The potential use of the Penicillium chrysogenum antifungal protein PAF, the designed variant PAF^opt and its γ‐core peptide Pγ^opt in plant protection

Abstract

The prevention of enormous crop losses caused by pesticide-resistant fungi is a serious challenge in agriculture. Application of alternative fungicides, such as antifungal proteins and peptides, provides a promising basis to overcome this problem; however, their direct use in fields suffers limitations, such as high cost of production, low stability, narrow antifungal spectrum and toxicity on plant or mammalian cells. Recently, we demonstrated that a Penicillium chrysogenum-based expression system provides a feasible tool for economic production of P. chrysogenum antifungal protein (PAF) and a rational designed variant (PAF opt), in which the evolutionary conserved c-core motif was modified to increase antifungal activity. In the present study, we report for the first time that c-core modulation influences the antifungal spectrum and efficacy of PAF against important plant pathogenic ascomycetes, and the synthetic c-core peptide Pc opt , a derivative of PAF opt , is antifungal active against these pathogens in vitro. Finally, we proved the protective potential of PAF against Botrytis cinerea infection in tomato plant leaves. The lack of any toxic effects on mammalian cells and plant seedlings, as well as the high tolerance to harsh environmental conditions and proteolytic degradation further strengthen our concept for applicability of these proteins and peptide in agriculture.

Biofungicidal Potential of Neosartorya (Aspergillus) Fischeri Antifungal Protein NFAP and Novel Synthetic γ-Core Peptides

“…So far, no adverse effects of the fungal APs and PDs on plants or fruits have been reported in the literature (Vila et al, 2001;Moreno et al, 2003Moreno et al, , 2006Theis et al, 2005;Barakat, 2014;Garrigues et al, 2018); however, little information is available on the induction of morphological aberrations when growing seedlings with these biomolecules. Exceptions are AFP that did not affect the growth of the tomato plant roots (Theis et al, 2005) or PAF and its derived variant and PD that did neither harm the leaves of barley (Barna et al, 2008) nor those of tomatoes nor affected the seedlings of M. truncatula (Tóth et al, 2020). In the present study, the vegetative growth and the morphology of the roots of the M. truncatula seedlings were not disturbed by NFAP (Figure 4).…”

“…As for the harmlessness in humans and animals, our proof-of-principle experiments indicated that NFAP and the PD γ NFAP -opt were not cytotoxic against keratinocytes, colonic epithelial cells, and monocytes and were not hemolytic in the antifungal effective concentration (Figure 3). Therefore, this N. fischeri protein and peptide exhibited harmless activity in vitro similar to other members of the fungal APs, such as PAF and AFP (Szappanos et al, 2005(Szappanos et al, , 2006Tóth et al, 2020), PAF γ-core PDs (Sonderegger et al, 2018;Tóth et al, 2020), and NFAP2 (Kovács et al, 2019). Importantly, experiments with a murine model for pulmonary fungal infection and fungal vulvovaginitis further proved the safety in vivo for PAF (Palicz et al, 2013(Palicz et al, , 2016 and NFAP2 (Kovács et al, 2019), respectively.…”

“…The suitability of APs to efficiently protect agriculturally important plants and crops against fungal infection and contamination by direct topical application was proved previously (Moreno et al, 2003;Theis et al, 2005;Barna et al, 2008;Barakat et al, 2010a,b;Barakat, 2014;Tóth et al, 2020). However, the introduction of APs into the biopesticide market requires high ecological compatibility and tolerance by the host (humans, animals, and plants).…”

“…Different APs from Penicillium or Aspergillus spp. can effectively inhibit the growth of several plant pathogens (Vila et al, 2001;Moreno et al, 2003Moreno et al, , 2006Theis et al, 2005;Barna et al, 2008;Garrigues et al, 2018;Tóth et al, 2020) and foodborne and postharvest mycotoxigenic fungi (Barakat et al, 2010a,b;Delgado et al, 2015Delgado et al, , 2017Delgado et al, , 2018Garrigues et al, 2016). Topical application of the Aspergillus giganteus AP (AFP) (Moreno et al, 2003) and the Penicillium chrysogenum AP (PAF) (Barna et al, 2008) on plant leaves proved to protect Pelargonium, barley and wheat, respectively, against fungal pathogens without causing any detrimental effects to the host.…”

“…In our previous study, we introduced a synthetic PD of the native PAF γ-core with anti-Candida activity and demonstrated that amino acid substitutions that elevate the positive net charge and hydrophilicity increased the antifungal potency (Sonderegger et al, 2018). And very recently, we described the potential of the P. chrysogenum PAF, its designed variant PAF opt , and γ-core peptide Pγ opt in plant protection (Tóth et al, 2020).…”