Synthesis and processing of killer toxin from Ustilago maydis virus P4

The synthesis of toxin protein from Ustilago maydis virus (UmV) strain P4 was studied in vitro and in vivo. The protein synthesized in vitro and in vivo has a molecular weight of approximately 30 kd whereas the native toxin has a molecular weight of about 12 kd. In the presence of protease inhibitors and glycosylation inhibitors, toxin protein synthesized in vivo showed higher molecular weight products that could be immunoprecipitated with toxin antibodies. These results suggest that the UmV P4 toxin protein is synthesized as a preprotein, which upon processing results in the 12 kd secreted form toxin.     

Structure and heterologous expression of the Ustilago maydis viral toxin KP4

Killer toxins are polypeptides secreted by some fungal species that kill sensitive cells of the same or related species, in the best-characterized cases, they function by creating new pores in the ceil membrane and disrupting ion fluxes. Immunity or resistance to the toxins is conferred by the preprotoxins (or products thereof) or by nuclear resistance genes. In several cases, the toxins are encoded by one or more genomic segments of resident double-stranded RNA viruses. The known toxins are composed of one to three polypeptides, usually present as multimers. We have further characterized the KP4 killer toxin from the maize smut fungus Ustilago maydis. This toxin is also encoded by a single viral double-stranded RNA but differs from other known killer toxins in several respects: it has no N-linked glycosylation either in the precursor or in the mature polypeptide, it is the first killer toxin demonstrated to be a single polypeptide, and h Is not processed by any of the known secretory protelnases (other than the signal peptidase). It is efficiently expressed in a heterologous fungal system.     

Isolation and characterization of an autonomously replicating sequence from Ustilago maydis.

DNA fragments that function as autonomously replicating sequences (ARSs) have been isolated from Ustilago maydis. When inserted into an integrative transforming vector, the fragments increased the frequency of U. maydis transformation several-thousandfold. ARS-containing plasmids were transmitted in U. maydis as extrachromosomal elements through replication. They were maintained at a level of about 25 copies per cell but were mitotically unstable. One ARS characterized in detail, which we called UARS1, was localized to a 1.7-kilobase fragment. UARS1 contained a cluster of active sequences. This element could be reduced further into three separate subfragments, each of which retained ARS activity. The smallest one was 383 base pairs (bp) long. Although not active itself in yeast, this small fragment contained seven 8-bp direct repeats, two contiguous 30-bp direct repeats, and five 11-bp units in both orientations with sequences similar but not identical to the consensus sequence found to be crucial for ARS activity in Saccharomyces cerevisiae.     

Ustilago maydis KP6 killer toxin: structure, expression in Saccharomyces cerevisiae, and relationship to other cellular toxins.

There are a number of yeasts that secrete killer toxins, i.e., proteins lethal to sensitive cells of the same or related species. Ustilago maydis, a fungal pathogen of maize, also secretes killer toxins. The best characterized of the U. maydis killer toxins is the KP6 toxin, which consists of two small polypeptides that are not covalently linked. In this work, we show that both are encoded by one segment of the genome of a double-stranded RNA virus. They are synthesized as a preprotoxin that is processed in a manner very similar to that of the Saccharomyces cerevisiae k1 killer toxin, also encoded by a double-strand RNA virus. Active U. maydis KP6 toxin was secreted from S. cerevisiae transformants expressing the KP6 preprotoxin. The two secreted polypeptides were not glycosylated in U. maydis, but one was glycosylated in S. cerevisiae. Comparison of known and predicted cleavage sites among the five killer toxins of known sequence established a three-amino-acid specificity for a KEX2-like enzyme and predicted a new, undescribed processing enzyme in the secretory pathway in the fungi. The mature KP6 toxin polypeptides had hydrophobicity profiles similar to those of other known cellular toxins.     

Virus-encoded toxin of Ustilago maydis: two polypeptides are essential for activity.

