Molecular and functional analysis of a brown planthopper resistance protein with two nucleotide binding site domains.

The brown planthopper (Nilaparvata lugens Stal, BPH) resistance gene BPH9 encodes an unusual coiled-coil (CC) nucleotide-binding leucine-rich repeat (LRR) protein with two NBS (Nuceotide binding site) domains. To understand how this CC-NBS-NBS-LRR protein regulates defense signaling and BPH resistance, we dissected each domain's functions. The CC domain of BPH9 self-associated and was sufficient to induce cell death. The region of 97-115 residues in CC domain is crucial for self-association and activation. NBS2, which contains a complete set of NBS function motifs and inhibits CC domain activation, rather than NBS1, acts as a molecular switch to regulate the activity of BPH9. We demonstrated that the CC domain, the NBS domain and LRR domains of BPH9 associate with each other and themselves in planta. Further domain swapping experiments revealed the CC domains of BPH9 and susceptible alleles were similarly competent to induce resistance and HR (Hypersensitive response), while the LRR domain of BPH9 confers resistance specificity to BPH. These findings provide new insights into the regulatory mechanisms governing the activity of CNNL proteins.