Host-induced gene silencing of the Verticillium dahliae thiamine transporter protein gene(VdThit)confers resistance to Verticillium wilt in cotton

Verticillium wilt(VW),induced by the soil-borne fungus Verticillium dahliae(Vd),poses a substantial threat to a diverse array of plant species.Employing molecular breeding technology for the development of cotton varieties with heightened resistance to VW stands out as one of the most efficacious protective measures.In this study,we successfully generated two stable transgenic lines of cotton(Gossypium hirsutum L.),VdThitRNAi-1 and VdThit-RNAi-2,using host-induced gene silencing(HIGS)technology to introduce double-stranded RNA(dsRNA)targeting the thiamine transporter protein gene(VdThit).Southern blot analysis confirmed the presence of a single-copy insertion in each line.Microscopic examination showed marked reductions in the colonization and spread of Vd-mCherry in the roots of VdThit-RNAi cotton compared to wild type(WT).The corresponding disease index and fungal biomass of VdThit-RNAi-1/2 also exhibited significant reductions.Real-time quantitative PCR(qRT-PCR)analysis demonstrated a substantial inhibition of VdThit expression following prolonged inoculation of VdThit-RNAi cotton.Small RNA sequencing(sRNA-Seq)analysis revealed the generation of a substantial number of VdThit-specific siRNAs in the VdThit-RNAi transgenic lines.Additionally,the silencing of VdThit by the siVdThit produced by VdThit-RNAi-1/2 resulted in the elevated expression of multiple genes involved in the thiamine biosynthesis pathway in Vd.Under field conditions,VdThit-RNAi transgenic cotton exhibited significantly enhanced disease resistance and yield compared with WT.In summary,our findings underscore the efficacy of HIGS targeting VdThit in restraining the infection and spread of Vd in cotton,thereby potentially enabling the development of cotton breeding as a promising strategy for managing VW.

Expression analysis of the R2R3-MYB gene family in upland cotton and functional study of GhMYB3D5 in regulating Verticillium wilt resistance

Improving plant resistance to Verticillium wilt(VW),which causes massive losses in Gossypium hirsutum,is a global challenge.Crop plants need to efficiently allocate their limited energy resources to maintain a balance between growth and defense.However,few transcriptional regulators specifically respond to Verticillium dahliae and the underlying mechanism has not been identified in cotton.In this study,we found that the that expression of most R2R3-MYB members in cotton is significantly changed by V.dahliae infection relative to the other MYB types.One novel R2R3-MYB transcription factor(TF)that specifically responds to V.dahliae,GhMYB3D5,was identified.GhMYB3D5 was not expressed in 15 cotton tissues under normal conditions,but it was dramatically induced by V.dahliae stress.We functionally characterized its positive role and underlying mechanism in VW resistance.Upon V.dahliae infection,the up-regulated GhMYB3D5 bound to the GhADH1 promoter and activated GhADH1expression.In addition,GhMYB3D5 physically interacted with GhADH1 and further enhanced the transcriptional activation of GhADH1.Consequently,the transcriptional regulatory module GhMYB3D5-GhADH1 then promoted lignin accumulation by improving the transcriptional levels of genes related to lignin biosynthesis(GhPAL,GhC4H,Gh4CL,and GhPOD/GhLAC)in cotton,thereby enhancing cotton VW resistance.Our results demonstrated that the GhMYB3D5 promotes defense-induced lignin accumulation,which can be regarded as an effective way to orchestrate plant immunity and growth.