FliCxoo糖基化修饰机制及其对水稻免疫反应激发功能的研究

It has been known that the bacterial flagellin functions as one of the pathogen-associated molecular patterns (PAMPs) to be specifically recognized by host receptors and elicit the host immune responses, i.e. the PAMP-triggered immunity (PTI). Flagellin glycosylation helps the bacterial pathogen evade host receptor recognition and suppress PTI, thereby promoting bacterial virulence in most cases. However, our previous study showed that glycosylated flagellin (FliCxoo) from Xanthomonas oryzae pv. oryzae, the bacterial blight pathogen of rice, induced higher levels of H2O2, a typical PTI in leaf tissues of rice than did the unglycosylated flagellins, and bacterial virulence on rice was significantly increased in the mutants with unglycosylated flagellin compared to the wildtype strain PXO99A with glycosylated flagellin (Yu et al., Molecular Plant Pathology, 2018). Based on these observations, we speculate that FliCxoo, which is glycosylated with an unknown unique mode, determines its functional outcomes in enhanced elicitation of PTI and bacterial impaired virulence in rice. In this proposal, we will further explore the glycosylation modes of FliCxoo, and its enhanced induction of PTI in rice as follows. (1) identifying the glycosylation sites and glycans of FliCxoo, (2) determining the roles of FliCxoo with different glycosylation forms in rice PTI induction and bacterial pathogenicity, and (3) characterizing regulatory functions of glycosylation of FliCxoo in induced resistances to the X. oryzae pv. oryzae infection and transcription responses in rice. The objectives of the proposal are to demonstrate biochemical and molecular characteristics of FliCxoo glycosylation, to reveal mechanisms underlying the elicitation of immune responses in rice by glycosylated FliCxoo, and to provide novel insights into the functionality of flagellin glycosylation in the host-pathogenic bacterium interactions.

细菌鞭毛素作为一类重要的病原物相关分子模式(PAMPs)，可被寄主受体特异性识别，从而激发寄主免疫反应(PTI)；糖基化修饰可使鞭毛素逃避寄主识别、PTI受到抑制，病菌毒性增强。然而，水稻白叶枯病菌鞭毛素FliCxoo糖基化后，诱导水稻H2O2产生能力明显增强，病菌致病性下降(Yu et al., Mol. Plant Pathol., 2018)。这些结果暗示糖基化的FliCxoo可能通过激发PTI提高水稻抗病性，从而降低病菌致病性。我们推测FliCxoo糖基化修饰机制可能决定了其PTI激发功能，本申请将对此进行深入探究，内容包括：(1)糖基化位点和糖苷鉴定；(2)糖基化位点和糖苷在水稻PTI诱导和病菌致病性中的作用；(3)糖基化调控水稻诱导抗性及其基因转录反应。本研究旨在揭示FliCxoo糖基化分子特征及其水稻PTI激发作用机理，为阐明鞭毛素糖基化功能机制可能存在的新模式进行理论探索。

细菌鞭毛素作为一类重要的病原物相关分子模式(PAMPs)，可被寄主受体特异性识别，从而激发寄主免疫反应(PTI)；糖基化修饰可使鞭毛素逃避寄主识别、PTI受到抑制，病菌毒性增强。然而，水稻白叶枯病菌鞭毛素FliCxoo糖基化后，诱导水稻H2O2产生能力明显增强，病菌致病性下降。本研究首先鉴定了FliCxoo糖基化位点，并对其进行突变验证，随后通过转录组分析了FliCxoo糖基化修饰诱导水稻免疫相关基因的表达，结果显示：1680个基因在接种野生型水稻中相对于突变体差异表达（DEGs）；其中1159个基因表达上调，521个基因表达下调。功能注释显示，上调的DEG参与细胞壁、脂质和二次代谢、防御反应和激素信号传导，而下调的DEG与光合作用有关。这些结果揭示了FliCxoo糖基化位点及其水稻PTI激发作用机理，为阐明鞭毛素糖基化功能机制奠定了基础。