Evolutionary and functional diversification of cork oak NLRs reveals RNL expansion and dual roles in biotic and abiotic stress

The Nucleotide-Binding Domain Leucine-Rich Repeat (NLR) gene family is a central component of plant immune systems, yet its diversity and evolutionary dynamics remain poorly characterized in long-lived tree species. Here, we performed a genome-wide analysis of the NLR gene family in Quercus suber (cork oak) using InterNLR, a new annotation tool, and explored their expression regulation in response to biotic and abiotic stresses. A total of 918 NLR and NLR-like genes were identified, encompassing both canonical and non-canonical members. Phylogenetic analyses based on the NB-ARC domain highlighted the distinct evolutionary trajectory of RNL proteins, which function as helper NLRs and show evidence of clade-specific gene duplication. Transcriptomic analyses revealed pronounced tissue-specific expression patterns, with RNLs exhibiting significantly elevated expression in xylem, suggesting a specialized role in this tissue. Under drought stress, seven NLR genes were differentially expressed and shared orthology with known abiotic stress-responsive genes. Notably, a CNL gene (LOC111996439) responded to both biotic and abiotic stresses, indicating a potential role as an integrative regulator of early defence responses, while an ADR1 orthologue (LOC112022539) suggests molecular crosstalk between stress signaling pathways. Population genetic analyses further revealed signatures of both positive and balancing selection acting on NLR genes. Together, these results provide new insights into the evolution, expression, and functional diversification of NLRs in cork oak. This work advances our understanding of immune gene architecture in an ecologically and economically important forest tree species.