糖基转移酶基因rbfCxoo缺失突变导致水稻白叶枯病菌毒性表达增强

Deletion mutation of rbfCxoo, encoding a putative glycosyltransferase in Xanthomonas oryzae pv. oryzae leads to enhanced virulence expression

摘要:

摘 要：【目的】阐明水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae，简称Xoo)基因组中推导的脂多糖O抗原合成蛋白基因rbfCxoo (XOO2599) 的结构和生物学功能。【方法】通过基因克隆、序列分析、缺失突变和表型测定，对rbfCxoo的分子特征及其功能进行了鉴定。【结果】 用特异性引物进行PCR扩增，从野生型菌株PXO99A基因组DNA中成功地获得了与己测序菌株KACC10331序列完全一致的全长基因序列； RbfCxoo序列N端和C端分别具有一个糖基转移酶的保守结构域(Glycos_transf_2)。用标记置换法获得了基因缺失突变体△rbfCxoo。与PXO99A相比，△rbfCxoo 脂多糖O抗原合成能力并未发生变化，但鞭毛素糖基化能力有所降低。此外，△rbfCxoo鞭毛运动性、生物膜形成和胞外纤维素酶和木聚糖酶活性都无明显改变，但对水稻的致病性显著增强，毒性相关基因的表达也有所增加。【结论】RbfCxoo可能与细菌鞭毛素糖基化修饰以及毒性表达有关。

Abstract:

Abstract: [Objective] To better understand the structure and biological function of rbfCxoo, a gene with the putative function in lipopolysaccharide O-antigen synthesis in Xanthomonas oryzae pv. oryzae (Xoo), the causal pathogen of bacterial blight of rice. [Methods] The molecular identification and function analysis of rbfCxoo were performed through gene cloning, sequencing and deletion analysis. [Results] The sequence of rbfCxoo cloned from the genomic DNA of wild-type PXO99A was the same as that of the sequenced strain KACC10331. There were glycosyltransferase domains (Glycos_transf_2) at N and C terminal of RbfCxoo respectively. The deletion mutant △rbfCxoo generated through a double crossover recombination and validated by PCR assay displayed the reduced flagellin glycosylation and no change in lipopolysaccharide O-antigen synthesis compared with PXO99A. Moreover, no significant changes in flagellar mobility, biofilm formation and production of extracellular cellulase and xylanase in vitro were observed in △rbfCxoo compared to PXO99A. Most importantly, the deletion mutation of rbfCxoo resulted in enhanced virulence and gene expression. [Conclusion] RbfCxoo might be related to flagellin glycosylation and virulence expression in Xoo.