Enabling the Study of Gene Function in Gymnosperms: VIGS in Ephedra tweedieana

ABSTRACTPremiseAs the sister clade to angiosperms, gymnosperms are key to enabling the reconstruction of ancestral gene regulatory networks for seed plants. However, tools to rapidly and efficiently investigate gene function in gymnosperms remain limited due to the challenges of long life cycles and large genome sizes. Species within the xerophytic genusEphedra(Gnetales) have comparatively smaller genomes and shrubby growth habits with shorter life spans, making them better suited for greenhouse cultivation and laboratory experiments.Methods and ResultsHere, we implement Virus-Induced Gene Silencing (VIGS) to manipulate gene expression inEphedra tweedieana. Agrobacterium-mediated vacuum infiltration of Tobacco Rattle Virus (TRV2 and TRV1) in seedlings resulted in highly efficient silencing of theE. tweedieana PHYTOENE DESATURASEorthologEtwPDS. The expected photobleaching phenotype was observed as early as two weeks. It lasted at least three months, in stems, shoot tips, leaves, axillary meristems, and lateral branches of treated plants.ConclusionsThis first report of transient transformation and targeted gene silencing in a gymnosperm will further enable functional studies of the genetic mechanisms underpinning adaptations in this important and underrepresented lineage of seed plants.