Conservation of the PBL-RBOH immune module in land plants.

The rapid production of reactive oxygen species (ROS) is a key signaling output in plant immunity. In the angiosperm model species Arabidopsis thaliana (hereafter Arabidopsis), recognition of non- or altered-self elicitor patterns by cell-surface immune receptors activates the receptor-like cytoplasmic kinases (RLCKs) of the AVRPPHB SUSCEPTIBLE 1 (PBS1)-like (PBL) family, particularly BOTRYTIS-INDUCED KINASE1 (BIK1).1,2,3 BIK1/PBLs in turn phosphorylate the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) to induce apoplastic ROS production.4,5 PBL and RBOH functions in plant immunity have been extensively characterized in flowering plants. Much less is known about the conservation of pattern-triggered ROS signaling pathways in non-flowering plants. In this study, we show that in the liverwort Marchantia polymorpha (hereafter Marchantia), single members of the RBOH and PBL families, namely MpRBOH1 and MpPBLa, are required for chitin-induced ROS production. MpPBLa directly interacts with and phosphorylates MpRBOH1 at specific, conserved sites within its cytosolic N terminus, and this phosphorylation is essential for chitin-induced MpRBOH1-mediated ROS production. Collectively, our work reveals the functional conservation of the PBL-RBOH module that controls pattern-triggered ROS production in land plants.

A phosphoinositide hub connects CLE peptide signaling and polar auxin efflux regulation.

Auxin efflux through plasma-membrane-integral PIN-FORMED (PIN) carriers is essential for plant tissue organization and tightly regulated. For instance, a molecular rheostat critically controls PIN-mediated auxin transport in developing protophloem sieve elements of Arabidopsis roots. Plasma-membrane-association of the rheostat proteins, BREVIS RADIX (BRX) and PROTEIN KINASE ASSOCIATED WITH BRX (PAX), is reinforced by interaction with PHOSPHATIDYLINOSITOL-4-PHOSPHATE-5-KINASE (PIP5K). Genetic evidence suggests that BRX dampens autocrine signaling of CLAVATA3/EMBRYO SURROUNDING REGION-RELATED 45 (CLE45) peptide via its receptor BARELY ANY MERISTEM 3 (BAM3). How excess CLE45-BAM3 signaling interferes with protophloem development and whether it does so directly or indirectly remains unclear. Here we show that rheostat polarity is independent of PIN polarity, but interdependent with PIP5K. Catalytically inactive PIP5K confers rheostat polarity without reinforcing its localization, revealing a possible PIP5K scaffolding function. Moreover, PIP5K and PAX cooperatively control local PIN abundance. We further find that CLE45-BAM3 signaling branches via RLCK-VII/PBS1-LIKE (PBL) cytoplasmic kinases to destabilize rheostat localization. Our data thus reveal antagonism between CLE45-BAM3-PBL signaling and PIP5K that converges on auxin efflux regulation through dynamic control of PAX polarity. Because second-site bam3 mutation suppresses root as well as shoot phenotypes of pip5k mutants, CLE peptide signaling likely modulates phosphoinositide-dependent processes in various developmental contexts.

The Arabidopsis E3 ubiquitin ligase PUB4 regulates BIK1 and is targeted by a bacterial type-III effector.

Plant immunity is tightly controlled by a complex and dynamic regulatory network, which ensures optimal activation upon detection of potential pathogens. Accordingly, each component of this network is a potential target for manipulation by pathogens. Here, we report that RipAC, a type III-secreted effector from the bacterial pathogen Ralstonia solanacearum, targets the plant E3 ubiquitin ligase PUB4 to inhibit pattern-triggered immunity (PTI). PUB4 plays a positive role in PTI by regulating the homeostasis of the central immune kinase BIK1. Before PAMP perception, PUB4 promotes the degradation of non-activated BIK1, while after PAMP perception, PUB4 contributes to the accumulation of activated BIK1. RipAC leads to BIK1 degradation, which correlates with its PTI-inhibitory activity. RipAC causes a reduction in pathogen-associated molecular pattern (PAMP)-induced PUB4 accumulation and phosphorylation. Our results shed light on the role played by PUB4 in immune regulation, and illustrate an indirect targeting of the immune signalling hub BIK1 by a bacterial effector.

Evolutionary analysis of the LORELEI gene family in plants reveals regulatory subfunctionalization.

