Periodic Reporting for period 4 - IMMUNO-PEPTALK (Regulation of plant receptor kinase-mediated immunity by endogenous peptides and their receptors)
Reporting period: 2022-12-01 to 2023-11-30
Multicellular organisms employ cell-surface receptors to survey the extracellular environment and adjust to changing conditions. Our work aims at deciphering the molecular mechanisms that control the function of these important sensors. In addition to providing important fundamental knowledge, this research ultimately has also the potential to inform strategies to develop better crops with increased yield and resilience to an ever-changing environment affected by global climate change. We are particularly interested to understand the regulatory function of some receptor kinases on others, and the role played by endogenous peptides in controlling such processes. As these receptors are embedded into the plasma membrane, we are interested to understand the role that the dynamic nanoscale organization of the plasma membrane plays in the regulation of receptor kinase complex assembly. Similarly, as plant cells are surrounded by a cell wall, we aim to understand the role played by the cell wall itself in regulating the perception of the external environment, as well as the mechanisms underlying the perception of cell wall properties. Our overall working model is that receptor kinases are an essential part of the continuum constituted by the external environment, the cell wall and the plasma membrane (and ultimately the interior of the cell), and that this continuum is shaped by the perception of endogenous peptides as part of a highly dynamic and interconnected biological system.
The first objective aimed at deciphering the regulation, function, and perception of RALF peptides by malectin-like receptor kinases (MLRKs) during immunity. We have re-annotated the Arabidopsis RALF family and analyzed their bioactivity and genetic dependence on FER (Abarca et al., Plant Physiol. 2021). Having determined the molecular basis of RALF23 perception by the heterotypic LLG1-FER complex (Xiao, Stegmann, Han et al., Nature 2019), we have shown that leucine-rich repeat extensin (LRX) proteins are also involved in the regulation of immune signaling by FER and RALF23 (Gronnier et al., eLife 2022; Noble et al., Plant Physiol. 2022). FER being a positive regulator of immune signaling, we have now identified several RALFs that positively regulate it and immune signaling (Fernández-Fernández, Abarca et al., in prep.). We have also identified additional RKs involved in RALF perception/signaling, which we are currently biochemically and genetically characterizing (Fernández-Fernández, Abarca et al., in prep.). In addition, In collaboration with colleagues in UK, we contributed to the characterization of additional MLRKs in other signaling pathways (Galindo-Trigo et al., EMBO Rep. 2020).
The second objective aimed at deciphering the formation, composition, and function of plasma membrane (PM) immune receptor nanoclusters. We showed that FER localizes to PM nanoclusters and regulates the lateral mobility of FLS2 and BAK1 within PM nanoclusters (Gronnier et al., eLife 2022). In collaboration with colleagues in France, we showed that FER regulates the localization of PM phosphatidylserine to regulate stress signaling (Smokvarska et al., Science Adv., 2023).
The third objective aimed to reveal the function of the LRR-receptor kinase MIK2 and its ligand(s) in immunity. Having identified MIK2 as the receptor for SCOOP peptides (Rhodes et al., Nature Comm. 2021), we now showed that the SCOOP family is much larger than anticipated (50 members) and identified the subtilases cleaving PROSCOOP proteins to generate bioactive peptides (Yang et al., Nature Plants, 2023; Rhodes & Zipfel, Methods Mol. Biol., 2024). Progress is being made to decipher MIK2 signaling in different pathways. In particular, we could identify that a single SCOOP peptide is linked to MIK2’s function in cell wall damage response (Zhai et al., in preparation), and we have identified several SCOOPs that might be involved in salt and drought responses. Finally, we recently pioneered a computational approach, combining phylogenomics and AI-based protein structural modeling, to infer the binding mechanisms of SCOOPs to MIK2 (Snoeck et al., bioRxiv 2024).
