Understanding cell-type specific immunity in plants

了解植物细胞类型特异性免疫

• 批准号: RGPIN-2020-04002
• 负责人: Germain, Hugo
• 金额: $ 3.42万
• 依托单位: Université du Québec à Trois-Rivières
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=739615
• 关键词: Understanding; cell; type; specific; immunity

The plant defence response system is described as a two-layer system. In the first layer, cell-surface pattern recognition receptors (PRR) detect conserved Pathogen-Associated Molecular Patterns (PAMPs). The second layer is composed of cytosolic Resistance (R) protein which detect pathogen-secreted effector from pathogenic microbes. However, plants lack adaptive immunity (antibodies) and it is generally assumed that they do not possess specialized immune cells as mammalian do, as such it is expected that all plant cells possess the same cell-surface receptors and R proteins repertoires. The latter is contradicted by transcriptomic data which demonstrates that different cell types, express PRRs at varying levels. In addition a recent report demonstrated that three different cell types display different transcriptomic responses to PRR activation. Our limited capacity to properly query individual plant cell type immune capacity has led researcher to devise generalist plant immunity models that assume that all plants cells are equally competent. The main goal of this project is to address a fundamental knowledge gap in plant biology: are all plant cells equally competent to launch an immune response (ETI and PTI). This single-cell type approach is a novel approach in plant biology but will bring a much deeper insight into the detailed mechanistic of plant immunity and has the potential to represent a major leap in plant immunity. In order to investigate the cell specificity of the immune response we are using a reporter line system that was first reported by Schürholz et al. (BioRxiv, 2018) which we have adapted and modified to our needs. Using this system, it is possible to use an inducer to turn on mTurquoise (a reporter gene) in a tissue specific manner; thus far we have 22 cell-type specific transgenic lines validated to express mTurquoise in 22 individual cell types (some from Wolf and some that we generated). In addition, we generated three additional constructs which harbor the pOp6 promoter driving the expression of three bacterial effectors (RSLM3, RPS4 and RPS1). Combining these systems, we have transformed the 22 transgenic lines with the effector-harbouring plasmid (pOp6-Effector) into protoplast made using the driver-lines. We can now express the three effectors in a cell-specific manner, and purify activated cells by FACS based on their mTurquoise fluorescence. The objectives that we want to achieve using this system are: 1) To monitor the functional capacity of the cell-specific protoplasts to respond to PTI or ETI induction 2) to perform a transcriptome profiling of specific cell-types in which we attempt to trigger ETI or PTI in which we do not detect an activation of the immune response. Hence, the two objectives will answer whether each cell-type is competent to respond to either the effectors or the PAMP and if they are not competent, the transcriptomic data will reveal the components of immunity which are missing.

植物防御响应系统被描述为两层系统。在第一层，细胞表面模式识别受体 (PRR) 检测保守的病原体相关分子模式 (PAMP)。第二层由胞质抗性（R）蛋白组成，可检测病原微生物分泌的病原体效应子。然而，植物缺乏适应性免疫（抗体），并且通常认为它们不像哺乳动物那样拥有专门的免疫细胞，因此预计所有植物细胞都拥有相同的细胞表面受体和 R 蛋白库。后者与转录组数据相矛盾，转录组数据表明不同的细胞类型以不同的水平表达 PRR。此外，最近的一份报告表明，三种不同的细胞类型对 PRR 激活表现出不同的转录组反应。我们正确查询单个植物细胞类型免疫能力的能力有限，这导致研究人员设计了通用植物免疫模型，假设所有植物细胞都具有同等能力。该项目的主要目标是解决植物生物学的基本知识空白：所有植物细胞是否都具有同等能力启动免疫反应（ETI 和 PTI）。这种单细胞类型的方法是植物生物学中的一种新颖方法，但将使人们更深入地了解植物免疫的详细机制，并有可能代表植物免疫的重大飞跃。为了研究免疫反应的细胞特异性，我们使用了 Schürholz 等人首次报道的报告系系统。 （BioRxiv，2018）我们已根据我们的需求进行了调整和修改。使用该系统，可以使用诱导剂以组织特异性方式开启 mTurquoise（报告基因）；到目前为止，我们已经验证了 22 种细胞类型特异性转基因系，可以在 22 种单独的细胞类型中表达 mTurquoise（一些来自 Wolf，一些是我们生成的）。此外，我们还生成了三个额外的构建体，其中包含驱动三个细菌效应子（RSLM3、RPS4 和 RPS1）表达的 pOp6 启动子。结合这些系统，我们将 22 个带有效应子携带质粒 (pOp6-Effector) 的转基因系转化为使用驱动系制成的原生质体。现在，我们可以以细胞特异性的方式表达这三种效应子，并根据激活的细胞的 mTurquoise 荧光通过 FACS 纯化它们。我们希望使用该系统实现的目标是：1) 监测细胞特异性原生质体对 PTI 或 ETI 诱导作出反应的功能能力 2) 对特定细胞类型进行转录组分析，其中我们尝试触发 ETI 或 PTI，但我们没有检测到免疫反应的激活。因此，这两个目标将回答每种细胞类型是否有能力对效应器或 PAMP 做出反应，如果它们没有能力，转录组数据将揭示缺失的免疫成分。