Table_3_Integrative Proteomic and Phosphoproteomic Analyses of Pattern- and Effector-Triggered Immunity in Tomato.XLSX

Plants have evolved a two-layered immune system consisting of pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). PTI and ETI are functionally linked, but also have distinct characteristics. Unraveling how these immune systems coordinate plant responses against pathogens is crucial for understanding the regulatory mechanisms underlying plant defense. Here we report integrative proteomic and phosphoproteomic analyses of the tomato-Pseudomonas syringae (Pst) pathosystem with different Pst mutants that allow the dissection of PTI and ETI. A total of 225 proteins and 79 phosphopeptides differentially accumulated in tomato leaves during Pst infection. The abundances of many proteins and phosphoproteins changed during PTI or ETI, and some responses were triggered by both PTI and ETI. For most proteins, the ETI response was more robust than the PTI response. The patterns of protein abundance and phosphorylation changes revealed key regulators involved in Ca2+ signaling, mitogen-activated protein kinase cascades, reversible protein phosphorylation, reactive oxygen species (ROS) and redox homeostasis, transcription and protein turnover, transport and trafficking, cell wall remodeling, hormone biosynthesis and signaling, suggesting their common or specific roles in PTI and/or ETI. A NAC (NAM, ATAF, and CUC family) domain protein and lipid particle serine esterase, two PTI-specific genes identified from previous transcriptomic work, were not detected as differentially regulated at the protein level and were not induced by PTI. Based on integrative transcriptomics and proteomics data, as well as qRT-PCR analysis, several potential PTI and ETI-specific markers are proposed. These results provide insights into the regulatory mechanisms underlying PTI and ETI in the tomato-Pst pathosystem, and will promote future validation and application of the disease biomarkers in plant defense.植物已进化出由模式触发免疫（pattern-triggered immunity, PTI）和效应子触发免疫（effector-triggered immunity, ETI）组成的双层免疫系统。PTI与ETI在功能上相互关联，但亦具备独特的分子特征。阐明这两类免疫系统如何协同调控植物对病原物的应答，对于解析植物防御的核心调控机制至关重要。本研究针对番茄-丁香假单胞菌（Pseudomonas syringae, Pst）致病互作系统，利用不同的Pst突变体实现对PTI与ETI的拆分解析，开展了整合蛋白质组学与磷酸化蛋白质组学分析。研究共鉴定到在Pst侵染番茄叶片过程中差异积累的225种蛋白质与79种磷酸肽。在PTI或ETI激活过程中，众多蛋白质及磷酸化蛋白质的丰度发生显著改变，部分免疫应答可同时被PTI与ETI触发。对于绝大多数蛋白质而言，ETI介导的应答强度显著高于PTI介导的应答。蛋白质丰度与磷酸化修饰的变化模式揭示了参与钙信号通路、丝裂原活化蛋白激酶级联反应、可逆蛋白质磷酸化、活性氧（reactive oxygen species, ROS）与氧化还原稳态、转录与蛋白质周转、转运与运输、细胞壁重塑、激素生物合成与信号转导的关键调控因子，提示它们在PTI和/或ETI中发挥共同或特异性的调控作用。此前转录组学研究中鉴定到的两个PTI特异性基因——NAC（NAM、ATAF与CUC家族）结构域蛋白与脂质颗粒丝氨酸酯酶，在蛋白质水平未被检测到差异调控，且未被PTI诱导表达。结合整合转录组学与蛋白质组学数据，以及实时定量逆转录PCR（qRT-PCR）分析，本研究提出了若干潜在的PTI与ETI特异性标记物。本研究结果为解析番茄-Pst致病互作系统中PTI与ETI的调控机制提供了全新视角，将推动植物防御相关疾病生物标记物的后续验证与应用推广。