Functional Characterization Of Secreted Proteins Specifically Expressed In Appressoria Of Magnorporthe Oryzae

Rice is one of the main food crops,with more than 50% of the population feeding on rice in the world.Rice blast caused by the ascomycete fungus Magnaporthe oryzae brings serious losses to rice production,and the annual crop yield loss caused by rice blast is 10%-30%,and even leads to harvest failure when the disease is serious.Therefore,the study on M.oryzae is the great significance for the prevention and management of diseases.In our previous study,a family specifically expressed in appressorium,called Mo Mas protein,was screened from 851 putative M.oryzae secreted proteins through bioinformatics analysis.In this study,the Mo Mas protein family including six members,which encoded by six genes,MGG＿12337(GAS1),MGG＿04202(GAS2),MGG＿00703(Mo MAS3),MGG＿09875(Mo MAS4),MGG＿02253(Mo MAS5),and MGG＿00992(Mo MAS6).GAS1 and GAS2 as two virulence factors that have been reported in 2002,but the functions of the other four genes are not yet known.So,this paper mainly studies the functions of Mo MAS3,Mo MAS4,Mo MAS5,and Mo MAS6.1)It was found that the four Mo Mas proteins are rich in alanine(Ala)and glycine(Gly),contain a signal peptide at the N-terminus,and all have a conserved DUF3129 domain with unknown function via bioinformatics analysis.2)△Momas3,△Momas4,△Momas5 and △Momas6 mutants were obtained by knocking out in the wild-type strain Guy11 using Split marker PCR and PEG-mediated protoplast transformation.Growth tests were performed on all mutants,and the results showed that none of the knockout mutants affected the fungal growth of M.oryzae.All mutants were sprayed inoculated noto rice leaves and found that only△Momas3 and △Momas5 mutants reduced virulence against CO-39 rice leaves,while △Momas4 and△Momas6 mutants did not change the virulence of M.oryzae.The appressorium formation and penetration rate of △Momas3 and △Momas5 mutants were tested,and the results showed that deletion of Mo MAS3 or Mo MAS5 did not alter the appressorium formation and penetration of M.oryzae.The morphology of infectious hyphae of △Momas3 and △Momas5 were observed during the biotrophic stage using rice leaf sheath inoculation,and found that infectious hyphae of △Momas3 and △Momas5 were restricted to expand to neighbor cells in rice.△Momas3/Momas5 double-knockout mutants were obtained by knocking out Mo MAS3 in the △Momas5 mutant background.Pathogenicity and phenotype analysis revealed that the△Momas3/Momas5 double deletion mutants had the same phenotype as the △Momas3 or △Momas5 single knockout mutants.3)Using real-time PCR to detect the expression patterns of Mo MAS3 and Mo MAS5 at different stages of M.oryzae,the results showed that both Mo MAS3 and Mo MAS5 had higher expression levels in the early stages of infection(18 and 24 hpi),followed by a dramatic decrease(42 hpi).The signal peptide activity of Mo Mas5 was tested using the yeast system,and the results showed that the signal peptides of Mo Mas5 had secreted activity.Localization of Mo Mas3 and Mo Mas5 from the spores to the germ tube,appressorium,and infection hyphae in the biotrophic stage was observed by fusing GFP protein at the Cterminus of these two proteins.The results showed that Mo Mas3 was localized in the septum and cytoplasm of the spores,as well as in the germ tube and hyphae.However,during the biotrophic phase,Mo Mas3 was localized in Extra-invasive hyphal membrane(Ei HM),completely outlining the infection hyphae.By contrast,the Mo Mas5 was localized to the appressorium and penetration pegs.The BFA treatment of Mo Mas3-infected leaf sheaths found that Mo Mas3 was inhibited to secrete into the apoplast space,resulting in the accumulation of green fluorescence inside the infectious hyphae.4)Analysis of rice leaf sheaths infected with △Momas3,△Momas5,and Guy11 showed that the invasive hyphae of the △Momas3 or △Momas5 mutants were restricted to the first invading cell,and black deposits were found around these restricted invasive hyphae within the infected cell.Using 3,3-diaminobenzidine(DAB)staining,ROS accumulation in rice cells infected with △Momas3 or △Momas5mutants were found;after(Diphenyleneiodonium Chloride)DPI treatment,it was able to inhibit the ROS accumulation in rice leaf sheath cells,restored the growth of infection hyphae and expanded to neighboring plant cells.The expression levels of plant disease resistance-related genes(PR1 and PBZ1)in △Momas3,△Momas5 mutants,wild-type strain Guy11 and complementary strains were detected by q RT-PCR,and it was found that △Momas3 and △Momas5 induced the expression of PR1 and PBZ1 genes in plants,thereby enhancing the resistance of host plants to the mutants.In a word,Mo MAS3 and Mo MAS5 not only participate in the pathogenic process of M.oryzae as two virulence factors but also inhibit the host-derived ROS and PR genes expression,thereby inhibiting the host’s innate immunity.