Enhanced Antifungal Activity of Engineered Proteins via Swapping between Thioredoxin H2 and H3

Thioredoxins (Trxs) are proteins that act as antioxidants by facilitating the reduction of other proteins and are highly conserved in all organisms. Plant H-type Trx isoforms have different structures and perform multiple functions. Previous studies have reported that the low molecular weight AtTrx-H2 acts as a disulfide reductase and the high molecular weight AtTrx-H3 functions as an oxidoreductase and a molecular chaperone. In this study, we compared the antifungal activities of Arabidopsis Trx-H2 and -H3 with engineered proteins 2N3C and 3N2C via domain-swapping between the N- and C-terminal regions of Trx-H2 and -H3. All AtTrx-H variant proteins inhibited cell growth of various pathogenic fungal strains at pH 5.2 and pH 7.2 and showed significant intracellular accumulation in the fungal cells. Interestingly, only two engineered proteins penetrated the fungal cell wall and membrane, indicating their ability to destabilize the fungal cell membrane before internalization into the cytosol. To our knowledge, this is the first study that demonstrates novel functions of plant antioxidants AtTrx-H2 and -H3 as antifungal proteins and shows their enhanced activity using the domain swapping technique.