Study of the roles and dynamics of plant processing bodies during bacterial infection

了解植物和病原体之间的分子对话对于制定有效和可持续的控制战略来防治目前威胁世界粮食安全的植物疾病至关重要。为了保护自己免受这些病原体的侵袭，植物的免疫在转录和转录后水平得到了有效的调节。MRNA被分割成翻译抑制的聚合体，称为加工体，是参与发育和胁迫反应的一个关键的转录后调控过程。来自宿主团队的初步结果表明，细菌植物病原体丁香假单胞菌(Pseudomonas syringae，PST)在感染时以效应器依赖的方式诱导PB的形成，并且PB缺陷的拟南芥对PST具有更强的耐受性。这表明PBS是植物免疫的负调节因子，可以作为细菌效应物的靶标。该项目的目的正是为了研究PBS作为植物免疫转录后调节因子的作用以及PST效应子对其进行调节的能力。为此，将采用遗传学、生物化学、蛋白质组学和细胞生物学相结合的方法来解决：1)PBS的动力学和作用，2)效应介导的PB形成的调节，以及3)PBS和自噬之间的相互作用，所有这些都是在兼容的植物-细菌相互作用的背景下进行的。拟议项目分为与上述三个目标相对应的三个工作包。在第一部分中，将研究PBS在感染时的动力学、参与免疫和组成。PB的形成动力学将通过共聚焦显微镜来确定；免疫的参与将通过PB缺陷突变体的防御反应和转录特征的表征来确定；以及纯化的PBS的蛋白质和RNA组成将分别通过质谱学和RNA测序来确定。在该项目的第二部分，将对PST效应器进行筛选，以了解它们单独调节PB形成、与PB标记共同定位以及与PB组分物理相互作用的能力。这种调节背后的机制将在HopM1的情况下进一步研究，HopM1是第一个被发现能够与PBS结合并调节其组装的效应器。在项目的第三部分，基于PBS与选择性自噬受体NBR1相互作用和共定位的初步结果，将探索PB形成和自噬之间的相互作用。为此，将产生表达PB标记和NBR1的转基因植物及其各自的突变体的不同组合，并对其进行鉴定，以从遗传上确定每个过程对彼此的贡献。总之，拟议的项目构成了一种创新和全面的战略，首次表征了PBS在植物-病原体相互作用中的作用，并确定了PBS和自噬之间的潜在相互作用。

Understanding the molecular dialogue between plants and pathogens is vital for developing effective and sustainable control strategies against the plant diseases that currently threaten food security worldwide. To protect themselves from these pathogens, plant immunity is efficiently regulated at the transcriptional and post-transcriptional level. The compartmentalization of mRNA into translationally repressed aggregates called processing bodies (PBs) is a key post-transcriptional regulatory process involved in development and stress responses. Preliminary results from the host team have shown that the bacterial plant pathogen Pseudomonas syringae (Pst) induces the formation of PB upon infection in an effector-dependent manner and that PB-defective Arabidopsis plants are more tolerant to Pst. This suggests that PBs are negative regulators of plant immunity that can be targeted by the bacterial effectors. The proposed project aims precisely at studying the role of PBs as post-transcriptional regulators of plant immunity and the ability of Pst effectors to modulate them. For this, a combination of genetic, biochemical, proteomic and cell biology approaches will be conducted to address: 1) the dynamics and roles of PBs, 2) the effector-mediated modulation of PB formation and 3) the interplay between PBs and autophagy, all in the context of a compatible plant-bacterium interaction. The proposed project is structured in three work packages corresponding to the three beforementioned objectives. In the first part, the dynamics, involvement in immunity and composition of the assembled PBs upon infection will be studied. The dynamics of PB formation will be determined by confocal microscopy; the involvement in immunity, by the characterization of defence responses and transcriptomic features of a PB-defective Arabidopsis mutant; and the protein and RNA composition of purified PBs, by mass spectrometry and RNA sequencing respectively. In the second part of the project, Pst effectors will be screened for their ability to singly modulate PB formation, co-localize with PB markers and physically interact with PB components. The mechanisms behind this modulation will be further studied in the case of HopM1, the first identified effector able to associate with PBs and modulate their assembly. In the third part of the project, the interplay between PB formation and autophagy will be explored based on the preliminary result that PBs interact and co-localize with the selective autophagy receptor NBR1. For this, different combination of transgenic plants expressing PB markers and NBR1 and the respective mutants will be generated and characterized to determine genetically the contribution of each process to each other. Altogether, the propose project constitutes an innovative and well-rounded strategy to characterize for the first time the role of PBs in a compatible plant-pathogen interaction and identify the potential interplay between PBs and autophagy.