The Cell Wall of Fusarium oxysporum

Sugar analysis of isolated cell walls from three formae speciales of Fusarium oxysporum showed that they contained not only glucose and (N-acetyl)-glucosamine, but also mannose, galactose, and uronic acids, presumably originating from cell wall glycoproteins. Cell wall glycoproteins accounted for 50–60% of the total mass of the wall. X-ray diffraction studies showed the presence of α-1,3-glucan in the alkali-soluble cell wall fraction and of β-1,3-glucan and chitin in the alkali-insoluble fraction. Electron microscopy and lectin binding studies indicated that glycoproteins form an external layer covering an inner layer composed of chitin and glucan.     

Three highly divergent subfamilies of the impala transposable element coexist in the genome of the fungus Fusarium oxysporum

The transposable element impala is a member of the widespread superfamily of Tc1-mariner transposons, identified in the genome of the plant pathogenic fungus Fusarium oxysporum. This element is present in a low copy number and is actively transposed in the F.␣oxysporum strain F24 that is pathogenic for melons. The structure of the impala family was investigated by cloning and sequencing all the genomic copies. The analysis revealed that this family is composed of full-length and truncated copies. Four copies contained a long open reading frame that could potentially encode a transposase of 340 amino acids. The presence of conserved functional domains (a nuclear localisation signal, a catalytic DDE domain and a DNA-binding domain) suggests that these four copies may be autonomous elements. Sequence comparisons and phylogenetic analysis of the impala copies defined three subfamilies, which differ by a high level of nucleotide polymorphism (around 20%). The coexistence of these divergent subfamilies in the same genome may indicate that the impala family is of ancient origin and/or that it arose by successive horizontal transmission events. Received: 2 December 1997 / Accepted: 28 April 1998     

Two xylanase genes of the vascular wilt pathogen Fusarium oxysporum are differentially expressed during infection of tomato plants

Two genes encoding putative family F xylanases from the tomato vascular wilt pathogen Fusarium oxysporum f.sp. lycopersici have been cloned and sequenced. The two genes, designated xyl2 and xyl3, encode proteins with calculated molecular masses of 33 and 39.3 kDa and isoelectric points of 8.9 and 6.7, respectively. The predicted amino acid sequences show significant homology to other family F xylanases. XYL3 contains a cellulose-binding domain in its N-terminal region. Southern analysis suggested that xyl2 and xyl3 homologs are also present in other formae speciales of F. oxysporum. Both genes were expressed during growth on oat spelt xylan and tomato vascular tissue in vitro. RT-PCR revealed that xyl3 is expressed in roots and in the lower stems of tomato plants infected by F. oxysporum f.sp. lycopersici throughout the whole disease cycle, whereas xyl2 is only expressed during the final stages of disease. Received: 1 June 1998 / Accepted: 25 December 1998     

skippy, a retrotransposon from the fungal plant pathogen Fusarium oxysporum

A retrotransposon from the fungal plant pathogen Fusarium oxysporum f. sp. lycopersici has been isolated and characterized. The element, designated skippy (skp) is 7846 by in length, flanked by identical long terminal repeats (LTR) of 429 by showing structural features characteristic of retroviral and retrotransposon LTRs. Target-site duplications of 5 bp were found. Two long overlapping open reading frames (ORF) were identified. The first ORF, 2562 by in length, shows homology to retroviral gag genes. The second ORF, 3888 bp in length, has homology to the protease, reverse transciptase. RNase H and integrase domains of retroelement pol genes in that order. Sequence comparisons and the order of the predicted proteins from skippy indicate that the element is closely related to the gypsy family of LTR-retrotransposons. The element is present in similar copy numbers in the two races investigated, although RFLP analysis showed differences in banding patterns. The number of LTR sequences present in the genome is higher than the number of copies of complete elements, indicating excision by homologous recombination between LTR sequences.     

