Download Aspergillus nidulans mating and analysis of meiotic progeny

Aspergillus nidulans mating and analysis of meiotic progeny
Appendix 15
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Aspergillus nidulans as an experimental system for genetic analyses
Aspergillus nidulans is a member of the Moniliaceae, the largest of the form families in the
Deuteromycetes. Aspergillus nidulans is commonly known as “green bread mould” but other
Aspergillus species have different characteristic spore colours. A. nidulans has a convenient
characteristic: it is homothallic, so theoretically any strain can be crossed to any other.
The “Glasgow wildtype” (strain A4 at the Fungal Genetics Stock Centre, U Kansas,
www.fgsc.net) has been used as the parental strain for many mutational analyses, leading to
literally thousands of distinct mutant strains separated by mutations in only one or a few genes.
Almost 1000 of these strains are stored at FGSC, and there are many more in collections around
the world. The power and value of this group of mutants is that they are isogenic. In the late
1960s scientist in Glasgow, John Clutterbuck, isolated a wild strain of A. nidulans that had a
mutation he called velvet. The velvet mutation is recessive (veA-) and confers two experimentally
useful phenotypes. Colonies will not overgrow each other, so distinct isolates can be grown in
close proximity. Strains will conidiate in darkness, simplifying growth conditions and leading to
a continuous lawn of conidiating hyphae, rather than the periodic conidiation we discussed in
lecture. Velvet has recently been shown to negatively influence mating, nevertheless, veA- strains
can be crossed sexually.
Mating A. nidulans strains (this will be done for you before the lab begins)

Strains must have complementary auxotrophies to form anastomoses efficiently – an
auxotrophy is a mutation that prevents synthesis of an essential nutrient. Anastomosis is
important for heterokaryon (and later for cleistothecium) formation for outcrossing.
However, prototrophs will also mate, albeit with lower efficiency, and strains can also be
mated to themselves. We always use different spore colours for the two parental strains,
since identifying outcrossed cleistothecia will be easier as their progeny will have a variety of
spore colours.

Begin by inoculating fresh spores in fully supplemented liquid medium (CM** is complete
medium with arginine, biotin, para-amino benzoic acid, pyridoxin, and pyrimidines, which
will support the growth of all the parental strains used) for 2d at 28°C. Aim to have similar
numbers of spores from each parent. Use a sterile 24-well tissue culture dish or sterile
Eppendorf tube with a hole poked in the lid using a hot needle. A. nidulans needs oxygen to
grow well!

Allow mycelium to grow for ~ 2 d, and then transfer the mats of hyphae to a minimal
medium plate using sterile forceps. Incubate at 28°C for 1-2 d. During this time, both strains
should conidiate, and so you should see colours of both the parents. Minimal medium does
not contain nutritional supplements, so it selects for heterokaryons formed between strains
with complementary auxotrophies.

