Functional Characterization Of Multiple Pathogenesis-related Signalings Mediated By ERD2 Orthologue Of Magnaporthe Oryzae

ERD2(ER-retention defective)has been found to be a transmembrane protein which is evolutionarily conserved in eukaryotic organism.This protein can recognize the carboxyl-terminal H/KDEL motif and anterograde H/KDEL-tagged proteins back into ER.ERD2 was originally identified in S.cerevisiae.Although the biological function and regulatory mechanism of ERD2 homologous proteins in animal cells have been studied in depth,the underlying mechanism in pathogenetic organism,especially in plant pathogen fungal is still unrevealed.Recently,a research team in South Korea reported that the ERD2 ortholog in rice blast plays important roles in mycelial growth,conidial production,and pathogenicity.In the laboratory previous work,one mutant with obvious defects in mycelium growth and conidial production was obtained by screening the insertional mutant library of Magnaporthe oryzae.Further analysis confirmed the T-DNA insertion site was located in the promoter region of reported ERD2 gene of M.oryzae,so we named the disrupted gene as MoErd2 and carried out knockout analysis for this gene subsequently.To our surprise,enlarged vacuoles with some contents in MoErd2 deletion mutant were observed by transmission electron microscopy and eighteen candidates which may interact with MoErd2 were obtained by split-ubiquitin yeast two hybridization.Based on previous work,in-depth research about the functional mechanism of MoErd2 was performed and the following results were obtained.Proteins MoErd2 and yeast ERD2 are highly conserved in amino acid sequence,so we speculate that MoErd2 and ERD2 may have similar functions.Therefore,MoErd2 was introduced into S.cerevisiae erd2 mutant and results indicated that MoErd2 could rescue the phenotypic defect of erd2 mutant.To investigate the regulatory mechanism,we engineered the MoErd2-GFP fusion construct and introduced it into MoErd2 deletion mutant.It was found that the MoErd2-GFP driven by its own native promoter could complement the phenotypic defect of MoErd2 knockout mutant.Microscopic observations showed that the fusion protein MoErd2-GFP was co-located in the endoplasmic reticulum(ER)with mCherry-HDEL which has been widely used as an ER localization marker.It was interesting to found proteins tagged with HDEL motif at its carboxyl terminus was located in vacuole rather than in ER in MoErd2 deletion mutant.In combination with the results of transmission electron microscopy,we assume that MoErd2 might be involved in the regulation of autophagy.So,we comparatively analyzed the localization and accumulation of autophagy marker protein GFP-MoATG8 in wild-type and MoErd2 knockout mutant.The results showed that under well-fed condition,GFP-MoATG8 in MoErd2 null mutant was accumulated around vacuole;under nutrition-lacking state,GFP-MoATG8 in MoErd2 knockout mutant could not located into vacuolar.At the same time,under normal culture conditions,GFP-MoATG8 was significantly decomposed in MoErd2 knockout mutant.These results suggested that MoErd2 participated in the regulation of autophagy in M oryzae.To reveal the functional mechanism of MoErd2 in M.oryzae,we carried out a follow-up study on the function of some proteins which was isolated in previous work and possibly interacted with MoErd2.The direct interaction between MoErd2 and Mps1 was established by split-ubiquitin yeast two-hybrid and co-immunoprecipitation experiments.In this study,It was found that the Mps1-GFP driven by its own native promoter could complement the phenotypic defect of Mps1 knockout mutant.Microscopic observation shown that Mps1 located in ER.At the same time,the Mps1-GFP was introduced into the MoErd2 knockout mutant,and it was striking to found that Mps1 was located in the vacuole,which indicated that the localization of Mps1 was dependent on MoErd2.Besides,the amount of phosphorylated Mps1 was evidently decreased in MoErd2 deletion mutant in comparison with that in wild-type under MM-N cultural condition.Furthermore,the degradation level of GFP-MoATG8 in Mps1 knockout was significantly higher than that in wild-type.Therefore,we speculated that Mps1 was a negative regulator of autophagy.Upon the results stated above,it suggested MoErd2 regulated autophagy through mediating the ER localization and phosphorylation of Mps1.To explore the effect of phosphorylation of Mps1 on biological function of M.oryzae,we performed mutation analysis for the key phosphorylation sites of Mps1.The results indicated that mutation of the phosphorylation site of Mps1 resulted in significantly decreased conidiation and seriously reduced pathogenicity.Furthermore,it was found that mutation of the phosphorylation site of Mps1 inhibited the expansion of infectious hyphae inside barley epidermal cells.Upon these results,it was indicated that phosphorylation of Mps1 affected the conidiation and pathogenicity of M oryzae.Moreover,another two MoErd2-interacting proteins MEI1 and DCR1 were also investigated.Co-localization observation and split-ubiquitin yeast two-hybrid experiments verified that both DCR1 and MEI1 located in ER and interacted with MoErd2,respectively.Further studies showed that the localization of DCR1 and MEI1 was also dependent on MoErd2.Taken together,it is a pioneering work to find that MoErd2 mediate the ER localization of many proteins without typical ER retention signal,and that these proteins play important roles in pathogenicity by regulating the autophagy.At the same time,we also discover that MoErd2 mediate several pathogenicity-related signaling pathways.These results provide a new perspective for us to understand the functions of RED2 and other ER proteins.Additionally,this study also provides many novel ideals for one to uncover the pathogenic mechanism,especially the secretory mechanism of effector protein.