7.9 Cytoplasmic segregations: mitochondria, plasmids, viruses and prions

7.9 Cytoplasmic segregations: mitochondria, plasmids, viruses and prions

There are a number of cytoplasmic factors that affect the fungal phenotype and which depend for their transmission upon some of the features of heterokaryosis we have described so far. Chief among these are the mitochondria. Mitochondrial genomes are independent of and quite distinct from the nuclear genome. Loss of function mutations in mitochondrial genes result in characteristic phenotypes in both Neurospora crassa and Saccharomyces cerevisiae. Mitochondrial DNA (mtDNA) of yeast usually makes up 18% of the total DNA, but it has a distinctive very high AT content (82%) so is relatively easy to separate from chromosomal DNA. The mtDNA is circular, of 25 nm circumference, and comprises about 7.5 × 105 base pairs. In yeast, mtDNA codes for three of seven polypeptides of the cytochrome c complex (the rest derive from nuclear genes), four polypeptide components of a mitochondrial ATPase, and one component of cytochrome b. Mutations in these genes can produce recognisable respiratory deficiency phenotypes (for example, mutants named petite in S. cerevisiae, poky in Neurospora crassa) and thereby provide mitochondrial mutants. Also, although chromosomal genes code mitochondrial ribosomal proteins, mtDNA determines mitochondrial ribosomal RNA (rRNA) and transfer RNAs (tRNAs).

Mitochondrial ribosomes are similar in size to prokaryotic ribosomes and share some other prokaryotic properties; in particular, protein synthesis on mitochondrial ribosomes is inhibited by chloramphenicol, erythromycin and several other antibacterials that have no effect on cytoplasmic (eukaryotic) ribosomes. Consequently, another kind of mutant phenotype due to mitochondrial mutation is resistance to inhibition by mitochondrion-specific drugs. Mitochondrial gene sequences are also similar to equivalent genes in prokaryotes. These features encouraged the endosymbiont theory of mitochondrial origin, which envisages mitochondria to be relics of ancient bacteria-like organisms that formed a symbiotic association that resulted in the ancestral ‘eukaryotic’ cell.

S. cerevisiae can have over 100 genomes per mitochondrion, corresponding to about 6500 in each yeast cell; some of the mitochondrial genomes are circular, others are linear. Oddly enough, when segregation of organelle genes is followed in genetic crosses, the segregation patterns are consistent with there being only one copy of the mitochondrial genome in the cell. The fact that this is clearly not the case indicates that we do not fully understand how organelle genomes are transmitted from parent to offspring. The basic procedure for making mitochondrial crosses involves making heterokaryons (diploids in yeast) between haploids carrying mitochondrial markers. After some vegetative growth of the heterokaryon or diploid, diploid daughter cells (of yeast) or spores or hyphal fragments (of filamentous fungi) are plated out and the resulting colonies are scored for the mitochondrial markers present in the original haploids.

In Neurospora crassa, phenotypes controlled by mitochondrial genes are generally transmitted through the female, that is the protoperithecial parent. This is vertical mitochondrial transmission, from one generation to the next. Horizontal transmission, which is between individuals of the same generation, occurs as a result of hyphal fusion. Although complementation and recombination can be detected between mitochondrial genomes, mycelia containing genetically different mitochondria (called heteroplasmons or heteroplasmic mixtures) tend to segregate the different mitochondria into different cells (Wilson & Xu, 2012).

Uniparental inheritance of mitochondrial phenotypes has been observed in yeast, which is isogamous (does not show a male/female differentiation). Mitochondrial genomes can segregate in association with mitosis and the consequential bud formation The propagation of mitochondria during cell division depends on replication and partitioning of mitochondrial DNA, cytoskeleton-dependent mitochondrial transport, intracellular positioning of the organelle, and a variety of activities that coordinate these processes (Westermann, 2014). In filamentous fungi, mitochondria are not closely associated with the mitotic spindle, so vegetative segregation may simply be a matter of random physical sorting. However, mitochondria interact with elements of the cytoskeleton in both Neurospora (Fuchs et al., 2002) and Aspergillus (Suelmann & Fischer, 2000), although the cytoskeleton is involved in determining mitochondrial morphology as well as mitochondrial movement. Nuclear and mitochondrial genes influence mitochondrial genome transmission and it is also affected by membrane chemistry. In some Ascomycota sub-cultured for a long time in the laboratory, altered mitochondrial DNAs due to molecular rearrangements have been associated with modifications in mycelial growth in Neurospora crassa and Neurospora intermedia, as well as in growth of yeast.

