Visual monitoring system for virus-induced gene silencing (VIGS) in pepper fruit

Abstract

After completion of sequencing multiple plant genomes, virus-induced gene silencing (VIGS) has become an option of functional genomics tool for specific plants, which are recalcitrant to Agrobacterium-mediated transformation such as pepper. Although VIGS in plants has being widely employed as a powerful tool for functional genomics, scattering phenotypic effects resulted by irregular gene silencing become a main problem to be overcome especially in fruit tissue. I improved VIGS system based on tobacco rattle virus (TRV) by using An2 MYB transcription factor, which is the genetic determinant of purple color by accumulation of anthocyanin in pepper fruit. Silencing of endogenous An2 in the anthocyanin-rich pepper with modified TRV vector for ligation-independent cloning (LIC) resulted in deficiency of the purple pigment in leaves, flowers and fruits. Infection with TRV-LIC including a tandem construct of An2 and Phytoene desaturase (PDS) resulted in a typical photobleaching in leaves without the purple pigment whereas the silencing of PDS alone lead to photobleached and purple-colored leaves. Plants infected with TRV-LIC vector including fragments of both An2 and pepper Golden-like 2 (CaGLK2) showed color change from green to pale green in fruits. Co-silencing of Capsaicin synthase with An2 in fruits resulted in decreased level of capsaicin and dihydrocapsaicin accumulation measured by HPLC analysis coupled with absence of the purple pigment in fruits. These results imply that VIGS with tandem constructs anchoring An2 as a visible reporter can accelerate a study about functions of metabolic genes in fruit development.