Functional Analysis On Regulatory Proteins Pams1and Clp1in The Development And Pathogenicity Of Magnaporthe Oryzae

Rice blast,caused by Magnaporthe oryzae（synonym Pyricularia oryzae）,severely threatens production security of global food crops.As a filamentous fungus,M.oryzae infects many cereal crops,such as rice and wheat,and is a model to study the interactions between pathogenic fungi and host plants.An appressorium is a functional structure for M.oryzae to infect plant cells.The appressorium formation is regulated by cellular activities such as sensing of plant surface signals,inward signal transduction,specific gene transcription,and autophagy.Therefore,the thorough research of appressorium formation,transcriptional regulation,and autophagy will promote a well understanding of the pathogenic mechanism of M.oryzae,and provide good ideas for scientific,efficient and durable control strategies of blast diseaes.In this study,we characterized the biological functions of the appressorium-specific expressed membrane protein Pams1 and a nuclear and PHD domain protein Clp1 on autophagy,appressorium formation and virulence in M.oryzae.Pams1（Pyricularia appressorium membrane-specific protein 1）is a membrane protein specifically expressed in the maturing appressoria of M.oryzae.The function of Pams1 and its homologous proteins have not been reported in organisms including animals,plants and microorganisms.Pams1 is initially localized in cell membranes during appressorium differentiation,and then gradually transfered to endosome membranes during appressorium maturation.Deletion of PAMS1 led to the formation of malformed appressoria.Δpams1 has two types of abnormal appressoria:melanized type I of appressoria is viable and has reduced virulence,while pale type II of appressoria is dead and has no penetration ability.Cutin monomers inhibited adenylate cyclase（Mac1）activity ofΔpams1 during appressorium maturation,and made the mutant to form a higher proportion of type II of appressoria.c AMP promotedΔpams1 to form higher proportion of melanized type I of appressoria.And H89-2HCl,a PKA inhibitor,promotedΔpams1 to form higher proportion of pale type II of appressoria.The evidences implyed that the c AMP-PKA pathway is impaired in maturing appressoria ofΔpams1.Activated Gα（a G protein alpha subunit）,activated Ras2（a small G protein）,forskolin（a Mac1 activator）,or cutin monomers promoted appressorium formation ofΔpams1 on hydrophilic surfaces,while did not promoteΔpams1 to form higher proportion of melanized type I of appressoria on hydrophobic surfaces.Protein-lipid overlay assay indicated that cutin monomers bind to the N terminal polypeptide of Mac1.Mac1 was localized in endosomes of an appressorium in the wild type strain,while Mac1 was separated from endosomes of an appressorium inΔpams1.Therefore,the plant surface signals activate Mac1 inΔpams1 during appressorium differentiation,while inhibit or do not activate Mac1 inΔpams1 during appressorium maturation.The N terminal peptides of Pams1（Pams1-N）is essential for its function.The pams1^ΔN mutant,in which Pams1-N is deleted,has phenotypes similar to those of theΔpams1 mutant.Yeast two-hybrid,Pull-down,and Co-IP methods confirmed the interaction between Pams1-N and Sep4.Fluorescent protein co-localization analysis showed that Pams1 and Sep4 are colocalized on the endosomal membrane of an appressorium.AndΔpams1 did not form normal septin ring in marture appressoria.Abnormal appressoria and abnormal septin ring are two of the reasons for the reduced virulence ofΔpams1.Totally,the c AMP-PKA pathway is independently activated twice during appressorium differentiation and maturation of M.oryzae,in which the membrane protein Pams1 stabilizes the normal function of Mac1 and Sep4 during appressorium maturation,thereby maintains the normal roles of mature appressoria.The PHD（plant homeodomain）domain protein Clp1 in M.oryzae is homologous to Saccharomyces cerevisiae Cti6（Cyc8-Tup1 interacting protein 6）.Cti6 is a nuclear protein that involved in transcriptional regulation in S.cerevisiae.In M.oryzae,the biological function of Clp1 has not been understood.Clp1 is mainly localized in the nucleus and partly in the cytoplasmic vesicles.Yeast two-hybrid and Pull-down experiments revealed that Clp1 interacts with the autophagy-related proteins Atg5,Atg7,Atg16,Atg24 and Atg28.Fluorescent protein colocalization analysis showed that Clp1 colocalizes with above-mentioned five autophagy-related proteins in the cytoplasmic vesicles.Deletion of CLP1 increased the fungal background autophagy level in M.oryzae.Clp1 is involved in the expression of some known genes required for conidiation and growth,such as CNF1,RSY1,ALB1,CREA.Δclp1 displayed reduced hyphal growth,hyperbranching,abnormal fungal morphology（including colony,spore,and appressorium）,hindered appressorial glycogen metabolism and turgor production,weakened plant infection,and decreased virulence.In addition,the PHD domain is indispensable for the function of Clp1.Hence,Clp1 regulates fungal development and pathogenicity by maintaining autophagy homeostasis and affecting gene transcription in M.oryzae.In conclusion,this study revealed the essential functions and regulatory mechanism of Pams1 and Clp1 in appressorium formation and maturation,fungal growth and development,virulence,gene transcription and autophagy in M.oryzae.