Westermann Jens, Koebke Eva, Lentz Roswitha, Hülskamp Martin, Boisson-Dernier Aurélien
Institute for Plant Sciences, Faculty of Mathematics and Natural Sciences, University of Cologne, Cologne, Germany.
Front Plant Sci. 2020 Oct 15;11:569194. doi: 10.3389/fpls.2020.569194. eCollection 2020.
Even though stable genomic transformation of sporelings and thalli of is straightforward and efficient, numerous problems can arise during critical phases of the process such as efficient spore production, poor selection capacity of antibiotics or low transformation efficiency. It is therefore also desirable to establish quick methods not relying on stable transgenics to analyze the localization, interactions and functions of proteins of interest. The introduction of foreign DNA into living cells via biolistic mechanisms has been first reported roughly 30 years ago and has been commonly exploited in established plant model species such as or . Here, we report the fast and reliable transient biolistic transformation of Marchantia thallus epidermal cells using fluorescent protein fusions. We present a catalog of fluorescent markers which can be readily used for tagging of a variety of subcellular compartments. Moreover, we report the functionality of the bimolecular fluorescence complementation (BiFC) in with the example of the p-body markers MpDCP1/2. Finally, we provide standard staining procedures for live cell imaging in , applicable to visualize cell boundaries or cellular structures, to complement or support protein localizations and to understand how results gained by transient transformations can be embedded in cell architecture and dynamics. Taken together, we offer a set of easy and quick tools for experiments that aim at understanding subcellular localization, protein-protein interactions and thus functions of proteins of interest in the emerging early diverging land plant model .
尽管对苔类植物的幼苗和叶状体进行稳定的基因组转化直接且高效，但在该过程的关键阶段仍可能出现许多问题，如高效孢子产生、抗生素选择能力差或转化效率低。因此，也需要建立不依赖稳定转基因的快速方法来分析目标蛋白的定位、相互作用和功能。大约30年前首次报道了通过生物弹道机制将外源DNA导入活细胞，并且该方法已在诸如拟南芥或烟草等成熟的植物模式物种中广泛应用。在此，我们报道了使用荧光蛋白融合对地钱叶状体表皮细胞进行快速可靠的瞬时生物弹道转化。我们展示了一系列荧光标记物目录，可用于标记各种亚细胞区室。此外，我们以p小体标记物MpDCP1/2为例报道了地钱中双分子荧光互补（BiFC）的功能。最后，我们提供了地钱活细胞成像的标准染色程序，适用于可视化细胞边界或细胞结构，以补充或支持蛋白质定位，并了解通过瞬时转化获得的结果如何嵌入细胞结构和动态变化中。综上所述，我们为旨在了解亚细胞定位、蛋白质-蛋白质相互作用以及因此了解新出现的早期分化陆地植物模式物种中目标蛋白功能的实验提供了一套简单快速的工具。
