Novel polyketide synthase from Nectria haematococca

We identified a polyketide synthase (PKS) gene, pksN, from a strain of Nectria haematococca by complementing a mutant unable to synthesize a red perithecial pigment. pksN encodes a 2,106-amino-acid polypeptide with conserved motifs characteristic of type I PKS enzymatic domains: beta-ketoacyl synthase, acyltransferase, duplicated acyl carrier proteins, and thioesterase. The pksN product groups with the Aspergillus nidulans WA-type PKSs involved in conidial pigmentation and melanin, bikaverin, and aflatoxin biosynthetic pathways. Inactivation of pksN did not cause any visible change in fungal growth, asexual sporulation, or ascospore formation, suggesting that it is involved in a specific developmental function. We propose that pksN encodes a novel PKS required for the perithecial red pigment biosynthesis.     

Novel Polyketide Synthase from Nectria haematococca

We identified a polyketide synthase (PKS) gene, pksN, from a strain of Nectria haematococca by complementing a mutant unable to synthesize a red perithecial pigment. pksN encodes a 2,106-amino-acid polypeptide with conserved motifs characteristic of type I PKS enzymatic domains: β-ketoacyl synthase, acyltransferase, duplicated acyl carrier proteins, and thioesterase. The pksN product groups with the Aspergillus nidulans WA-type PKSs involved in conidial pigmentation and melanin, bikaverin, and aflatoxin biosynthetic pathways. Inactivation of pksN did not cause any visible change in fungal growth, asexual sporulation, or ascospore formation, suggesting that it is involved in a specific developmental function. We propose that pksN encodes a novel PKS required for the perithecial red pigment biosynthesis.     

Sesquiterpene lactone glycosides from Crepis capillaris

Four new sesquiterpene lactone glucosides, in addition to known compounds, have been isolated from the roots of Crepis capillaris and their structures elucidated on the basis of spectral analysis and hydrolytic studies.     

Structure of chinensin: A new lignan lactone from Polygala chinensis

Chinensin, a 1-aryl-2,3-naphthalide lignan, was isolated from Polyqala chinensis. Chemical transformation and UV, IR, PMR and MS evidence established its structure as 6,7-methylenedioxv-]-(3′,4′-dimethoxyphenyl)-3-hydroxymethylnaphthalene-2-carboxylic acid lactone. The lignan has not be en encountered before in nature or prepared synthetically.     

A heptaketide naphthaldehyde produced by a polyketide synthase from Nectria haematococca

Bostrycoidin and fusarubin are biologically active fungal polyketides produced by Nectria haematococca. This azaanthraquinone and naphthoquinone are thought to be biosynthesized via formation of a C(14) heptaketide aldehyde as a common key intermediate. A BLAST search against the genome of N. haematococca revealed one candidate gene (NECHADRAFT_101778, NhPKS1), which encodes a multi-domain polyketide synthase (PKS) with a thiol reductase (TR) domain that would facilitate the reductive release of the intermediate to produce a free aldehyde. To investigate the possible involvement of NhPKS1 in the biosynthesis of bostrycoidin and fusarubin, NhPKS1 was heterologously expressed in Aspergillus oryzae, and shown to produce a heptaketide 3-acetonyl-1,6,8-trihydroxy-2-naphthaldehyde as a single product. Thus, NhPKS1 catalyzes a C-2/C-11 and C-4/C-9 aldol-type cyclization of a linear intermediate followed by a subsequent reductive product release to yield the naphthaldehyde. The results indicate NhPKS1 is the enzyme involved in the biosynthesis of bostrycoidin and fusarubin.     

Isodehydrocostus lactone and isozaluzanin C,two guaianolides from Saussurea lappa

Two new guaianolides have been isolated from Saussurea lappa as minor components and have been named isodehydrocostus lactone and isozaluzanin C. A structure has been assigned to isodehydrocostus lactone on the basis of spectral data and correlation with estafiatin. The ¹H NMR data and dehydrogenation studies show that the other highly crystalline guaianolide is isomeric with zaluzanin C. Earlier 3β-H-zaluzanin C has however been reported to occur as a colourless oil.     

