As shown in Fig. Observation of the hygromycin B-resistant transformants under the fluorescent microscope revealed that five transformants clearly displayed strong mCherry signals. Indeed, bp and 1. Generation of fluorescently labeled S. The plasmid construct was ectopically integrated into the S. The transformants and wild-type strain are shown in Lanes 1—5 and WT, respectively.
To examine the nuclear dynamics in different sexual structures, the ascospores of S. Approximately 1—2 h post-inoculation, the ascospores of the H2B-mCherry-labeled strain formed a germination pore at one end of the spore, followed by the formation of the germination vesicle, which allowed the nuclei to enter the vesicle Fig.
This differs from the germination behavior of N. The germ pore or emergence of the germination vesicle appeared to be located at the pointed terminus of the ascospore, while the opposite side of the germination pore seemed more rounded, indicating that S.
Moreover, the ascospores in Sordaria spp. Physically, the germination of S. In contrast, N. Moreover, the expansion of germination vacuoles was observed in the cytoplasm of the germination vesicle during the outgrowth of the germ tube, and the spore was eventually found to be empty Hackett and Chen At 6 h post-inoculation, various inclusions were visible in this germling, including a single large or multiple medium-sized lipid droplet vacuoles in the center of the germination vesicle.
Interestingly, lipid droplet vacuoles in the germinating ascospores of S. Live-cell imaging of nuclear distribution during the S. The nuclear distribution in a single germination vesicle and germ tube from an ascospore was observed at 2, 3, and 6 h post-inoculation hpi.
At 15 hpi, nuclear fluorescence was distributed in the mature vegetative hyphae and hyphal fusion regions, whereas the nuclei were not detected in the hyphal tip regions of the mature vegetative hyphae.
Nuclear distribution in the coil and ascogonia hyphae, including the early protoperithecia, was detected within 27 and 48 hpi, respectively. At approximately hpi, mature perithecia with asci containing black mature and multinucleated ascospores were observed. Live images were captured with the Deltavision core system. Nuclear distribution in each developmental stages of S.
White arrows and arrowheads indicate hyphal fusion and the extension tip of a vegetative hypha, respectively. Yellow asterisks indicate vacuoles. Nuclear distribution of germ tube and mature vegetative hyphal tip and representative hyphal fusion events.
The ascospores of the H2B-mCherry-labeled strain were grown on malt extract agar III under uniform constant white light. White arrows indicate differentiated sites of nuclear distribution among these fungal structures. White arrowheads indicate germination vesicles. After germination, the germ tube emerging from the vesicle continued elongating and branching to produce young vegetative hyphae.
Initially, a uniform nuclear distribution was displayed along the length of the vegetative hyphae Figs. However, at 15 h post-inoculation, the hyphal tips were devoid of nuclei Figs. Such a nuclei-devoid region near the hyphal tip has also been described in other fungal systems, including N. The pre-fusion interaction between vegetative hyphae was also observed at 15 h post-inoculation Figs. The main morphology types of the hyphal fusions were tip-to-side and tip-to-tip.
This observation indicated that the hyphal tip extension phase was replaced by hyphal orientation to form the mating structure Brand and Gow Some of the approaching hyphae showed slight swelling of the apex but also displayed nuclei at the tip ends Fig. As sexual development further proceeded, ascogonial coils, likely formed as side branches of vegetative hyphae ca.
At 48 h post-inoculation, in addition to ascogonial coil formation, multinucleate cellular compartments resulting from differentiated ascogenous hyphae were observed in this culture and defined as early protoperithecia Fig. These structures were ca. In fact, the protoperithecia developed from the ascogonium were formed by the aggregation of enveloping hyphae that emerged from either the ascogonial coil or neighboring hyphae, as previously described in detail Lord and Read Through the growth of the protoperithecium accompanied by septation and tissue differentiation, the dark-pigmented mature fruiting body perithecium with its neck was observed at approximately h post-inoculation, with a size of ca.
Moreover, an ascus rosette containing asci of various stages of maturity derived from the perithecium of the mCherry-labeled strain was observed.
The black, mature ascospore contained multiple nuclei derived from repetitive mitotic division, as shown in Fig. Overall, in the present study, we established a gene delivery system for S. Although much is known about nuclear distribution during hyphal extension and fusion in fungi such as N. Research on nuclear behavior using this tool could help unveil the complex pre- and post-fusion mechanisms, which would in turn provide valuable insights for understanding cell fusion in other eukaryotic species.
Moreover, the link between the hyphal fusion event and fruiting body development is one of the most important questions in fungal biology, and the model genetic system of S. Fungal Genet Biol — Article PubMed Google Scholar. Curr Opin Microbiol — Bretzloff CW Fungus fruiting in submerged culture.
Science — Mycol Res — Emerson MR Chemical activation of ascospore germination in Neurospora crassa. J Bacteriol — Emerson MR Some physiological characteristics of ascospore activation in Neurospora crassa.
