Elucidating Context-Specific FERONIA Receptor Kinase Signaling Project Summary/Abstract Understanding how cellular signaling pathways achieve specificity is an important question in developmental biology. FERONIA (FER) receptor kinase is universally expressed and a critical regulator in plant growth, development and stress responses. Loss-of-function fer mutant has pleotropic effects with reduced plant growth, fertility and altered responses to stress. The goal of the proposal is to understand how FER signaling achieves specificity using root as a system in genetic model plant Arabidopsis. The primary roots offer both spatial and temporal information of root development, and our preliminary single cell RNA-sequencing (scRNA- seq) showed that FER-regulated gene expression in root is high cell type-specific, which makes root an ideal system to study FER signaling specificity. Multiple lines of evidence indicated that FER phosphorylates transcription factors to regulate gene expression and various biological processes. First, recent multiomics study demonstrated that FER regulates the expression of thousands of genes including large number of transcription factors (TFs), suggesting that transcriptome reprogramming is critical for FER-mediated signaling. Second, it has been demonstrated that FER phosphorylates transcription factors, MYC2 and ABI5, to regulate plant immunity and seed germination, respectively. Third, scRNA-seq analysis with fer mutant revealed that FER affects the expression of ~3,000 genes in roots in a highly cell type- and developmental stage-specific manner. Fourth, AKS (Abscisic acid-responsive Kinase Substrate) family of TFs are FER kinase substrates. Further genetic and functional data showed that they are major transcription factors functioning downstream of FER and FER is required for their transcriptional activity. AKS genes have cell type- or developmental stage- specific expression patterns while FER has a more universal expression, suggesting that FER modulates these TFs in different cell types in regulating their target gene expression and FER signaling specificity. Based on these results, it’s hypothesized FER phosphorylates and modulates the AKS TFs in specific cell types and developmental stages to regulate their target gene expression in achieving FER signaling specificity. scRNA- seq analysis, tissue-specific gene manipulation by CRISPR/cas9 and complementation, target gene identifications and transcriptional network analysis are proposed to test the hypothesis. In Research strategy section1, the genetic, genomic and functional interactions of FER and AKSs will be characterized by constructing genetic mutants, phosphosite mutations and identifying cell type-specific transcriptional targets. In Research strategy section 2, scRNA-seq of fer and aks mutants under control and salt treatment will be performed. In addition to the cell type-specific FER CRISPR knockout mutants we have already successfully constructed, we will also generate cell type-specific FER complementation lines. Further characterization of the cell type-specific CRISPR knockouts and complementation lines along with the gene regulatory network analysis will provide unprecedent new insights into how FER achieves signaling specificity.

Elucidating Context-Specific FERONIA Receptor Kinase Signaling Project Narrative The proposed studies will provide unprecedent new insights into how FER receptor kinase achieves signaling specificity. Receptor kinases signaling pathways play critical roles in diverse cellular processes and deregulation of the pathways leads to serious human diseases such as cancer. Understanding the pathway specificity has been a major area of study in developing effective therapies. Better understanding cell signaling specificity of FER receptor kinase can help shed light on receptor kinase signaling pathways related to human health.