The O-glucosyltransferase Oglutl Is Critical For Infection Of The Rice Blast Fungus Magaraporthe Oryzae

Rice(Oryza sativa)is considered one of the world’s most valuable crops and has been grown worldwide in recent years.Magnaporthe oryzae,which is commonly known as rice blast fungi,is amongst the most severe problems in rice cultivation due to its high rate of infectivity and because of that,each year triggers a substantial amount of losses in rice yield.For a long time now M.oryzae is been used for the research purposes in terms of fungal-plant associations as a model fungus.So due to its economic importance,we need to identify the infection mechanism in this fungus to seek more suitable ways to address and overcome this problem.Protein O-glucosylation is a necessary form of O-glycosylation(a post-translation modification),which involves glucose(Glc)addition to a serine residue within a consensus sequence C1-X-S-X-P-C2 of epidermal growth factor(EGF)-like repeats found in several proteins.The growth and differentiation of eukaryotic cells are influenced by the protein Oglucosylation.This type of modification by O-Glut1 can regulate the degradation level of other proteins that are the targets of O-Glut1 in the endoplasmic reticulum localities and can affect their function.We found that O-glucosylation roles in plant pathogenic fungi are not well addressed in particular during the process of a fungal infection.In this study,we investigated the significance of the protein O-glucosylation in M.oryzae through functional analysis of the O-glucosyltransferase gene,OGLUT1.OGLUT1 is high-level expressed during appressorium formation,but low-level expressed during primary infection hyphae stage.Oglut1 protein is located in ER.By the targeted disruption of OGLUT1 gene,it was observed that its function was dispensable in terms of hyphal growth but was resulted in a drastic reduction in sporulation with abnormal conidial shape,and also a significant decrease in sensitivity to the oxidative stress was noticed.Most importantly,due to the loss of OGLUT1 virulence in the host plant was significantly reduced,which was due to the reasons that decreased in appressorium formation and invasive growth was observed in the Δoglut1.The Δoglut1 mutant is significantly reduced in appressorium formation.The ability of glycogen and lipid utilization was decreased in Δoglut1,indicating a role of OGLUT1 in functional appressorium formation.Inoculation of host plants with OGLUT1 knockout strain can induce rice reactive oxygen species(ROS)accumulation and defense gene expression,suggesting that OGLUT1 was involved in the interaction between M.oryzae and the host,including in ROS detoxification.Accordingly,the Δ oglut1 is less sensitive to different stresses,including H2O2.Dithiothreitol(DTT),an ER stress inducer,significantly reduced the conidiation and conidiophore formation of the OGLUT1 knockout mutants,it also evidently suppressed the appressorium formation and formation of invasive hyphae of the mutants,suggesting OGLUT1 is responsible for ER stress response during development and infection.Previous studies showed us that,in humans and Drosophila melanogaster,homologs of OGLUT1 modify targets at a consensus sequence C1-X-S-X-P-C2 in the EGF repeat domain.Here,in M.oryzae,we identified two candidate Oglut1 target proteins,Fbxl5 and Ddp1.Interestingly,both targets contain the C1-X-S-X-P-C2 binding sites,but do not contain an EGF repeats domain.This finding overturns previous reports that the target protein must contain an EGF repeat region.Western blotting analysis indicated that both target proteins were increased in protein levels and unknown modification levels,suggesting that O-glucosylation is interactive with other modifications.In sum,we found and predict that Oglut1-mediated O-glucosylation play key roles in M.oryze development,stress response and infection processes,and we also identified two important targets of O-glucosylation,which builds a foundation for the future regulatory mechanism study of this novel modification.