Isolation and characterization of novel genes involved during appressorium formation in the rice blast fungus, Magnaporthe grisea: From genes to genomics

The ascomycete Magnaporthe grisea, the causal agent of rice blast, is one of the most destructive plant pathogens in rice production worldwide. It penetrates the host cuticle directly by way of a highly melanized, specialized infection structure called an appressorium. cAMP and signal transduction pathways have been shown to be involved in appressorium formation. To further understand the cellular mechanisms in appressorium formation, I cloned a gene (MAC1) encoding adenylate cyclase, a membrane-bound enzyme that catalyze the production of cAMP from ATP. Transformants lacking MAC1 were unable to form appressoria on an inductive surface and were unable to penetrate rice. Appressorium formation was restored in the presence of exogenous cAMP treatment. These results confirm that cell signaling involving cAMP plays a central role in the development and pathogenicity of M. grisea.;Genes expressed during appressorium formation in the rice blast fungus M. grisea were identified by sequencing the 5' end of cDNA clones prepared from conidia germinated on a surface inductive for appressorium formation. A total of 2325 expressed sequence tags (ESTs) corresponding to 1430 unique sequence sets (307 contigs and 1123 singletons) were generated. 893 ESTs of 2325 (38.4%) showed significant matches (e value less than 10-5) to sequences in public databases. The 893 ESTs showing significant homology to known genes were assigned to 10 functional categories.;Differential hybridization and subtraction approaches were applied to identify genes showing appressorium stage specific expression pattern. From a cDNA library constructed from conidia germinated on an appressorium inductive surface, a number of genes were selected as up-regulated in appressorium formation compared to vegetative growth.;A 112 kb BAC clone located on chromosome 7 was sequenced and annotated. Gene prediction annotation software, GenScan, identified 36 putative ORFs. Analysis of ESTs for M. grisea chromosome 7 identified 13 ESTs that mapped onto the BAC clone. Northern blot analysis showed that several genes are differentially expressed during appressorium formation. These results demonstrate that the sequencing BAC clones when combined with expressed sequence tags (EST) analysis is a powerful and practical approach for sequencing the rice blast genome.