Wall Structure of the Neurospora Hyphal Apex: Immunofluorescent Localization of Wall Surface Antigens

Abstract

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.

Biosynthesis of the Fungal Wall-Mechanisms and Implications The First Fleming Lecture

“…In the fungal wall it forms microfibrils, which can be visualized in the electron microscope by chemically or enzymically removing overlying wall components such as glucans and proteins (Hunsley & Burnett, 1970). In the growing hyphal apex these microfibrils are randomly oriented and extend right over the tip (Hunsley & Kay, 1976) and, presumably, are largely responsible for the structural integrity of the apex. In some cases, the microfibrils are predominantly oriented in a particular direction, more reminiscent of the orientations of cellulose microfibrils in the walls of higher plants and some algae.…”

“…The First Fleming Lecture 3 represent the process leading to the increases in the size of the chitin microfibrils and in the thickness of the chitin layer that are observed in electron micrographs (Hunsley & Burnett, 1968;Hunsley & Kay, 1976). Non-apical deposition of chitin also occurs during septum formation, during formation of side branches, where apices must arise de novo in the lateral hyphal walls, and during the rare intercalary elongation of cells in such specialized structures as the stipes of Agaric fruit bodies (Gooday, 1977).…”

Use of Recombinant Cellulose-Binding Domains of Trichoderma reesei Cellulase as a Selective Immunocytochemical Marker for Cellulose in Protozoa

“…Apparently mature cysts pose a diffusion barrier. Such a masking of cellulose by noncellulose materials has also been suggested in previous studies (17,35). We tried trypsinization prior to incubation with D-CBD and anti-CBD antibody without success.…”

“…We tried trypsinization prior to incubation with D-CBD and anti-CBD antibody without success. Drastic chemical treatment may be necessary, as shown for the chitinous layer of Neurospora crassa hyphae (17), but preliminary experiments using 1 M sodium hydroxide caused severe morphological damage and nonspecific background staining. We therefore prepared frozen sections, which yielded reproducible results (Fig.…”

Protoplast Transformation for Genome Manipulation in Fungi

“…Due to plasticity of fungal cell walls, the structure varies during different growth phases. Newly synthesized portions of the walls are thin and smooth, whereas older portions have the primary wall covered with secondary layers composed of amorphous matrix material (Hunsley 1973 ;Hunsley and Gooday 1974 ;Hunsley and Kay 1976 ;Trinci 1978 ;Trinci and Coolinge 1975 ). Consequently, young mycelium is more vulnerable to enzymatic degradation than aged hyphae.…”