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: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=716762
• 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。在第一层中，检测到的细胞表面模式识别受体（PRR）构成了与病原体相关的分子模式（PAMP）。第二层由胞质抗性（R）蛋白组成，该蛋白从致病微生物中检测出病原体分泌的效应子。然而，植物缺乏适应性免疫学（抗体），通常认为它们不具有像哺乳动物的专门免疫细胞一样，因此可以预期所有植物细胞都具有相同的细胞表面受体和R蛋白质谱。后者与转录组数据相矛盾，这些数据表明，不同的细胞类型在不同水平上表达PRR。此外，最近的一份报告表明，三种不同的细胞类型对PRR激活显示不同的转录组响应。我们正确查询单个植物细胞类型免疫能力的能力有限，导致研究人员设计了通才植物免疫学模型，这些模型假定所有植物细胞均具有同样的能力。该项目的主要目标是解决植物生物学的基本知识差距：所有植物细胞都同样有能力发起免疫学反应（ETI和PTI）。这种单细胞类型的方法是植物生物学的一种新颖方法，但将对植物免疫学的详细机制有更深入的了解，并有可能代表植物免疫的重大飞跃。 为了研究免疫激素的细胞特异性，我们使用的是Schürholz等人首先报道的报告线系统系统。 （Biorxiv，2018）我们已经适应并根据我们的需求进行了修改。使用该系统，可以使用诱导的诱导以特定于组织的方式打开mturquoise（报告基因）。到目前为止，我们有22种细胞类型的特异性转基因线，以表达22种单个细胞类型的mturquoise（有些来自狼，有些是我们生成的）。此外，我们生成了另外三个构造，它们具有pop6启动子，驱动了三种细菌效应的表达（RSLM3，RPS4和RPS1）。结合了这些系统，我们将22条转基因线与效应子质质粒（POP6-效应器）转变为使用驱动器线制成的原生质体。现在，我们可以以细胞特异性的方式表达三种效果，并根据其Mturquoise荧光通过FACS纯化活化的细胞。我们要使用此系统实现的对象是：1）监测细胞特异性原生质体的功能能力，以响应PTI或ETI诱导2）执行特定细胞类型的转录组分析，我们试图触发ETI或PTI，在其中我们无法检测到免疫反应的激活。因此，这两个对象将回答每个细胞类型是否有能力响应效果或弹药症，如果它们不胜任，转录组数据将揭示缺失的免疫力组成部分。