FIG 3.

CBC in vitro (EMSA) and in vivo (ChIP-qPCR) binds to the promoters of genes related to asexual conidiation. (A) The purified HapB, HapC, and HapE proteins are shown by SDS-PAGE and Coomassie blue staining. HapB, HapC, HapE, and their mixture (1:1:1) are shown as labeled, respectively. (B to H) CBC binds to the promoters of target genes in vitro. EMSA of CBC binding to Cy5-labeled promoter fragments of brlA, fluG, flbD, and flbC. Two specific brlA probes (double-stranded DNA) were designed to target the CCAAT motif located at position bp 4421/3175 upstream of the brlA translational start site (−4421/−3175). Similarly, probes for fluG, flbD, and flbC targeting the CCAAT motif located at their own promoters were given. The specificity of EMSA binding was validated by adding specific competitors/cold probes (unlabeled probes) or substituting the Cy5-labeled mutant probe for the original Cy5-labeled probe with the CCAAT motif. (I) In vivo binding of the CBC was confirmed by comparing % recovery of DNA (ChIP-qPCR) from promoter fragments of conidiation-related genes to an unbound region of the AFUB_041590. ChIP-qPCR with IgG was performed as the control. Mean ± SD (n = 3). Unpaired t test. **, P < 0.01; *, P < 0.05; ns, not significant.