A widely conserved N-terminal motif in the coiled-coil domain of NLR immune receptors is required for activation of hypersensitive cell death

The majority of plant NLR immune receptors carry an N-terminal coiled-coil domain, which mediates self-association and is required or, in some cases, sufficient for activation of hypersensitive cell death. However, our understanding of how CC-NLRs trigger cell death remains limited. Here, we used the in vitroMu transposition system to generate a random truncation library and identify the minimal region required for CC-NLR-mediated cell death. We applied this method to NRC4—a helper NLR that functions with a multitude of sensor NLRs within a complex receptor network. This revealed that the N-terminal 29 amino acids of NRC4 are sufficient to induce cell death. Remarkably, this region is defined by a motif that follows the consensus MADAxVSFxVxKLxxLL, and is conserved not only in NRC proteins but also in ~20% of all CC-NLRs of dicot and monocot species. Motif swapping experiments revealed that this sequence is functionally conserved between NLRs from distantly related plant species. Interestingly, NRC-dependent NLRs and many other sensor NLRs lack the N-terminal motif which may have become non-functional over evolutionary time. We conclude that the evolutionarily constrained “MADA motif” is critical for the cell death inducing activity of CC domains from a significant fraction of plant NLR proteins.