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策略以应对目前威胁全球粮食安全的植物疾病至关重要。为了保护自身免受这些病原体的侵害，植物免疫在转录和转录后水平上被有效地调节。mRNA区室化成被称为加工体（PB）的被抑制的聚集体是参与发育和应激反应的关键转录后调节过程。来自宿主团队的初步结果表明，细菌性植物病原体假单胞菌（Pst）在感染后以效应子依赖的方式诱导PB的形成，并且PB缺陷型拟南芥植物对Pst更耐受。这表明PB是植物免疫的负调节剂，可以被细菌效应子靶向。拟议的项目的目的正是研究PBs作为植物免疫的转录后调节因子的作用，以及Pst效应子调节它们的能力。为此，将进行遗传学，生物化学，蛋白质组学和细胞生物学方法的组合，以解决：1）PB的动力学和作用，2）效应子介导的PB形成调节和3）PB和自噬之间的相互作用，所有这些都是在相容的植物-细菌相互作用的背景下进行的。拟议的项目分为三个工作包，分别对应于上述三个目标。在第一部分中，将研究感染后组装的PB的动力学、参与免疫和组成。PB形成的动力学将通过共聚焦显微镜来确定;参与免疫，通过表征PB缺陷型拟南芥突变体的防御反应和转录组学特征;以及纯化的PB的蛋白质和RNA组成，分别通过质谱和RNA测序。在该项目的第二部分中，将筛选Pst效应子，以确定其单独调节PB形成、与PB标记物共定位以及与PB组分物理相互作用的能力。这种调制背后的机制将进一步研究的情况下，HopM 1，第一个确定的效应器能够与PB和调节其组装。在该项目的第三部分中，PB形成和自噬之间的相互作用将基于PB与选择性自噬受体NBR 1相互作用和共定位的初步结果进行探索。为此，将产生并表征表达PB标记和NBRl的转基因植物和相应突变体的不同组合，以在遗传上确定每个过程对彼此的贡献。总而言之，拟议项目构成了一个创新和全面的战略，首次表征PB在相容的植物-病原体相互作用中的作用，并确定PB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.