Mechanism of flagellin perception in rice

单子叶蚜的寄主范围很广，但单个菌株只能感染一种或几种寄主。在培养的水稻细胞中，几种防御反应仅由水稻不相容菌株诱导。最近我们也报道了来自不相容的A.avenae的鞭毛蛋白参与了这些防御反应的诱导。为了阐明水稻中鞭毛蛋白感知的机制，我们试图利用大肠杆菌表达系统中产生的重组缺失蛋白来确定激活防御反应所需的鞭毛蛋白区域。我们发现含有c端区域的鞭毛蛋白片段在培养的水稻细胞中诱导活性氧的产生和防御相关基因的表达，而含有中端或n端区域的鞭毛蛋白片段没有引起任何防御反应。n端片段包含保守的22个氨基酸（flg22）的核心，在番茄和拟南芥中诱导防御反应。事实上，n端片段和flg22-avenae（包括N1141鞭毛蛋白的flg22位置）在培养的拟南芥细胞中诱导活性氧的产生。这些结果表明，水稻的鞭毛蛋白感知系统与拟南芥不同。接下来，我们尝试利用酵母-双杂交系统鉴定水稻中鞭毛蛋白感知分子。以水稻cDNA文库为先导、c端结构域为诱饵的双杂交筛选结果表明，FIP50（鞭毛蛋白相互作用蛋白）与鞭毛蛋白c端结构域具有特异性相互作用。FIP50是一种功能未知的蛋白，位于细胞质中。免疫沉淀实验显示重组FIP50蛋白在体外特异性结合鞭毛蛋白c端蛋白。这些结果与鞭毛蛋白处理下FIP50表达水平的下调表明，FIP50参与了水稻鞭毛蛋白感知和鞭毛蛋白感知信号的转导。少

The host range of Asidovorax avenae is wide among monocotyledonous but individual strains can infect only one or a few host species. Several defense responses in cultured rice cells were only induced by a rice incompatible strain. Recently we also reported that the flagellin from incompatible A.avenae was involved in the induction of these defense responses. To clarify the mechanism of flagellin perception in rice, we tried to define the region(s) of flagellin protein required for activation of defense response using the recombinant deleted proteins produced in an Escherichia coli expression system. We found that flagellin fragment containing the C-terminal region induced generation of reactive oxygen species and expression of defense-related genes in cultured rice cells, whereas the flagellin fragments containing middle or N-terminal region did not cause any defense responses. The N-terminal fragment comprises core of conserved 22 amino acids (flg22) which induce defense responses in … More tomato and Arabidopsis thaliana. In fact, the N-terminal fragment and flg22-avenae (comprising flg22 position of N1141 flagellin) induced generation of reactive oxygen species in cultured A.thaliana cells. These results suggest that the flagellin perception system in rice is different from that in A.thaliana. We next tried to identify the flagellin perception molecule in rice using yeast-two-hybrid system. Two-hybrid screening using rice cDNA library as play and C-terminal domain as bait showed that FIP50 (flagellin interacting protein) specifically interactes with C-terminal domain of flagellin. FIP50 was functional unknown protein and located in cytoplasm. Immune-precipitation assay revealed that recombinant FIP50 protein specifically binds with flagellin C-terminal protein in vitro. These results together with down regulation of FIP50 expression level by flagellin treatment suggest that FIP50 is involved in flagellin perception and transduction of flagellin perception signaling in rice. Less