Study On Molecular Mechanism Of Bph6 Mediated Resistance To BPH

The insects have been a great threat to food production.Rice(Oryza sativa L.)is one of the three major grain crops in the world and its cultivated area is the largest.The brown planthopper(Nilaparvata lugens St(?)l,BPH)is the most destructive pest in Asia.In recent decades,farmers used pesticide to controll the breakout of the brown planthopper.However,the brown planthopper has developed resistance to pesticide.One of the most effective and environmentally-friendly means to control this pest is to cultivate resistant rice varieties.Although multiple planthopper-resistance genes has been cloned,the molecular mechanis of their resistance to BPH is not very clear.The study on plant-insect interaction will provide important support for sustainable controll of pests in agricultural production.In previous work we have cloned a BPH resistance gene Bph6 that localized to chromosome 4.Bph6 encodes a protein of 981 amino acids.The subcellular localization result showed that BPH6 localized to the exocyst complex.BPH6 could promot releasing of Os Exo70E1 and Os SAMS2 to extracellular space.Besides,cytokinin also played an important role in Bph6-mediate resistance.On these reaserch basis,we screened for BPH6-interacting protein by yeast-2-hybrid system and TAP system.We found that BPH6-interacting protein NDU has274 amino acids with a ? ? ? ? ? ? ? structure in 125-233 aa and contains a thioredoxin-like domain.Insulin reduction assay showed that NDU-HIS possessed reductase activity.The supression of NDU expression in Nip-Bph6-NIL plants resulted in decreasing of plant resistance to BPH that indicate the participate of NDU in Bph6-mediate resistance.The subcellular localization result showed that NDU localized to mitochondria.NDU could co-localize with BPH6 in rice protoplasts.We detected the protein in mitochongdria and cytoplasm of NIP and NIP-Bph6-NIL plants,respectively.Westernblot results showed that NDU localizes to mitochondria in NIP plants while to mitochondria and cytoplasm in NIP-Bph6-NIL plants.These indicated that BPH6 promote the releasing of NDU to cytoplasm.We found the NDU-interacting protein NH5 in cytoplasm and knockout NH5 gene in NIP-Bph6-NIL plants.nh5 mutants showed obvious episperm dehiscence that suggest NH5's role in plant development.The BPH resistance assay will be carried out using the next generation plants.NH5 belongs to NPR1-like gene family and no evidence showed its correlation to resistance.While NH1 participate in rice resistance through regulating PR genes' expression.Gene expression analysis showed that the expression of R1 b,PR5 and PR10 remarkable increased in NIP-Bph6-NIL plants compared to NIP plants.We further explored the interaction beteewn BPH6 and NH proteins.Yeast two hybrid assays and Co IP assays showed that BPH6 interacts with NH3.nh3 mutants showed elevated resistance to BPH and brown lesions on the leaves.These results indicated that NH3 is a negative regulator of Bph6-mediated resistance.We further explored the mechanism of NH3-regulated resistance.Yeast two hybrid assays and Co IP assays showed that NH3 interacts with NH1 and promotes degradation of the latter.Meanwhile,we also found that BPH6 can reduce the content of NH3 protein corresponding the co-localize between BPH6 and NH3.These suggested that the regulation of BPH6 over NH3 is related to BPH6's transporting fuction in cell.The study on BPH6-intercting proteins contribute to the broaden of Bph6-mediate resistance research and provide new theoretical basis for insect-crop interaction.