HEALY, R.A., C.R. BRONSON, and H.T. HORNER.* Departments of Botany and Plant Pathology, Iowa State University, Ames, IA 50011-1020. - Visualization of the extracellular matrix of the pathogenic fungus Cochliobolus heterostrophus.
Many plant pathogenic fungi produce an extracellular matrix (ECM). The
ECM is known to play a role in pathogenesis through adhesion of the
conidiospore and/or germling to its host. The ECM may contribute to
pathogenesis in other ways once the fungus has entered the host
tissues. We suspect this is true for the maize pathogen Cochliobolus
heterostrophus because a mutant that lacks the full ECM is unable to
form normal lesions after it enters the leaf. Otherwise, it behaves
normally in culture and on the leaf surface. The ECM of the wild type
consists of two layers: an inner layer that binds to a wide variety of
stains; and an outer layer that appears in negative relief with India
ink. The mutant lacks the outer layer and this provides a good control
for testing the hypothesis that the outer layer plays a role in
pathogenesis within host tissues. Study of the outer ECM layer within
host tissues has been unsuccessful due to the difficulty of
visualizing it. Our research has focused on further characterizing the
entire ECM and rendering the outer layer visible using a variety of
staining techniques on germlings incubated on cellophane membranes,
and on hyphae within maize leaf lesions produced after conidiospore
inoculation. Both were studied in the fresh state, as well as after
conventional fixation and cryofixation followed by freeze
substitution. Visualization with light microscopy included nigrosin
and India ink, silver-enhanced, gold-labeled antibody and colloidal
gold staining, and differential interference contrast. Localization
techniques with the electron microscope included enbloc staining using
tannic acid and/or uranyl acetate, enbloc staining with ruthenium red
or alcian blue followed by ruthenium red and osmium tetroxide;
colloidal gold staining, Thiery technique, and antibody labeling. Our
results are presented pictorially.
Key words: Cochliobolus, ECM, LM, pathogenic fungus, ruthenium red, TEM