The tomato receptor CuRe1 senses a cell wall protein to identify Cuscuta as a pathogen

Parasitic plants of the genus Cuscuta penetrate shoots of host plants with haustoria and build a connection to the host vasculature to exhaust water, solutes and carbohydrates. Such infections usually stay unrecognized by the host and lead to harmful host plant damage. Here, we show a molecular mechanism of how plants can sense parasitic Cuscuta. We isolated an 11 kDa protein of the parasite cell wall and identified it as a glycine-rich protein (GRP). This GRP, as well as its minimal peptide epitope Crip21, serve as a pathogen-associated molecular pattern and specifically bind and activate a membrane-bound immune receptor of tomato, the Cuscuta Receptor 1 (CuRe1), leading to defense responses in resistant hosts. These findings provide the initial steps to understand the resistance mechanisms against parasitic plants and further offer great potential for protecting crops by engineering resistance against parasitic plants

Autophosphorylation-based calcium (Ca2+) sensitivity priming and Ca2+/Calmodulin inhibition of Arabidopsis thaliana Ca2+-dependent protein kinase 28 (CPK28)

Plant calcium (Ca2+) dependent protein kinases (CPKs) are composed of a dual specificity (Ser/Thr and Tyr) kinase domain tethered to a Calmodulin-like domain (CLD) via an autoinhibitory junction (J) and represent the primary Ca2+-dependent protein kinase activities in plant systems. While regulation of CPKs by Ca2+ has been extensively studied, the contribution of autophosphorylation in the control of CPK activity is less well understood. Furthermore, whether Calmodulin (CaM) contributes to CPK regulation, as is the case for Ca2+/CaM-dependent protein kinases (CaMKs) outside the plant lineage, remains an open question. We screened a subset of plant CPKs for CaM-binding and found that CPK28 is a high-affinity Ca2+/CaM-binding protein. Using synthetic peptides and native gel electrophoresis, we coarsely mapped the CaM-binding domain to a site within the CPK28 J domain that overlaps with the known site of intramolecular interaction between the J domain and CLD. Peptide kinase activity of fully dephosphorylated CPK28 was Ca2+-responsive and inhibited by Ca2+/CaM. Using in situ autophosphorylated protein, we expand on the known set of CPK28 autophosphorylation sites, and demonstrate that, unexpectedly, autophosphorylated CPK28 had enhanced activity at physiological concentrations of Ca2+ compared to dephosphorylated protein, suggesting that autophosphorylation functions to prime CPK28 for Ca2+-activation. Furthermore, CPK28 autophosphorylation substantially reduced sensitivity of the kinase to Ca2+/CaM inhibition. Overall, our analyses uncover new complexities in the control of CPK28 and provide mechanistic support for Ca2+ signaling specificity through Ca2+ sensor priming

Pathogen effector recognition-dependent association of NRG1 with EDS1 and SAG101 in TNL receptor immunity

Plants utilise intracellular nucleotide-binding, leucine-rich repeat (NLR) immune receptors to detect pathogen effectors and activate local and systemic defence. NRG1 and ADR1 “helper” NLRs (RNLs) cooperate with enhanced disease susceptibility 1 (EDS1), senescence-associated gene 101 (SAG101) and phytoalexin-deficient 4 (PAD4) lipase-like proteins to mediate signalling from TIR domain NLR receptors (TNLs). The mechanism of RNL/EDS1 family protein cooperation is not understood. Here, we present genetic and molecular evidence for exclusive EDS1/SAG101/NRG1 and EDS1/PAD4/ADR1 co-functions in TNL immunity. Using immunoprecipitation and mass spectrometry, we show effector recognition-dependent interaction of NRG1 with EDS1 and SAG101, but not PAD4. An EDS1-SAG101 complex interacts with NRG1, and EDS1-PAD4 with ADR1, in an immune-activated state. NRG1 requires an intact nucleotide-binding P-loop motif, and EDS1 a functional EP domain and its partner SAG101, for induced association and immunity. Thus, two distinct modules (NRG1/EDS1/SAG101 and ADR1/EDS1/PAD4) mediate TNL receptor defence signalling

Silencing of the Mitogen-Activated Protein Kinase MPK6 Compromises Disease Resistance in Arabidopsis

