The First Observation of the Filamentous Fungus Neurospora crassa Growing in the Roots of the Grass Brachypodium distachyon
<p>Growth of <span class="html-italic">B. distachyon</span> roots with (blue) and without (orange) <span class="html-italic">N. crassa</span> co-cultivation. The red dashed line represents the time point, at which <span class="html-italic">N. crassa</span> spatially reaches the roots in the co-cultivation experiment. Significant differences between roots in the same developmental stage (grouped by boxes) are depicted as *** (<span class="html-italic">p</span> < 0.005).</p> "> Figure 2
<p>The SEM visualization of <span class="html-italic">B. distachyon</span> roots infected with <span class="html-italic">N. crassa</span>. Roots are wrapped in a dense network of hyphae (<b>A</b>–<b>D</b>) and scattered with oval-shaped conidia (<b>C</b>–<b>H</b>). Hyphae of different thickness can be observed (<b>E</b>). The integrity of the root surface is preserved (<b>F</b>). Protoperithecia (encircled) form in the vicinity of a root (<b>B</b>,<b>D</b>,<b>G</b>). The Microconidia production of old hyphae (<b>H</b>). Asterisk (<b>*</b>): hypha; circle: protoperithecium; white arrow: root hair.</p> "> Figure 3
<p>Fluorescent images of longitudinal <span class="html-italic">B. distachyon</span> root sections co-cultivated with the <span class="html-italic">N. crassa</span> FGSC #9518 strain, expressing GFP-coupled histone1 protein. Additional green fluorescence results from the auto-fluorescing plant cell wall. (<b>A</b>) Confocal laser scanning images show apoplastic (red arrows) and subcellular (yellow arrows) growth of fungal hyphae. (<b>B</b>) Fluorescent microscopic images show <span class="html-italic">N. crassa</span> accumulation in single cortical plant cells. (<b><span class="html-italic">i–iii</span></b>) GFP channel, (<b><span class="html-italic">I–III</span></b>) corresponding brightfield images. (<b>C</b>,<b>D</b>) Spinning-disk confocal microscopy was used to present an overview of a larger root section area. Distinct sectors (depicted by red boxes) with invasive <span class="html-italic">N. crassa</span> growth (yellow arrows) are magnified. Blue arrows indicate the extensive hyphal network covering the root surface.</p> "> Figure 3 Cont.
<p>Fluorescent images of longitudinal <span class="html-italic">B. distachyon</span> root sections co-cultivated with the <span class="html-italic">N. crassa</span> FGSC #9518 strain, expressing GFP-coupled histone1 protein. Additional green fluorescence results from the auto-fluorescing plant cell wall. (<b>A</b>) Confocal laser scanning images show apoplastic (red arrows) and subcellular (yellow arrows) growth of fungal hyphae. (<b>B</b>) Fluorescent microscopic images show <span class="html-italic">N. crassa</span> accumulation in single cortical plant cells. (<b><span class="html-italic">i–iii</span></b>) GFP channel, (<b><span class="html-italic">I–III</span></b>) corresponding brightfield images. (<b>C</b>,<b>D</b>) Spinning-disk confocal microscopy was used to present an overview of a larger root section area. Distinct sectors (depicted by red boxes) with invasive <span class="html-italic">N. crassa</span> growth (yellow arrows) are magnified. Blue arrows indicate the extensive hyphal network covering the root surface.</p> "> Figure 4
<p>The histological analysis of <span class="html-italic">B. distachyon</span> roots infected with <span class="html-italic">N. crassa</span>. (<b>A</b>,<b>B</b>) Paraffin sections, 7 μm, stained with hematoxylin and eosin. Conidia outside the root (<b>A</b>,<b>B</b>). Hyphae (<b>*</b>) seen inside a few plant root cortical cells whereas the majority of root cortical cells do not appear to be infected (<b>A</b>). Hyphae (<b>*</b>) growing through the vascular bundle (<b>B</b>). (<b>C</b>,<b>D</b>) Epon sections, 1 μm, stained with Richardson’s solution. Hyphae (<b>*</b>) growing inside the vascular bundle of a moderately infected root with vascular elements preserved (<b>C</b>), and of a strongly infected root with disrupted vascular elements (<b>D</b>). Xylem elements can be recognized by secondary wall thickenings. Panels (<b>i</b>) display details of boxed areas. Asterisk (<b>*</b>): hypha.</p> "> Figure 5
<p>The TEM analysis of <span class="html-italic">B. distachyon</span> roots infected with <span class="html-italic">N. crassa</span>. Hyphae in epidermal cells (<b>A</b>,<b>B</b>), in cortical cells (<b>C</b>,<b>D</b>), in a moderately infected vascular bundle (<b>E</b>,<b>F</b>), and in a strongly infected vascular bundle (<b>G</b>–<b>J</b>). The plant cell cytoplasm/membrane (arrowheads) can be observed in infected cells and neighboring cells (<b>C</b>). Hyphae spread through the root by crossing plant cell walls (<b>D</b>,<b>F</b>). In a moderately infected root, plant vascular elements are largely preserved (<b>E</b>,<b>F</b>). In a strongly infected root, vascular elements are destroyed except for the xylem elements with secondary wall thickenings (<b>G</b>,<b>J</b>). Parallel, thin, black lines represent folds that were generated over these rigid cell walls during sectioning (<b>G</b>,<b>J</b>). Hyphae grow unrestricted (<b>H</b>) or inside the xylem vascular elements (<b>I</b>,<b>J</b>). The characteristic ultrastructure of fungal hyphae (<b>*</b>) includes the cell wall, septa (with pores) (arrows), and dense cytoplasm. Boxed areas (<b>A</b>,<b>E</b>,<b>G</b>,<b>J)</b> are shown as enlarged (<b>B</b>,<b>F</b>,<b>H</b>,<b>I</b>). Asterisk (<b>*</b>): hypha; white arrow: hyphal septum; black arrowhead: plant cell cytoplasm/membrane; Ep: epidermis; C: cortex; En: endodermis; Vb: vascular bundle; Cw: cell wall.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Strains, Growth Condition
2.2. Fixation, Sectioning, and Staining
2.3. Microscopy
2.4. Electron Microscopy
2.5. Determination of the Root Growth and the Extent of Infection
3. Results
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Kollath-Leiß, K.; Repnik, U.; Winter, H.; Winkelmann, H.; Freund, A.S.; Kempken, F. The First Observation of the Filamentous Fungus Neurospora crassa Growing in the Roots of the Grass Brachypodium distachyon. J. Fungi 2024, 10, 487. https://doi.org/10.3390/jof10070487
Kollath-Leiß K, Repnik U, Winter H, Winkelmann H, Freund AS, Kempken F. The First Observation of the Filamentous Fungus Neurospora crassa Growing in the Roots of the Grass Brachypodium distachyon. Journal of Fungi. 2024; 10(7):487. https://doi.org/10.3390/jof10070487
Chicago/Turabian StyleKollath-Leiß, Krisztina, Urska Repnik, Hannes Winter, Heinrich Winkelmann, Anna Sophia Freund, and Frank Kempken. 2024. "The First Observation of the Filamentous Fungus Neurospora crassa Growing in the Roots of the Grass Brachypodium distachyon" Journal of Fungi 10, no. 7: 487. https://doi.org/10.3390/jof10070487