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Plant pathogenic fungi are responsible for enormous crop losses worldwide. Overcoming this problem is challenging as these fungi can be highly resistant to approved chemical fungicides. There is thus a need to develop and introduce fundamentally new plant and crop protection strategies for sustainable agricultural production. Highly stable extracellular antifungal proteins (AFPs) and their rationally designed peptide derivatives (PDs) constitute feasible options to meet this challenge. In the present study, their potential for topical application to protect plants and crops as combinatorial biofungicides is supported by the investigation of two Neosartorya (Aspergillus) fischeri AFPs (NFAP and NFAP2) and their γ-core PDs. Previously, the biofungicidal potential of NFAP, its rationally designed γ-core PD (γNFAP-opt), and NFAP2 was reported. Susceptibility tests in the present study extended the in vitro antifungal spectrum of NFAP2 and its γ-core PD (γNFAP2-opt) to Botrytis, Cladosporium, and Fusarium spp. Besides, in vitro additive or indifferent interactions, and synergism were observed when NFAP or NFAP2 was applied in combination with γNFAP-opt. Except for γNFAP2-opt, the investigated proteins and peptides did not show any toxicity to tomato plant leaves. The application of NFAP in combination with γNFAP-opt effectively inhibited conidial germination, biofilm formation, and hyphal extension of the necrotrophic mold Botrytis cinerea on tomato plant leaves. However, the same combination only partially impeded the B. cinerea-mediated decay of tomato fruits, but mitigated the symptoms. Our results highlight the feasibility of using the combination of AFP and PD as biofungicide for the fungal infection control in plants and crops. [ABSTRACT FROM AUTHOR]

In the genome of Aspergillus nidulans, a defensin-like protein, Anisin1, was annotated with high homology to the mosquito defensin AaDefA1. So far, no studies exist on defensins from filamentous ascomycetes. Therefore, we characterized the anisin1 gene in A. nidulans and generated a deletion mutant, which suffered from a defect in mitospore development and produced less conidia at 42°C compared to the reference strain. In surface cultures of A. nidulans wild type, the anisin1 expression correlated with that of the central regulator for asexual development, brlA, and with the major scavanger of HO stress, catB, which is indicative for cell differentiation in developing fungi. Interestingly, brlA and anisin1 expressions were deregulated in a Δ srrA strain that covers a central role in the histidine-to-aspartate (His-Asp) phosphorelay signaling pathway and shows impaired asexual development and HO detoxification. In submers cultures of A. nidulans wild type and other mutants of the His-Asp phosphorelay signaling pathway, anisin1 was repressed, but derepressed in a Δ srrA background, and anisin1 transcription was further increased in this mutant by HO addition. We therefore conclude that the secreted protein Anisin1 contributes to the optimal development of A. nidulans and we further propose that it has a sensing/signaling function for elevated HO levels. [ABSTRACT FROM AUTHOR]

Certain filamentous fungi, such as the penicillin-producing strainPenicillium chrysogenum, secrete small, highly basic and cysteine-rich proteins with antifungal effects. Affected fungi include a number of important zoopathogens, including those infecting humans. Recent studies, however, have pointed to a membrane-perturbing effect of these antifungal compounds, apparent as a potassium efflux from affected fungal cells. If present on mammalian cells, this would severely hinder the potential therapeutic use of these molecules. Here we studied the effects of theP. chrysogenum-derived antifungal peptide (PAF) on a number of mammalian cells to establish whether the protein has any cytotoxic effects, alters transmembrane currents on excitable cells or activates the immune system. PAF, in a concentration range of 2-100 µg/ml, did not cause any cytotoxicity on human endothelial cells from the umbilical vein. Applied at 10 µg/ml, it also failed to modify voltage-gated potassium channels of neurones, skeletal muscle fibers, and astrocytes. PAF also left the hyperpolarization-activated non-specific cationic current (Ih) and the L-type calcium current unaffected. Finally, up to 2 µg/ml, PAF did not induce the production of pro-inflammatory cytokines such as IL-6, IL-8, and TNF-a. These results suggest that PAF should have only minor, if any, effects on mammalian cells in the intended therapeutic concentration range. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies (mABs) against various cell types of the basal free-living flatwormMacrostomumsp. were produced by immunising Balb/c mice with cell suspensions of disintegrated animals. We identified 360 positive supernatants with specific staining of various tissues, cell types, patterns or structures. Here we report immunocytochemical characterisation, histological stainings and isotyping of 11 mABs specific for muscle cells (MMu-1, MMu-2, MMu-3, MMu-4), digestive and prostate glands (MDr-1 and MDr-2, MPr-1), epidermal cells (MEp-1), the ventral nerve cord including neuron clusters (MNv-1), gastrodermal cells (MDa-1) and spermatids (MSp-1). Confocal microscopy, histological techniques, electron microscopy and immunoblotting were applied to demonstrate stainings in juveniles, adults, starved or well-fed animals. Considering the current lack of specific markers the obtained mABs will be particularly helpful studying embryonic and postembryonic development, pattern formation, cell differentiation, regeneration and reproductive allocation inMacrostomumsp., and possibly other basal flatworms. The small size, ease of culturing, short generation time, transparency and the basal phylogenetic position specifyMacrostomumsp. as a suitable model organism for comparative analyses within Platyhelminthes and toDrosophilaandC. elegans. [ABSTRACT FROM AUTHOR]

The filamentous fungus Penicillium chrysogenum abundantly secretes the small, highly basic and cysteine-rich protein PAF (Penicillium antifungal protein). In this study, the antifungal activity of PAF is described. PAF inhibited the growth of a variety of filamentous fungi, including opportunistic human pathogenic and phytopathogenic fungi, whereas bacterial and yeast cells were unaffected. PAF reduced the conidial germination and hyphal extension rates in a dose-dependent manner and induced severe changes in cell morphology that resulted in crippled and distorted hyphae and atypical branching. Growth-affected hyphae suffered from oxidative stress, plasma membrane leakage, and metabolic inactivity, which points to an induction of multifactorial effects in sensitive fungi. In contrast to other known antifungal proteins, the effects of PAF were only partially antagonized by cations. [ABSTRACT FROM AUTHOR]