A functional approach to profiling candidate genes in non model Brassicales

Obtaining functional data is an essential component in understanding mechanisms underlying morphological variation. Virus-induced gene silencing (VIGS) is a reverse genetic technique for identifying the function of target loci through viral mediated transcript knockdown. Here we introduce this technique to two plant systems Cleome violacea (Cleomaceae) and Erucaria erucarioides (Brassicaceae) chosen for their morphological traits and phylogenetic position. In both species, a construct with a heterologous sequence and a construct with an endogenous sequence of the visual marker PHYTOENE DESATURASE (PDS) were incorporated into separate viral constructs for downregulation. Downregulation using a heterologous sequence produced a comparable fold change in transcript abundance to the endogenous sequence in both species however C. violacea was more susceptible to this process. Additional targets FRUITFULL (FUL ) and a TCP1 homologue were targeted for knockdown within C. violacea to extend the application of this technique. A protocol for VIGS application is now available to both species for optimization and future functional analysis.