Eun-Sung Song

The genome sequence of Xanthomonas oryzae pathovar oryzae KACC10331, the bacterial blight pathogen of rice

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.

Whole genome and global gene expression analyses of the model mushroom Flammulina velutipes reveal a high capacity for lignocellulose degradation.

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.

Molecular analysis of the hrp gene cluster in Xanthomonas oryzae pathovar oryzae KACC10859.

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.

Functional analysis of the aroC gene encoding chorismate synthase from Xanthomonas oryzae pathovar oryzae

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.

Electrophoretic karyotyping and construction of a bacterial artificial chromosome library of the winter mushroom Flammulina velutipes.

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.

Transcriptome-Based Identification of Differently Expressed Genes from Xanthomonas oryzae pv. oryzae Strains Exhibiting Different Virulence in Rice Varieties

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight (BB) in rice (Oryza sativa L.). In this study, we investigated the genome-wide transcription patterns of two Xoo strains (KACC10331 and HB1009), which showed different virulence patterns against eight rice cultivars, including IRBB21 (carrying Xa21). In total, 743 genes showed a significant change (p-value < 0.001 in t-tests) in their mRNA expression levels in the HB1009 (K3a race) strain compared with the Xoo KACC10331 strain (K1 race). Among them, four remarkably enriched GO terms, DNA binding, transposition, cellular nitrogen compound metabolic process, and cellular macromolecule metabolic process, were identified in the upregulated genes. In addition, the expression of 44 genes was considerably higher (log2 fold changes > 2) in the HB1009 (K3a race) strain than in the Xoo KACC10331 (K1 race) strain. Furthermore, 13 and 12 genes involved in hypersensitive response and pathogenicity (hrp) and two-component regulatory systems (TCSs), respectively, were upregulated in the HB1009 (K3a race) strain compared with the Xoo KACC10331 (K1 race) strain, which we determined using either quantitative real-time PCR analysis or next-generation RNA sequencing. These results will be helpful to improve our understanding of Xoo and to gain a better insight into the Xoo–rice interactions.

PCR-based Assay for the Specific Detection of Pseudomonas syringae pv. tagetis using an AFLP-derived Marker

A PCR method has been developed for the pathovar-specific detection of Pseudomonas syringae pv. tagetis, which is the causal agent of bacterial leaf spots and apical chlorosis of several species within the Compositae family. One primer set, PSTF and PSTR, was designed using a genomic locus derived from an amplified fragment length polymorphism (AFLP) fragment produced a 554-bp amplicon from 4 isolates of P. syringae pv. tagetis. In DNA dot-blot analysis with the PCR product as probe, a positive signal was identified in only 4 isolates of P. syringae pv. tagetis. These results suggest that this PCR-based assay will be a useful method for the detection and identification of P. syringae pv. tagetis.