Past reports of dung fungi as endophytes would seem to challenge the view that their ascospores can be used as proxies for Quaternary herbivore activity. However, those reports have been questioned because surface-sterilization protocols could have failed to kill dung fungus ascospores stuck to the plant surface. Isolation into medium after surface-sterilization would have then misleadingly suggested to researchers that dung fungi were endophytes.
Our focus here was thus on effects of inoculation with dung fungi on plant growth and fecundity. We reasoned that significant effects on plant fitness could not result from ascospores merely stuck to the plant surface.
Now, having found that isolates of S. We hypothesized that fungi obligate to dung might be expected to influence plant growth in such a way as to increase palatability to herbivores. We now see that this hypothesis is without merit because the dung fungi that we tested were not obligate to dung. Spores of dung fungi in Quaternary sediments should not be taken as indisputable evidence of past herbivory.
Dung fungus ascospores may be more common in dung than on plant residues in nature, but that difference is unconfirmed. If equally true in the past, that difference might be the basis for continued use of dung fungus spores as bio-indicators of past herbivory. But that use would not be because these fungi have a life cycle obligate to dung. Support was provided by the U. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field.
Abstract Dung fungi, such as Sordaria fimicola , generally reproduce sexually with ascospores discharged from mammalian dung after passage through herbivores. Introduction The life cycle of many coprophilous fungi is thought to be obligate to dung. Materials and Methods Inoculation of Bromus tectorum seedlings with representative isolates of three dung fungi Greenhouse assays were conducted to determine whether B.
Inoculation of roots or leaves of B. Leaf inoculations. Effects of S. Completion of life cycle via passage through an herbivore and reproduction in dung In late winter, four open ewes of the Suffolk breed were placed in a 2 x 3 m indoor pen, with ad libitum access to water.
Completion of life cycle via infection of a plant and reproduction in senescent plant tissue Ten B. Reproduction in the absence of competitors in plant tissue Leaf tissue from five B. Preference experiment Six yearling Suffolk ewes provided by the University of Idaho Palouse Research, Education, and Extension Center were housed in a communal pen with ad libitum access to water and locally grown grass hay, comprised primarily of tall fescue Festuca arundinacea.
Ethics Statement Experiments with sheep were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Results Inoculation of Bromus tectorum seedlings with representative isolates of three dung fungi Inoculations resulted either in significant reductions in growth and fecundity or in insignificant effects; there were no significant increases in growth and fecundity as a result of inoculations with the three dung fungi Fig 1.
Download: PPT. Fig 1. Fecundity top row—seeds per plant and growth bottom row—grams of dry, aboveground, vegetative biomass per plant of some populations of Bromus tectorum were significantly reduced by inoculation with representative isolates of three genera of dung fungi i.
Table 1. Completion of life cycle via passage through a herbivore and reproduction in dung All sheep dosed with the S. Fig 2. The sexual state observed both after passage through a sheep, and after infection of a B. Table 2. Transit of S. Completion of life cycle via infection of a plant and reproduction in senescent plant tissue Two of the five B.
Reproduction in the absence of competitors in plant tissue Mycelia developed on sterilized B. Fig 4. No preference for plants infected with S. Discussion Dung fungi are associated with diverse plant taxa.
Supporting Information. S1 Table. Data from forage quality and preference experiments. References 1. European Journal of Cell Biology — Genetics 87— The American Biology Teacher — View Article Google Scholar 4.
Quaternary Science Reviews — View Article Google Scholar 5. London, UK. Quaternary Science Reviews 21— View Article Google Scholar 8. Cugny C, Mazier F, Galop D Modern and fossil non-pollen palynomorphs from the Basque mountains western Pyrenees, France : the use of coprophilous fungi to reconstruct pastoral activity. Vegetation History and Archaeobotany — View Article Google Scholar 9. PloS one 7: e Raper D, Bush M A test of Sporormiella representation as a predictor of megaherbivore presence and abundance.
Quaternary Research — View Article Google Scholar Herrera J, Poudel R, Khidir HH Molecular characterization of coprophilous fungal communities reveals sequences related to root-associated fungal endophytes. Microbial Ecology — Fungal Diversity — Fungal Biology — Mycological Research — Canadian Journal of Research — Sydowia 46— Petrini O, Fisher P, Petrini L Fungal endophytes of bracken Pteridium aquilinum , with some reflections on their use in biological control.
Sydowia — Carroll GC Fungal endophytes in stems and leaves: from latent pathogen to mutualistic symbiont. Ecology 2—9. Zak J, Wicklow D Response of carbonicolous ascomycetes to aerated steam temperatures and treatment intervals. Canadian Journal of Botany — Horie Y, Li D Five interesting ascomycetes from herbal drugs.
