Oostlander Anne Geertje, Brodde Laura, von Bargen Miriam, Slippers Bernard, Becker Yvonne, Brandt Ulrike, Klawonn Frank, Grobler Christiaan, Well Lucas, Stenlid Jan, Oliva Jonas, Elfstrand Malin, Fleissner Andre
Institute of Genetics, Technische Universität Braunschweig, Braunschweig, Germany.
Department of Forest Mycology and Plant Pathology, Swedish University of Agricultural Sciences, Uppsala, Sweden.
PLoS One. 2024 Dec 27;19(12):e0308794. doi: 10.1371/journal.pone.0308794. eCollection 2024.
Diplodia sapinea (Fr.) Fuckel is a widespread fungal pathogen affecting conifers worldwide. Infections can lead to severe symptoms, such as shoot blight, canker, tree death, or blue stain in harvested wood, especially in Pinus species. Its impact on forest health is currently intensified, likely due to climate change, posing an increasing threat to global ecosystems and forestry. Despite extensive and successful research on this pathogen system, fundamental questions about its biology and plant-associated lifestyle remain unanswered. Addressing these questions will necessitate the development of additional experimental tools, including protocols for molecular genetics and cell biology approaches. In this study, we continue to address this need by establishing an Agrobacterium-mediated genetic transformation protocol for D. sapinea, enabling targeted mutagenesis and heterologous gene expression. We utilized this methodology to localize the histone H2B by tagging it with the fluorescent protein mCherry. Additionally, we established a time- and space-efficient laboratory-scale infection assay using two-week-old Pinus sylvestris seedlings. Integrating these tools in a proof-of-concept study enabled the visualization of D. sapinea in planta growth through the fluorescently labeled reporter strain.
松色二孢菌（Diplodia sapinea (Fr.) Fuckel）是一种广泛分布的真菌病原体，影响着全球范围内的针叶树。感染会导致严重症状，如嫩梢枯萎、溃疡、树木死亡或采伐木材中的蓝变，尤其是在松属物种中。目前，其对森林健康的影响因气候变化而加剧，对全球生态系统和林业构成越来越大的威胁。尽管对该病原体系统进行了广泛且成功的研究，但关于其生物学特性和与植物相关的生活方式的基本问题仍未得到解答。要解决这些问题，需要开发更多实验工具，包括分子遗传学和细胞生物学方法的实验方案。在本研究中，我们通过建立一种用于松色二孢菌的农杆菌介导的遗传转化方案来继续满足这一需求，该方案能够实现靶向诱变和异源基因表达。我们利用这种方法通过用荧光蛋白mCherry标记来定位组蛋白H2B。此外，我们使用两周大的欧洲赤松幼苗建立了一种省时且高效的实验室规模感染测定法。在一项概念验证研究中整合这些工具，能够通过荧光标记的报告菌株观察松色二孢菌在植物体内的生长情况。
