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

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 cul-ture 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 degrees C (day, 12 h)/16 degrees 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.

Automated summary

A virus-induced gene silencing (VIGS) system based on tobacco rattle virus (TRV) was constructed and optimized for functional genomics studies in pecan. The optimized conditions included the use of a TRV-based vector with a 300 bp target sequence, syringe infiltration method, inoculation of 'Pawnee' or 'Shaoxing' seedlings, and exposure to a temperature cycle of 24 degrees C (day, 12 h)/16 degrees C (night, 12 h). The established VIGS system was successfully used for reverse genetic studies in pecan.