Project description:Rice blast caused by Magnaporthe oryzae is the most devastating disease of cultivated rice. Several protein kinase cascades have been known to be essential to pathogenesis or important for response to stress, mycelial growth and conidiation in M. oryzae. However, phosphoproteins and their phosphorylation sites (p-sites) in this important fungal pathogen remain largely to be identified. In this study, 8087 phosphopeptides corresponding to 9825 p-sites from 1147 phosphoproteins were identified in mycelia of M. oryzae under a false discovery rate of < 0.55% at the peptide level. Notably, 33 previously reported pathogenesis-related proteins were included in the phosphoproteins at the mascot delta score 10. Further analyses of 581 motif-containing phosphoproteins that met more stringent criteria revealed that the phosphoproteins shared 19 distinct phosphorylation motifs, including the motif RxxpSP that was newly identified in this study but is widely distributed in diverse organisms. These phosphoproteins were mapped into 81 biological pathways. A total of 82 acidic motif-containing phosphoproteins were identified. Surprisingly, none of them except one were mapped to any of the metabolic pathways. Furthermore, a prediction disclosed a total of 174 kinase-substrate specific interactions in mycelia of M. oryzae. This study also detected phosphorylation of the tyrosine phosphatase Pmp1 and 7 other proteins upstream of Pmk1, but not Pmk1 and its downstream transcription factors. These results prompted a necessary revision of Pmk1 MAPK cascade, in which dephosphorylation of Pmk1 by Pmp1 in mycelia may block the activation of downstream targets.
Project description:The dark-phase in light/dark cycle plays an important role in successful disease development in M. oryzae-Oryza sativa. We used microarrays to detail the global programme of gene expression in response to light-to-dark transition. We identified different groups of genes (based on GO categories) that are up- or down-regulated in response to light-to-dark transition. Keywords: time course Overall design: We performed a microarray experiment using microarray chips manufactured by Agilent Technologies Inc. This chip contains more than 13,000 M. oryzae probes and 7,000 rice probes. Our mycelial samples were grown in liquid media under constant white-light and then moved to constant darkness for 0.5, 2, 12, or 24 hours. Three biological samples per data point were collected in two separate experiments. Total RNA was extracted from the mycelial samples and labeled with Cy-3 dye. For normalization between slides we used a control RNA, which was prepared by pooling RNA samples from all of the time points. The control RNA was labeled with Cy-5. Hybridization and all the subsequent procedures were performed following the manufacturer’s recommendations.