Lack of evidence for antisense suppression in the fungal plant pathogen Ustilago maydis.

Modulation of the expression of the Ustilago maydis Pyr3 gene, encoding dihydroorotase (DHOase), through antisense RNA regulation has been explored. This was done by placing the gene in sense and antisense orientations under the control of an hsp70-like gene promoter in a high-copy number autonomously replicating expression vector. Cells transformed with the antisense vector contained similar levels of DHOase activity to those found in cells harboring the expression vector alone. Transformants containing the antisense vector did not exhibit uridine-dependent growth, which would be expected for a Pyr3-deficient phenocopy. This was despite detection of high levels of antisense RNA transcripts in cells transformed with the Pyr3 antisense vector.

Isolation of an Ustilago maydis ERG11 gene and its expression in a mutant deficient in sterol 14 alpha-demethylase activity.

A gene (ERG11) encoding cytochrome P450 sterol 14 alpha-demethylase (P450(14DM)) was isolated from the maize pathogen, Ustilago maydis, by amplifying part of the coding region of the gene using PCR and by employing the amplified DNA fragment as a hybridization probe to recover the complete gene from an U. maydis lambda EMBL3 genomic library. The deduced amino acid sequence of the U. maydis gene showed homology to P450(14DM)s from other organisms and contained specific motifs which were hallmarks of P450s. Expression of the gene in an U. maydis mutant (A20) deficient in P450(14DM) led to only a partial restoration of P450(14DM) activity. Accumulation of ergosta-7,22-dienol and ergost-7-enol in A20 transformants containing the ERG11 gene implied that an additional mutation affecting sterol delta 5,6-desaturase activity accompanied the P450(14DM) lesion.

A gene encoding gamma-adaptin is required for apical extension growth in Ustilago maydis.

The nucleotide (nt) sequence of an Ustilago maydis (Um) gene that complemented a partially osmotic-remedial temperature-sensitive (or-ts) mutant defective in apical extension growth has been determined. It contained a continuous open reading frame (OFR) predicted to encode a protein of 853 amino acids (aa). The deduced aa sequence was homologous to gamma-adaptin, a component of clathrin-coated vesicles derived from the Golgi.

Isolation of an Ustilago maydis gene encoding 3-hydroxy-3-methylglutaryl-coenzyme A reductase and expression of a C-terminal-truncated form in Escherichia coli.

A gene encoding 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase was isolated from the maize fungal pathogen Ustilago maydis. This was accomplished by identifying cDNA and genomic clones that hybridized to an internal fragment of the gene, amplified from U. maydis genomic DNA by PCR. The nature of the gene was determined by nucleotide sequence analysis, and by comparing the derived amino acid sequence of the gene with HMG-CoA reductases from yeast, and from other organisms. The hydrophobic nature of the N-terminal region of the deduced protein sequence also supported the view that this gene encoded HMG-CoA reductase. A C-terminal-truncated fragment of the U. maydis HMG-CoA reductase gene was shown to be expressed in Escherichia coli in a catalytically active form. The expressed protein was also shown to be sensitive to an inhibitor of mammalian HMG-CoA reductase activity.

Isolation of the ERG2 gene, encoding sterol delta 8-->delta 7 isomerase, from the rice blast fungus Magnaporthe grisea and its expression in the maize smut pathogen Ustilago maydis.

The Magnaporthe grisea ERG2 gene, encoding delta 8-->delta 7 sterol isomerase, was isolated from a genomic library by heterologous hybridization to a fragment of the Ustilago maydis ERG2 gene. The isolated gene contained a reading frame of 745 bp which encoded a protein of 221 amino acids. The coding region was interrupted by a single putative 79-bp-long intron. The deduced amino-acid sequence exhibited similarity to the ERG2 gene products of U. maydis and of Saccharomyces cerevisiae, particularly in the central region of the proteins. The NH2-terminal of all three proteins contained a long stretch of amino acids that were strongly hydrophobic, suggesting that they may function by anchoring the protein to a membrane surface. The M. grisea ERG2 gene complemented a U. maydis deletion mutant in which the ERG2 gene had been removed using a one-step gene replacement procedure. The delta 8-->delta 7 sterol isomerase produced by the M. grisea ERG2 gene exhibited a level of sensitivity to the sterol biosynthesis inhibitor, tridemorph, similar to that of the enzyme derived from the U. maydis ERG2 gene.

