J. Cell Sri. 50, 171-180 (1981)
171
Printed in Great Britain © Company of Biologists Limited xg8i
A SEARCH FOR SYNAPTONEMAL
COMPLEXES IN USTILAGO MAYDIS
H. L. FLETCHER*
Genetics Department, National Institute for Medical Research,
The Ridgeway, Mill Hill, London NW7 1AA, England
SUMMARY
Germinating spores, germ-tubes (promycelia), gall tissue and spores developing in the gall
were examined. No synaptonemal complexes (SCs) were found in any of these cell types. There
are 3 possible explanations for this: (1) Ustilago maydis does not have SCs. This is the case in
Schizosaccharomyces pombe (Olson, Eden, Egel-Mitani & Egel, 1978) and in many strains of
Saccharomyces cerevisiae (e.g. see Byers & Goetch, 1975). (2) The SC might occur in spores
either in the gall or before germination when the spore wall was too solid to allow examination
of the contents by the methods used. (3) The SC was present, but for a very short time, and
most of the cells examined were in any case not undergoing meiosis, so the SCs were not seen.
(Large numbers of spores were examined, but few spores germinate and undergo normal
meiotic division; also a minority of the cells in the gall are forming spores at any one time.)
The gall was found to contain uninucleate single cells (i.e. it is a yeast, as it is in artificial
culture medium) and virtually all these cells were haploid, 1 in 3000 recovered cells was diploid.
It appears that the haploids fuse to form a heterokaryotic or diploid cell immediately before
spore formation. A heterokaryotic phase is presumed to exist to establish and maintain the
infection.
INTRODUCTION
Ustilago maydis is a heterobasidiomycete that causes covered smut in maize.
Numerous mutants are known, and U. maydis is amenable to genetic and biochemical
analysis. The mechanisms that manipulate DNA during meiosis, especially those
leading to hybrid DNA formation and genetic conversion or recombination, are of
particular interest (Holliday, 1977). In order to facilitate the biochemical investigation of these processes it was desirable to examine microscopically the stages of
meiosis to correlate the physical and biochemical events of meiosis. Ultrastructural
investigation of the synaptonemal complex (SC) should indicate when the homologous
bivalents were synapsed. Reconstruction of the SCs from serial sections should also
reveal the number of bivalents for comparison with genetic linkage maps.
The vegetative phase of U. maydis is a budding yeast. Both haploid and diploid
cells are prototrophic and can be cultured in artificial medium. Haploids of opposite
mating type will fuse on special medium or in the host tissue to form a heterokaryon.
Diploids may be selectively recovered from gall tissue. The meiotic phase is only
initiated within the host. Meristematic host tissue is induced to proliferate and form a
gall, which is eventually digested by the U. maydis and becomes filled with teliospores.
• Present address: Genetics Subdepartment, David Keir Building, The Queen's
University of Belfast, Stranmillis Road, Belfast BT7 iNN, Northern Ireland.
172
H. L. Fletcher
These can be germinated on complete medium. The spore grows a promycelium or
germ-tube. The nucleus moves into this and then undergoes both meiotic divisions.
Cell walls partition the germ-tube between the nuclei, and then the 4 cells bud off
haploid, yeast-like basidiospores that reproduce by vegetative budding (Fischer &
Holton, 1957).
There is evidence that the temperature, pH and nutritional and ionic composition
of the medium on which the teliospores germinate all affect the frequency of recombination between the mating type locus and the centromere (Hiittig, 1931, 1933).
Recombination at other loci changes non-linearly with temperature (Holliday,
personal communication). Thus it seemed that meiotic recombination occurred
during germination and this was the first stage to examine for SCs. In many fungi
(e.g. Neottiella and Neurospora) meiosis occurs immediately after fusion of 2 haploid
nuclei to form a diploid. Developing teliospores of the Cedar Apple Rust fungus
Gymnosporangiumjumperi-virgimanae were found to contain SCs (Mims, 1977). This
species is also a heterobasidiomycete and the most closely related species to U. maydis
in which SCs have been found. In Blastocladiella emersonii (Phycomycete) SCs are
present in resting sporangia 28 to 52 h old (Olson & Reichle, 1978). It could be
advantageous to the fungus to start meiosis while in a secure position and so reduce the
time required for germination of the spore in a potentially hostile environment. The
developing spores in the gall are thus the second place in which to look for SCs.
Irradiation of diploid cells induces meiotic levels of recombination in surviving cells,
presumably as a result of the repair mechanisms. This was also a reasonable situation
in which to look for SCs.
