The appearance of the walls of apical and sub-apical regions of hyphae of predominantly 5 day cultures of Neurospora crassa, Schizophyllum commune and Phytophthora parasitica as seen with the electron microscope, employing shadowed or sectioned material, is illustrated and desCribed in detail. The appearance of untreated or control material in buffer is compared with that exposed to various single and sequential treatments with enzymes, including laminarinase, Pronase, cellulase and chitinase, as well as various chemical treatments.From these observations is inferred the co-axial distribution of polymers such as PI ,3-, PI ,6-glucans, glycoproteins, proteins, chitin and cellulose in the walls of each species. The distributions have both similarities and differences between the species. The significance of all these features for the growth, mechanical rigidity and integrity of a hypha is briefly discussed. I N T R O D U C T I O NThe growth of fungal hyphae is not well understood. One reason for this is that the molecular architecture of hyphal walls has not received sufficient attention. Chemical studies are beginning to reveal the complexity of hyphal wall components (e.g. review, Bartnicki-Garcia, I 968), while studies with enzymes have indicated that they are arranged in definite layers, e.g. in Neurospora crassa . Only a little information is available concerning wall structure as revealed by electron microscopy (e.g. review, Aronson, 1965) and this is either based mostly on residual shadowed material after drastic chemical treatments, or from thin sections which alone are not very informative.In this paper we describe the consequences of treating hyphae with enzymes, singly or successively, and then examining both shadow-cast and sectioned material with an electron microscope. The three species studied were selected because they differ in the chemistry of their cell walls, which was relatively well known from studies by other investigators. This new approach provides information on a comparative basis, concerning the distribution and arrangement of certain kinds of polymers within hyphal walls. M E T H O D SOrganisms. The organisms used were as follows : an ascornycete, Neurospora crassa, wild type, Emerson A from the Neurospora stock cultures; Schizophyllum commune, a basidiomycete, was isolated from damp plywood in Newcastle upon
SUMMARYAntisera have been raised in rabbits against three wall fractions from Neurospora crassa. Fractions were separated according to Mahadevan & Tatum ( 1 9 6 9 , i .e. fraction I, glucan-peptide-galactosamine complex; fraction 111, laminarin-like glucan ; and fraction IV, chitin. Distinct patterns of immunofluorescent staining were obtained using an indirect staining method. Hyphae stained with antiserum to fraction I showed maximum fluorescence in the apical and/or subapical regions : in both cases, fluorescence showed a sharp decrease with distance behind the subapical region. Hyphae stained with antiserum to fraction I11 showed faintly fluorescent tips with fluorescence increasing with distance from the tip. Hyphae stained with antiserum to fraction IV showed faint fluorescence, equivalent to leveis of autofluorescence, except at the sites of hyphal fractures. Antisera were also raised against whole walls from 24 and 120 h cultures. Hyphae stained with antisera against whole walls which had previously been absorbed to remove antibodies to fractions I, I11 and IV showed preferential staining of apices. The uncharacterized tip antigen(s) thus revealed was also demonstrated on immunodiffusion plates. This pattern of immunofluorescence was compared to the fluorescence of apices after staining with an optical brightener.Enzymic dissection procedures did not generally give reliable results with apices from 24 h cultures. Untreated apices appeared amorphous, while a drastic chemical treatment revealed randomly oriented microfibrils which were shown to be a-chitin. The apical hyphal walls were significantly thinner than those from more mature hyphal regions.
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