P-061 - Molecular Characterization of a Natural Resistance Gene Stack in Lettuce

Lettuce downy mildew (LDM), caused by Bremia lactucae, is a major threat to lettuce production worldwide. Resistance genes provide high levels of resistance but may only be effective for a limited time because B. lactucae rapidly evolves new virulence phenotypes. Stacking multiple resistance genes in a cultivar maximizes evolutionary hurdles for the pathogen, potentially providing more durable resistance. ViAE is a Lactuca sativa breeding line with resistance introgressed from L. virosa. In ViAE, recognition and cell death occur in response to seven B. lactucae effectors which is genetically linked to resistance on Chromosome 8.  This suggests ViAE has a natural gene stack that could provide durable resistance to LDM. A T2T genome assembly of ViAE was generated using PacBio HiFi and Oxford Nanopore reads. Comparison to the susceptible line Salinas revealed a highly variable 35.2 Mb region on Chromosome 8, within the 65 Mb introgression interval mapped using RILs. This region contains 27 NLRs, 5 RLKs, and 2 RLPs as candidate resistance genes. Highly expressed NLRs with all three domains (N-terminus, NBS, LRR) were prioritized for functional validation. Over half of these candidate NLRs have been cloned and co-agrotransformed with the seven effectors. Two NLRs recognized at least two effectors, with both recognizing an effector in common. Different combinations of CRISPR-mediated knockouts are being generated to test the number of effective NLRs in the resistance gene stack.