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Hyphal tip growth

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Title:
Hyphal tip growth molecular composition of elongating and non-elongating regions of Achlya cell wall
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Shapiro, Alexandra, 1971-
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English
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vi, 107 leaves : ill. ; 29 cm.

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Achlya ( jstor )
Cell growth ( jstor )
Cell walls ( jstor )
Chitin ( jstor )
Electron micrographs ( jstor )
Fungi ( jstor )
Hyphae ( jstor )
Mycology ( jstor )
Oomycetes ( jstor )
Turgor ( jstor )
Achlya -- Physiology ( lcsh )
Botany thesis, Ph.D ( lcsh )
Dissertations, Academic -- Botany -- UF ( lcsh )
Fungi -- Hyphae -- Growth ( lcsh )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph.D.)--University of Florida, 2000.
Bibliography:
Includes bibliographical references (leaves 94-106).
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Printout.
General Note:
Vita.
Statement of Responsibility:
by Alexandra Shapiro.

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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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Full Text
HYPHAL TIP GROWTH: MOLECULAR COMPOSITION OF ELONGATING AND
NON-ELONGATING REGIONS OF ACHLYA CELL WALL
By
ALEXANDRA SHAPIRO
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2000


TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS
iv
ABSTRACT,
CHAPTERS
> i
.v
1 INTRODUCTION 1
Apical Growth 1
The Organism 3
Class Oomycetes 5
2 LITERATURE REVIEW 9
Mechanism of Apical Growth 9
Structure of the Hyphal Wall 10
Electron Microscopic Studies of Fungal Cell Walls 14
Biosynthesis of the Fungal Cell Wall 21
Role of Turgor in Wall Expansion 25
Role of Cytoskeleton in Hyphal Growth 26
Cytology of Growing Hyphal Apices 2 8
Calcium Gradient 31
Ion Currents 32
3 HYPHAL GROWTH 3 3
Introduction 3 3
Materials and Methods 33
Results 35
Discussion 35
4 LOCALIZATION OF CELLULOSE IN THE CELL WALL
AS REVEALED BY ELECTRON MICROSCOPY AND
CYTOCHEMICAL TECHNIQUES 3 9
Introduction 3 9
Materials and Methods 40
Culture Methods and Microscopy Techniques 40
Electron Microscopy 40
Cellulose Localization Using Enzyme-Gold
Affinity Labeling 42
Cytochemical Controls 43
Cellulase Enzyme Activity during Labeling 44
Detection of Gold Particles with a
Backscatter Detector 45
Zymolyase Hydrolysis 45
Treatement of Growing Colonies with
Dichlorobenzoni trile 4 5
Results 46
Cellulose Localization 46
i i


Cytochemical Controls .. 47
Cellulase Activity during Labeling 48
Cellulose Localization on the Surface of
the Hyphae in Colonies Incubated
with Zymolyase 68
Hyphal Elongation, Spores Germination and
Cellulose Localization in the Presence
of DCB 68
Discussion 69
5 LOCALIZATION OF 1,3 -B-GLUCANS IN THE CELL
WALL AS REVEALED BY ELECTRON MICROSCOPY
AND CYTOCHEMICAL TECHNIQUES 7 5
Introduction 7 5
Materials and Methods 78
Culture Methods, Fixation and Microscopy
Techniques 7 8
Localization of 1,3-B-Glucans Using
Monoclonal Antibody 7 8
Cytochemical Controls 79
Results 80
Localization of 1,3-B-Glucans on Sections and
Hyphal Surfaces Using Monoclonal Antibodies 80
Cytochemical Controls 80
Discussion 86
6 LOCALIZATION OF CHITIN IN THE CELL WALL AS
REVEALED BY ELECTRON MICROSCOPY AND
CYTOCHEMICAL TECHNIQUES 87
Introduction 87
Materials and Methods 88
Chitin Localization Using Lectin 88
Cytochemical Controls 88
Results 89
Discussion 89
7 CONCLUSIONS 9 2
LIST OF REFERENCES 94
BIOGRAPHICAL SKETCH 107
iii


ACKNOWLEDGMENTS
I thank Drs. Tom Emmel, Greg Erdos and Alice Harmon for
serving as members of my supervisory committee, and for their
time and expertise.
I would like to express my appreciation to Dr. J.T.
Mullins, my supervisory chairman, for his support,
understanding, patience, interest in the research project and
his help and guidance throughout the work on my dissertation.
I also would like to thank Karen Vaughn of ICBR EM core
lab for her technical assistance. My special thanks go to
Scott Whittaker of the same lab for his help, time and
technical expertise.
The support of my family was very important for me during
these years. I am truly indebted to my parents and my
parents in-law for their love, constant encouragement and
inspiration, help with the kids and readiness to help anytime
I needed it.
Finally, my greatest gratitude goes to my husband, Andrei
Sourakov. His love, patience and help made the completion of
my dissertation possible.
IV


Abstract of Dissertation Presented for the Graduate
School of the University of Florida in Partial
Fulfillment of the Requirements for the Degree of Doctor
of Philosophy
HYPHAL TIP GROWTH: MOLECULAR COMPOSITION OF ELONGATING
AND NON-ELONGATING REGIONS OF ACHLYA CELL WALL
By
ALEXANDRA SHAPIRO
December 2000
Chair: J. Thomas Mullins
Major Department: Botany
Although apical growth is a widespread process in the
biological world and has been known for over a hundred
years, the mechanisms that underlie this process are not
yet understood. Knowledge of these mechanisms would allow
the development of techniques for inhibiting or
stimulating growth of medically or economically
important species. I approached the problem of hyphal tip
growth by comparing the cell wall composition of
elongating and non-elongating regions of the oomycete
Achlya bisexualis. Light microscope observations were
used to determine the growth rate and to distinguish
elongating and non-elongating hyphae for further EM
studies, because non-elongating hyphae often are found
among growing mycelia. I found that hyphal growth is a


discontinuous irregular process with periods of
elongation and no elongation. The elongation rate is not
steady, but instead fluctuates with periods of fast and
slow elongation. Both transmission and scanning electron
microscopes were used with a variety of cytochemical
labels, and several fixation techniques. Cellulose, the
microfibrillar component of the Achlya wall, was
identified with cellulase enzyme-gold affinity labeling.
Elongating hyphae have cellulose in mature and subapical
regions, but not at the apex. In non-elongating hyphae,
cellulose was found in all the regions including the
apex. These results suggest that the apices of elongating
hyphae lack cellulose. This contradicts the long-standing
hypothesis that the microfibrilar component is present in
the elongating hyphal apex. The 1,3-S-glucans, the major
matrix wall components, were immuno-localized in all
regions of elongating and non-elongating hyphae. A number
of cytochemical, biochemical and physiological controls
were performed to assure the reliability of these
findings. I suggest that in elongating regions, the
matrix is synthesized first and synthesis of
microfibrilar component follows. Another explanation for
these results is that localized apical cellulose
hydrolysis by endoglucanase creates plastic wall regions
consisting mainly of 1,3-E-glucans, which expand under
turgor and/or cytoskeleton pressure. Cellulose deposition
quickly follows to prevent "blowing out" of the hypha.
vi


CHAPTER 1
INTRODUCTION
Apical Growth
Hyphal tip growth is a hallmark of the fungi, even
though it also occurs in specialized plant cells (i.e.,
growth of pollen tube, root hair, moss protonema).
Diverse animal cells share this capacity to protrude
their cytoplasm and then move in that direction, a
process termed ameboid movement. The essential feature of
tip growth is that the tip of the hypha is protruded into
the enviroment from the subapical region. This protrusion
involves the synthesis and extension of the cell wall and
cytoplasm (with its contained organelles) The organism
is thus able to explore and exploit its environment.
Although apical growth is a widespread process in the
biological world and has been known for over a hundred
years, the mechanisms underlying this process are not yet
understood. Knowledge of these mechanisms would allow an
understanding of other related characteristics of fungi,
such as the influence of environmental factors on growth
and morphogenesis and the interaction between fungi and
other organisms. Ultimately, detailed knowledge of hyphal
tip growth would allow the development of techniques for


2
inhibiting or stimulating growth of medically or
economically important species.
Studying hyphal tip growth is a complex problem
because the apex represents only a tiny part of a hypha.
Most of the important growth events occur within 5
micrometers of the tip. On the other hand, the mature
part of the hypha is not inactive. In growing hyphae, the
wall synthesis per unit area is maximal at the tip. The
total amount of wall material synthesized subapically at
the same time is appreciable (Sietsma et al 1985). This
also contributes to the difficulty of studying tip
growth. Finally, not all of the hyphae in an actively
growing colony are growing (apically elongating) at a
given moment in time. Therefore, conventional
biochemical, autoradiographical and cytological
techniques must be adapted to the specificity of the
problem.
In this study I approached the problem of hyphal tip
growth by comparing cell wall architecture in elongating
versus non-elongating hyphal apices of an oomycete Achlya
bisexualis. Electron microscopy, both transmission and
scanning, was used with a variety of immunocytochemical
labeling of hyphae. Several fixation techniques were used
to ensure that the results were not only reproducible but
also not artifacts of the fixation procedure. The results
allowed me to propose a new hypothesis for the mechanism
of hyphal tip growth.


3
The Organism
Members of the genus Achlya grow as branched
coenocytic hyphae, which collectively are termed a
mycelium. Septa are formed only to delimit reproductive
structures, while vegetative growth occurs at the apex.
Achlya has both asexual and sexual cycles of
reproduction. Asexual reproduction occurs by
fragmentation, differentiation of resistant gemmae, or by
the differentiation of vegetative apices into sporangia
(Sparrow 1960). Achlya differs from related genera by the
fact that the primary zoospores immediately encyst in a
loose cluster at the orifice of the sporangium after
discharge (Johnson 1956).
Sexual reproduction occurs by gametangial contact.
The male gametes produced in an antheridium are
transported via fertilization tubes to female gametes
produced in an oogonium (Mullins 1994). Sexual
morphogenesis is initiated and sequentially controlled by
a series of diffusible steroid hormones (Raper 1939).
While most water molds are monoecious, bearing both male
and female reproductive structures on a single diploid
mycelium, some members of the genus Achlya are dioecious.
True "male" and "female" strains of dioecious species of
Achlya may exist, but the expression of mating type in a


4
strain depends on that of its mating partner (Raper
1939). The involvement of hormones in sexual reproduction
in this genus is very noteworthy, as species of Achlya
appear to be the most primitive eukaryotes known to
produce and respond to steroids.
Achlya has been proposed as a eukaryotic model
system for studying basic mechanisms of growth and
development. Species of Achlya have been used to
investigate the regulatory mechanisms of
steroid-hormone-induced and regulated sexual
differentiation (Thomas and Mullins 1967, Mullins and
Ellis 1974, Horgen 1977, Riehl and Toft 1984, Mullins
1994). They also served in studies on: (i) the
differentiation of vegetative hyphae into asexual
sporangia (Griffin and Breuker 1969, Thomas et al. 1974,
LeJohn et al. 1977, Kropf et al. 1983, Cottingham and
Mullins 1985); (ii) the mechanism of nutrient transport
in fungi (Cameron and LeJohn 1972, Manavathu and Thomas
1982, Kropf et al. 1984); (iii) the tropic responses to
nutrients and other chemoattractants (Musgrave et al
1977, Manavathu and Thomas 1985); (iv) the role of turgor
in hyphal tip growth (Money and Harold 1992, 1993); and
(v) ionic and electrical currents (Harold 1994). In this
study, I used Achlya bisexualis Coker and A. Couch (ATCC
accession number 14524) to investigate the mechanisms of
hyphal tip growth.


5
Class Oomvcetes
The genus Achlya is classified in the family
Saprolegniaceae, order Saprolegniales, class Oomycetes,
subdivision Mastigomycotina, division Eumycota of the
kingdom Fungi (Carlile and Watkinson 1994). The
subdivision Mastigomycotina contains organisms that
produce motile spores (zoospores). The subdivision is
divided into three classes, based on the morphology of
zoospores: Chytridiomycetes, Oomycetes, and
Hyphochytriomycetes. The first class is similar to other
Eumycota, while the latter two show similarities to some
protists rather than to fungi. In fact, the morphological
divergence of the Oomycetes has long been recognized
based on their morphology (Gaumann and Dodge 1928). Their
biochemical properties, such as L-lysine biosynthesis
(Vogel 1964), cell wall chemistry (Bartnicki-Garcia
1968), and tryptophan-pathway enzyme organization (Hutter
and DeMoss 1967) strongly support this view. More recent
ultrastructural (Beakes 1987) and molecular studies
(Lovett and Haselby 1971, Ohja et al. 1975, Kwok et al.
1986, Forster and Coffey 1990, Forster et al. 1990) also
confirmed the divergence of the Oomycetes. According to
Bartnicki-Garcia (1996), these biochemical and
morphological differences indicate that the Oomycetes and
the higher fungi probably arose from different ancestors.
However, the same author disagrees with the idea of
breaking up the kingdom Fungi based on these phylogenetic


6
considerations. In the past, the classes
Chitridiomycetes, Oomycetes, and Hyphochytriomycetes
often have been grouped with nonfungal organisms with
which they have very little in common, either on a
physiological, morphological, or ecological basis. For
example, the Oomycetes were lumped with heterokont algae
in the kingdom Chromista (Cavalier-Smith 1983,
Moore-Landecker 1996), or were placed with all zoosporic
fungi, protozoa and algae in kingdom Protoctista
(Margulis et al. 1990). An admittedly polyphyletic
kingdom Fungi is a more rational taxonomical solution
than the ones listed above. This solution allows us to
assemble and study the collection of organisms that share
key morphological, physiological and ecological
properties (Bartnicki-Garcia 1996).
Though my work does not concern systematics, an
understanding of the phylogenetic position of Achlya is
relevant to the problem of hyphal tip growth. Because the
Oomycetes could have evolved independently, their
mechanism of hyphal tip growth, despite its superficial
similarity to one of true fungi, could prove to be
different.
There are about 600 species of the Oomycetes. The
sexual phase of the Oomycetes has a clear differentiation
into large female and small male structures, termed
oogonia and antheridia. These are the sites of meiosis
and gametogenesis. Each oospore produced after


7
fertilization has a single diploid nucleus. When the
oospore germinates, it gives rise to a diploid vegetative
mycelium, in contrast to the haploid mycelium of most
fungi. Other characters of the Oomycetes that distinguish
them from the Eumycota are the biflagellate zoospore;
mitochondria with tubular cristae; Golgi bodies
consisting of multiple flattened cisternae; cellulose as
a microfibrillar component of the cell wall; the presence
of the amino acid hydroxyproline in cell wall
glycoproteins; and various other biochemical and
molecular characteristics (Carlile and Watkinson 1994).
True fungi have mitochondria with platelike cristae and
produce Golgi bodies that are very simple in structure,
often consisting of only a single cisternal element. Cell
walls of true fungi have chitin as the microfibrillar
component and do not contain hydroxyproline (Alexopoulos
et al. 1996).
The class Oomycetes consists of 5 orders:
Saprolegniales, Lagenidiales, Peronosporales, Rhipidiales
and Leptomitales (Alexopoulos et al. 1996). The order
Saprolegniales contains a single family Saprolegniaceae.
Usually these fungi occur in fresh water and in soil as
saprotrophs and play an important role in decomposition
and recycling of materials in aquatic ecosystems. Some,
however, are obligate parasites of plants, animals, or
other fungi. For example, some species of Saprolegnia,
Achlya, and Aphanomyces attack fish and their eggs


8
(Alexopoulos et al. 1996). The members of Saprolegniaceae
are often called water molds, are distributed
universally, and are among the easiest fungi to isolate
and cultivate in the laboratory.


CHAPTER 2
LITERATURE REVIEW
Mechanism of Apical Growth
The phenomenon of hyphal tip growth has been known
for over a hundred years (Reinhardt 1892). Its mechanism,
though, is not yet understood. Several theories of hyphal
tip growth dominate the literature. They are (1) the
delicate balance theory (Park and Robinson 1966,
Bartnicki-Garcia 1973); (2) the steady-state theory
(Wessels 1990); and more recently a combination of the
first two, (3) the hybrid theory (Johnson 1996). All
three imply that the wall of the apex is plastic, while
that of the subapical nongrowing area is rigid. They also
assume that the driving force for cell elongation is
turgor pressure and/or cytoskeleton.
The delicate balance theory assumes that the
plasticity of the hyphal apex is achieved by a constant
delicate balance between biosynthesis and hydrolysis of
wall components.
The steady-state theory suggests that the plastic
region at the tip contains a mixture of nonlinked wall
polymers that are being constantly synthesized, and the
rigid condition of the wall is established by chemical
crosslinking that is initiated at or near the tip and


10
continues progressively further back in the hyphal wall.
Presoftening of the apical wall is catalyzed by endolytic
enzymes that briefly initiate growth but do not sustain
it.
The hybrid model retains from the steady-state model
the constant exocytosis of a plastic mixture of wall
polymers at the tip and its rigidification via
crosslinking. Among the concepts retained from the
delicate balance model is continuous endoglycanolytic
activity expressed in proportion to the rate of tip
extension (Johnson 1996).
Thus these models suggest different mechanisms to
explain the events of wall growth, while agreeing on
other aspects such as the role of turgor and the
cytoskeleton.
Structure of the Hyphal Wall
Fungal cell walls have essential roles in the life
of the fungal cell, i.e., maintenance of cell shape,
plasticity, protection against unfavorable environmental
conditions, cellular recognition, immune response, and
host-parasite interaction (Rosenberger 1976, Wessels and
Sietsma 1979). The general organization of hyphal cell
walls comprises an inner layer of microfibrillar
polysaccharides overlaid by an outer layer of amorphous
polysaccharides (Burnett 1979).
In Oomycetes, these polysaccharides are,
respectively, cellulose and 1,3 3-glucans containing some


11
1,6-6 branches. Cellulose usually represents about 20%
(w/w), 1,3 -6-glucans about 80% (w/w) of the total wall
carbohydrates (Sietsma 1969, Burnett 1979).
In the hyphal wall of A. ambisexualis Raper
(Reiskind and Mullins 1981a), acid-soluble 1,3 -6-glucans
with single 1,6-6-linked residues as branches represents
40% (w/w) of the dry wall. An alkali soluble glucan, a
polymer of 1,3-6 and 1,4-6 linkages with occasional 1,6-6
glucosyl residues as side chains, represents 7% (w/w) of
the wall. Cellulose represents 21% (w/w) of the wall. An
insoluble residuum with a linkage pattern similar to the
alkali soluble fraction is present at 6% (w/w). An
insoluble component consisting of glucosamine represents
3% (w/w) of the wall. This insoluble fraction probably
represents chitin (Mullins et al 1984). Protein
containing hydroxyproline residue comprises 10% (w/w).
There is also a small amount of phosphorus.
In the study on the ultrastructural organization of
the hyphal wall of A. ambisexualis (Reiskind and Mullins
1981b) a model of the various layers in the wall was
proposed. The method used in this study of the hyphal
wall consisted of the sequential chemical or enzymatic
removal of the various fractions, followed by analysis
(with electron microscopy) of carbon-platinum replicas.
The model shows (a) a surface layer of amorphous
1,3-6-glucan hydrolyzed by acid or the enzyme
laminarinase; (b) another 1,3-6-glucan layer containing


12
some 1,4-B and 1,6-B linkages hydrolyzed by alkali or
laminarinase; (c) microfibrillar cellulose, removed by
cadoxen or the enzymes cellulase plus protease; and (d)
an innermost layer of insoluble residuum, faintly
microfibrillar.
The most abundant and most thoroughly studied
glucans from the fungal cell walls are B-glucans. These
1,3 -B-glucans are variable in degree of 1,6-B branching
and in the length of the branches.
Cellulose is a linear polysaccharide made of
glucosyl moieties joined through 1,4-B linkages. The
glucan chains in this polysaccharide associate through
hydrogen bonding to form microfibrils. According to chain
orientation, different crystalline structures exist. The
most prevalent form is Cellulose I, where glucose chains
are arranged in parallel fashion (the free reducing
groups are in the same end of the microfibrils, and the
nonreducing ends are in the opposite one). In this sense,
as demonstrated by X-ray diffraction analysis (Reiskind
and Mullins 1981a), and also apparently in size, fungal
cellulose is similar to the polysaccharide found in
plants (Ruiz-Herrera 1991).
Chitin is an unbranched polysaccharide containing
exclusively N-acetylglucosamine residues linked 1,4-B.
Three crystalline isoforms of the polysaccharide exist in
nature, according to the arrangement of the chains. These
forms can be recognized by X-ray diffraction. In fungi,


13
only alpha-chitin, characterized by the antiparallel
arrangement of the chains, has been detected (Sentandreu
et al. 1994).
Structural proteins present in the cell wall of
fungi are glycoproteins. They display a basic common
structure, consisting of protein with covalently bound
carbohydrate chains. In fungi, they are usually called
mannoproteins because the carbohydrate moiety mainly
consists of mannose units, although small amounts of
other sugars and phosphodiester groups are found (Peberdy
1990, Ruiz-Herera 1991). Hydroxyproline is reported as a
constituent of cell wall proteins in the Oomycetes
(Webster 1980, Reiskind and Mullins 1981a, Ruiz-Herrera
1991). The carbohydrate moieties are attached to the
protein through two types of linkages. One type is
O-glycosidic linkage between mannose or small
oligosaccharide chains and the hydroxy-amino-acids
(Nakajima and Ballou 1974, Sentadreu and Northcote 1969,
Tanner and Lehle 1987). The second type of linkage
(N- glycosidic) connects high molecular weight, highly
branched, mannan tufts to asparagine residues of the
protein, through diacetylchitobiose (Byrd et al. 1982,
Cohen and Ballou 1981, Tanner and Lehle 1987).
Studies of the structure of fungal cell walls by
cast shadowing or replica techniques have demonstrated
that their outer and inner surfaces appear different. The
outer surface is usually amorphous or finely granular,


14
whereas the inner face shows intertwining microfibrils of
different size, width and orientation. However, there is
evidence that some components such as microfibrils may-
escape observation because they are masked by the
presence of the large amounts of matrix compounds. From a
structural point of view, the fungal cell wall has been
compared to such manmade composites as reinforced
concrete or fiber-reinforced plastics which are formed by
two distinct elements: an elastic one, which in the cell
wall would be microfibrils of the structural
polysaccharides, and a plastic one, which would
correspond to the rest of the wall components, generally
referred as amorphous or cementing (Ruiz Herrera 1991).
Electron Microscopic Studies of Fungal Cell Walls
In thin sections, fixed and stained by the usual
standard method including glutaraldehyde and osmium
tetroxide, fungal cell walls appear multilayered. At
least two layers are observed in most walls: an outer one
which is electron dense; and an inner layer, thicker and
electron transparent. However, appearance of the cell
wall in sections may depend on the technique used for
fixation (Ruiz-Herrera 1991).
Variability in composition of the cell wall of fungi
does not allow the proposal of a single model of the wall
structure. In general, evidence suggests that fibrillar
polysaccharides are accumulated mostly in the inner
layers of the cell walls, while amorphous components are


15
more abundant in the external layers. The description of
wall structure observed in different genera of fungi
analyzed by various techniques may be more useful in
providing a general overview of fungal wall architecture
(Ruiz-Herrera 1991).
Early studies on the chemical characterization of
fungal cell wall layers were conducted by Hunsley and
Burnett (1970). They studied the wall structure of
Neurospora crassa, Schizophyllum commune and Phytophtora
parasitica after treatment with several hydrolytic
enzymes. The outer surface of N. crassa in shadow-cast
samples appeared amorphous. Laminarinase treatment
removed the amorphous coat revealing a layer of coarse
strands whose interstices were filled with amorphous
material, whereas treatment with both laminarinase and
pronase enhanced the reticular appearance. The
microfibrils were sensitive to chitinase. The authors
concluded that the external coat was made of amorphous
beta-glucans placed over a reticulum of glycoproteins.
More internally, it was suggested, a protein layer
followed in which chitin microfibrils were embedded. In
contrast to Neurospora, the cell wall of S. commune was
resistant to laminarinase, pronase and chitinase,
apparently due to the presence of superficially located
1,3 -alpha-glucan which prevented the access of the lytic
enzymes. After removal of this glucan layer by KOH,
laminarinase and pronase treatment gave rise to the


16
appearance of a fibrillar structure sensitive to
chitinase, suggesting that inner wall layers had a
chemical composition and organization similar to N.
crassa. Appearance of the wall from P. parasitica was not
affected by pronase, but laminarinase unmasked a
fibrillar layer sensitive to cellulase treatment. These
results were interpreted as suggesting the presence of
two layers rich in amorphous beta-glucans and cellulose,
respectively, in the wall of this fungus.
The cell of yeast and mycelial cells of Candida
albicans reveals four wall layers when treated by a
standard gluteraldehyde-osmium technique (Yamaguchi
1974). When stained by Thiery's technique, eight
different layers can be observed, depending on the
intensity of staining and their electron density. The
four outermost layers are PATAg positive, whereas layers
5 and 7 appear electron transparent and PATAg negative
(Poulain et al. 1978) The authors concluded that the
inner layers must be rich in chitin and 1,3 -B-glucan,
which are both electron transparent and PATAg negative.
Other outer layers must be rich in glucans and mannans.
The existence of mannans on the surface of the cell was
confirmed by Horisberger et al. (1975) who observed
binding of colloidal gold-tagged concanavalin A (ConA-Au)
by intact cells of the fungus. The presence of mannan in
two continuous layers at the periphery of blastospores
was demonstrated by staining ultrathin sections with


17
Concanavalin A-horseradish peroxidase 3,3'diamino
benzidine and HO (Tronchin et al. 1979). In this
technique, the lectin binds to the mannose residues of
the glycoprotein and it is recognized by peroxidase. The
peroxidase forms a dark product by the catalytic
decomposition of HO in the presence of an oxygen
acceptor. A similar method, which included treatment with
wheat germ lectin followed by chitibiosyl- horse radish
peroxidase or chitobiosyl- ferritin, was used to conclude
that chitin was located mostly in the inner layers of the
wall of C. albicans.
In related species Candida utilis, sections were
stained with ConA-Au and gold-labeled antimannan
antibodies. These techniques demonstrated that
mannoproteins were denser in the cell periphery although
labeling also was observed close to the plasmalemma
(Horisberger and Vonlanthen 1977). Similar results were
obtained with Saccharomyces cerevisiae by the same
authors (Horisberger and Vonlanthen 1977).
Lectins bound to fluorescein isothiocyanate (FITC)
were used to detect superficial polysaccharides in the
different yeasts by Barkai-Golan and Sharon (1978). The
authors observed that S. cerevisiae, S. bayanus and
Candida mycodema bound ConA only, suggesting the presence
of mannoproteins on the surface of the cells. On the
other hand, Schizosaccharomyces pombe did not bind ConA;
but it bound peanut lectin, which recognizes D-galactose,


18
indicating that the cell surface of fission yeast is
covered by a galactomannan, not by mannoproteins. Candida
rugosa and Sporobolomyces roseus bound both lectins. This
result may indicate the presence of both galactomannan
and mannoproteins on the surface of these cells.
Treatment of the cells with KOH resulted in a strong
reaction with wheat germ lectin which recognizes GlcNAc,
suggesting that chitin is located internally and is
covered by alkali soluble mannoproteins. The presence of
galactomannan on the surface of S. pombe was confirmed by
use of the lectin from Bandeiraea simplicifolia bound to
colloidal gold (Horisberger and Rosset 1977). This
lectin, which recognized alpha-galactopyranosyl residues
bound to the outer layer of the wall, and in minor
amounts was distributed evenly over the whole thickness
of the cell, including the fission scars. In a further
report, these authors demonstrated differential
distribution of galactomannan depending on the growth
stage of the cells (Horisberger et al. 1978).
Galactomannan appeared in the form of two layers of the
wall: one close to the plasmalemma, and another on the
surface of the cell. Labeling by the lectin occurred at
the cell periphery and at the growing end, but not on the
wall, formed after cell division. These results were
interpreted as meaning that the polysaccharide was
synthesized during cell extension, but not during septum
formation.


