Cloning And Disease Resistance Of SlNBRP1 Gene In Tomato

Crop disease is one of the main reasons for limiting agricultural production.In general,plants resist disease by two pathways: basic resistance(PTI)and gene-to-gene resistance(ETI).A major role in the ETI pathway is the resistance gene(R gene),which encodes an immune receptor in the ETI system.Breeding resistant varieties with R gene is the most direct,environmentally friendly and effective means of controlling crop diseases.Most R genes can encode a protein containing a coiled-coil nucleotide-binding site and leucine-rich-repeat domain(CC-NBS-LRR).Tomato is the most widely grown cash crop in the world and plays a vital role in the supply of fruits and vegetables.Bacterial spot disease poses a serious threat to the production of tomato.In-depth study of the pathogenesis of bacterial spot disease not only provides new clues for improving tomato breeding but also plays an important role in improving tomato quality.DDB1 in tomato,as the core component of CRL4 ubiquitin ligase complex,participates in the regulation of various biological functions,including chloroplast development,fruit size and disease resistance response,by targeting the abundance and activity of different substrate proteins.Previous studies have found that tomato DDB1 loss-of-function mutant hp1 shows high sensitivity to pathogens.To further investigate the disease resistance of DDB1 in tomato,we screened a tomato resistance gene SlNBRP1 by yeast two-hybrid assay using DDB1 as a bait.Through gene expression pattern analysis and subcellular localization experiments,we know that SlNBRP1 is expressed in roots,stems,leaves,flowers and fruits of tomato,and is located in the cytoplasm.By bioinformatic analysis of SlNBRP1,it was found to contain a highly conserved CC-NBS domain of the resistance protein.The interaction between SlNBRP1 and DDB1 was further confirmed by immunoprecipitation and yeast two-hybrid experiments.Through the protoplast transformation experiment of SlNBRP1,We know that SlNBRP1 is degraded by the ubiquitination of the CUL4-DDB1 complex.We hypothesized that SlNBRP1 can participate in the regulation of tomato immune response with DDB1.In order to verify the function of the resistance protein SlNBRP1,we constructed a plant expression vector pBI121-35S::SlNBRP1 by plant genetic engineering technology,and introduced the SlNBRP1 gene into the wild-type tomato genome by Agrobacterium-mediated transformation to obtain transgenic plants.Real-time PCR was used to analyze the expression of SlNBRP1 mRNA in transgenic plants.The results showed that the expression of SlNBRP1 was up-regulated and down-regulated in different degrees,that is,over-expression(OE)and co-suppression(COR).Infection of wild-type and transgenic plants with Pst DC3000 revealed that the resistance of co-suppression transgenic plants decreased and the amount of leaf bacteria increased,while the over-expressed lines showed stronger disease resistance than wild type.This indicates that the SlNBRP1 gene plays a positive regulatory role in the immune response of plant disease resistance.This study adds new molecular targets for genetic breeding to improve crop disease resistance.