MATERIALS AND METHODS
Spores
The most important criterion was a high level of synchronous germination. Spores from
cross Mi46
nari-12 niri-i inosi-3nici-2 + B2 b_2
nan-13 niri-i inosi-3 + tsdi-i ai bi
were used initially, then spores from a wild-type cross. Spore suspension was dropped onto
the surface of complete agar medium so that there was a spacing of around 30 /tm to 40 /im
between spores. If the spores were too close together it was difficult to section them. The
spores germinated in the plane of the surface, so sectioning in this plane consistently gave
sections of both spores and complete germ-tubes with buds at appropriate stages. Germination
was at temperatures between 25 and 35 °C. Germinating spores were enrobed by pouring a
thin layer of molten 2 % agar at 46 °C over them. Enrobed cells continued to grow if left
unfixed, so the few seconds at 46 °C did not harm them. Fixation was in 4 % glutaraldehyde in
o-i M-cacodylate or 0-05 M-phosphate buffer at pH 7 applied before, with, or after the agar.
Few cells could be sectioned unless the cell walls were eroded enzymically, but after penetrating the cell wall the enzymes attacked the cytoplasm. The mercaptoethanol/55 % gluculase
treatment of Zickler & Olson (1975) was used: o-i M-mercaptoethanol, 0-2 M-Tris, 0-02 MEDTA at pH 9 for 10 min at 35 °C; followed by o-i ml of o-i M-sodium citrate/phosphate
buffer, pH 5-8,+ 0-3 ml 1 M-KC1 + O-OI ml 1 M-MgCl + O'S ml gluculase for 3 h at 35 °C.
Cellulase (Okazaki) was also at between 1 and 5 % concentrations in o-i M-citrate/phosphate
at pH 5 (485 ml of 01 M-citric acid+ 51-5 ml 01 M-NajPO*) either alone or with gluculase.
Incubation times varied between 30 min and 4 h. Intracellular membranes did not stain
Synaptonemal complexes in Ustilago maydis
173
with osmium after gluculase treatment. Gluculase did not digest SCs in grasshopper testes.
Spores were usually postfixed in 1 % osmium tetroxide in sodium barbitone buffer, pH 7-2
(379 g/1 sodium barbitone, 2-42 g/1 sodium acetate, 0-0125 M-HC1) f° r 4 h or overnight.
Dalton's (1955) chrome/osmium was also used overnight. Spores were thoroughly washed,
dehydrated through an ethanol/water series or in acidified 2-2-dimethoxypropane for 5 min
(Muller & Jacks, 1975). Some samples were stained in uranyl acetate in 75%, 90% or 98%
ethanol for 1-2 h. Cells were infiltrated with Spurr' (1969) resin overnight and embedded.
Sections were cut with a glass knife, and were poststained with uranyl acetate/lead citrate
(Reynolds, 1963).
Gall tissues
Maize seedlings were inoculated with compatible wild-type strains of U. maydis. Galls
formed within a week, and spores then started to accumulate. Segments of gall just beginning
to produce spores were fixed in 4 % glutaraldehyde. The U. maydis cells were thin-walled but
embedded in a gel that collapsed when dehydrated and was impossible to infiltrate with resin.
The gel was removed by digestion with 2 % cellulase (Okazaki) for 10 min, a balance between
insufficient removal of gel and damage to cells. The gall tissue was postfixed overnight in
Dalton's osmium, washed, dehydrated in 2-2-dimethoxypropane for 5 min and embedded
in Spurr's resin. Sections were poststained in uranyl acetate/lead citrate.
Irradiated vegetative cells: diploid cells of strain d68
nari-12 n i n - i inosi-3nici-2
+ a2 b2
nari-13 niri-i inosi-3 + tsdi-i ai bi
were irradiated with 150, 300 and 450 krad of y from a cobalt 60 source. These doses produced
8, 92 and 200 nar + recombinants per io6 survivors, respectively. At i-h intervals from 1-4 h
cells were fixed in 4 % glutaraldehyde for at least 24 h. A few drops of a paste of settled cells
was put into a i-cm diameter round-bottomed test-tube and sufficient o-j-mm diameter
glass beads were added to appear just above the surface. The whole was whirlimixed for up
to 10 min, regularly monitoring the cell-wall removal with a phase-contrast microscope.
Refractivity changed when the wall ruptured. This method avoided damage from enzymes.
The cells were resuspended, decanted off the glass beads, stained with Dalton's (1955) chrome/
osmium, dehydrated for 3 min in 2-2-dimethoxypropane, infiltrated for 3-4 h with Spurr's
(1969) resin and embedded.