19
Four layers in the cell wall of Dictyostelium
discoideum spores were observed by freeze-etching and
replica (Hemmes et al. 1972). The innermost layer, which
appeared amorphous or slightly fibrillar, could be
eliminated by successive treatment with cellulase and
pronase, suggesting that it was constituted by a mixture
of cellulose and proteins. The middle layers (both
fibrillar) were removed by cellulase treatment alone,
indicating the cellulosic nature of the microfibrils. The
most superficial layer was resistant to both pronase and
cellulase treatment. Hydrolysis resulted in release of
galactose, suggesting that this is a major component of
the acidic polysaccharide present in the walls.
In sections of Agaricus bisporus spores treated with
the standard method, three layers could be recognized.
The authors concluded that the middle layer contained
protein because treatment with pronase increased the
fibrillar appearance of this layer. These fibrils
corresponded to 1,3 -B-glucans and chitin, as they were
removed by B-glucanase and chitinase treatment. The outer
layer was composed of melanins and 1,3 alpha -glucans,
which was deduced by chemical analyses and electron
microscopic observations. The thin inner layer was poorly
characterized, but the authors suggested it was of
mucilagenous nature (Rast and Hollenstain 1977).
The structure of the mycelial wall of the same
fungus was different (Michalenko et al. 1976). The outer


20
layer, which appeared amorphous in replicas, was made of
mucilage. The thin middle layer was made of amorphous
glucans. The innermost layer, which in replicas appeared
fibrillar, is probably made of a mixture of B-glucans
covering fibrillar chitin, since chitinase by itself
could not remove it, whereas the combined action of
B-glucanase and chitinase solubilized the layer. Staining
with silver hexamine suggested that proteins were present
in all layers of the cell wall of the fungus.
A similar approach was followed in the
characterization of the architecture of the wall from
microconidia of Trichophyton mentagrophytes. Three layers
were recognized in sections. The outer layer appeared
electron dense, and the innermost one appeared electron
transparent. The material extracted from the outer layer
contained a single glycoprotein. The median layer was
made of proteinaceous rodlets. The inner layer apparently
was composed of amorphous glucans and microfibrillar
chitin (Wu-Yuan and Hashimoto 1977).
Structure of the cell wall from Trichoderma
pseudokoningii was studied by treatment of intact cells
with different lytic enzymes (Jeenah et al. 1982).
Accordingly, the authors concluded that the outer layer
contained B-glucans, whereas the internal layer was
composed of chitin embedded in a protein matrix.


21
Biogenesis of the Fungal Cell Wall
Cell wall biosynthesis takes place in three sites:
cytoplasm, plasma membrane and the wall itself.
Structural polymers such as chitin and 1,3-B- and
1,4-6-linked glucans are synthesized vectorially at the
plasma membrane, by transmembrane synthases accepting
nucleotide sugar precursors from the cytosol and
extruding the polymerized chain into the wall (Cabib et
al. 1983, Shematek et al. 1980, Girard and Fevre 1984,
Jabri et al. 1991, Cabib et al. 1991, Hromova et al.
1989). Matrix polymers such as glycoproteins are
synthesized in the cytoplasmic secretory pathway of
endoplasmic reticulum through Golgi vesicles to secretory
vesicles. Wall assembly, involving activities such as
covalent crosslinking of polymers and modifications such
as deacetylation of chitin, takes place in the wall
itself (Gooday 1995).
Fungal wall 1,3-B-glucans are biosynthesized via the
nucleotide sugar, UDP-glucose. The glucan synthases are
intrisic proteins of the plasma membrane. Preparations of
membranes from Saprolegnia monoica, when provided with
UDP-glucose, produce polymers containing varying amounts
of 1,4-B and 1,3-B links (Girard and Fevre 1984) The
vectorial synthesis of 1,3-B-glucan chains allows only
linear molecules to be made and thus any 1,6-B branches
must be added in the wall (Gooday 1995). These


22
1,3 -13 glucan synthases are stimulated by the presence of
trypsin but inhibited by other proteases. Stimulation
occurs from the beginning of incubation in the presence
of the protease but prolonged action of trypsin leads to
inactivation of the glycosyl transferases. The
1,3 J3 glucan synthases, therefore, must exist in an
inactive state that can be activated by moderate
proteolysis. Such regulation, which also appears to
modulate plant glycosyl transferases (Girard and
Maclachlan 1987), characterizes the chitin synthase
system of various fungi (Cabib 1981).
The 1,4 -fi-glucan synthases, like 1,3 -fi-glucan
synthases, have a transmembrane orientation in the
plasmalemma, leading to a vectorial synthesis of
cellulose from UDP-glucose. These enzymes may have a
common structure or organization, as revealed by
preliminary immunological studies, but they are different
systems and can be separated by glycerol gradient
centrifugation (Fevre et al 1990). Cellulose synthases
from Saprolegnia are inactivated by trypsin, but
stimulated in the presence of certain nucleotides. Fungal
cellulose synthase enzymatic complex may resemble the
plant plasma membrane rosettes involved in cellulose
synthesis (Mullins and Ellis 1974, Muller and Brown 1980,
Montezinos 1982). Some proteins, sensitive to proteases
or capable of reacting with nucleotides, would be
involved in the regulatory processes. Other proteins


23
would be involved in UDP-glucose binding. Such an enzyme
seems to exist in plants (Delmer 1999). Cellulose
synthases may have a more complicated organization than
1,3-B-glucan synthases. Cellulose synthase activities of
cell free extracts are always much lower than
1,3-B-glucan synthase activities. It is possible that
cellulose synthases require a specific factor that is
lost in the course of isolation. The opposite behavior of
the synthases towards protease and nucleotides, and the
presence of a membrane bound activator of 1,3-6-glucan
synthase, may indicate a difference in the regulation of
their activities. This would have implications in the
cell wall assembly where the deposition of the different
polysaccharides during apical growth is coordinated in
time and space (Fevre et al 1990).
Chitin synthases, like glucan synthases, are
intristic proteins of plasma membrane. These enzymes
catalyze glycosidic bond formation from the nucleotide
sugar substrate, UDP-N-acetylglucosamine. Most chitin
synthase preparations are zymogenic, i.e., produced as
proenzymes requiring activation by specific proteases.
This proteolytic activation presumably plays a role in
the temporal and spatial regulation of the enzyme, by
locally activating it in the membrane when and where its
activity is required (Gooday 1995). As well as being in
zymogenic and active forms in the plasma membrane,
zymogenic chitin synthase also occurs in fungal cells as


24
chitosomes, which are membrane-bound microvesicles about
70 nm in diameter (Bartnicki-Garcia et al 1979, Kamada et
al 1991) After purification by differential
centrifugation, chitosomes can be activated by treatment
with proteolytic enzymes, and then produce chitin
microfibrils if incubated with UDP-GlcNAc (Gooday 1995) .
Wall glycoproteins are biosynthesized in the
secretory pathway: endoplasmic reticulum > Golgi bodies >
secretory vesicles > release at the plasma membrane.
Carbohydrate material detected in apical vesicles could
be the carbohydrate portion of glycoproteins. The
transmembrane stages in glycoproteins biosynthesis
involve sugar precursors linked to polyprenol dolichol,
the "lipid intermediates" (Lehle 1981, Cabib et al.
1988). In the O-linked chains, the first mannose unit is
linked to the protein via the precursor
dolichol-phosphomannose, in the endoplasmic reticulum.
The other mannose units are added via the nucleotide
sugar guanosine diphosphomannose, GDP-Man, in the Golgi
bodies. The N-linked chains are assembled by a more
complex scheme, giving a lipid intermediate
dolichol-diphospho-(GlnNAc)2-Man9-Glc3 which is N-linked
to asparagine in the protein in the endoplasmic
reticulum, with the release of the terminal four sugar
units, Man-Glc3. The outer chain of many mannose units is
added by several linkage-specific mannosyl transferases,
with Glc-Man as substrate, in the Golgi bodies (Gooday


25
1995). Some secreted enzymes, notably invertase, acid
phophatase and chitinase, are also mannoproteins,
synthesized and secreted in a similar fashion (Kuranda
and Robbins 1991) .
Once the cell wall components are synthesized and
secreted, they must be converted into an integrated
structure. This process includes covalent crosslinking,
hydrogen bonding, hydrophobic and electrostatic
interactions between different macromolecules
(Ruiz-Herrere 1991).
Role of Turgor in Wall Expansion
The difference in hydrostatic pressure between a
cell and its surroundings is called turgor pressure. This
actual pressure is thought to provide the driving force
for hyphal extension. Several observations and
measurements suggest that it is necessary for the apical
growth process (Robertson 1958, Park and Robinson 1966,
Robertson and Rizvi 1968). Osmometry has been used to
demonstrate a correlation between hyphal extension rates
and turgor pressure in many fungal species (Eamus and
Jennings 1986, Luard and Griffin 1981, Woods and Duniway
1986). Experiments have shown that filamentous fungi
respond to increases in external osmotic pressure by
accumulating compatible solutes, including potassium
ions, glycerol, mannitol, erythritol and arabitol (Luard
1982a,b,c; Pfyffer and Rast 1988, 1989).


26
The most detailed analyses of the relationship
between hyphal extension and turgor pressure have been
carried out on hyphae of Achlya bisexualis and
Saprolegnia ferax and these studies suggest that growth
can occur without significant turgor (Money and Harold
1992, Kaminskyj et al. 1992). The rate of growth under
these conditions is about half of the maximum rate.
Achlya continues to grow even after the turgor is
undetectable; however, its morphology is radically
altered. On solid medium it shows plasmodial- like growth.
In liquid medium of the same composition, it exhibited a
yeast-like morphology. Saprolegnia has a different
response to the absence of turgor, since it continues to
grow in the hyphal form. Both Achlya and Saprolegnia
appear not to respond to changes in external osmotic
pressure by controlling the concentration of internal
compatible solutes (regulation of turgor); instead, the
plasticity of the wall is modulated to balance the force
applied against it.
Role of Cvtoskeleton in Hyphal Growth
It seems very unlikely that the thin wall covering
the apices of extending cells has sufficient mechanical
strength to contain the turgor pressure of the cytoplasm.
It was suggested that other cellular components may play
a role in the regulation of tip expansion. The possible
existence of other factors is suggested by: (1) the
ability of mutants with abnormal cell wall composition to


27
generate relatively normal hyphae (Katz and Rosenberger
1970); (2) the poor correlation between growth rates and
turgor pressure (Kaminskyj et al. 1992); and (3) the
ability of some species to produce hyphae in the absence
of measurable turgor pressure (Money and Harold 1993) .
The apex might be stabilized by a fibrillar
cytoplasmic network similar to that found in other
cellular systems (amoebae, slime molds). The main
structural components of such a cytoskeleton are actin
filaments (F-actin), microtubules, and intermediate
filaments. Each of these are elongated polymers composed
primarily of globular proteins known as actins, tubulins,
and other unrelated units respectively (Heath 1994). The
presence of an array of F-actin was shown in growing tips
of Saprolegnia (Heath 1987). It was always present in
growing tips, but absent in nongrowing tips. None of
these observations proves a morphogenic role for F-actin,
because it is not possible to differentiate between
direct and indirect effects in the complex system of
hyphal tips, but they do suggest that F-actin has some
role (Heath 1994).
Another explanation of the presence of actin plaques
at the growing apices is vesicle and organelle traffic
control. There is evidence for the involvement of both
microtubules and F-actin in wall vesicle transport (Heath
1994) .


28
Cytology of Growing Hvphal Apices
A growing hypha consists of an apical region where
the extension takes place, a nonelongating subapical, and
mature regions, which were the sites of earlier growth.
This is reflected both in the structure of the wall and
the cytoplasm.
Older regions of the hyphal wall are rigid and
thick. The cytoplasm in these regions is restricted to a
thin layer between a large tonoplast and the plasma
membrane. This cytoplasm contains the usual variety of
eukaryotic organelles. The tonoplast in mature regions is
represented by a central vacuole whereas younger regions
contain many vacuoles of smaller size. The subapical
region has a thinner wall. The cytoplasm is nonvacuolated
and is particularly rich in organelles. At the very apex,
the hyphal wall is thin and the associated cytoplasm
lacks the usual organelles, containing only small
cytoplasmic vesicles of differing size (Shapiro 1995).
Based on the organelle distribution, three cytoplasmic
zones or regions are recognized: (1) an older, highly
vacuolated region; (2) a subapical organelle-rich region;
and (3) a terminal vesiculate region (Grove et al 1970).
Since one of the differences between the growing tip
region and the basal parts of the hypha is the abundance
of cytoplasmic vesicles, they are usually assumed to be
involved in the synthesis of the new wall. One
possibility would be that they carry wall polymers ready


29
for insertion in the growing wall. Intracellular
synthesis of wall polymers and their delivery to the wall
by vesicles occurs with pectin, hemicellulose, and
hydroxyproline-rich glycoproteins in plants (Northcote
1984), and to wall mannoproteins in yeast (Zlotnik et al.
1984). For filamentous fungi, there is no convincing
evidence for a similar process. Cytochemical staining
does detect polysaccharide material in some apical
vesicles (Grove 1978, Hill and Mullins 1980), but this
material may represent glycoproteins destined for
secretion. More likely, these vesicles contain precursors
of the cell wall, their membrane probably contributes to
the extending plasmalemma, and they may contain wall
synthase enzymes for insertion into the plasma membrane
(Heath 1994) .
Growing and nongrowing hyphae differ in the type of
wall material that covers their apices (Wessels 1986).
The absence of alkali insoluble beta-glucans at the very
apex of growing hyphae in Schizophyllum commune has been
demonstrated by light microscopic autoradiography
(Wessels et al. 1983). A subsequent study using electron
microscopic autoradiography on shadowed preparations
revealed that chitin in growing apices, though alkali
insoluble, is in a conformation state quite different
from that in nongrowing apices and subapical parts. In
contrast to the chitin in these older parts, the newly
synthesized chitin at apices appeared nonfibrillar, very


30
susceptible to chitinase degradation and partly soluble
in hot dilute mineral acid. Earlier observations had
indicated discontinuities in the presence of microfibrils
at hyphal apices (Strunk 1968). These have been
contradicted by other workers who showed a continuous
network of chitin microfibrils over the apex after
chemical treatments which removed a "matrix substance"
(Aronson and Preston 1960, Hunsley and Burnett 1970,
Bartnicki-Garcia 1973, Schneider and Wardrop 1979,
Burnett 1979, Aronson 1981). Wessels (1990), however,
suggested that these images showing apical microfibrils
probably represent nongrowing apices, which are known to
occur abundantly among growing hyphae.
There is a number of light microscopic studies using
fluorescently labeled probes which also suggest that the
wall covering the growing apex is different from that
covering a nongrowing apex or that of subapical regions.
In these studies fluorescently labeled antibodies (Fultz
and Sussman 1966, Marchant and Smith 1968, Hunsley and
Kay 1976), fluorescent brighteners such as calcofluor
(Gull and Trinci 1974), and fluorescently labeled wheat
germ agglutinin were used. This differential staining at
growing tips could result from the absence of outer wall
materials, or to a difference in the conformation of the
polymers that bind these probes.


31
Calcium Gradient
There is a tip-high calcium gradient in apically
growing cells. Free cytoplasmic calcium in the oomycete
Saprolegnia ferax is highest at the tip as demonstrated
using fluorescent dyes such as Indo-1 or Fluo-3 (Yuan and
Heath 1991, Jackson and Heath 1993 Garrill et al. 1993).
Studies using patch-clamp techniques suggest that the
tip-high gradient reflects a spatial organization of
calcium channels in the cell membrane. Using patch-clamp
electrophysiology, two types of channels were identified
in Saprolegnia ferax: (a) calcium-activated potassium
channels that were thought to be involved in turgor
regulation, but were not obligatory for growth; and (b)
stretch-activated calcium channels that were activated by
potassium ions and which may be essential for apical
extension (Garrill et al. 1992, 1993). The
stretch-activated channels were concentrated at the
hyphal apex and were blocked by Gd3 which also inhibited
hyphal extension and dissipated the tip-high calcium
gradient revealed by Indo-1 (Garrill et al. 1993). In
contrast to the stretch-activated channels, the
calcium-activated potassium channels were uniformly
distributed along the hyphal cell membrane. These could
be inhibited by tetraethylammonium, which only caused a
transient effect on growth. Stretch-activated calcium
channels have also been identified in the germ tubes
apices of the plant pathogen Uromyces appendiculatus


32
(Hoch et al. 1987, Zhou et al. 1991). These data suggest
that the tip-high calcium gradient is important for
polarized hyphal extension and is generated by a locally
high concentration of stretch-activated calcium channels
in the hyphal apex. It is presumed that the channels are
delivered to the surface in microvesicles. They may be
maintained there by anchoring them to the cytoskeleton or
by membrane recycling (Gow 1995) .
Ion Currents
The net flow of electrical current carried by the
circulating ions can be detected with an ultrasensitive
voltmeter called the vibrating microelectrode (Jaffe and
Nuccitelli 1974). In Achlya bisexualis and filamentous
fungi in general, a positive proton carried current
normally enters the growing apex. Inward current was
shown to be due to amino acid-proton co-transport
(symport) localized at the tip and the outward current
was due to electrogenic proton efflux via a plasma
membrane ATPase (Kropf et al. 1984, Gow et al. 1984, Gow
1984, Schreurs and Harold 1988). The proton current also
established an extracellular pH gradient around the
hypha, with the medium adjacent to the tip relatively
alkaline (Gow 1984). On the other hand, there are many
examples where there is no correlation between the
direction or magnitude of the current and the process of
tip growth (Gow 1995).


CHAPTER 3
HYPHAL GROWTH
Introduction
Until recently, hyphae were assumed to extend at a
constant linear rate when environmental factors are
favorable and stable, and nutrients are ample. Detailed
analysis of hyphal growth, however, reveals oscillating
elongation rates (Lopez Franco et al. 1994).
Materials and Methods
An isolate of Achlya bisexualis Coker & A. Couch
(ATCC 14524) was used in this study. Stock cultures were
maintained on corn meal agar. Mycelia were grown on corn
meal agar (CMA), prepared from 17 g of Difco corn meal
agar, 10 g of purified grade agar (Fisher Scientific) and
1 L of distilled water. Small plugs (approximately 1
millimeter) were removed from the edge of a 24-hour-old
CMA colony. The plugs were then placed in a 250 mL flask
containing 100 mL of liquid peptone-yeast extract glucose
(PYG) medium, pH 6.8 (Cantino and Horenstein 1953). The
PYG medium was prepared by combining 1.25 g of
bacto-peptone, 1.25 g of yeast extract (Sigma) and 3 g of
D-glucose with 1 L of distilled water. The culture was
incubated for 10 to 12 h until the hyphae had grown out
33


34
from the agar plugs to a distance of 2 to 5 mm and then
studied.
To obtain actively growing colonies, the 10 to 20 h
old cultures incubated in PYG were used. Based on dry
weight accumulation, colony size increase, glucose
incorporation, and cellulase secretion, these colonies
are in the midexponential stage of growth (Hill and
Mullins 1979).
Non-growing conditions were obtained by transferring
growing colonies from PYG to 0.2% glucose solution, by
incubation in glucose for about 48 h to cease elongation.
Procedure made it possible to find colonies with no
elongating hyphae. Such colonies were fixed and used for
studying non elongating hyphae. Screening for no
elongation is necessary, because there are hyphae in some
colonies that are still elongating at a slow rate.
Hyphal elongation was monitored with an Olympus BH-2
light microscope. The colonies were kept on small
depression slides with cover slips. Digital images of the
hyphal tips were taken every 10 min with a Pixera 120C
digital camera. The microscope light was turned off
between the measurements to avoid heating. Average
elongation rates were calculated using a stage
micrometer. One hundred hyphae from different growing
colonies and about 50 hyphae from non-growing colonies
were studied.


35
Results
These light microscopic observations suggested that
in colonies growing in PYG, the majority of the hyphae
are elongating and a small number of the hyphae is not.
When individual hyphae are monitored over a long period
of time (5 to 6 h), they go through alternating periods
of elongation and non elongation. The rate of elongation
is not steady, but fluctuates between periods of fast and
slow rates. The average rate is 3.6 pm/min, but it
fluctuates from 2 to 6 pm/min. The fastest rates are in
the middle of an elongating cycle, with lower rates at
the beginning and the end, resulting in a bell shaped
curve (Fig. 1). Elongating hyphae have sharp apices
(Fig. 2) .
The majority of the hyphae in the colonies incubated
in glucose-only medium are not elongating and a small
portion of the hyphae (about 5%) is elongating with an
average rate of 1 pm/min. In some colonies there are no
elongating hyphae at all. All the hyphae have rounded
apices (Fig. 3).
Discussion
Light microscopic observations suggest that hyphal
growth is a discontinuous, irregular process with periods
of elongation and no elongation (Fig. 1). The elongation
rate is not constant, but instead fluctuates with periods
of fast and slow elongation. During the elongation period
the higher rates are in the middle and the rate changes


36
in a bell shaped curve mode. A similar irregular mode of
hyphal tip growth was demonstrated by Lopez-Franco et al.
(1994). Growing hyphal tips were recorded with
video enhanced phase contrast microscopy at high
magnification, and digital images were measured at very
short time intervals (1 TO 5 s). The study was conducted
using fungi from several major taxonomic groups
(Oomycetes, Pythium aphaniderma turn and Saprolegnia ferax;
Zygomycetes, Gilbeltella persicaria; Deuteromycetes,
Trichoderma viride; Ascomycetes, Neurospora crassa and
Fusarium culmorum; Basidiomycetes, Rhizoctonia solani).
In all fungi, apparent steady growth of hyphal tips
revealed patterns of pulsed hyphal elongation. It was
shown that the hyphae do not grow continuously with a
steady rate but instead this rate fluctuates, with
alternating periods of fast and slow elongation. This
results in irregular pulses of growth. Pulsed growth was
observed in fungi differing in cell diameter, overall
growth rate, taxonomic position, and presence and pattern
of Spitzenkorper organization, thus suggesting that it is
a general phenomenon. The basis of these pulses was not
determined, it was proposed that their origin could be in
the pulsating mode of intracellular processes, especially
the secretory vesicle delivery/ discharge system.


elongation (|jm)
37
60
0 50 100 150 200 250
time (min)
Fig. 1. Elongation measurements of three individual
Achlya bisexualis hyphae growing in PYG medium.
300


38
Figs. 2-3. Scanning electron micrographs of hyphae of
Achlya bisexualis. 2. Elongating hypha from a colony
incubated in PYG. 3. Non-growing colony incubated in
glucose-only medium showing the hyphae with rounded
apices. Bars: 2 = 16.7pm; 3 = 27.3 pm.


CHAPTER 4
LOCALIZATION OF CELLULOSE IN THE CELL WALL AS REVEALED
BY ELECTRON MICROSCOPY AND CYTOCHEMICAL TECHNIQUES
Introduction
Cellulose, a crystalline 1,4-B-glucan, is the most
abundant biopolymer in nature. Its biomass makes it a
global carbon sink and renewable energy source, and its
crystallinity provides mechanical properties to cellulose
containing cell walls (Arioli et al. 1998) The
understanding of cellulose properties and metabolism is
important for understanding morphogenesis in plants and
certain fungi.
Traditionally, cell wall components have been
identified cytochemically by using indirect, extractive
methods. This approach can lead to problems such as
incomplete extraction and unseen effects of the
extraction procedure on cell wall ultrastructure.
Enzyme linked colloidal gold labeling is a
nondestructive, direct labeling technique and can be used
to localize cellulose on thin sections (Berg et al.
1988). This labeling technique can be also used to
localize cellulose on the surface of cells.
Previously cellulose was identified in Achlya cell
walls with cellulase-gold affinity labeling. Thin


40
sections of growing hyphae revealed labeling for
cellulose in mature and subapical regions, but not at the
apex (Shapiro 1995).
The present study was done to observe the
distribution of cellulose along the elongating and
non-elonging hyphae, as part of an overall examination of
hyphal tip growth.
Materials and Methods
Culture Methods and Microscopy Techniques
The general culture methods are described in Chapter
3. To induce sporulation, ten discs from the edge of a 48
h old colony growing on CMA plates were punched out with
a small cork border. The discs were incubated at room
temperature in 100 mL PYG liquid medium in a 250 mL flask
for 15 h with shaking at 110 rpm. Then they were washed
several times with 0.5 mM calcium chloride. At ths point,
they were left in fresh calcium chloride for 6 to 7 h to
induce sporulation.
Electron microscopy
For chemical fixation, the agar plugs bearing hyphae
were fixed for 30 min at room temperature with 4% (v/v)
glutaraldehyde in 0.05 M sodium cacodylate buffer, pH
7.2. After rinsing in 3 changes of buffer, the material
was postfixed in 1 % (w/v) osmium tetroxide in the same
buffer, for 30 min. Samples were again washed several
times in buffer, followed by dehydration in an ethanol
series, terminating in absolute acetone. For


41
freeze-substitution fixation colonies were frozen in
acetone at -80 C, then substituted with methanol at -80 C
for 72 h. The samples were warmed over 2 h at room
temperature and transferred into absolute acetone for TEM
or rehydrated in a methanol/water series to be labeled
with cellulase-gold complex and processed for SEM
(modified from Bourett et al. 1998) For methanol
fixation colonies were frozen in methanol at -80 C, then
warmed at room temperature over 2 h and transferred into
absolute acetone for TEM or rehydrated in an
methanol/water series. These samples were labeled with
cellulase-gold complex and processed for SEM. Material
from absolute acetone was infiltrated with an epoxy
embedding medium and polymerized at 60 C for 48 h in a
flat embedding mold. Epon 812 embedding medium was
prepared by combining 55 g of Epon 812, 35 g of DDSA and
21 g of NMA. The accelarator DMP-30 (0.2 mL per 10 mL of
the medium) was added right before embedding. Embedded
samples were sectioned on a Reichert Ultracut R (Leica).
Thin sections (75 to 80 nm) were collected on formvar
coated nickel grids and labeled with the cellulose-gold
complex.
For scanning electron microscopy the colonies were
fixed with 4% glutaraldehyde in 0.05 M sodium cacodylate
buffer and washed several times in buffer (osmium
tetroxide fixation was omitted). Then the colonies were
processed for cellulase-gold labeling, silver enhanced,


42
dehydrated in an ethanol series finally critically point
dried. For silver enhancement the colonies were placed in
a non-diluted mixture (1:1) of reagents from the Aurion
Silver Enhancement Kit for 5 min and washed several times
with water to stop the reaction (Scopsi et al. 1986).
Cellulose Localization Using Enzyme-Gold Affinity
Labeling
Colloidal gold of approximately 15 nm diameter was
made via reduction of chloroauric acid by sodium citrate
as described by Frens (1973). The enzyme cellulase was
purchased from Worthington Biochemical Corporation,
catalogue No. LS02601. This is chromatographically
"purified" cellulase isolated from cultures of a selected
strain of Tricoderma reesei. A second enzyme was also
used, endocellulase III (provided by Dr. Tim Fowler,
Genencore International, Inc). The solutions used for
conjugation with this enzyme were 5.5 rather than 4.5 for
the commercial cellulase. To coat the gold with
cellulase, the pH of 10 mL of 15 nm colloidal gold was
adjusted to 4.5 and then 1 mg of cellulase dissolved in
0.1 mL distilled water was added with stirring. After 5
minutes the enzyme-gold complex was further stabilized by
the addition of 0.5 mg/mL polyethylene glycol (molecular
weight 20,000). Then the solution was poured into a
centrifugation tube and 1.5 mLof 20% glycerol (in citrate
buffer pH 4.5) was carefully placed on the bottom of the
tube (glycerol was added for long-term storage at -80 C).


