Isolation of a carbon source-regulated gene from Ustilago maydis

We have isolated a carbon source-regulated gene from the phytopathogenic fungus Ustilago maydis by use of a promoter-probe vector. This gene, called crg1, is strongly induced by L-arabinose and efficiently repressed by D-glucose and D-xylose. The predicted 36.5-kDa mature crg1 gene product lacks similarity to known proteins but is likely to be secreted. Sequences required for regulated expression of a reporter gene are contained within a 3.6-kb fragment upstream of the crg1 gene. The promoter of crg1 fulfils requirements for an efficient controllable gene expression system in U. maydis.     

A maize-specifically expressed gene cluster in Ustilago maydis

The corn pathogen Ustilago maydis requires its host plant maize for development and completion of its sexual cycle. We have identified the fungal mig2-1 gene as being specifically expressed during this biotrophic stage. Intriguingly, mig2-1 is part of a gene cluster comprising five highly homologous and similarly regulated genes designated mig2-1 to mig2-5. Deletion analysis of the mig2-1 promoter provides evidence for negative and positive regulation. The predicted polypeptides of all five genes lack significant homologies to known genes but have characteristic N-terminal secretion sequences. The secretion signals of mig2-1 and mig2-5 were shown to be functional, and secretion of a full length Mig2-1-eGFP fusion protein to the extracellular space was demonstrated. The central domains of the Mig2 proteins are highly variable whereas the C-termini are strongly conserved and share a characteristic pattern of eight cysteine residues. The mig2 gene cluster was conserved in a wide collection of U. maydis strains. Interestingly, some U. maydis isolates from South America had lost the mig2-4 gene as a result of a homologous recombination event. Furthermore, the related Ustilago scitaminea strain, which is pathogenic on sugar cane, appears to lack the mig2 cluster. We describe a model of how the mig2 cluster might have evolved and discuss its possible role in governing host interaction.     

Glycolysis in Ustilago maydis

The kinetic parameters of the 10 glycolytic enzymes and glycolytic fluxes were determined for the first time in Ustilago maydis. Enzyme activities in yeast grown in minimal medium and harvested in the stationary stage were twofold higher than those from yeast grown in rich medium. In contrast, in yeast harvested in the exponential stage, the enzyme activities were higher in cells grown in rich medium. Phosphofructokinase activity was the lowest in the four culture conditions analyzed, suggesting that this enzyme is a flux-controlling step in U. maydis glycolysis. The V(max) and K(m) values of hexokinase and pyruvate kinase were similar under all conditions. The results revealed that U. maydis aldolase belongs to the class II type of metalo-aldolases. 3-Phosphoglycerate mutase (PGAM) activity was 2,3-bisphosphoglycerate cofactor independent, which contrasted with the cofactor dependency predicted by the amino acid sequence alignment analysis. Pyruvate was secreted by U. maydis yeast in the presence and absence of external glucose. The glycolytic enzyme activities in the U. maydis mycelial form were similar to those found in yeast, except for one order of magnitude higher phosphofructokinase and PGAM activities, thus suggesting differences in the glycolysis regulatory mechanisms between the two cellular forms.     

Molecular cloning of a gene required for DNA replication in Ustilago maydis

TSD2, a gene necessary for DNA synthesis in Ustilago maydis, was cloned by complementation of the temperature sensitive growth defect of a mutant known previously as pol1-1 and renamed here tsd2-1. Linkage analysis established that the cloned fragment contained an allele of tsd2-1 and not a suppressor. DNA sequence determination of the cloned DNA fragment indicated the presence of a single large uninterrupted open reading frame capable of encoding a protein of 845 amino acids without homology to any known gene involved in DNA synthesis. TSD2 was found to be cell cycle-regulated and mRNA levels peaked in early S or G₁ phase.     

The a locus governs cytoduction in Ustilago maydis.