Argophyllone-B,a sesquiterpene lactone from Helianthus argophyllus

The sesquiterpene lactone, 2-methyl-2-butenoic acid dodecahydro-4-(hydroxymethyl)-10a-methyl-8-methylene-3,7-dioxooxineno[5,6]cyclodeca[1,2-b]furan-9-yl ester [1aR^*-[1aS^*,4R^*,5aS^*,8aR^*,9R^*(E)]], argophyllone-B, was isolated from acetone extracts from the leaves of Helianthus argophyllus. Its structure has been determined by single crystal X-ray analysis. Complete ¹H NMR and ¹³C NMR assignments have been made.     

A new triterpene lactone from Gymnocladus dioica

2β,23-Dihydroxyacacic acid lactone was isolated from Gymnocladus dioica as an artifact from 2β, 3β 16β, 21β, 23-pentahydroxyolean-12-ene-28-oic acid.     

Argentilactone,a novel 5- hydroxyacid lactone from Aristolochia argentina

Argentilactone, a new constituent of the rhizomes of Aristolochia argentina, was isolated and characterized as the (?)-(5R)-δ-lactone of 5-hydroxydodeca-Z,Z-2,6-dienoic acid.     

Adhesion of Macroconidia to the Plant Surface and Virulence of Nectria haematococca

To study spore attachment of the cucurbit pathogen Nectria haematococca (anamorph, Fusarium solani f. sp. cucurbitae), mutants with adhesion-deficient macroconidia were isolated. The adhesion-deficient mutants were selected after treatment with N-methyl-N′ -nitro-N-nitrosoguanidine followed by repeated enrichment for macroconidia which did not attach to polystyrene. Two independently derived mutants produced macroconidia with an approximately 50% reduction in attachment to polystyrene and to zucchini fruits. When macroconidia were inoculated into wounded zucchini fruits, the adhesion-deficient mutants were as virulent as the wild-type strain. However, in disease assays in which macroconidia were deposited onto the surface of unwounded zucchini, the mutants were less virulent than the wild type. Thus, adhesion of N. haematococca macroconidia to its host surface appears to be a virulence factor.     

Three new sesquiterpene lactone dimers from Carpesium faberi

Three new 2,4-linked sesquiterpene lactone dimers (SLDs), faberidilactones F-H (1–3), have been isolated from Carpesium faberi. Unlike 1 and 2, 3 was a modified Diels-Alder adduct, characteristic by a 1′-OH and a Δ^5′(6′) double bond. Furthermore, the relative configuration of 1′-OH in 3 was assigned as β-configuration by comparison the experimental NOESY data with those of its two possible epimers. The exo/endo stereochemistry of 1–3 was determined by the spectrographic features of 2,4-linked SLDs that we discovered in our previous works. Compounds 1 and 2 showed potent cytotoxicity against human leukemia (CCRF-CEM) cells with IC₅₀ value of 5.62 and 3.74 μM, respectively.     

Autonomously replicating plasmids and chromosome rearrangement during transformation of Nectria haematococca.

A previously described, autonomously replicating plasmid was examined for its ability to replicate in the plant pathogenic fungus, Nectria haematococca (Nh). The plasmid, pFOLT4R4, replicates as a linear molecule, contains a subterminal inverted repeat, as well as the repeated hexanucleotide telomere consensus sequence, TTAGGG, at both ends, and increases frequency of fungal transformation approximately 100-fold compared to a similar integrative plasmid, pHRC. Transformation of Nh occurs by way of autonomous replication; the transformed, hygromycin B-resistant (HyR) phenotype is unstable without selection and in most cases pFOLT4R4 is maintained in the fungus, separate from chromosome-sized DNAs. Surprisingly, a non-autonomously replicating derivative of pFOLT4R4 (called pLD), lacking the subterminal inverted repeat and having the 5'-TTAGGG repeat in only one direction on the plasmid, transformed Nh at a rate as high as pFOLT4R4. Therefore, autonomous replication and high-frequency transformation are separable phenomena in Nh. In pLD transformants, plasmid sequences are integrated into chromosome-sized DNAs of Nh and these cultures generally have a stable HyR phenotype. Treatments involving ligation of Nh genomic DNA to pLD result in a lower frequency of transformation. In many cultures transformed with pLD plus genomic DNA, one wild-type chromosome-sized band is not visible, but another smaller chromosome-sized band is found. Mobility changes in some cases are consistent with deletions of over 1000 kb. Some HyS revertants of transformants appear to lack the entire chromosome into which integration had occurred. These results indicate that the Nh genome is extremely malleable and large portions may be non-essential for growth in culture.     

