Genetic analysis in Aspergillus nidulans Exercise 4 page 1 of 6

We have been studying fungal growth and response to changing environments, and mould isolation and identification. With few exceptions, none of the organisms in these labexercises were characterized beyond genus. In contrast, when fungi are used as experimentaltools they are generally very precisely identified and characterized.

Aspergillus nidulans as an experimental system for genetic analyses

Aspergillus nidulans is a member of the Moniliaceae, the largest of the form families in theDeuteromycetes. Aspergillus nidulans is commonly known as “green bread mould” but otheraspergilli have different characteristic spore colours. A. nidulans has a convenient characteristic:it is homothallic, meaning that there are no mating-type genes, so theoretically any strain can becrossed to any other.

The “Glasgow wildtype” (strain A4 at the Fungal Genetics Stock Centre, U Kansas) has beenused as the parental strain for many mutational analyses, leading to literally thousands of distinctmutant strains. Almost 1000 of these strains are stored at FGSC, and there are many more incollections around the world. The power and value of this group of mutants is that they areisogenic, so different strains vary only at specified loci. In the late 1960s scientist in Glasgow,John Clutterbuck, isolated a wild strain of A. nidulans that had a mutation he called velvet. Thevelvet mutation is recessive (veA-) and confers two experimentally useful phenotypes. Colonieswill not overgrow each other, so distinct isolates can be grown in close proximity. Strains willconidiate in darkness, simplifying growth conditions and leading to a continuous lawn ofconidiating hyphae, rather than the periodic conidiation we discussed in lecture.

Mating A. nidulans strains (this will be done for you before the lab begins)

• Strains must have complementary auxotrophies to form anastomoses efficiently – anauxotrophy is a mutation that prevents synthesis of an essential nutrient. Anastomosis isimportant for heterokaryon (and later for cleistothecium) formation for outcrossing.However, prototrophs will also mate, albeit with lower efficiency, and strains can also bemated to themselves. We always try to use different spore colours for the two parentalstrains, since identifying outcrossed cleistothecia will be easier as their progeny will have avariety of spore colours.

• Begin by inoculating fresh spores in fully supplemented liquid medium (CM** is completemedium with arginine, biotin, para-amino benzoic acid, pyridoxin, and pyrimidines, whichwill support the growth of all the parental strains used) for 2d at 28°C. Aim to have similarnumbers of spores from each parent. Use a sterile 24-well tissue culture dish or sterileEppendorf tube with a hole poked in the lid using a hot needle. A. nidulans needs oxygen togrow well!

• Allow mycelium to grow for ~ 2 d, and then transfer the mats of hyphae to a minimalmedium plate using sterile forceps. Incubate at 28°C for 1-2 d. During this time, bothstrains should conidiate, and so you should see colours of both the parents. Minimal mediumdoes not contain nutritional supplements, so it selects for heterokaryons formed betweenstrains with complementary auxotrophies.

Genetic analysis in Aspergillus nidulans Exercise 4 page 2 of 6

• 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, andstore in the dark for 2-3 weeks for the cleistothecia to mature.

Isolating and analyzing the progeny from a mating

Part 1

• The teleomorph of Aspergillus nidulans is Emericella nidulans, which produces cleistothecia.These contain asci, which in turncontain eight ascospores. E.nidulans cleistothecia are blackspheres, generally 100-300 µmin diameter, which are coveredwith pale yellow cells calledHülle cells.

• The pictures on the right show A.nidulans conidia andconidiogenous cells as seen withlight microscopy and scanningelectron microscopy. Belowthem are E. nidulans Hülle cellsand ascospores seen with lightmicroscopy. Notice that theshape of the ascopores differentfrom the conidia.

• The function of Hülle cells maybe to protect or nourish theyoung cleistothecium. WhetherHülle cells are viable is a matterof uncertainty, and may varybetween species. Since it is apossibility that would complicatelater work, and since the Hüllecell layer could trap conidia, theHülle cells are removed beforethe ascospore characteristicsare analyzed.

• Cleistothecia are cleaned by rolling them on a clean agar surface until the Hülle cellsare removed and the cleistothecium wall is shiny black. This takes patience. Cleistotheciaare delicate, so you must take care not to rupture them, releasing the crimson colouredascospores. This process is analogous tocleaning dusty soap bubbles by rolling themaround on a carpet with a baseball bat. Weuse agar containing diatomaceous earth as amild abrasive.

Genetic analysis in Aspergillus nidulans Exercise 4 page 3 of 6

• The cleaned cleistothecium is transferred to a labeled Eppendorf tube containing 200 µlsterile distilled water, and crushed to release the ascospores. These are dispersed byvigorous agitation.

• The matings for this lab were done between strains with different spore colours andnutritional auxotrophies (see table below). In order to test whether individual cleistotheciaresulted from an outcross, that is, between parental strains, a small volume of ascosporesuspension is spread on rich medium and incubated at 28°C for two to three days. Theascospore suspension is stored at 4°C, where the spores will remain viable for months toyears.

Cleistothecium amongst conidia Cleaned and broken open to liberate ascospores

Pictures taken my Dr. T Hill, Rhodes College, Memphis TN

Strain information will be supplied on Tuesday

Decoding the strain and gene names. Only the mutations are listed. Anything that is not listedis assumed to be "wildtype". Imagine you have mated the following strains – we will examinethis cross as way of showing how the analytical methods. You will be analyzing differentcrosses in lab, hypA::sepD and hypA::sepG

AKB9 bsh167; pyrG89, pabaA6, yA1, biA1; pyroA4, veA1ASK78 hypA1; wA3; argB2; veA1GR5 pyrG89, pyroA4, wA3; veA1

The strain names are AKB9 and ASK78, the 9th and 78th Aspergillus strains generated by KenBruno and Susan Kaminskyj, respectively. These strains were created to study two genesaffecting cell shape: bsh167 and hypA1. Gene designations are written in italics. bsh167 is a"working name" – it is the 167th strain isolated by KB that appeared to be a bypass suppressor ofsepH. The other gene designations are formal names – they have had more extensive geneticanalysis.

