Grant: Discovery Grants Program - Individual

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.