Jae-Soon Cha

Rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae produces multiple DSF-family signals in regulation of virulence factor production

BackgroundXanthomonasoryzae pv. oryzae (Xoo) is the causal agent of rice bacterial blight disease. Xoo produces a range of virulence factors, including EPS, extracellular enzyme, iron-chelating siderophores, and type III-secretion dependent effectors, which are collectively essential for virulence. Genetic and genomics evidence suggest that Xoo might use the diffusible signal factor (DSF) type quorum sensing (QS) system to regulate the virulence factor production. However, little is known about the chemical structure of the DSF-like signal(s) produced by Xoo and the factors influencing the signal production.ResultsXoo genome harbours an rpf cluster comprising rpfB, rpfF, rpfC and rpfG. The proteins encoded by these genes are highly homologous to their counterparts in X. campestris pv. campestris (Xcc), suggesting that Xcc and Xoo might use similar mechanisms for DSF biosynthesis and autoregulation. Consistent with in silico analysis, the rpfF mutant was DSF-deficient and the rpfC mutant produced about 25 times higher DSF-like activity than the wild type Xoo strain KACC10331. From the supernatants of rpfC mutant, we purified three compounds showing strong DSF-like activity. Mass spectrometry and NMR analysis revealed that two of them were the previously characterized DSF and BDSF; the third one was a novel unsaturated fatty acid with 2 double bonds and was designated as CDSF in this study. Further analysis showed that all the three DSF-family signals were synthesized via the enzyme RpfF encoded by Xoo2868. DSF and BDSF at a final concentration of 3 μM to the rpfF mutant could fully restore its extracellular xylanase activity and EPS production to the wild type level, but CDSF was less active than DSF and BDSF in induction of EPS and xylanase. DSF and CDSF shared a similar cell density-dependent production time course with the maximum production being detected at 42 h after inoculation, whereas the maximum production of BDSF was observed at 36 h after inoculation. When grown in a rich medium such as YEB, LB, PSA, and NYG, Xoo produced all the three signals with the majority being DSF. Whereas in nutritionally poor XOLN medium Xoo only produced BDSF and DSF but the majority was BDSF.ConclusionsThis study demonstrates that Xoo and Xcc share the conserved mechanisms for DSF biosynthesis and autoregulation. Xoo produces DSF, BDSF and CDSF signals in rich media and CDSF is a novel signal in DSF-family with two double bonds. All the three DSF-family signals promote EPS production and xylanase activity in Xoo, but CDSF is less active than its analogues DSF and BDSF. The composition and ratio of the three DSF-family signals produced by Xoo are influenced by the composition of culture media.

Virulence Reduction and Differing Regulation of Virulence Genes in rpf Mutants of Xanthomonas oryzae pv. oryzae

To define the functions of the rpf genes in Xanthomonas oryzae pv. oryzae (Xoo), which regulates pathogenicity factors in Xanthomonas campestris pv. campestris (Xcc), marker-exchange mutants of each rpf gene were generated. When the mutants were inoculated on a susceptible cultivar, the lesion lengths caused by the rpfB, rpfC, rpfF, and rpfG mutants were significantly smaller than those caused by the wild type, whereas those caused by the rpfA, rpfD, and rpfI mutants were not. Several virulence determinants, including extracellular polysaccharide (EPS) production, xylanase production, and motility, were significantly decreased in the four mutants. However, the cellulase activity in the mutants was unchanged. Complementation of the rpfB and rpfC mutations restored the virulence and the expression of the virulence determinants. Expression analysis of 14 virulence genes revealed that the expression of genes related to EPS production (gumG and gumM), LPS (xanA, xanB, wxoD, and wxoC), phytase (phyA), xylanase (xynB), lipase (lipA), and motility (pitA) were reduced significantly in the mutants rpfB, rpfC, rpfF, and rpfG. In contrast, the expression of genes related to cellulase (eglxob, clsA), cellobiosidase (cbsA), and iron metabolism (fur) was unchanged. The results of this study clearly show that rpfB, rpfC, rpfF, and rpfG are important for the virulence of Xoo KACC10859, and that virulence genes are regulated differently by the Rpfs.

Agrobacterium-mediated Transformation of the Winter Mushroom, Flammulina velutipes

Flammulina velutipes was transformed efficiently’ by Agrobacterium-medmted transformation system. The transformation frequency was about 16% with the gill tissues of the fungal fruiting body. Southern hybridization and genetic analysis suggest that the introduced DNA was inserted onto different locations of the fungal genome, and inherited stably to the next generation via basidiospores. Transformation or gene tagging with Agrobacterium T-DNA based vector should be useful for wide ranges of genetic or molecular biological studies of the mushroom.

Isolation of pathogen-induced Chinese cabbage genes by subtractive hybridization employing selective adaptor ligation.

