Isolation And Functional Analysis Of Potato Nbs Gene Conferring Resistance To Late Blight

Potato late blight, caused by the oomycete pathogen Phytophthora infestans, is the most seriousdisease in potato crops worldwide. The most effective and important way to control the disease is tobuild field polymorphism of resistance by discovering and isolating new genes, the ones conferringbroad-spectrum resistance, especially. In this study, based on repeat disease testing with three“super-race” isolates of P.infestans, we located and isolated a new gene with NBS profiling combinedwith homology cloning strategy. Our finding will provide new ideas for researches on genes isolatingand the mechanism with which they evolve. The main results were summarized as follows:1.Abtained new germplasm resource conferring resistance to super-race P.infestans. Based ondisease testing with twelve isolates of P.infestans, among which three are “super-race”(S08-02, CN152and NL10147), we discovered a novel gene termed as RD from potato wild species S.demissum.Interestingly and surprisingly, this gene is the first one from S.demissum and the third one afterRB/Rpi-blb1and Rpi-blb2for the whole potato family, which can confer broad-spectrum resistance tolate blight.2.Developed RD-specific marker and located this gene at chromosome4of potato genome.Resistance-related markers NBS2-AluI-210and NBS2-HaeⅢ-185/194were generated with NBSprofiling biotechnology. The BLASTn results against NCBI and PGSC database suggest that gene RD isclose to SCAR marker Th21, which was located at the “hot-spot” region on chromosome4.3.Abtained the full length DNA sequence of gene RD. Primer RDSP was designed with theBLASTn result of NBS sequence. Screening PCR lead to a resistance-linked band which share highsimilarity with R2, with a85bp deletion between RD fragments and R2. It’s indicated that gene RD isthe paralog of R2.Using long range PCR, a common sequence was identified from the resistantgenotypes but not in the susceptible ones. Analysis suggest that the sequence contain a complete ORF of2184bp and that it belong to the classic NBS-LRR resistant genes, containing the conserved motifs suchas kinase1a, kinase2, GLPL and LRR. Compared to R2, it occurs to RD that a308bp deletion includingthe leucine zipper (LZ) from the original initiation codon of R2was found. R2and RD exhibit76.77%identity at the nucleotide level and72.41%similarity at the amino acid level.4.Performed the transient expression of RD in Nicotiana tabacum. Two binary expression vectorspBI121: RDGH-2and pBINPLUS: RDGH-2were successfully constructed. With pBINPLUS inGV3101and pCLD04541:RB in AGL0as negative and positive control, respectively, localhypersensitive reaction (HR) was successfully initiated with the aid of agrobacterium-mediated transientgene expression in Nicotiana tabacum, which indicate that the cloned RD is indeed functional.