Activation Mechanisms Of MEK Kinase Mst11 And Functional Characterization Of Mobud7 In Magnaporthe Oryzae

Appressorium formation and invasive growth are two important steps in the infection cycle of Magnaporthe oryzae.It has been known that these two steps are regulated by the Mstll-Mst7-Pmkl MAP kinase pathway.However,the molecular mechanism involved in the activation of Mst11 MEK kinase is not clear in the rice blast fungus.In this study,the author functionally characterized the regulatory region of Mst11 and its self-inhibitory binding.Deletion of the middle region of Mst11 that contains the Ras-association（RA）domain and two conserved phosphorylation sites（S453 and S458）blocked Pmkl activation and appressorium formation.However,the RA domain deletion mutant MST11△RA transformant MRD-2 still formed appressoria but their tugor pressure was reduced,which might lead to reduced virulence in MRD-2.Interestingly,over 50%of its germ tubes branched and formed two appressoria by 48 h,which was suppressed by addition with exogenous cAMP.Ras2 is GTP-binding protein,and G18V mutation makes Ras2 dominant active.With the yeast two hybridization experiments,the author found that the G18V mutation enhanced the interaction of Ras2 with Mstll,suggesting that Mstll has stronger interactions with the activated Ras2.Furthermore,deletion and site-directed mutagenesis analyses indicated that phosphorylation at S453 and S458 of Mstl 1 is important for appressorium formation and required for the activation of Pmkl.We also showed that the N-terminal region of Mst11 directly interacted with its kinase domain,and the S789G mutation reduced their interactions.Expression of the MST11S789G allele rescued the defect of the mstll mutant in plant infection and resulted in the formation of appressoria on hydrophilic surfaces,suggesting the gain-of-function effect of the S789G mutation.Overall,our results indicate that the interaction of Mst11 with activated Ras2 and phosphorylation of S453/S458 play regulatory roles in Mst11 activation and infection-related morphogenesis,possibly by relieving its self-inhibitory interactionbetween its N-terminal region and the C-terminal kinase domain.In addition,binding of Mst11 to Ras2 may be involved in the feedback inhibition of cAMP signaling and further differentiation of germ tubes after appressorium formation.In addiation,the author functionally characterized Mobud7 gene in rice blast fungus,which was previously identified in our laboratory.In this study,the author found that the deletion mutant Mobud7 was defected in appressorium formation,which was rescured by addition with the cAMP inhibitor IBMX,suggesting that Mobud7 functions upstream of the cAMP pathway.Futhermore,deletion of Mobud7 affected the appressorial penetration and virlunce.Cytorrhysis assay and KI/I2 staining assay showed Mobud7 was important for torgor accumulation and glucogen degradation during the appressorial maturation.Further analyses indicated that Mobud7 was localized in cytoplasm and was highly expressed in appressorium,which was also confirmed by qRT-PCR.With the pull-down experiments and yeast two-hybrid assay,the author also found that Mobud7 may interact with MoArfl,MGG₀2799,and itself which was similar with the ChAPs protein family reported in budding yeast.These results suggested that Mobud7 may be involved in the transportation of glucogen degration releted proteins in M.oryzae which are important for appressorium formation and maturation.