INHIBITION OF THIGMOSTIMULATED CELL-DIFFERENTIATION WITH RGD-PEPTIDESIN UROMYCES GERMLINGS

Germlings of the plant pathogenic fungus Uromyces appendiculatus sense and respond to topographical signals of various substrata by undergoi ng a cell differentiation process that culminates in a structure terme d an appressorium. In some cell systems, recognition and mediation of extracellular signals is via transmembrane glycoproteins known as inte grins that often exhibit specific affinities to the tripeptide sequenc e Arg-Gly-Asp (RGD) found in several extracellular matrix components. Germlings grown on substrata inductive for appressorium formation in t he presence of buffered synthetic peptides containing the amino acid s equence RGD, e.g., RGD, RGDS, GRGD, and GRGDGSPK (0.5-2.0 mM), were in hibited from developing appressoria. Two non-RGD peptides (GGGG and RG ES) as well as two RGD peptides (GRGDS and RGDSPASSKP) did not inhibit appressorium formation. Germling growth was not significantly affecte d by any of the peptides. Furthermore, 0.5 mu m diameter micropipettes that are normally inductive for appressorium formation when positione d between the germling apex and the substratum did not induce appresso rium formation when coated with the RGD peptide. Silanized micropipett es left uncoated or coated with RGES were inductive for appressorium f ormation. Those observations lead to the hypothesis that an integrin-l ike protein may be involved in the process of signaling for initiation of appressorium formation in Uromyces. An RGDSPC-affinity column was used to isolate proteins from Uromyces germlings with affinity to the RGD sequence. Elution with RGD or EDTA, but not with RGES, yielded at least 12 proteins of which one protein (95 kDa) expressed affinity on immunoblots to two different antibodies of beta(1)-integrin; one to th e carboxyl-terminus of a synthetic peptide of integrin from chicken, a nd the other from the amino terminus of integrin from human placenta.