Harvey C. Hoch

The fungal spore and disease initiation in plants and animals.

I. Spore Attachment and Invasion.- 1. Adhesion of Fungi to the Plant Surface: Prerequisite for Pathogenesis.- 2. Signaling for Infection Structure Formation in Fungi.- 3. The Plant Cell Wall as a Barrier to Fungal Invasion.- 4. Rust Basidiospore Germlings and Disease Initiation.- 5. Attachment of Mycopathogens to Cuticle: The Initial Event of Mycoses in Arthropod Hosts.- 6. The Fate of Fungal Spores in the Insect Gut.- 7. Candida Blastospore Adhesion, Association, and Invasion of the Gastrointestinal Tract of Vertebrates.- 8. Infectious Propagules of Dermatophytes.- II. Fungal Spore Products and Pathogenesis.- 9. Melanin Biosynthesis: Prerequisite for Successful Invasion of the Plant Host by Appressoria of Colletotrichum and Pyricularia.- 10. The Plant Cuticle: A Barrier to Be Overcome by Fungal Plant Pathogens.- 11. Appearance of Pathogen-Related Proteins in Plant Hosts: Relationships between Compatible and Incompatible Interactions.- 12. The Role of Cuticle-Degrading Enzymes in Fungal Pathogenesis in Insects.- 13. Potential for Penetration of Passive Barriers to Fungal Invasion in Humans.- 14. Dihydroxynaphthalene (DHN) Melanin and Its Relationship with Virulence in the Early Stages of Phaeohyphomycosis.- III. Host Response to Early Fungal Invasion.- 15. Invasion of Plants by Powdery Mildew Fungi, and Cellular Mechanisms of Resistance.- 16. Induced Systemic Resistance in Plants.- 17. The Plant Membrane and Its Response to Disease.- 18. The Fungal Spore: Reservoir of Allergens.- 19. Conidia of Coccidioides immitis: Their Significance in Disease Initiation.- 20. Cell-Mediated Host Response to Fungal Aggression.- 21. Suppression of Phagocytic Cell Responses by Conidia and Conidial Products of Aspergillus fumigatus.- IV. Molecular Aspects of Disease Initiation.- 22. Molecular Approaches to the Analysis of Pathogenicity Genes from Fungi Causing Plant Disease.- 23. Current Status of the Molecular Basis of Candida Pathogenicity.- Taxonomic Index.

Signaling for Growth Orientation and Cell Differentiation by Surface Topography in Uromyces

The dimensions of the topographical signals for growth orientation and infection structure formation, a cell differentiation event that includes nuclear division, were determined for the stomatal penetrating rust fungus Uromyces appendiculatus. The differentiation signal was found to be a simple ridge on the substrate surface that had a markedly optimum height of 0.5 micrometer. Such ridges were microfabricated on silicon wafers by using electron-beam lithography. A similar ridge, in the form of a stomatal lip, was found associated with the stomatal guard cells of the bean (Phaseolus vulgaris) leaf. Ridge elevations greater than 1.0 micrometer or less than 0.25 micrometer did not serve as effective signals. Germ tubes of the fungus were highly oriented by ridge spacings of 0.5 to 6.7 micrometers. The data indicate that the fungus is able to distinguish uniquely minute differences in leaf surface topography in order to infect the host plant.

Upstream Migration of Xylella fastidiosa via Pilus-Driven Twitching Motility

Xylella fastidiosa is a xylem-limited nonflagellated bacterium that causes economically important diseases of plants by developing biofilms that block xylem sap flow. How the bacterium is translocated downward in the host plants vascular system against the direction of the transpiration stream has long been a puzzling phenomenon. Using microfabricated chambers designed to mimic some of the features of xylem vessels, we discovered that X. fastidiosa migrates via type IV-pilus-mediated twitching motility at speeds up to 5 mum min(-1) against a rapidly flowing medium (20,000 mum min(-1)). Electron microscopy revealed that there are two length classes of pili, long type IV pili (1.0 to 5.8 mum) and short type I pili (0.4 to 1.0 mum). We further demonstrated that two knockout mutants (pilB and pilQ mutants) that are deficient in type IV pili do not twitch and are inhibited from colonizing upstream vascular regions in planta. In addition, mutants with insertions in pilB or pilQ (possessing type I pili only) express enhanced biofilm formation, whereas a mutant with an insertion in fimA (possessing only type IV pili) is biofilm deficient.

Freeze-Substitution of Fungi

To critical cytologists, well-preserved cells are of the utmost importance. It is difficult, if not impossible, to accurately interpret cell structure and relate function to cell components if the cells have not been adequately preserved. For mycologists or cell biologists interested in fungal ultrastructure, cell preservation achieved with chemical fixatives at room temperatures has served as the basis for nearly all structural interpretations to date. Some fungi, particularly members of the Phycomycetes, appear to be preserved well enough with conventional fixation protocols that serious fixation artifacts are not usually recognized. Most members of the Ascomycotina and Basidiomycotina (and Fungi Imperfecti), in contrast, are not well fixed. The cytoplasm of these fungi often appears so muddled that one cannot easily distinguish between mitochondria, Golgi, and endoplasmic reticulum (cf. Brushaber and Jenkins, 1971; Hammill, 1974; Hoch, 1977a; Collinge and Markham, 1982; O’Donnell and McLaughlin, 1984). Unfortunately, mycological cytologists have become accustomed to this quality of preservation and too often either do not recognize the gross cytoplasmic distortions or tend to ignore such artifactual problems and interpret cell structure as best they can.