Furthermore, based on their transcriptional up-regulation upon immune signaling (like SCOOPs), we identified a novel family of phytocytonines conserved in Angiosperms, CTNIPs, and their cognate receptor, the LRR-RK HSL3 (Rhodes et al., eLife 2022). The characterization of CTNIPs and HSL3 is other plant species, such as grasses, legumes and Solanaceae is currently ongoing, as well as the analysis of Arabidopsis CTNIP-related peptides that we also identified based on bioinformatics searches.
The second objective aimed at deciphering the formation, composition, and function of plasma membrane (PM) immune receptor nanoclusters. We showed that FER localizes to PM nanoclusters and regulates the lateral mobility of FLS2 and BAK1 within PM nanoclusters (Gronnier et al., eLife 2022). In collaboration with colleagues in France, we showed that FER regulates the localization of PM phosphatidylserine to regulate stress signaling (Smokvarska et al., Science Adv., 2023).
The third objective aimed to reveal the function of the LRR-receptor kinase MIK2 and its ligand(s) in immunity. Having identified MIK2 as the receptor for SCOOP peptides (Rhodes et al., Nature Comm. 2021), we now showed that the SCOOP family is much larger than anticipated (50 members) and identified the subtilases cleaving PROSCOOP proteins to generate bioactive peptides (Yang et al., Nature Plants, 2023; Rhodes & Zipfel, Methods Mol. Biol., 2024). Progress is being made to decipher MIK2 signaling in different pathways. In particular, we could identify that a single SCOOP peptide is linked to MIK2’s function in cell wall damage response (Zhai et al., in preparation), and we have identified several SCOOPs that might be involved in salt and drought responses. Finally, we recently pioneered a computational approach, combining phylogenomics and AI-based protein structural modeling, to infer the binding mechanisms of SCOOPs to MIK2 (Snoeck et al., bioRxiv 2024).
Furthermore, based on their transcriptional up-regulation upon immune signaling (like SCOOPs), we identified a novel family of phytocytonines conserved in Angiosperms, CTNIPs, and their cognate receptor, the LRR-RK HSL3 (Rhodes et al., eLife 2022). The characterization of CTNIPs and HSL3 is other plant species, such as grasses, legumes and Solanaceae is currently ongoing, as well as the analysis of Arabidopsis CTNIP-related peptides that we also identified based on bioinformatics searches.
The major findings of this projet are:
1. Deciphering of RALF-binding mechanisms to FER (and other MLRKs) ((Xiao, Stegmann, Han et al., Nature 2019).
2. Genome-wide re-annotation and characterization of the RALF family in Arabidopsis ((Abarca et al., Plant Physiol. 2021; Fernández-Fernández, Abarca et al., in prep.).
3. Demonstration that FER regulates the nanoscale plasma membrane dynamics of immune receptor complexes (Gronnier et al., eLife 2022).
4. Identification of SCOOPs as MIK2 ligands, characterization of their perception mechanisms and roles in diverse signaling pathways (Rhodes et al., Nature Comm. 2021; Yang et al., Nature Plants, 2023; Snoeck et al., bioRxiv 2024; Zhai et al., in prep.).
5. Identification of CTNIP-HSL3 as a novel conserved stress responsive peptide-receptor module (Rhodes et al., eLife 2022).
A number of additional results are still being prepared for publication.
1. Deciphering of RALF-binding mechanisms to FER (and other MLRKs) ((Xiao, Stegmann, Han et al., Nature 2019).
2. Genome-wide re-annotation and characterization of the RALF family in Arabidopsis ((Abarca et al., Plant Physiol. 2021; Fernández-Fernández, Abarca et al., in prep.).
3. Demonstration that FER regulates the nanoscale plasma membrane dynamics of immune receptor complexes (Gronnier et al., eLife 2022).
4. Identification of SCOOPs as MIK2 ligands, characterization of their perception mechanisms and roles in diverse signaling pathways (Rhodes et al., Nature Comm. 2021; Yang et al., Nature Plants, 2023; Snoeck et al., bioRxiv 2024; Zhai et al., in prep.).
5. Identification of CTNIP-HSL3 as a novel conserved stress responsive peptide-receptor module (Rhodes et al., eLife 2022).
A number of additional results are still being prepared for publication.