Critical technologies development to enable the use of synthetic siRNAs in crop protection.

Abstract

The use of interfering RNAs is pointed out as a viable alternative to control the thousands of cases of resistance to chemical control agents and to sustainably manage pests, diseases, and weeds. The main justifications are the easy biodegradation, the great diversity of potential sites of action, and the possibility of obtaining highly selective RNAs that act on a single species or a single biotype. The specificity of siRNAs allows selective control in situations where chemical agents, which interact with proteins, cannot do so. When plants are genetically modified to produce RNAs, there are already several examples of success in plant protection, but there are high costs to develop GMOs and meet the requirements of regulatory systems. There are also hundreds of patents for synthetic RNAs that have potential use in plant protection but have not reached the market. The main limitations to the use of these synthetic RNAs refer to the stability of siRNAs in agricultural environments and the difficulty of developing delivery systems for these compounds that can overcome the barriers to entry of xenobiotics present in all species. Even when the purpose of RNAi is to control pests or diseases, there are situations in which plant penetration, stability, and movement are required. Even single-stranded RNAs with 21 nucleotides are huge molecules, with a molecular mass of more than 7,000 Daltons, which in the light of current knowledge have great difficulty entering the leaves and roots of plants without the occurrence of injury. The objective of this project is to obtain critical information to understand the dynamics and develop the technologies that can allow the application, penetration, movement, and action of RNAs in plants. A complex set of methodologies based on molecular biology, high-resolution or unit-resolution mass spectrometry, ion chromatography, fluorescence, and spectrophotometry (in plant solutions or tissues) will be used to directly detect and quantify RNAs and compounds or symptoms indicating their presence and action. The tetrapyrroles synthesis route was selected as a target considering: 1) the products of the route have potential for use and commercialization as photosensitizing agents for Photodynamic Therapy; 2) possibility of silencing more than one gene; 3) the genes associated with the enzymes -aminolevupupeneic acid dehydratase (non-toxic to plants and non-photoactive), protoporphyrinogen IX oxidase (extremely photoactive, reddish in color and toxic to plants) and Iron Chelatase 2 (FC2) were selected. The initial objective of the project is to determine whether small RNAs, with different constructions, can enter and act on plant leaves. A total of 10 siRNAs with 21 nucleotides were designed for each of the target genes (PPOX, HemB1, and FC2). To reduce project costs, three siRNAs for each target will be first evaluated, and other siRNAs will be synthesized if needed. (AU)