Key message

In the ros1-defective mutant, DREB1A repression by the transgene-induced promoter methylation of ice1-1 became inheritable across generations even in the absence of the causative transgene NICE1.

Abstract

Transgene silencing (TGS) is a widely observed event during plant bioengineering, which is presented as a gradual decrease in ectopic gene expression across generations and occasionally coupled with endogenous gene silencing based on DNA sequence similarity. TGS is known to be established by guided DNA methylation machinery. However, the machinery underlying gene recovery from TGS has not been fully elucidated. We previously reported that in ice1-1 outcross descendants, the expressional repression and recovery of DREB1A/CBF3 were instantly achieved by a newly discovered NICE1 transgene, instead of the formerly proposed ice1-1 mutation in the ICE1 gene. The plants harboring NICE1 produced small RNAs targeting and causing the DREB1A promoter to be hypermethylated and silenced. To analyze the role of the plant-specific active DNA demethylase REPRESSOR OF SILENCING 1 (ROS1) in instant DREB1A recovery, we propagated the NICE1-segregating population upon ros1 dysfunction and evaluated the gene expression and DNA methylation levels of DREB1A through generations. Our results showed that the epigenetic DREB1A repression was substantially sustained in subsequent generations even without NICE1 and stably inherited across generations. Consistent with the gene expression results, only incomplete DNA methylation removal was detected in the same generations. These results indicate that a novel inheritable epiallele emerged by the ros1 dysfunction. Overall, our study reveals the important role of ROS1 in the inheritability of TGS-associated gene repression.

中文翻译：

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DNA去甲基化酶ROS1通过转基因诱导的拟南芥ice1-1突变体中的启动子甲基化阻止可遗传的DREB1A / CBF3抑制

关键信息

在ros1缺陷突变体中，即使在没有致病转基因NICE1的情况下，转基因诱导的ice1-1启动子甲基化对DREB1A的抑制也可以跨代遗传。

抽象的

转基因沉默 (TGS) 是植物生物工程中广泛观察到的事件，表现为异位基因表达在几代人之间逐渐减少，并且偶尔与基于 DNA 序列相似性的内源基因沉默相结合。已知 TGS 是通过引导的 DNA 甲基化机制建立的。然而，从 TGS 中恢复基因的机制尚未完全阐明。我们之前报道过，在ice1-1异交后代中， DREB1A / CBF3的表达抑制和恢复是通过新发现的NICE1转基因立即实现的，而不是之前提出的ICE1基因中的ice1-1突变。藏身的植物NICE1产生靶向并导致DREB1A启动子高甲基化和沉默的小 RNA。为了分析植物特异性活性 DNA 去甲基化酶抑制因子 1 (ROS1) 在即时DREB1A恢复中的作用，我们在ros1功能障碍时繁殖NICE1分离种群，并评估DREB1A的基因表达和 DNA 甲基化水平。我们的研究结果表明，即使没有NICE1 ，表观遗传DREB1A抑制在后代中也基本持续并稳定代代相传。与基因表达结果一致，在同一代中仅检测到不完全的 DNA 甲基化去除。这些结果表明，ros1功能障碍出现了一种新的可遗传的外等位基因。总体而言，我们的研究揭示了 ROS1 在 TGS 相关基因抑制的可遗传性中的重要作用。