Functional analysis of Medicago -derived pathogen-induced gene promoters for usage in transgenic alfalfa

Three gene promoters from the model legume species Medicago truncatula and Medicago sativa (alfalfa) pathogenesis-related (PR) proteins (MtPR5, MtPR10, and MsPR10) were isolated and investigated using in silico and in situ approaches. For the functional analysis of these promoters, plant transformation vectors linking promoter sequences with the β-glucuronidase (GUS) reporter gene were constructed and utilized to generate transgenic alfalfa. Histochemical GUS staining established that the PR5 and both PR10 promoters were functional in alfalfa and induce GUS expression primarily in the root tissue. When transgenic alfalfa leaves were inoculated with a diverse set of alfalfa pathogens, either Phoma medicaginis, Colletotrichum trifolii, or Pseudomonas syringae pv. syringae, the promoters were responsive to induction by both bacterial and fungal pathogens. Gene expression analysis also indicated that all three PR promoters were pathogen-induced and upregulate transgene expression. Additionally, several putative transcription regulator elements (REs) responsible for the observed pathogen-induced promoter activity were predicted in each of the promoter sequences. These characterized Medicago PR promoters may be a unique and valuable tool for pathogen-induced transgenic expression of antimicrobial proteins or other genes enhancing disease resistance in alfalfa.