| Rape Sclerotinia rot (RSR), caused by Sclerotinia sclerotiorum, is one of the most destructive diseases with worldwide distribution. This fungus can infect more than 400 plant species and subspecies in 75 families. At present, application of fungicides is the principal tool for controlling RSR. Fludioxonil, a novel phenylpyrrole fungicide, exhibits excellent control efficacy on RSR. Up to now, fludioxonil has not been registered in controlling RSR in China. Moreover, action mechanism of fludioxonil against S. sclerotiorum is unclear. We have known that action mechanism of fludioxonil was associated with two-component signaling system, which has two important response regulators RRG-1 and RRG-2.Th.us, in this study, the conditions of protoplast preparation and PEG-mediated genetic transformation system were optimized further, biological function of two response regulators RRG-1 and RRG-2 were determined using the trandformation system described aboved. This will provide important reference information for managing RSRand developping original fungicides. The main results are as follow:1. Genetic transformation system is one of the most crucial techniques to study functional gene. In this study, the conditions of protoplast preparation and PEG-mediated genetic transformation system were firstly optimized. The optimal conditions of protoplast preparation were determined as follows:YEPD liquid medium was used for culturing hyphae for 36 h at 175 r/min and 25℃,0.1 g of hyphae was mixed with 10 ml of enzyme solution (1.5% lysing enzyme in 0.8 M mannitol and citric acid-sodium citrate buffer) and incubated for 2.5 h at 30℃.Protoplasts were centrifuged for 5 min at 1200 rpm, resuspended with enzyme solution and recentrifuged for 5 min at 1200 rpm, and repeated with STC.Then, protoplasts were resuspended until the consension is 108/μL.80μL protoplast suspensions and 20μL PTC were mixed and placed on the ice. Fragments containing hygromycin phosphoric acid enzyme gene (HPH) was transferred into protoplasts. The transformers were screened by 100μg/mL hygromycin B.2.Two-component signal system is involved with the regulation of osmotic and oxidative stress, mycelia growth, fungicide resistance, and pathogenicity. In this study, the function of two regulatory factors SsRRG-1 and SsRRG-2 were investigated by constructing deleted/complementary mutants via the PEG-mediated genetic transformation system described above. The results were as follows:Disruption of SsRRG-1 resulted in increased sensitivity to SDS, high sugar, H2O2, andfludioxonilThe SsRRG-1 deletion mutants had greater cell membrane permeability and increased hyphal branch, but showed a significant reduction in vegetative hyphal growth and virulence. In addition, The SsRRG-1the deleted mutants produced more and smaller sclerotia, and exhibited similarity in sensitivity to metal ions and glycerol content. All the defects were restored by genetic complementation of the SsRRG1 detetion mutant with the wild-type SsRRG1 gene.Disrution of SsRRG-2 resulted in resistance to fludioxonil and increased sensitivity to high salt, H2O2, SDS, Congo red, and Caffine. The SsRRG-2 deletion mutants had great cell membrane permeability, but showed a significant reduction in virulence. In addition, the top of the hyphae was distorted and oxalic acid and glycerol content were reduced significantly. Compared to the parent strain, the SsRRG-2 deletion mutants exhibited varied sensitivity to metal ions and produced abnormal sclerotia. When SsRRG-2 was disrupted, the expression level of downstream kinase genes SsOS4 and SsOS5 was increased dramatically, but no differ in SsHogl. |
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