Enabling the study of gene function in gymnosperms: Virus-induced gene silencing in Ephedra tweedieana

As the sister clade to angiosperms, extant gymnosperms are crucial for reconstructing ancestral gene regulatory networks in seed plants. This highlights the need for model systems representing each of their distinct lineages. However, tools to quickly and effectively investigate gene function in gymnosperms are still limited due to the challenges of long life cycles and large genome sizes. Species within the xerophytic genus Ephedra (Gnetales) have comparatively smaller genomes and shrubby growth habits with shorter life spans, making them better suited for greenhouse cultivation and laboratory experiments.

We implement virus-induced gene silencing (VIGS) to manipulate gene expression in Ephedra tweedieana via Agrobacterium-mediated vacuum infiltration of tobacco rattle virus (TRV1 and TRV2) into seedlings.

Treatment resulted in highly efficient gene silencing of the E. tweedieana PHYTOENE DESATURASE (PDS) ortholog EtwPDS. The expected photobleaching phenotype was observed as early as two weeks, and lasted at least five months in stems, shoot tips, leaves, axillary meristems, and lateral branches of treated plants.

We report on virus-induced targeted gene silencing of PDS in a Gnetales representative to further enable functional studies of the genetic mechanisms underpinning adaptations in gymnosperms, an important and underrepresented lineage of seed plants.