Percentages of uninucleate, binucleate, multinucleate (i.e., three or more nuclei), and anucleate hyphal compartments in <i>E</i>. <i>festucae</i> Fl1, <i>E</i>. <i>typhina</i> E1022, and the putative hybrid between Fl1 and E1022 grown in culture.

<p>Average ± S. D., three replicates of n = 150.</p><p>Percentages of uninucleate, binucleate, multinucleate (i.e., three or more nuclei), and anucleate hyphal compartments in <i>E</i>. <i>festucae</i> Fl1, <i>E</i>. <i>typhina</i> E1022, and the putative hybrid between Fl1 and E1022 grown in culture.</p

Histone H1-GFP-labeled nuclei in <i>E</i>. <i>festucae</i> Fl1 grown in culture.

<p>Nuclei in Fl1 appear as either round or elongated structures. Though most hyphal compartments are uninucleate, some compartments possess three or more nuclei (asterisk). Arrowheads indicate two septa at the end of the hyphal compartment. The bar represents 20 μm.</p

Vegetative hyphal fusion in <i>Epichloë</i> grown in culture.

<p>1) Species names are based on the revised nomenclature of <i>Epichloë</i> species [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121875#pone.0121875.ref023" target="_blank">23</a>].</p><p>2) NH; non-hybrid. H; hybrid.</p><p>3) Hyphal fusion</p><p>++, commonly undergoing vegetative hyphal fusion (the average number of vegetative hyphal fusion in the observed area in one set of experiments (8067.6 μm²) was ≥ 1, which corresponds to ca. > 10 vegetative hyphal fusion per mm²)</p><p>+, rarely undergoing vegetative hyphal fusion (the average number of vegetative hyphal fusion in the observed area was < 1)</p><p>ND, fusion not detected</p><p>4) Hyphal cords (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121875#pone.0121875.s002" target="_blank">S2 Fig</a>);</p><p>++, extensive cords of more than ten hyphae running side by side</p><p>+ minor cords of ca. five hyphae</p><p>-, no cords</p><p>Vegetative hyphal fusion in <i>Epichloë</i> grown in culture.</p

Different types of vegetative hyphal fusion in <i>Epichloë festucae</i> E2368 grown in culture.

<p>Confocal micrographs of DIC optics and Calcofluor White (CFW) staining showing lateral-to-lateral fusion <b>(A)</b>, apical-to-lateral fusion <b>(B)</b>, and subapical-to-lateral fusion <b>(C)</b>. (<b>D</b>) Though a majority of vegetative hyphal fusion occurred between two hyphae running side-by-side (asterisks), it also occurred when two hyphae came into close proximity (double asterisks). Bars represent 10 μm.</p

Nuclear distribution in a putative hybrid grown in culture.

<p>The putative hybrid was generated through a protoplast fusion experiment between <i>E</i>. <i>festucae</i> Fl1 expressing histone H1-GFP and <i>E</i>. <i>typhina</i> E1022 expressing histone H1-TagRFP. Most hyphal compartments are uninucleate, with nuclei possessing both GFP and TagRFP signals (asterisks) without any vegetative hyphal fusion nearby. The bar represents 20 μm.</p

Plasmids used in this study.

<p>Plasmids used in this study.</p

Vegetative hyphal fusion of <i>E</i>. <i>festucae</i> Fl1 in the tall fescue leaf sheath.

<p>Confocal micrographs of DIC optics, aniline blue staining showing fungal hyphae, Alexa Fluor 488-conjugated wheat germ agglutinin (WGA-AF) showing septa, and overlaid images with aniline blue staining pseudocolored in red. <b>(A)</b> Arrows point septa in two short hyphae connecting two hyphae extending in parallel. <b>(B)</b> Two hyphae (arrows) connected by a short hypha (arrowhead) which presumably underwent vegetative hyphal fusion. Locations of all three hyphae correspond to outlines of plant cells (compare DIC and aniline blue images). Bars represent 20 μm.</p

Vegetative Hyphal Fusion and Subsequent Nuclear Behavior in <i>Epichloë</i> Grass Endophytes

<div><p><i>Epichloë</i> species (including the former genus <i>Neotyphodium</i>) are fungal symbionts of many agronomically important forage grasses, and provide their grass hosts with protection from a wide range of biotic and abiotic stresses. <i>Epichloë</i> species include many interspecific hybrids with allodiploid-like genomes, which may provide the potential for combined traits or recombination to generate new traits. Though circumstantial evidence suggests that such interspecific hybrids might have arisen from nuclear fusion events following vegetative hyphal fusion between different <i>Epichloë</i> strains, this hypothesis has not been addressed empirically. Here, we investigated vegetative hyphal fusion and subsequent nuclear behavior in <i>Epichloë</i> species. A majority of <i>Epichloë</i> strains, especially those having a sexual stage, underwent self vegetative hyphal fusion. Vegetative fusion also occurred between two hyphae from different <i>Epichloë</i> strains. Though <i>Epichloë</i> spp. are uninucleate fungi, hyphal fusion resulted in two nuclei stably sharing the same cytoplasm, which might ultimately lead to nuclear fusion. In addition, protoplast fusion experiments gave rise to uninucleate putative hybrids, which apparently had two markers, one from each parent within the same nucleus. These results are consistent with the notion that interspecific hybrids arise from vegetative hyphal fusion. However, we also discuss additional factors, such as post-hybridization selection, that may be important to explain the recognized prevalence of hybrids in <i>Epichloë</i> species.</p></div

Primers used for this study.

<p>Bold letters indicate vector-derived sequences for <i>in vitro</i> recombination.</p><p>Underline indicates restriction sites.</p><p>Italic letters indicate template DNA-derived sequences for PCR.</p><p>Primers used for this study.</p

Vegetative hyphal fusion of <i>E</i>. <i>festucae</i> E2368 grown in culture establishing cytoplasmic continuity.

<p>Confocal micrographs showing DIC optics, mitochondria <b>(A)</b> or vacuoles <b>(B)</b>, CFW staining, and overlaid images. Arrowheads indicate fusion bridges through which organelles extend into a neighboring compartment. Bars represent 10 μm.</p