Yeast two-hybrid analyses—P. brasiliensis/S. cerevisiae gene interactions.

Colonies that grew on SD-Ade/-His/-Leu/-Trp drop-out plates and had α-galactosidase activity, as determined by a blue colouration of the colonies growing on α-X-gal supplemented media, are defined as positive (+) and those that did not as (-). All the gene sequences were swapped between the pGADT7 and pGBKT7 vectors and only those giving a positive-reaction in both vectors were scored as positive for an interaction. The ScCyr1(1–365), ScCyr1(350–480), ScCyr1(600–800) are the N-terminal, Gα and Ras association domains, respectively; whilst ScGpr1(679–961) the ScGpa2 association domain. A set of control reactions was undertaken at the same time to validate the test reactions: positive control, pGADT7- Ag with pGBKT7-P53; negative controls, pGBKT7-Lam with pGADT7, pGADT7-TPK21-270, pGADT7-TPK21-583, pGADT7-TPK2265-583 and pGADT7-GPG1, and pGBKT7-TPK1 with pGADT7, pGBKT7, pGADT7-Lam, pGBKT7-Lam and pGBKT7-PbActin. NT- Not tested.Yeast two-hybrid analyses—P. brasiliensis/S. cerevisiae gene interactions.

在SD-Ade/-His/-Leu/-Trp缺陷培养基平板上生长，且经添加α-X-gal的培养基中菌落呈蓝色判定具有α-半乳糖苷酶活性的菌落，被定义为阳性（+）；无此表现的菌落则记为阴性（-）。所有基因序列均在pGADT7与pGBKT7载体间进行了互换，仅在两种载体中均呈阳性反应的样本，才被判定为存在蛋白相互作用。ScCyr1(1–365)、ScCyr1(350–480)、ScCyr1(600–800)分别对应N末端结构域、Gα结构域与Ras结合结构域；而ScGpr1(679–961)则为ScGpa2结合结构域。同步设置了一组对照反应以验证测试反应的有效性：阳性对照为pGADT7-Ag与pGBKT7-P53；阴性对照包括pGBKT7-Lam与pGADT7、pGADT7-TPK21-270、pGADT7-TPK21-583、pGADT7-TPK2265-583及pGADT7-GPG1，以及pGBKT7-TPK1与pGADT7、pGBKT7、pGADT7-Lam、pGBKT7-Lam及pGBKT7-PbActin。NT：未进行检测（Not Tested）。酵母双杂交分析——巴西副球孢子菌（P. brasiliensis）/酿酒酵母（S. cerevisiae）基因相互作用。