Biological Function Analysis Of Phosphatidylcholine Biosynthesis-Related Genes In Magnaporthe Oryzae

Phosphatidylcholine（PC）is the most abundant phospholipid in eukaryotic cell membrane system.It plays an important role in maintaining the integrity and function of cell membrane,and participates in cell growth and differentiation,metabolism,signal recognition and transduction,autophagy,cell cycle and other important progress.There are two main pathways for PC synthesis:phosphatidylethanolamine（PE）methylation pathway and CDP-choline（CDP-Choline）pathway（also known as Kennedy pathway）.So far,PC synthesis pathways have been well investigated in the budding yeast Saccharomyces cerevisiae,but not in plant pathogenic fungi.Rice blast caused by Magnaporthe oryzae is one of the most serious diseases in rice caltivation.Understanding the molecular disease mechanism of M.oryzae is not only conducive to the prevention and control of rice blast,but also has important reference significance for understanding the pathogenesis of other plant pathogenic fungi.In this paper,the biological functions of methyltransferase coding genes MoCHO2 and MoOPI3 of PE methylation pathway,the key rate-limiting enzyme coding gene MoPCT1 of CDP-Cho pathway and the tanscription factor of phospholipid metabolism MoOPI1 were studied.The main results are as follows:1.PE methylation pathway is important for the growth and sporulation of M.oryzae,and can block disease cycle by affecting sporulation.1)MoCHO2 and MoOPI3 are essential for the growth and sporulation of M.oryzae.The radial growth ofΔMocho2 andΔMoopi3 mutants on CM medium was similar to that of the wild-strain Guy11 and complemented strains,but both of the mutants could hardly grow on MM medium which has no exogenous choline.In addition,few aerial mycelia and spores were produced on colony surface of theΔMocho2 andΔMoopi3 mutants.2)MoCHO2 and MoOPI3 have no effect on pathogenicity of conidia.By spray-inoculation with conidia（induced by adding PC）suspensions,bothΔMocho2 andΔMoopi3 mutants were full pathogenic to rice seedlings,respectively.However,when inoculated with the mycelia blocks,bothΔMocho2 andΔMoopi3 were nonpathogenic,indicating that the deletion of MoCHO2or MoOPI3 has no effect on pathogenicity of conidia,but could block disease cycle by affecting conidia formation in M.oryzae.3)The blockage of PE methylation pathway does not affect PC content under nutrient-rich conditions in M.oryzae.ΔMocho2andΔMoopi3 mutants showed limited phenotypic recovery by exogenous PC addition in CM medium,and the results of ELISA showed that the mutants had no significant difference in PC content compared to the wild-type strain under CM condition.4)MoOpi3 acts downstream of Mo Cho2 in PE methylation pathway.Exogenous addition of phosphatidyl-mono-methylethanolamine（PMME）,which is the first step product of PE methylation pathway,restored the growth ofΔMocho2,but notΔMoopi3on MM.In addition,overexpression of MoOPI3 inΔMocho2 partially recovered phenotypic defects ofΔMocho2,suggesting that MoOpi3 acts downstream of Mo Cho2.2.MoPCT1,a key rate-limiting enzyme encoding gene of CDP-Cho pathway,plays an important role in vegetative growth,sporulation,turgor accumulation and pathogenicity of M.oryzae and is involved in autophagy and histone H3methylation modification.1)MoPct1 plays an important role in vegetative growth and sporulation of M.oryzae.Compared with Guy11,vegetative growth and sporulation ofΔMopct1decreased significantly either on CM medium or on MM medium.2)MoPct1 is necessary for full pathogenicity of M.oryzae.The pathogenicity ofΔMopct1 to both barley and rice was reduced notably,and evenΔMopct1 could not infect wounded barley leaves.3)MoPct1 is associated with turgor accumulation of appressorium in M.oryzae.The proportion of collapsed appressoria inΔMopct1 was dramatically increased than that in the wild-type Guy11 under 1M glycerol,suggesting that deletion of MoPCT1 impairs appressorium turgor accumulation in M.oryzae.4)MoPct1 is localized to the nucleus.We observed under a fluorescence microscopy that MoPct1-GFP fusion protein was localized to nucleus.Furthermore,by deletion of nuclear localization signal（NLS）of MoPct1,GFP fluorescence signal of MoPct1^ΔNLS-GFP mutant was dispersed in cell cytoplasm,while the mutant was full pathogenic to host.5)MoPct1 is involved in regulation of autophagy.Compared with Guy11,the level of autophagy was increased inΔMopct1 under nutrition-rich conditions and consistently the expression of autophagy-related genes was also up-regulated,suggesting that Mopct1 negatively regulated autophagy of M.oryzae.6)Exogenous PC can not recover phenotypic defects ofΔMopct1,and PE methylation pathway may play a compensating role inΔMopct1.The phenotypic defects ofΔMopct1 could not be recovered by exogenous PC or CDP-Cho,suggesting that these phenotypic defects ofΔMopct1 may not be caused by lack of PC.ELISA assay showed the content of PC inΔMopct1 was even higher than that in the wild-type Guy11.Furthermore,q RT-PCR results showed that deletion of MoPCT1 significantly increased the expression levels of PE methylation pathway related genes MoCHO2 and MoOPI3 as well as upstream PE synthesis related gene MoPSD2,suggesting that the mutant may compensate for CDP-Cho pathway through activation of PE methylation pathway.7)MoPct1 is involved in methionine metabolic cycle,histone H3 methylation and transcriptional regulation of several key pathogenicity-related genes.The results of q RT-PCR assays showed that deletion of MoPCT1 resulted in higher expression of genes related to methionine metabolic cycle.Western blot shows that methylation level of histone H3 was increased,and the expression levels of transcriptome inΔMopct1were changed,especially several key pathogenicity-related genes in M.oryzae.3.MoOpi1 negatively regulates the expression of genes related to phospholipid metabolism and plays an important role in sporulation,appressorium formation and pathogenicity of M.oryzae.1)MoOPI1 plays an important role in sporulation and spore morphogenesis of M.oryzae.Compared with the wild-type strain,conidiation ofΔMoopi1 decreased significantly,and the proportion of malformed spores was higher.2)Deletion of MoOPI1 affects pathogenicity of M.oryzae.The pathogenicity ofΔMoopi1 decreased significantly,and the mutant was unable to recover infection on wounded barley leaves.3)MoOPI1 plays an important role in conidia germination,appressorium formation and turgor accumulation of M.oryzae.The spore germination rate,appressorium formation rate and appressorium turgor pressure ofΔMoopi1 were decreased significantly.4)MoOPI1 is associated with the response to oxidative and osmotic stress in M.oryzae.The sensitivity to oxidative stress ofΔMoopi1 was decreased,on the contrary,the sensitivity to high osmotic stress was increased.5)MoOPI1 is involved in multiple pathways related to fungal development and pathogenicity in M.oryzae.Transcriptome analysis ofΔMoopi1 showed that MoOPI1was associated with various biological processes such as sterol metabolism,peroxisome and fatty acid metabolism,oxidoreductase activity,glycerol metabolism and cell wall degradation.6)MoOPI1 negatively regulates the expression of phospholipid metabolism-related genes.We found that many genes related to phospholipid metabolism such as MoCHO2,MoOPI3,Mo EKI1,Mo EPT1 and Mo CPT1 were up-regulated inΔMoopi1.