Biological Function Of RNA-dependent RNA Polymerase(RdRPs) Gene In Valsa Mali

Apple Valsa Canker is a major branch disease caused by the infection of Valsa mali(V.mali),which seriously affects the healthy and sustainable development of apple industry.Systematic analysis of pathogenic mechanism of pathogens is of great guiding significance for developing new strategies for disease control.RNA interference(RNAi)is a conserved gene silencing mechanism mediated by non-coding small RNA(s RNAs)in most eukaryotes,which is widely involved in biological processes such as vegetable growth,development of organisms and stress response.There are three conserved core components in RNAi pathway:Dicer,Argonaute and RNA-dependent RNA Polymerases(RdRP).Dicer and Argonaute genes have been analyzed in the previous research,and their key biological functions have been revealed,but RdRP is not clear yet.Therefore,through gene deletion technology combined with phenotypic analysis,this study systematically revealed the biological functions of RdRPs in pathogen pathogenesis.The specific results are as follows:RdRP genes(Vm RdRP1-4)were isolated from the whole genome of V.mali.Firstly,the expression pattern of Vm RdRP1-4 in infection process was detected by q RT-PCR,and it was found that the expression level of Vm RdRP1-4 was significantly up-regulated in the early infection,suggesting that Vm RdRPs might be involved in the infection process.On this basis,the single gene deletion mutants(ΔVm RdRP1-4)of Vm RdRPs were created by gene deletion technology.Phenotypic analysis showed that the vegetative growth and pathogenicity ofΔVm RdRP2 andΔVm RdRP3 were significantly reduced compared with the wild type,whileΔVm RdRP1 andΔVm RdRP4 had no significant changes.At the same time,the sensitivity ofΔVm RdRP1-4 to Na~+and K~+stress increased significantly,and the number of pycnidium decreased.After complement,vegetative growth,pathogenicity,abbiotic stress response and pycnidiums production were restored to the level of the wild type,indicating that Vm RdRPs contribute to these processes of V.mali,and there may be functional differentiation in vegetative growth and pathogenicity.Furthermore,based on the analysis of evolutionary relationship,we constructed the close homology of Vm RdRP1,Vm RdRP2,Vm RdRP3,Vm RdRP4 double gene deletion mutants(ΔVm RdRP1RdRP2 andΔVm RdRP3RdRP4).Compared with the wild type,the vegetative growth ofΔVm RdRP1RdRP2 was slightly reduced,but the pathogenicity was significantly reduced.The vegetative growth ofΔVm RdRP3RdRP4 was not significantly changed,but the pathogenicity was significantly decreased.Therefore,Vm RdRP1 and Vm RdRP2 may play a more important role in pathogen infection,and there may be functional redundancy in different members of the Vm RdRPs family.Furthermore,it was found that a single Vm RdRP gene deletion would lead to up-regulated expression of other Vm RdRPs,confirming the existence of certain functional redundancy.In order to explore the relationship between Vm RdRPs and s RNAs generation and amplification,s RNAs libraries ofΔVm RdRP2(MR2),ΔVm RdRP3(MR3)and the wild type(MVm)were constructed respectively.Based on s RNAs abundance analysis,it was found that the deletion of Vm RdRP3 increased the abundance of s RNAs,while the deletion of Vm RdRP2 reduced,and the changes were mainly reflected in the abundance of 20-21nt s RNAs.Further analysis showed that the deletion of Vm RdRPs did not affect the production of endogenous mil RNAs in V.mali,but played an important role in regulating the generation of si RNAs.The deletion of Vm RdRP2 resulted in ten differentially expressed si RNAs,among which seven were down-regulated and three up-regulated.The deletion of Vm RdRP3resulted in five differentially expressed si RNAs,of which three were specifically expressed and two down-regulated.Based on differential si RNAs,Vm RdRP2-associated Vm R2-si R1and Vm RdRP3-associated Vm R3-si R5 were selected for further functional studies.Based on expression pattern analysis,it was found that both Vm R2-si R1 and Vm R3-si R5 were up-regulated with in 6-24 h of pathogen infection,suggesting that they were involved in the pathogenic process.Furthermore,target mimic vectors of Vm R2-si R1 and Vm R3-si R5 were constructed for genetic transformation respectively,and silenced strains of Vm R2-si R1 and Vm R3-si R5 were obtained(STTM-Vm R2-si R1 and STTM-Vm R3-si R5).Compared with the wild type,there was no significant difference in the vegetative growth of STTM-Vm R2-si R1and STTM-Vm R3-si R5,but pathogenicity was significantly reduced,suggesting that Vm R2-si R1 and Vm R3-si R5 contributed in the infection and pathogenicity of V.mali.In conclusion,RdRP genes of V.mali were functionally analyzed in this study,and it was found that they are widely involved in biological processes such as vegetative growth,pathogenicity,abbiotic stress response and pycnidiums production.In particular,it was found for the first time that Vm RdRPs were involved in the production of si RNAs in V.mali,and Vm R2-si R1 and Vm R3-si R5 which related to Vm RdRPs played an important role in pathogen infection and pathogenicity.