Functional Characterization Of Rice Gene Bphi008α

The brown planthopper (BPH) Nilaparvata lugens Stal is an important pest of rice. The BPH feeds mainly on the rice stems through its piercing mouthparts (stylet), and sucks assimilates from the phloem. BPH feeding affects plant development. Feeding by a large number of BPH may result in drying of the leaves and wilting of the tiller, a condition called’hopperburn’. But till now, little is known how rice defends BPH-feeding through its innate immune system. In the previous study, we obtained a BPH-feeding induced gene Bphi008a in Minghui 63 by method of SSH (Suppression Subtractive Hybridization), which is one copy in rice genome and can also be induced by wounding and ethephon while SA, MeJA and ABA can’t.To clarify the role of Bphi008a in BPH stress responses, Bphi008a was overexpressed (OE) or blocked via double-stranded RNA interference (RNAi) in rice (cv. Hejiang 19). Northern blot analysis indicated that the expression of Bphi008a gene was enhanced in OE lines without BPH treatment and the expression of Bphi008a was blocked in RNAi plants treated with BPH for 48h, compared with that in WT plants. In T1 progeny of OE lines, we selected a homozygous transgenic plant OE21-15 and the Southern blotting analysis indicated that all offspring of OE21-15 were single-copy insertion. Three kinds of experiments were carried out to evaluate the resistance of transgenic lines to BPH and we found the resistance to BPH was upgraded in overexpression lines and appreciably downgraded in RNAi lines. By method of particle gun-mediated system, we found Bphi008a protein was localized in plasma membrane and nucleus and its N-terminal twenty amino acids were involved in its localization of membrane. We found Bphi008a expression levels were the highest in root of seedling stage, and then reduced in leaf-blade and leaf-sheath of heading stage, leaf-sheath, leaf-blade and stem of seedling stage in sequence. In flower, we detected the lowest Bphi008a expression level.We firstly chose the best stable HK gene(s) for normalization in different BPH-feeding time materials in order to reflect the accurate gene expression changes in WT lines, transgenic lines and 1-MCP treated WT lines by real-time PCR. OsACS and OsACO gene family expression levels suggested that BPH-feeding behavior was a rapid process turning on ethylene biosynthesis. Combination with Bphi008a expression level changes in 1-MCP (an ethylene competitive inhibitor) treated WT plants, we thought Bphi008a should be a downstream gene of ethylene signaling pathway. Combination of bioinformatics and kinase assay in vitro, we found Bphi008a could be phosporlated by osMPK5 and phosphorylation site was localizated in its C-terminal proline-rich region. Yeast two-hybrid proved this direct interaction between osMPK5 and Bphi008a in vivo and BiFC results further confirmed this interaction was taken place in nucleus. Subsequently we found that Bphi008a overexpression and RNAi plants were accompanied with different transcript levels changes of OsMPK5, OsMPK12, OsMPK13 and osMPK17, which were also induced by both ACC (an Ethylene precursor) treatment and pathogen M.grisea infection; and we also found two AP2/ERF transcription factors OsERF1 and OsEREBP1 expression levels were enhanced in overexpression lines, which were directly activated by phosphorylation of osMPK5 and osMPK12 respectively. Several plant enzymes expression levels changed in transgenic plants and WT plants, such as Arginase, CysPI and LEA2 which could degrade essential amino acids in herbivore midgut. Finally, yeast two-hybrid screening showed that Bphi008a could interact with a b-ZIP transcription factor (OsbZIP60) and a RNA polymerase polypeptide (SDRP). Our results indicate that ethylene induces expression of Bphi008a, and the phosphorylated Bphi008a, associating with OsbZIP60 and SDRP to form a transcriptional complex, provides enhanced resistance to the BPH by regulating MAPKs as part of the innate immune system of rice.