Cloning And Functional Analysis Of Gene PlBZP32 In Peronophythora Litchii Pathogenesis

Litchii Downy Blight caused by Peronophythora litchii is a common occurrence of oomycete disease in litchi producing areas,and affects the yield and quality of litchi fruit.At present,there are few molecularstudies on the pathogenicity of Peronophythora litchii.Understanding the pathogenesis of the fungus at the molecularlevel could provide a theoretical basis for development of new control strategies.In this study,foreign-derived double-stranded RN A(ds RNA)was integrated into the genome of Peronophythora litchii by polyethylene glycol(PEG)-mediated protoplast transformation.Gene silencing was archieved and functional analysis to study the fungi-related genes and their effects on growth and pathogenicity was conducted and the main results are as follows:The wild-type strains of Peronophythora litchii were transferred to different concentrations of geneticin in solid carrot medium.According to their growth,primary and second screening concentrations were 10 ?g/mL and 30 ?g/mL,respectively.A gene with a transcription factor BZPc32 homologous to the Phytophthora sojae bZIP-PAS structure was identified as a gene named Peronophythora litchii BZP32(Pl BZP32)by comparing the biological information of the whole genome of Peronophythora litchi.The results showed that the transcription factors of b ZIP-PAS structure were only in the lower fungal organism such as oomycetes and algae and phylogenetic analysis was carried out by bioinformatics method.The Pl BZP32 gene was constructed in the p TOR::mRFP as the vector,and transformed with PEG-mediated protoplasts to obtain the trans formants with red fluorescence.The mycelia and sporangia were stained with DAPI,which was excited with ultraviolet light and green light.It was found that the nucleus position indicated by blue fluorescence may overlapped with the strong region of red fluorescence protein(RFP),which showed that the gene was located in the nucleus.The Pl BZP32 gene was amplified by PCR and then constructed onto pTO R::mRFP vector.The recombinant vector was transferred into protoplasts by PEG method.The silencing efficiencies of three transformants T21,T15 and T35 were 30.8%,35.9% and 38.9%,respectively,by PC R and qRT-PCR.Compared with the wild type SHS3,the silencing of the Pl BZP32 gene did not affect the growth rate of the colonies,but the cysts increased and the internode became shorter and the sporangia production increased significantly.The germination rate of the spore suspension was significantly decreased.The sensitivity toCalcoflour white and Congo red showed that the sensitivity of Pl BZP32 silenced transformants to their conventional concentrations was not significantly different from that of wild type.However,the sensitivity of the silenced transformants was enhanced when Sodium dodecyl sulfate(SDS)was added into plichs plates.The results showed that the transcriptional level of Pl BZP32 did not changed with the oxidative stress,but the sensitivity of Pl BZP32 to the oxidative stress was enhanced.The results of plate assay showed that the activities of extracellularperoxidase and laccase of PlBZP32 silenced transformants were lower than that of wild type.The results of the inoculation of litchi leaves in vitro showed that the lesions caused by PlBZP32 silenced transformants were significantly smaller than that of wild type,indicating that their pathogenicity was reduced.