Development of plants resistant to tomato geminiviruses using artificial trans‐acting small interfering RNA

Abstract

RNA interference (RNAi), a conserved RNA-mediated gene regulatory mechanism in eukaryotes, plays an important role in plant growth and development, and as an antiviral defence system in plants. As a counter-strategy, plant viruses encode RNAi suppressors to suppress the RNAi pathways and consequently down-regulate plant defence. In geminiviruses, the proteins AC2, AC4 and AV2 are known to act as RNAi suppressors. In this study, we have designed a gene silencing vector using the features of trans-acting small interfering RNA (tasiRNA), which is simple and can be used to target multiple genes at a time employing a single-step cloning procedure. This vector was used to target two RNAi suppressor proteins (AC2 and AC4) of the geminivirus, Tomato leaf curl New Delhi virus (ToLCNDV). The vector containing fragments of ToLCNDV AC2 and AC4 genes, on agro-infiltration, produced copious quantities of AC2 and AC4 specific siRNA in both tobacco and tomato plants. On challenge inoculation of the agro-infiltrated plants with ToLCNDV, most plants showed an absence of symptoms and low accumulation of viral DNA. Transgenic tobacco plants were raised using the AC2 and AC4 tasiRNA-generating constructs, and T1 plants, obtained from the primary transgenic plants, were tested for resistance separately against ToLCNDV and Tomato leaf curl Gujarat virus. Most plants showed an absence of symptoms and low accumulation of the corresponding viruses, the resistance being generally proportional to the amounts of siRNA produced against AC2 and AC4 genes. This is the first report of the use of artificial tasiRNA to generate resistance against an important plant virus.

Mungbean yellow mosaic Indian virus encoded AC2 protein suppresses RNA silencing by inhibiting Arabidopsis RDR6 and AGO1 activities

“…By using this method, we have successfully made tobacco transgenic generating Tomato leaf curl New Delhi encoded AC2 and AC4 specific tasiRNA. These transgenic plants were challenged separately against ToLCNDV and Tomato leaf curl Gujarat virus and showed absence of symptom and low accumulation of the corresponding viruses (Singh et al, 2015). The co-infiltration of this vector and the plasmid expressing AC2 protein in tobacco using the agroinfiltration technique reduced the formation of siRNAs (Fig.…”

Small RNAs in plants: recent development and application for crop improvement

“…TGBp1/p25 of Potato Virus X (PVX) was targeted in tobacco by expression of amiRNAs based on an A. thaliana miR159a, miR167b, and miR171a precursors ( Ai et al, 2011 ). Singh et al (2014) generated transgenic tobacco plants resistant to Tomato leaf curl New Delhi virus (ToLCNDV) by transformation with trans -acting siRNA generating constructs against RNAi suppressor proteins (AC2 and AC4) of the geminivirus. Another promising strategy to reduce the multiplication and spread of virus in the plant includes the use of amiRNAs targeting the viral genes involved in replication, transmission, and symptom development after viral infection.…”

The Search for Resistance to Cassava Mosaic Geminiviruses: How Much We Have Accomplished, and What Lies Ahead

“…This strategy has been used to successfully engineer resistance to plant viruses. For example, transgenic N. benthamiana harboring a TAS3 gene modified to contain tasiRNA from the AC2 and AC4 genes of Tomato leaf curl New Delhi begomovirus (ToLCNDV) exhibited strong resistance to ToLCNDV and to Tomato leaf curl Gujarat begomovirus (ToLCGV) (Singh et al, 2015). Also, transgenic Arabidopsis thaliana plants expressing TAS3 modified with tasiRNAs targeting Turnip mosaic potyvirus and CMV were highly resistant to both viruses (Chen et al, 2016).…”