The presence of unfolded proteins in the ER induces a cellular response called the Unfolded Protein Response (UPR). The HacA transcription factor plays a central role in the UPR pathway, resulting on the induction of a large number of genes, among them chaperones and foldases that assist in the protein folding in the ER. We were interested in studying the effects of the hacA and ireA deletions, as well as the constitutive hacA expression on the growth and morphology of A. niger. For the knock-out mutants, deletion cassettes were made containing 500bp of the 5’ and 3’ flanking regions of the respective gene, and pyrG to be used as a selection marker. For generating the constitutive hacA mutant, a hacA gene construct was made that lacked the 20 nt intron sequence and transformed to replace the wild type hacA gene in the genome. For the construction of the deletion strains, an A. niger strain with kusA (ku70) gene deleted was used. In this background, not only the targeting efficiency is high, but also in the case of essential genes, heterokaryons are obtained as primary transformants, as ireA turn out to be. In S. cerevisiae, ire1 deletion is not lethal and results in inositol auxotrophy. However, supplementing growth media with inositol did not allow the growth of A. niger ireA deletion strains. Southern blot analysis confirmed the heterokaryotic nature of ireA transformants obtained. In A. niger, 916;hacA is not lethal, but the mutant shows a severe growth defect leading to a small and compact colony phenotype. Primary transformants displaying the hacA growth phenotype were never observed, but hacA deletion strains were efficiently obtained after purification of spores from primary transformants. These results show that gene deletions mutants with a strong defect in growth can easily be obtained via heterokaryons in the kusA background. The constitutive hacA mutant also shows a growth defect when compared with the wild type. Both on solid or liquid medium, this mutant grows slower then the wt. Morphologically, hyphae of the constitutive hacA mutant grow on a curly way, resembling mutants with defect in the tubulin cytoskeleton. The effect of constitutive hacA expression on tubulins is currently investigated.
Full conference title:
9th EUROPEAN CONFERENCE ON FUNGAL GENETICS
- ECFG 9th (2008)