Identification Of The Effector Proteins Of Penicillium Italicum And Functional Analysis Of PiWSC1

Blue mold of citrus caused by Penicillium italicum is one of the most important postharvest diseases of citrus fruits.The interactions of pathogens with their hosts are complicated,and virulence factors that mediate pathogenicity have not yet been identified.Effector proteins are a class of proteins secreted by pathogens that can enhance their virulence during hosts infection and play essential role in the interaction between plants and pathogens.In this paper,based on the published whole genomic information of P.italicum,effector proteins were screened and identified by bioinformatics and RT-q PCR analysis.By analyzing the structural changes,growth characteristics and expression patterns of interaction with host during infection of core candidate effector gene Piwsc1 deletion mutants,the interaction mechanism of P.italicum effector proteins affecting citrus fruits infection was explored at the molecular level.The main results are as follows:1.Combined with common effector proteins characteristics and transcriptomic data from P.italicum-infected Navel orange tissue at 72 h,a prediction pipeline was designed to determine the repertoire of P.italicum candidate effector proteins.Three hundred and seventy-five secreted proteins of P.italicum were identified by using Signal P,Target P,TMHMM,Pred GPI and other softwares,some of which(29.07%)were enzymes for carbohydrate utilization and might be involved in the interaction between the pathogen and its host.Twenty-nine candidate effector proteins were further screened,and five out of them were core effectors that met four requirements of the criteria designed in the pipeline.Twelve randomly selected candidate effector genes were actively expressed during the early stages of growth on PDA and infection on Navel oranges.2.One of the five core candidate effectors,cell wall integrity and stress response component(WSC)related protein Pi WSC1,was characterized and functionally analyzed.Phylogenetic analysis revealed that Pi WSC1 was highly conserved in different Penicillium species and other fungi genera.The transcriptional level of Piwsc1 was down-regulated to varying degrees under high osmotic pressure,cell wall perturbing agents,oxidation and heat stress.Based on the principle of homologous recombination,the knockout strainΔPiwsc1,complementation strain ΔPiwsc1-co and overexpression strain OE-Piwsc1 of Piwsc1 gene were constructed.The micromorphology,mycelial growth,sporulation,environmental stress and pathogenicity of the mutants of Piwsc1 gene were studied.Compared with wild type(WT)P.italicum,the colony growth diameter of ΔPiwsc1decreased by 20.15% at 7 days post-inoculation(dpi),the colony morphology showed sparse hyphae,and the internal structure changed;the colony growth diameter of OEPiwsc1 increased by 8.22%,and the phenotype was not significantly difference compared with WT.The spore production of ΔPiwsc1 decreased by 35.31% and OE-Piwsc1 increased by 38.76%.The spore germination rate of ΔPiwsc1 decreased by 22.27% and OE-Piwsc1 increased by 16.08%.Piwsc1 can play a certain role in response to some environmental stresses.In addition,compared with WT,the deletion of Piwsc1 resulted in reduced virulence on citrus fruits,as presented by an approximate 50% reduction in the diameter of lesions.Cell wall degrading enzymes activity may be the reason for the decrease ofΔPiwsc1 virulence in the early stage of infection citrus fruits.3.Effect of effector protein Pi WSC1 on the interaction between P.italicum and citrus fruits by dual RNA-seq analysis.P.italicum differentially expressed genes are mainly related to cell membrane,active oxygen metabolism,autophagy and death and MAPK signal transduction pathway,while citrus fruits differentially expressed genes are mainly related to stress response and secondary metabolites.Therefore,the deletion of Piwsc1 may destroy the cell structure of P.italicum,hinder its own growth and induce plant defense responses such as the production of reactive oxygen species(ROS)to limit the growth of P.italicum and up-regulate the transcription level of secondary metabolite phenylpropane,so as to enhance the defense system of citrus fruits.In conclusion,a total of 29 candidate effector proteins were screened in P.italicum,of which one core effector protein Pi WSC1 not only affected the growth,development and virulence of P.italicum,but also specifically expressed by P.italicum during the early stages of growth on PDA and infection of Navel oranges,and was recognized by citrus fruits to cause defense response.