The Farnesyltransferase β-Subunit RAM1 Regulates Localization Of RAS Proteins To Facilitate Infection In Magnaporthe Oryzae

Rice is one of the most important crops all over the world with an increasing worldwide production during past years.Rice blast disease caused by the fungus Magnaporthe oryzae,one of the most series problems in rice production which causes critical damages in rice yield every year.M.oryzae has been served as a model fungus to study fungus-plant interactions.Therefore,In order to find better ways to control this disease we need widely discover it’s infection mechanism.Protein farnesylation plays important roles in the growth and differentiation of eukaryotic cells.This modification can regulate sub-cellular localization through providing hydrophobic moieties that lead to the attachment of the target protein to the inner side of the plasma membrane.The function of farnesylation was not well understood in the plant pathogenic fungi,especially during the fungal infection process.Here,through functional analyses of the farnesyltransferaseβ-subunit gene,RAM1,we examined the importance of protein farnesylation in M.oryzae.Targeted disruption of RAM1 resulted in a slight reduction in hyphal growth and sporulation,and a significant increase in sensitivity to distinct stresses,including the cell wall perturb reagents,osmotic stresses and oxidative stresses.Importantly,loss of RAM1also attenuated in virulence to plant host,characterized by decreased appressorium formation and invasive growth.Interestingly,the defective in appressoria formation inΔram1 mutant can be recovered by exogenous cAMP and IBMX,indicating RAM1functions in upstream of the cAMP signaling pathway.Yeast complementation assay showed that M.oryzae Ram1 could rescue deficiency of the S.cerevisiae ram1 null mutant,suggesting M.oryzae Ram1 also functions as aβ-subunit of the farnesyltransferase.Previous studies in other organisms have revealed functions of the RAS proteins can be regulated by farnesylation,we therefore tested this possibility.RAS1 and RAS2 proteins have been revealed to be involved in both cAMP/PKA and PMK1-MAPK signaling pathways,which are important for appressorium formation and maturation.Interestingly,both RAS1 and RAS2 contain putative farnesylation sites in their C-terminal CAAX motifs(C238 and C211,respectively).Yeast-two hybrid and co-immunoprecipitation assays indicated RAS1 and RAS2 could physically interact with RAM1.Western blot analysis showed noticeable changes of protein bands for both RAS1 and RAS2 proteins in the wild-type,which indicated changes in RAS protein migration when putative farnesylated RAS proteins are compared to unfarnesylated RAS proteins in theΔram1 mutant.Similarly,both of the GFP-RAS1~C238S238S and GFP-RAS2~C211S211S proteins in the wild-type strain were respectively present at comparable levels with the GFP-RAS1 and GFP-RAS2 proteins in theΔram1mutant,revealing that C-terminal modifications of RAS1 and RAS2 by farnesylation affect their protein abundance and/or stability.To further determine whether RAM1 can regulate functions of RAS1 and RAS2proteins,subcellular localization was observed.Both of the GFP-RAS1 and GFP-RAS2proteins were uniformly distributed throughout the plasma membrane in appressorium of the wild type strain.In contRASt,in theΔram1 mutant background,both of the GFP-RAS1 and GFP-RAS2 proteins were distributed in the cytoplasm,but not in the plasma membrane,indicating localization of both RAS proteins were regulated by RAM1.Further,both of the GFP-RAS1~C238S238S and GFP-RAS2~C211S211S proteins were also restricted to the cytoplasm in appressorium.These data confirmed that farnesylation is essential for plasma membrane localization of RAS1 and RAS2.We used Tipifarnib,a competitive inhibitor of farnesyltransferase(FTase),to test the effect of blocking farnesylation in M.oryzae.When we treated conidia of the wild-type strain during appressorium formation with different concentration of Tipifarnib,dramatic reduction of appressorium formation we observed as a concentration-dependent manner.This data showed that inhibition of farnesylation process blocks both of the appressorium formation and invasive growth processes during infection of M.oryzae.Further,the extracellular secretion of the RAS proteins was also blocked by Tipifarnib.Therefore,The development of the fungal-specific farnesyltransferase inhibitors should also be an effective strategy for fungal disease control.Collectively,our findings support that the Ram1-mediated farnesylation process plays important roles in development,environmental response and pathogenesis in M.oryzae.These findings suggest that blocking farnesylation process through FTases are potential strategies to control plant fungal diseases.