A RID-like putative cytosine methyltransferase homologue controls sexual development in the fungus Podospora anserina

DNA methyltransferases are ubiquitous enzymes conserved in bacteria, plants and opisthokonta. These enzymes, which methylate cytosines, are involved in numerous biological processes, notably development. In mammals and higher plants, methylation patterns established and maintained by the cytosine DNA methyltransferases (DMTs) are essential to zygotic development. In fungi, some members of an extensively conserved fungal-specific DNA methyltransferase class are both mediators of the Repeat Induced Point mutation (RIP) genome defense system and key players of sexual reproduction. Yet, no DNA methyltransferase activity of these purified RID (RIP deficient) proteins could be detected in vitro. These observations led us to explore how RID-like DNA methyltransferase encoding genes would play a role during sexual development of fungi showing very little genomic DNA methylation, if any. To do so, we used the model ascomycete fungus Podospora anserina. We identified the PaRid gene, encoding a RID-like DNA methyltransferase and constructed knocked-out ΔPaRid defective mutants. Crosses involving P. anserina ΔPaRid mutants are sterile. Our results show that, although gametes are readily formed and fertilization occurs in a ΔPaRid background, sexual development is blocked just before the individualization of the dikaryotic cells leading to meiocytes. Complementation of ΔPaRid mutants with ectopic alleles of PaRid, including GFP-tagged, point-mutated and chimeric alleles, demonstrated that the catalytic motif of the putative PaRid methyltransferase is essential to ensure proper sexual development and that the expression of PaRid is spatially and temporally restricted. A transcriptomic analysis performed on mutant crosses revealed an overlap of the PaRid-controlled genetic network with the well-known mating-types gene developmental pathway common to an important group of fungi, the Pezizomycotina.

DNA甲基转移酶（DNA methyltransferases）是一类广泛存在且在细菌、植物与后鞭毛生物（opisthokonta）中保守的酶。这类能够催化胞嘧啶甲基化修饰的酶参与诸多生物学过程，尤以发育过程为重。在哺乳动物与高等植物中，由胞嘧啶DNA甲基转移酶（DMTs）建立并维持的甲基化修饰模式，对于合子发育至关重要。在真菌中，一类高度保守的真菌特异性DNA甲基转移酶家族的部分成员，既是重复序列诱导点突变（Repeat Induced Point mutation，RIP）基因组防御系统的介导因子，也是有性生殖的关键调控因子。然而，在体外实验中并未检测到这些纯化的RID（RIP缺陷型，RIP deficient）蛋白具有DNA甲基转移酶活性。基于上述观察，我们着手探究类RID DNA甲基转移酶编码基因在基因组DNA甲基化水平极低（若存在甲基化的话）的真菌的有性发育过程中所发挥的作用。为此，我们选用了模式子囊真菌倒孢柄孢壳（Podospora anserina）作为研究对象。我们成功鉴定出了编码类RID DNA甲基转移酶的PaRid基因，并构建了ΔPaRid基因敲除缺陷突变体。涉及ΔPaRid突变体的倒孢柄孢壳（Podospora anserina）杂交组合表现为不育。我们的研究结果显示：尽管在ΔPaRid遗传背景中，配子可正常形成且受精过程顺利完成，但有性发育会在双核细胞（dikaryotic cells）个体化形成减数分裂细胞（meiocytes）之前被阻断。通过将包含GFP标记（GFP-tagged）、点突变（point-mutated）及嵌合等位基因（chimeric alleles）在内的PaRid异位等位基因回补至ΔPaRid突变体中，实验证实推定的PaRid甲基转移酶的催化基序（catalytic motif）对于保障正常有性发育至关重要，同时PaRid的表达具有严格的时空限制性。对突变体杂交样本进行的转录组分析（transcriptomic analysis）显示，PaRid调控的遗传网络与一类重要真菌——盘菌亚门（Pezizomycotina）共有的经典交配型基因发育通路存在重叠。