After 2 days, seal the plates with Parafilm (at least 2 layers) to reduce oxygen availability
(improves efficiency of cleistothecium formation) prevent the medium from drying out, and
store in the dark for 2-3 weeks for the cleistothecia to mature.
Mate strains by growing mixtures of spores in fully supplemented liquid medium for 2 days at
28°C. Strains must have complementary auxotrophies to form hyphal anastomoses
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efficiently for heterokaryon (and later for
cleistothecium) formation. However,
prototrophs can mate, and strains can selfmate. Where possible, use different spore
colors for the two
parent strains as this will make it easier to
check whether both parents are growing,
and later will be useful for identifying
outcrossed cleistothecia.
It is most convenient to use a 24 well
tissue culture dish for matings, but it is
also possible to use sterile Eppendorf
tubes. For tissue culture
dishes, use about 2 ml of fully
supplemented liquid medium per well.
Typically we use two wells for each
parental combination, adding 1 µl of each
conidial stock per well. Ideally,
similar numbers of fresh spores should be
used for each parent. If one strain does not grow adequately, it can be reinoculated into the
original well, and incubated another day.
Alternatively, additional wells can be prepared with increasing numbers of the lagging strain.
For Eppendorf cultures, co-inoculate the parental strains in 400 µl liquid medium, close tightly
and place loose in a small Erlenmeyer flask in a shaking incubator for 24 h at 28°. Remove tubes
to an Eppendorf rack, poke a hole in the lid using a hot needle, and incubate as a stationary
culture for another day.
When mating a cold-sensitive (cs) strain with wildtype, we have had good results from allowing
the cs strain to grow at 37° for 1-2 days before adding wildtype spores and shifting to 32° for
another day. Similarly, a poorly conidiating strain can be mated by growing hyphal explants with
spores from the other parent.
Allow the parental mycelium to grow just until it has pigmented spores. Transfer the hyphal mat
using forceps to an MM plate (if the parent strains share any auxotrophies, these must be
supplemented) and incubate at 28°C for 1-2 d. Removing the mat intact is easier if the forceps are
run around the edge of the well to loosen the hyphae. Cleistothecia form most abundantly where
the mat is in contact with the MM agar; we cut the edges so it will lay flat. Some people dip their
hyphal mats in water, and then drain/blot dry to remove nutrients, but we have not found this to
be necessary. We routinely put two or more (for poor-mating combinations) hyphal mats per MM
plate. After 2 days, seal the plate with Parafilm (fold a 3 cm wide piece of Parafilm in half and
seal with the double layer) to prevent the medium from drying out, and store in the dark for 2-3
weeks for the cleistothecia to mature. Cleistothecia become noticeably more fragile after 6-8
months, but the spores remain viable and can still be used for isolating double mutants.
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Outcrossed cleistothecia are found more reliably on the hyphal mat than in the surrounding
heterokaryon. They are large and found near conidia with each of the parental colours.
Sometimes it is possible to see both parental spore colors on a single conidial head of a
heterokaryotic strain. There is considerable variation in size and abundance of cleistothecia for
any one cross, and variation in average cleistothecium size for different strain combinations.
Cleistothecium amongst conidiophores
Cleistothecium broken open
Typically, mature A. nidulans cleistothecia have a shiny black peridium covered with a layer of
light yellow Hülle cells, which may be viable, and plus parental conidia. These are removed by
rolling the cleistothecium on a plate of 3% agar, a process made more efficient by adding 1%
diatomaceous earth as a mild abrasive. Once clean, the cleistothecium is transferred to an
Eppendorf tube containing 200µl water, crushed, and vortexed to release the ascospores. These
are crimson, and make a red spot on the bottom of the tube upon settling.
Before analysis, the ascospores from individual
cleistothecia should be tested for evidence of
outcrossing. A loopful of ascospore suspension is
spread over a small area of a fully supplemented
plate, and incubated until the colonies have
pigmented conidia. These should have at least both
parental colours, plus recombinants if possible from
the cross. See discussion of conidial color. Here are
test spreads from four cleistothecia of a green:white
mating. Although all appear to be outcrossed, given
the choice we would choose not to analyze the
cleistothecium whose spread in the 3 o’clock position
since the color does not appear to be segregating 1:1.
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This is a convenient stage to test complementation vs allelism. Genes from different
complementation groups will produce wildtype progeny with all possible colors under selective
conditions.
Isolating and analyzing the progeny from a mating

The teleomorph of Aspergillus nidulans is Emericella nidulans, which produces cleistothecia.
These contain asci, which in turn
contain eight ascospores. E.
nidulans cleistothecia are black
spheres, generally 100-300 µm
in diameter, which are covered
with pale yellow cells called
Hülle cells.

The pictures on the right show A.
nidulans conidia and
conidiogenous cells as seen with
light microscopy and scanning
electron microscopy. Below
them are E. nidulans Hülle cells
and ascospores seen with light
microscopy. Notice that the
shape of the ascopores different
from the conidia.

The function of Hülle cells may
be to protect or nourish the
young cleistothecium. Whether
Hülle cells are viable is a matter
of uncertainty, and may vary
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between species. Since it is a possibility that would complicate later work, and since the
Hülle cell layer could trap conidia, the Hülle cells are removed before the ascospore
characteristics are analyzed.

Cleistothecia are cleaned by rolling them on a clean agar surface until the Hülle cells are
removed and the cleistothecium wall is shiny black. This takes patience. Cleistothecia are
delicate, so you must take care not to rupture them, releasing the crimson coloured
ascospores. This process is analogous to
cleaning dusty soap bubbles by rolling them
around on a carpet with a baseball bat. We
use agar containing diatomaceous earth as a
mild abrasive.

