Comprehensive Characterization of Plant Defensins in Alfalfa Reveals Conserved Evolution and Functional Specialization of MsPDF2.1

Bacterial and fungal pathogens threaten alfalfa production, and although chemical control is widely used, its environmental risks highlight the need for sustainable alternatives. Plant defensins, with natural origin and broad antimicrobial activity, represent promising candidates for enhancing crop resistance. However, their roles in alfalfa remain largely unknown. Here, we conducted the first genome-wide identification and characterization of the PDF gene family in alfalfa, identifying 10 members grouped into two subfamilies (PDF1 and PDF2). These genes showed diverse structures, uneven chromosomal distribution, and salicylic acid-/jasmonic acid-responsive cis-elements, indicating involvement in biotic stress responses. Expression profiling revealed distinct tissue-specific patterns and pathogen-related induction. Comparative synteny analysis across 93 angiosperms demonstrated strong evolutionary conservation of PDF2, whereas PDF1 and PDF3 exhibited lineage-specific signatures. Among all MsPDFs, MsPDF2.1 emerged as a key candidate due to high expression in disease-relevant tissues, significant differential expression between resistant and susceptible germplasms, and strong evolutionary conservation. Functional assays confirmed that MsPDF2.1 confers broad antifungal and antibacterial activity and enhances pathogen resistance in Nicotiana benthamiana. Bimolecular fluorescence complementation indicated that MsPDF2.1 forms homomeric oligomers, consistent with a membrane-disrupting mechanism. Structural modeling revealed a conserved defensin fold with variation mainly in loop regions. This study establishes a comprehensive framework for understanding PDF diversity and identifies MsPDF2.1 as a promising resource for improving alfalfa disease resistance.