[Show abstract][Hide abstract] ABSTRACT: Flammulina velutipes is a fungus with health and medicinal benefits that has been used for consumption and cultivation in East Asia. F. velutipes is also known to degrade lignocellulose and produce ethanol. The overlapping interests of mushroom production and wood bioconversion make F. velutipes an attractive new model for fungal wood related studies. Here, we present the complete sequence of the F. velutipes genome. This is the first sequenced genome for a commercially produced edible mushroom that also degrades wood. The 35.6-Mb genome contained 12,218 predicted protein-encoding genes and 287 tRNA genes assembled into 11 scaffolds corresponding with the 11 chromosomes of strain KACC42780. The 88.4-kb mitochondrial genome contained 35 genes. Well-developed wood degrading machinery with strong potential for lignin degradation (69 auxiliary activities, formerly FOLymes) and carbohydrate degradation (392 CAZymes), along with 58 alcohol dehydrogenase genes were highly expressed in the mycelium, demonstrating the potential application of this organism to bioethanol production. Thus, the newly uncovered wood degrading capacity and sequential nature of this process in F. velutipes, offer interesting possibilities for more detailed studies on either lignin or (hemi-) cellulose degradation in complex wood substrates. The mutual interest in wood degradation by the mushroom industry and (ligno-)cellulose biomass related industries further increase the significance of F. velutipes as a new model.
PLoS ONE 04/2014; 9(4):e93560. DOI:10.1371/journal.pone.0093560 · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We describe the development of a polymerase chain reaction (PCR) method for the rapid, precise, and specific detection of the Xanthomonas oryzae pv. oryzae (Xoo) K3a race, the bacterial blight pathogen of rice. The specific primer set was designed to amplify a genomic locus derived from an amplified fragment length polymorphism (AFLP) specific for the K3a race. The 1024 bp amplicon was generated from the DNA of 13 isolates of Xoo K3a races out of 120 isolates of other races, pathovars, and Xanthomonas species. The assay does not require isolated bacterial cells or DNA extraction. Moreover, the pathogen was quickly detected in the rice leaf 2 days after inoculation with bacteria and at a distance of 8 cm from the rice leaf 5 days later. The results suggest that this PCR-based assay will be a useful and powerful tool for the detection and identification of the Xoo K3a race in rice plants as well as for early diagnosis of infection in paddy fields.
Journal of Microbiology and Biotechnology 02/2014; · 1.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Xanthomonas oryzae pv. oryzae causes bacterial blight in rice, and this bacterial blight has been widely found in the major rice-growing areas. We constructed a transposon mutagenesis library of X. oryzae pv. oryzae and identified a mutant strain (KXOM9) that is deficient for pigment production and virulence. Furthermore, the KXOM9 mutant was unable to grow in minimal medium lacking aromatic amino acids. Thermal asymmetric interlaced-PCR and sequence analysis of KXOM9 revealed that the transposon was inserted into the aroC gene, which encodes a chorismate synthase in various bacterial pathogens. In planta growth assays revealed that bacterial growth of the KXOM9 mutant in rice leaves was severely reduced. Genetic complementation of this mutant with a 7.9-kb fragment containing aroC restored virulence, pigmentation, and prototrophy. These results suggest that the aroC gene plays a crucial role in the growth, attenuation of virulence, and pigment production of X. oryzae pv. oryzae.
Microbiological Research 12/2011; 167(6):326-31. DOI:10.1016/j.micres.2011.11.002 · 1.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Xanthomonas oryzae pathovar oryzae (Xoo) causes bacterial blight disease in rice (Oryza sativa L.). For a study of function, we constructed a random insertion mutant library of Xoo using a Tn5 transposon and isolated the mutant strain (M11; aroK::Tn5) that had extremely low pigment production. In addition, M11 had decreased virulence against the susceptible rice cultivar IR24. Thermal asymmetric interlaced-PCR and sequence analysis of M11 revealed that the transposon was inserted into the aroK gene (which encodes a shikimate kinase). To investigate the expression patterns of the pigment- and virulence-deficient mutant, DNA microarray analysis was performed. In addition, reverse transcriptase-PCR was performed to confirm the expression levels of several genes, including the aro genes of the aroK mutant. Our findings reveal that several crucial genes for virulence, including cellulase and hypersensitive response and pathogenicity (hrp) genes, were regulated by mutations in the aroK gene.
[Show abstract][Hide abstract] ABSTRACT: An electrophoretic karyotype of Korean Flammulina velutipes was obtained using contour-clamped homogeneous electric field (CHEF) gel electrophoresis. The monokaryotic progenies (4019-20 and 4019-18) and a dikaryotic strain (4019-2018) had 6-8 chromosomes, with sizes ranging from 1.6 to 5.8 megabase (Mb) pairs. Among the 3 strains that were examined, strain 4019-20 resolved into at least 7 chromosomes, with a total genome size of approximately 26.7Mb. We selected several chromosome-specific genes from the cDNA library of F. velutipes using Southern hybridization analysis. In order to determine the whole genome sequence, we constructed a bacterial artificial chromosome (BAC) library of the 4019-20 strain. The BAC library comprised 3840 clones, and the insert size ranged from 60 to 228kb, with an average size of 136kb.
