Rice RS2-9, which is bound by transcription factor OSH1, blocks enhancer-promoter interactions in plants.

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.

CW198 acts as a genetic insulator to block enhancer-promoter interaction in plants.

Insulators in vertebrates play a role in genome architecture and orchestrate temporo-spatial enhancer-promoter interactions. In plants, insulators and their associated binding factors have not been documented as of yet, largely as a result of a lack of characterized insulators. In this study, we took a comprehensive strategy to identify and validate the enhancer-blocking insulator CW198. We show that a 1.08-kb CW198 fragment from Arabidopsis can, when interposed between an enhancer and a promoter, efficiently abrogate the activation function of both constitutive and floral organ-specific enhancers in transgenic Arabidopsis and tobacco plants. In plants, both transcriptional crosstalk and spreading of histone modifications were rarely detectable across CW198, which resembles the insulation property observed across the CTCF insulator in the mammalian genome. Taken together, our findings support that CW198 acts as an enhancer-blocking insulator in both Arabidopsis and tobacco. The significance of the present findings and their relevance to the mitigation of mutual interference between enhancers and promoters, as well as multiple promoters in transgenes, is discussed.

eCry1Gb.1Ig, A Novel Chimeric Cry Protein with High Efficacy against Multiple Fall Armyworm (Spodoptera frugiperda) Strains Resistant to Different GM Traits.

Spodoptera frugiperda (fall armyworm, FAW) is one of the most devastating insect pests to corn and soybean production in the Americas and is rapidly expanding its range worldwide. It is known to be hard to control either by chemical insecticide applications or by GM. Although the use of GM traits can be an effective way to control this pest, it is very rare to find native insecticidal proteins that provide the necessary level of FAW control in crop fields where FAW pressure and damage are high. Insecticidal Cry proteins sourced from Bacillus thuringiensis have been heavily utilized in the development of crops with GM traits; however, it is increasingly difficult to identify Cry proteins with unique modes of action. Protein engineering via a phylogenetically guided Cry protein domain swapping approach enabled us to discover novel chimeric Cry proteins engineered from inactive parent sequences. Some of these chimeras show excellent efficacy against key biotypes of FAW from Brazil and North America. In this study, we characterized a Cry-based chimera eCry1Gb.1Ig that is a very potent FAW toxin. eCry1Gb.1Ig showed high efficacy against multiple FAW strains that are resistant to various traits, including Cry1Fa, Vip3Aa and Cry1A.105/Cry2Ab. These results clearly indicate that the FAW strains resistant to Cry1Fa, Vip3Aa or Cry1A.105/Cry2Ab demonstrate no cross-resistance to eCry1Gb.1Ig and strongly suggest that eCry1Gb.1Ig acts through a novel mode of action compared to the existing traits. In addition to its FAW activity, eCry1Gb.1Ig has also been shown to control Chrysodeixis includens (soybean looper, SBL) and Anticarsia gemmatalis (velvetbean caterpillar, VBC), which are significant pests of soybean. When eCry1Gb.1Ig was introduced into corn and soybean crops, transgenic events showed strong efficacy against FAW, SBL and VBC, but no adverse plant phenotypes. This suggests that the in planta expression of the eCry1Gb.1Ig protein does not compromise plant growth or reproduction and can protect plants from FAW-related damage. Therefore, this valuable discovery will provide a differentiating FAW control trait that will give growers another tool to help them reduce yield loss due to FAW.