment. Analysis of the DNA sequence of the fragment identified 11 putative open reading frames, consisting of seven known and four previously unidentified pyrroloquinoline quinone (PQQ) biosynthetic genes. All of the pqq genes showed expression only in nutrient-limiting conditions in a PqqH-dependent manner. Electrospray ionization-mass spectrometry analysis of culture filtrates confirmed that wild-type B16 produces PQQ, whereas mutants defective in plant growth promotion do not. Application of wild-type B16 on tomato (Solanum lycopersicum) plants cultivated in a hydroponic culture system significantly increased the height, flower number, fruit number, and total fruit weight, whereas none of the strains that did not produce PQQ promoted tomato growth. Furthermore, 5 to 1,000 nm of synthetic PQQ conferred a significant increase in the fresh weight of cucumber (Cucumis sativus) seedlings, confirming that PQQ is a plant growth promotion factor. Treatment of cucumber leaf discs with PQQ and wild-type B16 resulted in the scavenging of reactive oxygen species and hydrogen peroxide, suggesting that PQQ acts as an antioxidant in plants.

Toxoflavin Produced by Burkholderia glumae Causing Rice Grain Rot Is Responsible for Inducing Bacterial Wilt in Many Field Crops

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.

The barley ERF-type transcription factor HvRAF confers enhanced pathogen resistance and salt tolerance in Arabidopsis

We isolated HvRAF (Hordeum vulgare root abundant factor), a cDNA encoding a novel ethylene response factor (ERF)-type transcription factor, from young seedlings of barley. In addition to the most highly conserved APETALA2/ERF DNA-binding domain, the encoded protein contained an N-terminal MCGGAIL signature sequence, a putative nuclear localization sequence, and a C-terminal acidic transcription activation domain containing a novel mammalian hemopexin domain signature-like sequence. Their homologous sequences were found in AAK92635 from rice and RAP2.2 from Arabidopsis; the ERF proteins most closely related to HvRAF, reflecting their functional importance. RNA blot analyses revealed that HvRAF transcripts were more abundant in roots than in leaves. HvRAF expression was induced in barley seedlings by various treatment regimes such as salicylic acid, ethephon, methyl jasmonate, cellulase, and methyl viologen. In a subcellular localization assay, the HvRAF-GFP fusion protein was targeted to the nucleus. The fusion protein of HvRAF with the GAL4 DNA-binding domain strongly activated transcription in yeast. Various deletion mutants of HvRAF indicated that the transactivating activity was localized to the acidic domain of the C-terminal region, and that the hemopexin domain signature-like sequence was important for the activity. Overexpression of the HvRAF gene in Arabidopsis plants induced the activation of various stress-responsive genes, including PDF1.2, JR3, PR1, PR5, KIN2, and GSH1. Furthermore, the transgenic Arabidopsis plants showed enhanced resistance to Ralstonia solanacearum strain GMI1000, as well as seed germination and root growth tolerance to high salinity. These results collectively indicate that HvRAF is a transcription factor that plays dual regulatory roles in response to biotic and abiotic stresses in plants.

Regulation of polar flagellum genes is mediated by quorum sensing and FlhDC in Burkholderia glumae.

The 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.

Amyloidogenesis of Type III-dependent Harpins from Plant Pathogenic Bacteria

Harpins are heat-stable, glycine-rich type III-secreted proteins produced by plant pathogenic bacteria, which cause a hypersensitive response (HR) when infiltrated into the intercellular space of tobacco leaves; however, the biochemical mechanisms by which harpins cause plant cell death remain unclear. In this study, we determined the biochemical characteristics of HpaG, the first harpin identified from a Xanthomonas species, under plant apoplast-like conditions using electron microscopy and circular dichroism spectroscopy. We found that His6-HpaG formed biologically active spherical oligomers, protofibrils, and β-sheet-rich fibrils, whereas the null HR mutant His6-HpaG(L50P) did not. Biochemical analysis and HR assay of various forms of HpaG demonstrated that the transition from an α-helix to β-sheet-rich fibrils is important for the biological activity of protein. The fibrillar form of His6-HpaG is an amyloid protein based on positive staining with Congo red to produce green birefringence under polarized light, increased protease resistance, and β-sheet fibril structure. Other harpins, such as HrpN from Erwinia amylovora and HrpZ from Pseudomonas syringae pv. syringae, also formed curvilinear protofibrils or fibrils under plant apoplast-like conditions, suggesting that amyloidogenesis is a common feature of harpins. Missense and deletion mutagenesis of HpaG indicated that the rate of HpaG fibril formation is modulated by a motif present in the C terminus. The plant cytotoxicity of HpaG is unique among the amyloid-forming proteins that occur in several microorganisms. Structural and morphological analogies between HpaG and disease-related amyloidogenic proteins, such as Aβ protein, suggest possible common biochemical characteristics in the induction of plant and animal cell death.