43
The enzyme-gold complex was pelleted at 12,100 rpm for 1
h. Successful coating was evident by a mobile pellet,
which was resuspended in 0.75 mL of 20% glycerol.
Sections, on grids, were preabsorbed for 5 min by
floating them face down on citrate buffer containing 0.5%
gelatin as a blocking agent. The labeling solution was a
1:10 dilution of the enzyme-gold stock with citrate
buffer. To label, grids with sections were floated on the
labeling solution for 30 min. The grids were then floated
on citrate buffer alone for 5 min and rinsed twice for 5
min in distilled water.
The colonies destined for SEM observations were
treated with the same series of solutions, but were
completely submerged, rather than floated.
Cvtochemical controls
A number of cytochemical controls were performed to
prove the specificity of the label. (1) Substrate
competition: as a control to determine that the
enzyme-gold probe was binding to cellulose the
cellulase-gold complex was incubated with 1 mg/mL
carboxy-methylcellulose (CMC) (sodium salt, medium
viscosity, Hercules CMC 7MF) for 30 min before the
labeling of sections or colonies. (2) Labeling with
nonenzymatic protein: any nonspecific protein binding
sites were determined by incubating the sections and
colonies with 18 nm Colloidal Gold-AffiniPure Goat


44
Anti-Mouse IgG (H+L) (Jackson ImmunoResearch
Laboratories, Inc.). (3) Substrate specificity check: two
substates similar to cellulose and present in the cell
wall of Achlya were tested to see if cellulase-gold bound
nonspecifically to them: the cellulase-gold complex was
incubated with 10 mg/mL laminarin (from Laminaria
digitata, Sigma) and 11.8 mg/mL re-acetylated glycol
chitosan (Sigma). (4) Cellulase pretreatment: to
determine the effect of predigestion by free cellulase
the sections and colonies were incubated with 1 mg/mL
cellulase in incubation buffer for 30 minutes prior to
labeling with cellulose-gold complex.
Cellulase Enzyme Activity during Labeling
To determine if the cellulase enzyme, when
conjugated to gold used for labeling, retained enzyme
activity the following experiment was done. The samples
were combined with 1 mL of gold-cellulase complex (1:10
dilution) and incubated at room temperature. A control
for each sample was prepared with substrate and 0.05 M
sodium citrate buffer pH 4.5. The citrate buffer alone
was used as a blank. After 30 min (the usual time of
labeling) and 3 h, the enzyme activity was checked by
Nelson-Somogyi method, using a standard curve obtained by
plotting optical density (at 520 nm) measured on Du-64
Spectrophotometer (Beckman) against known concentrations
of glucose. The samples included: 1 mg CMC, 1 mg of the
whole Achlya wall, small colony and 1 mL of


45
cellulase-gold complex alone.
Detection of Gold Particles with a Backscatter Detector
To assure that the particles observed on the hyphal
surfaces were actually the gold particles, the regular
secondary images were compared with the backscattering
images of the same regions. The samples were carbon
coated, instead of the usual gold coating. Backscatter
detector (GW Electronics, USA) was used to detect the
backscattering signal.
Zvmolvase Hydrolysis
Chemically fixed or live colonies were incubated at
room temperature with 0.05 mg/mL zymolyase 100 T
(Arthrobacter luteus) (Seikagaku Corporation) in 66 mM
sodium phosphate buffer pH 7.5. The hydrolysis was
monitored with light microscope. After 24 h the colonies
were fixed, labeled with cellulase-gold complex and
processed for SEM observations.
Treatment of Growing Colonies with Dichlorobenzonitrile
Small colonies were grown in 500 mL flasks
containing 250 mL of PYG. Each flask contained 10 small
colonies. After 12 h of incubation, dichlorobenzonitrile
(DCB) was added to the flasks. DCB was previously
dissolved in 100% DMSO. The concentrations of DCB were
10, 20, 30, 40, 50, 60 100, and 200 pM. The colonies were
incubated in DCB-containing medium and their growth was
monitored with light microscopy. After 36 h of
incubation, the colonies were chemically fixed, labeled


46
with cellulase-gold complex and processed for SEM
observations. The colonies grown in PYG only medium
served as a control in this experiment. The ability of
spores to germinate in the medium containing DCB was also
checked. For this 5 mL of fresh spore suspension were
added to PYG medium with and without DCB.
Results
Cellulose Localization
In growing colonies (incubation in PYG) cellulose is
found on the surface of mature and subapical regions on
the hyphae. In apical regions three patterns of labeling
are found in a single colony. Some of the hyphae
(approximately 5%) are not labeled at the apex and show a
sharp border between labeled and non-labeled regions. The
unlabeled area is approximately 2 to 4 pm in diameter
(Figs. 4-11). In the majority of the hyphae, there is a
gradual decrease of the labeling towards the apex (Figs.
12-19). Some of the hyphae (about 5%) are labeled at the
apex as intensively as in the mature regions (Figs.
20-27). Fifty colonies from different batches were
examined and all of them had this pattern and ratio of
labeling.
In contrast, the labeling of hyphae from colonies
incubated in glucose-only medium gave a different pattern
of cellulose labeling. In these colonies, all the hyphae
were labeled at the apices and the labeling was as
intensive as in mature regions (Figs. 28-35). Ten


47
colonies from different batches were analyzed. Light
microscopic analysis prior to fixation ensured that they
did not contain elongating hyphae.
The surface labeling of hyphae in the
freeze substituted and methanol -fixed colonies has the
same patterns and ratio as in chemically fixed ones (data
not shown).
Cellulase-gold affinity labeling of cross sections
localizes cellulose in the cell wall exclusively (Fig.
36). The distribution of the gold particles in the wall
is even. The level of nonspecific labeling is very low.
On the longitudinal sections of elongating hyphae the
label is present in mature and subapical regions but is
very low or absent in apical regions (Fig. 37) .
There is no labeling on the cross sections (Fig. 38)
or the hyphal surface (data not shown) when the sample of
endocellulase III from Dr. Fowler was used. The
conjugation was successful, based on the raspberry red
color of the enzyme-gold complex and the presence of the
mobile pellet. Negative staining of the enzyme-gold
complex confirmed successful conjugation (Fig. 39).
Cvtochemical controls
All the cytochemical controls support the view that
the cellulase-gold complex is a specific label for
cellulose in Achlya. The preabsorbtion of the labeling
solution with CMC results in the absence of the labeling


48
on the hyphal surface as well as on the cross sections
(Figs. 40, 41).
The incubation of the sections and the colonies with
gold-coupled Goat Anti-Mouse IgG results in the absence
of labeling as well (Figs. 42, 43).
Cellulase pretreatment of the sections and colonies
results in the absence of the labeling (Figs. 44, 45).
The labeling pattern is regular when the
cellulase-gold complex is incubated prior to the labeling
with laminarin or chitosan (Figs. 46-49).
Detection of gold particles on the hyphal surface
with a backscatter detector gave an identical particle
distribution on secondary and backscatter images (Figs.
50-55) .
Cellulase activity during labeling
Cellulase-gold complex shows no enzyme activity
during the labeling of the colonies or whole wall samples
as measured by the production of reducing sugar (Table
I). Absorbance of glucose is measured after 30 min (the
usual time of labeling with cellulase-gold complex) and 3
h. Cellulase-gold is diluted 1:10 with the buffer. Based
on glucose equivalents from a standard curve, cellulase
activity is very low in reactions with a whole wall
preparation or a colony. Thus, there are no additional
primers produced and they do not alter the results of
labeling. Enzyme activity of cellulase-gold complex
against cellulase itself is also low. Cellulase activity


49
Figs. 4-11. Scanning electron micrographs of elongating
hyphae of Achlya bisexualis showing cellulose surface
labeling with cellulase-gold. Note the unlabeled apices.
Bars: 4-9 = 2.00 pm; 10 = 2.31 pm; 11 = 3.00 pm.


50
Figs. 12-19. Scanning electron micrographs of elongating
hyphae of Achlya bisexualis showing surface labeling of
cellulose with cellulase-gold. Note the gradual decrease
of labeling towards the apices. Bars: 12 = 3.33 pm;
13 = 2.73 pm; 14 = 1.67 pm; 15-18 = 2.00 pm; 19 = 3.31
pm.


51
Figs. 20-27. Scanning electron micrographs of surface of
non elongating hyphae present in growing colonies of
Achlya bisexualis showing cellulose labeling with
cellulase-gold. Note the labeled apices. Bars:
20-22 = 3.00 pm; 23, 24 = 2.00 pm; 25 = 1.50 pm;
26 = 2.31 pm; 27 = 3.33 pm.


52
Figs. 28-35. Scanning electron micrographs of
non-elongating hyphae from non-growing colonies of Achlya
bisexualis, incubated in glucose-only medium showing
surface labeling of apices for cellulose with
cellulase-gold. Note the labeled apices. Bars:
28 = 2.31 pm; 29 = 4.29 pm; 30 = 5.00 pm; 31 =
32, 33 = 3.75 pm; 34, 35 = 3.00 pm.
1.50 pm;


53
Fig. 36. Cross section of Achlya bisexualis hypha showing
labeling of cellulose in the cell wall with
cellulase-gold complex. Bar=l pm.


54
Fig. 37. Longitudinal section of the apical region of an
elongating Achlya bisexualis hypha showing labeling of
cellulose with cellulase-gold. Bar=l pm.


55
Fig. 38. Cross section of Achlya
for cellulose with endocellulase
bisexualis hypha labeled
Ill-gold. Bar=0.5 pm.


56
Fig. 39. Negative staining of endocellulase Ill-gold
complex. Bar=200 nm.


57
Figs. 40-41. Electron micrographs showing the absence of
cellulose labeling with cellulase-gold in the cell wall
of Achlya bisexualis resulting from the preabsorption of
the labeling solution with CMC. Bars: 40=1 pm; 41=1.5 pm.


58
Figs. 42-43. Electron micrographs showing the absence of
gold label in the cell wall of Achlya bisexualis
resulting from the preincubation of the sections (TEM) or
colonies (SEM) with colloidal gold-affinipure goat
anti-mouse IgG. Bars: 42=1 pm; 43=1.5 pm.


59
Figs. 44-45. Electron micrographs showing the absence of
cellulose labeling with cellulase-gold in the cell wall of
Achlya bisexualis resulting from the pretreatment of the
sections (TEM) or colonies (SEM) with cellulase. Bars: 44=1
pm; 45=1.67 pm.


60
Figs. 46-47. Electron micrographs showing the regular
pattern of cellulose labeling with cellulase-gold in the
cell wall of Achlya bisexualis resulting from the
preincubation of the labeling solution with chitozan.
Bars: 46=1 pm; 47=1.2 pm.


61
Figs. 48-49. Electron micrographs showing the regular
pattern of cellulose labeling with cellulase-gold in the
cell wall of Achlya bisexualis resulting from the
preincubation of the labeling solution with laminarin.
Bars: 48=0.5 pm; 49=3 pm.


62
50
, *&:+*;*' i'K * v t v. t
% .**> jt
** .*% # :§i &
.p'v t if. > % *t \ */V *t v ;
T wi- ...
t ( MW ¡ :v ^
Jgf t V-V **
v,.-*. \ ^r*
.*£ v-i" **% ?* >
' -* U.a
m * Hi T me T
51
. *.'*'' ^ "*?-* Vf'> ? *
jfr ''If ^ JP * *
.. - 2. # V *4 f '*
: "la fL ** *7^^, :
' H tfgpi .v
V ' .. v .<-
# V p j, .* f ***
52
' 4 *
53
lb
' *r
* -* .* *
- >.. .
> ...' ^ ir*1; i i ' ^
7 -v-V _
v ~ !r.*r ¡i 17^ {
* ; *; > V':<' >k; >'-* >;v
fM **- V -5 ' it
. : v p
-. * * o wf 1
54
m

./ i
' *. *- -r'. '
# *
55
'' v* r . ^ **''*
. ., ''
jP *
* ** ,yk .. * ' v
g¡ y.
Figs. 50-55. Scanning electron micrographs showing the
regular pattern of cellulose labeling with cellulase-gold
on the hyphal surface of Achlya bisexualis. 50, 52, 54.
Secondary images. 51, 53, 55. Backscatter images of the
same regions. Bars: 50 and 51=1.5 pm; 52 and 53=3.00 pm;
53 and 55=857 nm.


63
Table 1. Glucose equivalent from standard curve showing
cellulase activity during labeling
Sample
Glucose equivalent from
standard curve(mg/ml)
Reaction time:
3 0 min
Reaction time:
3 hrs
Whole wall and cellulase-gold
0.009
0.009
Whole wall and buffer
0.005
0.006
Small colony and cellulase-gold
0.004
0.004
Small colony and buffer
0.002
0.002
Cellulase- gold
0.006
0.007
CMC and cellulase-gold
0.02
0.04


64
Figs. 56-57. Scanning electron micrographs showing
cellulose labeling with cellulase-gold of elongating
Achlya bisexualis hyphae that were hydrolyzed with
zymolyase before chemical fixation. Bars: 56=2.31 pm;
57=2.00 pm.


65
Figs. 58-59. Scanning electron micrographs showing
cellulose labeling with cellulase-gold of elongating
Achlya bisexualis hyphae that were hydrolyzed with
zymolyase after chemical fixation. Bars: 58=2.31 pm;
59=1.00 pm.


66
Figs. 60-61. Scanning electron micrographs showing
cellulose labeling with cellulase-gold of an Achlya
bisexualis hypha from a non-growing colony treated with
zymolyase. Bars: 60=3.33 pm; 61=857 nm (higher
magnification of the apex).


67
Fig. 62. Transmission electron micrograph of cross
section of Achlya bisexualis hypha from a colony
incubated in 100 pM DCB. Cell wall cellulose is labeled
with cellulase-gold complex. Bar=0.5 pm.


68
is relatively higher when the enzyme-gold complex reacted
with CMC.
Cellulose Localization on the Surface of the Hvphae in
Colonies Incubated with Zvmolvase
In the colonies, growing in PYG medium, that were
hydrolyzed with zymolyase prior to fixation, most of the
hyphal apices are intact and labeled. A small portion of
the hyphae has broken apices. In these hyphae the
remaining cell wall is labeled with cellulase-gold
complex (Fig. 56, 57).
In the growing colonies that were chemically fixed
first and then hydrolyzed with zymolyase, all the hyphae
have intact apices. Most of the apices are labeled, but
some are not (Fig. 58,59).
The non-growing colonies from glucose-only medium
were screened for the absence of elongating hyphae prior
to the treatment. Time of fixation, before or after
hydrolysis, did not make any difference in the results.
All the hyphae in these colonies are intact and are
labeled as intensively as the rest of the hyphal regions
(Fig. 60, 61).
Hyphal Elongation, Spore Germination and Cellulose
Localization in the Presence of DCB
The presence of DCB in the medium does not affect
the growth of Achlya. It does not affect the morphology
of the hyphae or the growth rate. The average hyphal
elongation rate is 3.6 pm/min. Spores germinate equally


69
well in PYG medium with and without DCB. There is no
difference in the cellulose surface labeling of hyphae
from the colonies treated with DCB and regularly growing
colonies. The labeling patterns and their ratio is the
same (data not shown). Cross sections of hyphae from the
colonies treated with DCB have expected pattern of
cellulose labeling (Fig. 62).
Discussion
The cellulase-gold affinity labeling is specific for
cellulose in the fungus Achlya, based on its
reproducibility and a large variety of controls.
Differences in the fixation techniques do not affect the
labeling pattern. Standard chemical fixation gave the
same results as freeze substitution and cold methanol
fixations. The results are highly reproducible and are
not artifacts of the fixation procedure. Thus this
labeling technique provides a specific and reliable
method for localizing cellulose on thin sections and
hyphal surfaces.
The fact that cellulose labeling was found on the
hyphal surface, may contradict a general assumption that
the microfibrilar component of the cell wall, cellulose
in the case of Achlya, is located next to the plasma
membrane and is covered by the matrix components of the
wall. The cell wall may not be arranged as layers of
components, but as a mixture. This would explain the
presence of some cellulose on the surface, this


70
explanation seems unlikely since Achlya secretes
cellulase during growth (Thomas and Mullins 1967, 1969),
and if cellulose was present on the surface, it could be
hydrolyzed. Furthermore, when cellulase is applied
exogenuously to the living Achlya cultures, it does not
destroy the integrity of the hyphae, nor does it change
their surface as revealed by shadow replicas (Reiskind
and Mullins 1981b). The unique structure of the cellulase
enzyme complex may explain the presence of cellulose
labeling on hyphal surface. The cellulolytic enzyme
complex from Trichoderma reesei used here for labeling
consists of a number of enzymes: endoglucanases (EG);
cellobiohydrolases (CBH); and cellobiase (CB); which work
synergistically. All these enzymes contain a small highly
homologous 36-residue region called the A domain,
connected to the globular enzymatically active core
domain by a threonine- and serine-rich sequence. The A
domain has no catalytic activity in CBH I and CBH II, but
it is thought to have a cellulose-binding function. The
core protein alone does not have full
cellulose-hydrolysing activity, but has normal activity
on small synthetic substrates (Rouveinen et al. 1990).
Perhaps cellulases are able to bind cellulose
microfibrils located inside the wall via the small
cellulose binding domains (CBD). CBD could penetrate the
wall and find the binding sites, while the catalytic
domains, conjugated to gold remain on the surface. The


71
results with EG III labeling indirectly prove the idea of
CBD penetrating the wall and leaving the catalytic
domains attached to gold on the surface. EG III provided
by Dr. Tim Fowler (Genencor International, Inc.) is a
genetically modified enzyme that does not have a
cellulose binding domain (personal communication).
Without the binding domain, this enzyme can not attach to
the cellulose microfibrils and it results in the lack of
EG Ill-gold affinity labeling.
The results of the experiments that measured
cellulase-gold activity during labeling also provide a
support for the idea of CBD penetrating the wall and
leaving the catalytic domain attached to gold on the
surface. The enzyme-gold does not show enzyme activity
against a whole wall sample or a colony, but is active
against the soluble cellulose derivative CMC. Perhaps, in
the case of whole wall and colony treatments the
cellulose binding domain finds the binding site by
penetrating the wall and then attaches to cellulose. The
catalytic domain conjugated to gold does not get access
to cellulose microfibrils surrounded by the matrix
material of the wall. Thus, the binding takes place
without hydrolysis. In the case of CMC the cellulose
microfibrils are not covered, they are available for the
catalytic domain. Therefore, in this case both binding
and hydrolysis take place.


72
According to the results of cellulase-gold labeling,
all the hyphae from non-growing colonies (glucose-only
medium) are evenly labeled in all regions, including the
apices. Light microscope screening prior to the EM
processing ensured that these hyphae are not elongating.
On the other hand, cellulase-gold labeling of the hyphae
from growing colonies (PYG medium) revealed three
patterns of cellulase-gold labeling at the apices:
labeled (small portion of hyphae), unlabeled and with the
decreasing label towards the apex. Light microscopic
observations prior to fixation revealed that a small
portion of hyphae in these colonies is not elongating,
but the majority of the hyphae are elongating. Based on
this, I propose that in non-elongating hyphae cellulose
is evenly distributed along the hypha and is present in
the apex. Elongating hyphae lack cellulose at the apices
or there is a gradual decrease in the amount of cellulose
toward the apices.
The results of the experiments with zymolyase
hydrolysis support the conclusion that in some hyphae in
growing colonies there is no cellulose in the apical cell
wall. In the colonies that were treated with zymolyase,
prior to fixation, these hyphae have broken apices. I
explain this by the fact that the wall in these apical
regions lacks cellulose and consists mainly of
1,3-fi-glucans. Zymolyase has both 1,3-B-glucanase and
protease activities. It hydrolyzes not only


73
1,3-B-glucans, but also structural wall proteins, cell
membrane proteins and cytoplasmic proteins. Thus, the
elongating regions have "hydrolyzed" apices. In the
colonies that were chemically fixed first and then
hydrolyzed with zymolyase, the elongating hyphae have
intact unlabeled. The apices are not broken as in the
previous case because chemical fixation crosslinks
proteins so they can not be hydrolyzed. As expected, in
both experiments, non elongating hyphae (glucose only
medium) have intact apices with cellulose labeling as
intensive as in the other regions of the hyphae.
The experiments with DCB gave an unexpected result.
In these experiments it was the intention to use a
different approach to show the absence of cellulose in
the wall of elongation regions. DCB is a classic
inhibitor of cellulose biosynthesis in higher plants
(Delmer 1999). It was expected that the hyphae would
continue to elongate by synthesizing 1,3 -B-glucans and
producing large regions of apical wall made mainly of
this component and that these hyphal regions lacking the
structural support of cellulose might not have a tubular
form. However, DCB had no inhibition effect on the growth
process of Achlya. There were no changes in hyphal
morphology as revealed by light microscopy, TEM or SEM
observations. Hyphal elongation rates in colonies
incubated with DCB were the same as in the regular growth
medium. Cellulose labeling of the cross sections and the


74
hyphal surface revealed no difference between the hyphae
grown in regular medium or the medium with DCB. Perhaps,
the cellulose biosynthesis system of Achlya is different
from that found in higher plants in the step that is
affected by DCB, or DCB molecules may not be able to
penetrate the Achlya wall. Similar results for the lack
of an inhibitory effect of DCB were found in the cellular
slime mold Dictyostelium (Blanton 1997, Blanton personal
communication). Actually, none of the three major
cellulose-synthesis inhibitors used in higher plants--
DCB, isoxaben, and pthoxazolin--had an effect in this
organism.


