Metabolomics On Appressorium Of Magnaporthe Oryzae And Functional Analysis Of Sphingolipid Pathway During Appressorium Development And Pathogenesis

Magnaporthe oryzae is the pathogenic fungi causing rice blast,which seriously threatens global rice yield and food security.M.oryzae has a typical infection cycle.It spreads disease by producing conidia.Conidia differentiate under suitable conditions to form heavily melanized,dome-shaped infection structure known as the appressorium,which forcibly ruptures the cuticle to enter the rice leaf tissue.The appressorium penetrates the host epidermis by generating enormous turgor pressure.Glycerol is known to be the main source of turgor pressure.However,how glycerol is produced,whether glycerol is the only substance that produces turgor,and the formation and maturation of appressorium are still not fully understood.Here,we used untargeted metabolomics analyses to profile the metabolome of developing appressoria.Based on the establishment of appressorium metabolites research system and databases,53 differential metabolites between conidia and 24-hour appressorium and significant changes in six key metabolic pathways including degradation of lipids,degradation of carbohydrates,arginine synthesis,sphingolipid synthesis,sterol synthesis,and phospholipid metabolism were identified.Early studies have shown that these six pathways are involved in important insights of appressorium such as energy provision,redox balance,signaling pathway messenger,and protection during infection.After discussion of the biological functions of these six metabolic pathways studied till now,we focused on the role of ceramide synthesis pathway during appressorium formation and infection.Small molecule inhibitors,genetic analysis and chemical complementary analysis have proved that ceramide is necessary for the normal development and pathogenesis of appressorium.The absence of ceramide resulted in appressorium morphological changes,decreased turgor pressure,hindered the formation of penetration pegs,and finally led to the loss of pathogenicity of rice blast.We further studied the molecular mechanism of ceramide during fungal infection,the experimental results proved that lack of ceramide hindered the energy utilization of glycogen and lipid drops,ceramide are also proved to regulate cell cycle and cytoskeleton remodeling during appressorium development,thereby regulate the PKC-phosphorylation level of CWI signal pathway.Ceramides are the precursors of glucosylceramide and inositol phosphorylceramide which are the end products of late sphingolipid biosynthesis,in order to specify the role of end products of sphingolipids pathway,lipidomic analysis were performed on ceramide knockout mutant ?Molag1.Analysis results show that the MoLAG1 produce complex sphingolipid glucosylceramide.We further demonstrated the importance of glucosylceramide during the pathogenic process of rice blast by means of genetic analysis.Glucosylceramide synthases knockout mutant ?Mocgt1 showed appressorium morphological defects,reduced turgor pressure,and loss of pathogenicity.Overall,our untargeted analysis of metabolomics throughout the course of pathogenic development gave us an unprecedented high-resolution view of major shifts in metabolism that occur in the topmost fungal pathogen that infects rice,wheat,barley,and millet.Guided by these metabolic insights,we demonstrated their practical application by using two different small-molecule inhibitors of sphingolipid biosynthesis enzymes to successfully block the pathogenicity of M.oryzae.Our study thus defines the sphingolipid biosynthesis pathway as a key step and potential target that can be exploited for the development of antifungal agents.Furthermore,future investigations that exploit such important metabolic intermediates will further deepen our basic understanding of the molecular mechanisms underlying the establishment of fungal blast disease in important cereal crops.