Study On Gene Expression In Magnaporthe Grisea By Expressed Sequence Tags And CDNA Array Monitoring

The fungus Magnaporthe grisea causes an important rice blast. The rice blast pathosystem as a model system for fungus-host interaction has been investigated intensively. Now studies on pathogenicity of the fungus have focused on appressorium initiation, formation and maturation and their development regulation by cell signaling. This study constructed an expressed sequence tag (EST) database of M. grisea. With the prepared cDNA arrays containing unique clones of M. grisea, we detected the expression profiles of the fungus at different development stages and gene expression of the mutants defective in mitogen-activated protein kinase (MAPK) signal transduction pathway.1. This study has constructed a mixed cDNA library with RNA samples from mycelia, conidia, germ tubes (2 h on hydrophobic surfaces), young appressoria (8 h on hydrophobic surfaces), different mature appressoria at 20 h and 30 h on hydrophobic surfaces of M. grisea Y34. From this cDNA library total 13057 ESTs of the 3' ends were obtained and have been submitted for dbEST of NCBI . These EST sequences assembly out 4756 tentative unique transcripts (TUTs) including 961 contigs and 3795 singletons. Among them, there were 2339 TUTs with no BLASTx hits (E>10~-5) and 2417 annotated TUTs. Among these annotated TUTs, only 36 TUTs were annotated as genes of M. grisea. BLASTn search indicates that 763 of 4756 TUTs of M. grisea Y34 could not be matched in genome sequences of M. grisea 70-15 (E value<10~-3).2. With the above the EST database we prepared cDNA array containing 4108 unique clones of M. grisea. With the cDNA arrays, we detected gene expression at above six development stages of M. grisea. Total 511 differentially expressed genes were identified among any two development stages by program Cyber-T (P<0.01, fold change>2). The reliability of cDNA array data was confirmed by quantitative real-time RT-PCR (Q-PCR) analysis. Compared with mycelia or conidia, a large number of genes showed high-level expression during appressorium initiation, formation and maturation, more genes showed differential expression in appressorium maturation than appressorium differentiation. There were 46 and 10 genes showing especially differential expression in mycelia and conidia respectively. Seven expression patterns were clustered and further analysed atgerm tubes, young appressoria and mature appressoria compared with conidia. This study also identified homologues of many important genes (Magnaporthe CBP1, CYP1, MAGB, PTH11, MPG1 and east Nifl, protein-ryrosine phosphatase gene etc.) at different development stages of M. grisea.3. These cDNA arrays of M. grisea was also used for studying gene expression in the mycelia of four gene deletion mutants (mst50, mstll, mst7 and pmkl), which involved in MAPK signal pathway cascades. Compared with wild types, differentially expressed genes were identified respectively in mutants mstSO, mstll, mst7 and pmkl (P<0.01, fold change>2), and the amount of differentially expressed genes was largest in ihepmkl. The reliability of cDNA array data was confirmed by Q-PCR analysis. Glucose repressible gene was found up-regulated in all four mutants. Genes mainly displayed up-regulated expression in mutant mst50, including genes encoding integral membrane protein, Ras-related protein involved in cell signalling. It indicated that regulatory gene MST50 might also participate in other cell signalling pathways. The disruptions of cascades gene MST11, MST7, PMK1 respectively could lead to the same 30 genes expressed differentially in mutants, including 28 down-regulated genes. It indicated that the MST11-MST7-PMK1 cascades play a role in mycelium development of M. grisea. But there were some different genes expressed differentially in mutants mstll, mst7 and pmkl respectively. Especially in mutant pmkl, there were some up-regulated transcription factors (zinc finger protein, stress response element binding protein and etc.) involved in cell signaling regulating stress response. It indicated that there was branch pathway of cell signaling through some genes involved in PMK1 MAPK pathway of M. grisea. Hierarchical clustering and Q-PCR analysis indicated there were at least two types of gene expression changes during PMK1 MAPK pathway regulating mycelium development. It was also deduced that there might be "crosstalk" between PMK1 MAPK pathway and other signaling pathways through gene PMK1 arid MST50 of M. grisea.