Location: Application Technology Research
Project Number: 5082-30500-001-079-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jun 30, 2026
End Date: Aug 31, 2027
Objective:
1) Determine the regions in the Tomato brown rugous fruit virus (ToBRFV) that best serve as RNAi targets; 2) Design the formulation of exogenous delivery of dsRNA into plants; 3) Develop a host-induced gene silencing (HIGS) approach to confer ToBRFV resistance in tomatoes.
Approach:
1. Identify optimal SIGS targets in ToBRFV
We will divide ToBRFV transcripts into 500-nt regions from 5' to 3' and design primers for in vitro transcription to produce dsRNA fragments. We will screen ~12 dsRNAs spanning ToBRFV genome. Fragments will be applied to seedlings inoculated with ToBRFV. Disease symptoms (mosaic, chlorosis, mottling, deformation) will be scored.
2. Formulate topical dsRNA delivery
To enhance RNA stability, we will prepare nanopolymers or carbon dots via co-precipitation or microwave/FPLC methods. We will also test artificial nanovesicles for dsRNA delivery. In parallel, selected dsRNA segments will be expressed by E. coli for mass production. We will also rescue the virus by producing an infectious clone to potentially develop a mild-strain. Virus inoculation on lower leaves will be scored for symptoms and
RT-qPCR quantification of titers on local and upper leaves.
3. Develop host-induced gene silencing (HIGS) approach
Hairpin RNAs selected from Objectives 1 and 2 will be introduced into tomato via Agrobacterium-mediated transformation using cotyledon and hypocotyl explants.
Rooted plants will be inoculated with ToBRFV to assess resistance.