CHAPTER 5
LOCALIZATION OF 1,3 -B-GLUCANS IN THE CELL WALL AS
REVEALED BY ELECTRON MICROSCOPY AND CYTOCHEMICAL
TECHNIQUES
Introduction
The term glucan applies to polysaccharides composed
of glucose units and they are divided into alpha- and
B-anomers according to their stereochemistry around the
anomeric carbon. The B-glucans include both
homopolysaccharides and heteropolysaccharides. Six
different types of B-glucans have been described in
fungi: linear 1,3-glucans; 1,3-glucans with occasional
1-6 single glucose branches, with or without phosphate;
1,3-glucans with significant amounts of 1,6-branches;
glucans containing mostly 1,6 linkages; glucans
containing 1,3-, 1,4- and 1,6- linkages (Ruiz Herrera
1991).
These B-glucans are getting attention because of
their potential application in chemical, pharmaceutical
and food industries. Pharmaceutically, 1,3-B-glucans that
have 6-glucopyranosyl units attached by l->6 linkages as
single unit branches have been shown to enhance the
immune system. This enhancement results in antitumor,
antibacterial, antiviral, anticoagulatory and wound
healing activities (Bohn and Bemiller 1995).
75


76
The 1,3 £ -glucans are important components of fungal
cell walls and they are also storage carbohydrates in
some fungi, especially in the Oomycetes and
Basidiomycota. Some ft-glucans are secreted in the form of
slimy material, and may protect cells from desiccation
and other harmful environmental conditions (Ruiz Herrera
1991). In the case of pathogenic fungi, ft-glucans are
important in cellular recognition, and in eliciting
defense responses of infected plants (Ryan 1987, Dixon
and Lamb 1990, Cote and Hahn 1994).
Storage glucans accumulate intracellularly and are
used as reserve material at critical stages of growth and
reproductive development (Wang and Bartnicki- Garcia 1980,
Lee and Mullins 1994). In Phytophthora, Wang and
Bartnicki- Garcia (1973) reported a phosphorylated
1,3-ft-glucan in sporangia, zoospores and cysts. This
phophorylated 1,3-ft-glucan contains one or two phosphate
residues as monoester linkages at the C-6 hydroxyl groups
of some glucose units. In Achlya, a phosphorylated
cytoplasmic 1,3-ft-glucan has been isolated and
characterized (Lee and Mullins 1994, Lee et al 1996),
containing 5% phosphate (w/w), and has both mono-and
diphosphoester linkages. The diester linkages are used to
form very large polymers from the smaller neutral forms.
Although the biological role of the reserve 1,3 ft-glucans
is most often suggested as a source of energy or carbon


77
or both, in Achlya it is also an important site of
phosphate storage (Lee and Mullins 1994).
The most general role of B-glucans is a structural
one, as the major component of fungal cell walls.
Inhibition of B-glucan synthesis in yeast leads to cell
lysis and often death, resulting from a weakening of the
cell wall (Perez et al. 1983, Miyata et al. 1985). Such
inhibitors are used as important antifungal compounds
against both plant and animal pathogens.
The 1,3-B-glucans were localized on cross sections
of Achlya (Shapiro and Mullins 1997). The method used
indirect immunolabeling with a commercial polyclonal
antibody specific for 1,3-B-D-glucopyranose linkages. The
glucans occurred in the cell wall, the large vesicles in
the organelle-rich areas of the hypha, and in the large
central vacuole in more mature areas. Preabsorption of
the antibody with either purified neutral or
phosphoglucan from Achlya completely eliminated
subsequent labeling of hyphal sections. No labeling of
the large population of apical vesicles was found,
suggesting that these reserve glucans are not directly
involved in apical growth. Since the labeling occurred in
large vesicles and the central vacuole and no other
cytoplasmic sites showed conjugation, the vesicle and
vacuole membranes probably contain the synthases
responsible for the biosynthesis of the reserve glucans.
The labeling of serial sections revealed the


78
1,3 -B-glucans in both mature and apical regions.
Additional labeling experiments have now been carried out
on hyphae that were first determined to be elongating, as
described in Chapter 3, to ensure that elongating apices
contained 1,3 6 -glucans. Recall that in Chapter 4,
evidence was presented that clearly demonstrated a lack
of cellulose in elongating apices.
Materials and Methods
Culture Methods, Fixation and Microscopy Techniques
The general culture methods, fixation and microscopy
techniques are described in Chapter 3 and Chapter 4.
Localization of 1,3-B-Glucans Using Monoclonal Antibody
The primary antibody, raised in mouse against a
laminarin-haemocyanin conjugate, was purchased from
Biosupplies Australia PtyLtd (Parkville Victoria,
Australia), catalogue number 400-2. This antibody
recognizes linear 1,3-B-oligosaccharide segments in
1,3 6 glucans The epitope includes at least five
1,3-B-linked glucopyranose residues. It has no cross
reactivity with 1,4 6-glucans or 1,3-6-, 1,4-B-glucans
(Meikle et al. 1991). It was diluted 1:100 in phosphate
buffered saline (PBS), pH 7.2 containing 0.5 % cold water
fish gelatin. The gold reagent, 18 nm Colloidal
Gold-Affinipure Goat Anti-Mouse IgG (H+L) was purchased
from Jackson ImmunoResearch Laboratories, Inc. (West
Grove, Pennsylvania), catalogue number 115-215-146. The
nickel grids with sections were floated on PBS containing


79
1% gelatin for 30 min to block non-specific labeling.
Then the grids were floated on the top of 20 pi drops of
the primary antibody solution for 1 h. After three washes
in PBS the grids were floated on a 1:20 dilution of the
gold reagent in PBS for 1 h. The solution was centrifuged
at 18 000 g in a microcentrifuge for 1 min before use.
Finally, the grids were washed: twice in PBS and twice in
water.
The colonies destined for SEM observations were
treated with the same series of solutions while
completely submerged into them, rather than floated.
Cvtochemical Controls
(1) Preabsorbtion of primary antibody with
laminarin: in order to determine that the primary
antibody binds to 1,3 6 linkages it was preabsorbed with
laminarin. Laminarin from Laminaria digitata was
purchased from Sigma (St. Louis, Missouri), catalogue
number L-9634. The grids with sections were blocked
against nonspecific labeling as described above. Primary
antibody stock solution, 10 pi, was incubated with 100 mg
of laminarin in 1 ml of PBS containing 0.5 % gelatin for
1 h. The grids were floated on a drop of this solution
for 1 h. The labeling procedure as described above was
then followed. (2) Omission of the primary antibody: the
procedure was the same as during the regular labeling,
but the incubation with the primary antibody was omitted.
(3) Replacement of the primary antibody with a non-


80
specific primary antibody raised in mouse: the procedure
was the same as during the regular labeling but the
primary antibody was replaced by an undiluted
non-specific antibody (HL 1099) raised against
neurofilaments in mouse. HL 1099 was provided by the
Hybridoma Laboratory, Interdisciplinary Center for
Biotechnology Research (Gainesville, Florida).
Results
Localization of 1,3-fi-Glucans on Sections and Hyphal
Surfaces Using Monoclonal Antibodies
On cross sections of Achlya the monoclonal antibody
detected 1,3-B-glucans in the wall, small vacuoles, and
the large central vacuole of mature regions (Figs. 63,
64). No cytoplasm-specific labeling was found.
Longitudinal sections of both elongating (Fig. 65) and
non elongating (data not shown) hyphae show antibody
labeling in the cell wall of the apices and all along the
hyphae. No surface labeling was found using SEM (Fig.
66) .
Cytochemical Controls
(1) Preabsorbtion of primary antibody with laminarin
resulted in the absence of labeling (Fig. 67). (2)
Omission of the primary antibody resulted in the absence
of the labeling (Fig. 68). (3) Replacement of the primary
antibody with a non-specific primary antibody raised in
mouse resulted in the absence of the labeling (Fig. 69).


81
Fig. 63. Transmission electron micrograph showing
localization of 1,3 -J3-glucans with monoclonal antibody on
cross section of Achlya bisexualis hypha. Bar=0.5 pm.


82
Fig. 64. Transmission electron micrograph showing
localization of 1,3-B-glucans with monoclonal antibody on
cross section of Achlya bisexualis hypha. Bar=1.00 pm.


83
Fig. 65. Transmission electron micrograph showing
localization of 1,3 J3 glucans with monoclonal antibody on
longitudinal section of an elongating Achlya bisexualis
hypha. Bar=1.00 pm.


84
Fig. 66. Scanning electron micrograph showing the absence
of 1,3-B-glucans labeling on the surface of Achlya
bisexualis hypha. Bar=2.31 pm.


85
Figs. 67-69. Transmission electron micrograph showing the
absence of 1,3 J3 glucans labeling on the cross sections
of Achlya bisexualis hyphae resulting from: 67.
preabsorbtion of the labeling solution with laminarin.
Bar=1.00 pm; 68. omission of the primary antibody.
Bar=1.00 pm; 69. replacement of the primary antibody with
a non-specific antibody raised in mouse. Bar=1.00 pm.


86
Discussion
Based on the results of the cytochemical controls,
the monoclonal antibody used in the labeling procedure is
specific for 1,3 6-glucans. Previous results of labeling
with a polyclonal antibody (Shapiro and Mullins 1997) are
identical to the results of monoclonal antibody labeling.
Both antibodies detect 1,3-J3-glucans in the cell wall and
vacuoles. Previously, serial of cross sections of apical,
subapical, and mature regions of a hypha were
immunostained with the polyclonal antibody and strong
labeling was found in the wall on all the sections
(Shapiro 1995) Based on the data presented in Chapter 3,
it can not be ascertained whether this hypha was
elongating or non elongating. In the present study,
however, the elongating hyphae are distinguished from
non elongating ones and the distribution of 1,3 -B-glucans
is compared. It is now possible to state that
1,3 J3 glucans are found in the apical wall of both
elongating and non elongating hyphae.


CHAPTER 6
LOCALIZATION OF CHITIN IN THE CELL WALL AS REVEALED BY
ELECTRON MICROSCOPY AND CYTOCHEMICAL TECHNIQUES
Introduction
Chitin is the most characteristic polysaccharide of
the fungal cell walls. It is an unbranched polysaccharide
made of N- acetylglucosamine (GlcNAc) joined through 1,4-B
bonds. It was once thought to be absent in fungi
containing cellulose, but a number of examples from all
orders of the Oomycetes have demonstrated a least traces
of chitin (Dietrich 1973, 1975). An insoluble fraction
from the hyphal wall of Achlya radiosa Maurizio was
characterized by x-ray and infrared analyses as chitin,
and represented about 4% of the total wall (Campos-Takaki
et al. 1982). The role of chitin in Oomycete cell wall
remains unclear, and it has been suggested in Saprolegnia
that chitin does not play an important role in
morphogenesis based on results using the chitin synthase
inhibitor polyoxin D (Bulone et al. 1992). An insoluble
residue representing about 3% of the wall and containing
glucosamine was reported in Achlya (Reiskind and Mullins
1981a). This fraction was identified as chitin by x-ray
analysis (Mullins et al. 1984), but had unusual
properties in that it is more highly crystalline than the
87


88
alpha-chitin normally observed in fungi and the
characteristic lattice spacing was not readily
perceptible. Thus chitin is clearly present in those
fungi having cellulose as the major microfibrillar
component; but its role is yet to be determined.
Materials and Methods
Chitin Localization Using Lectin
Tomato (Lycopersicon esculentum) lectin conjugated
to gold was purchased from EY Laboratories, Inc. (San
Mateo, California). The tomato lectin is described by the
manufacturer as being specific for oligomers of
1,4-B-linked N-acetylglucosamine, with the binding site
being able to accommodate up to 4 carbohydrate units and
these units do not have to be consecutive. The sections,
on grids, were pretreated with phosphate buffer saline
(PBS) containing 1% bovine serum albumin (BSA) at room
temperature for 30 minutes. Then they were floated on the
labeling solution for 30 minutes. The lectin-gold complex
was a 1:9 dilution of the stock solution in PBS. The
samples then were washed with PBS three times and rinsed
twice in distilled water.
Cvtochemical Control
To determine that the lectin-gold complex was
binding to chitin, the probe was pre-incubated with
re-acetylated glycol chitosan provided by Dr Michael N.
Horst (Mercer University, Macon, Georgia). The glycol
chitosan stock (0.118 g/100 mL) was diluted with PBS


89
(1:9). One part of the lectin-gold stock solution was
diluted with nine parts of glycol chitosan and incubated
for 30 min before labeling of the sections.
Results
Chitin is localized to the cell wall of Achlya
bisexualis with tomato lectin-gold conjugate, where it is
evenly distributed in the cross sections (Fig. 70). The
chitin labeling is absent when the labeling solution is
preincubated with glycol chitozan (Fig. 71).
Discussion
The tomato-lectin-gold conjugate appears to be a
specific label for chitin in the cell walls of Achlya,
based on the lack of labeling when the conjugate was
pre-incubated with re-acetylated glycol chitosan.
Previous studies on chitin (Campos-Takaki et al 1982,
Mullins et al 1984, Gay et al 1992) demonstrated its
presence in the cell walls of oomycetes with biochemical
and biophysical analyses. This is the first report of the
cytochemical localization and distribution of chitin in
the walls of this group. Bulone et al 1992 described
chitin as small globular particles in Saprolegnia, and
found that hyphal growth and morphology were not altered
when chitin synthesis was inhibited by polyoxin D. They
concluded that chitin did not seem to play an important
role in morphogenesis. Additional biophysical work on
Saprolegnia (Gay et al 1992) describe chitin as localized
small round granules of crystalline microfibrillar alpha


90
chitin. Chitin, however, synthesized in vitro appeared as
spindle-like particles, and was not a skeletal
polysaccharide involved in wall architecture. In
regenerating protoplast walls it might have a secondary
role in wall architecture, since it is microfibrillar.
Thus the full role of chitin is still to be determined.


Fig. 70. Transmission electron micrograph showing
localization of chitin with tomato lectin on cross
section of Achlya bisexualis hypha. Bar=0.5 pm.
Fig. 71. Transmission electron micrograph showing the
absence of chitin labeling on the cross sections of
Achlya bisexualis hyphae resulting from preincubation of
the labeling solution with re-acetylated glycol chitozan.
Bar=1.00 pm.


CHAPTER 7
CONCLUSIONS
The results of cellulose localization suggest that
in non-elongating hyphae, cellulose is evenly distributed
along the hypha and is present in the apex. Elongating
hyphae lack cellulose at the apices or there is a gradual
decrease of cellulose amount toward the apices. On the
other hand, the major matrix component of the wall,
1,3 B-glucan, is distributed evenly over the elongating
and non-elongating hyphae and is present in their apices.
Such distribution of these two major components of Achlya
cell wall suggests that in the elongation zone,
1,3-B-glucans are synthesized first and cellulose
deposition follows. This contradicts the idea shared by
the major theories of hyphal tip growth, that all the
wall components are present in the elongation zone.
The small diameters of the cellulose unlabeled
regions of elongating hyphae suggest that cellulose
deposition takes place almost immediately after the start
of 1,3-B-synthesis. The plastic wall that consists mainly
of 1,3-B-glucans and lacks cellulose support is stretched
under the turgor pressure and/or perhaps pressure of
cytoskeleton. The quickly following cellulose deposition
helps to maintain the tubular cell shape and prevents the


93
elongation regions from "blowing out" in balloon-like
structures. Cellulose is thought to provide mechanical
support for the cell wall. The initial cellulose
hydrolysis by cellulase in the growing apex is possible.
This could create new primers in existing cellulose
chains, as suggested by Maclachlan (1976) for higher
plants. It was found that the growing colonies of Achlya
secrete endocellulase (Thomas and Mullins 1967; 1969).
The authors suggested that endocellulase is important for
the wall softening since this fungus does not use
cellulose in nutrition. The recent evidence that activity
of the secreted endocellulase correlates with the tensile
strength of the apical hyphal wall support this idea
(Money and Hill 1997).
The results of cellulose and 1,3 6-glucans
distribution in the apical wall, combined with the
results of hyphal growth monitoring suggest a new aspect
of the hypothesis for hyphal tip growth. This hypothesis
would state that in the Achlya growth process, all hyphae
go through periods of elongation and no elongation
(dormancy). The elongation is not a steady process as it
is generally assumed. It consists of alternating periods
with fast and slow growth rates. Elongation starts with
synthesis of 1,3--glucans, which is quickly followed by
synthesis of cellulose.


LITERATURE CITED
Alexopoulos CJ, Mims CW, Blackwell M. 1996. Introductory-
Mycology. New York: John Wiley and Sons. 869 p.
Arioli T, Peng L, Betzner AS, Burn J, Wittke W, Herth W,
Camilleri C, Hofte H, Plazinski J, Birch R, Cork A,
Glover J, Redmond J, Williamson RE. 1998. Molecular
analysis of cellulose biosynthesis in Arabidopsis.
Science 279:717-720.
Aronson JM. 1981. Cell wall chemistry, ultrastructure and
metabolism. In: Cole GT, Kendrick B. eds. Vol. 2: Biology
of conidial fungi. New York: Academic Press, p 459-471.
Aronson JM, Preston RD. 1960. The microfibrillar
structure of the cell walls of the filamentous fungus,
Allomyces. J Biophys Biochem Cytol 8:245-256.
Barkai-Golan R, Sharon N.. 1978. Lectins as a tool for the
study of yeast cell walls. Exp Mycol 2:110-115.
Bartnicki-Garcia S. 1968. Cell wall chemistry,
morphogenesis, and taxonomy of fungi. Ann Rev Microbiol
42:57-69.
Bartnicki- Garcia S. 1973. Fundamental aspects of hyphal
morphogenesis. Pp. 245-267. In: Arthwoth JM, Smith E.
eds. Microbial differentiation. Symp Soc Gen Microbiol.
Cambridge: University Press, p 245-267.
Bartnicki- Garcia S. 1996. The hypha: unifying threads of
the fungal kindom. In: Sutton BC. Ed. A century of
mycology. Cambridge: University Press.
Bartnicki-Garcia S, Ruiz-Herrera J, Bracker CE. 1979.
Chitosomes and chitin synthesis. In: Burnett JH, Trinci
APJ. eds. Fungal walls and hyphal growth. Cambridge:
University Press, p 149-168.
Beakes GW. 1987. Oomycete phylogeny: ultrastructural
prospectives. In: Rayner ADM, Brasier CM, Moore D. eds.
Evolutionary Biology of Fungi. Cambridge: University
Press, p 405-421.
94


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HYPHAL TIP GROWTH: MOLECULAR COMPOSITION OF ELONGATING A D NON-ELONGATING REGIONS OF ACHL YA CELL WALL By ALEXANDRA SHAPIRO A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2000

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T AB LE O F CO N T EN TS P a ge ACKNOWLEDGMENT s ....................................................................................................................................... i V ABSTRACT ............................................................................................................................................................... v CHAPTERS 1 INTRO D U C TI O N .......................................................................................................................................... 1 Api cal G rowth ................................................................................................................................. 1 The Organi sm .................................................................................................................................... 3 Cla ss O o my cetes ........................................................................................................................... 5 2 L I T E R AT UR E REVI EW ........................................................................................................................... 9 Mech a ni sm of Apical Growth .......................................................................................... 9 S tr uc t ure of the Hyphal Wall ................................................................................. 10 El ectron Microscopic Studies of Fungal Cel l W a lls .................. 14 Bi os y nthesis of the Fungal Cell Wall ......................................................... 21 R o l e of Turgor in Wal 1 Exp ans ion ..................................................................... 2 5 Rol e o f C y toskeleton in Hyphal Grow t h .... ................................................. 26 Cy tolog y of Growing Hyphal Apices .................................................................. 2 8 Cal c iu m G r adient ..................................................................................................................... 31 Ion Curren ts ................................................................................................................................. 3 2 3 HY P H A L G R OWTH .................................................................................................................................... 3 3 Int ro d u c t ion ................................................................................................................................. 3 3 Ma t er i als and Methods ............................................................................................. 33 Resul ts ....................................................................................................................................... 3 5 Disc uss i on .............................................................................................................................. 3 5 4 LOCALIZATI O N OF C E LLULOSE IN THE CEL L WA LL AS REVE ALED BY ELECTRON MICROSCOPY AND CYTOCHEMI C AL T E C H NIQUES ............................ ............................................ ... .................. 3 9 In troduction ............................................................................................................................. ... 3 9 Materials an d Met ho d s ...................................................................................................... 40 Culture Met ho ds and M icr oscop y Te ch niques ................................... .4 0 Electron Microsc op y ............. ........................................................................................ 40 Cellulose Localization Using Enzyme-Gold Affinity Label ing ................................................................................................... 4 2 Cytochemical Control s ................................................................................................ 4 3 Cellulase Enzyme Activity during Labeling ................................... .44 Detection of Gold Particles with a Backscatter De tee tor .......................................................................................... 4 5 Zymolyase Hydrolysis ................................................................................................... 4 5 Treatement of Gro w in g C ol o nies w ith Di chl orobenzoni tr i 1e .......................................................................................... 45 R e sults ................................................................................................................................................ 46 Cellulose Localization ....................................................................................... 46 ii

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C y tochem i ca l Con tr ol s ............................................................................................. 4 7 Cel 1 ulase A ct i v ity dur i ng Label ing ...................................................... 4 8 Cel lu lose L o c a liz a t ion on the Surface of t he H y pha e in Co l on i es Incubated with Zyrnol y ase ................................................................................................... .... 6 8 H y phal Elongation, Spores Ge rmin a ti o n and Cellulose Localization in the Presen ce of DCB .................................................................................................................................... 68 Discussion ....................................................................................................................................... 69 5 LOCALIZATION OF 1,3-B-GLUCANS IN THE CELL WALL AS REVEALED BY ELECTRON MICROSCOPY A N D CYTOCHEMICAL TECHNIQUES .................................................................................... 7 5 In troduction ................................................................................................................................. 7 5 Materials and Methods ...................................................................................................... 7 8 Culture Methods, Fixation and Microscop y Te c hn i qu es ........................................................................................................................ 7 8 Localization of 1,3-B-Glucans Using Monoclonal Antibody .......................................................................................... 7 8 Cy to ch em i ca 1 Cont r o 1 s ............................................................................................. 7 9 Results ................................................................................................................................................ 8 0 Localization of 1,3-B-Glucans on Sections and Hyphal Surf aces Using Monoclonal An tibodies ..................... 8 0 Cytochemical Controls ............................................................................................. 80 Di s cu s s i on ....................................................................................................................................... 8 6 6 LOCALIZATION OF CHITIN IN THE CELL WALL AS REVEALED BY ELECTRON MICROSCOPY AND CYTOCHEMICAL TECHNIQUES ................................................................................................ 87 In troduc tion ................................................................................................................................. 8 7 Materials and Methods ...................................................................................................... 88 Chitin Localization Using Lectin ............................................................... 8 8 Cy to ch em i ca 1 Cont r o 1 s ................................................................................................ 8 8 Results ................................................................................................................................................ 8 9 Discussion ...................................................................................... ............................................. 89 7 CONCLUSIONS .......................................................................................................................................... 9 2 L I S T OF REFERENCES ............................................................................................... ................. ............ 9 4 BIOGRAPHICAL SKETCH ........................................................................................................................ 10 7 iii

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ACKNOWLEDGMENTS I thank Drs. Tom Emmel, Greg Erdos and Alice Harmon for serving as members of my supervisory committee, and for their time and expertise. I would like to express my appreciation to Dr. J.T Mullins, my supervisory chairman, for his support, understanding, patience interest in the research project and his help and guidance throughout the work on my dissertation I also would like to thank Karen Vaughn of ICBR EM core lab for her technical assistance My special thanks go to Scott Whittaker of the same lab for his help, time and technical expertise The support of my family was very important for me during these years. I am truly indebted to my parents and my parents-in-law for their love, constant encouragement and inspiration, help with the kids and readiness to help anytime I needed it Finally, my greatest gratitude goes to my husband, Andrei Sourakov. His love, patience and help made the completion of my d i ss e rtation possible. iv

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Abstract of Dissertation Presented for the Gradua t e School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Docto r of Philosophy HYPHAL TIP GROWTH: MOLECULAR COMPOSITION OF ELONGATING AND NON-ELONGATING REGIONS OF ACHLYA CELL WALL By ALEXANDRA SHAPIRO December 2000 Chair: J Thomas Mullins Major Department: Botany Although apical growth is a widespread process in the biological world and has been known for over a hundred years, the mechanisms that underlie this proce~s are not yet understood. Knowledge of these mechanisms would allow the development of techniques for inhibiting or stimulating growth of medically or economically important species. I approached the problem of hyphal tip growth by comparing the cell wall composition of elongating and non-elongating regions of the oomycete Achlya bisexualis. Light microscope observations were used to determine the growth rate and to distinguish elongating and non-elongating hyphae for further EM studies, because non-elongating hyphae often are f ound among growing mycelia. I found that hyphal growth is a

PAGE 6

discontinuous irregular process with periods of elongation and no elongation. The elongation rate is not steady, but instead fluctuates with periods of fast and slow elongation. Both transmission and scanning electron microscopes were used with a variety of cytochemical labels, and several fixation techniques. Cellulose, the microfibrillar component of the Achlya wall, was identified with cellulase enzyme-gold affinity labeling. Elongating hyphae have cellulose in mature and subap i cal regions, but not at the apex. In non-elongating hyphae, cellulose was found in all the regions including the apex. These results suggest that the apices of elongating hyphae lack cellulose. This contradicts the long-standing hypothesis that the microfibrilar component is present in the elongating hyphal apex. The 1,3-B-glucans, the major matrix wall components, were immuno-localized in all regions of elongating and non-elongating hyphae. A number of cytochemical, biochemical and physiological contro l s were performed to assure the reliability of these findings. I suggest that in elongating regions, the matrix is synthesized first and synthesis of microfibrilar component follows. Another explanation for these results is that localized apical cellulose hydrolysis by endoglucanase creates plastic wall regions consisting mainly of 1,3-B-glucans, which expand under turgor and/or cytoskeleton pressure. Cellulose deposition quickly follows to prevent "blowing out" of the hypha. vi

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CHAPTER 1 INTRODUCTION Apical Growth Hyphal tip growth is a hallmark of the fungi, even though it also occurs in specialized plant cells (i.e., growth of pollen tube, root hair, moss protonema). Diverse animal cells share this capacity to protrude their cytoplasm and then move in that direction, a process termed ameboid movement. The essential feature of tip growth is that the tip of the hypha is protruded into the enviroment from the subapical region. This protrusion involves the synthesis and extension of the cell wall and cytoplasm (with its contained organelles). The organism is thus able to explore and exploit its environment. Although apical growth is a widespread process in the biological world and has been known for over a hundred years, the mechanisms underlying this process are not yet understood. Knowledge of these mechanisms would allow an understanding of other related characteristics of fungi, such as the influence of environmental factors on growth and morphogenesis and the interaction between fungi and other organisms. Ultimately, detailed knowledge of hyphal tip growth would allow the development of techniques for

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2 inhibiting or stimulating growth of medically or economically important species. Studying hyphal tip growth is a complex problem because the apex represents only a tiny part of a hypha. Most of the important growth events occur within 5 micrometers of the tip. On the other hand, the mature part of the hypha is not inactive. In growing hyphae, the wall synthesis per unit area is maximal at the tip The total amount of wall material synthesized subapically at the same time is appreciable (Sietsma et al 1985). This also contributes to the difficulty of studying tip growth. Finally not all of the hyphae in an actively growing colony are growing (apically elongating) at a given moment in time. Therefore, conventional biochemical, autoradiographical and cytological techniques must be adapted to the specificity of the problem In this study I approached the problem of hyphal tip growth by comparing cell wall architecture in elongating versus non-elongating hyphal apices of an oomycete Achlya bisexualis Electron microscopy, both transmission and scanning was used with a variety of immunocytochemical labeling of hyphae. Several fixation techniques were used to ensure that the results were not only reproducible but also not artifacts of the fixation procedure The results allowed me to propose a new hypothesis for the mechanism of hyphal tip growth.

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3 The Organism Members of the genus Achlya grow as branched coenocytic hyphae, which collectively are termed a mycelium. Septa are formed only to delimit reproductive structures, while vegetative growth occurs at the apex. Achlya has both asexual and sexual cycles of reproduction Asexual reproduction occurs by fragmentation, differentiation of resistant gemmae, or by the differentiation of vegetative apices into sporangia (Sparrow 1960). Achlya differs from related genera b y the fact that the primary zoospores immediately encyst in a loose cluster at the orifice of the sporangium after discharge (Johnson 1956). Sexual reproduction occurs by gametangial contact. The male gametes produced in an antheridium are transported via fertilization tubes to female gametes produced in an oogonium (Mullins 1994). Sexual morphogenesis is initiated and sequentiall y controlled by a series of diffusible steroid hormones (Raper 1939). While most water molds are monoecious, bearing both male and female reproductive structures on a single diploid mycelium, some members of the genus Achlya are dioecious True "male" and "female" strains of dioeciou s species of Achlya may exist, but the expression of mating type in a

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4 strain depends on that of its mating partner (Raper 1939). The involvement of hormones in sexual reproduction in this genus is very noteworthy as species of Achlya appear to be the most primitive eukaryotes known to produce and respond to steroids. Achlya has been proposed as a eukaryotic model system for studying basic mechanisms of growth and development Species of Achlya have been used to investigate the regulatory mechanisms of steroid-hormone-induced and regulated sexual differentiation (Thomas and Mullins 1967, Mullins and Ellis 1974, Horgen 1977, Riehl and Toft 1984, Mullins 1994). They also served in studies on: (i) the differentiation of vegetative hyphae into asexual sporangia (Griffin and Breuker 1969, Thomas et al. 1974, LeJohn et al. 1977, Kropf et al. 1983, Cottingham and Mullins 1985) ; (ii) the mechanism of nutrient transport in fungi (Cameron and LeJohn 1972, Manavathu and Thomas 1982, Kropf et al. 1984); (iii) the tropic responses to nutrients and other chemoattractants (Musgrave et al 1977, Manavathu and Thomas 1985) ; (iv) the role of turgor in hyphal tip growth (Money and Harold 1992, 1993) ; and (v) ionic and electrical currents (Harold 1994). In this study I used Achlya bisexualis Coker and A Couch (ATCC accession number 14524) to investigate the mechanisms of hyphal tip growth.

