Search

In N. crassaand N. intermedia, the kalilo plasmid triggers senescence by insertion into mitochondrial DNA. To investigate the cell death pathway induced by this plasmid, juvenile and senescent subcultures of several senescent strains were examined by light and transmission electron microscopy, and at the DNA level. There were no signs of apoptotic events, such as shrinkage of the cytoplasm away from the cell wall, apoptotic bodies, internucleosomal DNA fragmentation or condensation of the cytoplasm while retaining mitochondria and endomembrane structure. Instead, swollen mitochondria lacking cristae and containing amorphous inclusions, and disruption of nuclear and mitochondrial membranes indicated a necrotic mode of cell death.

The widespread occurrence of vegetative incompatibility in fungi and the high level of incompatibility gene polymorphisms in fungal populations means that generally parents in a cross will be vegetatively incompatible. In Neurospora crassa the mating type locus even functions as a vegetative incompatibility locus assuring vegetative incompatibility in a cross. In this paper we have tested the effect of vegetative incompatibility on regulating transmission of mitochondrial plasmids and nuclear genes in sexual interactions of N. crassa. In the absence of mating type vegetative incompatibility between the parents, transmission of plasmids from the conidial paternal parent was found to be approximately ten times higher than in normal crosses. Control experiments in which conidia of a contaminating plasmid bearing strain of similar mating type as (and fully vegetatively compatible with) the established maternal culture were added together with the presumed paternal conidia (opposite mating type) showed plasmid transmission to ascospores. Since trichogynes do not fuse with conidia of the same mating type, it may be concluded that plasmids from the contaminating strain entered the maternal tissue by somatic fusion. The fate of conidial nuclei in such sexual interactions has been investigated as well, both under conditions of vegetative compatibility and incompatibility between the strains. These experiments demonstrated that conidia that are vegetatively compatible to the established protoperithecial strain manage to get access to the resources of the maternal culture and initiate new fruiting bodies. This type of nuclear parasitism was never observed when vegetatively incompatible conidia were used. We propose that vegetative incompatibility may function in sexual crosses to protect unfertilised cultures from looting of maternal resources.

Abstract: There are four different variants of the kalilo “family” of linear mitochondrial plasmids. This family is found in several heterothallic species and one pseudohomothallic species of Neurospora, as well as in one homothallic species of Gelasinospora. The mode of dispersal of these plasmids is not known. Horizontal transmission has proved difficult to demonstrate. Another possibility is transfer by introgression, and this is modelled in the present paper. We have used introgression and subsequent heterokaryosis to successfully transfer the LA-kalilo plasmid from a Haitian strain of Neurospora crassa to the standard Oak Ridge N. crassa background, the LA-kalilo plasmid from the pseudohomothallic Neurospora tetrasperma to N. crassa, and the kalilo plasmid from N. crassa to N. tetrasperma. Thus, introgression is shown to be a possible avenue of dispersal between species. The recipient strains were all senescent but the mechanism of this senescence is not known. It could be caused by the plasmids, but if so the mechanism is novel since plasmid/mtDNA junction fragments of the type found in the standard mode of mtDNA insertion could not be detected. However, mtDNA changes were observed in the senescent recipients.

Because of the ease with which fungal strains and life cycles can be manipulated, fungal research has made important contributions to the present understanding of mitochondrial inheritance generally. Several mitochondrial DNA molecules have been completely sequenced and this information has provided useful probes for study of other mitochondrial genomes. Mapping and sequencing has shown that the mtDNA is circular, butin vivomany linear derivatives are observed whose significance is unclear. The genes carried in mtDNA are for components of the electron transport system, ATP synthesis and mitochondrial protein synthesis. Multigenic transcripts are processed to arrive at mRNAs. The mitochondrial genetic code shows several differences compared to the universal code. MtDNA shows a high level of neutral variation due to length mutations and restriction site differences: these are useful in measuring genetic distances in evolutionary and taxonomic studies. Mutations affecting gene function lead to slow/erratic growth or drug resistance. Many mutations are suppressive, rapidly dominating the culture. Mitochondrial plasmids are common in natural isolates of fungi. Strains can contain mixtures of linear and circular types. Some plasmid families show widespread distribution, across different species and genera. Linear plasmids generally have genes for presumptive DNA and RNA polymerases. Circular plasmids have a single gene for a DNA polymerase or a reverse transcriptase. Several plasmids recombine with mtDNA; the most striking cases are the senescence plasmids, which integrate into mtDNA and cause death through insertional mutagenesis. Genetic elements in the mitochondria, and the phenotypes determined by them, are inherited maternally or uniparentally in sexual crosses. Mitochondrial elements and phenotypes are transmitted by cytoplasmic contact between cells. In heteroplasmons the mitochondrial mixtures often segregate during somatic propagation, resulting in pure derivatives. Heteroplasmons also offer the opportunity for recombination between different mtDNA molecules.

