Investigating the mechanism of a wheat CNL/MLKL pair that provides resistance to blast disease

The CC-type nucleotide-binding, leucine-rich repeat (NLR) immune receptor, RWT3, is part of a wheat resistance locus that prevents successful infection of M. oryzae strains carrying any of three diverse effectors: PWT3, PWT6 or PWT8. Unlike canonically paired NLRs (with one NLR to perceive effectors (sensor) and another to execute cell death (helper)), RWT3 is genetically linked to an MLKL protein RWT3 associated kinase (R3AK). MLKL (mixed lineage kinase like) domain architectures comprise an N-terminal coiled-coil (CC) fused to a kinase domain. Both RWT3 and R3AK are required for resistance in wheat, establishing a novel immune receptor pairing. Here, we investigate the molecular mechanisms underpinning the NLR/MLKL paired resistance. We recapitulated the system through Nicotiana benthamiana transient expression assays, confirming that both RWT3 and R3AK are necessary and sufficient to cause cell death in response to PWT3, PWT6 or PWT8. Further, we demonstrate the CC domain of R3AK is required for effector triggered cell death and that R3AK can be made auto-active via mutating the kinase catalytic residues. RWT3 is not actively involved in the execution of cell death, thus we hypothesise that R3AK acts as the helper in this paired system. Finally, we show that the R3AK protein family is limited to Poales and genetic linkage to NLRs is prevalent but phylogenetically scattered. Together, this study highlights a novel resistance mechanism involving a non-canonical NLR/MLKL system.