We examined ways in which the Brown planthopper induced008a (Bphi008a; AY256682) gene of rice (Oryza sativa) enhances the plant’s resistance to a specialist herbivore, the brown planthopper (BPH; Nilaparvata lugens). Measurement of the expression levels of ethylene synthases and of ethylene emissions showed that BPH feeding rapidly initiated the ethylene signaling pathway and up-regulated Bphi008a transcript levels after 6 to 96 h of feeding. In contrast, blocking ethylene transduction (using 1-methylcyclopropene) reduced Bphi008a transcript levels in wild-type plants fed upon by BPH. In vitro kinase assays showed that Bphi008a can be phosphorylated by rice Mitogen-activated Protein Kinase5 (OsMPK5), and yeast two-hybrid assays demonstrated that the carboxyl-terminal proline-rich region of Bphi008a interacts directly with this kinase. Furthermore, bimolecular fluorescence complementation assays showed that this interaction occurs in the nucleus. Subsequently, we found that Bphi008a up-regulation and down-regulation were accompanied by different changes in transcription levels of OsMPK5, OsMPK12, OsMPK13, and OsMPK17 in transgenic plants. Immunoblot analysis also showed that the OsMPK5 protein level increased in overexpressing plants and decreased in RNA interference plants after BPH feeding. In transgenic lines, changes in the expression levels of several enzymes that are important components of the defenses against the BPH were also observed. Finally, yeast two-hybrid screening results showed that Bphi008a is able to interact with a b-ZIP transcription factor (OsbZIP60) and a RNA polymerase polypeptide (SDRP).
我們通過多種檢測方法證明水稻褐飛虱誘導基因Bphi008a能夠增強植株對褐飛虱的抗性，測量乙烯合成及乙烯傳遞信號的相關(guān)基因的表達水平表明褐飛虱的取食可以迅速啟動乙烯信號通路，并在褐飛虱取食的6到96個小時之內(nèi)，Bphi008a表達上調(diào)。然而，使用乙烯信號轉(zhuǎn)導阻斷劑1-甲基環(huán)丙烯能下調(diào)Bphi008a在褐飛虱取食的野生型植株的表達水平。體外激酶試驗表明Bphi008a能通過絲裂原活化蛋白激酶OsMPK5實現(xiàn)磷酸化。酵母雙雜表明Bphi008a通過其C末端富含脯氨酸區(qū)域能直接與OsMPK5蛋白相互作用。雙分子熒光互補試驗進一步表明這種互作發(fā)生在細胞核中。隨后，我們也發(fā)現(xiàn)轉(zhuǎn)基因植株中Bphi008a上調(diào)或者下調(diào)，伴隨著OsMPK5, OsMPK12, OsMPK13, OsMPK17的轉(zhuǎn)錄水平的不同變化。蛋白質(zhì)免疫印跡分析也表明，Bphi008a過表達和RNAi抑制植株中，OsMPK5蛋白水平在稻飛虱取食之后分別增加和下降。在轉(zhuǎn)基因株系中，我們也能檢測到一些重要的抵御褐飛虱的酶，其表達水平發(fā)生變化。最后，酵母雙雜篩選表明Bphi008a 能夠與b-ZIP 轉(zhuǎn)錄因子OsbZIP60以及RNA聚合酶多肽SDRP分別發(fā)生互作。