大白菜-根肿菌互作中关键糖转运蛋白SWEETs的挖掘和功能分析

SWEETs are a class of proteins responsible for transporting sugars across cell membranes, being crucial to regulate carbon transport and allocation in the host plants and pathogen interaction. Multiple SWEET genes have been identified to be involved in plant and microbiome interaction. However, the regulatory mechanism of SWEET-mediated sugar transport and accumulation in galls caused by Plasmodiophora brassicae infection as a ‘Sink’ is very less known. Previously, we characterized 32 SWEET family genes and their expression pattern in Chinese cabbage, and also found that P. brassicae could promote sugar accumulation in the infected roots. In this study, we propose to further define the key SWEET genes involved in the interaction between Chinese cabbage and P. brassicae based on the expression profiling of 32 SWEET genes, changes of sugars contents in Chinese cabbage after infection by P. brassicae and the SWEET activity identified by others. We also propose to analyze the interactions between SWEET genes by in vivo and in vitro experiments after validation of SWEET protein activity for sugar transport. To understand the molecular mechanism of SWEET in sugar transport regulation during the Chinese cabbage-P. bassicae interaction, the response to P. brassica, sugar content and other importent traits will be analyzed by using Chinese cabbage gene-silencing transgenic plants and Arabidopsis gain-of-function mutants of the key SWEET genes. Our results would provide new insight into pathogenesis of P. bassicae and genetic improvement of clubroot resistance.

SWEET是植物中一类跨膜转运糖的易化扩散蛋白家族，对调控寄主-病原菌互作中的碳素转运与分配具有重要意义。目前已鉴定出参与植物与真菌和细菌等病原菌互作中的多个关键SWEET基因。但根肿菌（原生动物）侵染造成的根瘤这一“库”组织中SWEET对介导糖供给与积累的调控机理尚无报道。本项目在获得大白菜32个SWEET基因及其表达特性、根肿菌可促进糖在根系的积累等研究基础上，拟结合接种根肿菌后大白菜SWEET表达差异、含糖量变化和前人研究基础，明确参与大白菜-根肿菌互作中的关键SWEET；在验证SWEET蛋白糖转运活性的基础上，通过体内和体外试验分析基因之间的互作关系；在获得SWEET基因的大白菜沉默转基因植株和拟南芥功能获得突变体的基础上，通过分析根肿病抗性、含糖量和其它性状，明确在大白菜-根肿菌互作中SWEET调控糖转运的分子机理，为研究根肿菌致病机理和抗根肿病的遗传改良提供理论依据。

SWEET是植物中一类跨膜转运糖的易化扩散蛋白家族，对调控寄主-病原菌互作中的碳素转运与分配具有重要意义。目前已鉴定出参与植物与真菌和细菌等病原菌互作中的多个关键SWEET基因。但根肿菌（原生动物）侵染造成的根瘤这一“库”组织中SWEET对介导糖供给与积累的调控机理尚无报道。此前，我们已经鉴定了大白菜32个SWEETs基因及其表达特性，并发现根肿菌可促进糖在根系的积累，结合接种根肿菌后大白菜SWEETs基因表达差异，我们筛选到五个关键的SWEET基因（BrSWEET1a、2a、11a、12、14a），其在接种根肿菌后表达水平显著上调。拟南芥同源SWEET1、11、12基因的β-葡萄糖苷酸酶(Gus)表达分析表明根肿菌侵染显著增加了其在叶片和根中的表达水平。己糖功能缺陷型酵母EBY4000功能互补实验表明BrSWEET1a和BrSWEET11a是葡萄糖转运蛋白，BrSWEET11a也具有果糖转运活性。亚细胞定位结果显示5个关键BrSWEETs蛋白均定位于质膜上。利用膜蛋白的分裂泛素酵母双杂交 (mbSUS)系统和双分子荧光互补实验表明五种 BrSWEETs 可以像AtSWEETs 一样进行同源和异源寡聚化，表明 BrSWEET 寡聚化以执行糖运输功能。拟南芥sweet突变体在发病率和病害指数方面与野生型植物相比差异较小，而功能获得型植物的发病率显着高于野生型植物。对受感染根进行石蜡切片显微观察表明，接种后16天，sweet1、sweet12突变体和野生型根部均未出现根瘤形成，而在功能获得型植株中可观察到明显的瘿积累。四个阶段功能获得植物的相对病原体DNA含量在感染根中显着高于野生型。利用VIGS技术分别单基因沉默BrSWEET1a，11a及双基因沉默BrSWEET1a/11a,经沉默处理组植株的发病率及病情指数显著低于野生型。.综上，我们的研究结果表明，这五个 BrSWEETs 基因在根肿菌诱导的瘤形成中发挥了关键作用，这为非根肿病抗性 (CR) 基因育种提供了新的见解。