Regulation Of Microsclerotium Dormancy Of Verticillium Dahliae By VdToxD1 And VdToxD3

Microsclerotia are special dormant structures formed in the late stage of infection by Verticillium dahliae,which can survive for 14 years in soil as dormant structures and serve as the primary inocula on many hosts.But so far,the molecular mechanism of microsclerotia dormancy remains unclear.In this study,based on our previous transcriptome analysis for germinated and dormant microsclerotia of V.dahliae,VdToxD1 and VdToxD3 genes,which were found to be highly expressed in dormant microsclerotia,were selected to study their functions in the microsclerotia dormancy process.It is expected to lay a theoretical foundation for further revealing the molecular mechanism of microsclerotia dormancy of V.dahliae.The main results are as follows:VdToxD1 and VdToxD3 positively regulate microsclerotia dormancy in V.dahliae.VdToxD1 contains zinc-binding dehydrogenase and alcohol dehydrogenase domains,which have the highest amino acid sequence similarity with the zinc-binding dehydrogenase of Fusarium albosuccineum;VdToxD3 contains a zinc-binding dehydrogenase domain,which has the highest similarity with the amino acid sequence of Tox D protein of Aspergillus flavus.Using Agrobacterium-mediated genetic transformation,VdToxD1 and VdToxD3 gene knockout and complementation mutants were obtained.Results showed that the microsclerotium germination rates of VdToxD1 and VdToxD3 knockout mutant strains were87.3% and 86.0% respectively,significantly higher than that of 64.0% in the wild-type strain XJ592.The Vd Crz1 transcription factor binds to specific motifs in the promoter regions of VdToxD1 and VdToxD3 genes.The transcription factor Vd Crz1 has been reported to be involved in regulating the germination and dormancy of microsclerotia.In this study,electrophoretic mobility shift(EMSA)and yeast one-hybrid assays were used to prove that the transcription factor Vd Crz1 binds to the promoter regions of VdToxD1 and VdToxD3.In addition,the transcription factors Vd Crz1 binds to [5′-GGGGCTTGG-3′],[5′-GTGGCTGAC-3′],[5′-CTACGCCTC-3′],[5′-TGAAGCCAC-3′] and[5′-GCGGCGTAG′] motifs from-937 bp to-349 bp upstream of VdToxD1,and binds to[5′-TGCAGCCAC-3′] and [5′-CAATGCCAC-3′] motifs from-752 bp to-496 bp upstream of VdToxD3.Meanwhile,this study explored other biological functions of VdToxD1 and VdToxD3 by comparing the differences in phenotype and virulence between VdToxD1 and VdToxD3 mutants and wild-type strains.Results showed that VdToxD1 and VdToxD3 negatively regulated conidial production and germination of V.dahliae,and were involved in response to zinc sulfate and hydrogen peroxide stresses of V.dahliae.The results of virulence analysis showed that VdToxD1 did not affect the virulence of V.dahliae to cotton,while VdToxD3 negatively regulated the virulence of V.dahliae to cotton.