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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.

Hyphal fusion and sexual development in filamentous fungi rely on coordinated signaling of numerous conserved nodes such as the striatin interacting phosphatase and kinase (STRIPAK) complex or the pheromone response (PR) MAP kinase cascade (MIK2, MEK2, MAK2, HAM5). Here we used the homothallic ascomycete Sordaria macrospora (Sm) to screen for putative protein interactors of the SmSTRIPAK complex. Using the STRIPAK complex interactor 1 (SCI1) subunit of the complex as bait, we enriched and identified canonical SmSTRIPAK components and a determinant of communication (DOC) protein. The DOC proteins were previously described in the closely related and heterothallic species Neurospora crassa, functioning in allorecognition of germlings and hyphal fusions. We generated ΔSmdoc1, ΔSmdoc2 single deletion strains and the double deletion mutant ΔSmdoc1ΔSmdoc2 in S. macrospora. Deletion phenotypes were paradoxical: single knockouts (ΔSmdoc1 or ΔSmdoc2) were nearly sterile and sexual development was impaired, yet the double mutant (ΔSmdoc1ΔSmdoc2) exhibited wild-type fertility and development, demonstrating non-redundant and mutually antagonistic roles. Similarly, we demonstrated an impairment of the N. crassa Δdoc-2 mutant in sexual development. Using gene tagging at the native locus, we performed TurboID-based proximity mapping with SmDOC1 and SmDOC2 as bait proteins. This proximity mapping demonstrated close ties of SmDOC1/2 to components of the PR MAP kinase pathway and revealed mutual SmDOC1 - SmDOC2 proximity. Yeast Two-Hybrid experiments with SmDOC1 confirmed the direct interaction with the MAP kinases MEK2 and MAK2. Fluorescence microscopy revealed that SmDOC1-TagRFP-T localized to ring-like structures around septal pores. Our results demonstrate that the DOC system is not restricted to heterothallic N. crassa, but also plays an essential role in the development of fruiting bodies in the homothallic fungus S. macrospora. These findings suggest the DOC1/2 proteins as a novel system that integrates STRIPAK and PR pathways, providing a possible mechanistic explanation for their non-additive deletion strain phenotypes.

Denitrification, a key process in soil nitrogen cycling, occurs predominantly within microbial hotspots, such as those around particulate organic matter (POM), where denitrifiers use nitrate as an alternative electron acceptor. For accurate prediction of dinitrogen (N2) and nitrous oxide (N2O) emissions from denitrification, a precise quantification of these microscale hotspots is required. The distribution of POM is of crucial importance in this context, as the local oxygen (O2) balance is governed not only by its high O2 demand but also by the local O2 availability. Employing a unique combination of X-ray CT imaging, microscale O2 measurements, and 15N labeling, we were able to quantify hotspots of aerobic respiration and denitrification. We analyzed greenhouse gas (GHG) fluxes, soil oxygen supply, and the distribution of POM in intact soil samples from grassland and cropland under different moisture conditions. Our findings reveal that both proximal and distal POM, identified through X-ray CT imaging, contribute to GHG emissions. The distal POM, i.e. POM at distant locations to air-filled pores, emerged as a primary driver of denitrification within structured soils of both land uses. Thus, the higher denitrification rates in the grassland could be attributed to the higher content of distal POM. Conversely, despite possessing compacted areas that could favor denitrification, the cropland had only small amounts of distal POM to stimulate denitrification in it. This underlines the complex interaction between soil structural heterogeneity, organic carbon supply, and microbial hotspot formation and thus contributes to a better understanding of soil-related GHG emissions. In summary, our study provides a holistic understanding of soil-borne greenhouse gas emissions and emphasizes the need to refine predictive models for soil denitrification and N2O emissions by incorporating the microscale distribution of POM. N2O originates from hotspots at the microscale and thus is largely unpredictable We combined X-ray CT imaging, microscale O2 sensors, and 15N labeling to map hotspots The position of the POM separates enhanced aerobic respiration and denitrification Distal POM (to air-filled pores) as driver of denitrification (N2O+N2) in soils Including POM distribution in models will enhance accuracy for soil GHG predictions