Microbiological Research 09/2009; 165(4):321-8. DOI:10.1016/j.micres.2009.06.003 · 1.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Xanthomonas oryzae pathovar oryzae is the causal agent of rice bacterial blight. The plant pathogenic bacterium X. oryzae pv. oryzae expresses a type III secretion system that is necessary for both the pathogenicity in susceptible hosts and the induction of the hypersensitive response in resistant plants. This specialized protein transport system is encoded by a 32.18kb hrp (hypersensitive response and pathogenicity) gene cluster. The hrp gene cluster is composed of nine hrp, nine hrc (hrp conserved) and eight hpa (hrp-associated) genes and is controlled by HrpG and HrpX, which are known as regulators of the hrp gene cluster. Before mutational analysis of these hrp genes, the transcriptional linkages of the core region of the hrp gene cluster from hpaB to hrcC of the X. oryzae pv. oryzae KACC10859 was determined and the non-polarity of EZTn5 insertional mutagenesis was demonstrated by reverse transcription polymerase chain reaction. Pathogenicity assays of these non-polar hrp mutants were carried out on the susceptible rice cultivar, Milyang-23. According to the results of these assays, all hrp-hrc, except hrpF, and hpaB mutants lost their pathogenicity, which indicates that most hrp-hrc genes encode essential pathogenicity factors. On the other hand, most hpa mutants showed decreased virulence in a different pattern, i.e., hpa genes are not essential but are important for pathogenicity.
[Show abstract][Hide abstract] ABSTRACT: Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight of rice. A random insertional mutant library of Xoo KACC10331 was constructed using a Tn5-derived transposon, and the virulence of the mutants against the susceptible rice cultivar IR24 was assayed. After the virulence assay, the M793 (purD::Tn5) mutant that had reduced virulence against the rice plants was isolated. Thermal asymmetric interlaced-PCR and sequence analysis revealed that the transposon was inserted into the purD gene (encodes a phosphoribosylamine-glycine ligase) of the M793 mutant. The reverse transcriptase-PCR assay revealed that the mutation of the purD gene did not affect the expression of other purine biosynthesis genes. However, the M793 mutant required exogenous purines and thiamine for growth in minimal media. These results indicate that the purD gene plays a crucial role in the growth and virulence of Xoo.
[Show abstract][Hide abstract] ABSTRACT: Pseudomonas syringae pv. tagetis causes apical chlorosis of several plant species in the Asteraceae, including marigold. As a means to facilitate the isolation of pathogenicity genes and to characterize the genome of this bacterium, we have constructed a bacterial artificial chromosome library of P. syringae pv. tagetis strain LMG5090. The library consists of 1,536 clones with insert size ranging from 30 to 160 kb and an average size of 86 kb. Based upon colony hybridization, the BAC clone 420E23 containing the hrp/hrc gene cluster encoding the type III secretion system was identified from this library and subsequently shotgun sequenced. The hrp/hrc gene cluster of P. syringae pv. tagetis has a 23 kb sequence which contains 27 open reading frames. Comparative analysis of the hrp/hrc gene cluster of P. syringae pv. tagetis LMG5090, P. syringae pv. tomato DC3000, P. syringae pv. syringae B728a, and P. syringae pv. phaseolicola 1448A revealed that the entire hrp/hrc gene cluster of P. syringae pv. tagetis is conserved and identically arranged in all four pathovars.
[Show abstract][Hide abstract] ABSTRACT: The nucleotide sequence was determined for the genome of Xanthomonas oryzae pathovar oryzae (Xoo) KACC10331, a bacterium that causes bacterial blight in rice (Oryza sativa L.). The genome is comprised of a single, 4 941 439 bp, circular chromosome that is G + C rich (63.7%). The genome includes 4637 open reading frames (ORFs) of which 3340 (72.0%) could be assigned putative function. Orthologs for 80% of the predicted Xoo genes were found in the previously reported X.axonopodis pv. citri (Xac) and X.campestris pv. campestris (Xcc) genomes, but 245 genes apparently specific to Xoo were identified. Xoo genes likely to be associated with pathogenesis include eight with similarity to Xanthomonas avirulence (avr) genes, a set of hypersensitive reaction and pathogenicity (hrp) genes, genes for exopolysaccharide production, and genes encoding extracellular plant cell wall-degrading enzymes. The presence of these genes provides insights into the interactions of this pathogen with its gramineous host.
Nucleic Acids Research 02/2005; 33(2):577-86. DOI:10.1093/nar/gki206 · 8.81 Impact Factor