Ld cultures of AcOTAbZIP-1/3 and WT strains grown in liquid MM in darkness at 25 1 C (OTA inducing conditions, [14]) employing the RNeasy Plant Mini Kit (MNK MedChemExpress Qiagen, Milan, Italy) in line with the manufacturer’s guidelines. First-strand cDNA was synthesized from 1 of RNA utilizing M-MLV reverse transcriptase (Life Technologies, Milan, Italy) and random primers inside a volume of 20 , as outlined by the manufacturer’s directions. The expression of genes integrated inside the putative OTA gene cluster (AcOTApks, AcOTAnrps, AcOTAP450, and AcOTAhal) was assessed by using a real-Time PCR Detection Method CFX96TM (Bio-Rad Laboratories, Hercules CA, USA) within a volume of 25 containing 12.5 of iQ SYBR Green SuperMix (Bio-Rad Laboratories), 0.five of each and every primer and 1 on the reverse transcription reaction. All primer pairs were created together with the Primer3 computer software, and where possible, the forward ones had been made on the exon-intron junction websites to avoid amplification of feasible contaminant genomic DNA (Table S1). The circumstances for amplification have been as follows: 3 min denaturation at 95 C followed by 35 cycles of 95 C for 10 s and 60 C for 45 s. The gene encoding ubiquitin (ub; ID:393986) was utilized as a reference gene. Relative gene expression was calculated working with CFX Manager Computer software (Bio-Rad Laboratories) and also the 2-CT technique [46]. All samples have been analyzed in triplicate. For all analyzed genes, the ratio from the gene expression value (fold modify) in between every single deletion mutants and the WT strain was calculated.Supplementary Materials: The following are available on-line at https://www.mdpi.com/2072 -6651/13/2/111/s1, Table S1: Place with the putative-OTA-gene cluster inside the genome in the Aspergillus species and Penicillium nordicum. position of OTA-gene cluster inside the fungal genome ( genome.jgi.doe.gov) identified depending on homology with OTA putative gene cluster of A. carbonarius. Table S2: Features of BRLZ domains used in the Maximum Likelihood phylogenetic evaluation. Table S3: Detail in the Transcription issue binding motif (TFBM) identified by MEME within the OTA-gene cluster upstream, downstream, and intergenic sequences. Table S4: TOMTOM analysis representing the homology of TFBM identified by MEME with these of Saccharomyces cerevisiae. Name of transcription factor binding motif (TFBM) in accordance with the JASPAR database. Author Contributions: D.G., S.P., F.F., A.-R.B., R.M.D.M.A., and L.G.-C. conceived and made the experiments; D.G., F.G., and a.-R.B. performed the experiments; D.G., F.G., S.P., F.F., A.-R.B., and L.G.-C. analyzed the data; D.G., F.G., S.P., along with a.-R.B. wrote the paper, D.G., S.P., F.F., R.M.D.M.A., A.R.B., and L.G.-C. supervised the writing, D.G., S.P., F.F., R.M.D.M.A., A.-R.B., and L.G.-C. coordinated the collaboration of your authors. All authors have read and agreed towards the published version on the manuscript. Funding: The work was partially co-funded by the University of Bari Aldo Moro for the project “Epidemiology, genetics of plant pathogens and development of molecular diagnostic methods”, and in the Apulia Region, PO FESR 2007013–Axis I, Line of intervention 1.two., Action 1.2.1 for the project “Laboratory network for the choice, characterization, and conservation of germplasm and for stopping the spread of economically-relevant and quarantine pests (SELGE) No. 14” and by FEDER/Ministerio de Ciencia, Innovaci y Universidades–Agencia Estatal de Investigaci (AGL2017-28120-R and RTI2018-093392-A-I00). αIIbβ3 Storage & Stability Institutional R.
