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SUMMARY: Insulators characterized in Drosophila and mammals have been shown to play a key role in the restriction of promiscuous enhancer–promoter interactions, as well as reshaping the topological landscape of chromosomes. Yet the role of insulators in plants remains poorly understood, in large part because of a lack of well‐characterized insulators and binding factor(s). In this study, we isolated a 1.2‐kb RS2‐9 insulator from the Oryza sativa (rice) genome that can, when interposed between an enhancer and promoter, efficiently block the activation function of both constitutive and floral organ‐specific enhancers in transgenic Arabidopsis and Nicotiana tabacum (tobacco). In the rice genome, the genes flanking RS2‐9 exhibit an absence of mutual transcriptional interactions, as well as a lack of histone modification spread. We further determined that O. sativa Homeobox 1 (OSH1) bound two regions of RS2‐9, as well as over 50 000 additional sites in the rice genome, the majority of which resided in intergenic regions. Mutation of one of the two OSH1‐binding sites in RS2‐9 impaired insulation activity by up to 60%, whereas the mutation of both binding sites virtually abolished insulator function. We also demonstrated that OSH1 binding sites were associated with 72% of the boundaries of topologically associated domains (TADs) identified in the rice genome, which is comparable to the 77% of TAD boundaries bound by the insulator CCCTC‐binding factor (CTCF) in mammals. Taken together, our findings indicate that OSH1‐RS2‐9 acts as a true insulator in plants, and highlight a potential role for OSH1 in gene insulation and topological organization in plant genomes. Significance Statement: The role of insualtor in the regulation of enhancer‐promoter interaction and topological architecture of genomes has been well characterized in mammals but rarely in plants largely due to a lack of a true insualtor. We report the isolation and characterization of an insualtor from rice genome and its binding protein prominently associated with the boundaries of chromatin loops. Our findings are of significance in understanding the insualtor‐based gene regulation and genome demarcation in plants [ABSTRACT FROM AUTHOR]

Molecular stacking enables multiple traits to be effectively engineered in crops using a single vector. However, the co-existence of distinct plant promoters in the same transgenic unit might, like their mammalian counterparts, interfere with one another. In this study, we devised a novel approach to investigate enhancer-promoter and promoter-promoter interactions in transgenic plants and demonstrated that three of four flower-specific enhancer/promoters were capable of distantly activating a pollen- and stigma-specific Pps promoter (fused to the cytotoxic DT-A gene) in other tissues, as revealed by novel tissue ablation phenotypes in transgenic plants. The NtAGI1 enhancer exclusively activated stamen- and carpel-specific DT-A expression, thus resulting in tissue ablation in an orientation-independent manner; this activation was completely abolished by the insertion of an enhancer-blocking insulator ( EXOB) between the NtAGI1 enhancer and Pps promoter. Similarly, AGL8 and AP1Lb1, but not AP1La, promoters also activated distinct tissue-specific DT-A expression and ablation, with the former causing global growth retardation and the latter ablating apical inflorescences. While the tissue specificity of the enhancer/promoters generally defined their activation specificities, the strength of their activity in particular tissues or developmental stages appeared to determine whether activation actually occurred. Our findings provide the first evidence that plant-derived enhancer/promoters can distantly interact/interfere with one another, which could pose potential problems for the tissue-specific engineering of multiple traits using a single-vector stacking approach. Therefore, our work highlights the importance of adopting enhancer-blocking insulators in transformation vectors to minimize promoter-promoter interactions. The practical and fundamental significance of these findings will be discussed. [ABSTRACT FROM AUTHOR]