【目的】黄单胞菌细胞外泌蛋白质(Xanthomonas outer proteins, Xops)是植物病原黄单胞菌高度保守的毒性效应子或毒性辅助组分,通过细菌Ⅲ型分泌系统分泌到细菌细胞外部,然后转入植物细胞而发挥病理作用。水稻黄单胞菌水稻致病变种即水稻白叶枯病菌的标准菌株PXO99^A分泌的XopN是一种毒性效应子,通过影响寄主免疫反应而使水稻发病。但是,XopN对病菌毒性的影响是否因水稻品种的不同而异,还有待研究。【方法】利用同源双交换技术,先敲除了PXO99^A的XopN基因,获得了∆XopN突变体,又经过遗传互补,得到了回补菌株∆XopN/XopN。通过营养肉汤液体培养,测定了XopN对病菌繁殖能力的影响;根据文献选用14个水稻品种,通过接种实验,测定了PXO99^A对这些品种的毒性与XopN敲除或回补的影响;在XopN发挥毒性作用的水稻品种上,测定了隐性抗病基因OsSWEET11/xa13与显性感病基因OsSWEET11/Xa13受病菌侵染而表达的情况,分析了XopN敲除或回补的影响。【结果】在营养肉汤液体培养过程中,病菌突变体菌株∆XopN的繁殖速度明显低于野生型PXO99^A。PXO99^A、∆XopN和∆XopN/XopN接种水稻后,根据其在水稻叶片组织内的繁殖量及随后产生的白叶枯病症状的严重程度,将供试的14个水稻品种分为两种情况。一是感病程度与病菌XopN是否敲除或回补无关,这有10个水稻品种（IRBB1、IRBB3、IRBB8、IRBB10、IRBB14、IR24、IRBB203、IRBB204、IRBB205和IRBB211）,PXO99^A、∆XopN和∆XopN/XopN对它们的毒性无明显差别。二是XopN对病菌毒性发挥作用的水稻品种,包括高度感病的3个品种（IRBB208、Asominori和日本晴）和低感品种IRBB13。与PXO99^A或∆XopN/XopN相比,∆XopN对这4个水稻品种的毒性大为降低。在IRBB13上,病菌侵染对隐性抗病基因OsSWEET11/xa13在叶片内的表达发生抑制作用,这一效应与XopN的毒性功能相关。相反,日本晴显性感病基因OsSWEET11/Xa13却受病菌侵染的诱导,在叶片内的表达水平大幅度提高。IRBB208和Asominori携带OsSWEET11/Xa13同源基因,该同源基因在叶片内的表达水平也因病菌侵染而大幅提高。在这4个水稻品种上,PXO99^A和∆XopN/XopN能够诱导OsSWEET11/Xa13或其同源基因表达,但∆XopN无此作用。另外,XopN对病菌在非寄主植物烟草上诱发过敏反应有量变贡献,相比PXO99^A或∆XopN/XopN,∆XopN引起过敏反应的程度有所降低。【结论】XopN是一个有限广谱性效应子,在拥有OsSWEET11同源基因的水稻品种上发挥毒性作用。XopN也是病菌繁殖所需要的,对病菌在非寄主植物上诱导过敏反应有一定贡献。

【Objective】 Xanthomonas outer proteins (Xops) are conservatively virulent effectors or virulence-assistant components secreted by the type Ⅲ secretion system in plant-pathogenic bacteria that falls into the Xanthomonas genus, including the rice bacterial blight pathogen. Xops were secreted to the outside of bacterial cells, and then transported to plant cells to play a pathological role. One of these proteins, XopN, secreted by the standard strain PXO99^A of the rice pathogen, has been implicated in the bacterial virulence by subverting plant immunity, but the specificity in the virulence role of XopN is unclear. 【Method】 By using the double crossover homologous-recombination technique, the XopN gene-knockout mutant ∆XopN and genetically complemented strain ∆XopN/XopN were generated under the background of the bacterial wild-type strain PXO99^A. By comparing both recombinant strains with PXO99^A in terms of bacterial multiplication in nutrition broth, the role of XopN in the bacterial multiplication was evaluated. Based on literatures, 14 varieties of rice were chosen for experimental assessments on the contribution of XopN to the spectrum of the bacterial virulence. By quantitative analyses of gene expression, the virulence of XopN was related to the effects of bacterial infection on foliar expression of the OsSWEET11 gene or its homologs in different rice varieties. 【Result】 The bacteria gain better multiplication in the medium with the presence of the XopN gene, in contrast to the gene-knockout mutation, which highly impairs the bacterial propagation level. Based on bacterial populations of PXO99^A, ∆XopN and ∆XopN/XopN multiplied in rice leaves, as well as severities of subsequently developed leaf blight symptoms, the 14 rice varieties fall into two categories. 1) Ten of the rice varieties (IRBB1, IRBB3, IRBB8, IRBB10, IRBB14, IR24, IRBB203, IRBB204, IRBB205 and IRBB211) display similar susceptibility to the bacteria no matter whether the XopN gene is present or not. 2)By contrast, the bacteria depend on XopN to cause strong virulence on rice varieties IRBB208, Asominori and Nipponbare. The bacteria also depend on XopN to perform a relatively low level of virulence on the rice variety IRBB13. In IRBB13 leaves, the virulence role of XopN is coincident with a marked suppression of the recessive resistance gene OsSWEET11/xa13 by the bacterial infection. On the contrary, the bacterial infection causes enhanced expression of the dominant disease-susceptibility gene OsSWEET11/Xa13 in leaves of Nipponbare and enhanced expression of the gene homologs in IRBB208 and Asominori leaves. In essence, the transcriptional promotion or inhibition of the OsSWEET11 homologs is provided by the bacterial strains PXO99^A and ∆XopN/XopN, instead of ∆XopN, suggesting the critical role of XopN in inducing the gene expression. In addition, XopN is also required for the bacteria to effectively induce hypersensitive response in leaves of the non-host plant tobacco. While a high extent of the hypersensitive response is induced by PXO99^A or ∆XopN/XopN strain, the response incurs a substantial reduction by ∆XopN. 【Conclusion】 XopN is a multifunctional effector and plays a virulence role in rice varieties that possess OsSWEET11 or its homologs. XopN is also necessary to the bacterial multiplication and has an additive contribution to the induction of hypersensitive response by the bacteria in a non-host plant.