Functional Analysis Of An Sclerotinia Disease Resistance Gene

Oilseed rape (Brassica napus) is an important industrial crop and a major oil crop. It plays important roles in national economy and agricultural production. Sclerotinia stem rot caused by Sclerotinia sclerotiorum is the most devastating disease of oilseed rape in China. To date, no complete resistance has been found in rapeseed germlpasm and its relatives. Stellaria apetala ucria is a common weed in the field of canola, it has non-host resistance to Sclerotinia sclerotiorum. It is of very significance to understand the mechanisms from molecular level of the interactions between Brassica napus and Sclerotinia sclerotiorum and to isolate genes associated with susceptibility and resistance to the infection by Sclerotinia sclerotiorum, which should be helpful to protect plant from infection. Sspgld, one of endopolygalacturonases, is an important fungal effector secreted by the necrotrophic fungus Sclerotinia sclerotiorum during early infection. Using sspgld as bait, a membrane protein named ppn20 was identified by yeast two-hybrid screening of cDNA library of Stellaria apetala ucria. In previous study, we have demonstrated that ppn20 is a G protein-coupled receptor, and ppn20 transcription in Stellaria apetala leaf was induced by Sclerotinia sclerotioru. In this study, we want to further make clear its function. We use Agrobacterium-mediated transformation to obtain Brassica napus and Nicotiana Tabacum transgenic plants. Kanamicin resistance, PCR and RT-PCR were used to screen transgenic plants. The phenotype was analysed by inoculation with Sclerotinia sclerotiorum. Thus, plant expression vector ppn20-pBinplus was transformed into Agrobacterium. Agrobacterium-medialed transformation was used to transform Brassica napus and Nicotiana Tabacum. In total,10 positive Brassica napus plants were obtained by screening with kanamicin, of which 7 plants survived. Ppn20 can be detected in all of these positive plants by PCR and RT-PCR analysis. The resistance phenotype of these plants was assayed by inoculation with Sclerotinia sclerotiorum, the results showed that both of the control plants and the transgenic plants were easily infected with Sclerotinia sclerotiorum. But the fungus was highly restricted in the infection site of the transgenic Nicotiana tabacum, some plants even could not be infected by the fungus. Ppn20 was constructed in plant expression vector PVX, and which was transformed into Agrobacterium. Arobacterium infiltration assay was used to transiently express the ppn20 gene in N. benthamiana leaves. The resistance phenotype of the infiltration area was assayed by inoculating with Sclerotinia sclerotiorum, the results showed that the fungus has not restricted in the sites where ppn20 was infiltrated. Mating-based split-ubiquitin systems in yeast cells proved that there is no interaction between membrane protein ppn20 and the G protein of canola. This may be the reason that ppn20 gene does not show resistance to Sclerotinia sclerotiorum in Brassica napus.