Function Of Binding Protein Nbnrp1 Of The Fungal Elicitor PevD1 In Tobacco Disease Resistance

PevD1, a fungal protein elicitor,was secreted from Verticillium dahliae and could induce hypersensitive responses(HR)-like necrosis and systemic acquired resistances(SAR) in tobacco plant. But the mechanism of PevD1-induced the disease resistance is unknown. Previous researches had screened a PevD1 interaction protein Nbnrp1.Yeast two-hybrid(Y2H) and Bimolecular Fluorescence Complementation(BiFC) assay confirmed that Nbnrp1 protein was binding with PevD1. In this paper we verified the binding fragment of Nbnrp1 and PevD1 interactions in vivo and vitro assay through Y2 H and GST Pull-down technology. Subcellular Co-localization of Nbnrp1 and PevD1 was observed under fluorescence confocal microscopy. Nbnrp1 overexpression tobacco and Nbnrp1 silence tobacco were obtained. Compared with wild type tobacco, HR-like necrosis and disease resistance of these two kinds of transgenic plants make significantly change after PevD1 treatment. This paper will provide the basis for elucidation of molecular mechanism in Nbnrp1-mediaed protein elicitor PevD1-induced plant resistance in tobacco. The major results are as follows:1、The binding fragment DCD domain is required for Nbnrp1 and PevD1 interaction in vitro and in vivoWe verified the interaction between PevD1 and Nbnrp1 and in vivo by Y2 H and in vitro by GST Pull-down assay. Nbnrp1 can be divided into Nbnrp1△N(DCD domain) and Nbnrp1△C according to the bioinformatics analysis. Nbnrp1△N and Nbnrp1△C were built into the carrier pGADT7, respectively. The recombinant plasmid and pGBKT7-PevD1 were transformed into yeast cells. Y2 H assay showed Nbnrp1△N interacted with PevD1.To confirm the DCD domain bind to PevD1. The recombinant plasmid pGEX-6P-2-Nbnrp1△N was transformed into E. coli BL21(DE3) to express the recombinant protein GST-Nbnrp1△N. The recombinant protein was purified with GST affinity chromatography, desalting column and Q HP column. The SDS-PAGE analysis showed a single band molecular, and the weight of the recombinant protein is 41 kDa. The expression vector pET30-PevD1 was transformed into E. coli BL21(DE3) to express the recombinant protein His-PevD1, and high purity protein was obtained through His affinity chromatography and anion exchange chromatography. SDS-PAGE analysis showed the molecular weight of His-PevD1 protein was 18.2 kDa. The GST pull-down between His-PevD1 protein and GST-Nbnrp1△N protein showed His-PevD1 protein and GST-Nbnrp1△N protein could interact in vitro, and the DCD domain contribute to the interaction.2、Nbnrp1 Protein and the PevD1 Protein can overlap in tobacco cellThe recombinant vector pYBA1132-Nbnrp1 was constructed for transient expression. Agrobacterium GV3101 containing pYBA1132-Nbnrp1, pCAM1032-PevD1-RFP, P19 was used to infiltrate tobacco plants. Laser scanning confocal micrographs showed green fluorescence with Nbnrp1 and red fluorescence with PevD1 can overlap in tobacco cells and lead to emit yellow fluorescence, indicating Nbnrp1 and PevD1 can meet in tobacco nucleus and cytoplasm.3、Nbnrp1 overexpression transgene tobacco enhance PevD1-indued disease resistanceThe recombinant expression vector pBI121-Nbnrp1 was transformed into tobacco via the Agrobacterium-mediated method. 137 independent kanamycin-resistant Nbnrp1 overexpression transgene tobaccos were obtained. The PCR results show that 83 lines was positive in To plants and 34 positive plants, 13 negative plants of 47 T1 transgenic tobacco plants, the positive : negative ratio is about 3:1. Southern Blotting experiment showed that transgenic plants was single copy; Nbnrp1 overexpression plants showed earlier appearance of HR-like necrosis lesions, lower concentration required for necrosis lesions than wild-type plants. In addition, real time PCR showed expression level of HR marker gene HRS203 J increased. Nbnrp1 overexpression plants improved disease resistance against TMV and P. syringae pv.tabaci. The TMV inhibition increased by 41.2% and the resistance of P. syringae pv.tabaci increased by 91.1% post PevD1 treatment. And the resistance genes PR1-a and PR1-b were also up-regulated expression.4、Nbnrp1 silence transgene tobacco decreased PevD1-indued disease resistancepRNAi1017 was as RNAi interference vector and DCD domain was as interference fragment. The middle vector pRNAi1017-SA was constructed. The forward and reverse sequence of introns was inserted to plant expression vector pCAMBIA2300 and pCAMBIA-RNAi-Nbnrp1 was constructed. The recombinant vector was transformed into tobacco via the Agrobacterium-mediated method. 116 independent kanamycin-resistant Nbnrp1 silence transgene tobaccos were obtained. The PCR results showed that 56 lines was positive; The T1 generation of PCR detection shows 28 positive plants, 9 negative plants in 37 transgenic tobacco plants, the positive : negative ratio is about 3:1. The silence efficiency of Nbnrp1 was more than 80% in Nbnrp1 silence plants. Nbnrp1 silence plants showed less sensitive to PevD1-induced necrosis, including later necrosis appearance, high PevD1 concentration for necrosis than wild-type plants. In addition, the HR marker gene HRS203 J decreased. After PevD1 treatment, the resistance of TMV inhibition decreased by 71.7% and P. syringae pv.tabaci resistance decreased by 91.1% and the genes PR1-a and PR1-b were also down-regulated expression in Nbnrp1 silence lines.