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Molecular analysis of mating type loci from the mycophenolic acid producer Penicillium brevicompactum: Phylogeny and MAT protein characterization suggest a cryptic sexual life cycle
Fungal Biology ( IF 2.9 ) Pub Date : 2020-09-01 , DOI: 10.1016/j.funbio.2020.07.006
Yasaman Mahmoudjanlou 1 , Tim A Dahlmann 1 , Ulrich Kück 1
Affiliation  

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.

中文翻译:

来自霉酚酸生产者 Penicillium brevicompactum 的交配型基因座的分子分析:系统发育和 MAT 蛋白质表征表明一个神秘的性生命周期

产生霉酚酸的子囊菌 Penicillium brevicompactum 被认为是一种变形(无性)物种,从未观察到性周期。然而,由于最近关于其他无性繁殖丝状真菌的报道已经证明了由交配型基因座控制的性周期,我们对来自 P. brevicompactum 的交配型基因座进行了分子分析。使用来自广泛 DNA 测序分析的数据,我们确定了来自各种类型培养物保藏中心的 22 个菌株的交配型位点。我们发现 8 株携带 MAT1-1 基因座,编码 362 个氨基酸的 α 结构域转录因子。其他 14 个具有编码 298 个氨基酸的 HMG 结构域转录因子的 MAT1-2 基因座。cDNA 分析证实两个交配型基因座都被转录表达。使用轮廓钳位均匀电场 (CHEF) 电泳确定的六种选定菌株的核型显示出所研究菌株之间染色体大小和数量的明显差异。有趣的是,我们对来自 11 种不同青霉菌属的 72 株菌株的系统发育调查显示,MAT 基因可作为极好的分子标记来确定与 P. brevicompactum 密切相关的物种之间的系统发育关系。根据我们的测序结果,我们构建了转化载体,用于从两个选定的相反交配型菌株中进行交配型基因座的位点特异性缺失。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。这些菌株用于在含有或缺乏交配型基因的菌株之间进行比较表型分析。尽管所有 MAT1-2 菌株都无法区分,但 MAT1-1 和 MAT1-1-1 缺失菌株明显不同。MAT1-1-1 缺失菌株在固体培养基上产生更多的分生孢子,但在液体培养基中产生较小的颗粒。这可能是由于与野生型交配型菌株相比分生孢子管较少的结果。最后,我们表明 MAT-EGPF 融合蛋白定位于细胞核,并且可以通过西方分析在蛋白质样品中检测到。总之,我们的结果表明,无性繁殖真菌 P. brevicompactum 可能是一种具有隐秘性生命周期的异种物种。MAT1-1 和 MAT1-1-1 缺失菌株明显不同。MAT1-1-1 缺失菌株在固体培养基上产生更多的分生孢子,但在液体培养基中产生较小的颗粒。这可能是由于与野生型交配型菌株相比分生孢子胚管较少的结果。最后,我们表明 MAT-EGPF 融合蛋白定位于细胞核,并且可以通过西方分析在蛋白质样品中检测到。总之,我们的结果表明,无性繁殖真菌 P. brevicompactum 可能是一种具有隐秘性生命周期的异种物种。MAT1-1 和 MAT1-1-1 缺失菌株明显不同。MAT1-1-1 缺失菌株在固体培养基上产生更多的分生孢子,但在液体培养基中产生较小的颗粒。这可能是由于与野生型交配型菌株相比分生孢子胚管较少的结果。最后,我们表明 MAT-EGPF 融合蛋白定位于细胞核,并且可以通过西方分析在蛋白质样品中检测到。总之,我们的结果表明,无性繁殖真菌 P. brevicompactum 可能是一种具有隐秘性生命周期的异种物种。我们表明 MAT-EGPF 融合蛋白定位于细胞核,并且可以通过西方分析在蛋白质样品中检测到。总之,我们的结果表明,无性繁殖真菌 P. brevicompactum 可能是一种具有隐秘性生命周期的异种物种。我们表明 MAT-EGPF 融合蛋白定位于细胞核,并且可以通过西方分析在蛋白质样品中检测到。总之,我们的结果表明,无性繁殖真菌 P. brevicompactum 可能是一种具有隐秘性生命周期的异种物种。
更新日期:2020-09-01
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