The Genome of Nectria haematococca: Contribution of Supernumerary Chromosomes to Gene Expansion

The ascomycetous fungus Nectria haematococca, (asexual name Fusarium solani), is a member of a group of >50 species known as the “Fusarium solani species complex”. Members of this complex have diverse biological properties including the ability to cause disease on >100 genera of plants and opportunistic infections in humans. The current research analyzed the most extensively studied member of this complex, N. haematococca mating population VI (MPVI). Several genes controlling the ability of individual isolates of this species to colonize specific habitats are located on supernumerary chromosomes. Optical mapping revealed that the sequenced isolate has 17 chromosomes ranging from 530 kb to 6.52 Mb and that the physical size of the genome, 54.43 Mb, and the number of predicted genes, 15,707, are among the largest reported for ascomycetes. Two classes of genes have contributed to gene expansion: specific genes that are not found in other fungi including its closest sequenced relative, Fusarium graminearum; and genes that commonly occur as single copies in other fungi but are present as multiple copies in N. haematococca MPVI. Some of these additional genes appear to have resulted from gene duplication events, while others may have been acquired through horizontal gene transfer. The supernumerary nature of three chromosomes, 14, 15, and 17, was confirmed by their absence in pulsed field gel electrophoresis experiments of some isolates and by demonstrating that these isolates lacked chromosome-specific sequences found on the ends of these chromosomes. These supernumerary chromosomes contain more repeat sequences, are enriched in unique and duplicated genes, and have a lower G+C content in comparison to the other chromosomes. Although the origin(s) of the extra genes and the supernumerary chromosomes is not known, the gene expansion and its large genome size are consistent with this species'' diverse range of habitats. Furthermore, the presence of unique genes on supernumerary chromosomes might account for individual isolates having different environmental niches.     

Tissue-specific localization of pea root infection by Nectria haematococca. Mechanisms and consequences

Root infection in susceptible host species is initiated predominantly in the zone of elongation, whereas the remainder of the root is resistant. Nectria haematococca infection of pea (Pisum sativum) was used as a model to explore possible mechanisms influencing the localization of root infection. The failure to infect the root tip was not due to a failure to induce spore germination at this site, suppression of pathogenicity genes in the fungus, or increased expression of plant defense genes. Instead, exudates from the root tip induce rapid spore germination by a pathway that is independent of nutrient-induced germination. Subsequently, a factor produced during fungal infection and death of border cells at the root apex appears to selectively suppress fungal growth and prevent sporulation. Host-specific mantle formation in response to border cells appears to represent a previously unrecognized form of host-parasite relationship common to diverse species. The dynamics of signal exchange leading to mantle development may play a key role in fostering plant health, by protecting root meristems from pathogenic invasion.     

Fungal macrolides: Structure determination and biosynthesis of achaetolide,a lactone from Achaetomium cristalliferum

The structure of a new ten-membered lactone, achaetolide, isolated from cultures of Achaetomium cristalliferum is deduced from its mass and NMR spectra and from the study ofsomederivatives. The ¹³C NMR spectra of achaetolide enriched with [1-¹³C], [2-¹³C] and [1, 2-¹³C] acetate established its formation from eight intact acetate units via a precursor octaketide chain.     

Genetic and Biochemical Characterization of Nectria haematococca Strains with Adhesive and Adhesion-Reduced Macroconidia

A previous study reported the isolation of two mutants (LE1 and LE2) of the plant pathogenic fungus Nectria haematococca (anamorph, Fusarium solani f. sp. cucurbitae) with macroconidia with reduced ability to adhere (Att^-) to zucchini fruits and polystyrene. The adhesion-reduced-phenotype in LE1 and LE2 macroconidia is temperature sensitive and dependent on the concentration of nutrients. Classical genetic analysis of progeny derived from LE1 identified a mutation in a genetic locus, named Att1. The 90-kDa glycoprotein and macroconidial tip mucilage which were previously associated with the development of adhesion competence in Att⁺ macroconidia are specifically associated with macroconidia; neither is produced on microconidia, which are relatively nonadherent. However, macroconidia of both Att⁺ and Att^- strains produce the 90-kDa glycoprotein and the macroconidial tip mucilage.