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5 Class Oomycetes The genus Achlya i s classified in t he family Saprolegniaceae, order Saprolegniales, c l ass Oomycetes subdivision Mastigomycotina, division Eum y co t a o f the kingdom Fungi (Carlile and Watkinson 1994). The subdivision Mastigomycotina contains organisms that produce motile spores (zoospores). The subdi v ision is divided into three classes, based on the morpholog y o f zoospores : Chytridiomycetes, Oomycetes, and Hyphochytriomycetes The first class is similar to other Eumycota, while the latter two show similarities to some protists rather than to fungi. In fact, the morphological divergence of the Oomycetes has long been recognized based on their morphology (Gaumann and Dodge 1928). Their biochemical properties, such as L-lysine biosynthesis (Vogel 1964), cell wall chemistry (Bartnicki-Garcia 1968), and tryptophan-pathway enzyme organization (Hutter and DeMoss 1967) strongly support this view. More recent ultrastructural (Beakes 1987) and molecular studies (Lovett and Haselby 1971, Ohja et al. 1975, Kwok et a l 1986, Forster and Coffey 1990, Forster et al. 1990) a l so confirmed the divergence of the Oomycetes. According to Bartnicki-Garcia (1996), these biochemical and morphological differences indicate that the Oom y cetes and the higher fungi probably arose from different ancestors. However, the same author disagrees with the idea of breaking up the kingdom Fungi based on these phylogenetic

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6 considerations. In the past, the classes Chitridiomycetes, Oomycetes, and Hyphochytriomycetes often have been grouped with nonfungal organisms with which they have very little in common, either on a physiological, morphological, or ecological basis. For example, the Oomycetes were lumped with heterokont algae in the kingdom Chromista (Cavalier -S mith 1983, Moore-Landecker 1996), or were placed with all zoosporic fungi, protozoa and algae in kingdom Protoctista (Margulis et al. 1990). An admittedly polyphyletic kingdom Fungi is a more rational taxonomical solution than the ones listed above. This solution allows us to assemble and study the collection of organisms that share key morphological physiological and ecological properties (Bartnicki-Garcia 1996) Though my work does not concern systematics, an understanding of the phylogenetic position of Achlya is relevant to the problem of hyphal tip growth. Because the Oomycetes could have evolved independently, their mechanism of hyphal tip growth, despite its superficial similarity to one of true fungi, could prove to be different. There are about 600 species of the Oomycetes. The sexual phase of the Oomycetes has a clear differentiation into large female and small male structures, termed oogonia and antheridia These are the sites of meiosis and gametogenesis. Each oospore produced after

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7 fertilization has a singl e diploid nucleus When the oospore germinates it gives rise to a diploid vegetative mycelium in contrast to the haploid mycelium of most fungi Other characters of the Oomycetes that distinguish them from the Eumycota are the biflag ellate zoospore ; mitochondria with tubular cristae; Golgi bodies consi sting of multiple flattened cister nae ; cellulose as a microfibrillar component of the cell wall; the presence of the amino acid hydroxyproline in cell wa ll glycoproteins ; and various other biochemical and molecular characteristics (Carlile and Watkinson 1994) True fungi have mitochondria with platelike cristae and produce Golgi bodies that are very simple in structure, often consisting of only a single cisternal element. Cell walls of true fungi have chitin as the microfibrilla r component and do not contain hydroxyproline (Alexopoulos et al. 1996). The class Oomycetes consists of 5 orders: Saprolegniales, Lagenidiales, Peronosporales, Rhipid iales and Leptomitales (Alexopoulos et al. 1996). The order Saprolegniales contains a single family Saprolegniaceae. Usually these fungi occur in fresh water and in soil as saprotrophs and play an important role in decomposition and recycling of materials in aquatic ecosystems. Some, however, are obliga te parasites of plants, animals, or other fungi. For example, some species of Saprolegnia, Achlya, and Aphanomyces attack fish and their eggs

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8 (Alexopoulos et al 1996) The members of Saprolegniaceae are often called water molds are distributed universally, and are among the easiest fungi to isolate and cultivate in the laboratory

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CHAPTER 2 LITERATURE REVIEW Mechanism of Apical Growth The phenomenon of hyphal tip growth has been known for over a hundred years (Reinhardt 1892). Its mechanism, though, is not yet understood Several theories of hyphal tip growth dominate the literature. They are (1) the delicate balance theory (Park and Robinson 1966, Bartnicki-Garcia 1973) ; (2) the steady-state theory (Wessels 1990); and m o re recently a combination of the first two, (3) the hybrid theory (Johnson 1996). All three imply that the wall of the apex is plastic, while that of the subapical nongrowing area is rigid. They also assume that the driving force for cell elongation is turgor pressure and/or cytoskeleton. The delicate balance theory assumes that the plasticity of the hyphal apex is achieved by a constant delicate balance between biosynthesis and hydrolysis of wall components. The steady-state theory suggests that the plastic region at the tip contains a mixture of nonlinked wall polymers that are being constantly synthesized, and the rigid condition of the wall is established by chemical crosslinking that is initiated at or near the tip and

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10 continues progressively further back in the hyphal wall. Presoftening of the apical wall is catalyzed by endolytic enzymes that briefly initiate growth but do not sustain it. The hybrid model retains from the steady-state model the constant exocytosis of a plastic mixture of wall polymers at the tip and its rigidification via crosslinking. Among the concepts retained from the delicate balance model i s continuous endoglycanolytic activity express e d in proportion to the rate of tip extension (Johns o n 1996) Thus these models suggest different mechanisms to explain the events of w a ll growth, while agreeing on other aspects such as the role of turgor and the cytoskeleton Structure of the Hyphal Wall Fungal cell walls have essential roles in the life of the fungal cell, i.e., maintenance of cell shape, plasticity, protection against unfavorable environmental conditions, cellular recognition immune response, and host-parasite interaction (Rosenberger 1976, Wessels and Sietsma 1979). The general organization of hyphal cell walls comprises an inner layer of microfibrillar polysaccharides overlaid by an outer layer of amorphous polysaccharides (Burnett 1979). In Oomycetes these polysaccharides are, respectively cellulose and 1 3-B-glucans containing some

PAGE 17

11 1,6-B branches. Cellulose usual l y rep re se n ts about 20% (w/w), 1,3-B-glucans about 80% (w/w) of the total wall carbohydrates (Sietsma 1969, Burnett 1979). In the hyphal wall of A. ambisexualis Raper (Reiskind and Mullins 1981a), acid-soluble 1,3-B-gluca n s with single 1,6-B-linked residues as branches represents 40% (w/w) of the dry wall An alkali-soluble glucan, a polymer of 1,3-B and 1,4-B linkages with occasional 1,6-B glucosyl residues as side chains, represents 7% (w/w) of the wall. Cellulose represents 21% (w/w) of the wall. An insoluble residuum with a linkage pattern similar to the alkali-soluble fraction is present at 6% (w/w). An insoluble component consisting of glucosamine represents 3% (w/w) of the wall. This insoluble fraction probabl y represents chitin (Mullins et al 1984). Protein containing hydroxyproline r e sidue comprises 10% (w/w). There is also a small amount of phosphorus. In the study on the ultrastructural organization of the hyphal wall of A ambisexualis (Reiskind and Mullins 1981b) a model of the various layers in the wall was proposed. The method used in this study of the hyphal wall consisted of the sequential chemical or enzyma tic removal of the various fractions, followed by ana ly s i s (with electron microscopy) of carbon-platinum replicas. The model shows (a) a surface layer of amorphous 1,3-B-glucan hydrolyzed by acid or the enzyme laminarinase; (b) another 1,3-B-glucan l a y e r cont a ining

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12 some 1 ,4-B and 1,6-B linkages hydrolyzed by alkali or laminarinase ; (c) microfibrillar cellulose, removed by cadoxen or the enzymes cellulase plus protease; and (d) an innermost layer of insoluble residuum, faintly microfibrillar. The most abundant and most thoroughly studied glucans from the fungal cell walls are B-glucans These 1,3-B-glucans are variable in degree of 1,6-B branching and in the length of the branches Cellulose is a linear polysaccharide made of glucosyl moieties joined through 1 4-B linkages The gl ucan chains in this polysaccharide associate through hydrogen bonding to form microfibrils According to chain orientation, different crystalline structures exist. The most prevalent form is Cellulose I, where glucose chains are arranged in parallel fashion (the free reducing groups are in the same end of the microfibrils, and the nonreducing ends are in the opposite one) In this sense, as demonstrated by X-ray diffraction analysis (Reiskind and Mullins 1981a), and also apparently in size, fungal cellulose is similar to the polysaccharide found in plants (Ruiz-Herrera 1991). Chitin is an unbranched polysaccharide containing exclusively N-acetylglucosamine residues linked 1,4-B. Three crystalline isof orms of the polysaccharide exist in nature according to the arrangement of the chains These forms can be recognized by X-ray diffraction. In fungi

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13 only al pha-ch i tin, characte r ize d by the antiparallel a rra n gement of the chains, has been d etected (Sentandreu et al. 1994). Structural proteins present in the cel l w all o f f ungi are glycoproteins. They display a basic co mmon structure, consisting of protein with covale n t ly bound carboh y drate chains. In fungi, they are usua l l y ca lled mannoproteins because the carbohydrate moiet y m ai nly consists of mannose units, although small amo un ts of other sugars and phosphodiester groups are fo un d (Peberdy 1990, Ruiz-Herera 1991). Hydroxyproline is repo r ted as a constituent of cell wall proteins in the Oom yc etes (Webster 1980, Reiskind and Mullins 1981a, Ruiz-Herrera 1991). The carbohydrate moieties are attached to the protein through two types of linkages. One type is O glycosidic linkage between mannose or small oligosaccharide chains and the hydroxy-amino-acids (Nakajima and Ballou 1974, Sentadreu and Northcote 1 969, Tanner and Lehle 1987). The second type of linkage (N-glycosidic) connects high molecular weight, hig hly branched, mannan tufts to asparagine residues o f the protein, through diacetylchitobiose (Byrd e t al 1982, Cohen and Ballou 1981, Tanner and Lehle 1987). Studies of the structure of fungal cell wall s by cast-shadowing or replica techniques h a ve d emonstrated t hat their outer and inner surfac e s app e a r different The outer surface is usually amorphous o r fi nel y granular,

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14 whereas the inner face shows intertwining microfibrils of different size, width and orientation However, there is evidence that some components such as microfibrils may escape observation because they are masked by the presence of the large amounts of matrix compounds From a structural point of view, the fungal cell wall has been compared to such manmade composites as reinforced concrete or fiber-reinforced plastics which are formed by two distinct elements: an elastic one, which in the cell wall would be microfibrils of the structural polysaccharides, and a plastic one, which would correspond to the rest of the wall components, generally referred as amorphous or cementing (Ruiz-Herrera 1991). Electron Microscopic Studies of Fungal Cell Walls In thin sections, fixed and stained by the usual standard method including glutaraldehyde and osmium tetroxide fungal cell walls appear multilayered. At least two layers are observed in most walls: an outer one which is electron dense; and an inner layer, thicker and electron transparent However, appearance of the cell wall in sections may depend on the technique used for fixation (Ruiz-Herrera 1991). Variability in composition of the cell wall of fungi does not allow the proposal of a single model of the wall structure In general evidence suggests that fibrillar polysaccharides are accumulated mostly in the inner layers of the cell walls, while amorphous components are

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15 more abundant in the external layers The description of wall structure observed in di fferent genera of fungi analyzed by various techniques may be more useful in providing a general overview of fungal wall architecture (Rui z -Herrera 1991). Early studies on the chemical characterization of fungal cell wall layers were conducted by Hunsley and Burnett (1970). They studied the wall structure of Neurospora crassa, Schizophyllum commune and Phytophtora parasitica after treatment with several hydrolytic enzymes. The outer surface of N. crassa in shadow-cast samples appeared amorphous. Laminarinase treatment removed the amorphous coat revealing a layer of coa rse strands whose interstices were filled with amorphous material, whereas treatment with both laminarinase and pronase enhanced the reticular appearance. The microfibrils were sensitive to chitinase. The authors concluded that the external coat was made of amorphous beta-glucans placed over a reticulum of glycoproteins. More internally, it was suggested, a protein layer followed in which chitin microfibrils were embedded. In contrast to Neurospora, the cell wall of S. commune was resistant to laminarinase, pronase and chitinase, apparently due to the presence of superficia lly located 1,3-alpha-glucan which prevented the access of the lytic en zym es. After removal of this glucan layer by KOH laminarinase and pronase treatment gave rise to the

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16 appearance of a fibrillar structure sensitive to chitinase, suggesting that inner wall layers had a chemical composition and organization similar to N. crassa. Appearance of the wall from P. parasitica was not affected by pronase, but laminarinase unmasked a fibrillar layer sensitive to cellulase treatment. These results were interpreted as suggesting the presence of two layers rich in amorphous beta glucans and cellulose, respectively, in the wall of this fungus. The cell of yeast and mycelial cells of Candida albicans reveals four wall layers when treated by a standard gluteraldehyde-osmium technique (Yamaguchi 1974). When stained by Thiery's technique, eight different layers can be observed, depending on the intensity of staining and their electron density The four outermost layers are PATAg positive, whereas layers 5 and 7 appear electron transparent and PATAg negative (Poulain et al 1978) The authors concluded that the inner layers must be rich in chitin and 1,3-B-glucan, which are both electron transparent and PATAg negative. Other outer layers must be rich in glucans and mannans. The existence of mannans on the surface of the cell was confirmed by Horisberger et al. (1975) who observed binding of colloidal gold-tagged concanavalin A (ConA-Au) by intact cells of the fungus. The presence of mannan in two continuous layers at the periphery of blastospores was demonstrated by staining ultrathin sections with

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17 Concanavalin A-horseradis h peroxidase-3,3'diamino benzidine and HO (Tronchin et al 1979) In this technique, the lectin binds to the mannose residues of the glycoprotein and it is recognized by peroxidase. The peroxidase forms a dark product by the catalytic decomposition of HO in the presence of an oxygen acceptor. A similar method, which included treatment with wheat germ lectin followed by chitibiosyl-horse radish peroxidase or chitobiosyl-ferritin, was used to conclude that chitin was located mostly in the inner layers of the wall of C. albicans. In related species Candida utilis, sections were stained with ConA Au and gold-labeled antimannan antibodies. These techniques demonstrated that mannoproteins were denser in the cell periphery although labeling also was observed close to the plasmalemma (Horisberger and Vonlanthen 1977). Similar results were obtained with Saccharomyces cerevisiae by the same authors (Horisberger and Vonlanthen 1977). Lectins bound to fluorescein isothiocyanate (FITC) were used to detect superficial polysaccharides in the different yeasts by Barkai-Golan and Sharon (1978). The authors observed that S. cerevisiae, S. bayanus and Candida mycodema bound ConA only, suggesting the presence of mannoproteins on the surface of the cells. On the other hand, Schizosaccharomyces pombe did not bind ConA; but it bound peanut lectin, which recognizes D-galactose,

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18 indicating that the cell surface of fission yeast is covered by a galactomannan, not by mannoproteins Candida rugosa and Sporobolomyces roseus bound both lectins This result may indicate the presence of both galactomannan and mannoproteins on the surface of these cells Treatment of the cells with KOH resulted in a strong reaction with wheat germ lectin which recognizes GlcNAc, suggesting that chitin is located internally and is covered by alkali-soluble mannoproteins. The presence of galactomannan on the surface of S. pombe was confirmed by use of the lectin from Bandeiraea simplicifolia bound to colloidal gold (Horisberger and Rasset 1977). This lectin, which recognized alpha-galactopyranosyl residues bound to the outer layer of the wall, and in minor amounts was distributed evenly over the whole thickness of the cell, including the fission scars. In a further report, these authors demonstrated differential distribution of galactomannan depending on the growth stage of the cells (Horisberger et al 1978) Galactomannan appeared in the form of two layers of the wall : one close to the plasmalernrna, and another on the surface of the cell Labeling by the lectin occurred at t he c e ll periphery and at the grow i ng end, but not on t he w al l, formed after cell division These results were inter pr ete d as meaning that the polysaccharide was synt h esized during c el l extension, but not during septum formati o n

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19 Four layers in the cell wall of Dict yostelium discoideum spores were observed by freeze-etching and replica (Hemmes et al. 1972). The innermost layer which appeared amorphous or slightly fibrillar, could be eliminated by successive treatment with cellulase and pronase, suggesting that it was constituted by a mixture of cellulose and proteins. The middle layers (bo th fibrillar) were removed by cellulase treatment alone, indicating the cellulosic nature of the microfibrils. The most superficial layer was resistant to both pronase and cellulase treatment. Hydrolysis resulted in release of galactose, suggesting that this is a major component of the acidic polysaccharide present in the walls. In sections of Agaricus bisporus spores treated with the standard method, three layers could be recognized. The authors concluded that the middle layer contained protein because treatment with pronase increased the fibrillar appearance of this layer. These fibrils corresponded to 1,3-B-glucans and chitin, as they were removed by B-glucanase and chitinase treatment. The outer layer was composed of melanins and 1,3-alpha-g lucans, which was deduced by chemical analyses and electron microscopic observations. The thin inne r layer was poorly characterized, but the authors suggested it was of mucilagenous nature (Rast and Hollenstain 1977). The structure of the mycelial wall of the same fungus was different (Micha lenk o et al 1 976). The outer

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20 layer, which appeared amorphous in replicas, was made of mucilage The thin middle layer was made of amorphous glucans. The innermost layer, which in replicas appeared fibrillar, is probably made of a mixture of B-glucans covering fibrillar chitin, since chitinase by itself could not remove it, whereas the combined action of B-glucanase and chitinase solubilized the layer. Staining with silver hexamine suggested that proteins were present in all layers of the cell wall of the fungus. A similar approach was followed in the characterization of the architecture of the wall from microconidia of Trichophyton mentagrophytes. Three layers were recognized in sections The outer layer appeared electron dense, and the innermost one appeared electron transparent. The material extracted from the outer layer contained a single glycoprotein The median layer was made of proteinaceous rodlets. The inner layer apparently was composed of amorphous glucans and microfibrillar chitin (Wu-Yuan and Hashimoto 1977) Structure of the cell wall from Trichoderma pseudokoningii was studied by treatment of intact cells with different lytic enzymes (Jeenah et al 1982) Accordingly, the authors concluded that the outer layer contained B-glucans, whereas the internal layer was composed of chitin embedded in a protein matrix

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21 Biogenesis of the Fungal Cell Wall Cell wall biosynthesis takes place in t h ree s ites : cytoplasm, plasma membrane and the wall itself. Structural polymers such as chitin and 1,3-Band 1,4-B-linked glucans are synthesized vectorially at the plasma membrane, by transmembrane synthases accepting nucleotide sugar precursors from the cytosol and extruding the polymerized chain into the wall (Cabib et al. 1983, Shematek et al. 1980, Girard and Fevre 1984, Jabri et al. 1991, Cabib et al. 1991, Hromova et al. 1989). Matrix polymers such as glycoproteins are synthesized in the cytoplasmic secretory pathway of endoplasmic reticulum through Golgi vesicles to secretory vesicles. Wall assembly, involving activities such as covalent crosslinking of polymers and modifications such as deacetylation of chitin, takes place in the wall itself (Gooday 1995). Fungal wall 1,3-B-glucans are biosynthesized via the nucleotide sugar, UDP-glucose. The glucan synthases are intrisic proteins of the plasma membrane. Preparations of membranes from Saprolegnia monoica, when provided with UDP-glucose, produce polymers containing varying amounts of 1,4-B and 1,3-B links (Girard and Fevre 1984). The vectorial synthesis of 1,3 B-glucan chains allows on ly linear molecules to be made and thus any 1,6-B branches must be added in the wall (Gooday 1995). These

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22 1,3-B-glucan synthases are stimulated by the presence of trypsin but inhibited by other proteases. Stimulation occurs from the beginning of incubation in the presence of the protease but prolonged action of trypsin leads to inactivation of the glycosyl transferases. The 1,3-B-glucan synthases, therefore, must exist in an inactive state that can be activated by moderate proteolysis. Such regulation, which also appears to modulate plant glycosyl transferases (Girard and Maclachlan 1987), characterizes the chitin synthase system of various fungi (Cabib 1981). The 1,4-Bglucan synthases, like 1,3-B -glucan synthases, have a transmembrane orientation in the plasmalemma, leading to a vectorial synthesis of cellulose from UDP-glucose. These enzymes may have a common structure or organization, as revealed by preliminary immunological studies, but they are different systems and can be separated by glycerol-gradient centrifugation (Fevre et al 1990). Cellulose synthases from Saprolegnia are inactivated by trypsin, but stimulated in the presence of certain nucleotides. Fungal cellulose synthase enzymatic complex may resemble the plant plasma membrane rosettes involved in cellulose synthesis (Mullins and Ellis 1974, Muller and Brown 1980, Montezinos 1982). Some proteins, sensitive to proteases or capable of reacting with nucleotides, would be involved in the regulatory processes Other proteins

PAGE 29

23 would be involved in UDP-glucose binding Such an enzyme seems to exist in plants (Delmer 1999) Cellulose synthases may have a more complicated organization than 1,3-B-glucan synthases. Cel lulose synthase activities of cell free extracts are always much lower than 1,3-B-glucan synthase activities. It is possible that cellulose synthases require a specific factor that is lost in the course of isolation. The opposit e behavior of the synthases towards protease and nucleotides, and the presence of a membrane bound activator of 1,3-B-glucan synthase, may indicate a difference in the regulation of their activities. This would have implications in the cell wall assembly where the deposition of the diffe rent polysaccharides during apical growth is coordinated in time and space (Fevre et al 1990). Chitin synthases, like glucan synthases, are intristic proteins of plasma membrane. These enzymes catalyze glycosidic bond formation from the nucleotide sugar substrate, UDP-N-acetylglucosamine. Most chitin synthase preparations are zymogenic, i.e., produced as proenzymes requiring activation by specific proteases. This proteolytic activation presumably plays a role in the temporal and spatial regulation of the enzyme, by locally activating it in the membrane when and where its activity is required (Gooday 1995). As well as being in zymogenic and active forms in the plasma membrane, zymogenic chitin synthase also occurs in fungal cells as

PAGE 30

24 chitosomes, which are membrane-bound microvesicles about 70 nm in diameter (Bartnicki-Garcia et al 1979, Kamada et al 1991) After purification by differential centrifugation, chitosomes can be activated by treatment with proteolytic enzymes and then produce chitin microfibrils if incubated with UDP-GlcNAc (Gooday 1995) Wall glycoproteins are biosynthesized in the secretory pathway: endoplasmic reticulum> Golgi bodies> secretory vesicles> release at the plasma membrane Carbohydrate material detected in apical vesicles could be the carbohydrate portion of glycoproteins. The transmembrane stages in glycoproteins biosynthesis involve sugar precursors linked to polyprenol dolichol, the "lipid intermediates (Lehle 1981, Cabib et al 1988). In the O-linked chains, the first mannose unit is linked to the protein via the precursor dolichol-phosphomannose in the endoplasmic reticulum. The other mannose units are added via the nucleotide sugar guanosine diphosphomannose, GDP-Man, in the Golgi bodies. The N-linked chains are assembled by a more complex scheme, giving a lipid intermediate dolichol-diphospho(GlnNAc)2-Man9-Glc3 which is N-linked to asparagine in the protein in the endoplasmic reticulum, with the release of the terminal four sugar units, Man-Glc3. The outer chain of many mannose units is added by several linkage-specific mannosyl transferases, w it h Glc Man as substrate, in the Golgi bodies (Gooday

PAGE 31

25 1995). Some secreted enzymes, notabl y invertase, acid phophatase and chitinase, are also mannoproteins, synthesized and secreted in a similar fashion (Kuranda and Robbins 1991). Once the cell wall components are synthesized and secreted, they must be converted into an integrated structure. This process includes covalent crosslinking, hydrogen bonding hydrophobic and electrostatic interactions between different macromolecules (Ruiz-Herrere 1991) Role of Turqor in Wall Expansion The difference in hydrostatic pressure between a cell and its surroundings is called turgor pressure. This actual pressure is thought to provide the driving force for hyphal extension Several observations and measurements suggest that it is necessary for the apical growth process (Robertson 1958, Park and Robinson 1966, Robertson and Rizvi 1968). Osmometry has been used to demonstrate a correlation between hyphal extension rates and turgor pressure in many fungal species (Eamus and Jennings 1986, Luard and Griffin 1981, Woods and Duniwa y 1986). Experiments have shown that filamentous fungi respond to increases in external osmotic pressure by accumulating compatible solutes, including potassium ions, glycerol, mannitol erythritol and arabitol (Luard 1982a,b,c; Pfyffer and Rast 1988, 1989)

PAGE 32

26 The most detailed analyses of the relationship between hyphal extension and turgor pressure have been carried out on hyphae of Achlya bisexualis and Saprolegnia ferax and these studies suggest that growth can occur with out significant turgor (Money and Harold 1992, Kaminskyj et al. 1992). The rate of growth under these conditions is about half of the maximum rate Achlya continues to grow even after the turgor is undetectable; however, its morphology is radical ly altered. On solid medium it shows plasmodial-like growth. In liquid medium of the same composition, it exhibited a yeast-lik e morphology. Saprolegnia has a different response to the absence of turgor, since it continues to grow in the hyphal form. Both Achlya and Sapro legnia appear not to respond to changes in external osmotic pressure by controlling the concentration of internal compatible solutes (regulation of turgor); instead, the plasticity of the wall is modulated to balance the force applied against it. Role of Cytoskeleton in Hyphal Growth It seems very unlikely that the thin wal l covering the apices of extending cells has sufficient mechanical strength to contain the turgor pressure of the cytoplasm. It was suggested that other cellular components may play a role in the regulation of tip expansion The possible existence of other factors is suggested by: (1) the ability of mutants with abnormal cell wall composition to

PAGE 33

27 generate relatively normal hyphae (Kat z and R o senberger 1970); (2) the poor correlation between g r ow th r a tes and turgor pressure (Kaminskyj et al. 1992); and (3) the ability of some species to produce hyphae in the absenc e of measurable turgor pressure (Money and Harold 199 3) The apex might be stabilized by a fibrillar cytoplasmic network similar to that found in o t her cellular systems (amoebae, slime molds). The main structural components of such a cytoskeleton are actin filaments (F-actin), microtubules, and intermediate filaments Each of these are elongated polymers composed primarily of globular proteins known as actins, tubulins, and other unrelated units respectively (Heath 1994). The presence of an array of F-actin was shown in growing tips of Saprolegnia (Heath 1987). It was always present in growing tips, but absent in nongrowing tips. None of these observations proves a morphogenic role for F-actin, because it is not possible to differentiate between direct and indirect effects in the complex system of hyphal tips, but they do suggest that F-actin has some role (Heath 1994) Another explanation of the presence of actin plaques at the growing apices is vesicle and organelle traffic control There is evidence for the involvement of both microtubules and F-actin in wall vesicle transport (Heath 1994).

