Characterization and functional analysis of the Md-miR285N gene promoter in transgenic apple (Malus x domestica) and Arabidopsis thaliana

MicroRNAs are key regulators of plant physiological activities by functioning in RNA silencing and post-transcriptional regulation of gene expression. In apple, the uncharacterized Md-miR285N is predicted to target 35 RNA transcripts, mostly coding for TMV, SCN1 and CDPK proteins involved in plant defense to pathogens. In this work, ca. 2 Kb of the Md-miR285N promoter were isolated and cloned into an expression vector with its 3’-end fused to the ß-glucoronidase (GUS) reporter gene. The expression vector was used for Agrobacterium-mediated transformation of both Malus x domestica (cv. ‘Gala’) and Arabidopsis thaliana (ecotype Col-0). In transgenic apple plantlets a strong GUS activity was detected in stems, leaves and veins. In Arabidopsis seedlings the promoter was shown to be highly expressed in roots, shoot apex meristem, primary and secondary leaves and veins. To understand the functional response of Md-miR285N against pathogen infections, apple and Arabidopsis plantlets wereinoculated, respectively, with Erwinia amylovora strain Ea273 and Pseudomonas syringae pv. tomato DC3000. The GUS activity was qualitatively and quantitatively evaluated in treated plants at 12, 24, 36 and 48 hours post inoculation. A differential regulation of promoter expression was detected compared to untreated plants. These data suggest that Md-miR285N may act as important regulator of plant defense after bacteria infections.