An N-terminal motif in NLR immune receptors is functionally conserved across distantly related plant species

Abstract

The molecular codes underpinning the functions of plant NLR immune receptors are poorly understood. We used in vitro Mu transposition to generate a random truncation library and identify the minimal functional region of NLRs. We applied this method to NRC4—a helper NLR that functions with multiple sensor NLRs within a Solanaceae receptor network. This revealed that the NRC4 N-terminal 29 amino acids are sufficient to induce hypersensitive cell death. This region is defined by the consensus MADAxVSFxVxKLxxLLxxEx (MADA motif) that is conserved at the N-termini of NRC family proteins and ~20% of coiled-coil (CC)-type plant NLRs. The MADA motif matches the N-terminal α1 helix of Arabidopsis NLR protein ZAR1, which undergoes a conformational switch during resistosome activation. Immunoassays revealed that the MADA motif is functionally conserved across NLRs from distantly related plant species. NRC-dependent sensor NLRs lack MADA sequences indicating that this motif has degenerated in sensor NLRs over evolutionary time.

RefPlantNLR: a comprehensive collection of experimentally validated plant NLRs

“…Plant NLRs likely evolved from multifunctional receptors to specialized receptor pairs and networks (Adachi, Contreras, et al, 2019;Adachi, Derevnina, et al, 2019). NLRs which combine pathogen detection and immune signalling activities into a single protein are referred to as "functional singletons", whereas NLRs which have specialized in pathogen recognition or immune signalling are referred to as "sensor" or "helper" NLRs, respectively.…”

A vector system for fast-forwardin vivostudies of the ZAR1 resistosome in the model plantNicotiana benthamiana

“…We generated autoactive and inactive versions of full-length ZAR1 by mutating the MHD and MADA motifs, which are both conserved across ZAR1 orthologs (Figure 1-figure supplement 1). Whereas mutation from aspartic acid (D) to valine (V) in the MHD motif generally makes full-length NLRs autoactive (Tameling et al, 2006), mutations in the N-terminal MADA motif impair NLR cell death activity (Wang et al, 2019b;Adachi et al, 2019). We generated a mutant series of AtZAR1 and NbZAR1, namely MHD mutants (AtZAR1 D489V and NbZAR1 D481V ), MADA mutants (AtZAR1 L17E and NbZAR1 L17E ) and MADA/MHD mutants (AtZAR1 L17E/D489V and NbZAR1 L17E/D481V ).…”

“…In contrast, NbZAR1 caused macroscopic cell death when the MHD mutant NbZAR1 D481V was expressed in N. benthamiana leaves ( Figure 2D and 2E). NRC4 was used as a control MADA-CC-NLR, because its MHD mutant NRC4 D478V causes autoactive cell death in N. benthamiana leaves (Adachi et al, 2019).…”

“…The ZAR1 resistosome was proposed to mediate cell death directly by translocating into the plasma membrane through the funnel-like structure and perturbing membrane integrity similar to pore-forming toxins (Wang et al, 2019b). The N-terminal a1 helix matches a sequence motif known as MADA motif that is functionally conserved across ~20% of coiled-coil (CC)-type plant NLRs (Adachi et al, 2019). This indicates that the ZAR1 death switch mechanism may be widely conserved across plant species.…”

“…This indicates that the ZAR1 death switch mechanism may be widely conserved across plant species. Mutations in surface-exposed residues within the a1 helix/MADA motif impair cell death and disease resistance mediated by ZAR1 and other MADA-CC-NLRs (Wang et al, 2019b;Adachi et al, 2019).…”