C: RNA levels of PPG1 in mycelial phase G217B (n = 4), UC1 (n = 7), and UC26 (n = 4) compared to levels in strains Olaparib in vivo overexpressing MAT1-1-1 and BEM1 in the G217B background (n = 3). *** = p ≤ 0.001. UC1 as a tool to study cleistothecia formation Although the precise mechanisms by which UC1 gained the ability to form empty cleistothecia remained unclear, the strain provides an opportunity to study cleistothecia production in H. capsulatum. Since the pheromone response MAP kinase pathway plays a central role in the mating response of S. cerevisiae [12, 13], it was predicted to play a similar

role in the mating response of H. capsulatum. HMK1, a putative FUS3/KSS1 homolog, was silenced in UC1 to determine the role of the pheromone response pathway in cleistothecia formation of this strain. HMK1 RNA levels were reduced to 25% of the levels found in a control strain (Figure 6A). Silencing HMK1 had no effect on cleistothecia production when UC1 was paired INCB018424 manufacturer with UH3 (Figure 6B). This indicates that either the pheromone response pathway is not involved in formation of cleistothecia, or that low levels of HMK1 are still sufficient to support cleistothecia formation. Alternatively, the mechanisms that restored cleistothecia production in this strain could be suppressing the effects of silencing HMK1.

Figure 6 Effects of silencing HMK1 on cleistothecia formation. A: HMK1 RNA levels found in yeast phase of the silenced strain (UC1-HMK1-RNAi) compared to those

found in the empty vector control strain by qRT-PCR. HSP90 Values represent averages and standard error of triplicate samples. B: Number of cleistothecia counted from three pairings of UC1 + UH3, or UC1 with HMK1 silenced + UH3. To identify additional differences between UC1 and G217B that could play a role in cleistothecia formation, microarray analysis was performed comparing mycelial samples of UC26 and G217B. UC26 was used as the comparator to eliminate the differences attributable to hph activity. Seven hundred and forty one predicted transcripts demonstrated greater than three-fold altered Selleck CAL 101 expression in UC26 compared to G217B. Four hundred and thirty four transcripts were upregulated in UC26 compared to G217B while three hundred and nine transcripts were downregulated. Using Blast2Go for blast analysis and assignment of functional annotation and gene ontology, no specific patterns of biological processes could be discerned between up- or downregulated genes (Figure 7). Among genes with assigned molecular functions, genes associated with protein modification or gene regulation, such as transferases and phosphatases, accounted for 37% of downregulated genes in UC26 compared to G217B consistent with the suggestion that no single function results in the acquisition of the ability to form empty cleistothecia. Figure 7 Microarray analysis of UC26 and G217B gene expression.