PAGE 34

28 Cytology of Growing Hyphal Apices A growing hypha consists of an apical region where the extension takes place, a nonelongating subapical, and mature regions, which were the sites of earlier growth. This is reflected both in the structure of the wall and the cytoplasm. Older regions of the hyphal wall are rigid and thick. The cytoplasm in these regions is restricted to a thin layer between a large tonoplast and the plasma membrane. This cytoplasm contains the usual variety of eukaryotic organelles. The tonoplast in mature regions is represented by a central vacuole whereas younger regions contain many vacuoles of smaller size. The subapical region has a thinner wall. The cytoplasm is nonvacuolated and is particularly rich in organelles. At the very apex, the hyphal wall is thin and the associated cytoplasm lacks the usual organelles, containing only small cytoplasmic vesicles of differing size (Shapiro 1995) Based on the organelle distribution, three cytoplasmic zones or regions are recognized: (1) an older, highly vacuolated region; (2) a subapical organelle-rich region; and (3) a terminal vesiculate region (Grove et al 1970). Since one of the differences between the growing tip region and the basal parts of the hypha is the abundance of cytoplasmic vesicles, they are usually assumed to be involved in the synthesis of the new wall. One possibility would be that they carry wall polymers ready

PAGE 35

29 for insertion in the growing wall. Intracellular synthesis of wal l polymers and their delivery to the wall by vesicles occu rs with pectin, hemicellulose, and hydroxyproline-rich glycoproteins in plants (Northcote 1984 ), and to wall mannop roteins in yeast (Zlotnik et al 1984) For filamento us fungi, there is no convincing eviden ce for a similar process. Cytochemical staining does detect polysaccharide material in some apical vesicles (Grove 1978, Hill and Mullins 1980), but this material may represent glycoproteins destined for secretion. More likely, these vesicles contain precursors of the cell wall, their membrane probably contributes to the extending plasmalemma and they may contain wall synthase enzymes for insertion into the plasma membrane (Heath 1994). Growing and nongrowing hyphae differ in the type of wall material that covers their apices (Wessels 1986). The absence of alkali-insoluble beta-glucans at the very ape x of growing hyphae in Schizophyllum commune has been demonstrated by light microscopic autoradiogr aphy (Wessels et al 1983). A subsequent study using electron microscopic autoradiography on shadowed preparations revealed that chitin in growing apices, though alkali insoluble, is in a conformation state quite different from that in nongrowing apices and subapical parts. In contrast to the chitin in these older parts, the newly synthesized chitin at apices appeared nonfibrillar, very

PAGE 36

30 susceptible to chitinase degradation and partly soluble in hot dilute mineral acid. Earlier observations had indicated discontinuities in the presence of microfibrils at hyphal apices (Strunk 1968) These have been contradicted by other workers who showed a continuous network of chitin microfibrils over the apex after chemical treatments which removed a "matrix substance" (Aronson and Preston 1960 Hunsley and Burnett 1970, Bartnicki-Garcia 1973, Schneider and Wardrop 1979, Burnett 1979, Aronson 1981) Wessels (1990), however, suggested that these images showing apical microfibrils probably represent nongrowing apices, which are known to occur abundantly among grow i ng hyphae There is a number of light microscopic studies using fluorescently labeled probes which also suggest that the wall covering the growing apex is different from that covering a nongrowing apex or that of subapical regions. In these studies fluorescently labeled antibodies (Fultz and Sussman 1966, Marchant and Smith 1968, Hunsley and Kay 1976), fluorescent brighteners such as calcofluor (Gull and Trinci 1974), and fluorescently labeled wheat germ agglutinin were used. This differential staining at growing tips could result from the absence of outer wall materials, or to a difference in the conformation of the polymers that bind these probes.

PAGE 37

31 Calcium Gradient There is a tip-high calcium gradient in apicall y growing cells. Free cytoplasmic calcium in the oomycete Saprolegnia ferax is highest at the tip as demonstrated using fluorescent dyes such as Indo-1 or Fluo-3 (Yuan and Heath 1991, Jackson and Heath 1993, Garrill et al. 1993). Studies using patch-clamp techniques suggest that the tip-high gradient reflects a spatial organization of calcium channels in the cell membrane. Using patch-clamp electrophysiology, two types of channels were identified in Saprolegnia ferax: (a) calcium-activated potassium channels that were thought to be involved in turgor regulation, but were not obligatory for growth; and (b) stretch-activated calcium channels that were activated by potassium ions and which may be essential for apical extension (Garrill et al. 1992, 1993). The stretch-activated channels were concentrated at the hyphal apex and were blocked by Gd3 which also inhibited hyphal extension and dissipated the tip-high calcium gradient revealed by Indo-1 (Garrill et al. 1993). In contrast to the stretch-activated channels, the calcium-activated potassium channels were uniformly distributed along the hyphal cell membrane. These could be inhibited by tetraethylammonium, which only caused a transient effect on growth. Stretch-activated calcium channels have also been identified in the germ tubes apices of the plant pathogen Uromyces appendiculatus

PAGE 38

32 (Hoch et al 1987, Zhou et al. 1991). These data suggest that the tip-high calcium gradient is important for polarized hyphal extension and is generated by a locally high concentration of stretch-activated calcium channels in the hyphal apex. It is presumed that the channels are delivered to the surface in microvesicles They may be maintained there by anchoring them to the cytoskeleton or by membrane recycling (Gow 1995). Ion Currents The net flow of electrical current carried by the circulating ions can be detected with an ultrasensitive voltmeter called the vibrating microelectrode (Jaffe and Nuccitelli 1974). In Achlya bisexualis and filamentous fungi in general a positive proton-carried current normally enters the growing apex. Inward current was shown to be due to amino acid-proton co-transport (symport) localized at the tip and the outward current was due to electrogenic proton efflux via a plasma membrane ATPase (Kropf et al. 1984, Gow et al. 1984, Gow 1984, Schreurs and Harold 1988). The proton current also established an extracellular pH gradient around the hypha, with the medium adjacent to the tip relatively alkaline (Gow 1984). On the other hand, there are many examples where there is no correlation between the direction or magnitude of the current and the process of tip growth (Gow 1995)

PAGE 39

CHAPTER 3 HYPHAL GROWTH Introduction Until recently, hyphae were assumed to extend at a constant linear rate when environmental factors are favorable and stable, and nutrients are ample. Detailed analysis of hyphal growth, however, reveals oscillating elongation rates (Lopez-Franco et al. 1994). Materials and Methods An isolate of Achlya bisexualis Coker & A. Couch (ATCC 14524) was used in this study. Stock cultures were maintained on corn meal agar. Mycelia were grown on corn meal agar (CMA), prepared from 17 g of Difeo corn meal agar, 10 g of purified grade agar (Fisher Scientific) and 1 L of distilled water. Small plugs (approximately 1 millimeter) were removed from the edge of a 24-hour-old CMA colony. The plugs were then placed in a 250 mL flask containing 100 mL of liquid peptone-yeast extract-glucose (PYG) medium, pH 6.8 (Cantino and Horenstein 1953). The PYG medium was prepared by combining 1.25 g of bacto-peptone, 1.25 g of yeast extract (Sigma) and 3 g of D-glucose with 1 L of distilled water. The culture was incubated for 10 to 12 h until the hyphae had grown out 33

PAGE 40

34 from the agar plugs to a distance of 2 to 5 mm and then studied To obtain actively growing colonies, the 10 to 20 h old cultures incubated in PYG were used Based on dry weight accumulation colony size increase, glucose incorporation, and cellulase secretion, these colonies are in the midexponential stage of growth (Hill and Mullins 1979). Non-growing conditions were obtained by transferring growing colonies from PYG to 0.2% glucose solution, by incub ation in glucose for about 48 h to cease elongation. Procedure made it possible to find colonies with no elongating hyphae. Such colonies were fixed and used for studying non-elongating hyphae. Screening for no elongation is necessary, because there are hyphae in some colonies that are still elongating at a slow rate. Hyphal elongation was monitored with an Olympus BH-2 light microscope. The colonies were kept on small depression slides with cover slips. Digital images of the hyphal tips were taken every 10 min with a Pixera 120C digital camera The microscope light was turned off between the measurements to avoid heating. Average elongation rates were calculated using a stage micrometer One hundred hyphae from different growing colonies and about 50 hyphae from non -grow ing colonies were studied

PAGE 41

35 Results These light microscopic observation s suggested tha t in colonies growing in PYG, the majori ty of the hyphae are elongating and a small number of the hyphae is not. When individual hyphae are monitored over a long period of time (5 to 6 h), they go through alternating periods of elongation and non-elongation. The rate of elongatio n is not steady, but fluctuates between periods of fast and slow rates The average rate is 3.6 m/min, but it fluctuates from 2 to 6 m/min. The fastest rates are in the middle of an elongating cycle, with lower rates at th e beginning and the end, resulting in a bell shaped curve (Fig. 1) Elongating hyphae have sharp apices (Fig. 2) The majority of the hyphae in the colonies incubated in glucose-only medium are not elongating and a small portion of the hyphae (about 5%) is elongating with an average rate of 1 m/min In some colonies there are no elongating hyphae at all. All the hyphae have rounded apices (Fig. 3). Discussion Light microscopic observations suggest that hyphal growth is a discontinuous, irregular process with periods of elongation and no elongation (Fig. 1). The elongation rate is not constant, but instead fluctuates with periods of fast and slow elongation. During the elongation period the higher rates are in the middle and the rate changes

PAGE 42

36 in a bell shaped curve mode. A similar irregular mode of hyphal tip growth was demonstrated by Lopez-Franco et al. (1994). Growing hyphal tips were recorded with video-enhanced phase-contrast microscopy at high magnification, and digital images were measured at very short time intervals (1 TO 5 s). The study was conducted using fungi from several major taxonomic groups (Oomycetes, Pythium aphanidermatum and Saprolegnia ferax; Zygomycetes, Gilbeltella persicaria; Deuteromycetes, Trichoderma viride ; Ascomycetes, Neurospora crassa and Fusarium culmorum ; Basidiomycetes, Rhizoctonia solani) In all fungi, apparent steady growth of hyphal tips revealed patterns of pulsed hyphal elongation. It was shown that the hyphae do not grow continuously with a steady rate but instead this rate fluctuates, with alternating periods of fast and slow elongation. This results in irregular pulses of growth. Pulsed growth was observed in fungi differing in cell diameter, overall growth rate, taxonomic position, and presence and pattern of Spitzenkorper organization, thus suggesting that it is a general phenomenon The basis of these pulses was not determined it was proposed that their origin could be in the pulsating mode of intracellular processes, especially the secretory vesicle delivery/ discharge system

PAGE 43

37 60 50 I 40 E ::J.. .. t ._. I ~ '' I C ' 0 I \ ,' 30 +-ctS O') \ C 0 I 20 Q) I + ' I I ' I 10 I ' I I \ I \ : I : / 0 0 50 100 150 200 250 time (min) Fig. 1. Elongation measurements of three individual Achlya bisexualis hyphae growing in PYG medium 300

PAGE 44

38 Figs 2-3. Scanning electron micrographs of hyphae of Achlya bisexualis 2. Elongating hypha from a colony incubated in PYG 3. Non-growing colony incubated in glucose-only medium showing t he hyphae with rounded apices Bars : 2 = 16 7m; 3 = 27 3

PAGE 45

CHAPTER 4 LOCALIZATION OF CELLULOSE IN THE CELL WALL AS REVEALED BY ELECTRON MICROSCOPY AND CYTOCHEMICAL TECHNIQUES Introduction Cellulose, a crystalline 1,4-B-glucan, is the most abundant biopolymer in nature. Its biomass makes it a global carbon sink and renewable energy source, and its crystallinity provides mechanical properties to cellulose containing cell walls (Arioli et al. 1998). The understanding of cellulose properties and metabolism is important for understanding morphogenesis in plants and certain fungi. Traditionally, cell wall components have been identified cytochemically by using indirect, extractive methods. This approach can lead to problems such as incomplete extraction and unseen effects of the extraction procedure on cell wall ultrastructure. Enzyme-linked colloidal gold labeling is a nondestructive, direct labeling technique and can be used to localize cellulose on thin sections (Berg et al. 1988). This labeling technique can be also used to localize cellulose on the surface of cells. Previously cellulose was identified in Achlya cell walls with cellulase-gold affinity labeling. Thin

PAGE 46

40 sections of growing hyphae revealed labeling for cellulose in mature and subapical regions but not at the apex (Shapiro 1995) The present study was done to observe the distribution of cellulose along the elongating and non-elonging hyphae, as part of an overall examination of hyphal tip growth Materials and Methods Culture Methods and Microscopy Techniques The general culture methods are described in Chapter 3 To induce sporulation, ten discs from the edge of a 48 hold colony growing on CMA plates were punched out with a small cork border The discs were incubated at room temperature in 100 mL PYG liquid medium in a 250 mL flask for 15 h with sh a king at 110 rpm Then they were washed several times with 0 5 mM calcium chloride At ths point, they were left in fresh calcium chloride for 6 to 7 h to induce sporulation Electron microscopy For chemical fixation, the agar plugs bearing hyphae were fixed for 30 min at room temperature with 4% (v/v) glutaraldehyde in 0 05 M sodium cacodylate buffer, pH 7.2. After rinsing in 3 changes of buffer, the material was postfixed in 1 % (w/v) osmium tetroxide in the same buffer, for 30 min Samples were again washed several times in buffer, followed by dehydration in an ethanol series, terminating in absolute acetone. For

PAGE 47

41 freeze-substitution fixation colonies were frozen in acetone at -80 C, then substituted with methanol at -80 C for 72 h. The samples were warmed over 2 hat room temperature and transferred into absolute acetone for TEM or rehydrated in a methanol/water series to be labeled with cellulase-gold complex and processed for SEM (modified from Bourett et al. 1998). For methanol fixation colonies were frozen in methanol at -80 C, then warmed at room temperature over 2 hand transferred into absolute acetone for TEM or rehydrated in an methanol/water series. These samples were labeled with cellulase-gold complex and processed for SEM. Material from absolute acetone was infiltrated with an epox y embedding medium and polymerized at 60 C for 48 h in a flat embedding mold. Epon 812 embedding medium was prepared by combining 55 g of Epon 812, 35 g of DDSA and 21 g of NMA. The accelarator DMP-30 (0.2 mL per 10 mL of the medium) was added right before embedding. Embedded samples were sectioned on a Reichert Ultracut R (Leica). Thin sections (75 to 80 nm) were collected on formvar coated nickel grids and labeled with the cellulose-gol d complex. For scanning electron microscopy the colonies were fixed with 4% glutaraldehyde in 0.05 M sodium cacodylate buffer and washed several times in buffer (osmium tetroxide fixation was omitted). Then the colonies were processed for cellulase-gold labeling, silver enhanced,

PAGE 48

42 dehydrated in an ethanol series finally critically point dried. For silver enhancement the colonies were placed in a non-diluted mixture (1:1) of reagents from the Aurion Silver Enhancement Kit for 5 min and washed several times with water to stop the reaction (Scopsi et al 1986) Cellulose Localization Using Enzyme-Gold Affinity Labeling Colloidal gold of approximately 15 nm diameter was made via reduction of chloroauric acid by sodium citrate as described by Frens (1973). The enzyme cellulase was purchased from Worthington Biochemical Corporation, catalogue No. LS02601 This is chromatographically "purified" cellulase isolated from cultures of a selected strain of Tricoderma reesei. A second enzyme was also used, endocellulase III (provided by Dr Tim Fowler, Genenc ore International, Inc) The solutions used for conjugation with this enzyme were 5 5 rather than 4 .5 for the commercial cellulase To coat the gold with cellulase the pH of 10 mL of 15 nm colloidal gold was adjusted to 4.5 and then 1 mg of cellulase dissolved in 0 1 mL distilled water was added with stirring. After 5 minutes the enzyme-gold complex was further stabilized by the addition of 0.5 mg/mL polyethylene glycol (molecular weight 20 ,0 00) Then the solution was poured into a centrifugation tube and 1 5 mLof 20% glycerol (in citrate buffer pH 4 5) was carefully placed on the bottom of the tube (glycerol was added for long-term storage at -80 C)

PAGE 49

43 The enzyme-gold complex was pelleted at 12, 1 00 rpm for 1 h. Successful coating was evident by a mobile pe llet which was resuspended in 0.75 mL of 20% glycerol. Sections, on grids, were preabsorbed for 5 min b y floating them face down on citrate buffer containing 0.5% gelatin as a blocking agent. The labeling solution was a 1:10 dilution of the enzyme-gold stock with citrate buffer To label, grids with sections were floated on t he labeling solution for 30 min. The grids were then floated on citrate buffer alone for 5 min and rinsed twice for 5 min in distilled water. The colonies destined for SEM observations were treated with the same series of solutions, but were completely submerged, rather than floated. Cytochemical controls A number of cytochemical controls were performed to prove the specificity of the label. (1) Substrate competition : as a control to determine that the enzyme-gold probe was binding to cellulose the cellulase -gold complex was incubated with 1 mg/mL carboxy-methylcellulose (CMC) (sodium salt, medium viscosity, Hercules CMC 7MF) for 30 min before the labeling of sections or colonies. (2) Labeling with nonenzymatic protein: any nonspecific protein binding sites were determined by incubating the sections and colonies with 18 nm Colloidal Gold-AffiniPure Goat

PAGE 50

44 Anti-Mouse IgG (H+L) (Jackson ImmunoResearch Laboratories, Inc.) (3) Substrate specificity check: two substates similar to cellulose and present in the cell wall of Achlya were tested to see if cellulase-gold bound nonspecifically to them: the cellulase-gold complex was incubated with 10 mg/mL laminarin (from Laminaria digitata, Sigma) and 11.8 mg/mL re-acetylated glycol chitosan (Sigma). (4) Cellulase pretreatment: to determine the effect of predigestion by free cellulase the sections and colonies were incubated with 1 mg/mL cellulase in incubation buffer for 30 minutes prior to labeling with cellulose-gold complex. Cellulase Enzyme Activity during Labeling To determine if the cellulase enzyme, when conjugated to gold used for labeling, retained enzyme activity the following experiment was done. The samples were combined with 1 mL of gold-cellulase complex (1:10 dilution) and incubated at room temperature. A control for each sample was prepared with substrate and 0.05 M sodium citrate buffer pH 4.5 The citrate buffer alone was used as a blank. After 30 min (the usual time of labeling) and 3 h, the enzyme activity was checked by Nelson-Somogyi method, using a standard curve obtained by plotting optical density (at 520 nm) measured on Du-64 Spectrophotometer (Beckman) against known concentrations of glucose. The samples included : 1 mg CMC, 1 mg of the whole Achlya wall, small colony and 1 mL of

PAGE 51

45 cellulase-gold complex alone. Detection of Gold Particles with a Backscatter Det ector To assure that the particles obser v ed o n t he hyphal surfaces were actually the gold particles, the reg ula r secondary images were compared with the backscatte rin g images of the same regions. The samples were carbo n coated, instead of the usual gold coating. Backscatte r detector (GW Electronics, USA) was used to detect t he backscattering signal. Zymolyase Hydrolysis Chemically fixed or live colonies were incubated at room temperature with 0.05 mg/mL zymolyase 100 T (Arthrobacter luteus) (Seikagaku Corporation) in 66 mM sodium phosphate buffer pH 7.5. The hydrolysis was monitored with light microscope. After 24 h the colonies were fixed, labeled with cellulase-gold complex and processed for SEM observations Treatment of Growing Colonies with Dichlorobenzonitrile Small colonies were grown in 500 mL flasks containing 250 mL of PYG. Each flask contained 10 small colonies After 12 h of incubation, dichlorobenzonitr i le (DCB) was added to the flasks. DCB was previously dissolved in 100% DMSO. The concentrations of DCB were 10, 20, 30, 40, 50, 60 100, and 200 M. The colonies w e re incubated in DCB-containing medium and their growth was monitored with light microscopy. After 36 h o f incubation, the colonies were chemically fi xe d, la b eled

PAGE 52

46 with cellulase-gold complex and processed for SEM observations. The colonies grown in PYG only medium served as a control in this experiment. The ability of spores to germinate in the medium containing DCB was also checked. For this 5 mL of fresh spore suspension were added to PYG medium with and without DCB. Results Cellulose Localization In growing colonies (incubation in PYG) cellulose is found on the surface of mature and subapical regions on the hyphae. In apical regions three patterns of labeling are found in a single colony. Some of the hyphae (approximately 5%) are not labeled at the apex and show a sharp border between labeled and non-labeled regions. The unlabeled area is approximately 2 to 4 min diameter (Figs. 4-11). In the majority of the hyphae, there is a gradual decrease of the labeling towards the apex (Figs. 12-19). Some of the hyphae (about 5%) are labeled at the apex as intensively as in the mature regions (Figs. 20-27). Fifty colonies from different batches were examined and all of them had this pattern and ratio of labeling. In contrast, the labeling of hyphae from colonies incubated in glucose-only medium gave a different pattern of cellulose labeling. In these colonies, all the hyphae were labeled at the apices and the labeling was as i n t ensive as in mature regions (Figs. 28-35). Ten

PAGE 53

47 colonies from different batches were ana ly z e d. L i g ht microscopic analysis prior to fixation ensured t ha t they did not contain elongating hyphae. The surface labeling of hyphae in the freeze-substituted and methanol-fixed colonies has the same patterns and ratio as in chemically fixed ones (data not shown). Cellulase-gold affinity labeling of cross sections localizes cellulose in the cell wall exclusively (Fig. 36). The distribution of the gold particles in the wall is even. The level of nonspecific labeling is very low. On the longitudinal sections of elongating hyphae the label is present in mature and subapical regions but is very low or absent in apical regions (Fig. 37). There is no labeling on the cross sections (Fig. 38) or the hyphal surface (data not shown) when the sample of endocellulase III from Dr. Fowler was used. The conjugation was successful, based on the raspberry red color of the enzyme-gold complex and the presence of the mobile pellet. Negative staining of the enzyme-gold complex confirmed successful conjugation (Fig. 39). Cytochemical controls All the cytochemical controls support the view that the cellulase-gold complex is a specific label for cellulose in Achlya. The preabsorbtion of the labeling solution with CMC results in the absence of the labelin g

PAGE 54

48 on the hyphal surface as well as on the cross sections (F igs. 40, 41) The incubation of the sections and the colonies with gold-coupled Goat Anti-Mouse IgG results in the absence of labeling as well (Figs 42, 43). Cellulase pretreatment of the sections and colonies results in the absence of the labeling (Figs 44, 45). The labeling pattern is regular when the cellulase-gold complex is incubated prior to the labeling with laminarin or chitosan (Figs. 46-49). Detection of gold particles on the hyphal surface with a backscatt er detector gave an identical particle distribution on secondary and backscatter images (Figs 50-55) Cellulase activity during labeling Cellulase-gold complex shows no enzyme activity during the labeling of the colonies or whole wall samples as measured by the production of reducing sugar (Table I). Absorbance of glucose is measured after 30 min (the usual time of labeling with cellulase-gold complex) and 3 h Cellulase-gold is diluted 1 : 10 with the buffer. Based o n glucose equivalents from a standard curve, cellulase act i v it y is v ery low in reactions with a whole wall prep ara t ion or a colony. Thus, there are no additional pri m er s produced and they do not alter the results of labeling Enz ym e a ctiv i ty of cellulase-gold complex against ce l l ulase itself is also low Cellulase activity

PAGE 55

49 Figs. 4-11. Scanning electron micrographs of elongating hyphae of Achlya bisexualis showing cellulose surface labeling with cellulase-gold. Note the unlabeled apices. Bars: 4-9 = 2.00 m; 10 = 2.31 m; 11 = 3.00 m.

PAGE 56

50 Figs. 12-19 Scanning electron micrographs of elongating hyphae of Achlya bisexualis showing surface labeling of cellulose with cellulase-gold Note the gradual decrease of labeling towards the apices Bars : 12 = 3 33 m; 13 = 2 73 ; 14 = 1 67 m; 15-18 = 2 .00 ; 19 = 3 31

PAGE 57

51 Figs 20-27. Scanning electron micrographs of surface of non-elongating hyphae present in growing colonies of Achlya bisexualis showing cellulose labeling with cellulase-gold Note the labeled apices. Bars: 20-22 = 3.00 m; 23, 24 = 2 .00 m; 25 = 1.50 m; 26 = 2.31 m; 27 = 3.33 m.

PAGE 58

52 Figs 28-35 Scanning electron micrographs of non-elongating hyphae from non-growing colonies of Achlya bisexualis incubated in glucose-only medium showing surface labeling of apices for cellulose with cellulasegold Note the labeled apices Bars : 28 = 2.31 rn ; 29 = 4 29 rn ; 3 0 = 5 0 0 rn ; 31 = 1 50 rn ; 32 33 = 3 75 rn ; 34 35 = 3 00

PAGE 59

53 Fig 36. Cross section of Achlya bisexualis hypha showing labeling of cellulose in the cell wall with cellulase -gold complex. Bar=l m.

PAGE 60

54 37 Fig. 37. Longitudinal section of the apical region of an elongating Achlya bisexualis hypha showing labeling of cellulose with cellulase-gold. Bar=l m.

PAGE 61

55 Fig. 38. Cross section of Achlya bisexualis hypha labeled for cellulose with endocellulase III-gold. Bar=0.5 m.

PAGE 62

56 39 Fig. 39 Negative staining of endocellulase III-gold complex Bar=200 nm

PAGE 63

57 Figs. 40-41. Electron micrographs showing the a bsence of cellulose labeling with cellulase-gold in t he cell wall of Achlya bisexualis resulting from t he p r ea bsorption of the labeling solution with CMC. Bars: 4 0 =1 rn ; 41=1 5 rn.

PAGE 64

58 Figs 42-43 Electron micrographs showing the absence of gold label in the cell wall of Achlya bisexualis resulting from the preincubation of the sections (TEM) or colonies (SEM) with colloidal gold-affinipure goat anti-mouse IgG. Bars : 42=1 m; 43=1 5

PAGE 65

59 Figs. 44-45. Electron micrographs showing the absence of cellulose labeling with cellulase-gold in the cell wall of Achlya bisexualis resulting from the pretreatment of the sections (TEM) or colonies (SEM) with cellulase. Bars: 44=1 m; 45=1.67 m.

PAGE 66

60 Figs. 46-47. Electron micrographs showing the regular pattern of cellulose labeling with cellulase-gold in the cell wall of Achlya bisexualis resulting from the preincubation of the labeling solution with chito zan Bars : 46 =1 m; 47=1.2

PAGE 67

61 Figs. 48-49. Electron rnicrographs showing the regular pattern of cellulose labeling with cellulase-gold in the cell wall of Achlya bisexualis resulting from the preincubation of the labeling solution with larninarin. Bars: 48=0.5 rn; 49=3 rn.

PAGE 68

62 Figs. 50-55. Scanning electron micrographs showing the regular pattern of cellulose labeling with cellulase-gold on the hyphal surface of Achlya bisexualis 50, 52, 54. Secondary images. 51 53, 55 Backscatter images of the same regions. Bars: 50 and 51=1.5 m; 52 and 53=3.00 m; 53 and 55 = 857 nm.

