Heterokaryosis

A consequence of multinuclear condition is heterokaryosis. This term refers to the coexistence of two or more genetically different nuclei inside the same hyphal cell. If single spores of the fungus Botrytis cinerea taken from a natural substratum are grown on an agar medium, a circular colony grows which forms sectors, noticeable by poor or heavy sporulation (Figure 2.6). Single spores from each sector produced non-sectoring colonies whereas a spore from the main colony produced a culture that sectored like the parent. Two distinct types of nuclei coexisted in the hyphae and differing types of these nuclei were incorporated in the spores. The heterokaryotic condition can arise either by spontaneous mutation in some nuclei within an originally genetically homogeneous mycelium or by the fusion of genetically distinct hyphae followed by nuclear mixing. Because of their multinuclear condition, heterokaryosis is a unique feature of the fungi. It has been assumed to be an important mechanism in the adaptation of fungi to a fluctuating environment

Sporulation Fungi

Homokaryotic heavy sporulation

Heterokaryotic poor sporulation

Heterokaryotic intermediate sporulation

Single-spore derived cultures

Figure 2.6 Diagram of discovery of heterokaryosis. (A) A colony of Botrytis cinerea derived from a single spore producing sectors of poor and heavy spores. A single spore from each sector gave non-sectoring colonies (B) and (D) whereas a spore from the main colony gave a culture sectoring like the parent one (C). This condition is due to different types of nuclei represented by black and white. Redrawn from Ingold and Hudson, The Biology of Fungi (1993), Chapman and Hall. With permission of Kluwer Academic Publishers.

Homokaryotic heavy sporulation

Heterokaryotic poor sporulation

Heterokaryotic intermediate sporulation

Single-spore derived cultures

Figure 2.6 Diagram of discovery of heterokaryosis. (A) A colony of Botrytis cinerea derived from a single spore producing sectors of poor and heavy spores. A single spore from each sector gave non-sectoring colonies (B) and (D) whereas a spore from the main colony gave a culture sectoring like the parent one (C). This condition is due to different types of nuclei represented by black and white. Redrawn from Ingold and Hudson, The Biology of Fungi (1993), Chapman and Hall. With permission of Kluwer Academic Publishers.

through the alteration in nuclear ratios (the proportion of nuclear types). "A fungus which combines heterokaryosis with sexual reproduction provides both for the present and the future" (Burnett, 1976).

In some fungi, co-spotting a mixture of two mutant conidia of same mating type on unsupplemented agar media readily forms heterokaryons. The conidial germ tubes fuse and nuclear mixing occurs allowing heterokaryotic hypha to be formed (Figure 2.7). The formation of heterokaryotic mycelium is used to distinguish if two strains of the same phenotype have a mutation in the same gene or in two different genes in a biochemical pathway (test of allelism). If nuclei with nonallelic mutations coexist in a heterokaryon, the phenotype of heterokaryon is normal since what is lacking in one is present in the other and all functions can be performed. Allelic mutations fail to complement because neither nucleus can perform the vital function (Figure 2.8).

2.7.1 Sheltering of Lethal Mutation

Though the mutation rate is estimated to be on the order of one in one million nuclei, a multinuclear mycelium can over time accumulate lethal mutations. The mutant genes would be sheltered by their normal alleles in other nuclei in the cytoplasm and the lethal mutation can be undetected in multinucleate hypha. Nuclei can, however, be extracted

Heterokaryon

Figure 2.7 Formation of heterokaryon in Aspergillus. The two genetically different types of nuclei are shown as open and filled circles. The nuclear types are segregated during the budding of uninucleate spores from phialide cells. The conidia are uninucleate and on germination they produce homokaryotic hyphae that may fuse to give a heterokaryon. (From Ingold and Hudson, The Biology of Fungi (1993), Chapman and Hall. With permission of Kluwer Academic Publishers.)

Figure 2.7 Formation of heterokaryon in Aspergillus. The two genetically different types of nuclei are shown as open and filled circles. The nuclear types are segregated during the budding of uninucleate spores from phialide cells. The conidia are uninucleate and on germination they produce homokaryotic hyphae that may fuse to give a heterokaryon. (From Ingold and Hudson, The Biology of Fungi (1993), Chapman and Hall. With permission of Kluwer Academic Publishers.)

from mycelium in the form of uninucleate spores and these can be grown into a homokaryotic culture and examined for mutant phenotypes (Maheshwari, 2000). An example of this sheltering of a recessive lethal mutation in coenocytic hypha is given in Chapter 14.

