Antofine, a phenanthroindolizidine alkaloid, is a bioactive natural product isolated from milkweeds that exhibits numerous biological activities, including anticancer, antimicrobial, antiviral, and anti-inflammatory properties. However, the direct targets and mode of action of antofine have not been determined. In this report, we show that antofine displays antifungal properties against the phytopathogen Fusarium graminearum, the cause of Fusarium head blight disease (FHB). FHB does devastating damage to agriculture, causing billions of dollars in economic losses annually. We therefore sought to understand the mode of action of antofine in F. graminearum using insights from yeast chemical genomic screens. We used haploinsufficiency profiling (HIP) to identify putative targets of antofine in yeast and identified three candidate targets, two of which had homologs in F. graminearum. The Fusarium homologues of two targets, glutamate dehydrogenase (FgGDH) and resistance to rapamycin deletion 2 (FgRRD2), can bind antofine. Of the two genes, only the Fgrrd2 knockout displayed a loss of virulence in wheat, indicating that RRD2 is an antivirulence target of antofine in F. graminearum. Mechanistically, we demonstrate that antofine disrupts the interaction between FgRRD2 and FgTap42, which is part of the Tap42-phosphatase complex in the target of rapamycin (TOR) signaling pathway, a central regulator of cell growth in eukaryotes and a pathway of extensive study for controlling numerous pathologies. IMPORTANCE Fusarium head blight caused by the fungal pathogen Fusarium graminearum is a devastating disease of cereal crops worldwide, with limited effective chemical treatments available. Here we show that the natural alkaloid compound antofine can inhibit fusarium head blight in wheat. Using yeast genomic screening, we identified the TOR pathway component RRD2 as a target of antofine that is also required for F. graminearum pathogenicity.

The Marcgraviaceae is a neotropical plant family of lianas and shrubs distributed throughout Central and South America, mainly in evergreen and semi-evergreen tropical forests. In this study, extracts of 12 Marcgraviaceae species were assessed for inhibition of bacterial quorum sensing (QS) in Chromobacterium violaceum, bacterial biofilm formation in Pseudomonas aeruginosa PA14, and fungal growth in Saccharomyces cerevisiae. Six species showed promising inhibitory activities in at least one of the three bioassays (Marcgravia nervosa Triana & Planch., Marcgravia polyantha Delp., Marcgravia schippii Standl., Marcgraviastrum subsessile (Benth.) Bedell, Schwartzia brasiliensis (Choisy) Bedell ex Gir-Cañas, and Schwartzia costaricensis (Gilg.) Bedell). Analyses of the crude extract of the leaves of Marcgravia nervosa using HPLC–APCI–MS showed the presence of five pentacyclic triterpenes: ursolic acid (2), betulinic acid (3), α–amyrin (4), β–amyrin (5), lupeol (6). Bioassay-guided fractionation of this plant resulted in the isolation and identification of 2-methoxy-1,4-naphthoquinone (1) as the active principle with a minimum inhibitory concentration (MIC) of 85–100 μmol·L−1 against Saccharomyces cerevisiae BY4741 (haploid) and BY4743 (diploid).

Bioassay-guided fractionation of the crude extract (80% EtOH) of the leaves of Cestrum schlechtendahlii, a plant used by Q'eqchi' Maya healers for treatment of athlete's foot, resulted in the isolation and identification of two spirostanol saponins (1 and 2). Structure elucidation by MS, 1D-NMR, and 2D-NMR spectroscopic methods identified them to be the known saponin (25R)-1β,2α-dihydroxy-5α-spirostan-3-β-yl-O-α-L-rhamnopyranosyl-(1 → 2)-β-D-galactopyranoside (1) and new saponin (25R)-1β,2α-dihydroxy-5α-spirostan-3-β-yl-O-β-D-galactopyranoside (2). While 2 showed little or no antifungal activity at the highest concentration tested, 1 inhibited growth of Saccharomyces cerevisiae (minimum inhibitory concentration (MIC) of 15-25 μM), Candida albicans, Cryptococcus neoformans, and Fusarium graminearum (MIC of 132-198 μM).

