| How pathogens mediate host metabolism and inhibit plant defense responses through effector proteins has been a research foucus for understanding the plant-pathogen interaction.The most studied effector proteins of plant pathogens have N-terminal signal peptides,which are recognized as classically secreted proteins.And increasing evidence have showed that non-classically secreted proteins without signal peptides can also be transported to the extracellular space,and play an essential role in the interaction between the pathogen and its host.However,studies on the function of non-classically secreted effector proteins are relatively poor and backward.Powdery mildew disease of rubber tree(Hevea brasiliensis Muell)is one of the main leaf diseases of rubber tree,which is one of major factor affecting the yield of natural rubber.The pathogen of this disease is Erysiphe quercicola,an obligate obligate biotrophic fungus,which cannot be cultured in vitro.Therefore,it has not yet established an efficient and stable genetic transformation and host-mediated gene silencing system,leading the current understanding of the molecular pathogenesis of E.quercicola remains rather limited.To provide new ideas for understanding the pathogenic mechanism of obligate fungi,we aim to conduct an in-depth study of the function of non-classically secreted proteins of powdery mildew fungi in this study.Two recognized key enzymes in the phenylpyruvate pathway of fungi and plants are Chorismate mutase(Cmu)and Prephenate dehydratase(PDT).PDT catalyzes the synthesis of prephenate from Cmu catalyzed chorismate.It has been reported that the pathogen can secrete classically secreted Cmu and target the the chorismate in the phenylpyruvate pathway,thereby inhibiting plant salicylic acid(SA)synthesis,but no relevant reports have been reported on the inhibition of SA synthesis by PDT.In this study,the non-classically secreted Cmu and PDT genes with potential functions were first screened out through functional annotation of the classically and non-classically secreted proteins of four species of powdery mildew fungi.For easy-to-follow,the Cmu and PDT genes from E.quercicola were named as EqCmu and EqPDT in the following context.Then,the function of EqCmu and EqPDT were studied via enzymatic assays,protein secretion assays,phylogenetic analysis,genetic complementation analysis,temporal expression assays,subcellular localization analysis,and agrobacterium-mediated tobacco transient expression.The main results are summarized as follows:1.The study confirmed that the rubber powdery mildew fungus strain OH-73 belongs to E.quercicola,which is consistent with the reported classification through the pathogenicity determination,morphological observation,and molecular identification.After detailed and accurate observation of the infection process,the infection process of the strain OH-73 could be subdivided into six main stages: conidia germination(3-4 hpi),appressoria formation(7-8 hpi),primary infection(12-13 hpi),haustorial formation(13-24 hpi),secondary infection(24 hpi),and conidia formation(5-7 dpi).2.According to the the whole genome data of four strains of powdery mildew fungi,E.quercicola,Blumeria graminis f.sp.hordei,Blumeria graminis f.sp.tritici and Erysiphe necator,the classically and non-classically secreted proteins that they carried were predicted.The results showed that there are a large amount of non-classically secreted proteins in the genome of each powdery mildew fungi.For example,E.quercicola has 2,177 non-classically secreted proteins,accounting for 34.14% of the total protein;B.graminis f.sp.hordei has 2,110 non-classically secreted proteins,accounting for 29.64% of the total protein;B.graminis f.sp.tritici has 2,196 non-classically secreted proteins,accounting for 33.66% of the total protein;E.necator has 2,276non-classically secreted proteins,accounting for the total protein of 35.10%.The numbers of non-classically secreted proteins carried by each of fungi were two-five times larger than its relative classically secreted proteins.The results suggested that the non-classical secretion pathway might be an important pathway for protein secretion in powdery mildew fungi.3.Functional studies of EqCmu from E.quercicola.By analyzing the annotation results of the classically and non-classically secreted proteins of the above-mentioned four powdery mildew fungi,the classical secreting type Cmu was not found,but existed in the form of non-classical secreting type.It was confirmed that there is only one gene EqCmu encoding Cmu in the genome of E.quercicola.Despite the lack of a putatively signal peptide,EqCmu can transport the invertase which is missing the signal peptide to the extracellular,which is not agree with that Cmu is a classical secreted in previously reports.This is the first time that we found EqCmu harboring the characteristics of a non-classically secreted protein.Furthermore,our results showed that EqCmu could substitute for the cytoplasmic chorismate mutase of Saccharomyces cerevisiae through its Cmu enzyme activity,and could synthesize phenylalanine and tyrosine in the fungal cytoplasm.EqCmu can accumulate during primary infection,secondary infection and haustorial formation that were detected during interaction with the host.In addition,EqCmu was found to act on plant cytoplasm.Morever,the transient expression of EqCmu in N.benthamiana leaves tissues can promote infection by P.capsici,and suppress the synthesis of plant SA levels and the expression of PR1 gene induced by SA-mediated defense,thus promotes the infection of P.capsici.Furthermore,its enzyme activity plays a decisive role in the function of EqCmu in plant cytoplasm.Overall,EqCmu may have an action mechanism similar to that of classically secreted Cmu-targeting the plant cytoplasmic phenylpyruvate pathway and competing for the synthesis precursor of plant SA by catalyzing the cytoplasmic chorismate – transport from plant plastid chorismate,thereby inhibiting the synthesis of plant SA.4.Functional studies of EqPDT from E.quercicola.Similarily to the EqCmu,we found that PDT also existed in the form of non-classically secreted protein in the genomes of the four powdery mildew fungi detected above.Further,it was confirmed that E.quercicola EqPDT has the characteristics of a non-classically secreted protein.EqPDT can catalyze the synthesis of phenylpyruvate from prephenate in vitro and replace the cytoplasmic prephenate dehydratase from S.cerevisiae cytoplasmic PDT to synthesize phenylalanine through its PDT enzyme activity,suggesting that EqPDT has the function of synthesizing phenylalanine in the fungal cytoplasm.EqPDT mainly accumulates in the stages of secondary infection and haustorial formation that were observed during interaction with the host.EqPDT was detected to act on the cytoplasm of plant.The transient expression of EqPDT in N.benthamiana leaves tissues can suppress the synthesis of plant SA and the expression of PR1 gene induced by P.capsici infection via its PDT enzyme activity,thus promoting the infection of P.capsici.These results suggested that EqPDT may have a mechanism similar to EqCmu,which can target the plant cytoplasmic phenylpyruvate pathway and compete for the synthetic precursor of plant SA by catalyzing the plant cytoplasmic prephenate,and thereby inhibiting the synthesis of plant SA. |
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