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Long-Distance Translocation of Protein during Morphogenesis of the Fruiting Body in the Filamentous Fungus, Agaricus bisporus
Commercial cultivation of the mushroom fungus, Agaricus bisporus, utilizes a substrate consisting of a lower layer of compost and upper layer of peat. Typically, the two layers are seeded with individual mycelial inoculants representing a single genotype of A. bisporus. Studies aimed at examining th...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2011
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3232199/ https://www.ncbi.nlm.nih.gov/pubmed/22163014 http://dx.doi.org/10.1371/journal.pone.0028412 |
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author | Woolston, Benjamin M. Schlagnhaufer, Carl Wilkinson, Jack Larsen, Jeffrey Shi, Zhixin Mayer, Kimberly M. Walters, Donald S. Curtis, Wayne R. Romaine, C. Peter |
author_facet | Woolston, Benjamin M. Schlagnhaufer, Carl Wilkinson, Jack Larsen, Jeffrey Shi, Zhixin Mayer, Kimberly M. Walters, Donald S. Curtis, Wayne R. Romaine, C. Peter |
author_sort | Woolston, Benjamin M. |
collection | PubMed |
description | Commercial cultivation of the mushroom fungus, Agaricus bisporus, utilizes a substrate consisting of a lower layer of compost and upper layer of peat. Typically, the two layers are seeded with individual mycelial inoculants representing a single genotype of A. bisporus. Studies aimed at examining the potential of this fungal species as a heterologous protein expression system have revealed unexpected contributions of the mycelial inoculants in the morphogenesis of the fruiting body. These contributions were elucidated using a dual-inoculant method whereby the two layers were differientially inoculated with transgenic β-glucuronidase (GUS) and wild-type (WT) lines. Surprisingly, use of a transgenic GUS line in the lower substrate and a WT line in the upper substrate yielded fruiting bodies expressing GUS activity while lacking the GUS transgene. Results of PCR and RT-PCR analyses for the GUS transgene and RNA transcript, respectively, suggested translocation of the GUS protein from the transgenic mycelium colonizing the lower layer into the fruiting body that developed exclusively from WT mycelium colonizing the upper layer. Effective translocation of the GUS protein depended on the use of a transgenic line in the lower layer in which the GUS gene was controlled by a vegetative mycelium-active promoter (laccase 2 and β-actin), rather than a fruiting body-active promoter (hydrophobin A). GUS-expressing fruiting bodies lacking the GUS gene had a bonafide WT genotype, confirmed by the absence of stably inherited GUS and hygromycin phosphotransferase selectable marker activities in their derived basidiospores and mycelial tissue cultures. Differientially inoculating the two substrate layers with individual lines carrying the GUS gene controlled by different tissue-preferred promoters resulted in up to a ∼3.5-fold increase in GUS activity over that obtained with a single inoculant. Our findings support the existence of a previously undescribed phenomenon of long-distance protein translocation in A. bisporus that has potential application in recombinant protein expression and biotechnological approaches for crop improvement. |
format | Online Article Text |
id | pubmed-3232199 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-32321992011-12-09 Long-Distance Translocation of Protein during Morphogenesis of the Fruiting Body in the Filamentous Fungus, Agaricus bisporus Woolston, Benjamin M. Schlagnhaufer, Carl Wilkinson, Jack Larsen, Jeffrey Shi, Zhixin Mayer, Kimberly M. Walters, Donald S. Curtis, Wayne R. Romaine, C. Peter PLoS One Research Article Commercial cultivation of the mushroom fungus, Agaricus bisporus, utilizes a substrate consisting of a lower layer of compost and upper layer of peat. Typically, the two layers are seeded with individual mycelial inoculants representing a single genotype of A. bisporus. Studies aimed at examining the potential of this fungal species as a heterologous protein expression system have revealed unexpected contributions of the mycelial inoculants in the morphogenesis of the fruiting body. These contributions were elucidated using a dual-inoculant method whereby the two layers were differientially inoculated with transgenic β-glucuronidase (GUS) and wild-type (WT) lines. Surprisingly, use of a transgenic GUS line in the lower substrate and a WT line in the upper substrate yielded fruiting bodies expressing GUS activity while lacking the GUS transgene. Results of PCR and RT-PCR analyses for the GUS transgene and RNA transcript, respectively, suggested translocation of the GUS protein from the transgenic mycelium colonizing the lower layer into the fruiting body that developed exclusively from WT mycelium colonizing the upper layer. Effective translocation of the GUS protein depended on the use of a transgenic line in the lower layer in which the GUS gene was controlled by a vegetative mycelium-active promoter (laccase 2 and β-actin), rather than a fruiting body-active promoter (hydrophobin A). GUS-expressing fruiting bodies lacking the GUS gene had a bonafide WT genotype, confirmed by the absence of stably inherited GUS and hygromycin phosphotransferase selectable marker activities in their derived basidiospores and mycelial tissue cultures. Differientially inoculating the two substrate layers with individual lines carrying the GUS gene controlled by different tissue-preferred promoters resulted in up to a ∼3.5-fold increase in GUS activity over that obtained with a single inoculant. Our findings support the existence of a previously undescribed phenomenon of long-distance protein translocation in A. bisporus that has potential application in recombinant protein expression and biotechnological approaches for crop improvement. Public Library of Science 2011-12-06 /pmc/articles/PMC3232199/ /pubmed/22163014 http://dx.doi.org/10.1371/journal.pone.0028412 Text en Woolston et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Woolston, Benjamin M. Schlagnhaufer, Carl Wilkinson, Jack Larsen, Jeffrey Shi, Zhixin Mayer, Kimberly M. Walters, Donald S. Curtis, Wayne R. Romaine, C. Peter Long-Distance Translocation of Protein during Morphogenesis of the Fruiting Body in the Filamentous Fungus, Agaricus bisporus |
title | Long-Distance Translocation of Protein during Morphogenesis of the Fruiting Body in the Filamentous Fungus, Agaricus bisporus
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title_full | Long-Distance Translocation of Protein during Morphogenesis of the Fruiting Body in the Filamentous Fungus, Agaricus bisporus
|
title_fullStr | Long-Distance Translocation of Protein during Morphogenesis of the Fruiting Body in the Filamentous Fungus, Agaricus bisporus
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title_full_unstemmed | Long-Distance Translocation of Protein during Morphogenesis of the Fruiting Body in the Filamentous Fungus, Agaricus bisporus
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title_short | Long-Distance Translocation of Protein during Morphogenesis of the Fruiting Body in the Filamentous Fungus, Agaricus bisporus
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title_sort | long-distance translocation of protein during morphogenesis of the fruiting body in the filamentous fungus, agaricus bisporus |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3232199/ https://www.ncbi.nlm.nih.gov/pubmed/22163014 http://dx.doi.org/10.1371/journal.pone.0028412 |
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