Cells of Ustilago maydis containing double-stranded RNA viruses secrete a virus-encoded toxin to which other cells of the same species and related species are sensitive. Mutants affected in the expression of the KP6 toxin were characterized, and all were viral mutants. A temperature-sensitive nonkiller mutant indicated that the toxin consists of two polypeptides, 12.5K and 10K, that are essential for the toxic activity. The temperature-sensitive nonkiller mutant was affected in the expression of the 10K polypeptide, and its toxic activity was restored by the addition of the 10K polypeptide to its secreted inactive toxin. These results led to the reexamination of other mutants that were known to complement in vitro. Each was found to secrete one of the two polypeptides. Here we show for the first time that P6 toxin consists of two polypeptides that do not interact in solution, but both are essential for the toxic effect. Studies on the interaction between the two polypeptides indicated that there are no covalent or hydrogen bonds between the polypeptides. Toxin activity is not affected by the presence of 0.3 M NaCl in the toxin preparations and in the medium, suggesting that no electrostatic forces are involved in this interaction. Also, the two polypeptides do not share common antigenic determinants. The activity of the two polypeptides appears to be dependent on a sequential interaction with the target cell, and it is the 10K polypeptide that initiates the toxic effect. The similarity of the U. maydis virus-encoded toxin to that of Saccharomyces cerevisiae is discussed.     

DNA polymerase of Ustilago maydis: partial characterization of the enzyme and a pol 1 mutation.

The major DNA polymerase activity of wild-type U. maydis has been extensively purified. It possesses a molecular weight of about 150,000 daltons and appears to require a DNA primer with a 3'-hydroxyl terminus as well as a template. The polymerase activity has also been purified from the pol 1-1 strain, which is temperature sensitive fro growth and DNA synthesis, and which at the restrictive temperature contains only 10-25% levels of the DNA polymerase activity obtained from wild-type strains. It was similar in all properties studied, except that the activity was thermolabile at 40 degrees C compared to that from the wild-type strain. Physiological studies on the mutant showed that it was only slightly sensitive to UV, ionising radiation and nitrosoguanidine at the permissive temperature, and was proficient in genetic recombination. The results suggest that the pol 1-1 gene product does not play an important role in repair and recombination processes within the cell, and that its primary function lies in replication.     

The topoisomerase I gene from Ustilago maydis: sequence, disruption and mutant phenotype.

The Ustilago maydis genomic TOP1 gene encoding DNA topoisomerase I was cloned by amplifying a gene fragment using the polymerase chain reaction, and using this fragment to search a genomic DNA library by hybridization. The predicted peptide sequence exhibited 30-40% identity to other eukaryotic TOP1 genes, yet differed in several features. First, an unusually long acidic region was identified near the amino terminus (28/29 residues are acidic), which resembles other nucleolar peptide motifs. Second, an atypical carboxy-terminal 'tail', absent in other TOP1 genes, followed the active site tyrosine residue. A top1 gene disruption mutant was constructed by replacing the genomic TOP1 gene, with a top1::HygR null allele. This mutant lost the abundant topoisomerase I activity evident in wild-type U.maydis, and displayed a subtle coloration phenotype evident during cell senescence.     

Ferredoxin:NADP oxidoreductase of Cyanophora paradoxa: purification, partial characterization, and N-terminal amino acid sequence.

The ferredoxin:NADP+ oxidoreductase of the protist Cyanophora paradoxa, as a descendant of a former symbiotic consortium, an important model organism in view of the Endosymbiosis Theory, is the first enzyme purified from a formerly original endocytobiont (cyanelle) that is found to be encoded in the nucleus of the host. This cyanoplast enzyme was isolated by FPLC (19% yield) and characterized with respect to the uv-vis spectrum, pH optimum (pH 9), molecular mass of 34 kDa, and an N-terminal amino acid sequence (24 residues). The enzyme shows, as known from other organisms, molecular heterogeneity. The N-terminus of a further ferredoxin:NADP+ oxidoreductase polypeptide represents a shorter sequence missing the first four amino acids of the mature enzyme.     

