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

Abstract

Plants evolved intracellular immune receptors that belong to the NOD-like receptor (NLR) family to recognize the presence of pathogenderived effector proteins. NLRs possess an N-terminal Toll-like/IL-1 receptor (TIR) or a non-TIR domain [some of which contain coiled coils (CCs)], a central nucleotide-binding (NB-ARC) domain, and a C-terminal leucine-rich repeat (LRR). Activation of NLR proteins results in a rapid and high-amplitude immune response, eventually leading to host cell death at the infection site, the so-called hypersensitive response. Despite their important contribution to immunity, the exact mechanisms of NLR activation and signaling remain unknown and are likely heterogenous. We undertook a detailed structure-function analysis of the plasma membrane (PM)-localized CC NLR Resistance to Pseudomonas syringae pv. maculicola 1 (RPM1) using both stable transgenic Arabidopsis and transient expression in Nicotiana benthamiana. We report that immune signaling is induced only by activated full-length PM-localized RPM1. Our interaction analyses demonstrate the importance of a functional P-loop for in planta interaction of RPM1 with the small host protein RPM1-interacting protein 4 (RIN4), for constitutive preactivation and postactivation self-association of RPM1 and for proper PM localization. Our results reveal an additive effect of hydrophobic conserved residues in the CC domain for RPM1 function and RPM1 self-association and their necessity for RPM1-RIN4 interaction. Thus, our findings considerably extend our understanding of the mechanisms regulating NLR activation at, and signaling from, the PM.plant immunity | effector-triggered immunity | NLR receptor | oligomerization | Arabidopsis thaliana

Mechanism of plant immune activation and signaling: Insight from the first solved plant resistosome structure

“…This is similar to the wheel‐like structures of activated Apaf1 (Zhou et al ), NLRC4 (Hu et al ), or CED4 complexes (Qi et al ), suggesting that oligomerization might be a common theme in NLR activation. Consistent with this, the oligomerization of plant NLRs has been reported for CC‐NLRs such as Prf (Gutierrez et al ), RPM1 (El Kasmi et al ), RPS5 (Qi et al ), and MLA1 (Maekawa et al ), as well as for TIR‐NLRs such as N (Mestre and Baulcombe ), SNC1 (Xu et al ), and L6 (Bernoux et al ). The crystal structures of the CC or TIR domains of several plant NLRs also suggest these proteins undergo oligomerization; however, it is important to note that certain CC domains, such as that of Sr33, do exist as monomers (Casey et al ), although further experimentation is required to determine whether their full‐length NLRs form oligomers.…”

“…The authors showed this ZAR1 a-helix is required for its effector-enhanced association with the plasma membrane and is essential for ZAR1 activity. Other NLRs, such as RPS2 (Axtell and Staskawicz 2003), RPM1 (Gao et al 2011;El Kasmi et al 2017), RPS5 (Qi et al 2012), and Tm2 2 (Chen et al 2017), are also associated with the membrane; therefore, it will be interesting to test whether this membrane association is required for their activation. Recently, the association of the human NLRP3 inflammasome with the trans-Golgi membrane was shown to be essential for its activation (Chen and Chen 2018).…”

Structure-function analysis of ZAR1 immune receptor reveals key molecular interactions for activity

“…For ZAR1 CCH2b , the loop region between t1a and t1b was modified to be less prone in adopting a helical fold and therefore should be destabilizing. ZAR1 CCED targets the conserved EDVID motif (amino acids 73-77), which is found in many CC-type NLRs (Bai et al, 2012; Rairdan et al, 2008; Baudin et al, 2017; El Kasmi et al, 2017) (Figure S1A, S1C). Finally, we generated a shorter version of the CC domain, ZAR CC2 that can still dimerize in yeast (Baudin et al, 2017).…”

“…For RPM1 (RESISTANCE TO PSEUDOMONAS SYRINGAE PV. MACULICOLA 1), mutation of M34 or M41 weakened dimerization of the CC domain in coimmunoprecipitation assays, and mutation of 3 hydrophobic residues (I31E/M34E/M41E) was necessary to prevent CC dimerization in BiFC and coimmunoprecipitation assays (El Kasmi et al, 2017). It would be interesting to test the role of these residues in dimerization as ZAR1 contains hydrophobic residues at similar positions (Figure S1A).…”

“…In MLA10, the EDVID motif is surface-exposed and positioned at the dimer interface (Maekawa et al, 2011), suggesting that it contributes to CC homodimerization. RPM1, an NLR involved in the recognition of P. syringae effector AvrRpm1, also contains an EDVID motif that may contribute to the self-association of CC domains (El Kasmi et al, 2017). In agreement with previous studies, the EDVID motif in ZAR1 is predicted to be surface-exposed (Figure S1), and is involved in CC dimerization (Figure 1C) and in the CC-NBARC interaction (Figure 3C).…”