| Ras proteins are involved in a variety of signal-transduction processes. The switching between the inactive Ras-GDP and active Ras-GTP forms are regulated by guanine exchange factors (GEFs) and GTPase activating proteins (GAPs). The rice blast fungus Magnaporthe oryzae has two Ras genes. The RAS1 mutant had no obvious defects in appressorium formation and virulence, whereas deletion of RAS2 appears to be lethal. Expression of a RAS2DA dominant active allele results in elevated intracellular cAMP level, increased Pmk1 phosphorylation, and irregular appressorium formation, indicating that Ras2 functions upstream from both the Pmk1 and cAMP signaling pathways in M. oryzae. In this study, one RasGAP (GAP1) and two RasGEF ( GRF1) genes were characterized in M. oryzae. Deletion of GAP1 resulted in the production of abnormal conidia and long germ tubes during appressorium formation. The Deltagap1 mutant and RAS2DA transformant had similar defects in appressorium morphology and produced melanized conidia or aerial hyphal tips. The level of Pmk1 phosphorylation was increased in the Delta gap1 mutant. However, it had a normal intracellular cAMP level, indicating that other RasGAPs may be involved in the regulation of Ras2 for cAMP signaling. In addition, GAP1 was found to be required for the maintenance of polarity and organization of microtubules and actin cytoskeleton. For the two RasGEF genes characterized in this study, deletion of RGF2 had no significant effects on growth and plant infection. However, RGF2 is essential for appressorium formation and pathogenicity. The & delta;rgf1 mutant produced morphologically abnormal conidia that were defective in the surface attachment. Both Pmk1 phosphorylation and intracellular cAMP levels were reduced in the Delta gap1 mutant, which was suppressed by the expression ofRAS2 DA. Overall, these observations indicate that both GAP1 and RGF1 are functionally related to Ras2 and the activation of the Pmk1 or cAMP signaling pathway. |
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