Contrasting evolutionary patterns of helper and sensor NRC NLRs in lettuce reflect functional divergence following subfunctionalization.

Nucleotide-binding domain and leucine-rich repeat immune receptors (NLRs) are known for their rapid evolution, even at the intraspecific level, yet the rates of evolution differ significantly across NLRs. However, the degree to which evolutionary patterns reflect functional divergence remains poorly understood, notably in important crop species. Within the NRC (NLR Required for Cell Death) network in Asterids, sensor NLRs detect pathogen presence but require NRC helpers for signaling and to confer immunity. We conducted a comparative analysis of NLRs across 40 Solanales and 29 Asterales genomes to explore NRC network expansion and diversification within the less-studied Asterales order. Our findings reveal that the NRC network has expanded less in Asterales compared to Solanales. We functionally validated an Asterales NRC network with 2 helpers and 9 sensors in common lettuce (Lactuca sativa). Through selection analysis and structural modeling of NRC gene family in the Lactuca genus, we found distinct evolutionary trajectories between NRC helpers and sensors. Sensors reliant on the phylogenetically conserved helper NRC0 experience limited diversification, whereas sensors dependent on other NRC helpers show higher rates of positive selection and gene duplication. Our results highlight the lineage- and function-specific evolution of the NRC network, offering insights into the evolutionary pressures shaping plant immune receptor networks.