The information suggesting that S. schenckii is

diploid comes from early studies done by us comparing the DNA content of our strain (μg of DNA/cell) with that of a diploid Candida albicans and haploid S. cerevisiae. In these experiments the DNA content of our strain was similar to that of the diploid C. albicans and to twice that of the haploid S. cerevisiae (unpublished results). If our S. schenckii strain is diploid, one would have to effectively knockout both copies of a given gene using 2 markers to select the transformants. A variety of transformation systems have been developed for many fungi, being the most popular that of Ito and collaborators for S. cerevisiae [34]. Preliminary work done by us using this method showed that this transformation protocol was not useful

LY2874455 in vitro for S. schenckii yeast cells (unpublished results). In this paper we describe the adaptation of a method originally designed for the transformation of Ophiostoma ulmi by Royer et al., for the transformation of S. schenckii [33]. This method uses permeabilized cells and treatment with β-mercaptoethanol, both of these conditions have been observed by us to increase the success of transformation of S. schenckii, as is the case of Ophiostoma ulmi [33]. The frequency of transformation for all fungi is dependent mTOR inhibitor on a variety of different parameters such as the nature of the transforming DNA, the concentration of the transforming DNA and the selection agent, among others [[34–36]]. Our primary goal in this work was to obtain the greatest number of transformants; therefore a concentration of transforming DNA of the order of 10 μg per 108 cells was used. Having Inositol oxygenase used this amount of DNA, a frequency of transformation of approximately 24 transformants/μg of DNA was obtained. This number of transformants is within the range reported with other fungi specifically when unlinearized DNA is used [34]. After having a reliable transformation system for S. schenckii, the next goal was to inquire if RNAi was an option to study gene

function in this fungus. Due to the uncertainty as to the presence of the gene silencing mechanism in some fungi such as S. cerevisiae and Ustilago maydis [37], we identified the presence of one of the enzymes involved in processing RNAi in S. schenckii DNA, a Dicer-1 homologue. As stated previously, the Dicer enzymes are important components of the mechanism that processes double stranded RNA precursors into small RNAs [38]. In the filamentous fungi, one or two Dicer-like homologues have been described [[39–41]]. N. crassa is the fungus where quelling was first described and has been more thoroughly studied [42]. In this fungus two Dicer-like homologues, dcl-1 and dcl-2 genes have been described [39]. The double mutant dcl-1 and dcl-2 showed the suppression of the processing of dsRNA into siRNA in N. crassa.