Light microscopy
(1) Basic fuchsin: cells were fixed in Schaudin's solution (2 parts saturated mercuric chloride
solution : 1 part absolute alcohol) for 1 h, rehydrated, washed, hydrolysed in 1 M-HC1 at
60 °C for 10 min and stained in basic fuchsin. (2) Hoechst 33258 florochrome: cells were
either fresh or fixed with 4% glutaraldehyde. Hoechst 33258 was used in aqueous solutions
at concentrations between 1 /tg/ml and 50 fig/mX. Cells were examined in an ultraviolet
fluorescence microscope. At low concentration only the nuclei fluoresced, and they were always
blue. At progressively higher concentrations presumed cell-growth zones fluoresced yellow
and fungal cell walls and cytoplasm fluoresced green.
RESULTS
Germinating spores
No structures resembling SCs were seen at any stage in the germinating spores.
The sectioned nuclei generally had a uniform granular appearance (Fig. 1). with the
nucleolus showing up as a denser area. The germ-tube grows first, then the nucleus
migrates into it from the spore (Fig. 2, 3). Microtubular spindles were frequently
found with rather amorphous dense poles attached to the nuclear envelope (Fig. 4)
H. L. Fletcher
Synaptonemal complexes in Ustilago maydis
175
or to intranuclear intrusions of the nuclear membranes (Fig. 5). Spindles were found
in nuclei while they were still in the spore. By analogy with all other known cases,
the spindle forms after chromosome synapsis and formation of the SC. This suggests
that only the final division stages of meiosis occur in the germinating spore and germtube.
There is a resting phase between germ-tube growth and budding, during which
meiosis was thought to occur. Examination of individual germinating spores revealed
this to be a very variable stage, ranging from 15 min to more than 5 h, with a modal
time of 75 min at 32 °C and 100 min at 25 °C for strain M146. The reason for this is
not clear. The laboratory stocks are largely maintained by mitotic growth and may
have lost some meiotic functions, causing failure of meiosis in some spores. In
Saccharomyces cerevisiae, meiosis takes at least 4 h (7 h including induction), again
suggesting that only the later stages are possible in germinating U. maydis spores.
The major difficulty (apart from sectioning) was a low level of synchronous germination combined with the failure of a very large proportion of spores to complete
meiosis and produce buds. Between 20 and 95 % of germinating spores were still
plain germ-tubes when they were overgrown by colonies from the spores that germinated first. Calculation suggests that approximately 3 % of plated spores were in
any particular i-h stage of meiosis at any time. Several thousand spores were examined
and it is unlikely that normal SCs could have been overlooked if they had been there.
In a test sample of yeast in which 3 % of cells were sporulating, these cells were
easily found in every section (although SCs were not found).
In a few germ-tubes arrested before nuclear division the chromosomes condensed
and became visible by fluorescence microscopy after staining with Hoechst 33258.
There were at least 6, and perhaps 10,fluorescentstructures, although the smaller ones
were nearly invisible. The larger ones were clearly double structures, and while they
are presumed to have been bivalents they might have been chromosomes with visible
chromatids. Similar bodies were found in electron microscope sections (Fig. 6) spread
throughout a microtubular spindle. The sizes were from 0-25 /tm x 0-5 /fm to 0-5 /<m
x 0-9 /im, and the smaller ones would have been unresolvable by transmitted light
microscopy while inside the cell wall. They are visible as light sources when they
fluoresce.
Gall tissue
Most histological studies of Ustilaginales were made early in this century when, in
the absence of effective fungicides, they caused major crop diseases. The gall material
Figs. 1-6. Spore germination. Voids and unstained membranes are present in most
cells because of enzymic digestion. Fig. 1. Germinating spore showing a nucleus
with a nucleolus (arrow). Figs. 2, 3. Spores with germ-tubes containing nuclei with a
spindle (arrows), i.e. metaphase to telophase. Fig. 4. Spindle (Mx) showing dense
amorphous bodies at the poles (arrows). Fig. 5. Spindle fibres apparently attached to
intruded nuclear membrane (arrow). Fig. 6. Bodies presumed to be condensed
chromosomes (arrows) in a nucleus arrested in Mt from the germ-tube in Fig. 2.
