青枯菌假尿苷合成酶RluX的功能鉴定及其决定致病过程的分子机理研究

Ralstonia solanacearum is the causal agent of bacterial wilt and has been established as a model system to decipher the bacterial plant pathogenesis. The syringe-like type III secretion system (T3SS) plays a crucial role in the pathogenicity of R. solanacearum and the bacteria globally regulate the T3SS via a complex network. In the current research, we identified and confirmed that RluX can significantly affect the expression of T3SS and is essential for the pathogenicity of R. solanacearum toward host plants (tomato and tobacco plants), and the rluX mutant can also cause strong antagonism against the wild-type strain in tomato plant. According to the BLAST result through NCBI database, the RluX is suggested as a putative pseudouridine synthase of Rlu family, and it might be responsible for the pseudouridine modification in the large subunit of 23s rRNA in bacteria. The pseudouridine modification plays important roles during many biological processes among many species including eukaryotic and prokaryotic cells. But to date, it is barely reported that the pseudouridine modification can significantly affect the pathogenicity of pathogens toward host cells. Based on this interesting finding, we propose this research project to confirm the significant effect of pseudouridine modification on the pathogenicity of plant pathogens by the functional identification of RluX as pseudouridine synthase through the CMC specific modification and reverse transcription. This is the first time to report that pseudouridine modification will play crucial roles on pathogenesis of pathogens. We will identify the substrate RNA of RluX and reveal the working model of RluX to make pseudouridine modification on its substrate RNA. We will also reveal how RluX significantly affect the expression of T3SS, determines the pathogenicity of R. solanacearum toward host plants and makes strong antagonism against the wild-type strain. As a result, we will reveal the molecular mechanism how the pseudouridine modification by RluX determines the pathogenecity of R. solanacearum toward host plants.

青枯菌是研究植物病原细菌与寄主植物互作的模式系统之一，注射器状的三型分泌系统（T3SS）是其对寄主致病的一个决定因子，青枯菌运用精密网络对T3SS进行全局性调控。我们国内外首次证实青枯菌RluX显著影响其T3SS表达、决定其致病性且RluX在拮抗青枯病过程中起重要作用。经NCBI数据库比对，RluX可能是Rlu家族的假尿苷合成酶，负责对其23s rRNA大亚基进行假尿苷修饰，但迄今，关于假尿苷修饰严重影响病原菌致病性的研究，国内外少有报道。本研究拟通过CMC特异修饰-反转录技术等对RluX的假尿苷合成酶进行鉴定，确定假尿苷修饰决定病原菌致病性这一重大发现；鉴定RluX的底物RNA并探究其对底物进行假尿苷修饰的工作模式；研究RluX影响青枯菌T3SS表达的分子途径、探究RluX在青枯菌侵染寄主植物及拮抗青枯菌过程中的直接或间接作用，最终揭示假尿苷修饰决定青枯菌对寄主植物致病性的分子机理。

青枯菌是研究植物病原细菌与寄主植物互作的模式系统之一，注射器状的三型分泌系统（T3SS）是其对寄主致病的一个决定因子，青枯菌运用精密网络对T3SS进行全局性调控。青枯菌基因组中共有7个假定的假尿苷合成酶，我们国内外首次证实RluX显著影响其T3SS表达、决定其致病性，而其它六个假尿苷合成酶和T3SS表达、侵染过程无关。当该六个假尿苷合成酶全部敲除时，突变体展现出稍微减弱的致病性，但T3SS表达不受影响。进一步通过基因敲除、功能回补、CMC特异修饰-反转录等实验揭示RluX通过对23s rRNA大亚基进行假尿苷修饰、影响T3SS核心调控因子HrpG的转录表达强度、进而调控HrpB-T3SS的转录表达。此外，RluX也显著影响青枯菌生物膜形成、胞外多糖生成、泳动和颤动能力、植物体内生长繁殖及导管内迁移能力，这可能是其决定致病力的另一个主要原因。最终，基本揭示了RluX通过影响假尿苷修饰影响青枯菌T3SS表达、决定青枯菌对寄主植物致病性的分子机理。结果有助于进一步理解病原细菌假尿苷合成酶的生物学功能、青枯菌T3SS调控表达的全局性网络。