An efficient virus‐induced gene silencing (VIGS) system for gene functional studies in Miscanthus

• Published in: Global change biology. Bioenergy 2023-06, Vol.15 (6), p.805-820
• Main Authors: He, Guo, Zhao, Xuhong, Xu, Yan, Wang, Yu, Zhang, Zhihai, Xiao, Liang, Hudson, Matthew, Hu, Ruibo, Li, Shengjun
• Format: Article
• Language: English
• Subjects: Crops; Desaturase; Design factors; Design of experiments; Efficiency; Energy crops; Experimental design; Flowers & plants; Functional analysis; Gene silencing; Genetic transformation; Genomes; Genomics; Infiltration; Inoculation; Miscanthus; Optical density; Photobleaching; Phylogenetics; phytoene desaturase (PDS); Plant bacterial diseases; Plant species; Proteins; Renewable energy; Salinity tolerance; Salt tolerance; Seedlings; Seeds; sprout infiltration; Tobacco; tobacco rattle virus (TRV); transient gene silencing; Vacuum; Viral infections; Viruses; virus‐induced gene silencing (VIGS)
• ISSN: 1757-1693; 1757-1707
• DOI: 10.1111/gcbb.13051

Virus‐induced gene silencing (VIGS) is a powerful tool for transient gene functional analysis in plants, especially for monocot species (e.g., grasses) that are recalcitrant to transformation. Despite various VIGS systems that have been developed in different plant species, none was previously available for the bioenergy crop Miscanthus. Here, we report the establishment of an efficient and robust VIGS system mediated by Tobacco Rattle Virus (TRV) in Miscanthus. We first investigated the impact of various factors that may affect gene silencing efficiency using the Miscanthus sinensis Phytoene Desaturase (MsPDS) gene as a visual indicator of photobleaching. Then, we optimized the TRV‐elicited VIGS procedure using an orthogonal experimental design with four factors (sprout size, Agrobacterium concentration, vacuum infiltration time, and co‐incubation time) each at three levels. The following led to the highest silencing efficiency (~76%): inoculation of germinating seedlings (1.0–2.0 mm), Agrobacterium tumefaciens culture grown to optical density at 600 nm (OD600) of 0.4, vacuum infiltration for 90 min, and co‐incubation for 5 h. The VIGS system established was applicable for both M. sinensis and M. lutarioriparius, with comparable gene silencing efficiency. We verified the efficacy of the VIGS system via the functional characterization of the role of a MYB transcription factor, MsMYB112, in salt stress tolerance. Expression of MsMYB112 was successfully knocked down using the VIGS system, and this led to compromised salt tolerance in the silenced Miscanthus plants. The TRV‐based VIGS system established may, therefore, substantially facilitate functional genomic studies in Miscanthus. We established an efficient and convenient virus‐induced gene silencing (VIGS) system mediated by Tobacco Rattle Virus (TRV) using germinating sprouts in Miscanthus. The TRV‐mediated VIGS was successfully applied to knock‐down MsMYB112 expression to enhance salt stress tolerance in Miscanthus.