Fusarium verticillioides produces fumonisins, which are mycotoxins inhibiting sphingolipid biosynthesis in humans, animals, and other eukaryotes. Fumonisins are presumed virulence factors of plant pathogens, but may also play a role in interactions between competing fungi. We observed higher resistance to added fumonisin B₁ (FB₁) in fumonisin-producing Fusarium verticillioides than in nonproducing F. graminearum, and likewise between isolates of Aspergillus and Alternaria differing in production of sphinganine-analog toxins. It has been reported that in F. verticillioides, ceramide synthase encoded in the fumonisin biosynthetic gene cluster is responsible for self-resistance. We reinvestigated the role of FUM17 and FUM18 by generating a double mutant strain in a fum1 background. Nearly unchanged resistance to added FB₁ was observed compared to the parental fum1 strain. A recently developed fumonisin-sensitive baker’s yeast strain allowed for the testing of candidate ceramide synthases by heterologous expression. The overexpression of the yeast LAC1 gene, but not LAG1, increased fumonisin resistance. High-level resistance was conferred by FUM18, but not by FUM17. Likewise, strong resistance to FB₁ was caused by overexpression of the presumed F. verticillioides “housekeeping” ceramide synthases CER1, CER2, and CER3, located outside the fumonisin cluster, indicating that F. verticillioides possesses a redundant set of insensitive targets as a self-resistance mechanism.

Fumonisins, produced mainly by various Fusarium species, are sphinganine-analog mycotoxins and potent inhibitors of ceramide synthases in animals, plants, and other eukaryotes, leading to toxicity and programmed cell death. Wild-type baker’s yeast is naturally highly resistant to fumonisin B ₁ (FB1); on rich medium, it tolerates more than 1,000 µM. We report the construction of a fumonisin-sensitive Saccharomyces cerevisiae strain with six disrupted genes: SNQ2 , PDR12 , and YOR1 , coding for ATP-binding cassette transporters involved in active efflux; CKA2 , encoding casein kinase II, regulating ceramide synthase activity by phosphorylation; and LCB3 and VPS51 , encoding proteins involved in the biosynthesis and recycling of sphingolipid precursors, respectively. The constructed FB1-sensitive strain YTKT33 was tested on complex yeast extract-peptone-dextrose (YPD) and synthetic complete medium alongside the wild-type strain and strains with only three (YRU74) or five (YRU94ML) disrupted genes. YTKT33, exhibiting full growth inhibition at a concentration below 5 µM FB1 on synthetic complete agar lacking uracil (SC-URA), was used as a host for heterologous expression of previously reported fumonisin detoxification genes. Transformants expressing either the Sphingopyxis fumD esterase, which removes removing tricarballylic acid side chains, or an Aspergillus amine oxidase, which converts the amino- to a keto-group, can grow at FB1 concentrations inhibitory for transformants containing the empty vector. YTKT33 was inhibited by other type B fumonisins (B ₂ , B ₃ , B ₄ ) at concentrations very similar to FB1, allowing rapid evaluation of total fumonisin toxicity in crude fungal extracts. IMPORTANCE: Fumonisins can cause diseases in animals and humans consuming Fusarium -contaminated food or feed. The search for microbes capable of fumonisin degradation, or for enzymes that can detoxify fumonisins, currently relies primarily on chemical detection methods. Our constructed fumonisin B1-sensitive yeast strain can be used to phenotypically detect detoxification activity and should be useful in screening for novel fumonisin resistance genes and to elucidate fumonisin metabolism and resistance mechanisms in fungi and plants, and thereby, in the long term, help to mitigate the threat of fumonisins in feed and food.

Molecular mechanisms responsible for self-protection in fumonisin-producing fungi are still barely understood. A role for fumonisin in competition between fungi was proposed, particularly between Fusarium verticillioides and non-producing F. graminearum on maize. Recently, it was shown that the FUM-cluster associated ceramide synthase genes of F. verticilliodes (FUM17 and FUM18) are dispensable for the observed higher fumonisin resistance in F. verticillioides. To test the role of CER1, we have inactivated this gene in F. verticillioides (in the wild-type, fum1 and fum1 fum17-18 background). Despite the expected redundancy of ceramide synthases, inactivation of CER1 in F. verticillioides lead to inhibition of growth by 80-90% and FB₁ sensitivity at a concentration of only 10 μM. While the slow-growth phenotype could be complemented by expression of both a FvCER1 cDNA and the corresponding F. graminearum CER1 ortholog, this was not the case for the Saccharomyces cerevisiae LAG1 and LAC1 ceramide synthase genes. The F. graminearum CER1 not only complemented the growth defect but even conferred slightly higher FB₁ resistance. Heterologous expression in a sensitive yeast strain revealed that all F. verticillioides and also F. graminearum CER1, CER2 and CER3 genes confer FB₁ resistance, but CER2 and CER3 only against lower concentrations. However, overexpression of CER2 and CER3 is problematic due to toxicity in yeast leading to variable results. We hypothesize that F. graminearum ancestors may have lost the ability to produce fumonisin, but maintained the self-protection mechanism against sphinganine-analog mycotoxins based on target insensitivity.