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

Authors: SATHOFF, ANDREW - University Of Minnesota; Dornbusch, Melinda - Mindy; Miller, Susan; Samac, Deborah - Debby

Submitted to: Molecular Breeding
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/15/2020
Publication Date: 6/26/2020
Citation: Sathoff, A.E., Dornbusch, M.R., Miller, S.S., Samac, D.A. 2020. Functional analysis of Medicago-derived pathogen-induced gene promoters for usage in transgenic alfalfa. Molecular Breeding. 40(66):1-12. https://doi.org/10.1007/s11032-020-01144-6.
DOI: https://doi.org/10.1007/s11032-020-01144-6

Interpretive Summary: Genetic modification of crop plants to introduce novel traits requires isolation of DNA sequences called promoters for initiating and correctly regulating gene expression. Currently, there are few promoters available for expression of genes to achieve enhanced disease control. The ideal promoter for expression of genes to protect plant cells would be responsive to multiple types of pathogens. Three pathogenesis related gene promoters from barrel medic and alfalfa were isolated and tested in transgenic alfalfa for pathogen-induced expression of a marker gene. All three were functional in alfalfa and caused low levels of expression primarily in healthy root tissue. When leaves were infected by a bacterial pathogen there was rapid and strong localized increase in gene expression at the infection site. When leaves were infected by fungal pathogens, there was increased gene expression throughout the leaf. These promoters show promise for use in engineering disease resistance by specifically controlling gene expression during pathogen attack.

Technical Abstract: Three gene promoters from 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 ß-glucuronidase (GUS) reporter gene were constructed and utilized to generate transgenic alfalfa. Histochemical GUS staining established that both the PR10 and PR5 promoters were functional in alfalfa and cause 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, enhanced GUS staining was observed in the leaf tissue, which demonstrates that the promoters are responsive to both bacterial and fungal pathogens. Gene expression analysis using RT-qPCR also indicated that all three PR promoters were pathogen-induced with the highest up-regulation in GUS gene expression of 423-fold in response to P. medicaginis inoculation. Additionally, several putative transcription regulator elements (REs) responsible for the observed pathogen-induced promoter activity were identified in each of the promoter sequences. These characterized Medicago PR promoters may be valuable for pathogen-induced transgenic expression of antimicrobial peptides.