Gene Expression Research In Magnaporthe Grisea Gas1gas2 Double Mutant By Using CDNA Array

GAS1 and GAS2 were known genes located at downstreams of PMK1-dependent MAPK signal pathway. The main effects of Gas1 and Gas2 deletions was the drop of pathogenicity of Magnaporthe grisea. The cDNA array applied to detecting gene expression of gas1 gas2 double mutant in this research.1. Hybridized with cDNA array were performed respectively using gas1 gas2 double mutant and relevant wild type strain Guy11 as probes. The cDNA array contained 4108 Tutative unique transcript (TUT) of Magnaporthe grisea. After a series of process of data, 1269 differentially expressed genes were detected. In order to ensure the creditability, the quantitative real-time PCR assay were performed in this research.2. The 1269 differentially expressed genes which principally down-regulated were classified by MIPS. MIPS classification results demonstrated that the deletions of gas1 and gas2 was mainly affected substance and energy metabolism. Moreover, the mutation can slow down the TCA cycle which as a hinge of substance and energy metabolism.3. Many genes were down regulated contanied the genes relevanted to cell wall such as Chitin synthase A, Chitinase 1, Cell wall integrity protein scw1 and glucosidase CRH1. Hereby, we speculated that the drop of pathogenicity caused by double mutant was relevant to the damage of cell wall. Transcription factor pacC/RIM101, Sexual differentiation process protein isp4 and Cutinase transcription factor 1 beta were up regulated, which may relevant to normal sexual reproduction and spore formation of double mutant.4. The quantitative real-time PCR assay demonstrated that both PMK1 and MST12 of double mutant were up regulated obviously. The results demonstrated that PMK1-dependent GAS1/GAS2 signal pathway and PMK1-dependent MST12 signal pathway are really different in combination with the detecting data of cDNA array. Double mutant still had certain pathogenicity may relevant to compensation action of PMK1-MST12 pathway etc. Integrated down-regulated MPG1 and relevant information, we speculated that PMK1 -dependent GAS1/GAS2 signal pathway one by one are MPG1, MAGB, gene wich combined G protein and MAPKKK, MST11-MST7-PMK1, GAS1/GAS2 transcription factor and GA1/GAS2.5. Adenylate cyclase and cAMP-dependent protein kinase were down regulated in double mutant. These demonstrated that the level of cAMP was decreased, and cAMP-dependent PKA signal pathway was weakened which affected appressorium infection of double mutant directly. Because of the block of PMK1-GAS1/GAS2 signal pathway and influence of cAMP to PMK1 signal pathway, PMK1-MST12 signal pathway was up-regulated which was to compensate the influence of blocked PMK1-GAS1/GAS2.6. The homologous genes of Calmodulin, calcium influx promoting protein ehs1, 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase 1, were down-regulated expressed. but the homologous genes of calcium/calmodulin-dependent protein kinase were up-regulated expressed. These results indicated that the complicated Ca²⁺ signal transduction pathway existed in double mutant. We know that both GAS 1 and GAS2 had Protein kinase C (PKC) phosphorylation site which related to Ca²⁺ signal transduction pathway. And PKC participate in appressorium formation. Therefore, we speculated that complicated Ca²⁺ signal transduction pathway controlled appressorium formation of double mutant jointly, and led to normal formation of double mutant appressorium.7. The homologous genes of Sty1 and Wis4 were down regulated in double mutant, and the yeast sty1 signal pathway shown the highest homology to Magnaporthe grisea OSM1 signal pathway.therefor, OSM1 signal pathway was weakened and osmotic stress changed in double mutant. We speculated that the change of osmotic stress was related to the damage of cell wall. We also found that signal pathways of Ran, Ras, Rho and Sho were relevant to PMK1-GAS1/GAS2 signal pathway, which indicated that complicated signal transduction and control web was really existed in double mutant.In conclusion, the drop of pathogenicity of gas1 gas2 double mutant was due to large numbers of differentilly expressed genes. It were these differentilly expressed genes working together that contribute to the drop of pathogenicity of double mutant Among Signal pathways relevant to PMK1,they had complicated signal transduction and control web which were deserved more attention.