Development of an RNA virus vector for non-transgenic genome editing in tobacco and generation of Berberine Bridge Enzyme-Like mutants with reduced nicotine content

This study is led by Professor Zhenghe Li (State Key Laboratory of Rice Biology and breeding, Institute of Biotechnology, Zhejiang University, Hangzhou, China). The authors developed a non-transgenic genome editing approach in tobacco by delivering CRISPR/Cas9 with an engineered eggplant mottled dwarf virus (EMDV) vector, a negative-strand RNA virus belonging to the family Rhabdoviridae. Mutations induced in EMDV-infected somatic tissues during viral infection were integrated into germline cells through tissue culture, yielding a substantial proportion of tobacco regenerants with heritable edits. Most of these regenerants carried bi-allelic mutations in both homoeologs of this allotetraploid species.

Using this method, the authors targeted six berberine bridge enzyme-like protein (BBL) family genes for mutagenesis, which are implicated in the last steps of pyridine alkaloid biosynthesis in the commercial tobacco cultivar Hongda. A panel of 16 transgene-free mutant lines containing single, double, or higher-order homozygous mutations across the six BBL genes were generated and characterized by alkaloid profiling. Mutant lines homozygous for BBLa and BBLb mutations exhibited drastically reduced nicotine levels, while mutations of the other BBL members had a minor role in nicotine synthesis. The decline of nicotine content in these lines was accompanied by reductions in anatabine and cotinine levels but increases in nornicotine and its derivative myosmine.

Preliminary agronomic evaluation identified two ultra-low-nicotine lines (bbla/b/d1/d2/e and bbla/b/e) that exhibited growth phenotypes similar to wild-type plants under both greenhouse and field conditions. This work provides valuable genetic resources for breeding low-nicotine tobacco and contributes to a deeper understanding of alkaloid biosynthesis. Moreover, the results underscore the effectiveness of RNA virus-mediated genome editing in targeting multiple genes in polyploid plants and highlight the advantages of non-transgenic gene editing approaches in crop trait improvement, particularly in terms of development time and cost efficiency.

See the article:

Development of an RNA virus vector for non-transgenic genome editing in tobacco and generation of Berberine Bridge Enzyme-Like mutants with reduced nicotine content

https://link.springer.com/article/10.1007/s42994-024-00188-y