Structural requirements for activity of the pheromones of Ustilago hordei.

Ustilago hordei, the cause of barley-covered smut, initiates mating with pheromones. Gene sequence analysis suggested that these pheromones, Uhmfa1 and Uhmfa2, would be farnesylated peptides. Although isolation of mating-type-specific activity was rarely possible, chromatographic separations of culture supernatants yielded fractions that stimulated or inhibited mating. Based on predicted amino acid sequences and mass spectra of stimulating fractions, a series of pheromone analogs were synthesized and their activities were determined. Underivatized Uhmfa1 (PGKSGSGLGYSTC) or Uhmfa2 (EGKGEPAPYC) peptides were inactive, while peptides that were farnesylated and/or methyl esterified specifically induced conjugation tubes by cells of the opposite mating type. Uhmfa1 truncated from the amino terminus beyond the lysine lost activity, while truncated Uhmfa2 remained partially active. In mating bioassays, a pheromone concentration-dependent default mating response was observed. In competition studies, shorter Uhmfa1 peptides lacking pheromone activity inhibited activity of full-length peptides most effectively when both had the same functional groups.

Molecular analysis of the pheromone and pheromone receptor genes of Ustilago hordei.

Cell-cell signaling is an integral part of the sexual and disease cycles of the smut fungi, which must mate to be pathogenic. This study reports the cloning and characterization of the pheromone genes Uhmfa1 and Uhmfa2 from MAT-1 and MAT-2 mating types of U. hordei, respectively, and the pheromone receptor gene Uhpra2 from MAT-2 cells. Similar to other fungal pheromone genes, Uhmfa1 and Uhmfa2 encode precursor peptides. Uhpra2 encodes a protein with sequence similarity to the 7-transmembrane class of G-protein coupled receptors. Deletion of Uhmfa1 and Uhpra1, and their subsequent replacement, confirmed the role of these genes in initiation of the sexual cycle. Uhmfa1 and Uhmfa2 were differentially expressed in various cell types and when opposite mating-type cells were grown together. The predicted mature pheromones of each mating type were synthesized, and each specifically induced conjugation tube formation in cells of the opposite mating type.

Production of a Mating Inhibitor by Ustilago hordei.

ABSTRACT Ustilago hordei, the cause of barley covered smut, was found to produce a factor that inhibited its own mating. The mating inhibition factor (MIF) specifically inhibited mating of U. hordei and other Ustilago spp., but not teliospore germination or sporidial growth. MIF did prevent teliospore germination of Tilletia caries and T. contraversa. MIF was found at low levels in culture supernatants of either mating type of U. hordei grown separately, but at higher levels when both mating types were grown together, in the supernatants of MAT-1 mating type cells transformed with the MAT-1 pheromone gene mfa1 and of MAT-2 cells transformed with either mfa1 or the MAT-1 pheromone receptor gene pra1. Diploid cells produced no detectable inhibitor, nor did MAT-1 cells with a disrupted mating type locus that deleted both mfa1 and pra1. MIF production was restored when mfa1, but not pra1, was added back to the MAT-1Delta cells. MIF activity was altered by protease treatment. Highly purified MIF from MAT-1 cells contained cysteine methyl ester, farnesyl cysteine, farnesyl cysteine methyl ester, and a dodecapeptide with a mass consistent with that of MAT-1 pheromone lacking the terminal cysteine. Since smut fungi must first mate to become pathogenic, mating inhibition has the potential to be an effective method of disease control for these pathogens.

2,6-Dichlorophenolindophenol is a competitive inhibitor for xanthine oxidase and is therefore not usable as an electron acceptor in the fluorometric assay.

Xanthine oxidase has been recognized as an important source of oxygen free radicals in ischemia-reperfusion injury. In order to study this enzyme in biological tissues, the conversion of pterin (2-amino-4-hydroxypteridine) to isoxanthopterin provides the basis for a very sensitive fluorometric assay. Xanthine oxidase is typically assayed in the presence of pterin only, while an electron acceptor which replaces NAD+ is used to determine the combined xanthine dehydrogenase plus xanthine oxidase activity. 2,6-Dichlorophenol-indophenol has been used as an electron acceptor in this assay. However, it was found in this study that it acts as an effective competitive inhibitor for xanthine oxidase. We concluded that methylene blue is the electron acceptor of choice in the fluorometric assays for xanthine oxidase.