Figure 6. Co-occurrence of BasR and the orsellinic acid gene cluster in other fungi is linked to the S.rapamycinicus-triggered ors gene cluster activation.

(a) Phylogenetic analysis of BasR (AN7174; green) showing its position among other fungi. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The names of the selected sequences are given according to their UniProt accession numbers. A comprehensive phylogenetic tree is depicted in Figure 6—figure supplement 1. (b) Alignment of the orsellinic acid gene clusters in the fungal species containing a basR homologue (A. nidulans, A. sydowii and A. versicolor), where orsA encodes the polyketide synthase, whereas orsB-orsE code for tailoring enzymes. (c) Liquid chromatography–mass spectrometry (LC-MS)-based detection of orsellinic and lecanoric acid in monoculture of the A. sydowii basR overexpression strain following induction with doxycycline and during co-cultivation of A. sydowii and S. rapamycinicus. LC-MS profiles of the extracted ion chromatogram (EIC) are shown for m/z 167 [M – H]^–, which corresponds to orsellinate. Orsellinic (1) and lecanoric acid (2) were detected via their fragment ion orsellinate.

Figure 6—figure supplement 1. Molecular phylogenetic analysis of BasR (AN7174).

The tree reports distances between BasR-similar amino-acid sequences identified by BlastP analysis using the entire sequences. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The BasR proteins from A. nidulans, A. calidoustus, A. sydowii, A. versicolor, A. rambellii and A. ochraceoroseus form a separate clade (highlighted in green text), while the yeast Bas1p-related sequences are more distantly related to BasR (in red). The second similar Myb-like transcription factor from A. nidulans (AN8377) forms a clade with orthologs from A. calidoustus and A. versicolor (in blue), which seems to be more related to Bas1p than to BasR. The names of the selected sequences are given according to their UniProt accession numbers.

Figure 6—figure supplement 2. Deletion of the second putative bas1p homologous gene (AN8377) in A. nidulans and analysis of its impact on the ors gene cluster induction in response to S. rapamycinicus.

(a) Chromosomal organization of the A. nidulans AN8377 gene before and after deletion. The gene AN8377 was replaced by an argB cassette in A. nidulans wildtype strain A1153. Genomic DNA was digested with DraIII. A PCR fragment covering the downstream sequence of AN8377 was used as a probe (*). wt, wildtype strain used as a control. (b) LC-MS-based detection of orsellinic acid (1) and lecanoric acid (2) in the co-cultivation of the AN8377 deletion mutant with S. rapamycinicus.

Figure 6—figure supplement 3. Generation of the inducible basR-overexpression strain by ectopic integration of an additional copy of the basR gene in the A. sydowii wild type strain (wt).

The tet^On–basR construct was integrated ectopically into the wildtype genome, using the hph cassette as a selectable marker. For Southern blot analysis, transformant strains were checked with a probe (*) directed against a region flanking the tet^On cassette and the basR gene. The genomic DNA was digested with BamHI.