The multicomponent NADPH oxidase enzyme complex (Nox) converts molecular oxygen in a stepwise reduction to superoxides leading to the production of H2O2. This study characterized the role of the catalytic subunit (gp91(phox)) of Nox, NoxA and NoxB in F. graminearum. Targeted deletion of the genes NoxA and NoxB show that they differentially regulate the production of superoxides during mycelial development. The nitro blue tetrazolium staining method revealed that both the single noxA mutant and the double noxA/B mutant strains are restricted in the production of superoxides. This limitation, however, did not affect their ability to synthesize 15-ADON in culture. Deletion analyses also revealed that NoxA, but not NoxB, is involved in perithecia development and ascospore production. An in-vitro based cellophane breach assay indicated that both NoxA and NoxB contribute to virulence. The pathogenicity tests performed on a susceptible variety of wheat, Roblin', confirmed that these two genes act synergistically to promote virulence. Cumulatively, we provide evidence that the Nox homologues have non-redundant functions in F. graminearum. ResumeLe complexe a composantes multiples de l'enzyme NADPH oxydase (Nox) convertit l'oxygene moleculaire par reduction progressive en superoxydes, ce qui engendre la production de H2O2. Cette etude a caracterise le role de la sous-unite (gp91(phox)) de la Nox, de la NoxA et de la NoxB chez F. graminearum. La deletion ciblee des genes NoxA et NoxB montre qu'ils regulent differentiellement la production des superoxydes durant le developpement du mycelium. La methode de coloration au nitro bleu de tetrazolium a revele que les souches du mutant monogenique noxA et du mutant a deux genes noxA/B participent en nombre restreint a la production des superoxydes. Cette limitation, toutefois, n'a pas influence leur capacite a synthetiser le 15-ADON en culture. Les analyses des deletions ont egalement revele que le gene NoxA est implique dans le developpement des peritheces et dans la production des ascospores, mais pas le gene NoxB. Un essai in vitro relatif a la capacite de penetration de la cellophane a indique les genes NoxA et NoxB contribuent a la virulence. Les tests de pathogenicite effectues sur le cultivar de ble receptif Roblin' ont confirme que ces deux genes agissent de facon synergetique pour favoriser la virulence. Cumulativement, nous fournissons la preuve que les homologues de Nox ont des fonctions non redondantes chez F. graminearum.

The phytopathogenic fungus Fusarium graminearum is the principle cause of Fusarium Head Blight, a devastating disease of wheat, barley and other cereals. A commercial library of chemicals was tested against F. graminearum. Of the more than 500 compounds screened, 25 candidates were found to be inhibitory against F. graminearum growth. The compound Antofine was tested against a S. cerevisiae haploid single knock-out library and 30 mutants were shown to be hypersensitive to this compound. GeneMania, an online multiple association network integration algorithm and the Saccharomyces Genome Database Gene Ontology Term Finder search engine were used to uncover relationships between genes associated with Antofine sensitivity. The results suggested that Antofine likely perturbs genes involved in transcription regulation and mRNA processing.

We tested extracts of Vincetoxicum rossicum for inhibition of bacterial and fungal growth and for anti-insect activities that may account for the invasive characteristics of this introduced species in North America. Bioassay-guided fractionation was used to identify (−)-antofine as the principle inhibitor of bacteria and fungi in root extracts. This compound had especially pronounced antifungal activity, inhibiting the growth of diverse taxa that include yeast-like and filamentous fungi and, notably, broad-host-range plant pathogens. A second compound(s), that is as yet uncharacterized but distinct from (−)-antofine, was detected as having antifeedant activity against a larval hymenopteran, rose sawfly (Allantus cinctus), and toxicity to two larval lepidopterans, the masked birch caterpillar (Drepana arcuata) and the European corn borer (Ostrinia nubilalis). That V. rossicum contains potent inhibitors of plant pathogenic fungi, diverse bacteria, and herbivorous insects likely contributes to its success as an invasive species.