Rushil Patel, Satyanarayana Gouthu, and Laurent Deluc*
*Oregon State University, 2750 SW Campus Way, Room 4121, Corvallis, OR, 97331 (delucl@oregonstate.edu)

Genetic manipulation is a major tool in determining gene function and its relationship with traits of interest in plants. For any crop, it requires a tractable genetic transformation system and a reliable expression system to turn on or switch off expression of targeted genes. We implemented the microvine system for Agrobacterium tumefaciens-mediated transformation and a plant gene switch system to control the transgene expression by supplying transgenic greenhouse plants (through root-drenching) a chemical inducer, methoxyfenozide. As proof of principle, we conducted gain and loss of function studies on Auxin Responsive Factor 4, a transcription factor that may contribute to the timing of ripening initiation in grape berries. We developed three types of constructs. In construct 1, we aimed to induce over-expression of the native endogenous ARF4. In construct 2, we generated transgenic lines containing a synthetic ARF4 resistant to miRNA-mediated gene silencing (miRARF4). In construct 3, we created ARF4-silencing lines using artificial miRNA technology. To pinpoint plants with the most response to the chemical inducer, eight plants from each construct were grown to maturity and induced over a month with leaf collections at select points. Six collection times were chosen to comprehend both the short-term responses (days 0, 3, 7, and 10) and the reversibility of the system (days 14 and 31). The data extracted using qPCR allowed us to characterize the time period of greatest induced transgene expression. Expression levels of the most responsive plants were eight-fold in the native construct, 100-fold in miRARF4, and 1000-fold increase in the ARF4 silencing line. Ongoing experiments quantitatively compare expression levels in berry samples to verify the inducibility dynamics in the fruit. Once confirmed, the study intends to focus on comparing ARF4’s targetome among the three constructs by using genome-wide transcriptomic analyses.

Funding Support: Oregon Wine Board, Erath/Family Foundation, Oregon Wine Research Institute