Dynamics of interaction and internalisation of the antifungal protein PeAfpA into Penicillium digitatum morphotypes

Antifungal proteins (AFPs) as the highly active PeAfpA from Penicillium expansum or PdAfpB from Penicillium digitatum exert promising antifungal activity, but their mode of action is not fully understood. We characterised the interaction of PeAfpA against P. digitatum, comparing it to the less active PdAfpB. Despite similar effect on conidia germination, PeAfpA did not induce a burst of reactive oxygen species as PdAfpB. Live-cell fluorescence microscopy revealed complex dynamics of interaction and internalisation of both proteins with distinct P. digitatum morphotypes (quiescent conidia, swollen conidia, germlings and hyphae). Labelled PeAfpA co-localised at the cell wall of quiescent conidia, where its localisation was punctate and not uniformly distributed. This pattern changed during germination to a uniform distribution with increased intensity. Conidia from mutants of genes involved in melanin biosynthesis (pksP/alb1 or arp2) showed an altered distribution of PeAfpA but later mimicked the wild type trend of changes during germination. In swollen conidia and germlings, PeAfpA remained attached to the cell wall. In hyphae, PeAfpA was internalised through the growing hyphal tip after binding to the cell wall, in a non-endocytic but energy-dependent process that caused vacuolisation, which preceded cell death. These results may help the development of biofungicides based on AFPs.