A signaling complex comprising members of the LORELEI (LRE)-LIKE GPI-anchored protein (LLG) and Catharanthus roseus RECEPTOR-LIKE KINASE 1-LIKE (CrRLK1L) families perceive RAPID ALKALINIZATION FACTOR (RALF) peptides and regulate growth, reproduction, immunity, and stress responses in Arabidopsis (Arabidopsis thaliana). Genes encoding these proteins are members of multigene families in most angiosperms and could generate thousands of signaling complex variants. However, the links between expansion of these gene families and the functional diversification of this critical signaling complex as well as the evolutionary factors underlying the maintenance of gene duplicates remain unknown. Here, we investigated LLG gene family evolution by sampling land plant genomes and explored the function and expression of angiosperm LLGs. We found that LLG diversity within major land plant lineages is primarily due to lineage-specific duplication events, and that these duplications occurred both early in the history of these lineages and more recently. Our complementation and expression analyses showed that expression divergence (i.e. regulatory subfunctionalization), rather than functional divergence, explains the retention of LLG paralogs. Interestingly, all but one monocot and all eudicot species examined had an LLG copy with preferential expression in male reproductive tissues, while the other duplicate copies showed highest levels of expression in female or vegetative tissues. The single LLG copy in Amborella trichopoda is expressed vastly higher in male compared to in female reproductive or vegetative tissues. We propose that expression divergence plays an important role in retention of LLG duplicates in angiosperms.

Ca2+ signals in plant immunity.

Calcium ions function as a key second messenger ion in eukaryotes. Spatially and temporally defined cytoplasmic Ca2+ signals are shaped through the concerted activity of ion channels, exchangers, and pumps in response to diverse stimuli; these signals are then decoded through the activity of Ca2+ -binding sensor proteins. In plants, Ca2+ signaling is central to both pattern- and effector-triggered immunity, with the generation of characteristic cytoplasmic Ca2+ elevations in response to potential pathogens being common to both. However, despite their importance, and a long history of scientific interest, the transport proteins that shape Ca2+ signals and their integration remain poorly characterized. Here, we discuss recent work that has both shed light on and deepened the mysteries of Ca2+ signaling in plant immunity.

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.

Regulation of immune receptor kinase plasma membrane nanoscale organization by a plant peptide hormone and its receptors.

Spatial partitioning is a propensity of biological systems orchestrating cell activities in space and time. The dynamic regulation of plasma membrane nano-environments has recently emerged as a key fundamental aspect of plant signaling, but the molecular components governing it are still mostly unclear. The receptor kinase FERONIA (FER) controls ligand-induced complex formation of the immune receptor kinase FLAGELLIN SENSING 2 (FLS2) with its co-receptor BRASSINOSTEROID-INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1), and perception of the endogenous peptide hormone RAPID ALKALANIZATION FACTOR 23 (RALF23) by FER inhibits immunity. Here, we show that FER regulates the plasma membrane nanoscale organization of FLS2 and BAK1. Our study demonstrates that akin to FER, leucine-rich repeat (LRR) extensin proteins (LRXs) contribute to RALF23 responsiveness and regulate BAK1 nanoscale organization and immune signaling. Furthermore, RALF23 perception leads to rapid modification of FLS2 and BAK1 nanoscale organization, and its inhibitory activity on immune signaling relies on FER kinase activity. Our results suggest that perception of RALF peptides by FER and LRXs actively modulates plasma membrane nanoscale organization to regulate cell surface signaling by other ligand-binding receptor kinases.

Direct inhibition of phosphate transport by immune signaling in Arabidopsis.

Soil availability of inorganic ortho-phosphate (PO43-, Pi) is a key determinant of plant growth and fitness.1 Plants regulate the capacity of their roots to take up inorganic phosphate by adapting the abundance of H+-coupled phosphate transporters of the PHOSPHATE TRANSPORTER 1 (PHT1) family2 at the plasma membrane (PM) through transcriptional and post-translational changes driven by the genetic network of the phosphate starvation response (PSR).3-8 Increasing evidence also shows that plants integrate immune responses to alleviate phosphate starvation stress through the association with beneficial microbes.9-11 Whether and how such phosphate transport is regulated upon activation of immune responses is yet uncharacterized. To address this question, we first developed quantitative assays based on changes in the electrical PM potential to measure active Pi transport in roots in real time. By inserting micro-electrodes into bulging root hairs, we were able to determine key characteristics of phosphate transport in intact Arabidopsis thaliana (hereafter Arabidopsis) seedlings. The fast Pi-induced depolarization observed was dependent on the activity of the major phosphate transporter PHT1;4. Notably, we observed that this PHT1;4-mediated phosphate uptake is repressed upon activation of pattern-triggered immunity. This inhibition depended on the receptor-like cytoplasmic kinases BOTRYTIS-INDUCED KINASE 1 (BIK1) and PBS1-LIKE KINASE 1 (PBL1), which both phosphorylated PHT1;4. As a corollary to this negative regulation of phosphate transport by immune signaling, we found that PHT1;4-mediated phosphate uptake normally negatively regulates anti-bacterial immunity in roots. Collectively, our results reveal a mechanism linking plant immunity and phosphate homeostasis, with BIK1/PBL1 providing a molecular integration point between these two important pathways.

