The T-DNA Mutant Library Construction And The Function Study On Genes Involved In The Microsclerotia Formation And Pathogenicity

Sunflower yellow wilt caused by Verticillium dahliae could lead to severe yield loss on sunflower production, which is difficult to control. Microsclerotia is the main source of primary infection, which is able to survive in the soil for more than 10 years. However, there is few knowledge unravelling the mechanisms of the microsclerotia formation and involvement in the pathogenicity.The aims of this research are to understand the effects of exogenous cyclic adenosine monophosphate on the biological characteristics and pathogenicity of V. dahliae furthermore we also want to unravel the mechanisms of microsclerotia formation and the function of pathogenicity related genes by establishment of the T-DNA library, flanking sequence analysis of the mutant strains and the function of the candidates genes. We concluded as the followings:1. The application of exogenous cAMP could significantly promote the conidia production, however, it would inhibit the growth rate, spore germination, microsclerotia production, the ability of crude toxin production and pathogenicity of sunflower V. dahliae. Amongthese influences, the microsclerotia formation decreased the most dramatically.2. Using the Agrobacterium-mediated transformation （ATMT） mediated transformation, we transformed the binary vector contained the hygromycin resistance gene fused with the Green fluorescent protein gene （GFP） to the conidia of V. dahlia and obtained 800 positive tranformants through the hygromycin selection.3. Forty positive tansformants were randomly selected from T-DNA library which contained 800 positive transformants and used to characterize the colony morphology, growth rate, conidia production, toxic production and pathogenicity. The results showed among 42 selectived positive transformants, only two produced white aerial hyphae and no microsclerotia formation. Compared with control, the reduced growth rate of all the tested transformants was observed, but the conidia production ability of all the transformants is variable. Regarding to toxin production,5 transformants showed the increased tendency on the toxin secretion ability, however,27 transformants secreted less. The pathogenicity assay was also performed by roots dipping system, the pathogenicity of 4 transformants showed increased tendency, whereas,28 decreased; 10 showed the same pathogenicity as the control.3. HiTAIL-PCR was used to amplify the flanking sequence of 20 mutants. Twelve genes of which the size vary from 200 to 1200 bp were obtained. Two candidate genes, initiation-specific a-1,6-mannosyltransferase （VDAG₀2820） and aryl-alcohol dehydrogenase （VDAG₀3323） which related to the formation of microsclerotia was screened. We analyzed the function prediction and named them as VdOCH1 and VdAAD.4. Gene deletion and complementation experiments showed that VdOCHl not only played an important role in maintenance of cell wall integrity but also regulated the growth and microsclerotia formation. This genecould also influence the penetration capability of mycelia and the virulence of V. dahliae.5. The study of VdAAD showed that, VdAAD could regulate the growth and development of V. dahliae. It also regulated the microsclerotia formation, crude toxin secretion and virulence.