Construction of TptA point mutation strains

For site-directed mutagenesis, complementary primers, approximately 20 bp in length that included the desired mutation in the center position, were designed and synthesized. The plasmid pTptA-com-hph, harboring the wild-type A. fumigatus tptA gene, was used as a template. Two fragments containing the desired mutation were amplified using tptA-up-XbaI or tptA-down-HindIII with their respective primer pairs and the resulting PCR products were treated with DpnI to remove the template. The two fragments were then cloned into XbaI and HindIII digested pAN7-1 using the ClonExpressII One Step Cloning Kit (Vazyme). The related plasmids were transformed into the ΔtptA mutant to obtain the strains referred to as tptA^R53A, tptA^D60A, tptA^G153S, tptA^G205A, tptA^K255A and tptA^K315A.