The development of specialized infection structures including appressoria is a prerequisite for infection of plants by several pathogenic fungi. An appressorium is formed by the differentiation of a single-cell germ tube. Differentiation is induced by a thigomotropic (touch) stimulus. In the bean rust fungus, Uromyces appendiculatus, induction of differentiation requires contact with a specific topography of a physical surface such as a collodion membrane impregnated with oil or a 0.5 micrometer ridge on a plastic surface which resembles the surface of a host plant leaf. The over-all goal of this research is to understand how the touch stimulus initiates developmental events. Preliminary results have shown that the touch stimulus induces mitosis and expression of six differentiation-specific genes. Since it is difficult to characterize directly the "receptor" for the touch stimulus, the strategy has been to trace the steps of the signal transduction pathway backward, beginning from the documented effects of the stimulus, e.g. mitosis and specific gene-expression. As a first step, using polyclonal antibodies against the yeast cdc2 kinase, induction of a cdc2-like protein has been observed in differentiated germlings. On western blots, this protein is not detectable in undifferentiated germlings. The rust homologue of the cdc2 gene will be cloned and the temporal expression of mRNA for the protein will be studied. Since many cell cycle specific genes are regulated at the transcriptional level, the further goals of this project will be to identify trans-acting factors that bind to the promoter region of the gene. Further goals are to characterize the touch-specific gene, INF88, and localize the encoded protein in vivo. The sequence, CCGCGTTAA, has been identified about 300 bp from the transcription site(s) of INF24. This sequence is very similar to the sequence, ACGCGTTAA, present in several cell cycle specific genes including the thymidylate synthase (cdc21) gene in yeast. Starting from a restriction fragment containing this sequence, trans-acting factors binding to this region will be sought. Identification and characterization of these factors should reveal other components of the signal transduction pathway. Finally, to gain further insight into the function of the protein encoded by another differentiation-specific gene, INF24, the protein will be isolated from the differentiated germlings and its biochemcial and biophysical properties studied. The results of this research will further our understanding of how a touch stimulus initiates the developmental changes that allow this fungus to invade its plant host.
