Sheath blight disease (ShB), caused by the fungus Rhizoctonia solani Kühn, is one of the most destructive diseases of rice (Oryza sativa L.), causing substantial yield loss in rice. In the present study, a novel rice chitinase gene, LOC_Os11g47510 was cloned from QTL region of R. solani tolerant rice line Tetep and used for functional validation by genetic transformation of ShB susceptible japonica rice line Taipei 309 (TP309). The transformants were characterized using molecular and functional approaches. Molecular analysis by PCR using a set of primers specific to CaMv 35S promoter, chitinase and HptII genes confirmed the presence of transgene in transgenic plants which was further validated by Southern hybridization. Further, qRT-PCR analysis of transgenic plants showed good correlation between transgene expression and the level of sheath blight resistance among transformants. Functional complementation assays confirmed the effectiveness of the chitinase mediated resistance in all the transgenic TP309 plants with varying levels of enhanced resistance against R. solani. Therefore, the novel chitinase gene cloned and characterized in the present study from the QTL region of rice will be of significant use in molecular plant breeding program for developing sheath blight resistance in rice.

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Rice blast resistance gene, Pi54 provides broad-spectrum resistance against different strains of Magnaporthe oryzae. Understanding the cellular localization of Pi54 protein is an essential step towards deciphering its place of interaction with the cognate Avr-gene. In this study, we investigated the subcellular localization of Pi54 with Green Fluorescent Protein (GFP) as a molecular tag through transient and stable expression in onion epidermal cells (Allium cepa) and susceptible japonica cultivar rice Taipei 309 (TP309), respectively. Confocal microscopy based observations of the onion epidermal cells revealed nucleus and cytoplasm specific GFP signals. In the stable transformed rice plants, GFP signal was recorded in the stomata, upper epidermal cells, mesophyll cells, vascular bundle, and walls of bundle sheath and bulliform cells of leaf tissues. These observations were further confirmed by Immunocytochemical studies. Using GFP specific antibodies, it was found that there was sufficient aggregation of GFP::Pi54protein in the cytoplasm of the leaf mesophyll cells and periphery of the epidermal cells. Interestingly, the transgenic lines developed in this study could show a moderate level of resistance to Xanthomonas oryzae and Rhizoctonia solani, the causal agents of the rice bacterial blight and sheath blight diseases, respectively. This study is a first detailed report, which emphasizes the cellular and subcellular distribution of the broad spectrum blast resistance gene Pi54 in rice and the impact of its constitutive expression towards resistance against other fungal and bacterial pathogens of rice.

Cloning and functional characterization of plant pathogen inducible promoters is of great significance for their use in the effective management of plant diseases. The rice gene CYP76M7 was up regulated at 24, 48, and 72 hours post inoculation (hpi) with two isolates of Magnaporthe oryzae Mo-ei-11 and Mo-ni-25. In this study, the promoter of CYP76M7 gene was cloned from rice cultivar HR-12, characterized and functionally validated. The Transcription Start Site of CYP76M7 was mapped at 45 bases upstream of the initiation codon. To functionally validate the promoter, 5′ deletion analysis of the promoter sequences was performed and the deletion fragments fused with the β-glucuronidase (GUS) reporter gene were used for generating stable transgenic Arabidopsis plants as well as for transient expression in rice. The spatial and temporal expression pattern of GUS in transgenic Arabidopsis plants and also in transiently expressed rice leaves revealed that the promoter of CYP76M7 gene was induced by M. oryzae. The induction of CYP76M7 promoter was observed at 24 hpi with M. oryzae. We report that, sequences spanning -222 bp to -520 bp, with the cluster of three W-boxes, two ASF1 motifs and a single GT-1 element may contribute to the M. oryzae inducible nature of CYP76M7 promoter. The promoter characterized in this study would be an ideal candidate for the overexpression of defense genes in rice for developing durable blast resistance rice lines.