Part I

The Unique Features of Fungi

Chapter 1

The Hyphal Mode of Life

Fungi are composed of microscopic, tube-like structures called hyphae (singular hypha) which grow on substrates such as leaf litter, fallen timber and herbivore dung. The hypha has the shape of a cylindrical tube of even diameter with a tapering tip that branches subapically, with each branch having a tip of its own. By iteration of this modular unit comprising of a tip and a subapical branch (Figure 1.1), a radiating system of hypha called the mycelium is formed (Carlile, 1995). At places, hyphae become interconnected by short lateral outgrowths, bringing the entire mycelium into a protoplasmic continuity. The mycelium spreads over and penetrates into the substratum, secreting digestive enzymes which decompose the polymeric constituents of the substratum and absorbing the solubilized carbon and nitrogen and the phosphorus, potassium and sulfur compounds for its growth. This mode of fungal growth is inferred from observations of fungi growing in nature and by examination of cultures grown on nutrient medium solidified with agar (Figure 1.2).

1.1 FEATURES OF HYPHAE 1.1.1 Spread and Longevity

In the U.S. state of Michigan, a tree-root-infecting fungus, Armillaria bulbosa, which had colonized an extensive area of forests, was discovered (Smith et al., 1992). This fungus forms mushroom-like fruit bodies at the base of the tree trunk that are honey-colored, hence the fungus is commonly called the honey fungus. The question arose whether a wide area in the forest was infected by an individual fungus produced from a single spore but, since soil is an opaque medium, the area of spread of subterranean mycelium had to be estimated indirectly. In places, thousands of individual hyphae of this fungus aggregate and intertwine to form dark, macroscopic structures called rhizomorphs that look like shoelaces (http://helios.bto.ed.ac.uk/bto.microbes/armill.htm). The rhizomorphs have a large food base as they come out from the stump of a dead tree and are therefore able to extend through non-supportive terrain and infect roots of healthy trees (Figure 1.3). Formation of a rhizo-morph is a fungal strategy for bringing a large "starter" energy for overcoming the physical and chemical resistance of the plant host and colonizing it. Portions of rhizomorphs retrieved from the soil were placed on a nutrient agar medium to allow the mycelium to fan out. To determine if the rhizomorphs sampled from a large area of forest soil all belong to an individual fungus that developed from a single spore, an intercompatibility test was performed by pairing them in all combinations on nutrient medium in petri dishes—the rationale

Septal pore Septum Extension zone (<100 |m)

Septal pore Septum Extension zone (<100 |m)

Thetmaphillic Fungi

being that if the mycelial growth intermingled to form a continuous mat it would indicate that the isolates were genetically related, i.e., they belonged to an individual fungus. On the other hand, if the isolates showed a zone of aversion, i.e., an area between the paired mycelia not penetrated by hyphae (an incompatible reaction), it would indicate that they were genetically unrelated. Surprisingly, the results indicated that even though the subterranean mycelium of A. bulbosa may no longer have a continuous boundary due to the its fragmentation, the mycelial isolates belonged to an individual fungus (Figure 1.4).

1.1.2 Indeterminate Growth

The genetic relatedness of the mycelial isolates was confirmed by DNA fingerprinting method using the techniques of the restriction fragment length polymorphism (RFLP) and

Thermophilic Fungi
Figure 1.2 A two-day old Neurospora colony grown from a small amount of mycelium placed on the center of an agar medium in a petri dish.
Giant Mycelium Diagram
Figure 1.3 Hyphal strategy of attacking forest trees. From Ingold and Hudson, The Biology of Fungi (1993), Chapman and Hall, London. With permission of Kluwer Academic Publishers.

random amplification of polymorphic DNA (RAPD). Using RFLP, organisms can be differentiated from one another by analysis of patterns derived from cleavage of their DNA. If two organisms differ in the distance between sites of cleavage of a particular restriction endonuclease, the length of the resultant fragments will differ when the DNA is digested with a restriction enzyme and separated according to their size by electrophore-sis. If the pattern of DNA bands differs due to differences in their mobilities, the DNA is from different individuals. In the RAPD method, an arbitrarily designed ten-base-pair sequence is annealed to DNA isolated from the strains and a polymerase chain reaction is carried out. The product is separated on an agarose gel by electrophoresis and the pattern

Diagram Fungi

Figure 1.4 Diagram of stages in development of an individual colony of a fungus. (a) A germinating spore (black dot) that has produced a short hypha. (b) Branching to form a radially expanding mycelium. (c) An interconnected hyphal network formed by fusion of hyphae. (d) Disconnected mycelium of an individual fungal colony.

