In vivo characterization of plant promoter element interaction using synthetic promoters

Short directly-repeated (DR) DNA enhancer elements of plant viral origin were analyzed for their ability, both individually and in combination, to influence in vivo transcription when inserted upstream from a minimal CaMV35S promoter. Synthetic promoters containing multiple copies and/or combinations of DR cassettes were tested for their effect upon reporter gene (luciferase) expression using an Agrobacteria-based leaf-infiltration transient assay and within stably transformed plants (Nicotiana tabacum). Transgenic plants harboring constructs containing different numbers or combinations of DR cassettes were further tested to look for tissue-specific expression patterns and potential promoter response to the infiltration process employed during transient expression. Multimerization of DR elements produced enhancer activity that was in general additive, increasing reporter activity in direct proportion to the number of DR cassettes within the test promoter. In contrast, combinations of different DR cassettes often functioned synergistically, producing reporter enhancement markedly greater then the sum of the combined DR activities. Several of the DR constructs responded to Agrobacteria (lacking T-DNA) infiltration of transgenic leaves by an induction (2 elements) or reduction (1 element) in reporter activity. Combinations of DR cassettes producing the strongest enhancement of reporter activity were used to create two synthetic promoters (SynPro3 and SynPro5) that drive leaf reporter activities at levels comparable to the CaMV35S promoter. Characterization of these synthetic promoters in transformed tobacco showed strong reporter expression at all stages of development and in most tissues. The arrangement of DR elements within SynPro3 and SynPro5 appears to play a role in defining tissue-specificity of expression and/or Agrobacteria-infusion responsiveness.