High yield production of the antifungal proteins PeAfpA and PdAfpB by vacuole targeting in a TMV‐based expression vector

Antifungal proteins (AFPs) derived from filamentous fungi show great potential against economically significant fungi that cause plant diseases and consequently threat food safety and security. This study focuses on the Penicillium expansum PeAfpA and Penicillium digitatum PdAfpB proteins and their activity against several phytopathogens. The AFPs were synthesized through a highly productive tobacco mosaic virus-based expression vector in the fast-growing model plant Nicotiana benthamiana, combining signalling sequences for apoplastic and vacuolar compartmentalization to increase yields. Adding a vacuolar signalling peptide from a Nicotiana sylvestris chitinase at the C-termini of the AFPs in combination with an apoplastic N-terminal signalling peptide from N. benthamiana osmotin significantly enhanced AFP yields without altering functionality. Results showed an improvement of ninefold for PeAfpA and 3,5-fold for PdAfpB compared to constructs with only the apoplastic N-terminal signalling. Transmission electron microscopy and immunogold labelling confirmed the localization of AFPs in both the apoplast and the vacuole, highlighting its compatibility with vacuolar environments. In vitro and in vivo assessments against key pathogenic fungi, including Magnaporthe oryzae, Botrytis cinerea and Fusarium proliferatum, revealed that the activities of easily purified PeAfpA- and PdAfpB-enriched plant extracts closely mirrored those of their purified fungal counterparts. This innovative approach represents a notable advance towards the application of AFPs as effective, safe and environmentally friendly 'green biofungicides' for safeguarding crop and postharvest produce and could also be applied to control other pathogenic fungi that threat human health.