Development and application of a virus-induced gene silencing system for functional genomics in pecan (Carya illinoinensis)

Abstract

Pecan is a high-value nut tree that cultivated globally. Nowadays, in-depth studies of this species are often hampered by lacking of efficient molecular genetic tools. To facilitate functional genomics in pecan, a virus-induced gene silencing (VIGS) system based on tobacco rattle virus (TRV) was explored in this study. We used pecan magnesium chelatase subunit H (CiChlH) gene as a visual indicator to evaluate the effect of several factors including infiltration method, insert size, insert orientation, inoculation concentration, host genotype, and culture temperature on VIGS. Our results showed that the optimized conditions for pecan VIGS system were as followed: construction of TRV-based vector harboring ∼300 bp target sequence in sense or antisense orientation, adjustment of inoculation solution to a final OD600 of 0.7, use of syringe infiltration method, inoculation of ‘Pawnee’ or ‘Shaoxing’ seedlings at 2∼4 true-leaf stage with obvious shoot apical growth, and subjecting plants to a temperature cycle of 24 °C (day, 12 h)/16 °C (night, 12 h). Using the optimized system, we obtained an expected photobleaching at 4 weeks post infiltration. To further verify the stability of the system, a pecan phenylalanine ammonialyase (CiPAL) gene was used for silencing and we detected a significant reduction in total phenolics in silenced plants with respect to control seedlings, as anticipated. Altogether, the TRV-based VIGS system has been successfully established in pecan and could be used for future reverse genetic studies.