The farnesyltransferase β-subunit RAM1 regulates localization of RAS proteins and appressorium-mediated infection in Magnaporthe oryzae

Post-translational farnesylation can regulate subcellular localization and protein-protein interaction in eukaryotes. The function of farnesylation is not well identified in plant pathogenic fungi, particularly during the process of fungal Infection. Here, through functional analyses of the farnesyltransferase β-subunit gene, RAM1, we examine the importance of protein farnesylation in the rice blast fungus Magnaporthe oryzae. Targeted disruption of RAM1 resulted in the reduction of hyphal growth and sporulation, and an increase in the sensitivity to various stresses. Importantly, loss of RAM1 also led to the attenuation of virulence on the plant host, characterized by decreased appressorium formation and invasive growth. Interestingly, the defect in appressoria formation of the Δram1 mutant can be recovered by adding exogenous cAMP and IBMX, suggesting that RAM1 functions upstream of the cAMP signalling pathway. We found that two Ras GTPases, RAS1 and RAS2, can interact with Ram1, and their plasma membrane localization was regulated by Ram1 through their C-terminal farnesylation sites. Adding a farnesyltransferase inhibitor Tipifarnib can result in similar defects as in Δram1 mutant, including decreased appressorium formation and invasive growth, as well as mislocalized Ras proteins. Our findings indicate that protein farnesylation regulates the Ras protein-mediated signaling pathways required for appressorium formation and host Infection, and suggest that abolishing farnesyltransferase could be an effective strategy for disease control.