H. L. Fletcher
Synaptonemal complexes in Ustilago maydis
177
is difficult to work with, and the observations of various workers on different species
of Ustilago do not give a clear indication of the organization of a U. maydis gall
(Ainsworth & Sampson, 1950; Fischer & Holton, 1957). No mycelium was found in
any of the galls described here. Cells tended to form end-to-end chains and clusters
of attached buds. Most cells were uninucleate (Figs. 7, 8); the binucleate cells could
have been actively dividing cells or, in particular cases, haploids fusing prior to spore
formation. Many cells looked similar in shape to those grown on artificial medium
(Fig. 9), particularly during the early part of the gall's growth, and when proliferating
around wounds in the gall. As the gall increased in size large nodules of U. maydis
cells formed within it. The cells in the nodule were small and irregular. A gel was
deposited between the cells, which apparently pushed them apart (Fig. 10), leaving
separated cells with matching adjacent faces. The jelly collapsed and solidified when
it was dehydrated, and it had to be digested away with cellulase before electron
microscopy was possible. In more advanced nodules, spore walls started to form
around some larger cells. Among these young spores were large, rounded, thinwalled cells, and a few of these contained 2 nuclei (Fig. 11). This suggests that fusion
of haploids produces a diploid cell, which immediately forms a spore. This pattern
of behaviour is common in fungi.
All these observations were readily confirmed by incident light fluorescence with
Hoechst 33258 staining, which displayed well-separated uninucleate cells in large
undisturbed masses of gall tissue. Observations of such large pieces of gall was
impossible using transmitted light because of refraction. Only the nuclei of the maize
cells fluoresced (blue) but the U. maydis nuclei fluoresced blue while the cytoplasm
and cell wall fluoresced green. The gelatinous coat around each cell looked like a
continuation of the cytoplasm when viewed by visible light, and the interface of the
gel with surrounding water looked like a cell wall, giving a misleading appearance.
No definite synaptonemal complexes were seen. One large thin-walled cell (presumed to be a diploid spore-forming cell) had a short length of a structure like a very
faint SC but this was not continued through adjacent sections. Nuclei could be seen
in spores with substantially thickened walls, and the chromatin in these was partially
condensed producing electron-dense regions similar to those that characterize
condensed chromatin in higher plants and animals (Fig. 12). This was thought to be
a preparation for the dormant, resistant phase of the teliospore. It is possible that
SCs occurred in slightly older spores than those examined. The thickened spore wall
prevented examination of the contents.
Figs. 7-12. Gall cells. Figs. 7, 8. U. maydis cells within the gall are small and have
only 1 nucleus (arrows). Fig. 9. U. maydis cells growing in part of the gall cut and
damaged a day before. A mature spore (J) is visible. Fig. 10. Undisturbed gall showing
cells widely spaced by a gelatinous matrix (#) and 2 partially formed spores (1). Fig. 11.
Large binucleate cell (n,n) with a thin wall found in a spore-forming zone. This is
thought to be a heterokaryon after fusion of haploids and before spore formation.
Fig. 12. Incompletely formed spore (cf. Fig. 1) partially collapsed during preparation,
with condensed chromatin in the nucleus (arrow).
178
H. L. Fletcher
An attempt was made to monitor the course of cell fusion in the host as follows:
two compatible haploids carrying complementary auxotrophic markers were inoculated into a maize seedling. One week later a small piece of gall was removed and
incubated at 26 °C in complete medium in a shaker to produce a suspension of single
cells, which appeared after about 4 h and were then the typical shape of liquid-cultured
cells. The U. maydis cells embedded in the gall are assumed to have grown there,
they were not part of the original inoculation. No spores were visible; the first ones
would have been expected a few days later. Cells from the suspension were plated on
complete and minimal media to count all cells and diploids, respectively. The frequency of diploids was 288 in io 8 cells, less than 1 in 3000. Most gall cells obtained
were haploid. While these could have budded off from heterokaryons breaking down
in the medium, no such heterokaryons were seen and the simplest conclusion is that
the uninucleate U. maydis cells in the gall were haploid. This method could be used
to search for the occurrence of diploids and possible meiotic recombination if cells
beginning meiosis could be reverted to mitosis as has been done in the yeast S.
cerevisiae (Olson & Zimmermann, 1978 a).
Irradiated cells
Cells irradiated at 300 krad and 450 krad undergo recombination at a rate approaching that found in meiosis. This occurs within 4-5 h (Holliday, 1971) and appears to
be directly related to repair of double-strand damage. Vegetative cells were easy to
examine without treatment with enzyme but no ultrastructural differences were found
between irradiated cells and unirradiated controls. It seems that the SC is not necessary for recombination repair of DNA broken by irradiation.
DISCUSSION
Dr P. B. Moens (personal communication) has previously examined meiosis in
U. maydis. He did not find synaptonemal complexes either in germinating spores or
in the gall, where hyphae, binucleate cells, nuclear fusions and spore maturation
were traced from long series of sections. The results reported in this paper are in full
accord with those of Dr Moens: no conclusive evidence of SCs was found at any
stage of spore maturation or germination. The examination was difficult because of
the nature of the organism, and the stage at which SCs would be expected is not clear.