The linear autonomous form of kalilo DNA (previously called AR-kalDNA) is shown to be resident within mitochondria rather than nuclei, as had been suggested by previous experiments. This form has been renamed mtAR-kalDNA, to signify its mitochondrial location. Experiments are described that illustrate the inheritance and somatic transmission patterns of the mitochondrial kalilo plasmid and the mitochondrial inserted form of kalilo DNA (mtlS-kalDNA). Progeny of a cross with a pre-senescent subculture as the female parent inherited mtAR-ka1DNA only; mtIS-kalDNA was not transmitted sexually. During somatic propagation of the ascospore cultures, novel kalilo DNA inserts appeared and most of them persisted until death. We propose that these inserts originated from de novo integration of mtAR-kalDNA into the mitochondrial DNA. In two of the ascopore-derived series analyzed, the first inserts detected were seen only transiently and inserts appearing subsequent to the transient inserts were retained until death. We propose that these enduring inserts originated either from rearrangements of the transient inserts or from novel integration events, either from mtAR-kalDNA or from transposition of the transient inserts.

A strain of Neurospora intermedia from China contains five prominent extragenomic mitochondrial plasmids: three linear elements called zhisi plasmids, and two circular plasmids, Harbin-1 and -2. In one subculture, levels of four plasmids (all three zhisis and Harbin-1) fell to undetectable values and two novel linear plasmids appeared, Harbin-L and -L2, as well as a new small circular plasmid, Harbin-0.9. Cross-hybridization of restriction fragments and DNA sequencing showed that the Harbin-L plasmid was composed of parts of the circular Harbin-1 plasmid and of one of the linear zhisi plasmids. A model is presented in which the Harbin-1 and zhisi plasmids are present within the same mitochondrion, and crossovers at two separate 7 by sites of sequence identity effectively insert part of the circular Harbin-1 DNA into a zhisi linear plasmid, simultaneously deleting part of the zhisi element. The small plasmid Harbin-0.9 is a fragment of the Har-1 plasmid, and seems to be another product of the recombination process that created Har-L. Recombination of this type could have contributed to the wide array of mitochondrial plasmids found in natural populations of Neurospora.

Two Louisiana strains of Neurospora tetrasperma contain a linear plasmid (LA-kalDNA) with a restriction map identical to the Hawaiian Neurospora intermedia senescence plasmid, kalDNA, but with termini 100 nucleotide pairs shorter. One of these strains also bore a circular plasmid similar to the Hawaiian circular plasmid Hanalei-2. One species probably acquired both plasmids from the other by horizontal transfer, at a time sufficiently distant for sequence divergence to take place. Many LA-kalDNA-bearing derivative strains senesced, but this plasmid does not guarantee senescence. Furthermore, LA-kalDNA does not insert into mtDNA. One senescent strain showed no LA-kalDNA. The plasmids are effectively transmitted via the pseudohomothallic sexual cycle. Single mating-type derivatives transmit plasmids maternally.

Several field-collected strains of Neurospora crassa from the vicinity or Aarey, Bombay, India, are prone to precocious senescence and death. Analysis of one strain, Aarely-1e, demonstrated that the genetic determinants for the predisposition to senescence are maternally inherited. The senescence-prone strains contain a 7-kb, linear, mitochondrial DNA plasmid, maranhar, which is not present in long-lived isolates from the same geographical location. The maranhar plasmid has inverted terminal repeats with protein covalently bound at the 5' termini. Molecular hybridization experiments have demonstrated no substantial DNA sequence homology between the plasmid and the normal mitochondrial (mtDNA) and nuclear genomes of long-lived strains of N. crassa. Integrated maranhar sequences were detected in the mtDNAs of two cultures derived from Aarey-1e, and mtDNAs with the insertion sequences accumulated during subculturing. Nucleotide sequence analysis of cloned fragments of the two insertion sequences demonstrates that that they are flanked by long inverted repeats of mtDNA. The senescence syndrome of the maranhar strains, and the mode of integration of the plasmid, are reminiscent of those seen in the kalilo strains of N. intermedia. Nonetheless, there is no detectable nucleotide sequence homology between the maranhar and kalilo plasmids.