Here, we use a loss-of-function approach to demonstrate that the Arabidopsis (Arabidopsis thaliana) mitogen-activated protein kinase (MAPK) MPK6 plays a role in resistance to certain pathogens. MPK6-silenced Arabidopsis showed no apparent morphological phenotype or reduced fertility, indicating MPK6 is not required for development. However, resistances to an avirulent strain of Peronospora parasitica and avirulent and virulent strains of Pseudomonas syringae were compromised, suggesting that MPK6 plays a role in both resistance gene–mediated and basal resistance. Furthermore, this result demonstrates that MPK6's function cannot be fully complemented by other endogenous MAPKs. Although MPK6-silenced plants exhibited enhanced disease susceptibility, their ability to develop systemic acquired resistance or induced systemic resistance was unaffected. Expression of the pathogen-inducible gene VEGETATIVE STORAGE PROTEIN1 (VSP1) in MPK6-silenced plants was severalfold lower than in control plants, but the expression of other defense genes was comparable to the level observed in control plants. Taken together, these results provide direct evidence that a specific MAPK positively regulates VSP1 expression and resistance to a primary infection by certain pathogens, whereas systemic resistance and expression of several other defense genes appears to be mediated either by a functionally redundant MAPK(s) or independently from MPK6-dependent resistance

Phosphorylation-regulated activation of the Arabidopsis RRS1-R/RPS4 immune receptor complex reveals two distinct effector recognition mechanisms

The Arabidopsis RRS1-R/RPS4 immune receptor complex responds to bacterial effectors AvrRps4 and PopP2. Guo et al. show that effector-enhanced RRS1 N- and C-terminal proximity comprises a critical reconfiguration step involved in derepression of the complex. Further, RRS1-R C-terminal phosphorylation-dependent conformational reconfigurations distinguish responsiveness to the two effectors

A conserved module regulates receptor kinase signalling in immunity and development

Ligand recognition by cell-surface receptors underlies development and immunity in both animals and plants. Modulating receptor signalling is critical for appropriate cellular responses but the mechanisms ensuring this are poorly understood. Here, we show that signalling by plant receptors for pathogen-associated molecular patterns (PAMPs) in immunity and CLAVATA3/EMBRYO SURROUNDING REGION-RELATED peptides (CLEp) in development uses a similar regulatory module. In the absence of ligand, signalling is dampened through association with specific type-2C protein phosphatases. Upon activation, PAMP and CLEp receptors phosphorylate divergent cytosolic kinases, which, in turn, phosphorylate the phosphatases, thereby promoting receptor signalling. Our work reveals a regulatory circuit shared between immune and developmental receptor signalling, which may have broader important implications for plant receptor kinase-mediated signalling in general

Pathogen effector recognition-dependent association of NRG1 with EDS1 and SAG101 in TNL receptor immunity

Plants utilise intracellular nucleotide-binding, leucine-rich repeat (NLR) immune receptors to detect pathogen effectors and activate local and systemic defence. NRG1 and ADR1 “helper” NLRs (RNLs) cooperate with enhanced disease susceptibility 1 (EDS1), senescence-associated gene 101 (SAG101) and phytoalexin-deficient 4 (PAD4) lipase-like proteins to mediate signalling from TIR domain NLR receptors (TNLs). The mechanism of RNL/EDS1 family protein cooperation is not understood. Here, we present genetic and molecular evidence for exclusive EDS1/SAG101/NRG1 and EDS1/PAD4/ADR1 co-functions in TNL immunity. Using immunoprecipitation and mass spectrometry, we show effector recognition-dependent interaction of NRG1 with EDS1 and SAG101, but not PAD4. An EDS1-SAG101 complex interacts with NRG1, and EDS1-PAD4 with ADR1, in an immune-activated state. NRG1 requires an intact nucleotide-binding P-loop motif, and EDS1 a functional EP domain and its partner SAG101, for induced association and immunity. Thus, two distinct modules (NRG1/EDS1/SAG101 and ADR1/EDS1/PAD4) mediate TNL receptor defence signalling

Raw data for "Host-interactor screens of Phytophthora infestans RXLR proteins reveal vesicle trafficking as a major effector-targeted process"

This dataset contains raw and original images, phylogenetic tree files, sequence alignment files used for phylogenetic tree construction and unprocessed data for figures presented in the manuscript titled "Host-interactor screens of Phytophthora infestans RXLR proteins reveal vesicle trafficking as a major effector-targeted process". Each zip file contains raw data for each figure in the manuscript. A version of the manuscript is available on bioRxiv with doi.org/10.1101/2020.09.24.308585