Transplastomic tomato plants expressing insect-specific double-stranded RNAs: a protocol based on biolistic transformation
Kaplanoglu, E. , Kolotilin, I. , Menassa, R. , Donly, C. Transplastomic Tomato Plants Expressing Insect-Specific Double-Stranded RNAs: A Protocol Based on Biolistic Transformation. Methods in Molecular Biology. Volume 2360, Pages 235 - 252 (2022).
Plain language summary
Effective management of pest insects in agriculture requires ongoing development of novel tools and strategies to counter pest adaptation to existing methods. A highly promising new technology, called RNA interference, makes use of a unique version of the RNA molecules present in all living organisms to turn off essential genes in pests and deter feeding or even kill them. These double-stranded RNA molecules can be made right inside plants, using the plant’s chloroplasts as factories to produce the materials needed to protect them from pests. In this chapter, we describe the design of a system which can be used to stably transform the plastids of tomato plants for production and accumulation of dsRNAs that will target specific pest insects. In addition, we also describe a protocol for specific detection of full-length dsRNA molecules in plastids. These tools will enable the application of RNA interference technology in a broader range of cultivated plant species.
Expressing insecticidal double-stranded RNA (dsRNA) molecules in plant plastids is a novel approach for in-planta production of dsRNA that has enormous potential for developing improved plant-mediated RNA interference (RNAi) strategies for insect pest control. In this chapter, we describe the design of a transformation vector containing an expression cassette which can be used to stably transform plastids of tomato plants for production and accumulation of dsRNA. Such dsRNA can trigger the mechanisms of RNAi in pest insects and selectively suppress the expression of target genes, resulting in lethality. We also describe a protocol for detection of full-length dsRNA molecules in plastids using an RT-PCR-based method.