Functional Analysis And Potential Application Of AGC Kinases In Magnaporthe Oryzae

Rice blast is a fungal disease caused by Magnaporthe oryzae.It is widely distributed in various rice planting areas in the world.When it occurs,it seriously affects rice yield and causes serious economic losses.M.oryzae can occur in all development stages of rice,and the damage of neck blast is the most serious.M.oryzae has a typical infection cycle.Conidia are transmitted by wind and rainwater onto the plant surface,and form appressoria under appropriate temperature and humidity.Appressoria generate high turgor pressure which ruptures the host’s leaf cuticle,and form penetration pegs allowing M.oryzae to infect the leaf tissue.Infectious hyphae extend rapidly from one cell to the neighboring cells,causing lesions.These lesions are sites for fungal sporulation,allowing M.oryzae to spread to more plants.AGC kinases,a subgroup of Ser/Thr protein kinases,are highly conserved in eukaryotes.Many members of AGC kinases family are regulated by TOR,and play an important role in cell growth,development,and survival.The role of AGC kinases in the growth,development and pathogenicity are analyzed in this paper through gene knockout,phenotypic analysis,cytological analysis,and stress response in M.oryzae.With homologous sequence alignment,22 AGC kinases genes of M.oryzae were identified,and 12 gene-knockout vectors were constructed.Finally,7 gene-deletion mutants were obtained,ΔMo07012,ΔMo01260,ΔMo05074,ΔMo10181,ΔMo09519,ΔMo02757,ΔMo12408.Unfortunately,MGG₀0479,MGG₁5988,MGG₀1795,MGG₀6599 and MGG₀8689 could not be successfully knocked out.The four AGC kinases mutants（ΔMo02051,ΔMo02061,ΔMo04475,ΔMo06514）have been obtained previously in our laboratory.The phenotypes of the 11 gene-deletion mutants including mycelial growth,conidiation,pathogenicity,stress response and sensitivity to Rapamycin and Myriocin were analyzed systematically.As the result showed,theΔMo02757 mutatant had dense mycelia,and the growth rate of it reduced significantly.The conidiation ofΔMo07012,ΔMo02061,ΔMo05074,ΔMo06514 mutants were reduced compared with Guy11.Particularly,theΔMo07012 andΔMo05074 mutants showed rarely conidia,and the pathogenicity ofΔMo05074 mycelial to barley leaves were weakened while theΔMo07012 mycelia completely lost it.Analysis of the sensitivity to Rapamycin and Myriocin showed that theΔMo06514,ΔMo01260,ΔMo12408,ΔMo07012,ΔMo04475 mutants exhibited higher resistance to Rapamycin,whileΔMo02061,ΔMo10181,ΔMo09519,ΔMo02757 exhibited higher sensitivity to Rapamycin.The sensitivity ofΔMo12408 to Myriocin increased significantly,whileΔMo02051,ΔMo10181,ΔMo05074,ΔMo06514,ΔMo07012,ΔMo02757,ΔMo01260exhibited higher resistance to Myriocin.Taken together,these results illustrated the pleiotropic roles of AGC kinases in M.oryzae.We named MGG₀7012 as MoFpk1,which is a homologe of Saccharomyces cerevisiae Fpk1.Fpk1 is mainly involved in endocytosis,distribution of phospholipids asymmetry in the plasma membrane and cell wall integrity.It is important for intracellular homeostasis,cell growth,development and survival.Therefore,the functions of MoFpk1 are analyzed in detail.TheΔMofpk1 mutant was defective in conidia production.The degradation rates of lipid droplets and glycogen inΔMofpk1were slower than that in the wild-type strain Guy11.The cell wall integrity ofΔMofpk1was impaired.Appressorium-like structures formation was impaired,and mycelial pathogenicity was completely lost.However,the conidial pathogenicity of theΔMofpk1was similar to that of Guy11.TheΔMofpk1 mutant exhibited higher resistance to Myriocin and Rapamycin,and lipid fusion and transport had been changed on the CM plates supplemented with Myriocin and Rapamycin.Additionally,phenotypes of active site-directed mutagenesis strains MoFpk1^K230R230R and MoFpk1^D326A were similar toΔMofpk1,while MoFpk1^D354A was similar to Guy11.The appressorium-like structures formation related genes MAC1,MST12,and MSB2 were significantly down-regulated inΔMofpk1 through qRT-PCR analysis.The expression level of lipid biosynthesis related genes MGG₀0057,MGG₁0005,MGG₁3626 were significantly increased and MGG₀1925,MGG₁3793,MGG₀8074,MGG₀6888 were significantly decreased inΔMofpk1 compared to Guy11.The expression level of autophagy related genes MoATG3 and MoATG11 were upregulated,and MoATG1,MoATG15 and MoATG29 were down-regulated inΔMofpk1 than that in Guy11.These results indicate that MoFpk1 is required for growth,development,appressorium-like structures formation,pathogenicity and lipid fusion and transport in M.oryzae.MoFpk1 also has the potential to be a drug target for controlling rice blast.