Strategies for the transgenic manipulation of filamentous fungi
AssigneeThe Penn State Research Foundation
InventorsC. Peter Romaine, Carl David Schlagnhaufer, Benjamin Michael Woolston
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Abstract
Disclosed is a new technology that enables the transgenic modification of a mushroom-forming fungus to confer a transgenic genotype and/or phenotype. According to the invention herein, one can manipulate the fruiting body of a fungus conferring an altered phenotype, but a wild-type genotype. The fruiting body itself is devoid of any cognate transgene, and Applicants have discovered a new mechanism of protein synthesis and accumulation in fungi and a technique to independently control the genotype and phenotype of the fruiting body.
Claims27
A method of producing a filamentous fungus fruiting body to confer an altered phenotype, but a wild-type genotype comprising:
- seeding a lower nutrient substrate layer with a first filamentous fungal inoculant, so that said substrate becomes colonized with the mycelial inoculant,
- overlaying said lower nutrient substrate layer with an upper water-holding substrate layer that has been inoculated with a second transgenic filamentous fungal mycelial inoculant, wherein said second transgenic filamentous fungal mycelial inoculant incorporates a heterologous polynucleotide construct, and
- allowing fruiting bodies to form, wherein said formed fruiting bodies include the heterologous polynucleotide product from the second transgenic filamentous fungal mycelial inoculant.
A method of modulating the activity of an endogenous or heterologous protein in a fruiting body of a filamentous fungus comprising:
- seeding a lower nutrient substrate layer with a first transgenic filamentous fungal mycelial inoculant, so that said substrate becomes colonized with the mycelial inoculant,
- wherein said first transgenic filamentous fungal mycelial inoculant incorporates a heterologous polynucleotide construct,
- overlaying said nutrient substrate with an upper water-holding substrate layer that has been inoculated with a second non-transgenic filamentous fungal mycelial inoculant, and
- allowing fruiting bodies to form, wherein the polynucleotide construct from the first transgenic filamentous fungal inoculant construct provides modulation of an endogenous protein or heterologous protein in the formed fruiting bodies.
A method for transgenic manipulation of a filamentous fungus comprising:
- inoculating said fungus in an upper cultivation substrate wherein said inoculant is with a genotype desired in a fruiting body, and
- inoculating a lower cultivation substrate with a fungus that comprises an RNA and/or protein desired in said resulting fruiting body.
A method for the long-distance transport of protein and/or RNA transcript in a filamentous fungus comprising:
- exposing said fungus to a lower nutrient substrate layer comprising an inoculant with a heterologous polynucleotide construct operably linked to a promoter that expresses in the vegetative mycelium at levels sufficient for the transport of said RNA and or protein into the fruiting body of the filamentous fungus.
A method of producing a fruiting body that includes the genotype of one inoculant, and the phenotype of a second inoculant comprising:
- seeding a lower nutrient substrate layer with a first filamentous fungal mycelial inoculant, so that said substrate becomes colonized with the mycelial inoculant, the phenotype of said first inoculant being one that is desired in a produced fruiting body,
- overlaying said lower nutrient substrate layer with an upper water-holding substrate layer that has been inoculated with a second filamentous fungal mycelial inoculant, the genotype of said second inoculant being one which is desired in said produced fruiting body;
- and
- allowing fruiting bodies to form, wherein said formed fruiting bodies include the genotype of the second inoculant and the phenotype of the first inoculant.
A method for producing fruiting bodies of a filamentous fungus comprising:
- seeding a lower nutrient substrate layer with a transgenic mycelial inoculant, so that said substrate becomes colonized by the mycelial inoculant, and either
- overlaying said lower nutrient substrate with an upper water-holding substrate layer that has been seeded with the same transgenic mycelial inoculant used in the lower substrate layer or
- overlaying said lower nutrient substrate with an upper water-holding substrate layer that has not been seeded with a mycelial inoculant, but rather is allowed to become colonized by the transgenic mycelial inoculant used in the lower substrate layer, and
- allowing fruiting bodies to form, wherein said formed fruiting bodies possess a recombinant protein from the transgenic mycelial inoculant.
Dependencies are inferred from the claim text.