2.7.2 Nuclear Selection

Asynchronous nuclear division can result in varying proportions of nuclear types in multinuclear mycelium. Extreme nuclear disproportion can occur in certain genotypes in response to culture conditions. Ryan and Lederberg (1946) constructed a heterokaryon of N. crassa containing mutant leucineless (leu) nuclei and prototrophic (leu+) nuclei. When this heterokaryon is grown with leucine supplementation, the mutant nuclei are favored over the wild varieties; the degree of selection is so extreme that the mycelium becomes pure (homokaryotic) leucineless. Either the prototrophic leu+ nuclei were "inactivated" or eliminated.

Other instances of extreme nuclear disproportion have been discovered. In one experiment, heterokaryons of N. crassa having extremely disproportionate ratios of wild and mutant histidine nuclei were used to determine if the enzyme activity encoded by the wild allele is related to the dosage of wild nuclei. A [his-3 + his-3(EC)] heterokaryon was generated by transformation of a histidine auxotrophic strain (his-3) with a plasmid-containing a. Non-allelic mutations (gene-1 and gene-2 )

b. Allelic mutations (gene -2a and gene-2b)
Nucleus And Alleles
Figure 2.8 The heterokaryon test to determine whether two mutations are in the same gene. (a) Nuclei with non-allelic mutations complement each other, (b) nuclei with allelic mutations fail to complement. (From Davis (2003). With permission of the publisher.)

gene for histidinol dehydrogenase (Pitchaimani and Maheshwari, 2003). (Note that in genetic terminology, the genotype of strains combined in a heterokaryon is enclosed in parenthesis; a Neurospora gene that has been integrated ectopically by transformation is designated by appending "EC" to the gene symbol.) Extreme nuclear disproportion occurred in transformant grown in the presence of histidine but not when grown without histidine. Despite the drastic change in nuclear ratio, the activity of the enzyme histidinol dehydrogenase in mycelia grown in the two conditions was similar. The authors hypothesized that not all nuclei in hyphae are active at any given time, the rare active nuclei being sufficient to confer the wild phenotype through biosynthesis of enzyme (Pitchaimani and Maheshwari, 2003).

2.7.3 Nuclear Competence

The reaction of nuclei, even if they differ in a single gene, can be different when combined in a heterokaryon. A phenomenon described as nuclear competence illustrates this (Grote-lueschen and Metzenberg, 1995). As an example, in N. crassa mutant strain 1 is auxotrophic for markers X and Z, while strain 2 is auxotrophic for markers Y and Z. A heterokaryon formed by fusion of strains 1 and 2 is auxotrophic only for the Z marker his-3 al-1; mcm; inl;a (requires histidine for growth)

oo his-3 al-1+; mcm; pan; a (requires histidine for growth)

Electroporation with his-3+ plasmid jr*

Heterokaryon

(his-3 al-1; mcm; inl;a + his-3 al-1+; mcm; pan; a)

Induction of microconidia

Microconidium Nucleus

Plating of microconidia on media lacking histidine

Homokaryotic [ q colonies

Plating of microconidia on media lacking histidine

Homokaryotic [ q colonies

With inositol (white)

With pantothenic acid (orange)

Minimal medium

Figure 2.9 Diagram of discovery of nuclear competence. A histidine auxotroph was transformed with his-3+ plasmid and resolved into component nuclear types to determine into which nucleus or nuclei the plasmid had entered. Modified from Dev and Maheshwari (2002).

With inositol (white)

With pantothenic acid (orange)

Minimal medium

Figure 2.9 Diagram of discovery of nuclear competence. A histidine auxotroph was transformed with his-3+ plasmid and resolved into component nuclear types to determine into which nucleus or nuclei the plasmid had entered. Modified from Dev and Maheshwari (2002).

because of complementation of X and Y gene products. When a heterokaryon that contains two nuclear types (Figure 2.9) is transformed with a plasmid carrying an exogenous copy of the Z gene, the heterokaryon became prototrophic for the Z gene product and could be selected on a medium that allows growth only of the heterokaryotic cell. The heterokaryotic cell is resolved into component nuclear types by inducing it to produce uninucleate microconidia (Dev and Maheshwari, 2002). Analysis showed that at any given time, the transforming DNA randomly entered into only one type of nucleus that was "competent" but never into both nuclear types. The results show though in a common cytoplasm, the heterokaryotic nuclei respond differently, most likely because of their different nuclear division cycles where at any given time there is a very narrow window for the entry and integration of exogenously added DNA.

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