title: Synthetic TAL effectors for targeted enhancement of transgene expression in plants.
abstract: Transcription activator-like effectors (TALEs), secreted by the pathogenic bacteria Xanthomonas, specifically activate expression of targeted genes in plants. Here, we designed synthetic TALEs that bind to the flanking regions of the TATA-box motif on the CaMV 35S promoter for the purpose of understanding the engineerable 'hot-spots' for increasing transgene expression. We demonstrated that transient expression of de novo-engineered TALEs using agroinfiltration could significantly increase reporter gene expression in stable transgenic tobacco expressing the orange fluorescent protein reporter gene pporRFP under the control of synthetic inducible, minimal or full-length 35S promoters. Moreover, the additive effects of a combination of two different synthetic TALEs could significantly enhance the activation effects of TALEs on reporter gene expression more than when each TALE was used individually. We also studied the effects of the C-terminal domain and the activation domain of synthetic TALEs, as well as the best 'hot-spots' on the 35S promoter on targeted transgene activation. Furthermore, TALE activation of the Arabidopsis MYB transcription factor AtPAP1 (PRODUCTION OF ANTHOCYANIN PIGMENT 1) in stable transgenic tobacco gave rise to a dark purple colour on infiltrated leaves when driven by four copies of cis-regulatory elements of pathogenesis-related gene (PR1) with enhancer motifs B and A1 from the 35S promoter. These results provide novel insights into the potential applications of synthetic TALEs for targeted gene activation of transgenes in plants.
authors.affiliation: Department of Plant Sciences, The University of Tennessee, Knoxville, TN, USA.
authors.forename: Wusheng
authors.initials: W
authors.lastname: Liu
authors.forename: Mary R
authors.initials: MR
authors.lastname: Rudis
authors.forename: Yanhui
authors.initials: Y
authors.lastname: Peng
authors.forename: Mitra
authors.initials: M
authors.lastname: Mazarei
authors.forename: Reginald J
authors.initials: RJ
authors.lastname: Millwood
authors.forename: Jian-Ping
authors.initials: JP
authors.lastname: Yang
authors.forename: Wenzhi
authors.initials: W
authors.lastname: Xu
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Chesnut
authors.forename: Charles Neal
authors.initials: CN
authors.lastname: Stewart
mesh_terms: D029681:Arabidopsis Proteins / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D018744:DNA, Plant / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods*; D010858:Pigmentation; D018515:Plant Leaves / Q000502:physiology; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014026:Nicotiana / Q000235:genetics*; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation; D019076:Transgenes / Q000235:genetics*
chemical_list: D029681:Arabidopsis Proteins; D018744:DNA, Plant; D015534:Trans-Activators; D014157:Transcription Factors; C502612:AT1G56650 protein, Arabidopsis
keywords: AtPAP1; additive effects; pporRFP; synthetic TALEs; targeted transgene activation; transgenic plants