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)。第二层由胞质抗性(R)蛋白组成，检测病原菌分泌的效应物。然而，植物缺乏获得性免疫(抗体)，通常认为它们不像哺乳动物那样拥有专门的免疫细胞，因此所有植物细胞都拥有相同的细胞表面受体和R蛋白谱系。后者与转录数据相矛盾，后者表明不同的细胞类型在不同的水平上表达PRR。此外，最近的一份报告显示，三种不同类型的细胞对PRR激活表现出不同的转录反应。我们正确询问单个植物细胞类型免疫能力的能力有限，这使得研究人员设计了通用的植物免疫模型，该模型假设所有植物细胞都具有同等的能力。这个项目的主要目标是解决植物生物学中的一个基本知识鸿沟：所有植物细胞是否都具有同等的能力来发起免疫反应(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，但将使人们对植物免疫的详细机制有更深入的了解，并有可能代表着植物免疫方面的一次重大飞跃。为了研究免疫反应的细胞特异性，我们使用了由Schürholz等人最先报道的报告线系统。(BioRxiv，2018)，我们已经根据我们的需求进行了调整和修改。使用这个系统，可以使用诱导剂以组织特异性的方式打开m绿松石(一种报告基因)；到目前为止，我们已经有22个细胞类型特定的转基因株被验证在22种单个细胞类型中表达m绿松石(一些来自沃尔夫，一些是我们产生的)。此外，我们还生成了三个含有pOp6启动子的额外构建物，这些启动子驱动三个细菌效应物(RSLM3、RPS4和RPS1)的表达。结合这些系统，我们将22个含有效应子的转基因株系(pOp6-Effector)转化为利用驱动系制备的原生质体。我们现在可以以细胞特异性的方式表达这三种效应物，并根据它们的m绿松石荧光通过流式细胞仪纯化激活的细胞。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对PTI或ETI诱导的功能能力2)执行特定细胞类型的转录组分析，在该转录组中，我们试图触发ETI或PTI，在该转录组中，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.