Activation loop phosphorylation of a non-RD receptor kinase initiates plant innate immune signaling.

Receptor kinases (RKs) are fundamental for extracellular sensing and regulate development and stress responses across kingdoms. In plants, leucine-rich repeat receptor kinases (LRR-RKs) are primarily peptide receptors that regulate responses to myriad internal and external stimuli. Phosphorylation of LRR-RK cytoplasmic domains is among the earliest responses following ligand perception, and reciprocal transphosphorylation between a receptor and its coreceptor is thought to activate the receptor complex. Originally proposed based on characterization of the brassinosteroid receptor, the prevalence of complex activation via reciprocal transphosphorylation across the plant RK family has not been tested. Using the LRR-RK ELONGATION FACTOR TU RECEPTOR (EFR) as a model, we set out to understand the steps critical for activating RK complexes. While the EFR cytoplasmic domain is an active protein kinase in vitro and is phosphorylated in a ligand-dependent manner in vivo, catalytically deficient EFR variants are functional in antibacterial immunity. These results reveal a noncatalytic role for EFR in triggering immune signaling and indicate that reciprocal transphoshorylation is not a ubiquitous requirement for LRR-RK complex activation. Rather, our analysis of EFR along with a detailed survey of the literature suggests a distinction between LRR-RKs with RD- versus non-RD protein kinase domains. Based on newly identified phosphorylation sites that regulate the activation state of the EFR complex in vivo, we propose that LRR-RK complexes containing a non-RD protein kinase may be regulated by phosphorylation-dependent conformational changes of the ligand-binding receptor, which could initiate signaling either allosterically or through driving the dissociation of negative regulators of the complex.

Molecular mechanisms of early plant pattern-triggered immune signaling.

All eukaryotic organisms have evolved sophisticated immune systems to appropriately respond to biotic stresses. In plants and animals, a key part of this immune system is pattern recognition receptors (PRRs). Plant PRRs are cell-surface-localized receptor kinases (RKs) or receptor proteins (RPs) that sense microbe- or self-derived molecular patterns to regulate pattern-triggered immunity (PTI), a robust form of antimicrobial immunity. Remarkable progress has been made in understanding how PRRs perceive their ligands, form active protein complexes, initiate cell signaling, and ultimately coordinate the cellular reprogramming that leads to PTI. Here, we discuss the critical roles of PRR complex formation and phosphorylation in activating PTI signaling, as well as the emerging paradigm in which receptor-like cytoplasmic kinases (RLCKs) act as executors of signaling downstream of PRR activation.

Publisher Correction: The calcium-permeable channel OSCA1.3 regulates plant stomatal immunity.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The calcium-permeable channel OSCA1.3 regulates plant stomatal immunity.

Perception of biotic and abiotic stresses often leads to stomatal closure in plants1,2. Rapid influx of calcium ions (Ca2+) across the plasma membrane has an important role in this response, but the identity of the Ca2+ channels involved has remained elusive3,4. Here we report that the Arabidopsis thaliana Ca2+-permeable channel OSCA1.3 controls stomatal closure during immune signalling. OSCA1.3 is rapidly phosphorylated upon perception of pathogen-associated molecular patterns (PAMPs). Biochemical and quantitative phosphoproteomics analyses reveal that the immune receptor-associated cytosolic kinase BIK1 interacts with and phosphorylates the N-terminal cytosolic loop of OSCA1.3 within minutes of treatment with the peptidic PAMP flg22, which is derived from bacterial flagellin. Genetic and electrophysiological data reveal that OSCA1.3 is permeable to Ca2+, and that BIK1-mediated phosphorylation on its N terminus increases this channel activity. Notably, OSCA1.3 and its phosphorylation by BIK1 are critical for stomatal closure during immune signalling, and OSCA1.3 does not regulate stomatal closure upon perception of abscisic acid-a plant hormone associated with abiotic stresses. This study thus identifies a plant Ca2+ channel and its activation mechanisms underlying stomatal closure during immune signalling, and suggests specificity in Ca2+ influx mechanisms in response to different stresses.