Oostlander, Anne GeertjeBrodde, LauraVon Bargen, MiriamSlippers, BernardBecker, YvonneBrandt, UlrikeKlawonn, FrankGrobler, ChristiaanWell, LucasStenlid, JanOliva, JonasElfstrand, MalinFleissner, Andre2025-05-232025-05-232024-12-27Oostlander, A.G., Brodde, L, Von Bargen, M,, Slippers, B., Becker, Y., Brandt, U., et al. (2024) Development of a molecular genetics and cell biology toolbox for the filamentous fungus Diplodia sapinea. PLoS ONE 19(12): e0308794. https://doi.org/10.1371/journal.pone.0308794.1932-6203 (online)10.1371/journal.pone.0308794http://hdl.handle.net/2263/102496SUPPORTING INFORMATION : FILE S1. Laboratory protocol for the Agrobacterium-mediated transformation of Diplodia sapinea. TABLE S1. Oligonucleotides used in this study. MOVIE S1. Video of merged brightfield and fluorescence microscopy of D. sapinea expressing the H2B-mcherry. Merged brightfield and fluorescence microscopy of the transformed D. sapinea strain GD1_14 on VMM, expressing the H2B-mcherry fusion protein. The H2BmCherry fluorescence is triggered by excitation at 580 nm. RAW IMAGES S1. Original images of diagnostic PCRs and Southern blot image supporting Fig 2. PCR analysis of eight transformed strains was performed using primer pairs (A) 2245/2246 and (B) 2247/2248 to confirm homologous integration of the expression cassette at the niaD gene locus. (C) Southern blot analysis of eight strains transformed with Agrobacterium sp. AGL-1_AO11 was performed using HindIII-digested DNA from transformants and wild type (WT), with the entire T-DNA fragment as a probe. The Thermo Scientific GeneRuler 1 kb DNA Ladder was used as a marker in all gels.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.en© 2024 Oostlander et al. This is an open access article distributed under the terms of the Creative Commons Attribution License.InfectionsDiplodia sapineaPinus speciesClimate changeArticle