crassa strains were done as previously described [10]. N. crassa transformants were selected on medium containing 200 μg/ml of hygromycin B (Roche,
Mississauga, ON). un-24 constructs used in JIB04 in vivo incompatibility assays The un-24 OR or un-24 PA portions of the fusion genes were derived from standard N. crassa strains as described above. Fragments of un-24 were amplified with a forward primer that introduced a SpeI site allowing for an in-frame fusion with the hph marker, and a reverse primer that introduced a stop codon (or spanned the resident stop codon of un-24) as well as a flanking EcoRI site. All amplicons were cloned into pCR2.1. EcoRI and SpeI were then used to cut out the un-24 fragment and BglII and SpeI were used to cut out the hph fragment. The digests BTK inhibitor price were heat-inactivated, mixed and ligated before PCR amplification Selleckchem DMXAA using the primer that binds to the hph promoter and the appropriate un-24 reverse primer. The hph-un-24 fusion products were then cloned into pCR2.1. Our criteria for identifying incompatibility activity of OR and Panama (PA) constructs in N. crassa varies in accordance with the asymmetry of the system [15]. We recognized un-24 OR-associated incompatibility activity by a significant decrease (~95%) in the number of viable colonies
generated when the un-24 OR allele is transformed into the un-24 PA strain, in comparison to transformations with the vector control. In contrast, when un-24 PA is transformed into the un-24 OR strain, there is a modest (~20%) reduction in number of transformants recovered. However, 50 – 90% of the transformant colonies are small and have an irregular “star-like” growth form that contrasts with the wild-type “cloud-like” form of compatible transformants. Subcultures of the star-like colonies exhibit a self-incompatible phenotype as recognized by a slow growth rate and few aerial hyphae or conidia. This self-incompatible phenotype is inherently unstable and will spontaneously convert after about one week of continuous growth to near wild-type growth rate and morphology, a phenomenon called “escape” [11]. Therefore, to recognize un-24
PA-associated incompatibility activity we used three criteria: 1) more than half of colonies on the transformation plates displayed the self-incompatible morphology, 2) subcultures of PJ34 HCl these colonies had growth rates that were more than ten times lower than those of wild-type colonies and, 3) these subcultures subsequently escaped to a wild-type morphology and growth rate. Constructs were tested for incompatibility activity in at least three separate trials using transformation assays with strains C9-2 and C2(2)-1. Yeast Strains, media and growth conditions S. cerevisiae strains used in this study were derived from those listed in Additional file 2: Table S3 and were cultured by standard methods [59]. Selective plating of yeast transformants was performed with 100 μg/ml hygromycin B or 100 μg/ml nourseothricin (Werner Bioagents, Jena, Germany).