Production of huitlacoche, Ustilago maydis: timing inoculation and controlling pollination

Huitlacoche is the name given to young, fleshy, edible galls that form when ears of Zea mays are infected by Ustilago maydis. Huitlacoche is processed and sold fresh at markets in Mexico. Interest has increased recently in producing U. maydis as a specialty mushroom in the United States. Silk-channel inoculation methods developed to evaluate common smut resistance in maize can be used to produce huitlacoche commercially. This research assessed the effects of time of inoculation and preventing pollination on the severity of ear galls and yield of huitlacoche produced by inoculating silks with U. maydis. Yield of huitlacoche and severity of ear galls were closely related, as was evident from highly significant linear or curvilinear regressions. Severity and yield were greatest when ears were inoculated 4-8 d after the mid-silk growth stage. Ear galls were 5-15% more severe and yield of huitlacoche was 18-150% greater on ears that were not pollinated, compared to those that were pollinated. Maximum yield of huitlacoche was 131 g ear(-1) from unpollinated male-sterile field corn inoculated 6 d after the mid-silk growth stage and 92 g ear(-1) from detasseled sweet corn inoculated 6 d after mid-silk. About 25% of the total weight of ears consisted of marketable huitlacoche when yields were highest. Quality of huitlacoche was not affected by time of inoculation or pollination treatments, but quality of huitlacoche harvested 12-14 d after inoculation was unacceptable primarily due to lack of teliospores, which affected color and flavor.     

Production, characterization, and partial amino acid sequence of xylanase A from Schizophyllum commune.

Xylanase A, one of several extracellular xylanases produced by Schizophyllum commune strain Delmar when grown in submerged culture with spruce sawdust as carbon source, was purified 43-fold in 25% yield with respect to total xylanase activity. Although some polysaccharide was strongly bound to the purified enzyme, the complex could be dissociated by sodium dodecyl sulfate and appeared homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the protein, calculated from the electrophoretic mobility, was 33,000. The molecular activity of the purified xylanase A, determined with soluble larch xylan as substrate, was 1.4 X 10(5) min-1, with xylobiose and xylose as the major products. The enzyme had a pH optimum of 5.0 and a temperature optimum of 55 degrees C in 10-min assays. The acid hydrolysate of xylanase A was rich in aspartic acid and aromatic amino acids. The sequence of 27 residues at the amino terminus showed no homology with known sequences of other proteins.     

Production, characterization, and partial amino acid sequence of xylanase A from Schizophyllum commune.

Xylanase A, one of several extracellular xylanases produced by Schizophyllum commune strain Delmar when grown in submerged culture with spruce sawdust as carbon source, was purified 43-fold in 25% yield with respect to total xylanase activity. Although some polysaccharide was strongly bound to the purified enzyme, the complex could be dissociated by sodium dodecyl sulfate and appeared homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the protein, calculated from the electrophoretic mobility, was 33,000. The molecular activity of the purified xylanase A, determined with soluble larch xylan as substrate, was 1.4 X 10(5) min-1, with xylobiose and xylose as the major products. The enzyme had a pH optimum of 5.0 and a temperature optimum of 55 degrees C in 10-min assays. The acid hydrolysate of xylanase A was rich in aspartic acid and aromatic amino acids. The sequence of 27 residues at the amino terminus showed no homology with known sequences of other proteins.     

The O-Mannosyltransferase PMT4 Is Essential for Normal Appressorium Formation and Penetration in Ustilago maydis

