What's new in protein kinase/phosphatase signalling in the control of plant immunity?

Abstract

Plant immunity is crucial to plant health but comes at an expense. For optimal plant growth, tight immune regulation is required to prevent unnecessary rechannelling of valuable resources. Pattern- and effector-triggered immunity (PTI/ETI) represent the two tiers of immunity initiated after sensing microbial patterns at the cell surface or pathogen effectors secreted into plant cells, respectively. Recent evidence of PTI-ETI cross-potentiation suggests a close interplay of signalling pathways and defense responses downstream of perception that is still poorly understood. This review will focus on controls on plant immunity through phosphorylation, a universal and key cellular regulatory mechanism. Rather than a complete overview, we highlight "what's new in protein kinase/phosphatase signalling" in the immunity field. In addition to phosphoregulation of components in the pattern recognition receptor (PRR) complex, we will cover the actions of the major immunity-relevant intracellular protein kinases/phosphatases in the 'signal relay', namely calcium-regulated kinases (e.g. calcium-dependent protein kinases, CDPKs), mitogen-activated protein kinases (MAPKs), and various protein phosphatases. We discuss how these factors define a phosphocode that generates cellular decision-making 'logic gates', which contribute to signalling fidelity, amplitude, and duration. To underscore the importance of phosphorylation, we summarize strategies employed by pathogens to subvert plant immune phosphopathways. In view of recent game-changing discoveries of ETI-derived resistosomes organizing into calcium-permeable pores, we speculate on a possible calcium-regulated phosphocode as the mechanistic control of the PTI-ETI continuum.

Keywords: Phosphocode; calcium-dependent protein kinases; kinase; mitogen-activated protein kinases; phosphatase; signalling.

Figure 1. Overview of phospho-dependent immune signalling facilitated by endogenous protein kinases, edited by phosphatases, and manipulated by pathogen-derived effectors

At the surface of the plant cell, pathogen-derived molecules (e.g. fungal chitin or bacterial flg22/elf18; in red) are recognized by PRR complexes comprised of RLK receptors LYK5, FLS2, or EFR (light blue) and coreceptors CERK1 and BAK1 (dark blue) embedded in the plasma membrane (PM). The FLS2-BAK1/EFR-BAK1 complexes are also regulated through membrane nanodomains assisted by the FER/LLG1 scaffold in a RALF-dependent manner. On the cytoplasmic side, phosphorylation (orange circles marked with P) activates PRR signalling, leading to the phosphorylation and release of RLCKs of the VII subfamily, including BIK1 (purple rectangles). Subfamily VII RLCKs activate the MAPK cascade (green), while BIK1 phosphorylation of CNGC calcium channels (yellow) and RBOHD (fuchsia,) activates calcium (yellow orbs) influx and ROS (fuchsia stars) production, respectively. CPKs (yellow pentagon) sense and decode the calcium signals and write the phosphocode on diverse targets. On the PM, CPK5, as well as additional protein kinases (gray box, and BIK1), mediate phosphorylation of RBOHD to guarantee ROS production, which can induce further calcium influx and thus form feed-forward calcium-ROS amplification loops. In the nucleus, CPK5 targets transcription factors, some of which are commonly phosphorylated by MAPKs (e.g. WRKY33 or CAMTA3). The distinct phosphosite specificities of MAPKs and CPKs generate a phosphocode-defined ‘logic gate’ that dictates transcriptional reprogramming during defense. Protein phosphatases act in opposition to protein kinases at multiple levels of the immune phosphocascade (targets shown as green stars), erasing the phosphorylation marks. Effectors (red orbs) injected into the cytosol by pathogenic bacteria may be recognized in resistant plants through NLRs (blue). Recently it was shown that an effector-modified RLCK serves as a ligand to trigger oligomerization of a coiled-coil-type NLR into a calcium-permeable pore, further increasing cytosolic calcium flux. In susceptible plants, effector activity mimics or hijacks endogenous control mechanisms to rewrite the phosphocode and ultimately suppress immunity at all levels (example targets mentioned in this review are marked with red lightning bolts).

Figure 2. Regulatory phosphocode through multisite phosphorylation

Schematic models of how multisite phosphorylation on a single protein (conceptually valid for both protein kinases and their substrates) constitutes a regulatory phosphocode. (A) Sequential or consecutive phosphorylation by one or different protein kinases may generate a graded response that is tuneable by actions of opposing protein kinases and phosphatases present. An example is when all the phosphomarks result in the same outcome such as degradation (exemplified by MAPK substrates mentioned in this review), where the frequency of modified phosphodegron motifs will correlate with the likelihood of engagement by ubiquitin-proteasome machineries, and therefore increased removal as outcome. (B) Logic gating represents more complex decision-making that is important for phosphocode-dependent regulation of protein functions. Three hypothetical scenarios are illustrated here: (1) Outcome A arising if either phosphosite 1 OR 2 are modified; (2) Outcome B occurs only if both phosphosite 3 AND 4 are phosphorylated; or (3) Outcome C, a follow-up situation where, additionally, site 5 must NOT be phosphorylated. Phosphocode-dependent subfunctionalization of BAK1 or CPK28 or the convergence of MPK3/6 and CPK5 on transcription factors WRKY33 and CAMTA3 represent such situations. (C) Response safeguard represents the scenario where multiple independent protein kinases target one or more key phosphosite(s) required for full activity of the substrate protein. Using Outcome B (in b) as an example, if K1 is inactivated, a safeguarding second protein kinase (K2) with overlapping phosphosite specificity will ensure activity maintenance. An example for this scenario is the convergence of several protein kinases on RBOHD.