Synthetic TAL effectors for targeted enhancement of transgene expression in plants

Summary Transcription activator‐like effectors ( TALE s), secreted by the pathogenic bacteria Xanthomonas , specifically activate expression of targeted genes in plants. Here, we designed synthetic TALE s that bind to the flanking regions of the TATA ‐box motif on the Ca MV 35S promoter for the purpose of understanding the engineerable ‘hot‐spots’ for increasing transgene expression. We demonstrated that transient expression of de novo ‐engineered TALE s using agroinfiltration could significantly increase reporter gene expression in stable transgenic tobacco expressing the orange fluorescent protein reporter gene ppor RFP under the control of synthetic inducible, minimal or full‐length 35S promoters. Moreover, the additive effects of a combination of two different synthetic TALE s could significantly enhance the activation effects of TALE s 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 TALE s, as well as the best ‘hot‐spots’ on the 35S promoter on targeted transgene activation. Furthermore, TALE activation of the Arabidopsis MYB transcription factor At PAP 1 ( 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 ( PR 1 ) with enhancer motifs B and A1 from the 35S promoter. These results provide novel insights into the potential applications of synthetic TALE s for targeted gene activation of transgenes in plants.