Protoplast Transformation And Functional Analysis Of FreB Gene In Verticillium Dahliae

Verticillium dahliae is a soil-borne fungal pathogen,causing vascular wilt disease in a number of economically important crops.No treatments are available once the plants are infected by this fungus.Large efforts have focused on screening for genes involved in the virulence and pathogenicity of V.dahliae.Although Agrobacterium tumefaciens-mediated transformation?ATMT?has been widely used for gene screening,a quick and easy method has been needed to facilitate transformation.In this study,we isolated and transformed the protoplasts from V.dahliae in order to build an efficient transformation system.We also explored the role of FreB gene?VDAG₀6616?in the ferric reduction and virulence of V.dahliae by generating gene deletion and complementary mutants.Results are listed below in details:1.High-quality protoplasts,with excellent regeneration efficiency?65%?in TB3 broth,were generated using driselase?Sigma D-9515?and transformed with the GFP plasmid or linear GFP cassette using PEG or electroporation.PEG-mediated transformation yielded 600 transformants per microgram DNA for the linear GFP cassette and 250 for the GFP plasmid;electroporation resulted in 29transformants per microgram DNA for the linear GFP cassette and 24 for the GFP plasmid.2.To determine whether short interfering RNAs?siRNAs?can be delivered to the protoplasts and used for silencing genes,we targeted the GFP gene of Vd-GFP?V.dahliae GFP strain obtained in this study?by delivering one of four different siRNAs,19-nt duplex with 2-nt 3?overhangs?siRNA-gfp1,siRNA-gfp2,siRNA-gfp3 and siRNA-gfp4?,into the Vd-GFP protoplasts using PEG-mediated transformation.Up to 100%silencing of GFP was obtained with siRNA-gfp4;the other siRNAs were less effective?up to 10%silencing?.Verticillium transcription activator of adhesion?Vta2?gene of V.dahliae was also silenced with four siRNAs?siRNA-vta1,siRNA-vta2,siRNA-vta3 and siRNA-vta4?independently and together using the same approach;siRNA-vta1 had the highest silencing efficiency as assessed by colony diameter and quantitative real time PCR?qRT-PCR?analysis.3.We obtained the knockout mutants??FreB-1 and?Fre B-2?and complementary strains??FreB-C?using PEG-mediated protoplast transformation.Compared with the Vd-wt and?FreB-C,?Fre B-1and?FreB-2 were impaired in colony diameter and spore number on different carbon sources?starch,sucrose,galactose and xylose?,which suggest a tight association of FreB to utilizing carbon sources.Respectively cultured on media supplemented with FeSO₄,FeCl₃ and no iron,?FreB exhibited significantly reduced growth and spore production especially on media with no iron.4.Ferric reductase activity of?FreB was decreased up to 50%compared with Vd-wt.The activity was fully restored in?FreB-C.Meanwhile,the expression levels of other related genes?Frect-4,Frect-5,Frect-6 and Met?were obviously increased in?Fre B.?FreB-1 and?FreB-2 were also highly sensitive to oxidative stress as revealed by the plate diffusion assay when 100?M H₂O₂ was applied to the fungal culture.The sensitivity of?FreB-1 and?FreB-2 to hydrogen peroxide was about 25%higher as compared Vd-wt and?FreB-C.5.Through the statistical analysis of disease index,Nicotiana benthamiana plants inoculated with?Fre B exhibited less disease symptoms than Vd-wt and?FreB-C.The results demonstrate that the Fre B gene is related to the virulence of V.dahliae.In summary,protoplasts transformation system was optimized with high-efficiency to transform exogenous genes and investigate gene function in V.dahliae.Furthermore,it was revealed that FreB gene is essential for the growth,cell surface iron reduction,tolerance to oxidative stress and the virulence of V.dahliae and could be a potential target gene to control this fungus.