The cleaned cleistothecium is transferred to
a labeled Eppendorf tube containing 200 µl sterile distilled water, and crushed to release
the ascospores. These are dispersed by vigorous agitation.
Outcrossed cleistothecia are routinely analyzed by random ascospore analysis; some labs use
octad dissection, analogous to tetrad dissection in Saccharomyces. For random ascospore
analysis, we make a 2:200 dilution of the ascospore stock, and spread 30µl, 60µl and 90µl of this
on appropriately supplemented plates, as shown. This is a convenient stage at which to select
against unwanted nutritional markers. At least one of the spore concentrations should be
appropriate for picking isolated colonies. Spreading can be improved by adding 0.01% Triton X100; if so the diluted spores should be spread immediately as even overnight storage
compromises viability. These plates were spread without using Triton X-100.
Even with low spore concentrations and Triton X-100, some isolated colonies can grow from two
nearby ascospores, particularly if the asci weren’t disrupted thoroughly. These colonies are easy
to identify if they have two colors, but since neighboring spores might produce the same color
conidia, even single-colored colonies should be picked near an edge.
Random ascospore analysis
Single spore colonies are transferred using toothpicks to a gridded master plate. Typically this
has spaces for 50 progeny plus the two parental strains. A. nidulans strains with the veA1
mutation will not overgrow each other. Parental colonies are important controls when checking
for markers. Touch the toothpick to a clean, peripheral part of the agar to moisten it and
then gently touch/dimple the surface near the periphery of a colony. The toothpick will hold
enough conidia to inoculate at least two plates. Avoid creating drafts with your hands while
picking and making gridded plates since there is a risk of mixing spores between neighbouring
colonies. Replica plates with appropriate media are used for determining nutritional
phenotype. Theses are called dropout plates, containing MM with all the auxotrophies
supplemented except one. If inoculating multiple plates with one toothpicksworth of conidia, do
the positive control last.
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A grid needn’t be drawn onto each plate as long as the top is marked with an arrow for alignment
purposes – use a piece of card with Petri plate covers attached and copies of the grid
taped inside. Label the back of each plate with an alignment arrow, cross or experiment, date,
incubation temperature, medium type. The squares labeled P1 and P2 are for the parental
strains, a control to check if they are behaving as expected, and to aid in phenotype analysis
Strain information will be supplied. An example follows:
Decoding the strain and gene names. Only the mutations are listed. Anything that is not listed
is assumed to be "wildtype". Imagine you have mated the following strains – we will examine
this cross as way of showing how the analytical methods. You will be analyzing different
crosses in lab, hypA1::sepD3 These are allele names for the wildtype genes hypA+ and sepD+
The strain ASK78 was the 78th Aspergillus strain generated by Susan Kaminskyj, and most strain
names can be decoded to reveal their history. Typically the creator also has parental information.
These strains were created to study two genes affecting cell shape: bsh167 and hypA1. Gene
designations are written in italics. Unlike a strain name, bsh167 is a "working name" – it is the
167th strain in the study that appeared to be a bypass suppressor of sepH. Bsh167 was later given
a more formal isolate name AKB9, the 9th strain saved by Ken Bruno. The other gene
designations are formal names – they have had more extensive genetic analysis.
The nutritional marker genes in the list indicate auxotrophies. Examples include for
biosynthesis of arginine (argB2), biotin (biA1), para-amino benzoic acid (pabaA6), pyridoxin
(pyroA4), uridine and uracil (pyrG89, pyrimidines).
Spore colour in Aspergillus nidulans
Wildtype Aspergillus nidulans conidia are dark green. However, spore colour is due to the
function of a particular metabolic pathway. The first spore colour mutant had white spores (no
pigment) so the gene locus for colour is called WA for wildtype green, and wA for mutant white.
There are multiple alleles of wA: we are using wA3. Producing the wildtype green pigment takes
at least two steps, and a mutant was found that was blocked at an intermediate stage. It has
yellow spores due to a mutation in the gene called yA.
In the simplest pathway, spore colours mutations are white  yellow  green. A mutation in wA
prevents any colour; a mutation in yA prevents green colour. A wA strain might also have the yA
mutation, but it will be masked by wA – this is an example of epistasis. Colouration is a complex
trait it most species, and often there are colour modifier genes that "dilute" normal pigmentation.
An example of these produces the pale coat pattern seen in Siamese cats. In A. nidulans, the
analogous gene is called chaA, for chartreuse, a bright green colour. chaA will make a spore
colour lighter, so where yA spores are golden, yA, chaA spores would be light yellow.
What is veA1? Most experimental strains of A. nidulans carry this allele, which has two useful
phenotypes: it renders the strain “blind”, meaning that sporulation is continuous rather than
periodic, and colonies do not overgrow each other. Thus, we can plate many strains with slightly
different genotypes on the same plate, and still keep them separate for analytical purposes.
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For more information on Aspergillus and other filamentous fungi: see http://www.fgsc.net/
To check whether a particular cleistothecium was formed from a cross between two strains with
different spore colour, spread a loopfull of ascospores on a Petri plate containing a medium that
would support the growth of all the auxotrophic markers in the cross. Our medium is called
CM**, which contains complex organic nutrients like peptone and yeast extract, as well as
specific additions for the nutritional auxotrophies.
A. nidulans ascospores germinate within about two days at room temperature. The strains used in
this example had different spore colours: yellow for AKB9 and white for ASK78. yA and wA are
found at different genetic loci – they are not allelic. Therefore, it is possible for the progeny to
have any combination of yA, wA, or their corresponding wildtype alleles.
Imagine that we are examining only the inheritance of spore colour – this is often written as
follows, with the corresponding loci arranged vertically. The two parents are AKB9 and ASK78,
and an expanded genotype is written so that each parent’s allele is shown at each locus
AKB9
ASK78
yA
YA
WA
wA
or more briefly as
yA
+
+
wA
Since yA and wA are not alleles, there will be four types of progeny, with approximately equal
abundance
yA, + (yellow) : +, wA (white) : yA, wA (white from epistasis of wA over yA) :+,+ (green)
Depending on the parents in the cross, you will see different combinations of colours. For
example, if you mate a white and a green strain, you might get white / green progeny only or
additional colours as well. What are other possibilities? Why?