Amongst the Basidiomycota, mitochondrial inheritance has been studied in matings of Schizophyllum commune, Agaricus bitorquis, A. brunnescens, Coprinopsis cinerea, Lentinula edodes, Pleurotus ostreatus, plant pathogenic Armillaria species and Ustilago violacea, and in all these cases, hyphal anastomoses result in the production of mycelial colonies composed of sectors containing different mitochondrial DNAs (mitochondrial mosaics). In Agaricus bitorquis, A. bisporus, Armillaria bulbosa, P. ostreatus and U. violacea, dikaryons had mixed populations of mitochondria, which sometimes resulted in recombination between mitochondrial genomes; recombinant mitochondrial DNAs have also been recovered from dikaryons and dikaryotic protoplasts of Coprinopsis cinerea (Xu & Wang, 2015).

As well as the mitochondrial genome, mitochondria can also contain autonomously replicating DNA molecules, that are either derived from the mitochondrial DNA or represent true plasmids that show no homology with the mitochondrial chromosome. An increasingly important aspect of mitochondrial transmission is their content of plasmids. Amongst true plasmids at least three different categories can be recognised: (a) circular plasmids encoding a DNA polymerase; (b) linear plasmids with terminal inverted repeats encoding a DNA and an RNA polymerase and; (c) retroplasmids, which are linear or circular plasmids that encode a reverse transcriptase. DNA and RNA polymerases, or reverse transcriptase, encoded by plasmid DNA sequences are used to maintain and propagate the plasmid. True plasmids are mostly cryptic (that is, neutral) passengers in nature, but some linear plasmids (notably of Podospora anserina) insert into mitochondrial DNA and cause mycelial senescence. Most linear plasmids exhibit typical virus characteristics as far as structure, replication and function are concerned even to the extent that plasmid-free strains may contain plasmid remnants integrated into their mitochondrial DNA. These different groups of true plasmids probably arose independently of one another and may have a different evolutionary origin from that of the mitochondrial host-genome. They were either vertically transmitted from the original endosymbiont that gave rise to the mitochondrion or invaded the mitochondrion at various times during fungal evolution (Hausner, 2003).

Although most mitochondrial plasmids are cryptic and symptomless, cytoplasmic plasmid DNA is responsible for the killer phenomenon in the yeast Kluyveromyces lactis by coding for a killer toxin, which kills cells lacking the plasmid (cells hosting the killer plasmid are immune to the toxin). These plasmids reside in the cytoplasm and have an expression system independent of both nucleus and mitochondrion. Plasmids of Kluyveromyces lactis can be transferred to other yeasts (including Saccharomyces cerevisiae), conferring the killer/immunity phenotypes (Fichtner et al., 2003). This shows that the plasmids are autonomous replicons, which can be expressed in a wide range of host yeasts.

The Kluyveromyces lactis killer plasmid toxin is chemically and functionally different from killer toxins produced in Saccharomyces cerevisiae (especially wine yeasts), which are encoded by double-stranded RNA (dsRNA) virus. Different killer toxins, K1, K2, K28, and Klus, have been described; each being encoded by a 1.5- to 2.3-kb double-stranded M satellite RNA located in the cytoplasm. These M satellite dsRNAs require larger helper virus (generally called L-A virus) for maintenance; L-A belongs to the Totiviridae family, and its dsRNA genome of 4.6 kb codes for proteins that form the virions that encapsidate separately the L-A or M satellites (Rodríguez-Cousiño et al., 2011; Rodríguez-Cousiño & Esteban, 2017).

