A 1-kb Bacteriophage Lambda Fragment Functions as an Insulator to Effectively Block Enhancer–Promoter Interactions in Arabidopsis thaliana Stacy D. Singer & Jean-Michel Hily & Zongrang Liu Published online: 8 August 2009 # Springer-Verlag 2009 Abstract Enhancers are known to be capable of overriding the specificity of nearby promoters in a distance-dependent manner, which is problematic when multiple promoters coexist in a single transgene unit. In an attempt to deter- mine whether enhancer activation function is inversely related to its distance from the target promoter, we inserted 1-, 2-, and 4-kb bacteriophage λ fragments, respectively, between the cauliflower mosaic virus 35S enhancer and a flower-specific AGAMOUS second intron-derived promoter (AGIP) fused to the β-glucuronidase (GUS) coding region. In the absence of an insert sequence, the 35S enhancer activates AGIP-driven GUS expression in vegetative tissues of transgenic Arabidopsis thaliana lines. Moreover, neither the 2-kb nor the 4-kb λ fragment was able to block GUS expression in transgenic leaves, implying that the 35S enhancer can override a distance barrier of at least 4 kb in our system. Unexpectedly, insertion of the 1-kb λ insert into the same site resulted in diminished GUS expression in transgenic leaves. Our data indicate that this fragment functions as a true enhancer-blocking insulator that could potentially be utilized to minimize enhancer–promoter interference between multiple transcriptional units within a plasmid vector during plant transformation experiments. Keywords Arabidopsis thaliana . Bacteriophage lambda . Cauliflower mosaic virus 35S promoter . Enhancer-blocking insulator . Enhancer–promoter interaction Introduction Transgenic plant technology is an important tool for both basic plant biological research and the improvement of agronomic traits in a wide variety of crop species through the stable expression of foreign genes (Lanfranco 2003). Often, this necessitates the use of tissue-, organ-, or developmental stage-specific and strong constitutive pro- moters (Li et al. 2001; Liu and Liu 2008; Savidge et al. 1995) to drive transgene expression exclusively in targeted tissues. Until recently, the majority of transgenic research has been directed towards the improvement of a single trait (for example virus resistance, herbicide resistance) despite the fact that crops in field conditions must cope with diverse biotic and abiotic challenges and, thus, often require a comprehensive approach to enhance the performance of multiple traits. This demands the use of a transformation vector with the ability to harbor multiple transcriptional gene units, a feat that has been demonstrated to be feasible with the introduction of six transcriptional cassettes into plants using a single pPZP-based vector (Goderis et al. 2002). Unfortunately, the presence of multiple promoter and enhancer elements within a single vector might, due to the inherent orientation-independent nature of enhancers, pro- voke enhancer–promoter or promoter–promoter interference, thereby altering the specificity and strength of discrete promoters in transgenic plants. This crosstalk phenomenon is commonly observed in transgenic plant-bearing vectors in which the enhancer derived from the constitutive 35S promoter (Odell et al. 1988) is inserted near plant-derived S. D. Singer : J.-M. Hily : Z. Liu (*) USDA-ARS Appalachian Fruit Research Station, 2217 Wiltshire Road, Kearneysville, WV 25430, USA e-mail: zongrang.liu@ars.usda.gov Present Address: S. D. Singer : J.-M. Hily Department of Plant Pathology, New York State Agricultural Experiment Station, Cornell University, 630 West North Street, Geneva, NY 14456, USA Plant Mol Biol Rep (2010) 28:69–76 DOI 10.1007/s11105-009-0122-3