We have developed a subtractive cloning method in which target sequences are effectively enriched by selective adaptor ligation and PCR after hybridization. In this method both tester and driver DNAs are digested with RsaI, ligated with the linker DNA containing a KpnI recognition site, and amplified by PCR. The tester DNA samples are divided into two aliquots, each digested with either RsaI or KpnI. The two DNA samples are then combined and hybridized with an excess of the driver DNA retaining the linker. After hybridization, the DNA mixture is ligated to a new adaptor compatible only with double-stranded tester/tester DNAs. Therefore, only the tester/tester is selectively amplified in subsequent PCR. This also leads to complete elimination of the tester DNA hybridized with driver DNA from the tester DNA population. Although our protocol employs enzymatic treatments, the efficiency of the enzymatic treatments does not affect the subtraction efficiency. This new subtractive enrichment method was applied to isolate Chinese cabbage defense-related genes induced by Pseudomonas syringae pv. tomato (Pst), which elicits a hypersensitive response in Chinese cabbage. After two or three rounds of subtractive hybridization, the sequences of enriched DNAs were determined and examined by BLAST analysis. Northern blot hybridization showed that 12 of the 19 genes analyzed were strongly induced by Pst treatment. Among the 12 Pst-induced genes five represent pathogenesis-related genes encoding PR1a, two chitinases, a thaumatin-like protein, and a PR4 protein. Other Pst-induced genes include two cytochrome P450 genes responsible for glucosinolate biosynthesis, a disease resistance gene homolog, and several genes encoding proteins with unknown functions.

Characterization of salicylic acid-induced genes in Chinese cabbage

Salicylic acid is a messenger molecule in the activation of defense responses in plants. In this study, we isolated four cDNA clones representing salicylic acid-induced genes in Chinese cabbage (Brassica rapa subsp. pekinensis) by subtractive hybridization. Of the four clones, the BC5-2 clone encodes a putative glucosyltransferase protein. The BC5-3 clone is highly similar to an Arabidopsis gene encoding a putative metal-binding farnesylated protein. The BC6-1 clone is a chitinase gene with similarities to a rapeseed class IV chitinase. Class IV chitinases have deletions in the chitin-binding and catalytic domains and the BC6-1 chitinase has an additional deletion in the catalytic domain. The BCP8-1 clone is most homologous to an Arabidopsis gene that contains a tandem array of two thiJ-like sequences. These four cabbage genes were barely expressed in healthy leaves, but were strongly induced by salicylic acid and benzothiadiazole. Expression of the three genes represented by the BC5-2, BC5-3 and BCP8-1 clones were also induced by Pseudomonas syringae pv. tomato, a nonhost pathogen that elicits a hypersensitive response in Chinese cabbage. None of these four genes, however, was strongly induced by methyl jasmonate or by ethylene.

Characterization of phenotypic variants of Clavibacter michiganensis subsp. michiganensis isolated from Capsicum annuum

Phenotypic variants of Clavibacter michiganensis subsp. michiganensis (Cmm) were isolated from pepper fields and from pepper seeds during quarantine inspections. All strains isolated from pepper (pepper isolates) produced orange-coloured colonies with lower mucoidy than typical Cmm strains isolated from tomato (tomato isolates). However, the results of ELISA, fatty acid analysis, 16S rDNA sequencing, and PCR analysis showed that all pepper isolates were similar enough to be identified as Cmm. In addition to phenotypic variations, the pepper isolates showed different pathogenic and genetic characteristics from tomato isolates from the USA, Europe, or other countries. They could be clearly distinguished in terms of pathogenicity, as they showed increased pathogenicity to pepper but reduced pathogenicity to tomato. Tomato isolates caused strong wilting and canker in tomato, but caused only canker and no wilting in pepper and bell pepper. However, pepper isolates caused no wilting, even in tomato, and only caused canker in the three host plants. In addition, compared to tomato isolates, pepper isolates showed increased colonization efficiency and caused a greater reduction in shoot dry weight in pepper. Pepper and tomato isolates could be separated into two groups according to host origin on the basis of 16S rDNA and ITS sequence analysis. They also showed different rep-PCR genomic fingerprints. All pepper isolates showed higher cellulase activity than tomato isolates on M9CMC plates. However, two plasmid-borne virulence genes of Cmm, pat-1, and celA, were not detected in any pepper isolates by PCR. Furthermore, PCR for pathogenicity-related genes located on a pathogenicity island (PAI) revealed that all tomato isolates were positive for these genes, whereas the pepper isolates did not show any PCR products for the chpC, chpG, ppaA, or tomA genes. Therefore, we suggest that the pepper isolates may represent a separate Cmm population that has evolved within the limits of this host.

PCR assays for specific and sensitive detection of Pseudomonas tolaasii, the cause of brown blotch disease of mushrooms.

