Yang Kunlong, Liang Linlin, Ran Fanlei, Liu Yinghang, Li Zhenguo, Lan Huahui, Gao Peili, Zhuang Zhenhong, Zhang Feng, Nie Xinyi, Kalayu Yirga Shimuye, Wang Shihua
Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, Key Laboratory of Biopesticide and Chemical Biology of Education Ministry, and School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China.
Sci Rep. 2016 Mar 16;6:23259. doi: 10.1038/srep23259.
DNA methylation is essential for epigenetic regulation of gene transcription and development in many animals, plants and fungi. We investigated whether DNA methylation plays a role in the development and secondary metabolism of Aspergillus flavus, identified the DmtA methyltransferase from A. flavus, and produced a dmtA knock-out mutant by replacing the dmtA coding sequence with the pyrG selectable marker. The A. flavus dmtA null mutant lines produced white fluffy mycelium in liquid medium, and displayed a slightly flavescent conidial pigmentation compared with the normal yellow of the wild-type strain when grown on agar. The ΔdmtA lines exhibited decreased conidiation and aflatoxin (AF) biosynthesis, compared with the wild-type line, suggesting that the DmtA knock-out affected the transcriptional level of genes in the AF cluster. In particular, sclerotia development and host colonization were altered in the dmtA null mutants. Green fluorescent protein tagging at the C-terminus of DmtA showed that DmtA localized to the nucleus and cytoplasm. DNA methylation content measurements in the dmtA mutants revealed no widespread DNA methylation in the mutants or wild-type lines. Thus, our findings suggest that DmtA, apart from being a C-5 cytosine methyltransferase in A. flavus, contributes to asexual development, aflatoxin biosynthesis, sclerotial production and virulence.
DNA甲基化对于许多动物、植物和真菌中基因转录和发育的表观遗传调控至关重要。我们研究了DNA甲基化是否在黄曲霉的发育和次级代谢中发挥作用，鉴定了来自黄曲霉的DmtA甲基转移酶，并通过用pyrG选择标记替换dmtA编码序列产生了dmtA敲除突变体。黄曲霉dmtA缺失突变体在液体培养基中产生白色蓬松菌丝体，在琼脂上生长时，与野生型菌株正常的黄色相比，分生孢子色素沉着略显淡黄色。与野生型菌株相比，ΔdmtA突变体的分生孢子形成和黄曲霉毒素（AF）生物合成减少，这表明DmtA敲除影响了AF簇中基因的转录水平。特别是，dmtA缺失突变体的菌核发育和宿主定殖发生了改变。在DmtA的C末端进行绿色荧光蛋白标记表明，DmtA定位于细胞核和细胞质。对dmtA突变体的DNA甲基化含量测量显示，突变体或野生型菌株中均未发现广泛的DNA甲基化。因此，我们的研究结果表明，DmtA除了是黄曲霉中的一种C-5胞嘧啶甲基转移酶外，还对无性发育、黄曲霉毒素生物合成、菌核产生和毒力有贡献。
