Chalcone synthase as a reporter in virus-induced gene silencing studies of flower senescence

Abstract

Agrobacterium-mediated infection of petunia (Petunia hybrida) plants with tobacco rattle virus (TRV) bearing fragments of Petunia genes resulted in systemic infection and virus-induced gene silencing (VIGS) of the homologous host genes. Infection with TRV containing a phytoene desaturase (PDS) fragment resulted in reduced abundance of PDS transcripts and typical photobleaching of photosynthetic tissues. Infection with TRV containing a chalcone synthase (CHS) fragment resulted in silencing of anthocyanin production in infected flowers. The silencing phenotype ranged from scattered white spots on the normal purple background to entirely white flowers. Symptoms in the V26 cultivar were a diffuse mosaic, but infection of some purple-flowered commercial cultivars resulted in large white sectors and even entirely white flowers. Abundance of CHS transcripts in the white flowers was less than 4% of that in purple flowers on the same plant. Infection with TRV containing a tandem construct of PDS and CHS resulted in leaf photobleaching and white patterns on the flowers. Transcripts of CHS and PDS were reduced both in leaves and in flowers confirming simultaneous silencing of both genes by the tandem construct. We tested the effects of infection with TRV containing CHS and a fragment of a petunia gene encoding for 1-aminocyclopropane-1-carboxylate oxidase (ACO4) Abundance of transcripts encoding ACO4 and ACO1 were reduced (by 5% and 20%, respectively) in infected flowers. Whether the flowers were treated with ACC or pollinated, the white (silenced) flowers or flower sectors produced less ethylene and senesced later than purple (non-silenced) tissues. These results indicate the value of VIGS with tandem constructs containing CHS as reporter and a target gene as a tool for examining the function of floral-associated genes.

Visual Tracking of Plant Virus Infection and Movement Using a Reporter MYB Transcription Factor That Activates Anthocyanin Biosynthesis

“…Silencing of phytoene desaturase also served as reporter in such carotenoidrich organs as tomato fruits (Fu et al, 2005;Orzaez et al, 2006). Similarly, silencing of chalcone synthase, a key enzyme in anthocyanin biosynthesis, has been used as an endogenous reporter for gene silencing in such anthocyanin-rich organs as flower petals (Chen et al, 2004). Silencing of the transgenically expressed transcription factors Ros1 and Delila was also used as a virus-induced gene silencing reporter in purple tomatoes (Orzaez et al, 2009).…”

Recruitment of CRABS CLAW to promote nectary development within the eudicot clade

“…A previously described Tobacco Rattle Virus (TRV)-based VIGS system was used to silence CRC in Petunia and Nicotiana (Liu et al, 2002;Chen et al, 2004). A 575 bp fragment of the petunia CRC gene corresponding to entire coding region was cloned into pTRV2 to form pTRV2 CRC.…”

A Visual Reporter System for Virus-Induced Gene Silencing in Tomato Fruit Based on Anthocyanin Accumulation

“…Recently, the use of internal markers for VIGS based on pigment accumulation in petunia (Petunia hybrida), soybean (Glycine max), and tomato has proven very successful for empowering the method as a reverse genetics tool (Chen et al, 2004b;Nagamatsu et al, 2007;Spitzer et al, 2007;Jiang et al, 2008). Tomato introgression lines accumulating anthocyanins in their fruit have been described, but this accumulation is restricted to the epidermis; therefore, the use of these lines is restricted to analysis of gene function in this tissue.…”