Leveraging prime editing technologies and executor resistance mechanisms for next-generation rice improvement
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Rice is the major food crop across the globe and provides almost one-fifth of total caloric intake. Rice production remains under a continuous threat imposed by Xanthomonas oryzae pv. oryzae (Xoo) leading to bacterial blight (BB) disease. Breeding for BB resistance is one of the major objectives in rice breeding programs. Advanced approaches, such as genome editing, will be needed to generate novel resistant alleles or recapitulate the known resistance alleles against the evolving Xoo strains. Understanding the mechanisms of known BB resistance genes and deciphering the genetic factors required for resistance will aid in the future programs to generate novel alleles as well their breeding in elite cultivars. In this dissertation research, I touch upon both aspects to generate novel alleles against BB resistance using the latest genome editing, prime editing, and also to functionally characterize the role of a novel gene, EXR1, required for Xa7-mediated BB resistance in rice. In Chapter 2, we applied an improved prime editing system to develop two novel strategies for bacterial blight (BB) resistance. Inserting TAL effector binding elements (EBE) from SWEET14 into the xa23 promoter achieved 47.2% editing efficiency, including 18% biallelic edits, enabling inducible, TALE-dependent resistance. Editing TFIIAγ5, essential for BB susceptibility, mirrored xa5 resistance with 88.5% efficiency and 30% biallelic editing. Engineered plants resisted multiple Xoo strains in the T1 generation. Whole-genome sequencing confirmed no off-target effects. This study marks the first use of PE for biotic stress resistance and efficient 30-nt cis-element knock-in, promising durable BB protection. Further in chapter 3 and 4, we developed a modular assembly-based multiplex prime editing (PE) system in rice, enabling simultaneous editing of up to four genes in a single transformation. The duplex PE (DPE) system achieved 46.1% co-editing efficiency in T0 plants, converting TFIIAγ5 to xa5 and xa23 to Xa23SW11, conferring broad-spectrum resistance to Xoo in T1. We also edited OsEPSPS1 for herbicide tolerance and OsSWEET11a for Xoo resistance, reaching 57.14% co-editing efficiency. Using the quadruple PE (QPE) system, we targeted OsEPSPS1, OsALS1, TFIIAγ5, and OsSWEET11a, achieving 43.5% efficiency for all four genes. Additionally, we tested five more constructs, including two triplex (TPE) and three QPE systems, each targeting different gene sets. Our modular system delivered high editing efficiencies and simplified reagent assembly, making multiplex PE more accessible to researchers. These advances offer powerful tools for plant genome engineering and hold great potential for future crop improvement. Lastly, in chapter 5, in this study, we identified and cloned Executor Xa7-resistance required 1 (EXR1), the first genetic determinant essential for Xa7-mediated immunity in the indica rice variety IRBB7. Using a multiplexed CRISPR-Cas9 approach, EXR1 was pinpointed within a fine-mapped locus. It encodes an ER membrane-localized protein with 10 predicted transmembrane domains that interacts with XA7. AlphaFold modeling suggested a pore-like dimer structure, and both self-interaction and interaction with XA7 were confirmed. Transcriptome analysis of exr1 mutants highlighted its role in defense activation and potential transport functions. Notably, Xa7 and EXR1 regulate ER-tocytosol calcium fluxes, as shown by GCaMP calcium sensor lines, suggesting EXR1 mediates calcium signaling critical for the Xa7 resistance response. This discovery provides crucial insights into the molecular mechanism of Xa7-mediated immunity and offers valuable knowledge for breeding disease-resistant indica and japonica rice varieties.
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Ph. D.