We have developed a cytoduction assay to measure cell fusion quantitatively in the basidiomycete corn smut fungus Ustilago maydis. This assay employs a mutation conferring resistance to oligomycin that exhibits non-Mendelian inheritance and presumably affects the mitochondrial genome. After auxotrophic olir cells are mixed with prototrophic olis cells, prototrophic olir cells can be detected at a significant frequency after several hours of incubation, reaching a maximum of 10% of the total prototrophs in the mixture after 18 h. We demonstrate that this cell fusion event occurs only if the mating partners have different alleles of the a mating-type locus and is not influenced by the b locus. These studies support the view that the a locus but not the b locus controls establishment of the filamentous, pathogenic state.     

Isolation and characterization of a Ustilago maydis glyceraldehyde-3-phosphate dehydrogenase-encoding gene

The complete nucleotide sequence of the glyceraldehyde-3-phosphate dehydrogenase gene from the corn smut fungus Ustilago maydis is reported. The gene encodes a 337-amino acid protein, parts of which show sequence identity to corresponding regions of GAPDH-encoding genes from other organisms. A single, putative 407-bp intron interrupts the tenth codon. Codon usage is highly biased for codons ending in cytosine.     

Pheromones trigger filamentous growth in Ustilago maydis.

Cell recognition and mating in the smut fungus Ustilago maydis have been proposed to involve specific pheromones and pheromone receptors. The respective structural genes are located in the a mating type locus that exists in the alleles a1 and a2. We demonstrate that binding of pheromone to the receptor can induce a morphological switch from yeast-like to filamentous growth in certain strains. Using this as biological assay we were able to purify both the a1 and a2 pheromone. The structure of the secreted pheromones was determined to be 13 amino acids for a1 and nine amino acids for a2. Both pheromones are post-translationally modified by farnesylation and carboxyl methyl esterification of the C-terminal cysteine. An unmodified a1 peptide exhibits dramatically reduced activity. The pheromone alone is able to induce characteristic conjugation tubes in cells of opposite mating type and confers mating competence; even cells of the same mating type undergo fusion. We discuss the role of pheromones in initiating filamentous growth and pathogenic development.     

Ustilago maydis, the delightful blight.

Recent studies of the corn smut fungus life cycle and its regulation by two mating type loci and other genes provide a cornucopia of challenges in cell biology, genetics and protein structure. The fungus can exist in two states: nonpathogenic and pathogenic. The change from one state to the other is accompanied by a change in morphology (yeast-like to filamentous) and growth properties (saprophytic to parasitic).     

Mutation avoidance and DNA repair proficiency in Ustilago maydis are differentially lost with progressive truncation of the REC1 gene product.

The REC1 gene of Ustilago maydis has an uninterrupted open reading frame, predicted from the genomic sequence to encode a protein of 522 amino acid residues. Nevertheless, an intron is present, and functional activity of the gene in mitotic cells requires an RNA processing event to remove the intron. This results in a change in reading frame and production of a protein of 463 amino acid residues. The 3'-->5' exonuclease activity of proteins derived from the REC1 genomic open reading frame, the intronless open reading frame, and several mutants was investigated. The mutants included a series of deletions constructed by removing restriction fragments at the 3' end of the cloned REC1 gene and a set of mutant alleles previously isolated in screens for radiation sensitivity. All of these proteins were overproduced in Escherichia coli as N-terminal polyhistidine-tagged fusions that were subsequently purified by immobilized metal affinity chromatography and assayed for 3'-->5' exonuclease activity. The results indicated that elimination of the C-terminal third of the protein did not result in a serious reduction in 3'-->5' exonuclease activity, but deletion into the midsection caused a severe loss of activity. The biological activity of the rec1-1 allele, which encodes a truncated polypeptide with full 3'-->5' exonuclease activity, and the rec1-5 allele, which encodes a more severely truncated polypeptide with no exonuclease activity, was investigated. The two mutants were equally sensitive to the lethal effect of UV light, but the spontaneous mutation rate was elevated 10-fold over the wild-type rate in the rec1-1 mutant and 100-fold in the rec1-5 mutant. The elevated spontaneous mutation rate correlated with the ablation of exonuclease activity, but the radiation sensitivity did not. These results indicate that the C-terminal portion of the Rec1 protein is not essential for exonuclease activity but is crucial in the role of REC1 in DNA damage repair.     

