The cassettes

were PCR-amplified from these Entinostat cost plasmids and used for transformation of Aspergilli according to the procedure of Osmani et al. [59]. Transformants were scored for their ability to grow on minimal medium. PCR or Southern blot GSK1904529A supplier analyses were used throughout of the manuscript to demonstrate that the transformation cassettes had integrated homologously at the targeted A. fumigatus or A. nidulans loci. The A. fumigatus alcA::AfcrzA and A. nidulans alcA::AncrzA constructions were performed by amplifying by PCR 5′-end fragments (for A. fumigatus, 1084-bp from the start codon of the ORF with the primers Afcrz1 AscI: 5′-GGCGCGCCAATGGCTTCACAGGAGATGTTCC-3′ and Afcrz1 PacI: 5′-CCTTAATTAAGCACATTGGGCATCATTTCCTGTCC-3′; and for A. nidulans, 1068 bp from the start codon of the ORF with the primers AncrzA AscI 5′-GGCGCGCCAATGGATCCTCAAGATACGCTGCAGG-3′ and AncrzA PacI 5′-CCTTAATTAACATCTGTGACGCTTGCCCGATATC-3′), digesting them with PacI and AscI, and cloning them in the

corresponing PacI and AscI restriction sites of the pMCB17-apx plasmid. The fragment of the ORF is under the control of the A. nidulans Protein Tyrosine Kinase inhibitor alcA promoter and after homologous integration the translation produces an N-terminal fusion protein. A. fumigatus and A. nidulans pyrG – strains were transformed with the corresponding vectors pMCB17-apx-crzA and after homologous recombination the alcA::gfp::crzA construction and a truncated crzA non-coding gene were generated. All the transformants were confirmed by PCR using specific primers. Acknowledgements We would like to thank the Laboratories of Confocal Microscopy and Electronic Microscopy from the Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Brazil, for the use of the confocal microscope, and the four anonymous reviewers for their suggestions. This research was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil, and John Simon Guggenheim Memorial

Foundation, USA. Electronic supplementary material Additional file 1: MycoClean Mycoplasma Removal Kit Genes less expressed after Aspergillus fumigatus ΔcrzA mutant CaCl 2 200 mM exposition for 10 and 30 minutes. List of the genes identified in the microarray experiment as less expressed. (PDF 54 KB) Additional file 2: Genes more expressed after Aspergillus fumigatus ΔcrzA mutant CaCl 2 200 mM exposition for 10 and 30 minutes. List of the genes identified in the microarray experiment as more expressed. (PDF 87 KB) Additional file 3: The fungal RcnAs form a distinct clade. Phylogenetic analysis was carried out using the MEGA-2 (Molecular Evolutionary Genetics Analysis version 3.1) software (18, 2001; http://​www.​megasoftware.​net).