Unravelling the role of conserved plant defense gene EDS1 in lettuce using CRISPR/Cas9
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[EMBARGOED UNTIL 08/01/2026] ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) is a conserved regulator of effector-triggered immunity (ETI), specifically required for signaling through TIR-domain NLRs (TNLs). While EDS1 is well studied in Arabidopsis thaliana, its role in lettuce (Lactuca sativa), which possesses multiple EDS1 paralogs, remains unclear. This dissertation aims to elucidate the function of LsEDS1 paralogs (LsEDS1A, LsEDS1B, and LsEDS1C) in lettuce by combining lettuce transformation, CRISPR/Cas9 genome editing, and immune response assays. At first, an optimized regeneration protocol was developed and used to screen six lettuce cultivars. Two readily transformation-amenable cultivars, Valmaine and PI251246, were selected. Next, a CRISPR platform was established using PHYTOENE DESATURASE (PDS) as a proof-of-concept target to validate editing efficiency. And finally, individual knockouts of LsEDS1A, LsEDS1B, and LsEDS1C, along with LsEDS1B/C double mutants, were generated and confirmed through sequencing. To assess immune function, mutant lines were infiltrated with Agrobacterium expressing the bacterial effector AvrRps4N. LsEDS1A mutants showed markedly reduced HR (hypersensitive response), while LsEDS1B mutants retained full HR, similar to wild type. LsEDS1C mutants have been generated and are awaiting phenotyping. However, double LsEDS1B/C mutants that I preliminarily genotyped as homozygous retained HR responses. These results demonstrate that among the three paralogs, LsEDS1A is likely the key signaling node in AvrRps4N -triggered ETI. This work not only advances our understanding of immune gene evolution and functional diversification in crop species, but also provides a valuable platform for future gene function studies and disease resistance engineering in lettuce and related Asteraceae crops.
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Ph. D