Structure-function Analysis of Rice Immune Receptor BPH14 Reveals Planthopper-resistance Mediated through Protein-protein Interaction with WRKY46/72. Oryza sativa

BPH14 is the first planthopper-resistance gene which encodes a coiled-coil–nucleotide-binding site–leucine-rich repeat (CC-NB-LRR) protein. Here, we analysed the function of BPH14 domains and characterized the possible molecular mechanisms underlying BPH14-mediated BPH resistance. In this study, we found BPH14 form homo-complexes and interacted with WRKY46 and WRKY72, to enhance WRKYs accumulation and increase WRKYs-dependent transactivation activity. We performed chromatin immunoprecipitation (ChIP) analysis to identify the candidate promoters that interacted with the WRKY46 or WRKY72 directly in rice protoplasts. After the ChIP test, the enrichment of specific DNA fragments in the immune precipitant were determined by ChIP sequencing. Two candidate genes RLCK281 and SCalS5 were confirmed by yeast one-hybrid system and EMSAs. These data would advance our understanding about the mechanisms underlying the characteristics of BPH14-dependent BPH resistance in the future. Overall design: The chromatin immunoprecipitation experiment was performed, following a previously reported protocol (Gendrel et al., 2005) with several modifications. Approximately 9 × 106 cells in each sample from rice protoplasts transfected with WRKY46-Myc or WRKY72-Myc were used as a starting material and proteins were immunoprecipitated using antibodies against Myc (MBL, M192-3). The precipitated DNA was purified with the Qia quick PCR purification kit (Qiagen, Germany) and processed further for ChIP-seq data processing and analysis.