BUSCADOR RECOLECTA

[EN] 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., This work was supported by grant PID2021-125858OB-I00, and the Severo Ochoa Excellence Program CEX2021-001189-S, funded by MCIN/AEI/ 10.13039/501100011033 and by ERDF, a way of making Europe . M.G.L. and C.R.P. were recipients of predoctoral grants FPU19/02066 and ACIF/2021/364 funded by MCIN/AEI/10.13039/501100011033 and by ESF-Investing in Your future and by Conselleria d'Educacio¿ (Generalitat Valenciana, Comunitat Valenciana, Spain), respectively. M.G.L. international exchange was supported by EST23/00233 grant (funded by MCIN/AEI/10.13039/ 501100011033 and by ESF-Investing in Your future) and SEG10314 EMBO Scientific Exchange Grant. S.G. holds a Juan de la Cierva Incorporacion grant (IJC2020-042749-I) from the Spanish Ministerio de Ciencia e Innovacion, ¿ funded by the European Union, NextGenerationEU PRTR. Work undertaken with EMB and DDT was funded by the Medical Research Council Centre for Medical Mycology at the University of Exeter (MR/N006364/2 and MR/V033417/1), that forms part of the National Institute for Health and Care Research (NIHR) Exeter Biomedical Research Centre (BRC), and the BBSRC project grant BB/ V017004/1 to EMB. We thank Jose Mª Coll (IATA) for his technical help in microscopy and Dr. Amparo Tarrega ¿ (IATA) for her expert advice in the statistical analyses.

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., This work was supported by grant PID2021-125858OB-I00, and the Severo Ochoa Excellence Program CEX2021-001189-S, funded by MCIN/AEI/ 10.13039/501100011033 and by “ERDF, a way of making Europe”. M.G.L. and C.R.P. were recipients of predoctoral grants FPU19/02066 and ACIF/2021/364 funded by MCIN/AEI/10.13039/501100011033 and by ESF-Investing in Your future and by “Conselleria d'Educació” (Generalitat Valenciana, Comunitat Valenciana, Spain), respectively. M.G.L. international exchange was supported by EST23/00233 grant (funded by MCIN/AEI/10.13039/ 501100011033 and by ESF-Investing in Your future) and SEG10314 EMBO Scientific Exchange Grant. S.G. holds a Juan de la Cierva Incorporación grant (IJC2020-042749-I) from the Spanish Ministerio de Ciencia e Innovación, funded by the European Union, NextGenerationEU PRTR. Work undertaken with EMB and DDT was funded by the Medical Research Council Centre for Medical Mycology at the University of Exeter (MR/N006364/2 and MR/V033417/1), that forms part of the National Institute for Health and Care Research (NIHR) Exeter Biomedical Research Centre (BRC), and the BBSRC project grant BB/V017004/1 to EMB., With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2021-001189-S), Peer reviewed