Highly efficient generation of bacteria leaf blight resistance and transgene-free rice using genome editing and multiplexed selection system

Background: Rice leaf blight is a worldwide devastating disease caused by bacteria Xanthomonas oryzae pv. Oryzae ( Xoo ). The UPT (up-regulated by transcription activator-like 1 effector) box in promoter region of the rice Xa13 gene played a key role in Xoo pathogenicity. Mutation of key bacterial protein binding site in UPT box of Xa13 to abolish PXO99-induced Xa13 expression is a way to improve rice resistant to bacterial.Highly efficient generation and selection transgene-free, edited plants helpful to shorten and simple the gene editing breeding process. Selective elimination of transgenic pollen of E0 plants can enrich proportion of E1 transgene-free offspring and expression of the color mark gene in seeds makes the selection of E2 plants is very convenient and efficient. In this study, a genome editing and multiplexed selection system was used to generate bacteria leaf blight resistance and transgene-free rice plants. Results: We introduced site specific mutations into the UPT box using CRISPR/Cas12a technology to hamper TAL (Transcription-Activator Like effectors) protein binding and gene activation, and generated genome edited rice with improved bacteria blight resistance. Transgenic pollens of E0 plants were eliminated by pollen specific expression of α-amylase gene Zmaa1 , the proportion of transgene-free plants were enriched from 25% to 50% in single T-DNA insertion events in E1 generation. Transgenic seeds were visually identified and discarded by specific aleuronic expression of DsRed, which reduced 50% cost and achieved up to 98.64% of accuracy for selection of transgene-free edited plants. Conclusion: We demonstrated core nucleotide deletion in the UPT box of Xa13 promoter conferred resistance to rice blight and selection of transgene-free plants were boosted by introducing multiplexed selection. The combination of genome editing and transgene-free selection is an efficient strategy to accelerate functional genomic research and plant breeding.