In Saccharomyces cerevisiae, the PMT, KRE2/MNT1, and MNN1 mannosyltransferase protein families catalyze the steps of the O-mannosylation pathway, sequentially adding mannoses to target proteins. We have identified members of all three families and analyzed their roles in pathogenesis of the maize smut fungus Ustilago maydis. Furthermore, we have shown that PMT4, one of the three PMT family members in U. maydis, is essential for tumor formation in Zea mays. Significantly, PMT4 seems to be required only for pathogenesis and is dispensable for other aspects of the U. maydis life cycle. We subsequently show that the deletion of pmt4 results in a strong reduction in the frequency of appressorium formation, with the few appressoria that do form lacking the capacity to penetrate the plant cuticle. Our findings suggest that the O-mannosylation pathway plays a key role in the posttranslational modification of proteins involved in the pathogenic development of U. maydis. The fact that PMT homologs are not found in plants may open new avenues for the development of fungal control strategies. Moreover, the discovery of a highly specific requirement for a single O-mannosyltransferase should aid in the identification of the proteins directly involved in fungal plant penetration, thus leading to a better understanding of plant–fungi interactions.     

The PAK family kinase Cla4 is required for budding and morphogenesis in Ustilago maydis

The phytopathogenic basidiomycete Ustilago maydis displays a dimorphic switch between budding growth of haploid cells and filamentous growth of the dikaryon. In a screen for mutants affected in morphogenesis and cytokinesis, we identified the serine/threonine protein kinase Cla4, a member of the family of p21-activated kinases (PAKs). Cells, in which cla4 has been deleted, are viable but they are unable to bud properly. Instead, cla4 mutant cells grow as branched septate hyphae and divide by contraction and fission at septal cross walls. Delocalized deposition of chitinous cell wall material along the cell surface is observed in cla4 mutant cells. Deletion of the Cdc42/Rac1 interaction domain (CRIB) results in a constitutive active Cla4 kinase, whose expression is lethal for the cell. cla4 mutant cells are unable to induce pathogenic development in plants and to display filamentous growth in a mating reaction, although they are still able to secrete pheromone and to undergo cell fusion with wild-type cells. We propose that Cla4 is involved in the regulation of cell polarity during budding and filamentation.     

Purification, characterization, and partial amino acid sequence of nerve growth factor from cobra venom.

The nerve growth factor (NGF) from Naja naja (cobra) venom has been purified and its structure compared to the NGF from mouse submaxillary gland. A two-step purification procedure has been devised, consisting of a gel filtration step in 1 M acetic acid followed by chromatography of the active pool on carboxymethylcellulose at pH 5. The molecular weight of the native protein was found to be 28000, and this value was reduced by approximately one-half under denaturing conditions. These values are comparable to those obtained for mouse 2.5S or betaNGF. Tryptic peptide maps of S-[14C]carboxymethyl NGF gave the number of labeled peptides expected for a structure composed of two identical or very similar subunits. Thus, the quaternary structures of mouse and cobra NGF are the same. Cyanogen bromide (CNBr) treatment of Naja naja NGF produced three fragments, of which two were purified to homogeneity. These fragments and the whole protein were analyzed in the automated protein Sequencer. The amino-terminal CNBr fragment of the protein was also subjected to digestion by thermolysin and the resultant peptides were purified and characterized. These data plus those from the characterization of the tryptic peptides provided the basis of the construction of a tentative primary structure of Naja naja NGF which is approximately 60% identical with mouse NGF.     

Rat muscle acylphosphatase: Purification, amino sequence, and immunological characterization

Acylphosphatase was purified from rat skeletal muscle essentially by gel filtration and high-performance ion-exchange chromatography. The complete amino acid sequence was reconstructed by using the sequence data obtained from tryptic, peptic, andS. aureus V8 protease peptides. The protein consists of 96 amino acid residues and is acetylated at the NH₂-terminus. The immunological cross-reactivity of acylphosphatase from rat and horse skeletal muscle was examined by ELISA. The reaction with rabbit antiserum revealed the presence of at least five antigenic sites on rat enzyme, two of which are common to horse muscle enzyme. Anti-rat antibodies also recognize the peptide that corresponds to the initial part of the molecule, which varies greatly from equine enzyme. Two completely new antigenic sites are herein described: the first can be considered the main antigenic site and is located within positions 21–36, the second is in the COOH-terminal part of the molecule. A mixture of immunoreactive peptides gives strong antibody-antigen reaction inhibition (94%).