Figure 1.4 Diagram of stages in development of an individual colony of a fungus. (a) A germinating spore (black dot) that has produced a short hypha. (b) Branching to form a radially expanding mycelium. (c) An interconnected hyphal network formed by fusion of hyphae. (d) Disconnected mycelium of an individual fungal colony.

of DNA fragments compared. The finding was that DNA fingerprints of fungal isolates from an extensive area were identical. It was inferred that all isolates sampled from an area approximately 15 hectares belonged to the same individual fungus.

Having ascertained the individuality of this giant fungus colony, the question arose of determining its total mass. From the average weight of the rhizomorph and their numbers in a representative area of the forest soil, it was estimated that this colony of A. bulbosa contained at least 10,000 kg of biomass. Furthermore, from the growth rate of the fungus on wooden stakes buried in the soil as well as on nutrient medium, the age of the fungal colony was estimated to be around 1500 years and this large and aged living organism may still be growing. Although less publicized, a clone of A. ostoyae discovered in Oregon has a mycelium spread over 890 hectares and is 2400 years old (http://www.anbg.gov.au/fungi/mycelium.html). Since hyphae are capable of potentially unlimited growth, fungi are regarded as immortal organisms.

1.1.3 Apical Extension and Synchronized Growth

For a fungal hypha, life is at its tip.

Early in the last century, measurements over a period of the distance between the hyphal tip and the first septum (Figure 1.1) and distances between the successive segments showed that while the former increased the latter remained constant, thereby demonstrating that the growth of hypha is confined to the tip. When 14C-labeled N-acetylglucosamine, a radioactive precursor of chitin—a structural component of the fungal cell wall—was fed to a growing mycelium and its site of incorporation in hyphae was determined by autoradiographic imaging of the whole mycelium, the incorporation of the label was observed only in the terminal region of the hypha. This observation confirmed that a very small terminal region (less than 100 ^m) is the growing region of the hypha (Wösten et al., 1991). Cell wall and even organelles in the distal region of the hypha may be broken down by controlled autolysis and the solubilized nutrients translocated to the growing tip for addition into the cell membrane and cell wall at the tip and perpetuation of its growth. The strategy of fungal growth is to keep the tip extending by the active forward movement of the protoplasm. The hallmark of hypha is its growth in one direction only, i.e., its growth is polarized. However, the mechanism by which the protoplasm is drawn toward the tip, leaving an empty tube behind, remains a mystery.

Since the directionality of growth is established at the hyphal tip, this is the likely place where clues to polarized growth of hypha could be found. A variety of approaches are being used: electron microscopy; video-enhanced microscopy of movement of green fluorescent protein (GFP)-tagged organelles in living hypha; fluorescence imaging of distribution of ions; micro-electrode measurement of pH along hypha; patch-clamp detection of ion channels; immunofluorescence detection of enzyme distribution; and measurement of turgor by the plasmolysis method. However, there is no understanding as yet of how the growth of hypha is polarized but some of the pertinent findings are given below.

1.1.4 Spitzenkörper

By phase contrast microscopy and vital staining of living hyphae with a membrane-selective fluorescent dye, a cluster of small vesicles with no clear boundary was observed just beneath the plasma membrane of the hyphal tip (Figure 1.5). This apical body is called

Pollen Tube Growth Apical

Cell wall

Plasma membrane

Spitzenkörper

Actin

Vesicle

Microtubule

Vesicle

Mitochondria Endoplasmic reticulum

Microtubule

Golgi

Nucleus

Endoplasmic reticulum

Vacuole

Golgi

Figure 1.5 Diagram of a longitudinal section of hypha. Adapted from Ingold and Hudson (1993).

Cell wall

Plasma membrane

Spitzenkörper

Actin

Vesicle

Microtubule

Vesicle

Mitochondria Endoplasmic reticulum

Microtubule

Golgi

Nucleus

Endoplasmic reticulum

Vacuole

Golgi

Figure 1.5 Diagram of a longitudinal section of hypha. Adapted from Ingold and Hudson (1993).

