Long-Distance Translocation of Protein during Morphogenesis of the Fruiting Body in the Filamentous Fungus, Agaricus bisporus

Abstract

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.

Complex multicellularity in fungi: evolutionary convergence, single origin, or both?

“…complex multicellular structures and organs are formed by the aggregation, elongation and specialization of hyphae, which has implications for the evolution of complex multicellularity. For example, there is no need for a qualitatively new mechanism for long‐distance distribution of nutrients or O 2 (Woolston et al , ), as seen in complex animals and plants. It should be noted, however, that it has been hypothesized that in the most complex fruiting bodies of Basidiomycota air channels are formed by the deposition of hydrophobins along the cell walls.…”

Live visualizations of single isolated tubulin protein self-assembly via tunneling current: effect of electromagnetic pumping during spontaneous growth of microtubule

“…), using particular resonance frequency of tubulin 22 (Some resonance peaks for tubulins are: [37, 46, 91, 137, 176, 281, 430] MHz; [9, 19, 78, 160, 224] GHz; [28, 88, 127, 340] THz (see Movie 1 online for quantum tunneling images); Some microtubule resonance peaks 15 16 are: [120, 240, 320] kHz; [12, 20, 22, 30, 101, 113, 185, 204] MHz; [3, 7, 13, 18] GHz, see Movie 2 online for quantum tunneling images) which is consistent with earlier findings 23 24 , that challenged the role of GTP 25 . By collecting tubulin samples from species that radically differs in genetic signature [porcine (brain neuron), human (MCF 7 active breast cancer cell), fungi 26 (Agaricus bisporus mashroom) and plant (six days old soybean germ lings)] we have experimentally determined the particular frequency domain that activates the protein molecules for a particular species. Such a sharing of the frequency-space is unprecedented.…”

The biotechnology of higher fungi - current state and perspectives

“…However, there are described and patented several methods for genome manipulations in higher fungi e.g. (Zhang et al 2002, Romaine 2011. Particularly intensive studies concern edible mushrooms.…”

“…Agaricus bisporus is one of the most intensively studied species. Despite more than 60 years of scientific investigation, advances in the genetic enhancement of this mushroom species has been impeded by its difficult genetics (Summerbell et al 1989, Van Griensven 1991, Romaine 2011. Modifications of the genetic characteristics of homobasidiomycetes such as Agaricus bisporus via treatment with donor DNA, fusions using protoplasts and via matings between strains are patented (Huizing et al 1995, Mikosh et al 2001.…”

“…These methods may be used in order to improve commercial characteristics of edible mushrooms and to commercially produce enzymes and metabolites in modified strains The use of transgenic basidiomycetes as a recombinant expression system for the production of a mucosal vaccine was also described (Florack & Rouwendal 2007). The first description of long-distance movement of a fully functional protein in a mushroom was given by Woolston et al (2011) (Feng et al 2010, Luo et al 2009. Similar experiments were also successfully conducted in our Department (Malinowska et al 2009b).…”