2.4. Molecular cloning

The plasmid p‐Ama1‐P_srbA‐gfp‐srbA for labeling SrbA with GFP was constructed as follows: using primers Ama1‐srbA‐F and Ama1‐srbA‐R, the P_srbA‐gfp‐srbA fragment containing the srbA promoter, GFP and srbA ORF was amplified from the gDNA of an N‐tagged GFP‐SrbA strain and then subcloned into the BamHI site of the plasmid prg3‐AMAI‐NotI, generating the plasmid p‐Ama1‐P_srbA‐gfp‐srbA.

For colocalization analysis of GFP‐SrbA with the nucleus, the plasmid p‐Ama1‐P_srbA‐gfp‐srbA‐P_gpdA‐rfp‐H2A was generated as follows: the fragment P_srbA‐gfp‐srbA was amplified from p‐Ama1‐P_srbA‐gfp‐srbA with primers Ama1‐srbA‐F and gpd‐srbA‐R. The fragment P_gpdA‐rfp‐H2A was amplified from pBARGPE‐P_gpdA‐RFP‐H2A using primers gpd‐F and Ama1‐trpC‐R. Then, the two fragments were cloned into the BamHI site of the plasmid prg3‐AMAI‐NotI, yielding p‐Ama1‐P_srbA‐gfp‐srbA‐P_gpdA‐rfp‐H2A.

To analyze the localization of truncated SrbA (SrbA^T, it contains residues 1 to 380 of the N‐terminus of SrbA), the plasmid p‐Ama1‐P_srbA‐gfp‐srbA^T‐P_gpdA‐rfp‐H2A was generated as follows: The fragment P_srbA‐gfp‐srbA^T was amplified from p‐Ama1‐P_srbA‐gfp‐srbA with primers Ama1‐srbA‐F and Ama1‐srbA^T‐R. The fragment P_gpdA‐rfp‐H2A was amplified from pBARGPE‐P_gpdA‐RFP‐H2A using primers gpd‐srbA^T‐R and Ama1‐trpC‐R. Then, the two fragments were cloned into the BamHI site of the plasmid prg3‐AMAI‐NotI, yielding p‐Ama1‐P_srbA‐gfp‐srbA^T‐P_gpdA‐rfp‐H2A.

The plasmid p‐Ama1‐P_srbA‐srbA^F/srbA^T was generated as follows: the fragment P_srbA‐ srbA^F/P_srbA‐srbA^T was amplified from the gDNA of A. fumigatus with primers Ama1‐srbA‐F and Ama1‐srbA‐R/Ama1‐srbA^T‐R. Then, the fragment was subcloned into the BamHI site of the plasmid prg3‐AMAI‐NotI, yielding p‐Ama1‐P_srbA‐srbA^F/srbA^T.

The above plasmids were transformed into different background strains, which are listed in data Table ​TableA1A1.