Plasmodium cynomolgi: the hsp 70 gene.

The hsp 70 gene of Plasmodium cynomolgi was isolated and characterized. As expected the gene is highly similar to that of the hsp 70 gene of Plasmodium falciparum (98% at the protein level, 82% at the nucleotide level). Surprisingly, the hsp 70 gene appears to be present in a single copy in all the P. cynomolgi strains tested, a finding that has implications for the parasite's ability to undergo a heat shock response.     

Cloning and disruption of Ustilago maydis genes.

We have demonstrated that genes from Ustilago maydis can be cloned by direct complementation of mutants through the use of genomic libraries made in a high-frequency transformation vector. We isolated a gene involved in amino acid biosynthesis as an illustrative example and showed that integrative and one-step disruption methods can be used to create null mutations in the chromosomal copy of the gene by homologous recombination. The results of this investigation make it clear that one-step gene disruption will be of general utility in investigations of U. maydis, since simple, precise replacement of the sequence under study was readily achieved.     

The Gbeta-subunit-encoding gene bpp1 controls cyclic-AMP signaling in Ustilago maydis

In the phytopathogenic fungus Ustilago maydis, fusion of haploid cells is a prerequisite for infection. This process is controlled by a pheromone-receptor system. The receptors belong to the seven-transmembrane class that are coupled to heterotrimeric G proteins. Of four Galpha subunits in U. maydis, only gpa3 has a function during mating and cyclic AMP (cAMP) signaling. Activation of the cAMP cascade induces pheromone gene expression; however, it does not lead to the induction of conjugation tubes seen after pheromone stimulation. To investigate the possibility that a Gbeta subunit participates in pheromone signaling, we isolated the single beta subunit gene, bpp1, from U. maydis. bpp1 deletion mutants grew filamentously and showed attenuated pheromone gene expression, phenotypes associated with deltagpa3 strains. In addition, a constitutively active allele of gpa3 suppressed the phenotype of the bpp1 deletion strains. We suggest that Bpp1 and Gpa3 are components of the same heterotrimeric G protein acting on adenylyl cyclase. Interestingly, while deltagpa3 strains are impaired in pathogenicity, deltabpp1 mutants are able to induce plant tumors. This could indicate that Gpa3 operates independently of Bpp1 during pathogenic development.     

The REC2 gene encodes the homologous pairing protein of Ustilago maydis.

Amino acid sequence analysis has established that the homologous pairing protein of Ustilago maydis, known previously in the literature as rec1, is encoded by REC2, a gene essential for recombinational repair and meiosis with regional homology to Escherichia coli RecA. The 70-kDa rec1 protein is most likely a proteolytic degradation product of REC2, which has a predicted mass of 84 kDa but which runs anomalously during sodium dodecyl sulfate-gel electrophoresis with an apparent mass of 110 kDa. To facilitate purification of the protein product, the REC2 gene was overexpressed from a vector that fused a hexahistidine leader sequence onto the amino terminus, enabling isolation of the REC2 protein on an immobilized metal affinity column. The purified protein exhibits ATP-dependent DNA renaturation and DNA-dependent ATPase activities, which were reactions characteristic of the protein as purified from cell extracts of U. maydis. Homologous pairing activity was established in an assay that measures recognition via non-Watson-Crick bonds between identical DNA strands. A size threshold of about 50 bp was found to govern pairing between linear duplex molecules and homologous single-stranded circles. Joint molecule formation with duplex DNA well under the size threshold was efficiently catalyzed when one strand of the duplex was composed of RNA. Linear duplex molecules with hairpin caps also formed joint molecules when as few as three RNA residues were present.