Heritable virus-induced germline editing in tomato

Area of Science:

• Plant biotechnology
• Genome editing
• Molecular biology

Background:

• Genome editing offers precise genetic modification in plants.
• Virus-induced genome editing (VIGE) provides a transient method for gene modification.
• Establishing efficient heritable VIGE in tomato is crucial for crop improvement.

Purpose of the Study:

• To implement and optimize heritable virus-induced genome editing (VIGE) in tomato (Solanum lycopersicum).
• To evaluate the efficiency of different promoters (35S, SlRPS5A, SlYAO) for Cas9 expression in VIGE.
• To investigate the impact of environmental conditions on heritable editing frequencies.

Main Methods:

• Generated transgenic tomato lines expressing Streptococcus pyogenes Cas9 (SpCas9) under three different promoters.
• Utilized a tobacco rattle virus (TRV)-based system with guide RNAs (gRNAs) fused to mobile RNA sequences for gene targeting.
• Targeted Phytoene desaturase (SlPDS) and Downy mildew resistance 6 (SlDMR6) genes for editing.
• Assessed somatic and heritable editing efficiencies in SpCas9-expressing tomato lines.
• Optimized environmental conditions, including reduced light intensity, to enhance editing frequencies.

Main Results:

• Significant somatic editing efficiency was observed in all tested tomato lines targeting SlPDS and SlDMR6 genes.
• Heritable monoallelic mutations were detected in progenies from SlYAO promoter-driven SpCas9 lines (3% frequency).
• Optimization of environmental conditions, particularly reduced light intensity, dramatically increased heritable editing frequencies (up to 86% for SlPDS and 100% for SlDMR6).
• Biallelic mutations were successfully generated under optimized conditions.

Conclusions:

• The choice of promoter and optimized environmental conditions are critical for enhancing heritable VIGE efficiency in tomato.
• VIGE offers a promising strategy for generating stable, heritable mutations in tomato without requiring extensive tissue culture or transformation post-establishment of Cas9-expressing lines.
• This method facilitates efficient mutant generation in established tomato lines, accelerating crop breeding and research.