Polyacetylenic natural products are a substantial class of often unstable compounds containing a unique carbon-carbon triple bond functionality, that are intriguing for their wide variety of biochemical and ecological functions, economic potential, and surprising mode of biosynthesis. Isotopic tracer experiments between 1960 and 1990 demonstrated that the majority of these compounds are derived from fatty acid and polyketide precursors. During the past decade, research into the metabolism of polyacetylenes has swiftly advanced, driven by the cloning of the first genes responsible for polyacetylene biosynthesis in plants, moss, fungi, and actinomycetes, and the initial characterization of the gene products.The current state of knowledge of the biochemistry and molecular genetics of polyacetylenic secondary metabolic pathways will be presented together with an up-to-date survey of new terrestrial and marine natural products, their known biological activities, and a discussion of their likely metabolic origins.

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Wood decay mechanisms in Agaricomycotina have been traditionally separated in two categories termed white and brown rot. Recently the accuracy of such a dichotomy has been questioned. Here, we present the genome sequences of the white rot fungus Cylindrobasidium torrendii and the brown rot fungus Fistulina hepatica both members of Agaricales, combining comparative genomics and wood decay experiments. Cylindrobasidium torrendii is closely related to the white-rot root pathogen Armillaria mellea, while F. hepatica is related to Schizophyllum commune, which has been reported to cause white rot. Our results suggest that C. torrendii and S. commune are intermediate between white-rot and brown-rot fungi, but at the same time they show characteristics of decay that resembles soft rot. Both species cause weak wood decay and degrade all wood components but leave the middle lamella intact. Their gene content related to lignin degradation is reduced, similar to brown-rot fungi, but both have maintained a rich array of genes related to carbohydrate degradation, similar to white-rot fungi. These characteristics appear to have evolved from white-rot ancestors with stronger ligninolytic ability. Fistulina hepatica shows characteristics of brown rot both in terms of wood decay genes found in its genome and the decay that it causes. However, genes related to cellulose degradation are still present, which is a plesiomorphic characteristic shared with its white-rot ancestors. Four wood degradation-related genes, homologs of which are frequently lost in brown-rot fungi, show signs of pseudogenization in the genome of F. hepatica. These results suggest that transition towards a brown rot lifestyle could be an ongoing process in F. hepatica. Our results reinforce the idea that wood decay mechanisms are more diverse than initially thought and that the dichotomous separation of wood decay mechanisms in Agaricomycotina into white rot and brown rot should be revisited.

SummaryAntifungal defensins, MsDef1 and MtDef4, from Medicago spp., inhibit the growth of a fungal pathogen, Fusarium graminearum, at micromolar concentrations. However, molecular mechanisms by which they inhibit the growth of this fungus are not known. We have characterized a functional role of the fungal sphingolipid glucosylceramide in regulating sensitivity of the fungus to MsDef1 and MtDef4. A null mutation of the FgGCS1 gene encoding glucosylceramide synthase results in a mutant lacking glucosylceramide. The DFggcs1-null mutant becomes resistant to MsDef1, but not to MtDef4. It shows a significant change in the conidial morphology and displays dramatic polar growth defect, and its mycelia are resistant to cell wall degrading enzymes. Contrary to its essential role in the pathogenicity of a human fungal pathogen, Cryptococcus neoformans, GCS1 is not required for the pathogenicity of F. graminearum. The DFggcs1 mutant successfully colonizes wheat heads and corn silk, but its ability to spread in these tissues is significantly reduced as compared with the wild-type PH-1 strain. In contrast, it retains full virulence on tomato fruits and Arabidopsis thaliana floral and foliar tissues. Based on our findings, we conclude that glucosylceramide is essential for MsDef1-mediated growth inhibition of F. graminearum, but its role in fungal pathogenesis is host-dependent.

