DIADE, University of Montpellier, CIRAD, IRD, Montpellier, France.
Universidad Veracruzana, INBIOTECA, Xalapa, Veracruz, Mexico.
PLoS One. 2022 Aug 30;17(8):e0273695. doi: 10.1371/journal.pone.0273695. eCollection 2022.
Although paramutation has been well-studied at a few hallmark loci involved in anthocyanin biosynthesis in maize, the cellular and molecular mechanisms underlying the phenomenon remain largely unknown. Previously described actors of paramutation encode components of the RNA-directed DNA-methylation (RdDM) pathway that participate in the biogenesis of 24-nucleotide small interfering RNAs (24-nt siRNAs) and long non-coding RNAs. In this study, we uncover an ARGONAUTE (AGO) protein as an effector of the RdDM pathway that is in charge of guiding 24-nt siRNAs to their DNA target to create de novo DNA methylation. We combined immunoprecipitation, small RNA sequencing and reverse genetics to, first, validate AGO104 as a member of the RdDM effector complex and, then, investigate its role in paramutation. We found that AGO104 binds 24-nt siRNAs involved in RdDM, including those required for paramutation at the b1 locus. We also show that the ago104-5 mutation causes a partial reversion of the paramutation phenotype at the b1 locus, revealed by intermediate pigmentation levels in stem tissues. Therefore, our results place AGO104 as a new member of the RdDM effector complex that plays a role in paramutation at the b1 locus in maize.
虽然在玉米中几个涉及花青素生物合成的标志性基因座上，已经对前馈突变进行了深入研究，但该现象背后的细胞和分子机制在很大程度上仍不清楚。先前描述的前馈突变因子编码 RNA 指导的 DNA 甲基化 (RdDM) 途径的组成部分，该途径参与 24 核苷酸小干扰 RNA (24-nt siRNA) 和长非编码 RNA 的生物发生。在这项研究中，我们发现 ARGONAUTE (AGO) 蛋白是 RdDM 途径的效应因子，负责指导 24-nt siRNA 到其 DNA 靶标，以创建新的 DNA 甲基化。我们结合免疫沉淀、小 RNA 测序和反向遗传学，首先验证了 AGO104 是 RdDM 效应复合物的成员，然后研究了它在前馈突变中的作用。我们发现 AGO104 结合 RdDM 中涉及的 24-nt siRNA，包括在 b1 基因座前馈突变中所需的 siRNA。我们还表明，ago104-5 突变导致 b1 基因座前馈突变表型的部分回复，表现在茎组织中出现中间色素沉着水平。因此，我们的结果将 AGO104 定位为 RdDM 效应复合物的新成员，它在玉米中 b1 基因座的前馈突变中起作用。