Classic SCs should have been found if they had been present in only 1 % of any of
the cell types examined. The SC of S. cerevisiae offers an interesting comparison.
Moens & Rapport (1971) said that the absence of conventional SCs was the most
striking feature of meiotic prophase nuclei. Zickler & Olson (1975) found SCs and
have continued to do so. Byers & Goetsch (1975) satisfactorily resolved the problem.
They examined a temperature-sensitive cell-cycle mutant cdc-\. When homozygous
it produces continuous SCs in meiotic cells at the permissive temperature, and
intermittent SCs at the restrictive temperature. No SCs were found in the heterozygous a/r-4/wild-type strain. Thus the extent ot the SC may be a variable feature
found only in particular strains. Olson et al. (1978) have also reported the absence
Synaptonemal complexes in Ustilago maydis
179
of SCs during meiosis in Schizosaccharomyces pombe and Olson & Zimmermann
(1978 a) have shown that gene conversion (which requires chromosome pairing and
hybrid DNA formation) occurs before SCs form during meiosis in S. cerevisiae. This
also suggests that there may not be an absolute requirement for SCs during meiosis
in these fungi.
Some other fungi lack SCs for a more specialized reason: they have localized or
restricted recombination. Zickler (1973) reported an absence of normal SCs in
Podospora anserina and P. sertosa (Ascomycetes). P. anserina normally produces 4
binucleate spores, the nuclei being of opposite mating type. The restricted SCs are
apparently related to the presence of a single crossover per arm, which causes the
mating type loci to segregate at the second meiotic division, and presumably facilitates the inclusion of nuclei of opposite mating type into the same spore. The cultivated mushroom Argaricus bisporus (Basidiomycete) produces pairs of dikaryotic
spores and 70 % of these produce homothallic mycelia, suggesting that they may be
heterozygous for mating type alleles (see Evans, 1959) in a system analogous to that
of P. anserina. A. bisporus also has several very short pieces of SC (personal observation) and this may be usual in species producing homothallic dikaryotic spores.
No SCs were found in mitotic U. maydis cells undergoing meiotic levels of recombination to repair DNA damaged by radiation (see Holliday, 1971). S. cerevisiae can
also undergo recombination repair of irradiated DNA without SCs (Olson & Zimmermann, 19786). In order for recombination repair of double-strand breaks in DNA it
may be necessary for the homologous chromosomes to be paired as intimately as they
are in meiosis. Diploid U. maydis suffers around 30 % mortality after 450 krad of
y irradiation, and genes altered by the recombination repair process in survivors can
be expressed by the production of active enzyme 4-5 h after irradiation (Holliday,
1971). G2 diploid cells can repair around 100 double-strand breaks per haploid
genome. It can be calculated that death only occurs when all 4 chromatids (cells are
in G2 phase) are broken in a short region approximately the length of a gene or
operon (Fletcher, 1981). The rapid formation of hybrid DNA during repair suggests
that the homologues are paired in mitotic cells. This close-pairing may be facilitated
by the small size of the chromosomes, the average 5. cerevisiae chromosome is
1/6th the size of the genome of Escherichia coli. It seems to be possible for some fungi
to undergo meiotic recombination and segregation without the complication of an
SC.
The discovery of almost entirely haploid single cells in the gall tissue was surprising.
Infection with 2 haploids differing at 2 loci (mating type and infectivity) is necessary
to produce a gall in the host. Diploid strains that are heterozygous at these 2 loci are
solopathogenic: they do not need a complementary strain to become infectious.
Compatible haploid cells will fuse on mating medium. Mass matings will produce
multinucleate cells. Some singleton nuclei in these cells may move into buds and
become haploid again. True heterokaryotic cells form very long 'infection' hyphae.
These will slowly grow and divide on medium containing charcoal. They do not have
clamp connections and readily revert to haploid yeast on other media (Day & Anagnostakis, 1971). The charcoal is thought to absorb a product of the U. maydis, which
180
H. L. Fletcher
would promote the change to the yeast form. The observations reported here suggest
that a limited amount of heterokaryon is formed, which infects the host and induces
the galls. The heterokaryon may then break down, at least partially, to give equal
numbers of the 2 haploid types, which may proliferate in the gall. Diploid sporeforming cells appear to be produced by the fusion of haploid cells.
My thanks are due to Dr R. Holliday for guidance and discussion and to Dr P. R. Day
for criticism of the manuscript. The work was supported by an M.R.C. Training Fellowship.
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