Department of Chemical Engineering, Pennsylvania State University, University Park, Pennsylvania, United States of America.
PLoS One. 2011;6(12):e28412. doi: 10.1371/journal.pone.0028412. Epub 2011 Dec 6.
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.
商业种植双孢蘑菇（Agaricus bisporus）使用由下层堆肥和上层泥炭组成的基质。通常，这两层分别用单个菌丝体接种体接种，代表 A. bisporus 的单一基因型。旨在研究该真菌物种作为异源蛋白表达系统的潜力的研究揭示了菌丝体接种体在子实体形态发生中的意外贡献。这些贡献是通过使用双重接种体方法阐明的，其中两层分别用转基因β-葡糖苷酸酶（GUS）和野生型（WT）系接种。令人惊讶的是，在下层基质中使用转基因 GUS 系和在上层基质中使用 WT 系产生了表达 GUS 活性但缺乏 GUS 转基因的子实体。GUS 转基因和 RNA 转录物的 PCR 和 RT-PCR 分析结果分别表明，GUS 蛋白从殖民下层的转基因菌丝体易位到仅由殖民上层的 WT 菌丝体发育的子实体。GUS 蛋白的有效易位取决于在下层使用转基因系，其中 GUS 基因受营养菌丝体活性启动子（漆酶 2 和 β-肌动蛋白）控制，而不是子实体活性启动子（疏水蛋白 A）控制。缺乏 GUS 基因的 GUS 表达子实体具有真实的 WT 基因型，这通过其衍生的担子孢子和菌丝体组织培养物中稳定遗传的 GUS 和潮霉素磷酸转移酶选择标记活性的缺失得到证实。用不同组织偏好启动子控制的单个携带 GUS 基因的系分别接种两层基质，导致 GUS 活性增加了约 3.5 倍，比单个接种体获得的活性增加了约 3.5 倍。我们的发现支持了在 A. bisporus 中存在以前未描述的长距离蛋白易位现象，这在重组蛋白表达和作物改良的生物技术方法中具有潜在应用。
