Identification Of Mycelial Cell Wall Proteins And Functional Characterization Of A Splicing Factor MoSrp1 In Magnaporthe Oryzae

Cell wall is the outermost structure that play an important role in mediating fungal adhesion,phagocytosis and signal conduction.As a component of the cell wall,cell wall proteins（CWPs）may act as virulence factors of plant fungal pathogens to initiate infection of host plants.Identification of CWPs in Magnaporthe oryzae will not only help elucidate molecular mechanisms of the fungal pathogenesis,but also provide candidate targets for designing green agrochemicals.Although there are many studies on the identification and functional characterizations of genes that are important for the fungal pathogenesis,systemic studies are lacking on CWPs of M.oryzae.In this study,the author therefore isolated and identified CWPs in mycelia of M.oryzae,and functionally characterized some of the protein-encoding genes.With differential centrifugation,the author firstly obtained cell wall fractions from mycelia of two M.oryzae strains,the wild type P131 and a deletion mutant of ALG3 that encodes anα-1,3-mannosyltransferase involved in protein N-glycosylation.Then,proteins were subsequentially extracted from the mycelial cell wall fractions by using calcium chloride and lysing enzyme approaches,and were identified with LC-MS.The identified proteins were further filtered by predicted extracellular localization and the presence of signal peptides as candidate CWPs.With three duplicated experiments,the author eventually obtained 109 and 107 CWPs from the P131 and the Δalg3 strains,respectively.Interestingly,78 proteins were present in both the P131 and the Δalg3 strains,and therefore total 138 proteins were identified.These 138 CWPs were classified into 14 groups in accordance with their biological functions,including carbohydrate metabolic process,oxidation-reduction process,proteolysis,and response to stimulus.Among these overlapped proteins,four of the fiveβ-1,3-glucanosyltransferases encoded by the fungal genome were detected,except MGG₀3208 that was expressed in mycelial at a trace amount as confirmed by qRT-PCR,indicating that the approaches used in this study to identify CWPs is sensitive.However,31 proteins were found specifically present in the wild type P131,and 29 proteins were in the Δalg3 mutant.Of the 31 P131-specific proteins,29 were predicted to have N-glycosylation sites.Further,the author functionally characterized Cwg1 and Echt1,both of which are two CWPs expressed in mycelia of the two strains.Phenotype assays showed that deletion of CWG1 affected mycelial growth and virulence of the rice blast fungus,and that Echtl is likely a negative regulator of mycelial growth.The author also identified Lvh1,a levanse specifically detected in cell wall of P131,and found that it was important for the fungal conidiation.Notably,Lvhl could also be expressed,but its subcellular localization was no longer in cell wall of the Δalg3 mutant.Taken together,identification of cell wall proteins in this study provided important information for further investigation on their roles in asexual developments and pathogenesis of the rice blast fungus.In this study,the author also functionally characterized MoSRP1,which was previously identified as a novel gene important for colony growth of M.oryzae but whose molecular function is unknown.By assaying phenotypes of MoSRP1 deletion mutants including srplkol,the author found that MoSRP1 is important for the fungal virulence and the attenuated virulence in the mutants could be resulted from deficiency in invasive growth within host cells.The MoSRP1 deletion mutant,srp1kol expressing MoSrpl-eGFP,exhibited the wild type phenotypes and nuclear localization of the GFP signals,indicating that MoSrpl is localized in the nucleus.Moreover,MoSrpl interacted with MoRnps1,MoGrpl,and MoThoc1,These three proteins have an RNA binding domain and were revealed to localize to the nucleus.Notably,orthologues of the three proteins in the yeast are known to be involved in mRNA splicing.Therefore,MoSrpl is likely a splicing factor.In addition,the author generated a construct with deletion of the RRM domain and introduced it into srp1ko1.Phenotype assays showed that transformants with MoSrp1ΔRRM were similarly deficient as srp1ko1,indicating that the RRM domain is required for MoSrpl to be functiona1.Interestingly,the RRM domain was also found essential to the interactions of MoSrp1 with MoRnps1,MoGrp1,and MoThoc1.From previous mycelial phosphoproteomics analyses,117th,119th,and 193th serine were detected as phosphorylation sites.The author generated mutation of these sites and found that they can affect colony growth and pathogenicity to some extent.Importantly,the author found that MoSrpl could have capability to bind "GUAG" motif in mRNA with the RIP-seq followed by RNA-EMSA confirmation.Since orthologue of MoSrpl is absent from the budding yeast but present in the fission yeast,the author proposed that MoSrp1 may play an important role in alternative splicing,and therefore compared mycelial RNA-seq data from srp1kol with that of the wild type P131.The analyses showed that splicing efficiency of 2571 introns from 1750 genes was evidently reduced in srp1ko1.Among the 2571 introns with reduced splicing efficiency,there are 996 introns with the "GUAG" motif.Moreover,29 introns from 23 putative splicing factors are found in the 2571 introns.RT-PCR confirmed that intron splicing efficiency of MoATF1 and MoBRE1 were reduced in srp1kol compared to the P131.The two genes have "GUAG" motif in their introns and exons,suggesting that MoATF1 and MoBRE1 may be direct targets of MoSrp1.In addition,the author found that splicing of many introns without the "GUAG"motif was also affected in srp1kol,including those in MoMBF1,MoPUP1,and MoSKP1.At the same time,365 predicted nuclear protein-encoding genes were significantly down regulated in srp1ko1 compared with the wild type P131,including 24 putative splicing factors.Taken together,these data suggest that MoSrpl may regulate splicing efficiency of introns without the "GUAG" motif through regulating expression or splicing of other splicing factors.Further investigation will be required to clarify this conclusion.