Heterocaryosis and the Parasexual Cycle in Aspergillus

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(above diagrams from MycoAlbum CD by George Barron)

Left hand diagram:  Diagram shows the morphology of Aspergillus.  Aspergillus is one of the more sophisticated of the spore producing fungi in the Hyphomycetes (asexual or anamorphic fungi).  A swollen vesicle is borne at the apex of a stout conidiophore.    Large numbers of conidiogenous cells (phialides) are produced over the surface of the vesicle.   Each phialide produces a chain of conidia (phialospores) so that eventually thousands of conidia are produced on  a single head of the fungus.   Each phialide contains one haploid nucleus.  This divides by mitosis and one of the daughter nuclei moves into the developing conidium.   Thus, in the beginning, each conidium contains a single nucleus although the hyphal cells in Aspergillus can be multinucleate.  The colour of the pigment in the conidium in Aspergillus is controlled by the nucleus it contains.    The wild type colour in A. nidulans is green.  As it is the conidia that contain this pigment the colour of the colony on agar is green.    With simple techniques (to be explained elsewhere), using ultraviolet radiation, the sequence of biological steps during the formation of the pigment can be blocked at any point  resulting in intermediate colours along the pathway.  Some block pigment synthesis completely and the conida lack  pigment (heads appear white).   With Aspergillus nidulans, yellow and white or brown are useful 'morphological' mutants to use to study heterocaryosis and the parasexual cycle.  Each morphological mutant must now be reirradiated to produce a 'biochemical' mutant (auxotroph) that interferes with the synthesis of one of the essential vitamins or amino acids produced by the fungus (method of production and selection to be explained elsewhere).  Mutants affecting  vitamin synthesis are preferred for class purposes as they are easy to produce, detect, and isolate.  Also colonies grow normally when the missing component is supplied in minimal medium.   Amino acids are good alternatives.

In this type of work you require a minimal medium (e.g. synthetic Czapek's medium)  on which the mutant conidia cannot even germinate bevause their vitamin requirement is not present.   However,  if you add the defficient vitamin to the minimal medium (MM) conidia will germinate and produce a normal (wild type) colony.   You also require a complete medium (CM)  that contains everything you can think of that will cover any possible biochemical mutant you might produce during the irradiation procedures.   However, for demonstration purposes since you are working with known strains of the fungus(i.e. identified to requirement)  you can use a much simplified CM. 

Right hand diagram:  As you can see in the diagram we have produced a yellow thiamine requiring strain (y thi-) and a brown biotin  requiring strain (br bio-) of  Aspergillus nidulans.   Neither of these strains can grow on MM.   However, if we can create a heterocaryon (dikaryon) containing both of these nuclear types then the yellow strain has the wild type gene for biotin synthesis and the brown strain has the wild type gene for thiamine synthesis and such a heterocaryon can now grow on minimal medium.

Creation of Heterocaryon:    Streak a line of conidia from each strain a few millimetres apart on CM.   Both strains will germinate and grow on CM.  When the hyphae of the strains meet there will be anastomoses and nuclei will migrate from one hyphal system to the other and heterocaryotic hyphae will be established.

To recover the heterocaryonTake tiny pieces of agar from this 'heterocaryotic' zone between the two strains on CM and plate them on MM.   The parent strains if present cannot grow and will not survive.   Only the heterocaryon, that has the full complement of  wild type loci,  will grow on minimal medium.  The two nuclei are spatially separated but they behave as heterozygous diploid.( y thi- + +/+ +br bio- ) i.e. the heterocaryon has the wild type gene at all four  loci involved.   In the formation of the head both nuclear types might be present but only one haploid nucleus goes into each phialide (spore mother cell) and the spore chain from this will beeither yellow or brown as the colour of the spore is dependent on the gene in the nucleus of the spore then spores are identical to one or other of the parent strains.    This will give a variegated head where there are two colours of chains in the head (see diagram)   So!  In a colony from a heterocryon, (dependent on how the nuclei have segregated in the formation of the fruiting structure) you will find yellow heads, brown heads or variegated heads.      You can confirm this by streaking the spores from a chain on MM. where they will not grow  or on CM they will give a pure culture of one of the parent starins.   The heterocaryon has  broken down at spore formation.  

Synthesis of a  Heterozygous Diploid: 

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     The mechanics of diploidization in a haploid thallus is not clear.     A possible method is suggested in the sequence above of a multinucleate cell in Aspergillus. The nuceli shown in Figure A divide mitotically.   In Figure B the mitotoic division is now at early telophase.   The line between the chromosome clumps is the position of the original metaphase plate.    Occasionally the spindles of the mitotic divisions overlap.  In rare cases, as the nucelar envelop develops to form the daughter nuclei, it encompasses both sets of chromosomes, as indicated by the arrow in Figure C.

The diploid nucleus will now divide mitotically for a number of generations and form a diploid thallus.   The diploid thallus is heterozygous at all loci and will be phenotypically wild type.   In a heterocaryotic colony of  brown and yellow heads on complete medium (CM) the rare diploid will form green heads.   

 It can grow on minimal medium (lacking  biotin and thaimine supplements).  As it develops, the diploid thallus produces conidiophores and conidia containing diploid nuceli.    The nuclei in the conidia are heterozygous at all loci and the conidia will, therefore, be phenotypically wild type and green in colour.   The green conidia can be readily located under a dissecting scope, picked off with a fine needle, and streaked on minimal medium to give a pure culture of the heterozygous diploid.

So on minimal medium we get a diploid colony and the heterozygous for all the auxotrophic loci (green and nutritionally independent).

Diploid Colonies:

A diploid nucleus in Aspergillus is not a natural state.   The diploid nucleus is unstable and breaks down.  Aspergillus normally has four chromosomes and the diploid nucleus has eight.  The unstable nucleus breaks down by throwing off chromosomes randomly until it reaches the stable haploid state (four chromosomes).   Random loss of chromosomes results in various reassortments of the original eight (i.e. recombination).  Thus,  in the hypothetical  case  above, we could recover brown thiamine-requiring   strains and yellow biotin-requiring strains.

To recover these strains we could grow the heterozygous diploid on complete medium.   As the diploid broke down we would see yellow and brown sectors in the colony.   We could recover these and analyse them for vitamin requirements. 

Mitotic Crossovers:

Superimposed on all of this we have mitotoc crossovers.     Similar chromsomes in the diploid nucleus line up and rare crossovers occur between the chromosomes resulting in a recombination of those genes that are on the same chromosome.

Conclusion:

Between rasndom assortment of chromsomes and rare mitoic crossovers (somatic recombination in higher plants) we can have a reassortment of genetic material in certain fungi that mimics sexual recombination.

Important Caveat:

CHANCE