Pycnial nectar of rust fungi induces cap formation on pycniospores of opposite mating type

Pycnial nectar (including pycniospores) transferred between pycnia of opposite mating type (as indicated by subsequent aecium formation) induced formation of a cap on one end of pycniospores. The polar caps developed with seven species of Puccinia and four of Uromyces, but not with P. helianthi, Tranzschelia pruni-spinosae, or U. vignae. Caps were induced equally in reciprocal transfers of nectar between pycnia of opposite mating type. The caps stained with India ink, or labeled with colloidal gold or wheat germ agglutinin conjugated with fluorescein isothiocyanate (WGA-FITC), but were not visible in unstained preparations. Cap formation started within 10 min of nectar transfer and was completed in 20-60 min. Nectar retained cap-inducing activity after pycniospores were removed by centrifugation, whereas pycniospores washed free of nectar did not induce caps. Pycniospores that were removed from a pycnium and induced to form caps by pycniospore-free nectar of opposite mating type did not induce aecia when returned to the original pycnium, showing that cap formation alone was not sufficient for completion of mating processes. Caps were removed by treatment with proteinase K, sodium dodecyl sulfate (SDS), or 0.01 N HCl, indicating the presence of protein. Labeling by WGA-FITC suggested the presence of polysaccharides or glycoproteins containing N-acetylglucosamine. The cap-inducing activity of nectar was lost if the nectar was boiled or autoclaved. Cap-inducing nectar contained a complex of high molecular weight proteins larger than 100 kDa as shown by native polyacrylamide gel electrophoresis analysis. This protein complex had cap-inducing activity as determined by placing pycniospores directly on gels after electrophoresis. In SDS gels, 6-7 polypeptides ranging in size from 14-70 kDa were observed, but bioassays of these polypeptides for cap induction were negative. The results indicate that pycnial nectar of several rust species contains high molecular weight cap-inducing proteins which are mating type-specific and induce pycniospore cap formation as an early event associated with processes leading to fertilization.