Potential Role of Transposable Elements in the Rapid Reorganization of the Fusarium oxysporum Genome

The activity of several families of transposable elements (TEs) in the genome of Fusarium oxysporum represents a potential source of karyotypic instability. We investigated transposon-mediated chromosome rearrangements by analyzing the karyotypes of a set of strains in which transposition events had occurred. We uncovered exceptional electrophoretic karyotype (EK) variability, in both number and size of chromosomal bands. We showed that EK differences result from chromosomal translocations, large deletions, and even more complex rearrangements. We also revealed many duplicated chromosomal regions. By following transposition of two elements and analyzing the distribution of different families of TEs on whole chromosomes, we find (i) no evidence of chromosomal breakages induced by transposition, (ii) a clustering of TEs in some regions, and (iii) a correlation between the high level of chromosomal polymorphism and the concentration of TEs. These results suggest that chromosome length polymorphisms likely result from ectopic recombination between TEs that can serve as substrates for these changes.     

Possible involvement of G-proteins and cAMP in the induction of progesterone hydroxylating enzyme system in the vascular wilt fungus Fusarium oxysporum

Fungi present the ability to hydroxylate steroids. In some filamentous fungi, progesterone induces an enzyme system which converts the compound into a less toxic hydroxylated product. We investigated the progesterone response in the vascular wilt pathogen Fusarium oxysporum, using mass spectrometry and high performance liquid chromatography (HPLC). Progesterone was mainly transformed into 15α-hydroxyprogesterone, which was found predominantly in the extracellular medium. The role of two conserved fungal signaling cascades in the induction of the progesterone-transforming enzyme system was studied, using knockout mutants lacking the mitogen-activated protein kinase Fmk1 or the heterotrimeric G-protein β subunit Fgb1 functioning upstream of the cyclic adenosine monophosphate (cAMP) pathway. No steroid hydroxylation was induced in the Δfgb1 strain, suggesting a role for the G-protein β subunit in progesterone signaling. Exogenous cAMP restored the induction of progesterone-transforming activity in the Δfgb1 strain, suggesting that steroid signaling in F. oxysporum is mediated by the cAMP-PKA pathway.     

Determination of the trichothecene mycotoxin chemotypes and associated geographical distribution and phylogenetic species of the Fusarium graminearum clade from China

A large number of isolates from the Fusarium graminearum clade representing all regions in China with a known history of Fusarium head blight (FHB) epidemics in wheat were assayed using PCR to ascertain their trichothecene mycotoxin chemotypes and associated phylogenetic species and geographical distribution. Of the 299 isolates assayed, 231 are from F. asiaticum species lineage 6, which produce deoxynivalenol and 3-acetyldeoxynivalenol (3-AcDON); deoxynivalenol and 15-acetyldeoxynivalenol (15-AcDON); and nivalenol and 4-acetylnivalenol (NIV) mycotoxins, with 3-AcDON being the predominant chemotype. Ninety-five percent of this species originated from the warmer regions where the annual average temperatures were above 15 °C, based on the climate data of 30 y during 1970–1999. However, 68 isolates within F. graminearum species lineage 7 consisted only of 15-AcDON producers, 59 % of which were from the cooler regions where the annual average temperatures were 15 °C or lower. Identification of a new subpopulation of 15-AcDON producers revealed a molecular distinction between F. graminearum and F. asiaticum that produce 15-AcDON. An 11-bp repeat is present in F. graminearum within their Tri7 gene sequences but is absent in F. asiaticum, which could be directly used for differentiating the two phylogenetic species of the F. graminearum clade.     