Both budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe) have been used for studies of plant, animal and human viruses. Many RNA viruses and some DNA viruses replicate in yeasts. As many of the fundamental eukaryotic cell functions are highly conserved from yeasts to higher eukaryotes, these easily-cultivated fungi offer many unique advantages in virus research over ‘higher’ eukaryotes and are particularly suited to study the impact of viral activities on cell function during virus-host interactions (Zhao, 2017).

Virus‑like particles (VLPs) have been observed in electronmicrographs of many fungi. They are very similar in appearance to small spherical RNA viruses, but there is little evidence that these particles are effective in hypha‑to‑hypha infection. Many of the observed VLPs are presumably degenerate or defective viruses that can only be transmitted by hyphal fusions. No vectors are known for fungal viruses; transmission seems to depend on hyphal fusions. Unexpectedly, virus infections of fungi usually cause no recognisable phenotype. The exception is a mycovirus of Agaricus bisporus, which causes La France disease and ruins the crop. Diseased crops contain three virus particles and require up to 10 different RNA molecules to produce infective particles; as though some are defective viruses and others are helper viruses, or perhaps different viruses perform complementary, but essential, functions (Frost & Passmore, 1980). In a study of the infection on commercial mushroom farms in Poland, the virus particles were found in 120 of 200 samples tested; this level of La France disease could be a threat to the mushroom industry (Borodynko et al., 2010).

Saccharomyces cerevisiae also harbours retrovirus-like elements, as retrotransposons (now called transposable elements or TEs) able to integrate into the nuclear genome by targeting particular chromatin structures. The transposable elements of Saccharomyces cerevisiae consist of LTR (Long Terminal Repeat) retrotransposons called Ty elements belonging to five families, Ty1 to Ty5. They take the form of either full-length coding sequences or non-coding solo-LTRs corresponding to remnants of former transposition events. The first cytoplasmic plasmid to be observed wass the so-called two-micron DNA of Saccharomyces (Ty1). The name refers to the contour length of the circular DNA molecules in electronmicrographs; it has a base composition similar to nuclear DNA and quite different from mtDNA. There can be 50 to 100 two-micron DNA molecules per diploid cell, amounting to something like 3% of the nuclear DNA. The two-micron DNA molecules are transmitted to buds independently of both nuclei and mitochondria. The two-micron circular DNA carries inverted repeat sequences at either end of two different unique sequence segments; this structure implies that it inserts itself as a whole into the yeast chromosome (Bleykasten-Grosshans & Neuvéglise, 2011; Bleykasten-Grosshans et al., 2013; Stukenbrock & Croll, 2014).

So far we have described nucleic acid molecules that encode features segregating in the cytoplasm. In the final decade of the twentieth century, however, great attention was given (and continues to be given) to a proteinaceous hereditary element, called a prion protein. The attention devoted to prions derives from their ability to cause diseases in mammals: scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle; and in humans, kuru and new variant Creutzfeldt-Jakob disease (nvCJD). In these cases, the pathogenic agent is a variant of a normal membrane protein (the prion protein) that is encoded in the mammalian genome. Prions are infectious proteins, which means that they are altered forms of a normal cellular protein that may have lost their normal function but have acquired the ability to modify the normal form of the protein into the same abnormal configuration as themselves. The variant prion protein folds abnormally and in addition causes normal prion proteins to fold abnormally so that the proteins aggregate in the central nervous system and cause the encephalopathy; the aggregated proteins are called amyloids.

Several prion-forming proteins have been identified in fungi, mostly in the yeast Saccharomyces cerevisiae. We have already referred briefly (see Section 7.5 and Table 3) to the infectious het-s (heterokaryon incompatibility) phenotype of Podospora anserina as a protein that adopts a prion-like form to function properly as part of the self/nonself recognition system that ensures that only related hyphae share resources. The prion form of het-s can convert the non-prion form of the protein in a compatible mate after hyphal anastomosis. However, when an incompatible mycelium mates with a prion-containing mycelium, the prion causes the incompatible hyphal compartments to die when a programmed cell death response is triggered by interaction between specific het alleles (Paoletti & Clavé, 2007; Bidard et al., 2013; Paoletti, 2016).

