Optimization of Virus-induced Gene Silencing to Facilitate Evo-devo Studies in the Emerging Model SpeciesMimulus guttatus(Phrymaceae)¹

Abstract

Mimulus guttatus DC. (yellow monkey-flower; Phrymaceae) is an important model species for ecological and evolutionary studies, being locally adapted to a wide range of elevation, moisture and temperature gradients, soil types, and pollinator availabilities. In order to advance this species as a model for evolutionary genetic studies, we have developed virus-induced gene silencing (VIGS) using the tobacco rattle virus (TRV) to assay gene function. We demonstrate the effectiveness of Agrobacterium-mediated VIGS in two divergent populations of M. guttatus, Iron Mountain 767 (IM767) and Point Reyes (PR). Plants infected with a fragment of the carotenoid biosynthesis pathway gene PHYTOENE DESATURASE (PDS) cloned into the TRV2 vector exhibited endogenous PDS silencing and photobleached phenotypes. We further assayed for VIGS-induced floral phenotypes by silencing paralogous genes putatively affecting floral symmetry, CYCLOIDEA1 (CYC1) and CYCLOIDEA2 (CYC2). Simultaneous silencing of CYC1 and CYC2 resulted in organ number defects in the petal and stamen whorls; silencing of CYC1 affected petal margin growth; and silencing of CYC2 had no effect on flower development. Infection with TRV2 and TRV1 is significantly higher and more pervasive in the IM767 versus the PR population and is more efficient after vacuum infiltration. These results demonstrate the efficacy of VIGS for determining the function of developmental genes, including those involved in ecologically important reproductive traits.

Evolving Ideas on the Origin and Evolution of Flowers: New Perspectives in the Genomic Era

“…VIGS has been applied successfully in a diverse range of eudicot and monocot species ( Becker and Lange 2010 ), including functional investigations of genes involved in floral organ identity and symmetry in basal eudicots and core eudicots ( e.g. , Gould and Kramer 2007 ; Sharma et al 2011 ; Hidalgo et al 2012 ; Gonçalves et al 2013 ; Preston et al 2014 ).…”

Geometric morphometrics reveals shifts in flower shape symmetry and size following gene knockdown of CYCLOIDEA and ANTHOCYANIDIN SYNTHASE

“…Natural diversity in gene expression, divergence, and function can now be examined in non-model organisms and across clades [ 1 , 2 ] with the advent of new molecular tools and improved genetic resources (e.g., transcriptomes and an increasing number of genomes). In angiosperms, a growing number of non-model plants are emerging to characterise the role(s) of specific genes, or gene copies following polyploidization, in the evolution of morphological novelty, especially as it pertains to floral development (e.g., flexibility in floral structures in Nigella (Ranunculaceae) [ 3 ]; inflated calyx syndrome in Physalis (Solanaceae) [ 4 ]; and pollinator shifts in Mimulus (Phrymaceae) [ 5 ]). Examining gene function relies on assaying phenotypic shifts following experimental changes in the amount, location, or timing of candidate gene expression.…”

Genomic studies on the nature of species: adaptation and speciation in Mimulus

“…) and M. guttatus (Preston et al . ). Overall, these approaches add to the available molecular toolkit for evolutionary studies in the genus and represent important progress in understanding the functional genetic basis of complex phenotypes in Mimulus .…”

“…Yaowu Yuan (University of Connecticut) discussed how his work in M. lewisii with Toby Bradshaw (University of Washington) uses in planta stable transformation protocols (Yuan et al 2013b) and mutagenesis experiments (Yuan et al 2013a) to confirm the function of flower colour and floral scent genes (Byers et al 2014). Protocols for stable transformation and virus-induced gene silencing (VIGS) are also available for M. aurantiacus Susi c et al 2014) and M. guttatus (Preston et al 2014). Overall, these approaches add to the available molecular toolkit for evolutionary studies in the genus and represent important progress in understanding the functional genetic basis of complex phenotypes in Mimulus.…”