Purification and properties of two exo-cellobiohydrolases from a cellulolytic culture of Fusarium lini

Two distinct exo-cellobiohydrolases (1,4-β-d-glucan cellobiohydrolase, EC 3.2.1.91) have been isolated from culture filtrates of Fusarium lini by repeated ammonium sulphate fractionation and isoelectric focusing. The purified enzymes were evaluated for physical properties, kinetics and the mechanism of their action. The results of this work were as follows. (1) A two-step enzyme purification procedure was developed, involving isoelectric focusing and ammonium sulphate fractionation. (2) Yields of pure cellobiohydrolases I and II were 45 and 36 mg l^?1 of culture broth, respectively. (3) Both enzymes were found to be homogeneous, as determined by ultracentrifugation, isoelectric focusing, electrophoresis in polyacrylamide gels containing SDS and chromatography on Sephadex. (4) The molecular weights of the two cellobiohydrolases, as determined by gel filtration and SDS gel electrophoresis, were 50 000–57 000. (5) Both cellobiohydrolases had low viscosity-reducing and reducing sugar activity from carboxymethyl cellulose and high activity with Walseth cellulose and Avicel. (6) The enzymes produced only cellobiose as the end product from filter paper and Avicel, indicating that they are true cellobiohydrolases. (7) Cellobiohydrolase I hydrolysed d-xylan whereas cellobiohydrolase II was inactive towards d-xylan. (8) There was a striking synergism in filter paper activity when cellobiohydrolase was supplemented with endo-1,4-β-d-glucanase [cellulase, 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] and β-d-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21).     

卢氏香茶菜乙素的结构

从卢氏香茶菜叶的乙醚提取物中分得一个新的二萜化合物,卢氏香茶菜乙素(ludongnin B),其结构经各项光谱数据确定为(1)式。     

TRI12, a trichothecene efflux pump from Fusarium sporotrichioides : gene isolation and expression in yeast

Many of the genes involved in trichothecene toxin biosynthesis in Fusarium sporotrichioides are present within a gene cluster. Here we report the complete sequence for TRI12, a gene encoding a trichothecene efflux pump that is located within the trichothecene gene cluster of F. sporotrichioides. TRI12 encodes a putative polypeptide of 598 residues with sequence similarities to members of the major facilitator superfamily (MFS) and is predicted to contain 14 transmembrane-spanning segments. Disruption of TRI12 results in both reduced growth on complex media and reduced levels of trichothecene production. Growth of tri12 mutants on trichothecene-containing media is inhibited, suggesting that TRI12 may play a role in F. sporotrichioides self-protection against trichothecenes. Functional analysis of TRI12 was performed by expressing it in yeast strains that were co-transformed with a gene (TRI3) encoding a trichothecene 15-O-acetyltransferase. In the presence of the TRI3 substrate, 15-decalonectrin, cultures of yeast strains carrying TRI12 and TRI3 accumulated much higher levels of the acetylated product, calonectrin, than was observed for strains carrying TRI3 alone. PDR5, a transporter of the ABC superfamily, which is known to mediate trichothecene resistance in yeast, increased calonectrin accumulation in TRI12/TRI3 yeast strains but not in TRI3 strains. These results confirm the involvement of TRI12 in the trichothecene efflux associated with toxin biosynthesis, and demonstrate the usefulness of yeast as a host system for studies of MFS-type transporters. Received: 4 September 1998 / Accepted: 14 April 1999     

鲁山冬凌草戊素的结构

从鲁山冬凌草叶中分得一个新的二萜化合物,鲁山冬凌草戊素(lushanrubesensin E),其结构经各项光谱数据确定为对映-2α,11β-二羟基-3α,6β-二乙酰氧基-16-贝壳杉烯-15-酮。     

Enzymatic synthesis of trisaccharides and alkyl β-d-glucosides by the transglycosylation reaction of β-glucosidase from Fusarium oxysporum

Purified β-glucosidase from Fusarium oxysporum catalyses hydrolysis and transglycosylation reactions. By utilizing the transglycosylation reaction, trisaccharides and alkyl β-d-glucosides were synthesized under optimal conditions in the presence of various disaccharides and alcohols. The yields of trisaccharides and alkyl β-d-glucosides were 22–37% and 10–33% of the total sugar, respectively. The enzyme retained 70–80% of its original activity in the presence of 25% (w/v) methanol, ethanol and propanol. Thus, β-glucosidase from F. oxysporum appears to be an ideal enzyme for the synthesis of useful trisaccharides and alkyl β-d-glucosides.     

