SOLO1: Single-gene NLR receptors for engineering plant disease resistance

Plants use intracellular immune receptors called nucleotide-binding leucine-rich repeat proteins (NLRs) to detect pathogen-secreted effectors. Upon activation, these receptors can assemble into oligomeric complexes called resistosomes, which promote immune signalling and cell death. Some NLRs function as singletons, whereas others function in pairs or networks. Plant NLRs with integrated domains (NLR-IDs) are generally regarded as sensor NLRs that require partner helper NLRs to trigger immunity. Among such paired NLRs, the rice Pik system has provided a powerful platform for engineering novel immune receptors by replacing its integrated HMA domain with effector-binding modules. However, whether some NLR-IDs function as singletons remains unclear. Here, we used AlphaFold 3 to model resistosome structures for 296 NLR-IDs from 180 diverse plant species and identified a singleton NLR-ID (sNLR-ID) clade in Poaceae (the grass family) based on high-confidence structural predictions. The NLR-IDs in this clade triggered cell death in autoactive mutant backgrounds upon agroinfiltration in Nicotiana benthamiana. Intriguingly, they carry diverse C-terminal IDs, including DUF761, NUDIX, and HMA, suggesting recurrent natural domain shuffling. Using an Aegilops tauschii sNLR-ID carrying a DUF761 domain as a scaffold, we replaced the native integrated domain region with either HIPP43, which binds the Magnaporthe oryzae effector PWL2, or the GFP-binding nanobody Enhancer. In transient expression assays in N. benthamiana, these engineered receptors showed elicitor-dependent cell-death. Our results identify a naturally modular and engineerable singleton NLR-ID scaffold, expanding immune receptor design from paired sensor-helper systems to self-contained modular receptors.