Occurrence and function of fungal antifungal proteins: a case study of the citrus postharvest pathogen Penicillium digitatum
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Antifungal proteins (AFPs) of fungal origin have been described in filamentous fungi. AFPs are small, highly stable, cationic cysteine-rich proteins (CRPs) that are usually secreted in high amounts and show potent antifungal activity against non-self fungi. The role of AFPs in the biology of the producer fungus remains unclear. AFPs have been proposed as promising lead compounds for the development of new antifungals. The analyses of available antifungal CRP sequences from fungal origin and their phylogenetic reconstruction led us to propose a new classification of AFPs in three distinct classes: A, B and C. We initiate for the first time the characterization of an AFP in a fungal pathogen, by analysing the functional role of the unique afpB gene in the citrus fruit pathogen Penicillium digitatum. Null ΔafpB mutants revealed that this gene is dispensable for vegetative growth and fruit infection. However, strains that artificially express afpB in a constitutive way (afpB C) showed a phenotype of restricted growth, distortion of hyphal morphology and strong reduction in virulence to citrus fruits. These characteristics support an antifungal role for AfpB. Surprisingly, we did not detect the AfpB protein in any of the P. digitatum strains and growth conditions that were analysed in this study, regardless of high gene expression. The afpB C phenotype is not stable and occasionally reverts to a wild type-like phenotype but molecular changes were not detected with this reversion. The reduced virulence of afpB C strains correlated with localized fruit necrosis and altered timing of expression of fruit defence genes.
KeywordsAntifungal peptide Antifungal protein Antifungals Penicillium digitatum Postharvest pathology Citrus
We acknowledge the suggestions by Dr. Lourdes Carmona (IATA, Valencia, Spain) and the help in the microscopy experiments of José M. Coll-Marqués (IATA, Valencia, Spain). We truly acknowledge the excellent technical assistance of Cristina Font and Shaomei Xu (IATA, Valencia, Spain). We thank Lluís Giménez and members of Cooperativa Vinicola de Lliria S.C.V. (Lliria, Valencia, Spain) for providing orange fruits for infection assays.
Compliance with ethical standards
This work was funded by grants BIO2012-34381 from the “Ministerio de Economía y Competitividad” (MINECO, Spain) and PROMETEOII/2014/027 from “Conselleria d’Educaciò” (Generalitat Valenciana, Comunitat Valenciana, Spain). SG was the recipient of a predoctoral scholarship within the FPU programme from “Ministerio de Educación Cultura y Deporte” (MECD).
Conflict of interest
The authors declare that they have no competing interests.
This article does not contain any studies with human participants or animals performed by any of the authors.
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