The Study On The Pathogenicity Mediated By Dicer-like Of Penicillium Italicum

Citrus blue mold is one of the main diseases of post-harvest citrus,and its pathogenic strain is Penicillium italicum.SmallRNA(smallRNA,sRNA)is a non-codingRNA molecule that is cleaved from double-strandedRNA or single-strandedRNA with a stem-loop structure by Dicer and otherRNAIII enzymes to a length of 20～30 nucleotides.sRNA can regulate gene expression by mediating post-transcriptional level mRNA degradation or suppressing post-transcriptional translation of genes in a sequence-specific manner.As gene regulators,sRNAs in organisms play an important role in biological processes such as its growth and development,its biological and abiotic stress response.In this study,the function of the Dicer-like gene that induces sRNA production in P.italicum and its biological effects on targeting and regulating the immune-related gene in Citrus sinensis were verified experimentally,and the species and characteristics of P.italicum sRNA were revealed.Our research results have enriched the connotation of “ cross-kingdomRNAi”in plant pathogens,and these results were helpful to understand the molecular pathogenic mechanism of P.italicum deeply,all of which provided a certain theoretical basis for post-harvest preservation and breeding of citrus.In order to address the biological issu es such as the production and function of P.italicum sRNA,first of all,the P.italicum was used to infect citrus and was cultured in PDA plate,then the changes of the main components,related enzyme activities and gene expression levels of the peel cell wall of citrus fruit after infection of P.italicum were determined.At the same time,the expression of genes(DCL,AGO,Rdrp)related to the regulation of sRNA synthesis in P.italicum were also analyzed during infection process.Secondly,the gene silencing vectors for the DCL genes were constructed and transfected into P.italicum by protoplast transformation followed by the transformants obtained were screened and identified.The transformants mycelial and spore biomass and pathogenicity were determined.Finally,the wild-type and transformants were subjected to sRNA sequencing,which were analyzed by bioinformatics.The target gene of miRNA in the Valencia orange genome was predicted.The biological interactions between miRNAs and target genes were further verified by tobacco co-transfection assays.The main results of the study were as follows:(1)P.italicum infected citrus through modulating cell wall structure,and the cell wall degradation might be attributed to higher accumulation of ROS production caused by inhibited antioxidant activity and lower As A accumulation in citrus peel.Moreover,enhancement of enzymatic degradation of citrus peel cell wall was observed after P.italicum infection.Also,the higher expression of expansin together with lower expression of GLP1 also contributed to the non-enzymatic degradation of the cell wall in citrus peel.(2)Comparing to the P.italicum which inoculated on PDA plates,the expression levels of genes(DCL1,DCL2,AGO1,AGO2,AGO3,AGO4,and Rdrp1)associated with the regulation of synthesis sRNA were significantly up-regulated at 1～3 days after inoculation of citrus,among which the expression levels of DCL2,AGO3,AGO4 and Rdrp1 of P.italicum inoculated with citrus were significantly higher than that of PDA plate culture during the whole cultivation process.(3)After transfecting the RNAi vector of the DCL gene into the P.italicum via the method of protoplast transformation,the positive transformants p S-DCL1-3 and p S-DCL2-7 were identified and screened.Following by the measurement of the transformants biomass,it was found that the silencing of the DCL1 and DCL2 genes promoted the vegetative growth of P.italicum,but it had no significant effect on its reproductive growth.Therefore,it was indicated that the silence of DCL2 gene was closely related to the pathogenicity of P.italicum.Using spray-mediatedRNAi technology,a method was developed to directly us e exogenous ds DCL2 to mediate DCL2 gene silencing in P.italicum,which proved that exogenous dsRNA spray had the potential to be used in the prevention and treatment of postharves diseases of citrus.(4)RNA-seq sequencing of the sRNA were performed in the P.italicum wild type,as well as its DCL1 RNAi transformants and DCL2 RNAi transformants.The sequencing results were bioinformatically analyzed to predict a total of 12 novel microRNAs(miRNAs).The target genes of the novel miRNA were predicted.Howerve,there were only 4 novel miRNAs(Pit-novel1,Pit-novel7,Pit-novel11,and Pit-novel14)obtained with 5 potential target genes in the Valencia orange genome.(5)Stem-loop qPCR was used to verify the expression levels of 12 novel miRNAs predicted in P.italicum.It was found that among the DCL2 RNAi transformants,the expression level of remaining 11 novel miRNAs in DCL2 RNAi transformant were significantly lower than that of wild type while the expression level of novel2 miRNA were significantly higher than that of wild type.Furthermore,it was showed that the expression level of novel miRNAs in P.italicum had significant differences under the conditions of infecting citrus and plates.The expression levels of Pit-novel7 and Pit-novel14 were significantly up-regulated when P.italicum infected citrus,while the expression of their potential target genes was significantly reduced.The miRNA and its target genes were verified in vivo by co-transfecting tobacco with Agrobacterium-mediated method.The result was proved that Pit-novel7 targeted Valencia orange Cs7g19510 and Pit-novel14 targeted Valencia orange Cs7g09590.