Functional Analysis Of GF14? In Arabidopsis Thaliana Against Sclerotinia Sclerotiorum

The white mold fungus Sclerotinia sclerotiorum is one of the most devastating necrotrophic pathogens with a wide host range of over 400 plant species,including soybean,sunflower,and Brassicaceae.Canola(Brassica napus)is a major edible oil crop worldwide.Canola yield is significantly affected by sclerotinia stem rot disease caused by S.sclerotiorum.Due to the lack of resistant sources and the fact that the resistance is possibly mediated by multiple genes,it is necessary to identify and explore the genes that contribute to plant resistance.Therefore,we use the pathosystem between Arabidopsis thaliana and S.sclerotiorum to help us investigate genes involved so as to lay a foundation for the interaction mechanism between oilseed rape and S.sclerotiorum.Oxalic acid(OA)has been proved to be an important pathogenicity determinant of S.sclerotiorum.To further understand the molecular mechanism of OA in fungal pathogenesis,OA-insensitive mutants(about 6000 lines)from a chemical inducible Arabidopsis mutant library were screened on medium containing OA in our lab.The results showed that the T-DNA junctions of four of the five mutants screened were all inserted in the same site of the first intron of At5g10450,suggesting a role in S.sclerotiorum resistance.At5g10450 encodes a 14-3-3 family protein,GF14?,and there are many reports of 14-3-3 protein participating in plant response to abiotic and biotic stress.On the basis of this,the function of this gene against S.sclerotiorum is further explored in this thesis.First,we constructed a constitutive overexpression vector and a RNAi vector under the control of a 35S promoter.The transgenic plants were screened on soil containing 0.03%basta or MS medium containing 50mg/L hygromycin,and we had abtained more than 10 and 30 positive plants respectively.The trangenic plants were also further confirmed at DNA,RNA and protein level.Upon inoculation,overexpression lines showed enhanced resistance and inhibition of S.sclerotiorum mycelium growth.The result of NBT staining indicated that may play a role in clearing superoxide radical.Besides,that the transgenic plants showed tolerance to OA is also consistant with previous results.Moreover,we had also tested the expression level of plant defense related genes in transgenic plants and found that overexpression of AtGF14 ? activates the expression level of plant defense related genes without stress treatment,especially PR 1(about 50 times).However,the transcripts of these two genes were reduced to about zero in RNAi plants.Thus we speculate that AtGF14? may function by interacting with other proteins and enhance plant resistance by activating the expression of defense related genes.