Versicolorin B synthase catalyzes the side chain cyclization of racemic versiconal hemiacetal (7) to the bisfuran ring system of (؊)-versicolorin B (8), an essential transformation in the aflatoxin biosynthetic pathway of Aspergillus parasiticus. The dihydrobisfuran is key to the mutagenic nature of aflatoxin B 1 (1). The protein, which shows 58% similarity and 38% identity with glucose oxidase from Aspergillus niger, possesses an aminoterminal sequence homologous to the ADP-binding region of other flavoenzymes. However, this enzyme does not require flavin or nicotinamide cofactors for its cyclase activity. The 643-amino acid native enzyme contains three potential sites for N-linked glycosylation, Asn-Xaa-Thr or Asn-Xaa-Ser. The cDNA and genomic clones of versicolorin B synthase were isolated by screening the respective libraries with random-primed DNA probes generated from an exact copy of an internal vbs sequence. This probe was created through polymerase chain reaction by using nondegenerate polymerase chain reaction primers derived from the amino acid sequences of peptide fragments of the enzyme. The 1985-base genomic vbs DNA sequence is interrupted by one intron of 53 nucleotides. Southern blotting, nucleotide sequencing, and detailed restriction mapping of the vbscontaining genomic clones revealed the presence of omtA, a methyltransferase active in the biosynthesis, 3.3 kilobases upstream of vbs and oriented in the opposite direction from vbs. The presence of omtA in close proximity to vbs supports the theory that the genes encoding the aflatoxin biosynthetic enzymes in A. parasiticus are clustered.

SummaryThe fungal elicitor-induced ELI12 gene from parsley has been previously shown to encode a divergent form of the D 12-oleic acid desaturase. In this report, we show that the ELI12 gene product is a fatty acid acetylenase or a triple-bond-forming enzyme. Expression of this enzyme in transgenic soybean seeds was accompanied by the accumulation of the D 12-acetylenic fatty acids, crepenynic and dehydrocrepenynic acids. Using PCR with degenerate oligonucleotides, we also show that homologs of the ELI12 gene are present in other members of the Apiaceae family. In addition, cDNAs for divergent forms of the D 12-oleic acid desaturase were detected among the expressed sequence tags (ESTs) from English ivy, an Araliaceae species, and sun¯ower, an Asteraceae species. As with the ELI12 gene, expression of these cDNAs in transgenic soybean embryos was accompanied by the accumulation of crepenynic and dehydrocrepenynic acids. Homologs of the sun¯ower acetylenase gene were also detected in other Asteraceae species, as revealed by PCR analysis of isolated genomic DNA. Results from Northern blot and EST analyses indicated that the expression of the sun¯ower gene, like ELI12, was induced by fungal elicitation. Overall, these results demonstrate that expressed genes for D 12-fatty acid acetylenases occur in at least three plant families, and are responsive to fungal pathogenesis. Natural products derived from crepenynic and dehydrocrepenynic acids that display antifungal, insecticidal, and nematicidal properties are distributed through at least 15 plant families. The acetylenases described here provide probes for chemotaxonomists, and facilitate functional genomic and molecular investigations of these defensive mechanisms.

Triterpenes are structurally complex plant natural products with numerous medicinal applications. They are synthesized through an origami-like process that involves cyclization of the linear 30 carbon precursor 2,3-oxidosqualene into different triterpene scaffolds. Here, through a forward genetic screen in planta, we identify a conserved amino acid residue that determines product specificity in triterpene synthases from diverse plant species. Mutation of this residue results in a major change in triterpene cyclization, with production of tetracyclic rather than pentacyclic products. The mutated enzymes also use the more highly oxygenated substrate dioxidosqualene in preference to 2,3-oxidosqualene when expressed in yeast. Our discoveries provide new insights into triterpene cyclization, revealing hidden functional diversity within triterpene synthases. They further open up opportunities to engineer novel oxygenated triterpene scaffolds by manipulating the precursor supply.terpenes | natural products | plant defense | cyclization | mutants

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