Genome-wide analysis of cork oak NLRs reveals RNL-associated duplications and immune receptor candidates associated with biotic and abiotic stress response.

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 repertoire in Quercus suber (cork oak) using a newly developed annotation tool-InterNLR-which identifies and classifies NLRs as canonical and non-canonical. We identified a total of 374 canonical NLRs, and 544 non-canonical NLRs-which may represent pseudogenes, gene fragments or evolutionary intermediates. 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 significant differences in gene-level expression among NLR classes in cork oak stem tissues, mainly driven by contrasts involving the non-canonical N class. Seven cork oak NLR genes, sharing putative orthology with abiotic stress-responsive NLRs found in other species, were also differentially expressed in stems under drought conditions. From these, four candidate NLRs arise as candidate components of shared or convergent responses to drought and P. cinnamomi infection. Population-genetic analyses of RNA-seq-derived variants from expressed NLR loci in two Iberian populations further revealed heterogeneous patterns among expressed NLR loci, with a predominance of positive Tajima's D values. 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.