Optimization of virus-induced gene silencing in Catharanthus roseus

Catharanthus roseus (periwinkle) contains abundant alkaloids and is the only commercial source of two cancer drugs, vinblastine and vincristine. The plant is also used as an experimental host for phytoplasmas because it can be infected by most phytoplasmas and shows distinctive symptoms. Abundant gene sequences for periwinkle have become available through high-throughput transcriptome sequencing, so periwinkle has become an ideal model for studies of alkaloid metabolism and plant-phytoplasma interactions. Tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) for functional genomics studies has been established, but the duration of the system was too short for plant-phytoplasma interaction studies. To improve the system, several factors were analysed, including culture temperature, plant age, leaf age, organ preferences and virus distribution, for their effects on VIGS efficacy. A new agroinfiltration method was also used for inoculating entire seedlings. High temperature (30 degrees C) strongly inhibited VIGS, but low temperature (16 degrees C) inhibited plant growth. VIGS efficacy was reduced in old leaves but was not affected by plant age. VIGS occurred in all examined organs, including roots, stems, leaves and flowers, but was most effective in leaves and flowers. The TRV-based system required the existence of TRV2 to maintain gene silencing, so TRV2 concentration may be used as an indicator of successful gene silencing. The optimized VIGS system was useful for studies of plant-phytoplasma interactions and conferred gene silencing for more than 6 months.