An Optimized Protocol to Increase Virus-Induced Gene Silencing Efficiency and Minimize Viral Symptoms in Petunia.
Virus-induced gene silencing (VIGS) is used to down-regulate endogenous plant genes. VIGS efficiency depends on viral proliferation and systemic movement throughout the plant. Although tobacco rattle virus (TRV)-based VIGS has been successfully used in petunia (Petunia×hybrida), the protocol has not been thoroughly optimized for efficient and uniform gene down-regulation in this species. Therefore, we evaluated six parameters that improved VIGS in petunia. Inoculation of mechanically wounded shoot apical meristems induced the most effective and consistent silencing compared to other methods of inoculation. From an evaluation of ten cultivars, a compact petunia variety, 'Picobella Blue', exhibited a 1.8-fold higherCHSsilencing efficiency in corollas. We determined that 20 °C day/18 °C night temperatures induced stronger gene silencing than 23 °C/18 °C or 26 °C/18 °C. The development of silencing was more pronounced in plants that were inoculated at 3-4 versus 5 weeks after sowing. While petunias inoculated with pTRV2-NbPDSor pTRV2-PhCHSshowed very minimal viral symptoms, plants inoculated with the pTRV2 empty vector (often used as a control) were stunted and developed severe necrosis, which often led to plant death. Viral symptoms were eliminated by developing a control construct containing a fragment of thegreen fluorescent protein(pTRV2-sGFP). These optimization steps increased the area ofchalcone synthase(CHS) silencing by 69 % andphytoene desaturase(PDS) silencing by 28 %. This improved VIGS protocol, including the use of the pTRV2-sGFPcontrol plants, provides stronger down-regulation for high-throughput analyses of gene function in petunia.
Shaun R
SR
Broderick
Michelle L
ML
Jones
Chalcone synthase; Green fluorescent protein; Necrosis; Phytoene desaturase; Solanaceae; Tobacco rattle virus