Oschib1 gene encoding a GH18 chitinase confers resistance against sheath blight disease of rice caused by Rhizoctonia solani AG1-IA.

Sheath blight disease of rice caused by Rhizoctonia solani AG1-IA, is a major fungal disease responsible for huge loss to grain yield and quality. The major limitation of achieving persistent and reliable resistance against R. solani is the governance of disease resistance trait by many genes. Therefore, functional characterization of new genes involved in sheath blight resistance is necessary to understand the mechanism of resistance as well as evolving effective strategies to manage the disease through host-plant resistance. In this study, we performed RNA sequencing of six diverse rice genotypes (TN1, BPT5204, Vandana, N22, Tetep, and Pankaj) from sheath and leaf tissue of control and fungal infected samples. The approach for identification of candidate resistant genes led to identification of 352 differentially expressed genes commonly present in all the six genotypes. 23 genes were analyzed for RT-qPCR expression which helped identification of Oschib1 showing differences in expression level in a time-course manner between susceptible and resistant genotypes. The Oschib1 encoding classIII chitinase was cloned from resistant variety Tetep and over-expressed in susceptible variety Taipei 309. The over-expression lines showed resistance against R. solani, as analyzed by detached leaf and whole plant assays. Interestingly, the resistance response was correlated with the level of transgene expression suggesting that the enzyme functions in a dose dependent manner. We report here the classIIIb chitinase from chromosome10 of rice showing anti-R. solani activity to combat the dreaded sheath blight disease.

Genome-Wide Expression Profiling of Small RNAs in Indian Strain of Rhizoctonia solani AG1-1A Reveals Differential Regulation of milRNAs during Pathogenesis and Crosstalk of Gene Regulation.

Rhizoctonia solani AG1-1A is a necrotrophic fungus that causes sheath blight disease in rice. The reliable resistant source against this phytopathogenic fungus is not available in the gene pool of rice. Better understanding of pathogen genomics and gene regulatory networks are critical to devise alternate strategies for developing resistance against this noxious pathogen. In this study, miRNA-like RNAs (milRNAs) of an Indian strain of R. solani were identified by deep sequencing of small RNAs. We identified 128 known and 22 novel milRNAs from 20,963,123 sequence reads. These milRNAs showed 1725 target genes in the fungal genome which include genes associated with growth, development, pathogenesis and virulence of R. solani. Notably, these fungal milRNAs showed their target genes in host (rice) genome also which were later verified by qRT-PCR. The host target genes are associated with auxin metabolism, hypersensitive response, defense genes, and genes related to growth and development of rice. Osa-vacuolar-sorting receptor precursor: Rhi-milR-13, Osa-KANADI1:Rhi-milR-124, Osa-isoflavone reductase: Rhi-milR-135, Osa-nuclear transcription factor Y:Rhi-milR-131, Osa-NB-ARC domain containing protein: Rhi-milR-18, and Osa-OsFBX438: Rhi-milR-142 are notable potential regulons of host target genes: fungal milRNAs that need to be investigated for better understanding of the crosstalk of RNAi pathways between R. solani and rice. The detailed expression analysis of 17 milRNAs by qRT-PCR was analysed during infection at different time points of inoculation, at different growth stages of the host, in four different genotypes of the host, and also in four different strains of fungi which revealed differential regulation of milRNAs associated with pathogenesis and virulence. This study highlights several important findings on fungal milRNAs which need to be further studied and characterized to decipher the gene expression and regulation of this economically important phytopathogen.

A Comprehensive Gene Expression Profile of Pectin Degradation Enzymes Reveals the Molecular Events during Cell Wall Degradation and Pathogenesis of Rice Sheath Blight Pathogen Rhizoctonia solani AG1-IA.

Sheath blight disease of rice caused by Rhizoctonia solani Kühn (teleomorph: Thanatephorus cucumeris) remains a global challenge due to the absence of reliable resistance genes and poor understanding of pathogen biology. Pectin, one of the most vital constituents of the plant cell wall, is targeted by pectin methylesterases, polygalacturonases, and few other enzymes of fungal pathogens. In this study, we catalogued the expressed genes of the fungal genome from RNAseq of R. solani infected four rice genotypes. Analysis of RNAseq revealed 3325 pathogen genes commonly expressed in all rice genotypes, in which 49, 490, and 83 genes were specific to BPT5204, Tetep, and Pankaj genotypes, respectively. To identify the early and late responding genes of R. solani during plant cell wall degradation, a real-time PCR analysis of 30 pectinolytic enzymes was done at six different time points after inoculation. The majority of these genes showed maximum induction at the 72 h time point, suggesting that it is the most crucial stage of infection. Pankaj showed lesser induction of these genes as compared to other genotypes. Leaf-blade tissue and 45 days old-growth stage are more favorable for the expression of pectin degradation genes of R. solani. Additionally, the expression analysis of these genes from four different strains of R. solani suggested differential regulation of genes but no distinct expression pattern between highly virulent and mild strains. The implications of the differential regulation of these genes in disease development have been discussed. This study provides the first such comprehensive analysis of R. solani genes encoding pectin degrading enzymes, which would help to decipher the pathogen biology and sheath blight disease development.

Proteomic and transcriptomic approaches to identify resistance and susceptibility related proteins in contrasting rice genotypes infected with fungal pathogen Rhizoctonia solani.

The devastating sheath blight disease caused by Rhizoctonia solani Kuhn (teleomorph: Thanatephorus cucumeris) causes major yield loss in most rice growing regions of the world. In this study, two moderately tolerant and four susceptible genotypes of rice were selected for R. solani induced proteome analysis using two-dimensional polyacrylamide gel electrophoresis. Forty five differentially expressed proteins (DEPs) were identified and analyzed by Mass Spectrometry. Based on their functions, these proteins were classified into different groups, viz., photosynthesis, resistance and pathogenesis, stress, cell wall metabolism and cytoskeleton development associated proteins, and hypothetical or uncharacterized proteins. Expression of 14 genes encoding DEPs was analyzed by quantitative PCR which showed consistency in transcripts and genes expression pattern. Furthermore, the expression of 16 other genes involved in diverse biological functions was analyzed. Up-regulation of these genes in the tolerant genotype Pankaj during sheath blight disease suggested efficient genetic regulation of this cultivar under stress. Also, expression analysis of conserved microRNAs (miRNAs) and their target genes revealed important role of miRNAs in post-transcriptional gene regulation during development of rice sheath blight disease. Genome-wide discovery of miRNAs and further characterization of DEPs and genes will help in better understanding of the molecular events during sheath blight disease development in rice.