| Phospholipase C (PLC) and Diacylglycerol kinase (DGK) play a key role in lipid signalings. PLC can hydrolyze phosphatidylinositol4,5-bisphosphate (Phosphatidylinositol-4,5-bisphosphate of PIP2) to produce inositol1,4,5-triphosphate (inositol1,4,5-trisphosphate, IP3), and diacylglycerol (Diacylglycerol, DAG). While DGK produce phosphatidic acid (PA) by phosphate DAG DAG, IP3and PA are key molecules mediate plant disease resistance. Although several studies have elucidate the tight link between elicitor treatment and ROS burst, evidence are still lacking in the role of PLC/DGK medicate defense responses. Fortunately, Genome sequence data from M. oryzae and rice (O. sativa spp. japonica and spp. indica) are available. M. oryzae is a tractable organism and has become the model fungi for studying plant-pathogen interactions.In this paper, we demonstrate that:rice mutant dgkl, plc2and plc3are more susceptible to rice blast fungi than wildtype. Those indicate that OsDGK1, OsPLC2and OsPLC3may play a positive role in rice defense responses.To visilize the infect hypha, we transformate M. oryzae usuing pBV13plasmid which contain an EYFP gene. High quantum efficiency of fluorescent transformants was used in futher studies. We investigated the mycelium growth pattern in rice leaf sheath tissues and found that invasive hypha growth more rapidly in mutant tissues. Statistical analyze at24and40hours post inoculation review a remarkable diffences were located in24-40hpi. A DGK inhibitor R59022was futher used to verify the mutant effect, so did the neomycin sulfate (PLC inhibitor). Either shows a similar result to their mutants. Futher studies show that callose deposition occurs at the intercellular crossing point of the IH of wildtype as indicated by aniline blue dye. ROS accumulate near the infection site were determind by fluorescent dye H2DCF-DA. Results suggest that ROS bursts earlier in wildtype than in mutant.Oxidative burst, a rapid, transient, production of huge amounts of reactive oxygen species (ROS), is one of the earliest observable aspects of a plant’s defence strategy. We use elicitor-treat protoplast to investigate ROS production as indicated by ROS fluorescent dye H2DCF-DA. Results suggest that wildtype have a significant ROS burst15min after xylanase were added, while ROS burst in mutant were dampend. We futher anlyse the role of PA by adding16:018:2PA to mutant cells. Result show that PA can restore the burst of ROS in the mutant cells. Therefore, the inhibition of ROS caused by the knockout OsDGK1, OsPLC2OsPLC3may lead to the susceptible phenotype of mutant. Futher studies show that complement H2O2in mutant tissue can restore the inhibition of IH expansion. This indicated that PLC-DGK derived PA and its product ROS plays a positive role in resistance to rice blast. We also studied the relative expression levels of OsDGK1, OsPLC2, OsPLC3, OsPR1b, OsPBZ1, OsNPR1, OsRbohA during rice blast infection. Hypothetical signal transduction pathway and disease resistance mechanisms of rice cell were also demonstrated. |
