Functional Analysis Of Genes Related To Microsclerotial Development In Verticillium Dahlia

Verticillium dahliae is a worldwide soil-borne plant pathogenic fungus that could infect more than 200 dicotyledonous plants,resulting in verticillium wilt in cotton,eggplant,tomato and other crops,causing serious economic losses.The dormant body of the sclerotium-type Verticillium dahliae is microsclerotia,which could survive in the soil for 14 years.Clarifying the mechanism of microsclerotia formation and development is important to study epidemic regularty of verticillium wilt and constitute prevention and control measures.At present,the molecular mechanism of the microsclerotia development of Verticillium dahliae is obviously lagging behind compared with other phytopathogenic fungi,and the mechanism for regulating the formation and development of microsclerotia is poorly understood.By studying the function of microsclerotial development-related genes,we could provide theoretical basis for the analysis of the molecular mechanism of microsclerotia development and offer new thought for research and development of new fungicides and prevention and control of verticillium wilt.Two T-DNA insertion mutants 6I7,2H3 that showed abnormal microsclerotia development were selected from the T-DNA insertion mutant library of a Verticillium dahliae sclerotium-type strain V08DF1.Single T-DNA integration event occurs in the genome of 6I7 and 2H3.The inserted gene of 6I7,VdVTA1,encodes a transcription factor containing Zn(II)2Cys6 protein,and the inserted gene of 2H3,VdPKS,encodes a conidial yellow pigment biosynthesis polyketide synthetase.Based on these results,two VdVTA1 deletion mutants,ΔVdVTA1-2,ΔVdVTA1-10 and two VdPKS deletion mutants,ΔVdPKS1,ΔVdPKS2 were obtained by homologous recombination gene deletion technology.Three VdVTA1 complementary mutants,6I7-VTA1-1,6I7-VTA1-4,6I7-VTA1-5,6I7-VTA1-6 and three VdPKS complementary mutants,2H3-PKS6,2H3-PKS8,2H3-PKS9 were obtained by gene complementary technique.After culturing on PDA medium for two weeks,the wild type strain could produce a large amount of melanin.ΔVdVTA1-2 and ΔVdVTA1-10 could also produce melanin,but the quantities of black microsclerotia were significantly less than the wild type strian.The melanin accumulations of 6I7-VTA1-1,6I7-VTA1-4and 6I7-VTA1-5 were significantly more than VdVTA1 deletion mutants.Microscopic observation showed that ΔVdVTA1-2 could form black microsclerotia under PDA culture conditions,but the formation time was prolonged compared with the wild type strain.While it could only form bead-like cell-expanded structure in Czapek medium.There was no accumulation of melanin,and ΔVdVTA1-2 does not form a mature black microsclerotia structure.The results of q RT-PCR showed that VdVTA1 was highly expressed in the conidia stage and spore germination stage,and the expression level was gradually down-regulated during the formation of microsclerotia.These results indicated that VdVTA1 is involved in the regulation of melanin biosynthesis and microsclerotia formation under different nutrient conditions.There was no significant difference among the pathogenicity on cotton of the deletion mutants and the wild type strain.The result indicated that VdVTA1 had nothing to do with the pathogenicity.ΔVdPKS1 and ΔVdPKS2 could not produce melanin on PDA medium,but2H3-PKS6,2H3-PKS8 and 2H3-PKS9 all recovered the ability to produce melanin.Microscopic observation revealed that ΔVdPKS1 could only form a dense cell-expanded structure and could not form a mature black microsclerotia structure.VdPKS was gradually up-regulated during the development of microsclerotia.When melanin began to accumulate,the expression reaches the highest.After that,the expression was gradually reduced.The results revealed that VdPKS is involved in the formation of microsclerotia malanin,which is an essential gene for melanin synthesis.There was no significant difference among the pathogenicity on cotton of the deletion mutants,the wild type strain and the complementary mutants.The results indicated that VdPKS had nothing to do with the pathogenicity.In addition,the inhibition rate of azoxystrobin and fludioxonil on the mycelial growth of ΔVdPKS1 and ΔVdPKS2 was significantly higher than that of the wild type strain.There were no significant differences between the complementary mutants and the wild type strain.The result showed that VdPKS was related to the stress resistance.