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 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

Canning Tests on Mushroom Strain

Canning tests were carried out on 5 mushroom strains from 2 flushes of 4 separate crops. Particular attention was given to the ratio of whole closed canned mushrooms to that of whole closed canned mushrooms plus canned stems and pieces - i.e. total yield. Factors considered in the tests included grading prior to processing, blanching and retort losses, shrinkage in size and mushroom whiteness. Cream and off-white strains had the highest level of open and misshapen mushrooms prior to processing and a brown strain the lowest. Blanching losses, which comprised shrinkage and mushrooms that opened during blanching, ranged from 37.8% for the cream strain to 22.7% for the brown strain. Values for shrinkage during blanching were similar for the 5 strains with a mean of 19.2% loss in weight. Shrinkage during retorting ranged from 15.0% for the off-white-2 strain to 11.4% for the cream strain. Strains which showed a relatively low level of shrinkage during blanching had a relatively high shrinkage during retorting and combined shrinkage values for blanching and retorting were similar, ranging from 31.5 to 34.0%. The brown strain gave the highest yield of whole closed canned mushrooms at 54.8% and the cream strain the lowest at 45.0%. Corresponding yields of canned stems and pieces were 16.0 and 24.8% respectively. Total yield values for all strains were similar, ranging from 69.7% to 70.8%. Most shrinkage in mushroom size took place during blanching. Colour of the canned mushrooms of the 5 strains was considered acceptable by a sensory panel.Deposited by bulk impor

Prepacking and Shelf Life of Mushrooms

Mushrooms covered with the PVC film Resinite in a Hartmann Foodtainer dish had a shelf life of 5 to 7 days when stored at 15° to 21°C. Uncovered mushrooms had a shelf life of 2 to 4 days under similar conditions. Treatment of mushrooms with solutions of antioxidants followed by prepackaging with Resinite gave a shelf life of only 3 to 5 days. The shear press and reflectometer were found suitable for measuring texture and whiteness of mushrooms. Toughness of covered and uncovered mushrooms increased over a 5-day period. Uncovered mushrooms lost 31.6 percent of their original whiteness after 4 days while covered mushrooms lost 18.8 percent. The corresponding moisture losses were 68.3 and 10.8 percent.Deposited by bulk impor

Commercial mushroom production

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