Isolation and characterization of STE20 homologues from Ustilago maydis and Microbotryum violaceum

The mitogen-activated protein kinase (MAPK) pathways are conserved from fungi to humans and have been shown to play important roles in mating and filamentous growth for both yeast and dimorphic fungi, and in infectivity for pathogenic fungi. Upstream of the typical 3-kinase module, STE20 , encodes a protein kinase of the PAK family that regulates more than one of these cascades in yeasts. We hypothesized that a STE20 homologue would play a similar role in the dimorphic plant pathogens U. maydis and M. violaceum. Using degenerate PCR primers, portions of the genes for STE20 homologues were amplified from genomic DNA of both fungi and the resulting fragments were sequenced to confirm their identities. The full-length copy of the U. maydis gene was obtained from a genomic library and was found to contain a 2,238 bp coding region, yielding a predicted protein of 746 amino acids. Two regions of the predicted protein were particularly conserved compared to other STE20 proteins: one in the N-terminal portion, expected to be the binding region for a regulatory factor and the other in the C-terminal portion, corresponding to the kinase catalytic region in the S. cerevisiae protein. No intron was found in this gene. The sequence was also obtained for 1299 bp of the coding region for the STE20 homologue from M. violaceum, predicted to encode 433 amino acids, which also showed homology to the STE20 proteins in Genbank.;A knock-out construct was made for the U. maydis homologue. To decrease the likelihood of non-homologous recombination, it was used as a PCR product containing only the STE20 gene and a selectable marker that is inserted within the gene to disrupt its function.