An Ancient Integration in a Plant NLR is Maintained as a Trans-species Polymorphism

The origin of genetic diversity in a species is a product of novel mutation and alleles maintained during speciation. Identification of alleles that are derived from a common ancestor is critical for understanding the selective processes experienced during speciation events. Here, we report on the inter- and intra-specific diversity of plant immune receptors in the Mla locus across Poaceae species. These immune receptors belong to the class of cytoplasmic-nuclear localized nucleotide-binding leucine-rich repeat (NLR) proteins. Plant immune receptors are under constant selective pressure to maintain resistance to plant pathogens, and novel mutations leading to improved immune responses are selected. We discovered a fusion of the plant immune receptor RGH2 with a component of the exocyst complex (Exo70). Phylogenetic analysis of Exo70 gene families from Poaceae species found that the integrated Exo70 was derived from Exo70F1. To identify the origin of this gene fusion, we assembled leaf transcriptomes from diverse Poaceae species. Inter-specific conservation in the RGH2- Exo70F1 gene fusion was found in several Pooideae species, whereas Brachypodium distachyon RGH2 is fused to a receptor-like kinase. To establish the time of gene fusion, we performed phylogenetic analysis using non-integrated and integrated Exo70F1. We found a diphyletic tree composed of non-integrated Exo70F1 and integrated Exo70F1, with Exo70F1 from B. distachyon and B. stacei forming an outgroup. This indicates that a single gene fusion event occurred after the speciation of Brachypodieae, but prior to Poeae-Triticeae radiation. These results, coupled with the observation of inter- and intra-specific variation in RGH2, demonstrate the maintenance of RGH2-Exo70F1 as a trans-species polymorphism over 24 My.