Bing Yang

Summary Using genetic resistance against bacterial blight (BB) caused by Xanthomonas oryzae pathovar oryzae ( Xoo ) is a major objective in rice breeding programmes. Prime editing (PE) has the potential to create novel germplasm against Xoo . Here, we use an improved prime‐editing system to implement two new strategies for BB resistance. Knock‐in of TAL effector binding elements (EBE) derived from the BB susceptible gene SWEET14 into the promoter of a dysfunctional executor R gene xa23 reaches 47.2% with desired edits including biallelic editing at 18% in T 0 generation that enables an inducible TALE‐dependent BB resistance. Editing the transcription factor TFIIA gene TFIIAγ5 required for TAL effector‐dependent BB susceptibility recapitulates the resistance of xa5 at an editing efficiency of 88.5% with biallelic editing rate of 30% in T 0 generation. The engineered loci provided resistance against multiple Xoo strains in T 1 generation. Whole‐genome sequencing detected no OsMLH1dn ‐associated random mutations and no off‐target editing demonstrating high specificity of this PE system. This is the first‐ever report to use PE system to engineer resistance against biotic stress and to demonstrate knock‐in of 30‐nucleotides cis‐regulatory element at high efficiency. The new strategies hold promises to fend rice off the evolving Xoo strains and protect it from epidemics.