Using genetic resistance against bacterial blight (BB) caused by
- PAR ID:
- 10483170
- Publisher / Repository:
- ELSEVIER
- Date Published:
- Journal Name:
- Plant Communications
- Volume:
- 4
- ISSN:
- 2590-3462
- Page Range / eLocation ID:
- 100741
- Subject(s) / Keyword(s):
- bacterial blight herbicide tolerance multiplex genome editing prime editing rice.
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Summary Xanthomonas oryzae pathovaroryzae (Xoo ) is a major objective in rice breeding programmes. Prime editing (PE) has the potential to create novel germplasm againstXoo . 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 geneSWEET14 into the promoter of a dysfunctional executorR genexa23 reaches 47.2% with desired edits including biallelic editing at 18% in T0generation that enables an inducible TALE‐dependent BB resistance. Editing the transcription factor TFIIA geneTFIIAγ5 required for TAL effector‐dependent BB susceptibility recapitulates the resistance ofxa5 at an editing efficiency of 88.5% with biallelic editing rate of 30% in T0generation. The engineered loci provided resistance against multipleXoo strains in T1generation. Whole‐genome sequencing detected noOsMLH1dn ‐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 evolvingXoo strains and protect it from epidemics. -
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Abstract Adenine base editors (ABEs) are valuable, precise genome editing tools in plants. In recent years, the highly promising ADENINE BASE EDITOR8e (ABE8e) was reported for efficient A-to-G editing. However, compared to monocots, comprehensive off-target analyses for ABE8e are lacking in dicots. To determine the occurrence of off-target effects in tomato (Solanum lycopersicum), we assessed ABE8e and a high-fidelity version, ABE8e-HF, at 2 independent target sites in protoplasts, as well as stable T0 lines. Since ABE8e demonstrated higher on-target efficiency than ABE8e-HF in tomato protoplasts, we focused on ABE8e for off-target analyses in T0 lines. We conducted whole-genome sequencing (WGS) of wild-type (WT) tomato plants, green fluorescent protein (GFP)–expressing T0 lines, ABE8e-no-gRNA control T0 lines, and edited T0 lines. No guide RNA (gRNA)–dependent off-target edits were detected. Our data showed an average of approximately 1,200 to 1,500 single-nucleotide variations (SNVs) in either GFP control plants or base-edited plants. Also, no specific enrichment of A-to-G mutations were found in base-edited plants. We also conducted RNA sequencing (RNA-seq) of the same 6 base-edited and 3 GFP control T0 plants. On average, approximately 150 RNA–level SNVs were discovered per plant for either base-edited or GFP controls. Furthermore, we did not find enrichment of a TA motif on mutated adenine in the genomes and transcriptomes in base-edited tomato plants, as opposed to the recent discovery in rice (Oryza sativa). Hence, we could not find evidence for genome- and transcriptome-wide off-target effects by ABE8e in tomato.
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