First among several prions identified in Saccharomyces cerevisiae is the PSI+ form of the Sup35p protein. Sup35p is an essential yeast protein involved in the termination of translation. In the [PSI+] state, Sup35p adopts the structural conformation that causes it to direct the refolding of native molecules into a form that can aggregate into filaments of discarded nonfunctional protein. This depletes the cytoplasm of functional translation terminator and results in translation errors. This is the [PSI+] phenotype, which is inherited by daughter cells following budding, and is infectious following cell fusion, in which case it propagates by autocatalytic conversion of the normal form of the protein.

The prion phenotype is officially described as ‘increased levels of nonsense suppression’ because the prion suppresses nonsense-mutations by allowing the mutant genes to produce functional proteins; that is, the translation error is to translate the mutant code as working protein. Another unusual trait identified in yeast in the 1990s was called [URE3], which results from the prion form of the normal cellular protein Ure2p, which is a nitrogen catabolite repressor. Note that the names for yeast prions are normally shown in square brackets to indicate that they segregate in a non-mendelian manner. Two more recent discoveries are [MOT3+], the prion form of a nuclear transcription factor, which as a prion causes transcriptional derepression of anaerobic genes; and [GAR+], the normal function of the proteins being as components of plasma membrane proton pumps but as a prion it causes resistance to glucose-associated repression. For a list of prions refer to the Wikipedia page Fungal prion [at https://en.wikipedia.org/wiki/Fungal_prion], which seems to be regularly updated.

The part of the Sup35p protein that makes it a prion (the prion determining domain) is a glutamine/asparagine-rich amino-terminal region that contains several oligopeptide repeats. Removal of these repeats eliminates the ability of Sup35p to propagate PSI+ and expanding the repeat region increases the spontaneous occurrence of PSI+. Although deleting the analogous repeats from BSE prion protein does not prevent prion propagation and transmission in experimental mice, expansion of the repeat region does increase the spontaneous appearance of spongiform encephalopathies by several orders of magnitude in humans. It is likely that the oligopeptide repeats give the prion protein the intrinsic tendency to acquire a conformation that enables the protein to refold and self-propagate its conformation, so effectively polymerising with sister molecules to form the fibrous proteinaceous deposits called amyloids. Database searches for regions with amino acid content comparable to the yeast prions has revealed numerous such domains in eukaryotes, but these are lacking from prokaryotes (Alberti et al., 2009; King et al., 2012).

Fungal prions are generally benign; indeed, some confer a potential advantage to the fungus. Although it has been claimed that [URE3] and [PSI+] are ‘diseases’ of yeast caused by laboratory cultivation, some [PSI+] cells actually fare better than their prion-free siblings when subjected to adverse conditions. When 700 wild strains of Saccharomyces were genetically screened for unknown prion elements, one-third of the strains were found to harbour them (Halfmann et al., 2012). These ‘natural’ prions created diverse and often beneficial phenotypes. Evidently, fungal prions govern heritable traits in nature, but in a manner that confers the capacity on the fungus to adapt quickly and reversibly to variable environments. It has been suggested that the amyloid-folding promoted by fungal prions is a widespread and evolutionarily conserved mode of signal transduction, which is based on the transmission of an amyloid-fold from a STAND receptor protein to an effector protein (Daskalov et al., 2012). Fungal prions potentially provide a model for understanding disease-causing prions of humans and animals, particularly by identifying the sequence features and mechanisms that enable prion domains to switch between functional and amyloid-forming states [for information about disease-causing prions of humans and animals refer to the U.S. Department of Health & Human Services Centers for Disease Control and Prevention website at https://www.cdc.gov/prions/index.html].

Updated July, 2018