We analysed the distribution of mitochondrial plasmids among 82 Neurospora intermedia isolates from Hawaii; 74% of the isolates carried the neutral circular plasmid Han-2, whereas 38% contained the linear senescence-causing plasmid kalDNA. The distributions of the two plasmids are independent. There is no significant difference between the Kauaian population of 1972 and that of 1976. To further examine the reasons for this frequency distribution we studied the transmission of both Hawaiian plasmids through the maternal parent in a large series of crosses using non-Kalilo isolates as conidial parents. Plasmids can be lost during the sexual cycle. The Han-2 plasmid is transmitted more efficiently than kalDNA. No clear cases of autonomous or non-autonomous plasmid suppression were observed, so loss can be considered accidental. One Kalilo strain proved to be ineffectual as a maternal parent, and this reduced its ability to transmit kalDNA to the next generation. The dynamic balance of plasmids in natural populations over time is probably a result of the interplay of many forces, including those described in this work and those from several other studies on Neurospora plasmids.

The nuclear photosynthetic mutant, hcf2, of Arabidopsis thaliana was isolated by screening M2 seedlings for abnormally-high chlorophyll fluorescence (hcf), indicative of a block in photosynthetic electron transport. Fluorescence induction kinetics, photosynthetic electron transport activity assays and immunoblotting revealed that all the complexes involved in photosynthetic electron transport were affected to some extent. The most striking effect of the mutation was on the relative steady state levels of the petA transcript (encoding the apoprotein of cytochrome f) which were more than five-times higher in the mutant plants than in their wild-type siblings.

Heterokaryotic transmission is one of the major techniques for the study of cytoplasmic inheritance and here we have applied it to the senescence-determining plasmids kalilo (Hawaiian) and maranhar (Indian). We have shown that kalilo-based senescence is effectively transmitted by cytoplasmic contact, both in N. crassa and in N. intermedia. In the first place, the heterokaryons themselves are senescent, confirming the suppressivity of the senescence phenotype in mixtures of normal and senescent cytoplasms. Second, senescence is found in new nuclear associations, as shown by analysis of conidial isolates and meiocytes stemming from the heterokaryons. In addition, the free plasmid AR-kalDNA, and its form that is inserted into mtDNA, (mtIS-kalDNA), are both transmitted to new nuclear associations. In a transient fusion between senescent N. intermedia and nonsenescent N. crassa cells, AR-kalDNA was transmitted to N. crassa and mtIS-kalDNA was transmitted to N. crassa mtDNA. A cryptic mitochondrial plasmid, not associated with senescence, was also transmitted very efficiently to N. crassa mitochondria. In mixed kalilo/maranhar fusions, both plasmids coexisted, approximately equally, in the heterokaryons themselves, and in conidial isolates. However, in sexual derivatives, AR-marDNA was in an excess and AR-kalDNA was sometimes absent. The efficient heterokaryotic transmission of these elements suggests that this is one of their natural modes of spread in populations.

We have shown that senescence in Kalilo strains of Neurospora, caused by a linear mitochondrial plasmid called kalDNA, is suppressible by existing variants of the nuclear genome. The suppressors are manifested by 4:4 segregation of senescence and immortality in asci from crosses between senescent female strains and males chosen from non-senescent candidate stocks. In one case of suppression, the asci also show segregation at the plasmid level. There is a reduction of kalDNA to barely detectable levels in the four ascospores showing immortality, so this suppressor evidently influences the maintenance of the plasmid itself. In the other case of suppression, the phenotypic segregation is not correlated with segregation at the plasmid level, and all eight ascospores in the asci show both free and inserted forms of kalDNA. This suggests that the suppression genotype provides a way of tolerating the presence of the plasmid rather than diminishing it. However, the allele f, which provides an analogous kind of suppression for the cytoplasmic mutation poky, does not suppress Kalilo or Maranhar senescence. Suppression is hence shown to be a possible option for host strains to combat the plasmid in nature, but no examples of suppressors were found in a limited survey of natural isolates. In addition, we have shown that long-lived, presumably non-senescent, strains do not arise by suppressor mutation, but lose senescence plasmid DNA by another mechanism.