Structure of the phosphopeptidomannans from flocculent and non-flocculent yeast Kluyveromyces lactis

After extraction from whole cells, and purification by gel filtration, the chemical composition and molecular mass estimation of the cell-wall phosphopeptidomannan (PPM) showed no significant difference respectively between flocculent, weakly, very weakly and non-flocculent Kluyveromyces lactis yeast strains. However, when PPMs were tested as ligands of a lectin, extracted from the flocculent strain, the PPM isolated from the flocculent and weakly flocculent strain were recognized to a higher degree than those isolated from the non and very weakly flocculent strains. Acetolysis of PPM extracted from the four strains produced five oligosaccharide fractions corresponding to mono-, di-, tri-, penta-and hexa-saccharides. The flocculent strain was characterised by a high content of di-and penta-saccharides. The ¹H NMR analysis of the oligosaccharides demonstrated that the flocculent strain contained equivalent levels of the two mannobioses: Man( 1 → 2)Man and Man( 1 → 3)Man and of the two mannotrioses Man( 1 → 2)Man( 1 → 2)Man and Man( 1 → 3)Man( 1 → 2)Man. In contrast, the non-flocculent and the very weakly flocculent strains contained a single type of mannobiose Man( 1 → 2)Man and one type of mannotriose Man( 1 → 2)Man( 1 → 2)Man.     

Infraspecific chemical variability of the leaf essential oil of Juniperus phoenicea var. turbinata from Portugal

The essential oil composition of 68 individual plants of Juniperus phoenicea from Portugal was investigated by GC, GC-MS and ¹³C NMR. -Pinene, β-phellandrene, -terpinyl acetate and myrcene were found to be the main constituents. Botanical and chemical data as well as phytogeographical distribution indicate J. phoenicea var. turbinata as the unique subspecies occurring in Portugal. Nevertheless, this taxon exhibits chemical polymorphism. The results of the oil compositions were processed by hierarchical clustering and principal component analysis allowing to establish three groups of essential oils differentiated by the content of -pinene, β-phellandrene and -terpinyl acetate.     

Composition and infraspecific variability of essential oil from Thymus herba barona Lois

The composition of the essential oils of individual plants of Thymus herba barona Lois. growing wild in Corsica was investigated by GC, GC/MS an carbon-13 NMR. Eight groups of essential oil were distinguished: (i) thymol, (ii) carvacrol, (iii) linalool, (iv) geraniol, (v) -terpenyl acetate, (vi) terpinen-4-ol, (vii) carvone and cis-dihydrocarvone. Three chemotypes -thymol, carvacrol and linalool – are common in the genus Thymus, two others – geraniol, -terpenyl acetate – are scarce, while the latest three ones – terpinen-4-ol, carvone and cis-dihydrocarvone are quite original. It is the first time that the cis-dihydrocarvone chemotype is described in the genus Thymus     

Possible role of region 152-156 in the structural duality of a peptide fragment from sheep prion protein

The conformational conversion of the nonpathogenic "cellular" prion isoform into a pathogenic "scrapie" protease-resistant isoform is a fundamental event in the onset of transmissible spongiform encephalopathies (TSE). During this pathogenic conversion, helix H1 and its two flanking loops of the normal prion protein are thought to undergo a conformational transition into a beta-like structure. A peptide spanning helix H1 and beta-strand S2 (residues 142-166 in human numbering) was studied by circular dichroism and nuclear magnetic resonance spectroscopies. This peptide in aqueous solution, in contrast to many prion fragments studied earlier (1) is highly soluble and (2) does not aggregate until the millimolar concentration range, and (3) exhibits an intrinsic propensity to a beta-hairpin-like conformation at neutral pH. We found that this peptide can also fold into a helix H1 conformation when dissolved in a TFE/PB mixture. The structures of the peptide calculated by MD showed solvent-dependent internal stabilizing forces of the structures and evidenced a higher mobility of the residues following the end of helix H1. These data suggest that the molecular rearrangement of this peptide in region 152-156, particularly in position 155, could be associated with the pathogenic conversion of the prion protein.