Comparative Analysis of the Flax Immune Receptors L6 and L7 Suggests an Equilibrium-Based Switch Activation Model

Abstract

NOD-like receptors (NLRs) are central components of the plant immune system. L6 is a Toll/interleukin-1 receptor (TIR) domain-containing NLR from flax (Linum usitatissimum) conferring immunity to the flax rust fungus. Comparison of L6 to the weaker allele L7 identified two polymorphic regions in the TIR and the nucleotide binding (NB) domains that regulate both effector ligand-dependent and -independent cell death signaling as well as nucleotide binding to the receptor. This suggests that a negative functional interaction between the TIR and NB domains holds L7 in an inactive/ADP-bound state more tightly than L6, hence decreasing its capacity to adopt the active/ATP-bound state and explaining its weaker activity in planta. L6 and L7 variants with a more stable ADP-bound state failed to bind to AvrL567 in yeast two-hybrid assays, while binding was detected to the signaling active variants. This contrasts with current models predicting that effectors bind to inactive receptors to trigger activation. Based on the correlation between nucleotide binding, effector interaction, and immune signaling properties of L6/L7 variants, we propose that NLRs exist in an equilibrium between ON and OFF states and that effector binding to the ON state stabilizes this conformation, thereby shifting the equilibrium toward the active form of the receptor to trigger defense signaling.

Signaling from the plasma-membrane localized plant immune receptor RPM1 requires self-association of the full-length protein

“…1F and SI Appendix, SI Materials and Methods). The loss-of-function phenotype of the cis double mutant (26) is likely due to a continual requirement for the P-loop in ATP binding and turnover (22,23). Thus, we interpret our results as follows: RPM1 selfassociation depends on nucleotide binding, and a P-loop mutation alters NB-ARC domain structure or intramolecular domain interactions in such a way that both resting-state and active-state RPM1 self-association is inhibited or blocked.…”

“…The current model is that NLRs exist in an equilibrium between a "resting/off" ADP-bound state and an "active/on" ATP-bound state in which the "off" state is strongly favored (22,23). NLRs can be activated either directly by binding an effector protein or indirectly by monitoring an effector-specific modification of a host target (or a host decoy of a true target) (24,25).…”

“…Effector recognition/binding presumably leads to the release of negative intramolecular regulation and conformational changes allowing the exchange of ADP for ATP, thereby tipping the balance toward the ATP-bound "on" state. Recent work on the flax TNLs L6 and L7 suggests that, at least in cases where NLRs directly bind effectors, the effector preferably binds to the ATP-bound "on" state of the receptor, stabilizing this state and preventing recycling to the "off" state (22). In this model, ATP hydrolysis drives a return to the "off" state, thus regulating the switch.…”

The CC domain structure from the wheat stem rust resistance protein Sr33 challenges paradigms for dimerization in plant NLR proteins

“…We propose a similar model of signaling for the Sr33, Sr50, and MLA10 CC-NLRs, in which the transient self-association of the CC domain is stabilized by the full-length NLR to achieve the activated state. These associations would presumably facilitate the recruitment of downstream signaling molecules, as is the case in animal NLRs (19)(20)(21)41) and Toll-like receptors (42), and resemble the mechanism proposed for TIR-NLRs (5,8).…”

“…The central NB domain appears to control the activation of the protein through NB and nucleotide exchange (6)(7)(8). The LRR domain plays a role in effector recognition specificity for a number of plant NLRs, and, in some cases, it is implicated in effector binding (9)(10)(11).…”