Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, 44780 Bochum, Germany.
Fungal Biol. 2020 Sep;124(9):821-833. doi: 10.1016/j.funbio.2020.07.006. Epub 2020 Jul 29.
The mycophenolic acid producing ascomycete Penicillium brevicompactum is considered to be an anamorphic (asexual) species, for which a sexual cycle was never observed. However, since recent reports of otherwise asexually propagating filamentous fungi have demonstrated a sexual cycle controlled by mating type loci, we carried out a molecular analysis of mating type loci from P. brevicompactum. Using data from extensive DNA sequencing analysis, we determined the mating type loci from 22 strains derived from various type culture collections. We found 8 strains carrying a MAT1-1 locus encoding a 362 amino acid alpha domain transcription factor. The other 14 possessed a MAT1-2 locus encoding a 298 amino acid HMG domain transcription factor. cDNA analysis confirmed that both mating type loci are transcriptionally expressed. The karyotype of six selected strains, determined using contour-clamped homogeneous electric field (CHEF) electrophoresis, demonstrated distinct differences in size and numbers of chromosomes between the strains investigated. Interestingly, our phylogenetic survey of 72 strains from 11 different Penicillium species revealed that MAT genes serve as excellent molecular markers to determine phylogenetic relationships among species closely related to P. brevicompactum. Based on our sequencing results, we constructed transformation vectors for site-specific deletion of mating type loci from two selected strains of opposite mating type. Complementation strains were constructed containing both the mating type locus deletion cassette and a MAT-egfp fusion gene. These strains were used for comparative phenotypic analyses between strains containing or lacking the mating type gene. Whereas all MAT1-2 strains were indistinguishable, the MAT1-1 and MAT1-1-1 deletion strains differed distinctly. The MAT1-1-1 deletion strain produced more conidiospores on solid media, but smaller pellets in liquid media. This is probably the consequence of fewer conidial germ tubes than with the wild type mating type strain. Finally, we showed that the MAT-EGPF fusion protein is localized to the nuclei and detectable in protein samples by Western analysis. Together, our results suggest that the asexually propagating fungus P. brevicompactum might be a heterothallic species with a cryptic sexual life cycle.
产麦角酸的曲霉 Penicillium brevicompactum 被认为是一种无性（无性）物种，从未观察到其有性周期。然而，由于最近有报道称，原本无性繁殖的丝状真菌的性周期受到交配型基因座的控制，我们对 P. brevicompactum 的交配型基因座进行了分子分析。利用广泛的 DNA 测序分析数据，我们从来自各种类型培养物收集的 22 个菌株中确定了交配型基因座。我们发现 8 株携带 MAT1-1 基因座，该基因座编码一个 362 个氨基酸的α结构域转录因子。其他 14 株含有 MAT1-2 基因座，该基因座编码一个 298 个氨基酸的 HMG 结构域转录因子。cDNA 分析证实这两个交配型基因座均转录表达。使用连续限制均匀电场（CHEF）电泳对 6 个选定菌株的染色体组型进行分析，结果表明，所研究菌株之间的染色体大小和数量存在明显差异。有趣的是，我们对来自 11 种不同青霉属物种的 72 株菌的系统发育调查表明，MAT 基因是确定与 P. brevicompactum 密切相关的物种之间系统发育关系的极好分子标记。基于我们的测序结果，我们构建了用于从两个具有相反交配型的选定菌株中定点删除交配型基因座的转化载体。构建了包含交配型基因座缺失盒和 MAT-egfp 融合基因的互补菌株。这些菌株用于比较含有或缺乏交配型基因的菌株之间的表型分析。虽然所有 MAT1-2 菌株都无法区分，但 MAT1-1 和 MAT1-1-1 缺失菌株则明显不同。MAT1-1-1 缺失菌株在固体培养基上产生的分生孢子更多，但在液体培养基中形成的菌球更小。这可能是由于分生孢子发芽管比野生型交配型菌株少的结果。最后，我们证明 MAT-EGFP 融合蛋白定位于细胞核，并可通过 Western 分析在蛋白质样品中检测到。综上所述，我们的研究结果表明，无性繁殖的真菌 P. brevicompactum 可能是一种异型交配的物种，具有隐生的有性生命周期。