Genetic analysis in Aspergillus nidulans Exercise 4 page 4 of 6

The nutritional marker genes in the list indicate auxotrophies for biosynthesis of arginine(argB2), biotin (biA1), para-amino benzoic acid (pabaA6), pyridoxin (pyroA4), uridine and uracil(pyrG89, pyrimidines).

Wildtype Aspergillus nidulans conidia are dark green. However, spore colour is due to thefunction of a particular metabolic pathway. The first spore colour mutant had white spores (nopigment) 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 takesat least two steps, and a mutant was found that was blocked at an intermediate stage. It hasyellow spores due to a mutation in the gene called yA.

In the simplest pathway, spore colours mutations are white ‡ yellow ‡ green. A mutation inwA prevents any colour; a mutation in yA prevents green colour. A wA strain might also have theyA mutation, but it will be masked by wA – this is an example of epistasis. Colouration is acomplex trait it most species, and often there are colour modifier genes that "dilute" normalpigmentation. 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 willmake a spore colour lighter, so where yA spores are golden, yA, chaA spores would be lightyellow.

What is veA1? Most experimental strains of A. nidulans carry this allele, which has two usefulphenotypes: it renders the strain “blind”, meaning that sporulation is continuous rather thanperiodic, and colonies do not overgrow each other. Thus, we can plate many strains with slightlydifferent genotypes on the same plate, and still keep them separate for analytical purposes.For more information on Aspergillus and other filamentous fungi: see http://www.fgsc.net/

Part 2

To check whether the cleistothecium was formed from a cross between two strains with differentspore colour, you spread a loopfull of ascospores on a Petri plate containing a medium thatwould support the growth of all the auxotrophic markers in the cross. This medium is calledCM**, which contains complex organic nutrients like peptone and yeast extract, as well asspecific additions for the nutritional auxotrophies.

A. nidulans ascospores germinate within about two days at room temperature. The strains usedin this example had different spore colours: yellow for AKB9 and white for ASK78. yA and wAare found at different genetic loci – they are not allelic. Therefore, it is possible for the progenyto 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 asfollows, 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 yA WA or more briefly as yA +ASK78 YA wA + wA

Genetic analysis in Aspergillus nidulans Exercise 4 page 5 of 6

Since yA and wA are not alleles, there will be four types of progeny, with approximately equalabundanceyA, + (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. Forexample, if you mate a white and a green strain, you might get white / green progeny only oradditional colours as well. What are other possibilities? Why?

Analyzing the progeny of an outcrossed cleistothecium

Just learning that you have an outcrossed cleistothecium doesn’t get you very far. Generally youare trying to do two things – learn about a genetic interaction which sometimes requires isolatingstrains that have particular combinations of mutations, and determining the nutritionalrequirements of those strains. To accomplish these aims, you need single-spore progeny. Theoriginal spread of your newly isolated ascospores was done at high spore density. All you neededthen was to distinguish between outcrossed and self-crossed cleistothecia.

Part 3:

The next objective requires more precision: today you will be spreading a diluted sporesample on more CM+ plates, and next time you will be picking those progeny for analysis.

Preparation: make a glass spreader. Get a 5 1/4inch Pasteur pipette. Hold the narrow end in aBunsen burner flame until it seals and droopsslightly. Pull the pipette out of the flame, androtate it about 90° until the droopy tip is pointingtowards you. Then, flame again so that the pipettebends about an inch from the tip. Remove andplace in an Eppendorf rack to cool.

Examine the Eppendorf tubes that have your ascospore suspension: you should be able tosee a dark spot at the bottom, which is the mass of ascospores originally in the cleistothecium.Obviously, these spores are heavier than water. To spread plates for isolated colonies (likely tohave been produced from a single spore), thoroughly mix your original ascospore suspension andmake a 1:100 dilution in sterile distilled water. Use 2µl of ascospores in 200 µl of water. Mixagain, and them pipette 30µl, 60µl and 90µl of this suspension onto three labeled plates of CM+agar. Spread the suspension evenly over the surface of the plate using a sterile spreader. Dip yourspreader in alcohol, flame briefly, dip again in alcohol, flame to dry and let cool. Test thecoolness by touching the agar. Leave the plates right side up for 30-60 min to allow the water inthe suspension to absorb into the agar. Tape shut and invert. Store at room temperature for two tothree days. A. nidulans ascospores take longer to germinate than do conidia.

Genetic analysis in Aspergillus nidulans Exercise 4 page 6 of 6

Strains for today’s exercise:

ASK 357 hypA7, wA3, bi A1, paba A6SO 59.............nimX2cdc2 F233L, yA2, nicB8, pyroA4SO 60.............nimX3cdc2 Y305H, nicB8, pyroA4, riboA1

ASK 357 has the wA3 mutation, so it has white spores; SO59 has the yA2 mutation, so it hasyellow spores; SO60 has wildtype green spores.

Today you’ll be cleaning Aspergillus cleistothecia as described in the lab handout, suspendingthe ascospores in sterile distilled water, and plating a sample of spores to test whether they camefrom a cross between the two parents. Aspergillus can mate with itself, so this is not a trivial test.However, we will pick cleistothecia that are likely to be outcrossed by choosing large onesformed close to conidia that have both parental spore colours. The conidia will be plated onmedium that will support most of the auxotrophies, enough that we will be bale to tell outcrossesfor next time.