Isolation, characterization and sequence of a gene conferring resistance to the systemic fungicide carboxin from the maize smut pathogen, Ustilago maydis.

A gene which confers resistance to the systemic fungicide carboxin (Cbx) has been isolated from the maize pathogen, Ustilago maydis, by transferring a plasmid gene library from a Cbx-resistant mutant strain into a sensitive strain and selecting for expression of the resistance gene. Five plasmids, rescued from transformants which exhibited enhanced resistance to Cbx, were shown to have DNA inserts with common restriction enzyme fragments. All the plasmids transformed a sensitive U. maydis strain to Cbx resistance. The gene (Cbxr), sub-cloned on a 3.2 kb EcoR1-HindIII fragment, transformed U. maydis to Cbx resistance at frequencies similar to those obtained with the bacterial Hygromycin B resistance (HygBr) gene. The sequence of the Cbxr gene showed a high degree of homology to succinate dehydrogenase (EC 1.3.99.1) iron-sulphur subunit genes from other organisms.

Purification and characterization of two endopolygalacturonases from Sclerotinia sclerotiorum.

The endopolygalacturonase (EC 3.2.1.15) enzyme produced in vitro by Sclerotinia sclerotiorum were found to consist of numerous isoforms covering a broad pI range. Two of the isoforms, labelled PG2 and PG3, were purified using gel-filtration chromatography, isoelectric focusing and anion-exchange chromatography. The pIs of PG2 and PG3 were, respectively, 4.8 and 4.9. Their molecular weights were similar. Both enzymes hydrolysed 0.9% of the bonds in reaching a 50% reduction in viscosity. However, their enzymic parameters were different. Their amino acid compositions differed only in the aspartic acid-asparagine content. The N-terminal sequences differed at the fourth amino acid only. PG2 and PG3 exhibited a high level of glycosylation compared to a similar enzyme isolated from Aspergillus niger. Antibody raised against PG3 was shown to crossreact with PG2, but not with the enzyme purified from Aspergillus niger.

Common amino acid domain among endopolygalacturonases of ascomycete fungi.

The endopolygalacturonase (EC 3.2.1.15) enzymes produced in vitro by three ascomycete fungi, Aspergillus niger, Sclerotinia sclerotiorum, and Colletotrichum lindemuthianum were studied by using thin-layer isoelectric focusing and activity stain overlay techniques. The polygalacturonases from A. niger and S. sclerotiorum consisted of numerous isoforms, whereas the endopolygalacturonase from C. lindemuthianum consisted of a single protein species. The most abundant endopolygalacturonase isoform produced by each of these organisms was purified and characterized. Biochemical parameters, including molecular weight, isoelectric point, kinetic parameters, temperature and pH optima, and thermal stability, were determined. Considerable differences in physical and chemical properties were demonstrated among these fungal polygalacturonases. Antibodies raised against individual proteins exhibited little cross-reaction, suggesting that these enzymes differ structurally as well as biochemically. In contrast, the analysis of the N-terminal amino acid sequences of the three proteins showed extensive homology, particularly in a region labeled domain 1 in which 84% of the amino acids were conserved.

The binding of isolated mesophyll cells from barley leaves to hyphae of Pyrenophora teres.

Live mesophyll cells from barley leaves were isolated and used to investigate contact phenomena between these cells and hyphae of the barley pathogen Pyrenophora teres. When incubated with colonies of P. teres, the isolated cells became attached to the hyphae. The binding of the cells may have been mediated by an affinity between cellulose in the mesophyll cell wall and extracellular material produced by the fungal hyphae. Different fungi exhibited a wide range of affinities for the mesophyll cells. It is suggested that isolated mesophyll cells may be a useful tool in the study of host-pathogen interfaces.