Virus-induced Gene Silencing as a Tool for Functional Analyses in the Emerging Model Plant Aquilegia (Coumbine, Ranunculaceae)

Abstract

Background: The lower eudicot genus <i>Aquilegia</i>, commonly known as columbine, is currently the subject of extensive genetic and genomic research aimed at developing this taxon as a new model for the study of ecology and evolution. The ability to perform functional genetic analyses is a critical component of this development process and ultimately has the potential to provide insight into the genetic basis for the evolution of a wide array of traits that differentiate flowering plants. <i>Aquilegia</i> is of particular interest due to both its recent evolutionary history, which involves a rapid adaptive radiation, and its intermediate phylogenetic position between core eudicot (e. g., <i>Arabidopsis</i>) and grass (e. g., <i>Oryza</i>) model species. Results: Here we demonstrate the effective use of a reverse genetic technique, virus-induced gene silencing (VIGS), to study gene function in this emerging model plant. Using Agrobacterium mediated transfer of tobacco rattle virus (TRV) based vectors, we induce silencing of <i>PHYTOENE DESATURASE</i> (<i>AqPDS</i>) in Aquilegia vulgaris seedlings, and <i>ANTHOCYANIDIN SYNTHASE</i> (<i>AqANS</i>) and the B-class floral organ identity gene <i>PISTILLATA</i> in <i>A. vulgaris</i> flowers. For all of these genes, silencing phenotypes are associated with consistent reduction in endogenous transcript levels. In addition, we show that silencing of <i>AqANS</i> has no effect on overall floral morphology and is therefore a suitable marker for the identification of silenced flowers in dual-locus silencing experiments. Conclusion: Our results show that TRV-VIGS in <i>Aquilegia vulgaris</i> allows data to be rapidly obtained and can be reproduced with effective survival and silencing rates. Furthermore, this method can successfully be used to evaluate the function of early-acting developmental genes. In the future, data derived from VIGS analyses will be combined with large-scale sequencing and microarray experiments already underway in order to address both recent and ancient evolutionary questions.