PAGE 69

63 Table 1. Glucose equivalent from standard curve showing cellulase activity during labeling Glucose equivalent from Sample standard curve (mg /ml) Reaction time: Reaction time: 30 min 3 hrs Whole wall and cellulase-gold 0.009 0.009 Whole wall and buffer 0.005 0.006 Small colony and cellulase-gold 0.004 0.004 Small colony and buffer 0.002 0.002 Cellulase-gold 0.006 0.007 CMC and cellulase -gold 0.02 0 04

PAGE 70

64 Figs 56-57 Scanning electron micrographs showing cellulose labeling with cellulase-gold of elongating Achlya bisexualis hyphae that were hydrolyzed with zymolyase before chemical fixation Bars : 56=2 31 m; 57=2 .00 m.

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65 Figs. 58-59. Scanning electron micrographs showing cellulose labeling with cellulase-gold of elongating Achlya bisexualis hyphae that were hydrolyzed with zymolyase after chemical fixation. Bars: 58=2.31 m; 59=1 .00

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66 Figs 60-61. Scanning electron micrographs showing cellulose labeling with cellulase-gold of an Achlya bisexualis hypha from a non-growing colony treated with zymolyase Bars : 60 = 3 33 ; 61=857 nm (higher magnification of the apex)

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67 :. 62 Fig. 62. Transmission electron micrograph of cross section of Achlya bisexualis hypha from a colony incubated in 100 M DCB. Cell wall cellulose is labeled with cellulase-gold complex. Bar=0.5 m.

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68 is relatively higher when the enzyme-gold complex reacted with CMC. Cellulose Localization on the Surface of the Hyphae in Colonies Incubated with Zymolyase In the colonies growing in PYG medium, that were hydrolyzed with zymolyase prior to fixation, most of the hyphal apices are intact and labeled. A small portion of the hyphae has broken apices. In these hyphae the remaining cell wall is labeled with cellulase-gold complex (Fig 56, 57) In the growing colonies that were chemically fixed first and then hydrolyzed with zymolyase, all the hyphae have intact apices Most of the apices are labeled, but some are not (Fig 58 59). The non-growing col o nies from glucose-only medium were screened for the absence of elongating hyphae prior to the treatment. Time of fixation, before or after hydrolysis, did not make any difference in the results All the hyphae in these colonies are intact and are labeled as intensively as the rest of the hyphal regions (Fig. 60, 61). Hyphal Elongation, Spore Germination and Cellulose Localization in the Presence of DCB The presence of DCB in the medium does not affect the growth of Achlya. It does not affect the morphology of the hyphae or the growth rate. The average hyphal elongation rate is 3.6 m/min. Spores germinate equally

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69 well in PYG medium with and without DCB. There is no difference in the cellulose surface labeling of hyphae from the colonies treated with DCB and regularly growing colonie s. The labeling patterns and their ratio is the same (data not shown) Cross sections of hyphae from the colonies treated with DCB have expected pattern of cellulo se la beling (Fig. 62). Discussion The cellulase-gold affinity labeling is specific for cellulose in the fungus Achlya, based on its reproducibility and a large variety of controls. Differences in the fixation techniques do not affect the labeling pattern. Standard chemical fixation gave the same results as freeze-substitution and cold methanol fixations. The results are highly reproducible and are not artifacts of the fixation procedure. Thus this labeling technique provides a specific and reliable method for localizing cellulose on thin sections and hyphal surfaces. The fact that cellulose labeling was foun d on the hyphal surface, may contradict a general assumption that the microfibrilar component of the cell wall, cellulose in the case of Achlya, is located next to the plasma membrane and is covered by the matrix components of the wall. The cell wall may not be arranged as layers of components, but as a mixture. This would explain the presence of some cellulose on the surface, this

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70 explanation seems unlikely since Achlya secretes cellulase during growth (Thomas and Mullins 1967, 1969), and if cellulose was present on the surface, it could be hydrolyz ed. Furthermore, when cellulase is applied exogenuously t o the living Achlya cultures, it does not destroy the integrity of the hyphae, nor does it change their surface as revealed by shadow replicas (Reiskind and Mullins 1981b) The unique structure of the cellulase enzyme complex may explain the presence of cellulose labeling on hyphal surface. The cellulolytic enzyme complex from Trichoderma reesei used here for labeling consists of a number of en zymes : endoglucanases (EG); cellobiohydrolas es (CBH); and cellobiase (CB); which work synergistically All these enzymes contain a small highly homologous 36-residue region called the A domain, connected to the globular enzymatically active core domain by a threonineand serine-rich sequence. The A domain has no catalytic activity in CBH I and CBH II, but it is thought to have a cellulose-binding function The core protein alone does not have full cellulose-hydrolysin g activity, but has normal activity on small synthetic substrates (Rouveinen et al. 1990). Perhaps cellulases are able to bind cellulose microfibrils located inside the wall via the small cellulose binding domains (CBD). CBD could penetrate the wall and find the binding sites wh ile the catalytic domains, conjugated to gold remain on the surface The

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71 results with EG III labeling indirectly prov e t he idea of CBD penetrating the wall and leaving the catal ytic domains attached to gold on the surface. EG II I p rovided by Dr. Tim Fowler (Genencor International, Inc.) is a genetically modified enzyme that does not have a cellulose binding domain (personal communication). Without the binding domain, this enzyme can not attach to the cellulose microfibrils and it results in the lack of EG III-gold affinity labeling. The results of the experiments that measured cellulase-gold activity during labeling also provide a support for the idea of CBD penetrating the wall and leaving the catalytic domain attached to gold on the surface. The enzyme-gold does not show enzyme activity against a whole wall sample or a colony, but is active against the soluble cellulose derivative CMC. Perhaps, in the case of whole wall and colony treatments the cellulose binding domain finds the binding site by penetrating the wall and then attaches to cellulose. The catalytic domain conjugated to gold does not get access to cellulose microfibrils surrounded by the matrix material of the wall. Thus, the binding takes place without hydrolysis. In the case of CMC the cellulose microfibrils are not covered, they are available for the catalytic domain. Therefore, in this case both binding and hydrolysis take place.

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72 According to the results of cellulase-gold labeling, a ll the hyphae from non-growing colonies (glucose-only me diu m) are evenly labeled in all regions, including the a p ices. Light microscope screening prior to the EM pr ocessing ensured that these hyphae are not elonga ti ng. O n the other hand, cellulase-gold labeling of the hyphae from growing colonies (PYG medium) revealed three patterns of cellulase-gold labeling at the apices: labeled (small portion of hyphae), unlabeled and with the decreasing label towards the apex. Light microscopic observations prior to fixation revealed that a small portion of hyphae in these colonies is not elongating, but the majority of the hyphae are elongating. Based on this, I propose that in non-elongating hyphae cellulose is evenly distributed along the hypha and is present in the apex. Elongating hyphae lack cellulose at the apices or there is a gradual decrease in the amount of cellulose t oward the apices. T he results of the experiments with zymolyase hy dro ly s i s support the conclusion that in some h y phae in growin g c olon i es t here is no cellulose in the ap i cal ce ll wall I n t he colonies that were treated with zymolyase, prior t o fixati on, t h e se hyphae ha ve b r o k en a p ice s. I explain this by the fact that th e w al l i n these ap i cal regions lacks c e llu los e and consis t s main l y o f 1 3-Bg lucans Z ym ol y as e has both 1,3-B glucanase and protease activities It h y d r ol yz es not onl y

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73 1,3-B-glucans, but also structural wall proteins, cell membrane proteins and cytoplasmic proteins. Thus, the elongating regions have ''hydrolyzedn apices. In the colonies that were chemically fixed first and then hydrolyzed with zymolyase, the elongating hyphae have intact unlabeled. The apices are not broken as in the previous case because chemical fixation crosslinks proteins so they can not be hydrolyzed. As expected, in both experiments, non-elongating hyphae (glucose-onl y medium) have intact apices with cellulose labeling as intensive as in the other regions of the hyphae. The experiments with DCB gave an unexpected result. In these experiments it was the intention to use a different approach to show the absence of cellulose in the wall of elongation regions. DCB is a classic inhibitor of cellulose biosynthesis in higher plants (Delmer 1999). It was expected that the hyphae would continue to elongate by synthesizing 1,3-B-glucans and producing large regions of apical wall made mainly of this component and that these hyphal regions lacking the structural support of cellulose might not have a tubular form. However, DCB had no inhibition effect on the growth process of Achlya. There were no changes in hyphal morphology as revealed by light microscopy, TEM or SEM observations. Hyphal elongation rates in colonies incubated with DCB were the same as in the regular growth medium. Cellulose labeling of the cross sections and the

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74 hyphal surface revealed no difference between the hyphae grown in regular medium or the medium with DCB. Perhaps, the cellulose biosynthesis system of Achlya is different from that found in higher plants in the step that is affected by DCB, or DCB molecules may not be able to penetrate the Achlya wall. Similar results for the lack of an inhibitory effect of DCB were found in the cellular slime mold Dictyostelium (Blanton 1997, Blanton personal communication) Actually, none of the three major cellulose-synthesis inhibitors used in higher plantsDCB, isoxaben, and pthoxazolin--had an effect in this organism.

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CHAPTER 5 LOCALIZATION OF 1,3-B-GLUCANS IN THE CELL WALL AS REVEALED BY ELECTRON MICROSCOPY AND CYTOCHEMICAL TECHNIQUES Introduction The term glucan applies to polysaccharides composed of glucose units and they are divided into alphaand B-anomers according to their stereochemistry around the anomeric carbon. The B-glucans include both homopolysaccharides and heteropolysaccharides. Six different types of B-glucans have been described in fungi: linear 1,3-glucans; 1,3-glucans with occasional 1-6 single glucose branches, with or without phosphate; 1,3-glucans with significant amounts of 1,6-branches; glucans containing mostly 1,6-linkages; glucans containing 1,3-, 1,4and 1,6linkages (Ruiz-Herrera 1991). These B-glucans are getting attention because of their potential application in chemical, pharmaceutical and food industries. Pharmaceutically, 1,3-B-glucans that have B-glucopyranosyl units attached by 1->6 linkages as single unit branches have been shown to enhance the immune system. This enhancement results in antitumor, antibacterial, antiviral, anticoagulatory and wound healing activities (Bohn and Bemiller 1995). 75

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76 The 1,3-B-glucans are important components of fungal cell walls and they are also storage carbohydrates in some fungi, especially in the Oomycetes and Basidiomycota. Some B-glucans are secreted in the form of slimy material, and may protect cells from desiccation and other harmful environmental conditions (Ruiz-Herrera 1991). In the case of pathogenic fungi, B-glucans are important in cellular recognition, and in eliciting defense responses of infected plants (Ryan 1987, Dixon and Lamb 1990, Cote and Hahn 1994). Storage glucans accumulate intracellularly and are used as reserve material at critical stages of growth and reproductive development (Wang and Bartnicki-Garcia 1980, Lee and Mullins 1994) In Phytophthora, Wang and Bartnicki-Garcia (1973) reported a phosphorylated 1,3-B-glucan in sporangia, zoospores and cysts. This phophorylated 1,3-B-glucan contains one or two phosphate residues as monoester linkages at the C-6 hydroxyl groups of some glucose units. In Achlya, a phosphorylated cytoplasmic 1,3-B-glucan has been isolated and characterized (Lee and Mullins 1994, Lee et al 1996), containing 5% phosphate (w/w), and has both mono-and diphosphoester linkages. The diester linkages are used to form very large polymers from the smaller neutral forms. Although the biological role of the reserve 1,3-B-glucans i s most often suggested as a source of energy or carbon

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77 or both, in Achlya it is also an important site of phosphate storage (Lee and Mullins 1 994). The most general role of B-glucans is a structural one, as the major component of fungal cell walls. Inhibition of B-glucan synthesis in yeast leads to cell lysis and often death, resulting from a weakening of the cell wall (Perez et al. 1983, Miyata et al. 1985). Such inhibitors are used as important antifungal compounds against both plant and animal pathogens. The 1,3-B-glucans were localized on cross sections of Achlya (Shapiro and Mullins 1997). The method used indirect irnrnunolabeling with a commercial polyclonal antibody specific for 1,3-B-D-glucopyranose linkages. The glucans occurred in the cell wall, the large vesicles in the organelle-rich areas of the hypha, and in the large central vacuole in more mature areas. Preabsorption of the antibody with either purified neutral or phosphoglucan from Achlya completely eliminated subsequent labeling of hyphal sections. No labeling of the large population of apical vesicles was found, suggesting that these reserve glucans are not directly involved in apical growth. Since the labeling occurred in large vesicles and the central vacuole and no other cytoplasmic sites showed conjugation, the vesicle and vacuole membranes probably contain the synthases responsible for the biosynthesis of the reserve glucans. The labeling of serial sections revealed the

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78 1 3-B-glucans in both mature and apical regions. Additional labeling experiments have now been carried out on hyphae that were first determined to be elongating, as described in Chapter 3, to ensure that elongating apices contained 1,3-B-glucans. Recall that in Chapter 4 evidence was presented that clearly demonstrated a lack of cellulose in elongating apices. Materials and Methods Culture Methods, Fixation and Microscopy Techniques The general culture methods, fixation and microscopy techniques are described in Chapter 3 and Chapter 4. Localization of 1,3-B-Glucans Using Monoclonal Antibody The primary antibody, raised in mouse against a laminarin-haemocyanin conjugate, was purchased from Biosupplies Australia PtyLtd (Parkville Victoria, Australia), catalogue number 400-2. This antibod y recognizes linear 1,3-B-oligosaccharide segments in 1,3-B-glucans. The epitope includes at least five 1,3 -B-l inked glucopyranose residues. It has no cross reactivity with 1,4-B-glucans or 1,3-B-, 1 ,4-B-glu cans (Meikle et al. 1991). It was diluted 1:100 in phosphate buffered saline (PBS), pH 7.2 containing 0.5 % cold water fish gelatin The gold reagent, 18 nm Colloidal Gold Affinipure Goat Anti-Mouse IgG (H+L) was purchased from Jackson ImrnunoResearch Laboratories, Inc. (West Grove Pennsylvania) catalogue number 115-215-146. The nickel grids with sections were floated on PBS containing

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79 1% gelatin for 30 min to block non-sp ecific labeling Then the grids were floated on the top of 20 l drops of the primary antibody solution for 1 h. After three washes in PBS the grids were floated on a 1:20 dilution of the gold reagent in PBS for 1 h. The solution was centrifuged at 18 000 gin a microcentrifuge for 1 min before use. Finally, the grids were washed: twice in PBS and twice in water. The colonies destined for SEM observations were treated with the same series of solutions while completely submerged into them, rather than floated. Cytochemical Controls (1) Preabsorbtion of primary antibody with laminarin: in order to determine that the primary antibody binds to 1,3-B-linkages it was preabsorbed with laminarin. Laminarin from Laminaria digitata was purchased from Sigma (St. Louis, Missouri), catalogue number L-9634. The grids with sections were blocked against nonspecific labeling as described above. Primary antibody stock solution, 10 l, was incubated with 100 mg of laminarin in 1 ml of PBS containing 0.5 % gelatin for 1 h. The grids were floated on a drop of this solution for 1 h. The labeling procedure as described above was then followed. (2) Omission of the primary antibod y : the procedure was the same as during the regular labeling, but the incubation with the primary antibody was omitted. (3) Replacement of the primary antibod y with a non

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80 specific primary antibody raised in mouse: the procedure was the same as during the regular labeling but the primary antibody was replaced by an undiluted non -speci fic antibody (HL 1099) raised against neu rofilaments in mouse. HL 1099 was provided by the Hybridoma Laboratory, Interdisciplinary Center for Biotechnology Research (Gainesville, Florida). Results Localization of 1,3 B-Glucans on Sections and Hyphal Surfaces Using Monoclonal Antibodies On cross sections of Achlya the monoclonal antibody detected 1,3-B-glucans in the wall, small vacuoles, and the large central vacuole of mature regions (Figs. 63, 64). No cytoplasm-specific labeling was found Longitudinal sections of both elongating (Fig. 65) and non-elongating (data not shown) hyphae show antibody labeling in the cell wall of the apices and all along the hyphae No surface labeling was found using SEM (Fig. 6 6) Cytochemical Controls (1) Preabsorbtion of primary antibody with laminarin resulted in the absence of labeling (Fig. 67). (2) Omission of the primary antibody resulted in the absence of the labeling (Fig. 68). (3) Replacement of the primary antibody with a non-specific primary antibody raised in mouse resulted in the absence of the labeling (Fig. 69).

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81 Fig. 63. Transmission electron micrograph showing localization of 1,3-B-glucans with monoclonal antibody on cross section of Achlya bisexualis hypha. Bar=0.5 m.

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82 Fig. 64. Transmission electron micrograph showing localization of 1 3-B-glucans with monoclonal antibody on cross section of Achlya bisexualis hypha. Bar=l.00 m.

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83 .. Fig. 65. Transmission electron micrograph showing localization of 1,3-B-glucans with monoclonal antibody on longitudinal section of an elongating Achlya bisexualis hypha Bar=l.00 m.

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84 Fig. 66. Scanning electron micrograph showing the absence of 1,3-B-glucans labeling on the surface of Achlya bisexualis hypha. Bar=2 31 m.

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85 Figs. 67-69. Transmission electron micrograph showing the absence of 1,3-B-glucans labeling on the cross sections of Achlya bisexualis hyphae resulting from: 67. preabsorbtion of the labeling solution with laminarin. Bar=l.00 m; 68. omission of the primary antibody. Bar=l.00 m; 69. replacement of the primary antibody with a non-specific antibody raised in mouse. Bar=l.00 m.

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86 Discussion Based on the results of the cytochemical controls, the monoclonal antibody used in the labeling procedure is specific for 1,3-B-glucans. Previous results of labeling with a polyclonal antibody (Shapiro and Mullins 1997) are identical to the results of monoclonal antibody labeling. Both antibodies detect 1,3-B-glucans in the cell wall and vacuoles. Previously, serial of cross sections of apical, subapical, and mature regions of a hypha were irnrnunostained with the polyclonal antibody and strong labeling was found in the wall on all the sections (Shapiro 1995). Based on the data presented in Chapter 3, it can not be ascertained whether this hypha was elongating or non-elongating. In the present study, however, the elongating hyphae are distinguished from non-elongating ones and the distribution of 1,3-B-glucans is compared. It is now possible to state that 1,3-B-glucans are found in the apical wall of both elongating and non-elongating hyphae

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CHAPTER 6 LOCALIZATION OF CHITIN IN THE CELL WALL AS REVEALED BY ELECTRON MICROSCOPY AND CYTOCHEMICAL TECHNIQUES Introduction Chitin is the most characteristic polysaccharide of the fungal cell walls It is an unbranched polysaccharide made of N-acetylglucosamine (GlcNAc) joined through 1,4-B bonds. It was once thought to be absent in fungi containing cellulose, but a number of examples from all orders of the Oomycetes have demonstrated a least traces of chitin (Dietrich 1973, 1975) An insoluble fraction from the hyphal wall of Achlya radiosa Maurizio was characterized by x-ray and infrared analyses as chitin, and represented about 4% of the total wall (Campos-Takaki et al. 1982). The role of chitin in Oomycete cell wall remains unclear, and it has been suggested in Saprolegnia that chitin does not play an important role in morphogenesis based on results using the chitin synthase inhibitor polyoxin D (Bulone et al. 1992). An insoluble residue representing about 3% of the wall and containing glucosamine was reported in Achlya (Reiskind and Mullins 1981a). This fraction was identified as chitin by x-ray analysis (Mullins et al. 1984), but had unusual properties in that it is more highly crystalline than the 87

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88 alpha-chitin normally observed in fungi and the characteristic lattice spacing was not readily perceptible. Thus chitin is clearly present in those fungi having cellulose as the major microfibrillar component; but its role is yet to be determined. Materials and Methods Chitin Localization Using Lectin Tomato (Lycopersicon esculentum) lectin conjugated to gold was purchased from EY Laboratories, Inc. (San Mateo, California) The tomato lectin is described by the manufacturer as being specific for oligomers of 1,4-B-linked N-acetylglucosamine, with the binding site being able to accommodate up to 4 carbohydrate units and these units do not have to be consecutive. The sections, on grids, were pretreated with phosphate buffer saline (PBS) containing 1% bovine serum albumin (BSA) at room temperature for 30 minutes. Then they were floated on the labeling solution for 30 minutes. The lectin-gold complex was a 1:9 dilution of the stock solution in PBS. The samples then were washed with PBS three times and rinsed twice in distilled water. Cytochemical Control To determine that the lectin-gold complex was binding to chitin, the probe was pre-incubated with re-acetylated glycol chitosan provided by Dr Michael N. Horst (Mercer University, Macon, Georgia). The glycol chitosan stock (0.118 g/100 mL) was diluted with PBS

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89 (1:9). One part of the lectin-gold stock solution was diluted with nine parts of glycol chitosan and incuba ted for 30 min before labeling of the sections. Results Chitin is localized to the cell wall of Achlya bisexualis with tomato lectin-gold conjugate, where it is evenly distributed in the cross sections (Fig. 70). The chitin labeling is absent when the labeling solution is preincubated with glycol chitozan (Fig. 71). Discussion The tomato-lectin-gold conjugate appears to be a specific label for chitin in the cell walls of Achlya, based on the lack of labeling when the conjugate was pre-incubated with re-acetylated glycol chitosan. Previous studies on chitin (Campos-Takaki et al 1982, Mullins et al 1984, Gay et al 1992) demonstrated its presence in the cell walls of oomycetes with biochemical and biophysical analyses. This is the first report of the cytochemical localization and distribution of chitin in the walls of this group. Bulone et al 1992 described chitin as small globular particles in Saprolegnia, and found that hyphal growth and morphology were not altered when chitin synthesis was inhibited by polyoxin D. They concluded that chitin did not seem to play an important role in morphogenesis. Additional biophysical work on Saprolegnia (Gay et al 1992) describe chitin as localized small round granules of crystalline microfibrilla r alpha

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90 chitin. Chitin, however, synthesized in vitro appeared as spindle-like particles, and was not a skeletal polysaccharide involved in wall architecture. In regenerating protoplast walls it might have a secondary role in wall architecture, since it is microfibrillar. Thus the full role of chitin is still to be determined.

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91 Fig. 70. Transmission electron micrograph showing localization of chitin with tomato lectin on cross section of Achlya bisexualis hypha. Bar=0.5 m. Fig. 71. Transmission electron micrograph showing the absence of chitin labeling on the cross sections of Achlya bisexualis hyphae resulting from preincubatio n of the labeling solution with re-acetylated glycol chi t o z a n Bar=l.00 rn.

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CHAPTER 7 CONCLUSIONS The results of cellulose localization suggest that in non -elongating hyphae, cellulose is evenly distributed along the hypha and is present in the apex. Elongating hyphae lack cellulose at the apices or there is a gradual decrease of cellulose amount toward the apices. On the other hand, the major matrix component of the wall, 1,3-B-glucan is distributed evenly over the elongating and non-elongating hyphae and is present in their apices. Such distribution of these two major components of Achlya cell wall suggests that in the elongation zone, 1,3-B-glucans are synthesized first and cellulose deposition follows This contradicts the idea shared by the major theories of hyphal tip growth, that all the wall components are present in the elongation zone. The small diameters of the cellulose unlabeled regions of elongating hyphae suggest that cellulose deposition takes place almost immediately after the start of 1 3-B-synthesis The plastic wall that consists mainly of 1 3-B-glucans and lacks cellulose support is stretched under the turgor pressure and/or perhaps pressure of cytoskeleton The quickly following cellulose deposition helps to maintain the tubular cell shape and prevents the

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93 elongation regions from ublowing outu in balloo n-like structures. Cellulose is thought to provide mechanical support for the cell wall. The init ial cellulose hydrolysis by cellulase in the growing apex is possible. This could create new primers in existing cellulose chains, as suggested by Maclachlan (1976) for higher plants It was found that the growing colonies of Achl ya secrete endocellulase (Thomas and Mullins 1967; 1969). The authors suggested that endocellulase is important for the wall softening since this fungus does not use cellulose in nutrition. The recent evidence that activit y of the secreted endocellulase correlates with the tensile strength of the apical hyphal wall support this idea (Money and Hill 1997). The results of cellulose and 1,3-B-glucans distribution in the apical wall, combined with the results of hyphal growth monitoring suggest a new aspect of the hypothesis for hyphal tip growth. This hypothesis would state that in the Achlya growth process, all hyphae go through periods of elongation and no-elongation (dormancy) The elongation is not a steady process as it is generally assumed. It consists of alternating periods with fast and slow growth rates. Elongation starts with synthesis of 1,3-B-glucans, which is quickly followed by synthesis of cellulose

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LITERATURE CITED Alexopoulos CJ, Mims CW, Blackwell M. 1996. Introductory Mycology. New York: John Wiley and Sons. 869 p. Arioli T Peng L, Betzner AS, Burn J, Wittke W, Herth W, Camilleri C, Hofte H, Plazinski J, Birch R, Cork A, Glover J, Redmond J, Williamson RE. 1998. Molecular analysis of cellulose biosynthesis in Arabidopsis. Science 279:717-720. Aronson JM. 1981. Cell wall chemistry, ultrastructure and metabolism In : Cole GT, Kendrick B. eds. Vol. 2: Biology of conidial fungi. New York: Academic Press. p 459-471. Aronson JM, Preston RD. 1960. The microfibrillar structure of the cell walls of the filamentous fungus, Allomyces. J Biophys Biochem Cytol 8:245-256. Barkai-Golan R, Sharon N . 1978. Lectins as a tool for the study of yeast cell walls. Exp Mycol 2:110-115. Bartnicki-Garcia S. 1968. Cell wall chemistry, morphogenesis, and taxonomy of fungi. Ann Rev Microbial 42:57-69 Bartnicki-Garcia S. 1973. Fundamental aspects of hyphal morphogenesis. Pp. 245-267. In: Arthwoth JM, Smith E. eds Microbial differentiation. Syrop Soc Gen Microbial. Cambridge : University Press. p 245-267 Bartnicki-Garcia S. 1996. The hypha: unifying threads of the fungal kindom In : Sutton BC. Ed. A century of mycology. Cambridge : University Press. Bartnicki-Garcia S, Ruiz-Herrera J, Bracker CE. 1979. Chitosomes and chitin synthesis. In: Burnett JH, Trinci APJ. eds Fungal walls and hyphal growth. Cambridge: University Press p 149-168 Beakes GW. 1987 Oomycete phylogeny: ultrastructural prospectives. In: Rayner ADM, Brasier CM, Moore D. eds Evolutionary Biology of Fungi Cambridge: University Press p 405-421. 94

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95 Berg RH, Erdos GW, Gritzali M, Brown RD Jr 1988. Enzyme gold affinity labeling of cellulose. J Elect Microscop Tech 8 : 371-379. Blanton RL. 1997. Cellulose biogenesis in Dictyostelium disdodeum. In: Mnaeda Y, Inouye K, Takeuchi K eds Dictyistelium, A Model System for Cell and Developmental Biology. Frontiers Science Series No 21 Tokyo: Universal Academy Press. p.379-391. Bohn CFC, Bemiller JN. 1995. Beta(1-3) -D-glucans as biological response modifiers: a review of structure functional activity relationship. Carb Polym 28:3-14. Bourett TM, Czymmek KJ, Howard RJ. 1998. An improved method for affinity probe localization in whole cells of filamentous fungi. Fungal Gen Biol 24:3-13. Burnett JH. 1979. Aspects of the structure and growth of hyphal walls. In: Burnett JH, Trinci APJ. eds. Fungal Walls and Hyphal Growth. Cambridge: University Press. p 1-25. Byrd JC, Tarentino AL, Maley F, Atkinson PH, Trimble RB. 1982. Glycoprotein synthesis in yeast. J Biol Chem 257: 14657-14666. Cabib E. 1981. Chitin: structure metabolism, and regulation of biosynthesis. In: Tanner W, Loewus TA. eds. Plant carbohydrates II. Ency Plant Physiol Vol. 13. Berlin: Springer. p 395-415. Cabib E, Bowers B, Roberts RL. 1983. Vectorial synthesis of a polysaccharide by isolated plasma membranes. Proc Nat Acad Sci USA 80:3318-3321. Cabib E, Bowers B, Sburlati A, Silverman SJ. 1988. Fungal cell wall synthesis: the construction of a biological structure. Microbial Sci 5:370-375. Cabib E, Silverman SJ, Shaw JA, Gupta SD, Park H, Mullins JT. 1991. Carbohydrates as structural constituents of yeast cell wall and septum. Pure Appl Chem 63:483-489. Cameron LE, LeJohn HB. 1972. On the involvement of calcium in amino-acid transport and growth of the fungus Achlya. J Biol Chem 247:4729-4739. Cantino EC, Horenstein EA. 1953. Carotenoids and oxidative enzymes in the aquatic Phycomycetes Blastocladiella and Rhizophlyctis. Amer J Bot 40:688-694.

