Layered double hydroxide (LDH)-mediated topical delivery of dsRNA for protection against Tomato yellow leaf curl virus (TYLCV) in Nicotiana benthamiana

Cell wall is the major barrier in the delivery of biomolecules such as nucleic acids into the plant cell. Biological (bacteria or viruses) and biolistic (particle-based) methods are used to deliver nucleic acids into the plant cell. However, these methods have significant limitations when it comes to species range, scalability, and field assays. In this work, we report the use of layered double hydroxide (LDH) topically applied to deliver RNA molecules into the plant cell. LDH were assembled by methanol-based co-precipitation of magnesium and aluminum nitrate solution with sodium hydroxide and finally dispersed in deionized water. The assembled LDH were physically characterized by AFM, zeta-sizer and their binding to RNA was confirmed by gel electrophoresis. LDH complexed with double stranded RNA (dsRNA) was topically applied to Nicotiana benthamiana leaves. As a model system, virus specific dsRNA-LDH complexes were used to activate cellular RNAi machinery against Tomato Yellow leaf Curl Virus (TYLCV) in N. benthamiana plants. Our results demonstrated that topical application of the TYLCV specific dsRNA-LDH complexes reduce viral genome accumulation and viral symptoms development. Similarly, dsRNA-LDH protected plants produce typical leaves, flowers, and seeds, confirming efficient virus resistance compared unprotected TYLCV infected plants. Topical application and noninvasive delivery of nucleic acid has several advantages, as these methods are specie independent, easy to scale up, applied with low-pressure spray, requires no tissue culture and no sophisticated equipment. The LDH based noninvasive delivery of nucleic acids has the capability to overcome the cell wall barrier limitations and will open new opportunities to exploit the full potential of cellular machinery to produce resilient plants and insure sustainable food production.