Canadian Forest Service Publications
Transient gene expression in western white pine using agroinfiltration. 2019. Ma, Z., Liu, J-J, Zamany, A., Williams, H. J. For. Res.
Issued by: Pacific Forestry Centre
Catalog ID: 39932
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A method for transient gene expression was developed for western white pine (WWP, Pinus monticola Dougl. ex D.Don) using reporter gene uidA encoding β-glucuronidase (GUS). GUS was transiently expressed in cross sections of primary and secondary needles, cotyledons, and current and second year stems of WWP via vacuum-infiltration with Agrobacterium tumefaciens. Histochemical assays of cross sections of secondary needles showed stronger blue color indicating GUS expression at day 1 and 2 than on other days post agroinfiltration (dpa). GUS activity expressed inside WWP cells was confirmed using light microscopy. In fluorometric assays, GUS expression was high at 1 dpa and lasted until 4 dpa in detached secondary needles, while similarly high expression levels only lasted until 2 dpa in attached secondary needles then dropped significantly. Although the length of GUS-staining zones varied among different WWP organs and between growth and dormant seasons, all tested WWP tissues using the protocol had high levels of transient GUS expression. Thus, heterologous candidate genes or endogenous silencing can be expressed in various WWP tissues or organs using this agroinfiltration approach. The current protocol for efficient transient gene expression will aid functional genomics study of WWP and its pathogens and related conifer species.
Plain Language Summary
We developed a methodology for transient gene expression for functional genomics studies in western white pine (Pinus monticola, Dougl. ex D. Don) using report gene uidA encoding β-glucuronidase (GUS). We found that GUS can be transiently expressed in multiple western white pine organs, including primary and secondary needles, cotyledons, and current and second year stems via Agroinfiltration under vacuum. The report gene expression was confirmed by both histochemical GUS-staining assay and fluorometric assay. We found that the transient gene expression lasted time significantly longer (p < 0.01) in the detached needles and attached ones, but there was no highly significant difference of gene expression levels between growth and dormant seasons. To the best of our knowledge, western white pine is the first gymnosperm species on which Agroinfiltration has been successfully utilized. The current protocol reported here will aid functional genomics studies on western white pine, its pests/pathogens, and related conifer species, especially on the molecular interactions of white pine-blister rust.