Understanding cell-type specific immunity in plants

植物防御反应系统被描述为一个双层系统。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（PAMPs）。第二层由胞质耐药蛋白（cytosolic Resistance， R）组成，用于检测病原微生物的病原分泌效应物。然而，植物缺乏适应性免疫（抗体），一般认为它们不像哺乳动物那样具有特化的免疫细胞，因此，预计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，不同的细胞类型表达不同水平的PRRs。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。由于我们对单个植物细胞类型免疫能力的查询能力有限，研究人员设计了通用的植物免疫模型，假设所有植物细胞都具有同等的能力。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：是否所有植物细胞都具有启动免疫反应（ETI和PTI）的能力。这种单细胞类型的方法是植物生物学中的一种新方法，但将使我们对植物免疫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。为了研究免疫反应的细胞特异性，我们使用了sch<s:1> rholz等人（BioRxiv, 2018）首次报道的报告系系统，并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定的方式打开mTurquoise（一种报告基因）；到目前为止，我们已经有22个特定细胞类型的转基因系被验证可以在22个单独的细胞类型中表达mTurquoise（一些来自Wolf，一些来自我们自己）。此外，我们还生成了另外三个结构体，其中包含pOp6启动子，驱动三种细菌效应物（RSLM3， RPS4和RPS1）的表达。结合这些系统，我们将22个携带效应质粒（pOp6-Effector）的转基因系转化为利用驱动系制造的原生质体。我们现在可以以细胞特异性的方式表达这三种效应器，并根据它们的绿松石荧光用FACS纯化活化细胞。我们希望利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诱导反应的功能能力2)对特定细胞类型进行转录组分析，我们试图触发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物或PAMP作出反应，如果它们没有能力，转录组学数据将揭示缺失的免疫成分。

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.