The Penicillium chrysogenum tom1 Gene a Major Target of Transcription Factor MAT1-1-1 Encodes a Nuclear Protein Involved in Sporulation

Abstract

Fungal mating-type loci (MAT) encode transcription factors (TFs) MAT1-1-1 and MAT1-2-1, which govern sexual reproduction as well as other developmental processes. In Penicillium chrysogenum, the major producer of the beta-lactam antibiotic penicillin, a recent chromatin immunoprecipitation followed by sequencing (ChIP-seq) analysis identified 254 genes as direct targets of MAT1-1-1, many of which encode thus far uncharacterized proteins. Here, we characterized one of the major targets of MAT1-1-1, the tom1 gene, which encodes a protein highly conserved within the group of Eurotiomycetes fungi. Using fluorescence microscopy, we demonstrated binding of MAT1-1-1 to the tom1 promoter by reporter gene analysis. Extensive electrophoretic mobility shift assays (EMSAs) further showed that the promoter sequence of tom1 is bound in vitro by both MAT1-1-1 and MAT1-2-1. This indicated an interaction between the two TFs, which was verified by yeast two-hybrid analysis. The sequence of tom1 carries a nuclear localization sequence, and indeed its nuclear localization was verified by fluorescence microscopy. The in vivo function of tom1 was investigated using tom1 deletion strains, as well as a complementing strain where the wild-type tom1 gene was reintroduced. We found a clear sporulation defect in the deletion strain, which became more evident when the fungi were grown at an elevated temperature of 31°C.

Keywords: Mating-type transcription factor; Penicillium chrysogenum; electrophoretic mobility shift assay; fluorescence microscopy; sporulation; yeast two-hybrid analysis.

Figure 1

Identification of tom1 as an in vivo target of MAT1-1-1 (A) Schematic representation of the tom1 gene promoter region fused to the DsRed reporter gene. The consensus motif that binds MAT1-1-1 in vitro is indicated by orange bar, while other putative MAT1-1-1 motifs (p<0.001) identified by FIMO (Grant et al., 2011) are shown as green bars. The full-length Ptom1_{(0-965 bp)} and truncated variants Ptom1_{(-149-965 bp)} and Ptom1_{(-222-965 bp)} are given as black bars. (B) In vivo association of MAT1-1-1 with the tom1 promoter variants. Note, the egfp-MAT1-1-1 is expressed (green fluorescence) by recipient MAT1-ChIP strain. Red fluorescence indicates the expression of the DsRed gene under the control of the tom1 promoter. Scale bars, 20 μm.

Figure 2

EMSAs show sequence-specific binding of the MAT1-1-1 and MAT1-2-1 proteins to the tom1-2. (A) EMSA image showing in vitro binding of GST-MAT1-1-1 to tom1-2 probe in presence of increasing competitor, nonspecific kex2-3 (left) and specific tom1-2 (right). Equal molar amounts (2.5 µM) of protein was mixed with unlabled competitor DNA in pmol, as indicated. (B) EMSA image showing in vitro binding of GST-MAT1-2-1 to tom1-2 probe in presence of nonspecific kex2-3 (left) and specific tom1-2 (right) competitor probe. Equal molar amounts (7 µM) protein was mixed with unlabled competitor DNA in pmol, as indicated. In (A, B) homodimers and heterodimers of GST-MAT1-1-1 in complex with tom1-2 are indicated by open and black arrowheads respectively. (C) EMSA showing cooperative binding of MAT1-1-1 and MAT1-2-1 to the tom1-2 derived from the promoter of the tom1 gene. Equal molar amounts (4 µM) of each protein were co-incubated with tom1-2 and applied to the gel. The arrowheads indicate formation of GST-MAT1-1-1/GST-MAT1-2-1/tom1-2 (open arrowhead) and GST-MAT1-1-1/MAT1-2-1/tom1-2 (black arrowhead) complexes.

Figure 3

Proteins MAT1-1-1 and MAT1-2-1 physically interact. (A) Schematic representation showing truncated fragments of MAT1-1-1 and MAT1-2-1 TFs. The truncated fragments were fused to Gal4 AD (pGADT7 derivatives; MAT1-NtBD, MAT1-CtBD, MAT2, MAT2-Nt) and to Gal4 BD (pGBKT7 derivatives; MAT1-Nt, MAT1-NtBD, MAT2-Nt, MAT2-NtBD). Gal4 AD, Gal4 activation domain; Gal4 BD, Gal4 binding domain. (B) Yeast two-hybrid analysis of MAT proteins shows that MAT1-1-1 and MAT1-2-1 form homo- and heterodimer complexes. Yeast transformants expressing the Gal4 AD and Gal4 AD fusion constructs were selected on SD-Leu-Trp-His-Ade medium (left panel in black and white). The right panel shows α-galactosidase activity of yeast transformants selected on SD-Leu-Trp-His-Ade medium containing X-α-Gal. Blue color indicates positive protein interactions. Yeast transformants carrying empty plasmids pGADT7 and pGBKT7 were used as a negative control.

Figure 4

Differential interference contrast (DIC) and fluorescence microscopy to localize EGFP-Tom1 in subcellular structures. P2niaD18::EGFP-Tom1 transformants show nuclear localization of EGFP-Tom1 in mycelial cells. Arrowheads point to nuclei, which were identified by staining with 4′,6-diamidino-2-phenylindole (DAPI). Merge: merged images of DAPI and GFP fluorescence. Scale bars, 20 μm.

Figure 5

Quantitative assay of conidiation in Δtom1. Quantification of conidia formation in the recipient ΔPcku70, the deletion Δtom1 and corresponding complementation Δtom1::EGFP-Tom1 strains. Strains were grown on solid CCM for 168 h exposed to either constant light or constant dark at 27°C (A) and 31°C (B). Light blue/light orange columns represent exposure to light and dark blue/dark orange columns exposure to dark. Error bars indicate representing mean ± SD (n = 3) values for three independent experiments. Statistical analysis was performed using t-tests for comparisons to the control ΔPcku70 strain (***P< 0.005, **P < 0.05, *P < 0.5).