Assessing Adhesion Forces of Type I and Type IV Pili of Xylella fastidiosa Bacteria by Use of a Microfluidic Flow Chamber

ABSTRACT Xylella fastidiosa, a bacterium responsible for Pierces disease in grapevines, possesses both type I and type IV pili at the same cell pole. Type IV pili facilitate twitching motility, and type I pili are involved in biofilm development. The adhesiveness of the bacteria and the roles of the two pili types in attachment to a glass substratum were evaluated using a microfluidic flow chamber in conjunction with pilus-defective mutants. The average adhesion force necessary to detach wild-type X. fastidiosa cells was 147 ± 11 pN. Mutant cells possessing only type I pili required a force of 204 ± 22 pN for removal, whereas cells possessing only type IV pili required 119 ± 8 pN to dislodge these cells. The experimental results demonstrate that microfluidic flow chambers are useful and convenient tools for assessing the drag forces necessary for detaching bacterial cells and that with specific pilus mutants, the role of the pilus type can be further assessed.

Use of fluorochromes in the study of fungal cytology and differentiation

This review provides a general overview of a range of fluorochromes available with a brief description of their properties, uses and limitations in the study of fungi and other cells. Attention is also drawn to ways of optimizing visualization of images in the fluorescent microscope as well as to new developments in this field

Inhibition of thigmostimulated cell differentiation with RGD-peptides inUromyces germlings

SummaryGermlings of the plant pathogenic fungusUromyces appendiculatus sense and respond to topographical signals of various substrata by undergoing a cell differentiation process that culminates in a structure termed an appressorium. In some cell systems, recognition and mediation of extracellular signals is via transmembrane glycoproteins known as integrins that often exhibit specific affinities to the tripeptide sequence Arg-Gly-Asp (RGD) found in several extracellular matrix components. Germlings grown on substrata inductive for appressorium formation in the presence of buffered synthetic peptides containing the amino acid sequence RGD, e.g., RGD, RGDS, GRGD, and GRGDGSPK (0.5–2.0 mM), were inhibited from developing appressoria. Two non-RGD peptides (GGGG and RGES) as well as two RGD peptides (GRGDS and RGDSPASSKP) did not inhibit appressorium formation. Germling growth was not significantly affected by any of the peptides. Furthermore, 0.5 μm diameter micropipettes that are normally inductive for appressorium formation when positioned between the germling apex and the substratum did not induce appressorium formation when coated with the RGD peptide. Silanized micropipettes left uncoated or coated with RGES were inductive for appressorium formation. Those observations lead to the hypothesis that an integrin-like protein may be involved in the process of signaling for initiation of appressorium formation inUromyces. An RGDSPC-affinity column was used to isolate proteins fromUromyces 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 β1-integrin; one to the carboxyl-terminus of a synthetic peptide of integrin from chicken, and the other from the amino terminus of integrin from human placenta.

Cell-substratum adhesive protein involved in surface contact responses of the bean rust fungus☆

Abstract Uredospore germlings of the bean rust fungus Uromyces appendiculatus display two contact-sensitive responses on leaves or certain synthetic surfaces: a specific orientation of germ tubes and an induction of appressoria at leaf stomates or scratched surfaces. Germ tube orientation, nuclear division (used as a marker for appressorium formation) and germling adhesion on scratched “Parafilm” was reduced by the proteolytic enzyme, pronasc E (ED 50 = 20–50 μg ml −1 ), but not by the heat denatured proteolytic enzyme. Forty micrograms of pronase E per millilitre significantly reduced nuclear division of germlings incubated on either a hydrophilic or a hydrophobic surface. Pronase E reduced adhesion regardless of whether the enzyme was applied during growth or after attachment had occurred, although higher concentrations were required after attachment had occurred. The data suggest that extracellular protein is required for germling adhesion to a substratum and that adhesion is required for germ tube orientation and thigmodifferentiation. We partially characterized the composition of the bean rust extracellular material and analyzed for proteins on SDS-polyacrylamide gels. Intact germlings have six predominant extracellular peptides detected by 125 I labelling. Spent culture fluid contains several different peptides; four of the five major peptides in the spent culture fluid are glycosylated.

Visualization of the extracellular matrix surrounding pycnidiospores, germlings, and appressoria of Phyllosticta ampelicida

The presence and extent of the extracellular matrix surrounding pycnidiospore germlings and appressoria of Phyllosticta ampelicida was assessed using cryo-scanning and light microscopy. Conidia of ...

Mutations in Type I and Type IV Pilus Biosynthetic Genes Affect Twitching Motility Rates in Xylella fastidiosa

Xylella fastidiosa possesses both type I and type IV pili at the same cell pole. By use of a microfluidic device, the speed of twitching movement by wild-type cells on a glass surface against the flow direction of media was measured as 0.86 (standard error [SE], 0.04) microm min(-1). A type I pilus mutant (fimA) moved six times faster (4.85 [SE, 0.27] microm min(-1)) and a pilY1 mutant moved three times slower (0.28 [SE, 0.03] microm min(-1)) than wild-type cells. Type I pili slow the rate of movement, while the putative type IV pilus protein PilY1 is likely important for attachment to surfaces.