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.

Polymerase Chain Reaction Detection of Ustilago hordei in Leaves of Susceptible and Resistant Barley Varieties.

ABSTRACT Although Ustilago hordei infects barley seedlings, symptoms of the disease covered smut are not visible until heading. Natural or artificial inoculation usually results in inconsistent infection, even in highly susceptible lines. Thus, breeding for resistance to covered smut is time consuming and difficult. The ribosomal DNA internal transcribed spacer (ITS) regions of U. hordei were sequenced and a primer pair was developed for polymerase chain reaction (PCR). These primers amplified a 574-bp fragment from DNA of Ustilago spp., but did not amplify DNA from barley or other common barley pathogens. DNA extracted from as few as four U. hordei sporidia was detected by this method. U. hordei DNA was amplified from leaf tissue of inoculated susceptible and resistant plants at different stages of plant development in differential varieties. Growth of the fungus in different leaves of an individual plant was variable. Several highly resistant varieties were shown to contain U. hordei DNA in the first three to four leaves, but not in later leaves. Thus, although the fungus can infect many resistant plants, the plants remain symptomless. Detection of U. hordei prior to heading should assist efforts for breeding for resistance and provide clues concerning the mechanisms of resistance employed by different resistance genes.