TOPP4与WAT1相互作用调控赤霉素和生长素信号通路互作

The cross talking between gibberellin and auxin signaling pathways is one of the hot topics in plant biology research, but their mechanism is still poorly understood. In our previous studies, we identified that the protein phosphatase TOPP4 regulates gibberellin signaling by dephosphorylating DELLA proteins. Based on these findings, we used topp4-1 mutant as background material to find out its suppressors by EMS mutagenesis. We found that topp4-1 wat1 double mutant can recover the gibberellin defect phenotypes of topp4-1 mutant. WAT1 encodes a vacuolar auxin transport facilitator. WAT1 protein specificly localizes to the vacuolar membrane of the cell. TOPP4 protein ubiquitously localizes to the cytoplasm, nucleus and plasma membrane. While co-expressing of the two proteins showed that WAT1 can change the subcellular localization of TOPP4 to the vacuolar membrane. And protein interaction assays showed that WAT1 can directly interact with TOPP4. The present project plans to take advantage of the genetic and molecular interaction between WAT1 and TOPP4 as the key to identify the possible function and position of WAT1 played in GA signaling and to further identify that WAT1 is a new factor in GA signaling by constructing double mutants with gibberellin biosynthesis and signal transduction mutants. The main goal is to reveal whether WAT1 is a substrate protein or a regulatory subunit of TOPP4 by dephosphorylation analysis and phosphatase activity assay based on the protein interaction. Through these experiments, we plan to find the mechanism of wat1 mutation restoring the phenotypes of topp4-1 mutant and the role of TOPP4 played in auxin signaling pathway. The project is expected to elucidate a new cross talking mechanism between gibberellin and auxin signaling pathways through WAT1 and TOPP4 proteins.

赤霉素和生长素之间的信号互作是植物学领域的研究热点，但二者之间的作用机理还知之甚少。我们的研究已经揭示了蛋白磷酸酶TOPP4通过对DELLA蛋白的去磷酸化调控了GA信号通路。在此基础上，前期实验通过筛选topp4-1 突变体的抑制子，发现生长素液泡膜转运蛋白WAT1的突变可以恢复其GA缺陷表型，WAT1能改变TOPP4的亚细胞定位，二者共定位并在液泡膜上相互作用。项目拟以WAT1和TOPP4的遗传与分子互作为切入点，通过WAT1与GA生物合成及信号转导通路突变体的遗传关系，揭示WAT1在其中的功能，确定WAT1为GA信号通路下游的新组分；通过TOPP4和WAT1蛋白互作、WAT1蛋白去磷酸化分析、TOPP4酶活性检测等，确定WAT1是作为TOPP4的底物蛋白还是调节亚基来恢复topp4-1突变体表型的机理，阐明TOPP4在生长素信号通路中的作用，揭示GA与生长素信号通路互作的新的分子机理。

磷酸化和去磷酸化修饰是蛋白质十分重要的修饰方式，广泛参与植物的多个发育和生理过程。拟南芥蛋白磷酸酶PP1家族基因TOPP4点突变体topp4-1矮化，高温培养可以恢复其表型。细胞死亡、活性氧水平及抗性基因PR的表达检测结果均表明topp4-1发生了免疫自激活，进一步的研究显示topp4-1是在细胞质中激活了免疫。与免疫通路关键基因突变体进行的遗传分析发现，topp4-1的免疫激活依赖于水杨酸途径和抗病蛋白，且TOPP4可与抗病信号通路的MAPK蛋白互作。.筛选topp4-1表型抑制子鉴定到SUT1基因，其编码一个未报道的CC类R蛋白。SUT1基因在幼嫩的组织中广泛表达，随着植物不断成熟，SUT1表达逐渐下降，在新生真叶中不再表达；SUT1蛋白主要定位在细胞质和细胞膜上。超表达SUT1蛋白CC结构域可激活免疫；SUT1蛋白通过CC结构域互作形成二聚体。蛋白互作和遗传分析确定SUT1可与TOPP4互作，且二者均可与调节CC类R蛋白活性和成熟的HSP90-SGT1b-RAR1复合体中的两个成员HSP90和RAR1在蛋白水平和遗传水平上互作，表明TOPP4和SUT1共同依赖于HSP90-SGT1b-RAR1复合体参与植物免疫。.此外，以35S-TOPP4-GFP/topp4-1恢复株系为材料，通过高通量亲和免疫共沉淀法，利用质谱鉴定到TOPP4的潜在调节亚基PP1R3，其抑制TOPP4蛋白磷酸酶活性，PP1R3功能缺失突变体对ABA敏感。酵母双杂筛选到PP1R3的互作蛋白MYBx，MYBx与TOPP4及PP1R3均互作，且MYBx受到MAPK3/6磷酸化调节。由此推测，TOPP4-PP1R3全酶与MAPK3/6相互拮抗，通过调节MYBx的磷酸化水平参与ABA信号通路。以上这些研究拓展了蛋白磷酸酶TOPP4的功能，揭示了TOPP4在植物抗病和ABA信号通路中的作用模式。.本项目执行期间，在国际刊物上发表 SCI 论文 5篇，中文核心期刊2篇，均已标注本项目基金号。在本项目资助下，共毕业研究生 21 名，其中博士生 8名，硕士生 13 名。期间1名博士留校任教，1名博士转为师资博士后。项目负责人于2018年被评为教育部“长江学者奖励计划”特聘教授。