Spitzenkörper in German, and advances continuously as the hypha elongates (Figure 1.6). Video microscopy and image analysis of living hyphae showed a close correlation of Spitzenkörper trajectory and the direction of growth of the hypha (Riquelme et al., 1998). Electron microscopy of hyphal tips showed exocytosis of Golgi-derived vesicles to growing tips. From its position and behavior, the Spitzenkörper is considered to be a collection center of vesicles containing enzymes and preformed polysaccharide precursors for cell

Figure 1.6 Video-imaging of Spitzenkörper trajectory in hypha. Tracings from photographs of Trichoderma viride taken during 9 min interval (Bartnicki-Garcia, 2002).

wall synthesis, allowing their localized delivery and polarized growth of the hypha (Bartnicki-Garcia, 2002). Once the Spitzenkörper has discharged its contents, a new Spitzenkörper is reformed. The cycle of collection and discharge of vesicles is consistent with the observation that growth of fungal hypha occurs in pulses (Lopez-Franco et al., 1994). The association of the Spitzenkörper with a meshwork of microtubules and microfilaments suggests that its polarized trajectory is determined by the growing scaffolding of microtubules. Supporting this is the observation that benomyl, an inhibitor of microtubule assembly, markedly disturbed the directionality of hyphal growth of wild-type Neurospora crassa whereas a benomyl-resistant mutant was not affected.

The evidence for collection and discharge of vesicles containing membrane and cell wall precursors by the Spitzenkörper is circumstantial. However, their polarized delivery and insertion into the plasma membrane at the tip could explain the generation of hyphal shape and polarity of the hypha. The vesicle membrane and plasma membrane at the tip may have specific proteins which tether the communicating membranes very close for docking and driving their fusion as postulated in animal cells (Rothman, 1994). At the core of pairing between the fusions of vesicle with its target membranes lies an interaction between homologous vesicle and target membrane proteins called v- and t-SNARES. Using specific-antibodies, a tip-high gradient of t-SNARES in Neurospora hypha has been demonstrated (Gupta and Heath, 2002). The location of Spitzenkörper at the tip and of tip-high gradient of SNAREs may together bolster rapid apical growth of the tip.

1.1.5 Tip-High Calcium

As in the other tip-extending cells—for example, the plant root hair, pollen tube, rhizoid cell of the alga Fucus—the fungal hypha also contains a tip-high gradient of calcium ions (Levina et al., 1995). In hyphae of Saprolegnia ferax (an aquatic mold) and Neurospora crassa (a terrestrial mold), the cytosolic calcium was measured by ratio imaging emission intensities of the Ca2+-sensitive fluorescent dyes fluo-3 and fura red by confocal microscopy (Hyde and Heath, 1997; Levina et al., 1995; Silverman-Gavrila and Lew, 2000, 2001). Fluorescence emission was localized in the 10-^m region surrounding the tip of growing hyphae but not in the non-growing hyphae. Hyphal elongation was inhibited by microinjection of Ca2+

chelators, suggesting that the tip-high gradient of free calcium is required for tip growth due to an unknown mechanism.

1.1.6 Large Surface Area

Hyphae are generally 5 to 10 ^m in diameter. This implies that a hypha has a large surface area in relation to the total mass of protoplasm. The large surface area of hypha maximizes its contact with environment for uptake of raw materials for biosynthesis, for gas exchange and for the release of by-products of metabolism. A large surface area is advantageous in other ways, too. For example, sugars and metabolizing enzymes are stored in the space between the plasma membrane and the cell wall (intramural space), which the hypha can apparently use as the energy and carbon source during exploration of the surrounding area for nutrients. This strategy is manifest in the high respiratory rate of mycelium in the absence of exogenously supplied respiratory substrate (endogenous respiration)—a feature that undoubtedly contributes to tolerance to adverse conditions for extended periods.

The large surface area of the hypha is not without its disadvantages. Because of the single-cell thickness of the hypha, the environment has a direct effect on it, rendering the thin-walled hypha vulnerable to desiccation. Fungi, therefore, must grow either in aqueous media or in a very humid atmosphere. In adverse conditions, the tip perceives a signal and apparently produces a conidiation-inducing factor that diffuses behind it, inducing formation of double septa along the length of hypha. The cells disarticulate and function as propagules called arthroconidia (Figure 1.7). Arthroconidia are the simplest type of spores formed by hypha.

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Responses

  • topi
    How to observe fungal morphology in the petridish using light microscope.?
    6 years ago
  • olga
    How does pollen tube tip growth in plants?
    6 years ago

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