Functional Studies Of Three Appressorium Formation And Infection-Required Genes PoTEA1, PoRAL2 And PoSPF1 In Pyricularia Oryzae

Pyricularia oryzae(synonym Magnaporthe oryzae)has been a model organism that widely used in fungal molecular biology studies.In the process of its infection cycle,P.oryzae penetrates the epidermis of rice via the infection structure,an appressorium,and causes rice blast that is highly harmful and infectious.Therefore,it is of great significance to study its appressorium development,infection and pathogenic mechanisms in P.oryzae for the control of the rice blast.In this study,we analyzed the roles of two kelch domain proteins PoTea1 and PoRal2,and the P5-ATPase PoSpf1 in the growth,sporulation,appressorium formation and infection of P.oryzae,and discussed their functions in the cAMP-PKA pathway,Pmk1 MAPK pathway,endocytosis,endoplasmic reticulum stress response or unfolded protein response(UPR).Tea1(Tip elongation aberrant protein 1)proteins are a kind of cell-end markers that determine the aggregation of microtubules and actin at growth sites and regulate cell polarity.PoTea1 polymerizes with itself via its N-terminus kelch domains and C-terminus coiled-coil domains,and locates at the tips of spore,mycelium and germ tube,and the base of initial appressorium.?Potea1 has several mutant phenotypes that are different from the wild-type strain:the branching degree of the mutant's hyphae is increased,the cell separation of spores is reduced,the germ tubes formed by germinated spores is longer,the appressorial formation rate is reduced,the diameter of appressorium is reduced,and the degradation of glycogen and lipid droplets were delayed.In addition,?Potea1's ability to respond to exogenous cAMP was weakened during appressorium differentiation on hydrophilic surfaces,and the Pmk1phosphorylation level of the mutant's hyphae was increased.The subcellular location of PoTea1 does not depend on its physically interacting protein PoTeaR,but is influenced by microtubules and actin integrity.Deletion of Po TEA1 did not affect actin location,however,the central hole of Sep4 ring on appressorium pore became blurred,and the aggregation of myosin?Myo5 on mycelial tips was weakened.Furthermore,the endocytosis efficiency of FM4-64 by germ tubes during the appressorium differentiation was weakened,indicating that PoTea1 may be involved in the transport of secretory vesicles,and then affect the endocytosis of P.oryzae.Ral2 is a Ras like protein,and PoRal2 is a kelch domain protein that regulates the sporulation,appressorium formation,infection and pathogenic process of P.oryzae.?Poral2 germinates longer germ tubes,and cannot differentiate into appressoria on the non-inductive hydrophilic membranes by addition of exogenous cAMP or the expression of activated Ras2^G18V.In initial appressoria formed by?Poral2,the expression level of many appressorium formation regulatory genes,such as MPG1,PTH11,WISH,PDEH and RAS2 are abnormal,and the phosphorylation level of Pmk1in?Poral2's hyphae was decreased.Yeast two-hybrid,Pull-down or Co IP experiment results confirmed that PoRal2 interacts with Mst50,Scd1 and Smo1,in addition,the overexpression of SCD1 in?Poral2 aggravated the defects in appressorium formation.These results indicate that PoRal2,together with Mst50,Scd1,and Smo1,act on the complex of Ras2 and Cdc42,thereby regulating Pmk1 activation and controlling the appressorium formation and infection process of P.oryzae.P-type ATPase is a kind of kinetic pumps located on the plasma membrane that can be driven and phosphorylated by ATP.PoSpf1 is a member of the P5-ATPase family,and co-locates with Lhs1 in the endoplasmic reticulum(ER).After Po SPF1 was deleted in P.oryzae,fungal growth was slowed down,sporulation was reduced,appressorium formation was delayed,the growth of infective hyphae was slowed,and virulence on rice was weakened.?Pospf1 was sensitive to tunicamycin(TUNI),but its hyphal growth was improved by dithiothreitol(DTT),hydrogen peroxide and paraquat.The expression of genes such as SCJ1,ERV29,SEC61,PDI1,OST1,PMT1,KAR2 and PMR1 in mycelium is up-regulated,indicating that PoSpf1 is involved in the process of endoplasmic reticulum stress response and unfolded protein response(UPR).