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.

植物防御反应系统被描述为一个两层系统。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，其检测来自病原微生物的病原体分泌的效应物。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具有特化的免疫细胞，因此预期所有植物细胞都具有相同的细胞表面受体和R蛋白库。后者与转录组学数据相矛盾，转录组学数据表明不同的细胞类型以不同的水平表达PRR。此外，最近的报告表明，三种不同的细胞类型显示不同的转录组反应PRR激活。我们有限的能力，以适当查询个别植物细胞类型的免疫能力，导致研究人员设计的通才植物免疫模型，假设所有的植物细胞是同样的能力。该项目的主要目标是解决植物生物学中的一个基本知识缺口：所有植物细胞都同样有能力发起免疫反应（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。为了研究免疫应答的细胞特异性，我们使用了Schürholz等人（BioRxiv，2018）首次报告的报告细胞系系统，我们根据需要对其进行了调整和修改。使用该系统，可以使用诱导剂以组织特异性方式打开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，我们产生了三种另外的构建体，其具有驱动三种细菌效应子（RSLM 3、RPS 4和RPS 1）表达的pOp 6启动子。结合这些系统，我们已经转化了22个转基因株系与效应子携带质粒（pOp 6-Effector）到原生质体使用驱动线。我们现在可以以细胞特异性方式表达这三种效应物，并通过基于其mTurquoise荧光的FACS纯化活化细胞。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响应PTI或ETI诱导的功能能力2）进行特定细胞类型的转录组分析，其中我们尝试触发ETI或PTI，其中我们没有检测到免疫应答的激活。因此，这两个目标将回答每种细胞类型是否有能力响应效应物或PAMP，如果它们没有能力，转录组学数据将揭示缺失的免疫组分。