Virus-induced multiple gene silencing to study redundant metabolic pathways in plants: Silencing the starch degradation pathway in Nicotiana benthamiana

• Published in: Biotechnology journal 2012-07, Vol.7 (7), p.884-890
• Main Authors: George, Gavin M., Bauer, Rolene, Blennow, Andreas, Kossmann, Jens, Lloyd, James R.
• Format: Article
• Language: English
• Subjects: DPE1; DPE2; Gene Knockout Techniques - methods; Gene Silencing; Glycogen Debranching Enzyme System - genetics; Malto-oligosaccharides; Metabolic Networks and Pathways - genetics; Nicotiana - genetics; Nicotiana - metabolism; Phenotype; Plant Leaves - genetics; Plant Leaves - metabolism; Plants, Genetically Modified - genetics; Plants, Genetically Modified - metabolism; RNA Viruses - genetics; Starch - genetics; Starch - metabolism; Starch degradation; VIGS
• ISSN: 1860-6768; 1860-7314
• DOI: 10.1002/biot.201100469

Virus‐induced gene silencing (VIGS) is a rapid technique that allows for specific and reproducible post‐transcriptional degradation of targeted mRNA. The method has been proven efficient for suppression of expression of many single enzymes. The metabolic networks of plants, however, often contain isoenzymes and gene families that are able to compensate for a mutation and mask the development of a silencing phenotype. Here, we show the application of multiple gene VIGS repression for the study of these redundant biological pathways. Several genes in the starch degradation pathway [disproportionating enzyme 1; (DPE1), disproportionating enzyme 2 (DPE2), and GWD] were silenced. The functionally distinct DPE enzymes are present in alternate routes for sugar export to the cytoplasm and result in an increase in starch production when silenced individually. Simultaneous silencing of DPE1 and DPE2 in Nicotiana benthamiana resulted in a near complete suppression in starch and accumulation of malto‐oligosaccharides. Virus‐Induced Gene Silencing (VIGS) is an effective tool for ‘loss‐of‐function’ studies when multiple genes are targeted. To advance the understanding of starch degradation, in this article, the authors show the application of multiple gene VIGS repression for the study of the redundant biological pathways in the leaf metabolism of Nicotiana benthamiana. They demonstrate the feasibility for multiple VIGS to be used as reverse genetics tool, which can assess redundant enzyme systems typical for the plant kingdom.