Roles Of A Rice Dacylglycerol Kinase Gene OsDAGK1 And Phospholipase C/diacylglycerol Kinase-mediated Signalling In Rice Disease Resistance Responses

Phospholipase C (PLC)/diacylglycerol kinase (DAGK)-mediated signalling is known to play important roles in plant growth and development, stress and hormone response, and disease resistance. In this study, we cloned and identified a novel rice DAGK gene, OsDAGKl, and found that expression of OsDAGKl was specifically activated in disease resistance response induced by benzothiadiazole (BTH), that overexpression of OsDAGKl in transgenic tobacco plants led to an enhanced disease resistance against infection caused by Phytophthora parasitica var. nicotianae and tobacco mosaic virus, and that the PLC/DAGK-mediated signalling was involved in the regulation of hypersensitive cell death, oxidative burst and expression of defense-related genes induced by BTH.Previously, we have isolated and identified hundhreds of differentially expressed cDNA clones that were associated with BTH-induced resistance response through suppression subtractive hybridization. Similarity search against database revealed that the insert in one of the differentially expressed cDNA clones showed high level of similarity to genes encoding DAGK and therefore this clone may be a fragment of a rice gene encoding DAGK. We have cloned and identified full-length cDNA of this putative DAGK gene by rapid amplification of cDNA end (RACE) and designated as OsDAGKl. The full-length cDNA of the OsDAGKl was 2012 bp with a predicted open reading frame of 1500 bp in length, which predicted to encode a putative DAGK protein with 499 amino acid residues. The OsDAGKl gene was mapped to chromosome 4 of the rice genome and is composed of 12 exons and 11 introns. With OsDAGKl as starting point, we also identified other 7 putative DAGK genes in the rice genome. The predicted protein sequences of the rice DAGKs have a conserved diacylglycerol kinase catalytic domain of -300 amino acid residues in length. Expressed sequence tags (ESTs) matching to the sequences of the rice DAGKgenes were retrieved from database and their expression patterns were analyzed by RT-PCR, Northern blot and in silico analysis. Results from these analysis revealed that the rice DAGK genes have tissue-specific expression patterns, showing high level of expression in leaves, relatively low level in root and seeds, and almost no expression in stem and flowers, and that expression of DAGK genes in leaf tissues was induced after treatment with cold or pathogen infection. Expression of OsDAGK1 was rapidly activated within 6 h after inoculation with Magnaporthe grisea in BTH-treated rice seedlings and maintained a high level of expression for a relatively long time period. These results suggest that OsDAGKl plays an important role in rice disease resistance responses.To better understand the function of OsDAGKl in rice disease resistance response, we cloned the OsDAGKl coding sequence into a plant binary vector, CHF3, under control of the CaMV 35S promoter and transformed tobacco leaf discs by Agrobacterium-mediated transformation. Twelve kanamycin-resistant OsDAGKl transgneic tobacco lines were obtained and confirmed by PCR detection. Southern blotting analysis confirmed that the OsDAGKl gene was integrated into tobacco genome. A specific hybridization band with expected size of the mRNA was detected in Northern blotting analysis, indicating that OsDAGKl gene was transcribed in the transgenic tobacco plants. We evaluated disease resistance of the transgenic lines and found that the transgenic plants showed enhanced disease resistance against tobacco mosaic virus and Phytophthora parasitica var nicotianae.Using the interaction of rice and Xanthomonas oryzae pv.oryzae as a model, we further studied the possible roles of PLC/DAGK-mediated signalling in regulation of active oxygen species (AOS), hypersensitive cell death and expression of the PLC/DAGK pathway genes and some defense-related genes. Production of superoxide anion and hydrogen peroxide were detected in suspension-cultured rice cells 3 hours after BTH treatment and hypersensitive cell death was observed 8 hours after BTH treatment.