Bacterial quorum sensing, cooperativity, and anticipation of stationary-phase stress

Acyl-homoserine lactone–mediated quorum sensing (QS) regulates diverse activities in many species of Proteobacteria. QS-controlled genes commonly code for production of secreted or excreted public goods. The acyl-homoserine lactones are synthesized by members of the LuxI signal synthase family and are detected by cognate members of the LuxR family of transcriptional regulators. QS affords a means of population density-dependent gene regulation. Control of public goods via QS provides a fitness benefit. Another potential role for QS is to anticipate overcrowding. As population density increases and stationary phase approaches, QS might induce functions important for existence in stationary phase. Here we provide evidence that in three related species of the genus Burkholderia QS allows individuals to anticipate and survive stationary-phase stress. Survival requires QS-dependent activation of cellular enzymes required for production of excreted oxalate, which serves to counteract ammonia-mediated alkaline toxicity during stationary phase. Our findings provide an example of QS serving as a means to anticipate stationary phase or life at the carrying capacity of a population by activating the expression of cytoplasmic enzymes, altering cellular metabolism, and producing a shared resource or public good, oxalate.

Mutational Analysis of Xanthomonas Harpin HpaG Identifies a Key Functional Region That Elicits the Hypersensitive Response in Nonhost Plants

HpaG is a type III-secreted elicitor protein of Xanthomonas axonopodis pv. glycines. We have determined the critical amino acid residues important for hypersensitive response (HR) elicitation by random and site-directed mutagenesis of HpaG and its homolog XopA. A plasmid clone carrying hpaG was mutagenized by site-directed mutagenesis, hydroxylamine mutagenesis, and error-prone PCR. A total of 52 mutants were obtained, including 51 single missense mutants and 1 double missense mutant. The HR elicitation activity was abolished in the two missense mutants [HpaG(L50P) and HpaG(L43P/L50P)]. Seven single missense mutants showed reduced activity, and the HR elicitation activity of the rest of the mutants was similar to that of wild-type HpaG. Mutational and deletion analyses narrowed the region essential for elicitor activity to the 23-amino-acid peptide (H2N-NQGISEKQLDQLLTQLIMALLQQ-COOH). A synthetic peptide of this sequence possessed HR elicitor activity at the same concentration as the HpaG protein. This region has 78 and 74% homology with 23- and 27-amino-acid regions of the HrpW harpin domains, respectively, from Pseudomonas and Erwinia spp. The secondary structure of the peptide is predicted to be an alpha-helix, as is the HrpW region that is homologous to HpaG. The predicted alpha-helix of HpaG is probably critical for the elicitation of the HR in tobacco plants. In addition, mutagenesis of a xopA gene yielded two gain-of-function mutants: XopA(F48L) and XopA(F48L/M52L). These results indicate that the 12 amino acid residues between L39 and L50 of HpaG have critical roles in HR elicitation in tobacco plants.

Genetic Diversity and Distribution of Korean Isolates of Ralstonia solanacearum

Genetic diversity among 478 isolates of Ralstonia solanacearum collected from various plants in Korea between 1997 and 2005 was determined based on biovar, pathogenicity, amplified fragment length polymorphism (AFLP), 16S rRNA, endoglucanase, hrpB, and mutS gene sequence analyses. Of the isolates, 440 belonged to biovars 1, 3, or 4, and 38 belonged to biovar 2. Biovar N2 isolates were not found. The biovar 1 and 2 isolates were found mainly in southern Korea, whereas the biovar 3 and 4 isolates were widely distributed throughout all nine provinces. AFLP analysis divided the 109 representative Korean isolates into six clusters that were distinct from most of the foreign isolates. Grouping of 8 representative isolates based on their 16S rRNA gene sequences indicated that biovars 1, 3, and 4 belonged to division 1, while biovar 2 belonged to subdivision 2b. Sequence analysis of the endoglucanase, hrpB, and mutS genes from the same isolates indicated that the biovar 1, 3, and 4 isolates belonged to phylotype I, while the biovar 2 isolate belonged to phylotype IV. This study is the first comprehensive analysis of genetic diversity among Korean isolates of R. solanacearum.