The realm of antimicrobial proteins in plants is extensive but remains relatively uncharted. Understanding the mechanisms underlying the action of plant antifungal proteins (AFPs) holds promise for antifungal strategies. This study aimed to bridge this knowledge gap by comprehensively screening Arabidopsis thaliana species to identify novel AFPs. Using MALDI-TOF analysis, we identified a member of the TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR1 (TCP) family of transcription factors as a novel AFP, A. thaliana TCP21 (AtTCP21; accession number NP_196450). Bacterially purified recombinant AtTCP21 inhibited the growth of various pathogenic fungal cells. AtTCP21 was more potent than melittin, a well-known AFP, in combating Colletotrichum gloeosporioides. Growth inhibition assays against various fungal pathogens and yeasts confirmed the pH-dependent antimicrobial activity of AtTCP21. Without inducing any membrane alterations, AtTCP21 penetrates the fungal cell wall and membrane, where it instigates a repressive milieu for fungal cell growth by generating intracellular reactive oxygen species and mitochondrial superoxides; resulting in morphological changes and apoptosis. Our findings demonstrate the redox-regulating effects of AtTCP21 and point to its potential as an antimicrobial agent.

The rapid induction of drug resistance is considered a fatal drawback of conventional antibiotics and requires the continuous development of new antibiotics. Accordingly, antibacterial peptides (AMPs) have attracted interest as next-generation antibiotics and many studies have been conducted. However, much remains unknown regarding the mechanism of AMPs and the effects of amino acid sequence changes. We compared the structures and antifungal effects of HnMc-W (F1W substitution, straight alpha-helical structure), HnMc-WP1 (S9P substitution, bending alpha-helical structure), and HnMc-WP2 (addition of the PXXP motif, helix-to-helix structure) to those of a parent hybrid AMP (HnMc) regarding their mechanism of action. The most active was HnMc-WP2, which exhibited an antifungal effect via membranolytic action on the fungal cell membrane. The others inhibited fungal growth by inducing apoptosis through reactive oxygen species production caused by mitochondrial damage. This study proposes the addition of the ‘PXXP’ motif in the design of AMPs acting on cell membranes.