PopW具有激活植物免疫，促进植物抗病抗旱及植物生长等多方面的作用。作为一种harpin蛋白，在农作物的绿色防控方面有着非常广泛的应用前景。因此，解析PopW的调控机制，能够为其生防制剂应用提供重要的理论依据。实验室前期研究表明，PopW可诱导植物内源脱落酸（abscisic acid, ABA）的积累，并且能够通过气孔免疫抵御黄单胞菌Xanthomonas euvesicatoria（Xe）85-10的侵染。进一步筛选得到PopW互作蛋白SlSec13，发现其能够介导PopW诱导的气孔关闭，并参与免疫反应。本实验基于以上研究基础，筛选到了SlSec13互作蛋白SlPYR1，能够介导植物的ABA信号。以SlPYR1蛋白为主要研究对象，探索其在PopW诱导的植物免疫及抗病抗旱过程中的作用。具体研究结果如下：

1  ABA受体SlPYR1正向参与PopW诱导的早期免疫，并促进植物抗病抗旱

实验初期，对番茄SlSec13的互作蛋白进行筛选，获得了蛋白SlPYR1。通过酵母双杂、双分子荧光互补、免疫共沉淀、萤火素酶互补实验等实验方法验证了二者互作关系的稳定性。利用农杆菌介导的瞬时过表达技术，发现SlPYR1的过表达可以促进PopW诱导的植物早期免疫相关过程，即过表达SlPYR1能够对PopW诱导的气孔关闭、活性氧迸发、胼胝质沉积都产生促进作用。同时，利用病毒介导的基因沉默技术，沉默烟草相关同源基因NbPYR1-like、NbSec13b。在对照（pTRV2-GUS）和沉默植株上检测PopW诱导植物抗病性，结果表明，PopW处理后，与对照植株相比，NbPYR1-like沉默组植株仍能抑制Phytophthora capsici、Pseudomonas syringae pv. tomato（Pst）DC3000、Xe 85-10的侵染，但抗病性明显减弱。同样，在沉默植株上PopW诱导的抗旱能力也显著变弱，丙二醛含量升高，脯氨酸含量降低，表明PYR1对PopW诱导的植物抗旱十分重要。

2  PopW参与植物ABA信号的传递

免疫共沉淀实验发现PopW能够影响SlPYR1的二聚化状态。通过微量分子热涌动实验，证实了PopW可以促进SlPYR1与ABA的结合，并促进植物ABA复合体ABA-SlPYR1-SlABI1的形成。核质分离实验发现，PopW能够促进SlPYR1向细胞核内转移。PopW处理使SlOST1与SlABI1结合能力减弱，表明SlOST1被SlABI1释放。磷酸化实验显示，SlOST1磷酸化转录因子SlABF2能力增强。进一步利用荧光素酶报告基因系统，证明了PopW处理会促进SlABF2结合AREB序列，提高SlABF2的转录活性及稳定性。基于上述结果，我们推测了PopW通过调控ABA信号诱导植物免疫的模型：PopW处理使得植物内源ABA含量升高，促进了SlPYR1蛋白与ABA结合，促使SlABI1与SlPYR1、ABA形成复合体。并通过这种方式，释放SlOST1激酶，进一步激活转录因子SlABF2，从而启动ABA应答基因表达。

PopW has many functions such as activating plant immunity, promoting plant disease resistance and drought tolerance, and promoting plant growth. As a harpin protein, it has a very wide application prospect of crops in the green house. Therefore, it is very important to analyze its regulatory mechanism and provide a theoretical basis for its application as a biocontrol agent. Previous laboratory studies have shown that PopW induced the accumulation of endogenous abscisic acid in plants and can defend against pathogen Xanthomonas euvesicatoria (Xe) 85-10 infection through stomatal immunity. Screening of its interacting proteins revealed that SlSec13 can mediate PopW-induced stomatal closure and immune responses. Based on the above research basis, the specific regulatory mechanism was preliminarily explored, and the downstream interaction protein SlPYR1 was screened, which can mediate ABA signaling in plants. In this study, SlPYR1 protein was used as the main research object to explore its role in PopW-induced immune regulation and disease and drought tolerance. The specific results of the study are as follows:

1  ABA receptor PYR1 is positively involved in PopW-induced early immunity and promotes plant disease and drought tolerance

At the beginning of the experiment, the interaction protein of tomato SlSec13 was screened, and its downstream interaction protein SlPYR1 was clarified. The stability of the interaction between the two was verified by Yeast double-hybrid (Y2H), Bimolecular fluorescence complementation (BiFC), Co-precipitation for immunoprecipitation (CoIP), Luciferase complementation assay (LCA) and other experiments. Through Agrobacterium-mediated transient overexpression techniques, overexpression of SlPYR1 was found to promote PopW-induced plant immune-related processes. That is, overexpression of SlPYR1 can promote PopW-induced stomatal closure, burst of reactive oxygen species, and callus deposition. At the same time, virus-mediated gene silencing (VIGS) technology was used to silence tobacco-related homologous genes NbPYR1-like and NbSec13b. Inoculation of P. capsici, Pst DC3000 and Xe 85-10 on silent plants showed that NbPYR1-like silencing could lead to faster infection by P. capsici and also affect the colonization of Pst DC3000 and Xe 85-10 inhibited by PopW. Then, the drought simulation experiment was carried out with silent plants, and the drought tolerance of silent plants was significantly weakened, the malondialdehyde content increased, and the proline content decreased, indicating that PYR1 is very important for PopW-induced plant drought tolerance.

2  Positively modulation of ABA signaling by PopW

Immunoprecipitation experiments revealed that PopW could affect the dimerization state of SlPYR1. The MST assay confirmed that PopW could promote the binding of SlPYR1 to ABA and the formation of the plant ABA complex ABA-SlPYR1-SlABI1. Nucleoplasmic separation experiments revealed that PopW was able to promote the translocation of SlPYR1 into the nucleus. PopW treatment diminished the binding ability of SlOST1 to SlABI1, indicating that SlOST1 was released by SlABI1. Phosphorylation experiments showed that SlOST1 phosphorylation of the transcription factor SlABF2 was enhanced. At the same time, using the luciferase reporter system, it was proved that PopW treatment could promote the binding of SlABF2 to the AREB sequence and improve the transcriptional activity and stability of SlABF2. Based on the above results, we speculated that PopW regulated the model of ABA immunity in plants: PopW treatment increased the content of endogenous ABA in plants, promoted the binding of SlPYR1 protein to ABA, and promoted the formation of a complex between PP2C protein SlABI1 and SlPYR1 and ABA. Thereby the SlOST1 kinase is released, which further phosphorylates the transcription factor SlABF2, thereby activating ABA-responsive gene expression.

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