Aims: The present study describes PCR assays to detect specifically Pseudomonas tolaasii from various samples. 
Methods and Results: Two sets of PCR primers were developed to amplify genes required for tolaasin production. Only a PCR product of 449 bp or 249 bp was produced in PCR reactions with the Pt‐1A/Pt‐1D1 or Pt‐PM/Pt‐QM primer sets, respectively, and DNA and cells of Ps. tolaasii. Nested and immunocapture‐nested PCR could detect to 3 cells of Ps. tolaasii and amplify the Ps. tolaasii‐specific DNA from a sample containing 10 000 times more other bacterial cells than Ps. tolaasii, respectively. 
Conclusions: The PCR assays are simple, rapid and reliable methods for detection and identification of Ps. tolaasii. 
Significance and Impact of the Study: The protocols can effectively distinguish Ps. tolaasii from other bacteria and detect Ps. tolaasii from various samples for studying ecology of the bacterium and preventing the use of contaminated water or spawn or medium in mushroom cultivation.

Clavibacter michiganensis subsp. capsici subsp. nov., causing bacterial canker disease in pepper.

Clavibacter michiganensis is a Gram-stain-positive bacterium with eight subspecies. One of these subspecies is C. michiganensis subsp. michiganensis, which causes bacterial canker disease in tomato. Bacterial strains showing very similar canker disease symptoms to those of a strain originally classified as C. michiganensis have been isolated from pepper. In this paper, we reclassified strains isolated from pepper. On the basis of phylogenetic analysis with 16S rRNA gene sequences, the strains isolated from pepper were grouped in a separate clade from other subspecies of C. michiganensis. Biochemical, physiological and genetic characteristics of strain PF008T, which is the representative strain of the isolates from pepper, were examined in this study. Based on multi-locus sequence typing and other biochemical and physiological features including colony color, utilization of carbon sources and enzyme activities, strain PF008T was categorically differentiated from eight subspecies of C. michiganensis. Moreover, genome analysis showed that the DNA G+C content of strain PF008T is 73.2 %. These results indicate that PF008T is distinct from other known subspecies of C. michiganensis. Therefore, we propose a novel subspecies, C. michiganensis subsp. capsici, causing bacterial canker disease in pepper, with a type strain of PF008T (=KACC 18448T=LMG 29047T).

Biological characterization of various strains of Pseudomonas tolaasii that causes brown blotch disease

Brown blotch disease of cultivated mushrooms is caused by Pseudomonas tolaasii, which secretes the bacterial toxin, tolaasin. Tolaasin is a peptide toxin that causes pore formation in the plasma membrane of mushroom cells. Forty-two strains of pathogenic bacteria causing brown blotch or similar diseases were isolated from mushrooms showing disease symptoms. To characterize these bacteria, the genes of 16S rRNA were sequenced and analyzed. Thirty-three strains were identified as five different species of Pseudomonas. Of these, 23 were identified as P. tolaasii and named as P1-type pathogens. Because the strains identified as P. tolaasii were major pathogens that cause the brown blotch disease, phylogenetic analyses of these pathogens were conducted by the neighbor-joining method. These strains comprised three phylogenetic subtypes, P1α (6 strains), P1β (16 strains), and P1γ (1 strain). Biological characterizations of the isolated bacteria were performed and confirmed that all three subtypes were able to cause the disease by forming blotches on the surface of the mushroom tissue. However, hemolytic activities were observed in the P1α and P1γ strains, but not in the P1β strains. These results imply that remarkable diversity exists among the various strains of P. tolaasii, each strain showing distinct biological characters.

Development of PCR and TaqMan PCR Assays to Detect Pseudomonas coronafaciens, a Causal Agent of Halo Blight of Oats

Pseudomonas coronafaciens causes halo blight on oats and is a plant quarantine bacterium in many countries, including the Republic of Korea. Using of the certificated seed is important for control of the disease. Since effective detection method of P. coronafaciens is not available yet, PCR and TaqMan PCR assays for specific detection of P. coronafaciens were developed in this study. PCR primers were designed from the draft genome sequence of P. coronafaciens LMG 5060 which was obtained by the next-generation sequencing in this study. The PCR primer set Pc-12-F/Pc-12-R specifically amplified 498 bp from the 13 strains of P. coronafaciens isolated in the seven different countries (Canada, Japan, United Kingdom, Zimbabwe, Kenya, Germany, and New Zealand) and the nested primer set Pc-12-ne-F/Pc-12-ne-R specifically amplified 298 bp from those strains. The target-size PCR product was not amplified from the non-target bacteria with the PCR and nested primer sets. TaqMan PCR with Pc-12-ne-F/Pc-12-ne-R and a TaqMan probe, Pc-taqman, which were designed inside of the nested PCR amplicon, generated Ct values which in a dose-dependent manner to the amount of the target DNA and the Ct values of all the P. coronafaciens strains were above the threshold Ct value for positive detection. The TaqMan PCR generated positive Ct values from the seed extracts of the artificially inoculated oat seeds above 10 cfu/ml inoculation level. PCR and TaqMan PCR assays developed in this study will be useful tools to detect and identify the plant quarantine pathogen, P. coronafaciens.