schenckii, the sscmk1 gene was targeted using

RNAi directed to knockdown the expression of this gene. S. schenckii yeast cells were first transformed with pSD2G-RNAi1 containing a segment of the 3′ end of the sscmk1 gene. The size of the sscmk1 insert used for transformation was in the range used for other fungal RNAi transformations [43, 44]. Real-time PCR (qRT-PCR) confirmed that the levels Crenigacestat ic50 of sscmk1 transcript were lower for the cells transformed with the pSD2G-RNAi1 than for the cells transformed with the empty plasmid at 35°C. The pSD2G-RNAi1 transformants grew from the beginning as mycelium type colonies in the selection plates at 35°C. Later when cultivated in liquid medium with aeration at 35°C, the growth observed, if any, was scarce and had the appearance of mycelium clumps with very few yeast cells. Upon further transfers to fresh medium, some of the conidia lost the capacity to grow at 35°C but could grow as mycelia when these

same cultures were transferred to 25°C, as stated previously. The inability to grow at 35°C could be due to a gradual lowering Ralimetinib cost of the intracellular SSCMK1 levels and the resulting impairment of thermotolerance in these cells, not viability. The fact that the conidia from some pSD2G-RNAi1 transformants could not grow at 35°C but if transferred to 25°C developed into mycelia and grew almost as abundantly as the wild type reinforces our previous results that suggest that SSCMK1 is Etomidate necessary for the development of the yeast form of the fungus. In order

to dismiss the possibility that the morphological effects could be due to an off-target effect, a second transformation was done using a different insert, this time from the 5′ end of the sscmk1 gene. The same abnormal morphology and growth at 35°C was observed when pSD2G-RNAi2 was used for transformation. The growth phase affected by silencing the sscmk1 gene was that of the yeast form of the fungus. In S. schenckii, the development of the yeast form of this fungus is favoured by increasing the temperature to 35°C. The capacity to tolerate temperatures between 35-37°C is essential for S. schenckii to grow in the human host. Some other species of the Ophiostomaceae that are plant pathogens, can produce yeast cells but most lack the ability to grow at 35-37°C and are non-pathogenic to humans [1]. Previous results using CaMK inhibitors pointed to the role of SSCMK1 for the proliferation of the yeast cells induced to re-enter the cell cycle and for the maintenance of the yeast morphology in S. schenckii. In this work, we observed these same results but we also observed that the actual effect could lie in the loss of thermotolerance by the fungus when sscmk1 was silenced.