Rhizoctonia solani is a major fungal pathogen of rice (Oryza sativa L.) that causes great yield losses in all rice-growing regions of the world. Here we report the draft genome sequence of the rice sheath blight disease pathogen, R. solani AG1 IA, assembled using next-generation Illumina Genome Analyser sequencing technologies. The genome encodes a large and diverse set of secreted proteins, enzymes of primary and secondary metabolism, carbohydrate-active enzymes, and transporters, which probably reflect an exclusive necrotrophic lifestyle. We find few repetitive elements, a closer relationship to Agaricomycotina among Basidiomycetes, and expand protein domains and families. Among the 25 candidate pathogen effectors identified according to their functionality and evolution, we validate 3 that trigger crop defence responses; hence we reveal the exclusive expression patterns of the pathogenic determinants during host infection.

The WRKY family of transcription factors (TFs) functions as transcriptional activators or repressors in various signaling pathways. In this study, we discovered that OsWRKY62 and OsWRKY76, two genes of the WRKY IIa subfamily, undergo constitutive and inducible alternative splicing. The full-length OsWRKY62.1 and OsWRKY76.1 proteins formed homocomplexes and heterocomplexes, and the heterocomplex dominates in the nuclei when analyzed in Nicotiana benthamiana leaves. Transgenic overexpression of OsWRKY62.1 and OsWRKY76.1 in rice (Oryza sativa) enhanced plant susceptibility to the blast fungus Magnaporthe oryzae and the leaf blight bacterium Xanthomonas oryzae pv oryzae, whereas RNA interference and loss-offunction knockout plants exhibited elevated resistance. The dsOW62/76 and knockout lines of OsWRKY62 and OsWRKY76 also showed greatly increased expression of defense-related genes and the accumulation of phytoalexins. The ratio of full-length versus truncated transcripts changed in dsOW62/76 plants as well as in response to pathogen infection. The short alternative OsWRKY62.2 and OsWRKY76.2 isoforms could interact with each other and with full-length proteins. OsWRKY62.2 showed a reduced repressor activity in planta, and two sequence determinants required for the repressor activity were identified in the amino terminus of OsWRKY62.1. The amino termini of OsWRKY62 and OsWRKY76 splice variants also showed reduced binding to the canonical W box motif. These results not only enhance our understanding of the DNA-binding property, the repressor sequence motifs, and the negative feedback regulation of the IIa subfamily of WRKYs but also provide evidence for alternative splicing of WRKY TFs during the plant defense response.

Metabolomic and transcriptomic approaches were used to dissect the enhanced disease resistance in the plants harbouring a RNA interfering construct of OsWRKY62 and OsWRKY76 (dsOW62/76) genes. The primary metabolic pathways were activated in dsOW62/76 compared with wild-type (ZH17) plants, revealed by increased accumulation of amino acids and constituents of citric acid cycle etc. Contents of phenolic acids derived from phenylpropanoid pathway were elevated in dsOW62/76 plants. Importantly, phenolamides, conjugates of the phenolic acids with amines, were detected in large number and mostly at higher levels in dsOW62/76 compared with ZH17 plants; however, the free pools of flavonoids were mostly decreased in dsOW62/76. Salicylic acid (SA) and jasmonic acid (JA)/JA-Ile contents were increased in dsOW62/76 and knockout lines of individual OsWRKY62 and OsWRKY76 genes. Transcription of isochorismate synthase (OsICS1) gene was suppressed in dsOW62/76 and in MeJA-treated rice plants, whereas the transcription level of cinnamoyl-CoA hydratase-dehydrogenase (OsCHD) gene for β-oxidation in peroxisome was increased. The calli with OsCHD mutation showed markedly decreased SA accumulation. These results indicate that OsWRKY62 and OsWRKY76 function as negative regulators of biosynthetic defense-related metabolites and provide evidence for an important role of phenylpropanoid pathway in SA production in rice.

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