Alfredo D. Martínez-Espinoza

MAP kinase and cAMP signaling pathways modulate the pH-induced yeast-to-mycelium dimorphic transition in the corn smut fungus Ustilago maydis.

Acid pH induces the yeast-to-mycelium transition in haploid cells of Ustilago maydis. We tested two signal transduction pathways known to be involved in dimorphism for roles in acid-induced filamentation. In wild-type cells intracellular cAMP levels were reduced under acid growth. A mutant defective in the regulatory subunit of PKA, ubc1, failed to respond to acid induction on solid medium, but in liquid medium showed a mycelial phenotype at acid pH. Mutants in the pheromone-responsive MAP kinase pathway lost the capacity to grow as mycelium at acid pH, while a mutant in the pheromone response-transcriptional regulator, prf1, behaved as wild-type. Filamentation by both ubc1 and prf1 mutants was inhibited by addition of cAMP. A putative MAP kinase cascade adaptor protein gene, ubc2, complemented a previously identified myc mutant strain defective in pH-induced myceliation. These results indicate that pH-dependent dimorphism is regulated by two known signaling pathways but that an effector for cAMP signaling alternative to Ubc1 is present in U. maydis and that Prf1 is not the sole downstream target of MAP kinase signaling.

Monomorphic Nonpathogenic Mutants of Ustilago maydis

ABSTRACT We have developed conditions which promote the dimorphic transition of haploid cells of Ustilago maydis in vitro by controlling the pH of the media. At low pH (below 5.0) mycelial growth occurs, whereas at neutral pH yeastlike growth takes place. We screened for mutants unable to form mycelium at low pH and obtained 26 mutants. These mutants have been characterized by their cell and colony morphology in different media. Mutations in 18 strains were found to be recessive when these strains were crossed with the wild type. Other crosses indicated that they were affected in genes other than a and b. Crosses between mutants suggest that the mutations fall in at least two complementation groups. In addition, mutants were characterized by their pathogenicity to corn seedlings. Mutations which were recessive for pathogenicity were also recessive for morphogenesis in vitro.

Ubc2, an Ortholog of the Yeast Ste50p Adaptor, Possesses a Basidiomycete-Specific Carboxy Terminal Extension Essential for Pathogenicity Independent of Pheromone Response

Proteins involved in the mitogen-activated protein (MAP) kinase pathway controlling mating, morphogenesis, and pathogenicity have been identified previously in the fungus Ustilago maydis. One of these, the Ubc2 adaptor protein, possesses a basidiomycete-specific structure. In addition to containing sterile alpha motif (SAM) and ras association (RA) domains typical of Ste50-like adaptor proteins found in the fungal phylum Ascomycota, Ubc2 also contains two C-terminal SH3 domains. Yeast two-hybrid assays indicated that Ubc2 interacts with the MAP kinase-kinase kinase Ubc4 via the SAM domains at each of their respective N-termini. Site-directed mutagenesis of ubc2 and complementation analyses revealed that the SAM and RA domains of Ubc2 are essential for filamentous growth. These data support a role for the ascomycete-like N-terminus of Ubc2 in regulating pheromone-responsive mating and morphogenesis analogous to the role of Ste50p in Saccharomyces cerevisiae. In contrast, C-terminal deletion mutants were fully capable of filamentous growth and mating. However, surprisingly, these strains were nonpathogenic. Further, directed mutagenesis of the C-terminus revealed that both SH3 domains are required for pathogenicity. These results suggest that the Basidiomycota have retained the mating and morphogenetic functions of Ste50-type proteins in the N-terminal half of their Ubc2-type adaptors but, additionally, have integrated C-terminal SH3 domains that are critical for additional signal transduction mechanisms, including those that lead to pathogenesis.

Cloning and expression analysis of the ornithine decarboxylase gene (PbrODC) of the pathogenic fungus Paracoccidioides brasiliensis

We describe the isolation and sequencing of PbrODC, the gene encoding ornithine decarboxylase (ODC) in Paracoccidioides brasiliensis. The gene contains a single open reading frame made of 1413 bp with a single intron (72 bp), and encodes a 447 amino acid polypeptide with a predicted molecular weight of 50.0 kDa, an isoelectric point of 4.9 and a high similarity to other fungal ornithine decarboxylases. Functionality of the gene was demonstrated by transformation into a Saccharomyces cerevisiae odc null mutant. A phylogenetic tree generated with several fungal ODCs provided additional evidence to favour a taxonomic position for P. brasiliensis as an ascomycetous fungus, belonging to the order Onygenales. Expression of the PbrODC gene was determined by Northern analyses during growth of the mycelial and yeast forms, and through the temperature‐regulated dimorphic transition between these two extreme phases. Expression of PbrODC remained constant at all stages of the fungal growth, and did not correlate with a previously observed increase in the activity of ornithine decarboxylase at the onset of the budding process in both yeast growth and mycelium‐to‐yeast transition. Accordingly, post‐transcriptional regulation for the product of PbrODC is suggested. The PbrODC gene sequence is available at the GenBank database under Accession No. AF212867. Copyright

Integration of the gene for carboxin resistance does not impact the Ustilago maydis-maize interaction.

A previous report indicated that insertion of the carboxin resistance (cbxR) gene into the Ustilago maydis genome impaired the pathogenic ability of the fungus towards Zea mays, the corn host. Because we had anecdotal evidence from work in our laboratory that this was not necessarily the case we decided to determine how general was the observation of reduced pathogenicity associated with cbxR. To accomplish this we tested the pathogenicity of several strains that had been transformed with the cbxR gene and compared them with non-transformed strains or strains transformed with the gene conferring hygromycin resistance which is a commonly used selectable marker in this fungus. Our results indicate that carboxin resistance does not significantly alter pathogenicity and is therefore a suitable marker for use in genetic analysis of U. maydis.