Moreover, the expansion of germination vacuoles was observed in the cytoplasm of the germination vesicle during the outgrowth of the germ tube, and the spore was eventually found to be empty Hackett and Chen At 6 h post-inoculation, various inclusions were visible in this germling, including a single large or multiple medium-sized lipid droplet vacuoles in the center of the germination vesicle. Interestingly, lipid droplet vacuoles in the germinating ascospores of S. Live-cell imaging of nuclear distribution during the S.
The nuclear distribution in a single germination vesicle and germ tube from an ascospore was observed at 2, 3, and 6 h post-inoculation hpi. At 15 hpi, nuclear fluorescence was distributed in the mature vegetative hyphae and hyphal fusion regions, whereas the nuclei were not detected in the hyphal tip regions of the mature vegetative hyphae. Nuclear distribution in the coil and ascogonia hyphae, including the early protoperithecia, was detected within 27 and 48 hpi, respectively.
At approximately hpi, mature perithecia with asci containing black mature and multinucleated ascospores were observed. Live images were captured with the Deltavision core system. Nuclear distribution in each developmental stages of S.
White arrows and arrowheads indicate hyphal fusion and the extension tip of a vegetative hypha, respectively. Yellow asterisks indicate vacuoles. Nuclear distribution of germ tube and mature vegetative hyphal tip and representative hyphal fusion events. The ascospores of the H2B-mCherry-labeled strain were grown on malt extract agar III under uniform constant white light. White arrows indicate differentiated sites of nuclear distribution among these fungal structures.
White arrowheads indicate germination vesicles. After germination, the germ tube emerging from the vesicle continued elongating and branching to produce young vegetative hyphae. Initially, a uniform nuclear distribution was displayed along the length of the vegetative hyphae Figs.
However, at 15 h post-inoculation, the hyphal tips were devoid of nuclei Figs. Such a nuclei-devoid region near the hyphal tip has also been described in other fungal systems, including N. The pre-fusion interaction between vegetative hyphae was also observed at 15 h post-inoculation Figs.
The main morphology types of the hyphal fusions were tip-to-side and tip-to-tip. This observation indicated that the hyphal tip extension phase was replaced by hyphal orientation to form the mating structure Brand and Gow Some of the approaching hyphae showed slight swelling of the apex but also displayed nuclei at the tip ends Fig. As sexual development further proceeded, ascogonial coils, likely formed as side branches of vegetative hyphae ca.
At 48 h post-inoculation, in addition to ascogonial coil formation, multinucleate cellular compartments resulting from differentiated ascogenous hyphae were observed in this culture and defined as early protoperithecia Fig. These structures were ca.
In fact, the protoperithecia developed from the ascogonium were formed by the aggregation of enveloping hyphae that emerged from either the ascogonial coil or neighboring hyphae, as previously described in detail Lord and Read Through the growth of the protoperithecium accompanied by septation and tissue differentiation, the dark-pigmented mature fruiting body perithecium with its neck was observed at approximately h post-inoculation, with a size of ca.
Moreover, an ascus rosette containing asci of various stages of maturity derived from the perithecium of the mCherry-labeled strain was observed. The black, mature ascospore contained multiple nuclei derived from repetitive mitotic division, as shown in Fig. Overall, in the present study, we established a gene delivery system for S. Although much is known about nuclear distribution during hyphal extension and fusion in fungi such as N.
Research on nuclear behavior using this tool could help unveil the complex pre- and post-fusion mechanisms, which would in turn provide valuable insights for understanding cell fusion in other eukaryotic species.
Moreover, the link between the hyphal fusion event and fruiting body development is one of the most important questions in fungal biology, and the model genetic system of S.
KK and W-CS conceived the ideas and designed the experiments. KK performed the experiments and analyzed data. KK and W-CS contributed critically to the drafts and gave final approval for publication. Both authors read and approved the final manuscript.
We thank Ms. This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Electronic supplementary material. National Center for Biotechnology Information , U.
Journal List Bot Stud v. Bot Stud. Published online Jul 3. Kulsumpun Krobanan and Wei-Chiang Shen. Author information Article notes Copyright and License information Disclaimer. Corresponding author. Received Apr 18; Accepted Jun This article has been cited by other articles in PMC. Nuclear behavior in the hyphal cells of H2B-mCherry-labeled strain.
Nuclear trafficking in a germ tube emerging from a single germ pore displayed a uniform distribution throughout the structure. The video shows a period of 20 min with a time lapse of two frames per minute.
Additional file 2: Video S2. Nuclei absent from the extension tip regions of vegetative hyphae. Abstract Background The coprophilous ascomycete Sordaria fimicola usually reproduces sexually. Results Live imaging revealed that the nuclei in the germlings and fusion hyphae during the pre-contact interaction are located adjacent to the tip. Conclusions This is the first report of the application of a fluorescence labeling technique in S. Electronic supplementary material The online version of this article
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