鞭毛素蛋白FliCxcv对水稻免疫反应的诱导功能鉴定

 为揭示来源于番茄细菌性斑点病菌(Xanthomonas campestris pv. vesicatoria, Xcv)菌株XV18鞭毛素(FliCxcv)作为一种病原相关分子模式(PAMP)诱导水稻免疫反应的功能,本研究对FliCxcv编码基因flicxcv进行了基因克隆、序列分析、原核表达、蛋白纯化和诱导活性测定。结果表明,通过PCR特异性扩增,从Xcv菌株XV18中克隆了1 200 bp的fliCxcv基因片段,其序列与GenBank中己测序菌株的完全一致。在大肠杆菌中对该基因全长、N端和C端截短序列进行了原核表达,并获得了纯化的FliCxcv全长及其截短蛋白。将纯化蛋白浸润接种到水稻品种日本晴叶片组织,发现FliCxcv全长及其截短蛋白均能诱导水稻叶片细胞死亡、H₂O₂产生以及防卫基因(OsPAL和OsPR1b)表达等免疫反应,但诱导活性存在差异。因此,本研究验证了FliCxcv具有激发水稻细胞免疫反应的PAMP功能,为水稻免疫诱导制剂的研发提供了材料。

The Xanthomonas campestris pv. vesicatoria flagellin FliCxcv functions as a PAMP to trigger immunity responses in rice leaves

To elucidate whether the flagellin (FliCxcv) from Xanthomonas campestris pv. vesicatoria(Xcv) act as a PAMP to induce PTI (PAMP-triggered-immunity) in rice, the gene cloning, sequence analysis, prokaryotic expression, protein purification and PAMP activity assay of FliCxcv and truncated proteins were performed in this study. The results show that the fliCxcv gene was cloned through PCR specific amplication from the genomic DNA of Xcv strain XV18, the sequence of which is identical to that of the sequenced strain in the GenBank. Moreover, the full-length, N- and C-terminal domains were expressed in Escherichia coli respectively and purified to get FliCxcv and truncated proteins FliCxcv-N and FliCxcv-C. The immunity responses including hypersensitive cell death, H₂O₂ production and expression of defense-related genes, such as OsPAL and OsPR1b were observed in rice leave tissues infiltrated by these purified proteins. These results suggested that Xcv flagellin was an effective PAMP to elicit PTI in rice, which can be used for the development of novel inducing agent of rice disease resistance.