Plant NLR immunity activation and execution: a biochemical perspective

Abstract

Plants deploy cell-surface and intracellular receptors to detect pathogen attack and trigger innate immune responses. Inside host cells, families of nucleotide-binding/leucine-rich repeat (NLR) proteins serve as pathogen sensors or downstream mediators of immune defence outputs and cell death, which prevent disease. Established genetic underpinnings of NLR-mediated immunity revealed various strategies plants adopt to combat rapidly evolving microbial pathogens. The molecular mechanisms of NLR activation and signal transmission to components controlling immunity execution were less clear. Here, we review recent protein structural and biochemical insights to plant NLR sensor and signalling functions. When put together, the data show how different NLR families, whether sensors or signal transducers, converge on nucleotide-based second messengers and cellular calcium to confer immunity. Although pathogen-activated NLRs in plants engage plant-specific machineries to promote defence, comparisons with mammalian NLR immune receptor counterparts highlight some shared working principles for NLR immunity across kingdoms.

Keywords: Ca2+; EDS1; TIR domain; helper NLR; resistosome; ribosylated nucleotide.

Figure 1.

Different plant NLR resistosomes converge on Ca²⁺ influx in immunity. Left and centre: sensor CNLs are conformationally activated by direct or indirect effector recognition, which leads to formation of a pentameric CNL resistosome. The CNL resistosome has a funnel-like structure (scarlet) which creates an autonomous Ca²⁺-permeable channel at the plasma membrane to promote immunity and cell death by increasing Ca²⁺ influx to the cytoplasm. CNLs of the NRC family serve as ‘helper’ or signalling NLRs following effector perception by sensor CNLs, which are not part of an NRC resistosome-like complex. Induced NRC oligomers might also be plasma membrane-bound Ca²⁺-permeable channels potentiating immune responses. Right: direct effector detection by a sensor TNL (eg. RPP1) leads to assembly of a tetrameric TNL resistosome with TIR-domain (dark blue) encoded NADase activity. The induced TNL NADase enzyme generates nucleotide-based small molecules, some of which are bound by EDS1 heterodimers to conformationally induce their association with co-functioning CC^HeLo-domain helper NLRs. EDS1 dimer-activated CC^HeLo-NLRs likely also mobilize immunity by forming resistosome-like membrane channels that promote Ca²⁺ influx into cells. (Displayed CNL pentameric resistosome is based on ZAR1 structure PDB: 6J5T. TNL resistosome is based on RPP1 structure PDB: 7CRC). Three-dimensional models were generated with UCSF Chimera software (www.rbvi.ucsf.edu/chimera). Figure was generated with Biorender.com.

Figure 2.

TIR-domain enzymatic activities at the heart of PTI and ETI responses. Two sets of TIR-domain catalysed ribosylated nucleotides conformationally activate two related but functionally distinct EDS1 heterodimer complexes. Specific nucleotide binding to EDS1-PAD4 or EDS1-SAG101 complexes promotes their association, respectively, with ADR1- or NRG1-family CC^HeLo-NLRs. The EDS1 dimer-activated ADR1 or NRG1 probably form CNL resistosome-like pentamers with Ca²⁺-permeable channel activities at the plasma membrane. Evidence suggests additional nuclear EDS1 dimer–CC^HeLo-NLR pools contribute to the immune response either as a Ca²⁺-permeable ion channel/pore at the nuclear membrane (shown) or as a functionally different nuclear complex promoting transcriptional reprogramming. In Arabidopsis, mutual defence potentiation between cell-surface PRR-triggered immunity (PTI) and intracellular effector-triggered immunity (ETI) machineries might be facilitated by transcriptionally or otherwise mobilized TNL- and TIR-only enzymes generating phosphoribosylated nucleotide intermediates pRib-ADP/AMP and ADPr-ATP/di-ADPR which, respectively, activate the EDS1-PAD4-ADR1 and EDS1-SAG101-NRG1 immunity branches. Question marks (?) indicate putative routes to transcriptional reprogramming. Figure was generated with Biorender.com.

Figure 3.

A broader role for TNLs in local to systemic immunity transmission. The scheme depicts a model for the spatial regulation of NLR-triggered immunity promoted by TNL-activated EDS1-family dimers with CC^HeLo-domain helper NLRs. In cells directly responding to pathogen attack through effector recognition (infected cell), sensor CNLs (autonomously or with CNL helpers) and sensor TNLs (via the EDS1-SAG101-NRG1 node) promote an ETI response leading to death of the responding cell. This reaction produces immunogenic signals and resistance-potentiating ROS and Ca²⁺ waves. Bystander cells (local cell) in the vicinity of the ETI-triggered cell perceive mobile defence signals such as endogenously generated peptides (phytocytokines) recognized by cell-surface receptors. These reactions create further defence propagation via the EDS1-PAD4-ADR1 node, the TNL SADR1 and likely other TNL and TIR-domain protein activities. EDS1-PAD4-ADR1 also promotes immunity in distal tissues (distal cell) through NHP and salicylic acid (SA) synthesis, together with actions of a ROS gradient (H₂O₂). How EDS1-PAD4-ADR1 would be activated in distal tissues is not known. Further TNLs and TIR-domain proteins might help to transduce and propagate immunogenic signals in systemic tissues. It is also possible that immunogenic molecules generated by locally triggered cells travel via the symplast (plasmodesmata cell-to-cell connectors) to activate defence in more distal cells. Figure was generated with Biorender.com.