Virus-induced systemic and heritable gene editing in pepper (Capsicum annuum L.)

Area of Science:

• Plant biotechnology
• Molecular biology
• Agricultural science

Background:

• CRISPR/Cas genome editing accelerates plant breeding by introducing desired traits.
• Conventional Agrobacterium tumefaciens-mediated transformation and regeneration methods hinder gene editing in pepper (Capsicum annuum L.).

Purpose of the Study:

• To optimize and apply a virus-induced gene editing (VIGE) system for efficient gene editing in pepper.
• To overcome limitations of traditional transformation methods in pepper breeding.

Main Methods:

• Optimized a VIGE system using tobacco rattle virus 2 (TRV2) vectors expressing single guide RNAs (sgRNAs) targeting pepper genes.
• Modified TRV2 vectors with hammerhead ribozyme (HH) and tested alternative mobile elements (tRNA^Ile, tRNA^Met) for enhanced sgRNA mobility.
• Cultivated plants at 20°C to increase TRV persistence and spread, facilitating systemic editing.

Main Results:

• Achieved 36.3% systemic editing efficiency, observed via photobleaching phenotypes in systemic leaves.
• Confirmed 8.5% of progeny from plants with pTRV-HH-CaPDS-sgRNA-FT construct carried mutations at the CaPDS locus.
• Successfully generated fasciculate mutants by editing the FASCICULATE gene using the VIGE system.

Conclusions:

• The optimized VIGE system effectively bypasses tissue culture for gene editing in pepper.
• This VIGE approach offers a powerful tool for molecular studies and accelerates breeding programs in pepper (Capsicum annuum L.).