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96 Carlile MJ, Watkinson SC. 1994. The Fungi. London: Academic Press. 482 p. Cavalier-Smith T. 19 83. A 6-kingdom classification and a unified phylogeny. In: Schenk HEA, Schwemmler W. eds. Endocytobiology II. Berlin: DeGruyter. p 1027-1034 Cohen RE, Ballou CE. 1981. Mannoproteins : structure. In: Tanner W, Loewus FA. eds Plant carboh ydrates II. Vol 13B. Berlin: Springer. p 441-458. Cote F, Hahn MG. 1994. Oligosaccharins: str uctures and signal transduction. Plant Mol Biol 26:1379-1411. Cottin gham CK, Mullins JT. 1985. The preliminar y characterization of the beta-glucosidases in Achlya ambisexualis. Mycologia 77:381-389. Delmer DP. 1999. Cellulose biosynthesis: exciting times for a difficult field of study. Annu Rev Plant Physiol Plant Mol Biol 50:245-276. Dietrich SMC. 1973. Carbohydrates from the hyphal walls of some Oomycetes. Biochim Biophys Acta 313:95. Dietrich SMC. 1975. Comparative study of hyphal wall components of Oomycetes: Saprolegniaceae and Pythiciceae. Ann Acad Bras Ciencas 47:155-162. Dixon RA, Lamb CJ. 1990. Molecular communication in interaction s between plants and microbial pathogens. Ann Rev Plant Physiol Plant Mol Biol 41:339-367. Eamus D, Jennings DH. 1986. Turgor and fungal growth: studies on water relations of mycelia of Serlupa lacrimans and Phallus impudicus Trans Brit Mycol Soc 86:527-535. Fevre M, Girard V, Nodet P. 1990 Cel lulose and beta glucan synthesis in Saprolegnia. In: Kuhn PJ, Trinci, APJ, Jung MJ Goosey MW, Copping LG. eds. Biochemistry of cell walls and membranes in fungi. Berlin: Springer. p 97-107 Forster H Coffey MD. 199 0. Sequence analysis of the small subunit ribosomal RNAs of three zoosporic fungi and implications of fungal evolution Mycologia 82:306-312. Forster H, Oudemans P Coffey MD. 1990 Mitochondrial and nuclear DNA diversity within six species of Phytophtora Exp Mycology 14 : 18-31

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97 Frens G. 1973. Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions. Nature Phys Sci 241:20-22. Fult z SA, Sussman AS. 1966. Antigenic differences in the surface of hyphae and rhizoids in Allomyces. Science 152: 785-786. Garrill A, Lew RR, Heath IB. 1992. Stretch-a ctivated Ca 2 + and ca 2 + -activated K+ channels in the hyphal tip plasma membrane of the oomycete Saprolegnia ferax. J Cell Sci 101: 721-730. Garrill A, Jackson SA, Lew RR, Heath IB. 1993. Ion channel activity and tip growth: tip-localized stretch-activated channels generate an essen ti al Ca 2 + gradient in the oomycetes Saprolegnia ferax. Eur J Cell Biol 60:358-365. Gaumann EA, Dodge CW. 1928. Comparative Morpholog y of Fungi. New York: McGraw-Hill. 701 p. Girard V, Fevre M. 1984. Beta-1-4and beta-1-3-gluca n synthases are associated with the plasma membrane of the fungus Saprolegnia. Planta 160:400-406. Girard V, Maclachlan G. 1987. Modulation of pea membrane beta-glucan synthase activity by calcium, pol yc ations, endogenous protease, and protease inhibitors. Plant Physiol 85:131-136. Gooday GW. 1995. Cell walls. In: Gow NAR, Gadd GM. eds. The growing fungus. London: Chapman and Hall. p 43-62. Gow NAR 1984 Transhyphal electrical currents in fungi. J Gen Microbial 130:3313-3318. Gow NAR. 1995. Tip growth and polarity. Pp. 277-300. In: Gow NAR, Gadd GM. eds. The growing fungus. London: Chapman and Hall. P 277-300. Gow NAR Kropf DL, Harold FM. 1984. Growing hyphae of Achlya bisexualis generate a longitudinal Ph gradient in the surrounding medium. J Gen Microbial 130:2967-2974. Griffin DH, Breuker C. 1969. Ribonucleic acid synthesis during the differentiation of sporangia in the water mold Achlya. J Bacterial 98:689-696. Grove SN. 1978. The cyto lo gy of hyphal tip growth. In: Smith JE, Berry DR eds. The filamentous fungi New York : John Wiley and Sons. p 28-50

PAGE 104

98 Grove SN Bracker CE, Moore DJ 1970. An ultrastructural basis for hyphal tip growth in Pithium ultimum. Arner J Bot 57:245-266. Gull K, Trinci APJ. 1974 Detection of areas of wall differentiation in fungi using fluorescent staining. Arch Microbial 96 : 53-57. Harold FM. 1994. Ionic and electrical dimensions of hyphal growth. In: Wessels JGH, Meinhardt F. eds. The mycota. I. Growth, Differentiation, and Sexualit y Germany: Springer-Verlag. p 89-110. Heath IB 1987 Preservation of a labile cortical array of actin filaments in growing hyphal tips of the fungus Saprolegnia ferax. Eur J Cell Biol 44:10-16. Hea th IB. 1994. The cytoskeleton. In: Wessels, JGH, Meinhardt, F. eds. The mycota. I. Growth, Differentiation, and Sexuality. Germany: Springer-Verlag, p 43-66. Hemmes DE, Kojima-Baddenhagen ES, Hohl HR 1972. Structural and enzymatic analysis of the spore wall layers in Dictiostelium discoideum. J Ultrastruct Res 41 : 406-410 Hill TH, Mullins JT. 1979 Hyphal tip growth in Achlya: enzyme activities in mycelium and medium Can J Bot 57 : 2145-2149 Hill TH, Mullins JT. 1980. Hyphal tip growth in Ach lya I Cytoplasmic organisation. Can J Microbial 26:11321140. Hoch HC, Staples RC, Whitehead B. 1987 Signaling for growth orientation and cell differentiation by surface topography in Uromyces. Science 235:1659-1662. Horgen PA. 1977. Steroid induction of differntiatio n : Achlya as a model system. In: O'Day DH, Horgen PA. eds. Eukaryotic microbes as model developmental s ystems New York : Marcel Dekker. p 272 -292. Horisberger M Rasset J 1977 Loca lization of alpha-galacto mannan on the surface of Schizosaccharomyces pombe cells by scanning electron microscopy Arch Microbial 112 : 123-125 Horisberger M, Rasset J Bauer H. 1975 Colloidal gold granules as markers for cell surface receptors in the scanning electron microscope. Experientia 31 : 1147.

PAGE 105

99 Horisberger M, Vonlanthen M. 1977. Localization of mannan and chitin on thin sections of budding yeast with gold markers. Arch Microbial 115:1-12. Horisberger M, Vonlanthen M, Rasset J. 1978. Localization of alpha-galacto mannan and of weat germ agglutinin receptors in Schizosaccharomyces pombe. Arch Microbial 119:107-111. Hromova M, Taft 0, Selitrennikoff CP. 1989. Beta(1-3) -D-glucan syntase of Neurospora crassa: partial purification and characterization of solubilized enzyme activity. Exp Mycol 13:129-139. Hunsley D, Burnett JH. 1970. The ultrastructu ral architecture of the walls of some fungi. J Gen Microbial 62:203-218. Hunsley D, Kay D. 1976. Wall structure of the Neurospora hyphal apex: irnmunofluorescent localization of wall surface antigens. J Gen Microbial 95:233-248. Hutter R, DeMoss JA. 1967. Organization of tryptophan pathway: a phylogenetic study of the fungi. J Bacterial 94:1896-1907. Jabri E, Quigley DR, Alders M. 1991. Beta(1-3) -glucan synthesis of Neurospora crassa. Current Microbial 19:153-161. Jackson SL, Heath IB. 1993. Roles of calcium ions in hyphal tip growth. Microbial Rev 57:367-382. Jaffe LF, Nuccitelli R. 1974. An ultrasensetive vibrating probe for measuring steady intracellular currents. J Cell Biol 63:614-628. Jeenah M, Davidson BE, Boothby D. 1982. Layered cell wall of Trichoderma cell walls. Arch Microbial 133:330-332. Johnson BF, Calleja CB, Yoo BY. 1990. A new model for hyphal tip extension and its application to differentia l fungal morphogenesis. In: Chiu S-W, Moore D. eds. Patterns in fungal development. Cambridge: Universit y Press. p 37-69. Johnson TW. 1956. The genus Achlya. Morphology and taxonomy. Ann Arbor: University of Michigan Press. 180 p. Kamada T, Bracker CE, Bartnicki-Garcia S. 1991 Chitosomes and chitin synthase in the asexual life cycle of Mucor rouxii. J Gen Microbial 137 : 1241-1252

PAGE 106

100 Kaminsskyj SGW, Garrill A, Heath IB. 1992. The re lation between turgor and tip growth in Saprolegnia ferax: turgor is necessary, but not sufficient to explain apical extension rates. Exp Mycol 16:64 75. Katz D, Rosenberger RF. 1970 A mutation in Aspergillus nidulans producing hyphal walls which lack chitin. Biochim Biophys Acta 208:452-460. Kropf DL, Caldwell JH, Gow NAR, Harold FM. 1984. Transcellular ion currents in the water mold Achlya ambisexualis amino-acid proton symport as a mechanism of current entry J Cell Biol 99:486-496. Kropf DL, Lupa MDA, Caldwell JH, Harold FM. 1983. Cell polarity: endogenous ion currents precede and predict branching in the water mold Achlya. Science 220 : 13851387 Kuranda MJ, Robbins PW. 1991. Chitinase is requiered for cell separation during growth of Saccharomyces cerevisiae. J Biol Chem 266:19758-19767. Kwok S, White TJ, Taylor JW. 1986. Evolutionar y relationships between fungi, red algae, and other simple eucaryotes inferred from total DNA hybridizations to a cloned basidiomycete ribosomal DNA. Exp Mycol 10 : 196-204. Lee JH, Mullins JT. 1994. Cytoplasmic water-so luble beta-glucans in Achlya: response to nutrient limitation. Mycologia 86:235-241. Lee JH, Mullins JT, Grander JE 1996. Water-soluble reserve polysaccharides from Achlya are beta(1->3) -glucans. Mycologia 88:254-270. Lehle L 1981. Biosynthesis of mannoproteins in fungi. In : Tanner W, Loewus FA. eds. Plant carbohydrates II Berlin : Springer. p 459-483. LeJohn HB Klassen GR McNaughton DR, Cameron LE, Goh SH, Meuser RU 1977 Unusual phosphorylated compounds and transcroptional control in Achlya and other aquatic molds Pp 69-76 In : O Day DH, Horgen PA eds Eukaryotic microbes as model developmental systems. New York : Marcel Dekker p 69-76. Lopez-Franco R Bartnicki-Garcia S, Bracker CE 1994 Pulsed growth of fungal hyphal tips. Proc Natl Acad Sci USA 91 : 12228-12232

PAGE 107

101 Lovett JS, Haselby JA. 1971. Molecular weigh ts of the ribosomal ribonucleic acids of fungi Archiv fur Microbiologie 80 : 191-204. Luard EJ. 1982a Accumulation of intracellula r solutes by two filamentous fungi in response to growth at low steady state osmotic potential. J Gen Microbial 128:2563-2574. Luard EJ 1982b. Effects of osmotic shock on some intracellular solutes in two filamentous fungi. J Gen Microbial 128:2575-2581. Luard EJ. 1982c. Growth and accumulation of so lutes by Phytophtora cinnamoni and other lower fungi in response to changes in external osmotic potential. J Gen Microbial 128:2583 2590 Luard EJ, Griffin DM. 1981. Effects of water potential on fungal growth and turgor. Trans Brit Mycol Soc 76:33-40. Maclachlan GA 1976 A potential role for endo-cellulase in cellulose boisynthesis. Appl Polymer Symp 28:645-658. Manavathu EK Thomas DdesS. 1982. The uptake of S-adenosyl-L-methionine in the aquatic fungus Achlya ambisexualis FEBS Letters 137 : 14-18. Manavathu EK, Thomas DdesS 1985 Chemotropism of Achlya ambisexualis to methionine and methionyl compounds. J Gen Microbial 131 : 751 756. Marchant R, Smith DG 1968. A serological investigation of hyphal growth in Fisarium culmorum. Arch Microbial 63:85-94 Margulis L, Corliss JO, Melkonian M, Chapman DJ. 1990. Handbook of Protoctista. Boston: Jones and Bartlett. Meikle PJ, Bonig I, Hoogenraad NJ, Clarke AE, Stone BA. 1991. The location of (1->3) -beta-glucans in the walls of pollen tubes of Nicotiana alata using a (1->3) -beta-glucan-specific monoclonal antibod y Planta 185:1-8. Miyata M, Kanbe T, Tanaka K. 1985 Morphological alternations of the fission yeast Schizosaccharom ycetes pombe in the presence of aculeacin A: spherical wall formation. J Gen Microbial 131 : 611-621. Michalenko GO, Hohl HR, Rast D. 1976. Chemistry and architecture of the mycelial wall of Agaricus bisporus. J Gen Microbial 92 : 251-256.

PAGE 108

102 Money NP Harold FM 1 992. Extension growth of the water mold Achlya: int erplay of turgor and wall strength. Proc Natl Acad Sci USA 89:4245-4249. Money NP Harol d FM 1993. Two water molds can grow without measurable turgor pressure. Planta 190:426-430. Money NP, Hill TW. 1997. Correlation between endoglucanas e secretion and cell wall strength in oomycete hyphae : implications for growth and morphogenesi s. Mycologia 89: 777-785. Montezinos B. 1982. The role of the plasma membrane in cellulose microfibril assembly. In: Lloyd CW. Ed The cytoskeleton in plant growth and development. London: Academic Press. p 141-162. Moore-Lan decker E. 1996. Fundamentals of the Fungi. Prentice New Jersey: Prentice Hall. 574 p. Muller SC, Brown RM Jr. 1980. Evidence for an intramembrane component associated with a cellulose microfibril synthesizing complex in higher plants. J Cell Biol 84:315-326. Mullins JT 1994. Hormonal control of sexual dimorphism. Pp. 413-422 In: Wessels JGH, Meinhardt F. eds. The mycota. I. Growth, Differentiation, and Sexuality. Germany : Springer-Verlag, Germany. p 413-422. Mullins JT, Bertke CC, Aronson JM. 1984. An unusual form of chitin in Achlya ambisexualis? Mycol Soc Arner Newslett er 35 : 36 Mullins JT, Ellis EA. 1974. Sexual morphogenesis in Achlya: ultrastructural basis for the hormonal induction of antheridial hyphae Proc Natl Acad Sci USA 71:1347-1350. Musgrave A, Ero L, Scheffer R, Oehlers E. 1977. Chemotropism of Achlya bisexualis germ hyphae to casein hydrolysate and amino-acids. J Gen Microbial 101:65-60. Nakajima T Ballou CE. 1974. Characterization of the carbohydrate fragments obtained from Saccharomyces cerevisiae mannan by alkaline degradation. J Biol Chem 249 : 5798-5801 Northcote DH. 1984. In : Dugger WM, Bartnicki-Garcia S. eds Structure function and biosynthesis of plant cell walls Baltimore: Waverly Press p 222-234

PAGE 109

103 Ojha M, Dutta SK, Turian G 1975. DNA nucleo ti d e seq uense homologies between some zoosporic fungi. Mol G e ner Genetics 136:151-165. Park D, Robinson PD 1966 Aspects of hyphal morphogenesis in fungi In: Cutter EG. ed. Trends in plant morphogenesis. London: John Wiley and Sons. p 27-44. Peberdy JF. 1990. Fungal cell walls a review. In: Kuhn PJ, Trinci APJ, Jung MJ, Goosey WM, Copping LG eds. Biochemistry of cell walls and membranes in fungi. Berlin: Springer. p 5-30. Perez P, Garcia-Acha I, Duran A. 1983. Effect of papulacandin Bon the cell wall and growth of Geotricum lactis. J Gen Microbial 129:245-250. Pfyffer GE, Rast DM. 1988. The polyol pattern of fungi as influenced by the carbohydrate nutrient source. New Phytologist 109:321-326. Pfyffer GE, Rast DM. 1989. Accumulation acyclic polyols and trehalose as related to growth form and carbohydrate source in the dimorphic fungi Mucor rouxii and Candida albicans. Mycopathologia 105: 25-33. Poulain D, Tronchin G, Dubrernetz JF, Biguet J. 1978. Ultrastructure of the cell wall of Candida albicans blastospores: study of its constitutive layers by the use of a cytochernical technique revealing polysaccharides. Ann Microbial Inst Pasteur 129A:141. Raper JR. 1939. Role of hormones in the sexual reaction of heterothallic Achlya. Science 89:321-322. Rast D, Hollenstein GO. 1977. Architecture of the Agaricus bisporus spore wall. Can J Bot 55:2251-2255. Reinhardt MO. 1892. Das Wachsthurn der Pilzhyphen. Jahrb Wiss Bot 23:479-566 Reiskind JB, Mullins JT. 1981a. Molecular archtecture of the hyphal wall of Achlya ambisexualis Raper. I.Chemical analyses. Can J Microbial 27:1092-1099. Reiskind JB, Mullins JT. 1981b. Molecular architecture of the hyphal wall of Achlya ambisexualis Raper. II. Ultrastructural analysis and a proposed model. Can J Microbial 27:1100-1105. Rielh RM, Toft DD. 1984. Analysis of steroid recepto r o f Achlya ambisexualis. J Biol Chern 259:15324-15330.

PAGE 110

104 Robertson NF. 1958. Observations of the effect of water on the hyphal apices of Fusarium oxysporum. Ann Botan y (London) N.S. 22:159-173. Robertson NF, Rizvi SRH. 1968. Some observations on the water relations of the hyphae of Neurospora crassa. Ann Bot 32:279-291. Rosenberger RF. 1976. The cell wall. In: Smith JE, Berr y DR. eds. The filamentous fungi. New York : John Wiley and Sons p 328-344. Rouvinen J, Bergfors T, Teeri Y Knowles JKC, Jones TA 1990 .Three-dimensional structure of cellobiohydrolase. Science 24 9:380-386. Ruiz-Herrera J. 1991. Fungal cell wall: structure, synthesis, and assembly. Boca Raton: CRC Press. 248 p. Ryan CA. 1987 Oligosaccharide signaling in plants. Ann Rev Cell Biol 3:295-317. Schneider EF, Wardrop AB. 1979. Ultrastructural s tudies on the cell walls in Fusarium sulphureum. Can J Microbial 25: 75-85. Schreurs WJA, Harold FM. 1988. Transcellular proton current in Achlya bisexualis hyphae: relationship to polarized growt. Proc Natl Acad Sci USA 85:1534-1538. Scopsi L, Larsson L, Bastholm L, Nielsen WH 1986. Silver enhance colloidal gold probes as markers for scanning electron microscopy Histochem 86:35-43. Shapiro A. 1995 Hyphal tip growth of Achlya bisexua lis : microarchitecture of the apical dome. MS Thesis. Gainesville Florida : Univ Florida. 67 p. Shapiro A, Mullins JT. 1997 Localization of cytoplasmic water-soluble reserve (1->3) -beta-glucans in Achlya with immunostaining Mycologia 89 : 89-91 Sentandreu R, Mormeneo S, Ruiz-Herrera J 1994 Biogenesis of the Fungal Cell Wall. In : Wessels JGH, Meinhardt F eds The mycota I. Growth differentiation, and sexuality Germany : Springer-Verlag. p 111-124. Sentandreu R Northcotr DH. 1969 The characterization of glycosaccharides attached to threonine and serine in a mannan glycopeptide obtained from the cell wall of yeast Carbohydr Res 10 : 584-585

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105 Shematek EM, Braatz JA, Cabib E 1980. Biosynthesis of yeast cell wall. I. Preparation and properties of beta(l-3)glucan synthase. J Biol Chem 255 : 888-894 Sietsma JH. 1969. Protoplast formation and cell wall composition of some oomycete species. PhD Dissertation. Univ Amsterdam. Sietsma JH, Sonnenberg ASM, Wessels JGH. 1985. Localization by autoradiography of synthesis of beta(1->3) and beta(1->6) linkages in a wall glucan during hyphal growth of Schizophyllum commune. J Gen Microbial 131:1331-1337. Sparrow FK. 1960. Aquatic phycomycetes. 2 n d ed. Ann Arbor: The University of Michigan Press. 1187 p. Strunk C. 1968. Demonstration of the apical pore in Polystictus versicolor. Arch Microbial 60:255-261. Tanner Wand Lehle L. 1987. Protein glycosilation in yeast Biochim Biophys Acta 906:81-89. Thomas DdesS, Lutzas M, Manavathu EK. 1974. Cytochalasin selectivity inhibits synthesis of a secretory protein cellulase in Achlya. Nature 429:140-142. Thomas DdesS, Mullins JT. 1967. Role of enzymatic wall softening in plant morphogenesis: hormonal induction in Achlya. Science 156:84-85. Tronchin G, Poulain D, Herbaut J. 1979. Etudes cytochemiques et ultrastracturals de la paroi de Candida albicans. I. Localisation des mannanes a utilisation deconcanavaline A surcoupes ultrafines. Arch Microbial 26 : 121 Vogel HJ. 1964. Distribution of lysine pathwa ys among fungi: Evolutionary implications. American Naturalist 68:435-436. Wang MC, Bartnicki-Garcia S. 1980. Distribution of mycolaminarin and cell wall beta-glucans in the life cycle of Phytophtora. Exp Mycol 4:269-280. Webster J. 1980. Introduction to Fungi. 2~ edition. Cambridge: University Press. 669 p. Wessels JGH. 1986. Cell wall synthesis in api cal hyphal growth. Int Rev Cytol 104:37-79.

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106 Wessels JGH. 1990. Role of wall architecture in fungal tip growth generation. In: Heath IB. ed. Tip growth in plant and fungal cells. San Diego: Academic Press. p 1-29 Wessels JGH, Sietsma JH. 1979. Wall structure and growth in Schizophyllum commune. In: Burnett JH, Trinci APJ. eds. Fungal walls and hyphal growth. Cambridge: University Press. 27-48 p. Wessels JGH, Sietsma JH, Sonnenberg ASM. 1983. Wall synthesis and assembly during hyphal morphogenesis in Schizophyllum commune. J Gen Microbial 129:1607-1616. Woods DM, Duniway JM. 1986. Some effects of water potential on growth, turgor, and respiration of Phytophthora cryptogea and Fusarium moniliforme. Phytopathol 76:1248-1254. Wu-Yuan CD, Hashimoto T. 1977. Architecture and chemistry of microconidial walls of Trichophyton mentagrophytes. J Bacterial 129:1584-1589. Yamaguchi H. 1974. Effect of biotin insufficiency on composition and ultrastructure of cell wall of Candida albicans in relation to mycelial morphogenesis. J Gen Appl Microbial 20:217. Yaun S, Heath IB. 1991. Chlortetracycline staining patterns of growing hyphal tips of the oomycete Saprolegnia ferax. Exper Mycol 15:91-102. Zhou X-L, Stumpf A, Hoch HC, Kung C. 1991. A mechanosensetive channel in whole cells and in membrane patches of the fungus Uromyces. Science 253:1415-1417. Zlotnik H, Fernandes MP, Bowers B, Cabib E. 1984. Saccharomyces cerevisiae mannoproteins form an external cell wall layer that determines wall porosity. J Bacterial 159 : 1018-1026.

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BIOGRAPHICAL SKETCH Alexandra Shapiro was born in Moscow, Russia, on January 9, 1971. She graduated from high school in Moscow in 1988 In 1992, after four years of studies at the Moscow State University, she entered graduate school at the University of Florida in Gainesville. In 1995 Alexandra Shapiro received an MS in the Department of Botany and in 1996 she started her PhD program. She is married to Andrei Sourakov and has two daughters born in 1995 and 1997. 107

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholar ly p resentation and is fully adequate, in scope and qual ity as a dissertation for the degree of Doctor 9f Philosophy :=?5, -_ l4t l) [/J /)i~if I t0 J l Thomas Mullins, Chair Professor of Botany I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and sialit, as a dissertation for the degree of Doctor of P 1, lo f ophy ~I( / // [ / ff7 Gl'.?eg w Er OS / Asso ~ at / Scientist Interdisciplinary Cente r for Biotechnology Research I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Alice Harmon Associate Professor of Botany I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doc or of Phil ~ sophy C. 0 vnJ Thomas C Emmel Professor of Zoology This dissertation was submitted to the Graduate Facult y of the Department of Botany in the College of Liberal Arts and Sciences and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. December 2000 Dean Graduate Schoo l

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UNIVERSITY OF FLORIDA II I II IIIII I Ill I l l ll l ll l l lll I I I II I II I I II I II I II I I I I II II l l ll l l Ill I I 3 1262 08554 457 4


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