A new senescence-inducing mitochondrial linear plasmid in field-isolated Neurospora crassa strains from India

Summary 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.     

Vegetative incompatibility in Neurospora: its effect on horizontal transfer of mitochondrial plasmids and senescence in natural populations

We have investigated the horizontal transfer of two mitochondrial plasmids and the Kalilo senescence phenotype in the fungus Neurospora without the use of heterokaryon-forcing markers. The Kalilo senescent state was only transferred between fully-compatible N. crassa strains, but not between strains differing at any of the loci het-c, het-d, het-e or mating-type. However, the linear plasmid kalDNA and the circular plasmid Han-2 were transferred following incompatible vegetative interactions. Our data suggest that vegetative incompatibility due to allelic differences at het-c is more effective in preventing transfer than that due to het-d, het-e or mating-type. Based on these observations we have developed a novel test for assessing vegetative incompatibility between Kalilo and non-Kalilo field isolates of N. intermedia. In this procedure combinations of Kalilo and non-Kalilo field isolates of N. intermedia were grown together and tested for senescence. Compatibility is inferred if the young non-Kalilo strain dies along with the senescent Kalilo strain, whereas incompatibility is inferred when the Kalilo strain dies without imposing its senescent state onto the non-Kalilo strain. Our results suggest that each of the nine Kalilo strains tested is incompatible with each of 20 non-Kalilo isolates from the same N. intermedia population of the Hawaiian island of Kauai. However, the observed incompatibility did not completely prevent cytoplasmic exchange, and in several cases plasmid transfer could be detected.     

Association between variant plasmid formation and senescence in retroplasmid-containing strains of Neurospora spp.

Serial transfer of Neurospora strains harboring the Mauriceville and Varkud mitochondrial retroplasmids frequently displays erratic growth and senescence. Growth impairment is associated with the formation of variant forms of the retroplasmids that can integrate into the mitochondrial genome, resulting in mtDNA rearrangements and eventual loss of respiratory function. Here, we evaluate the rate at which variant plasmids arise in subcultures of the Mauriceville strain of N. crassa and their association with the senescent phenotype. Although variant plasmid formation preceded senescence, subcultures were found to tolerate variant plasmids for variable lengths of time and no correlation could be made between the specific sequence inserted in the plasmids and the rate or frequency of senescence. In addition, many cultures were found to contain more than one variant plasmid. The lack of concordance between the timing of variant plasmid formation and growth cessation distinguishes these two events, and provides additional insight into the etiology of senescence. We also detected differences in the frequency of senescence between retroplasmid-containing strains of N. crassa and N. intermedia and report the isolation of a strain in which senescence occurs in the absence of variant plasmid formation or detectable alterations in mtDNA. Our findings indicate there are multiple pathways that lead to senescence and suggest there are host-specific mechanisms that suppress the deleterious effects of the variant plasmids. Received: 8 August 2000 / Accepted: 17 November 2000     

A kalilo-like linear plasmid in Louisiana field isolates of the pseudohomothallic fungus Neurospora tetrasperma

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.     

Transfer of Neurospora kalilo plasmids among species and strains by introgression

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. Received: 15 February / 24 June 1999     

Genetic organization and structural features of maranhar,a senescence-inducing linear mitochondrial plasmid of Neurospora crassa

Summary The nucleotide sequence of maranhar, a senescence-inducing linear mitochondrial plasmid of Neurospora crassa, was determined. The termini of the 7-kb plasmid are 349-bp inverted repeats (TIRs). Each DNA strand contains a long open reading frame (ORF) which begins within the TIR and extends toward the centre of the plasmid. ORF-1 codes for a single-subunit RNA polymerase that is not closely related to that encoded by another Neurospora plasmid, kalilo. The ORF-2 product may be a B-type DNA polymerase resembling those encoded by terminal protein-linked linear genetic elements, including linear mitochondrial plasmids and linear bacteriophages. A separate coding sequence for the terminal protein could not be identified; however, the DNA polymerase of maranhar has an amino-terminal extension with features that are also present in the terminal proteins of linear bacteriophages. The N-terminal extensions of the DNA polymerases of other linear mitochondrial plasmids contain similar features, suggesting that the terminal proteins of linear plasmids may be comprised, at least in part, of these cryptic domains. The terminal protein-DNA bond of maranhar is resistant to mild alkaline hydrolysis, indicating that it might involve a tyrosine or a lysine residue. Although maranhar and the senescence-inducing kalilo plasmid of N. intermedia are structurally similar, and integrate into mitochondrial DNA by a mechanism thus far unique to these two plasmids, they are not closely related to each other and they do not have any nucleotide sequence features, or ORFs, that distinguish them clearly from mitochondrial plasmids which are not associated with senescence and most of which are apparently non-integrative.     

Characterization and prevalence of a circular mitochondrial plasmid in senescence-prone isolates of Neurospora intermedia

Genetic and molecular analyses of the phenomenon of senescence—i.e., irreversible loss of growth and reproductive potential upon subculturing—in Neurospora intermedia strain M1991-60A, collected from Maddur in southern India, showed the presence of plasmid pMaddur1, which is homologous to the senescence-inducing circular mitochondrial plasmid, pVarkud. Maternal inheritance of senescence in M1991-60A correlated to the formation of variant pMaddur1, its subsequent insertion into mitochondrial (mt)DNA and the accumulation of defective mtDNA with the pMaddur1insert. PCR-based analyses for similar plasmids in 147 natural isolates of Neurospora from Maddur showed that nearly 40% of the strains had pMaddur1 or pMaddur2 that shared 97–98% sequence homology with pVarkud and pMauriceville. Nearly 50% of the strains that harbored either pMaddur1 or pMaddur2, also contained a circular Varkud satellite plasmid (pVS). Size polymorphism maps to the cluster of PstI sites in the non-coding region. Whereas senescence of nearly 40% of N. intermedia strains may be due to pMaddur, the presence in seven strains of pVS but not pMaddur and the absence of either of these two plasmids in other senescence-prone isolates suggests yet undiscovered mechanisms of senescence in the Maddur strains.     

The dynamics of mitochondrial plasmids in a Hawaiian population of Neurospora intermedia

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.     

Kalilo plasmids are a family of four distinct members with individual global distributions across species

Kalilo is a linear 9-kb plasmid, isolated originally from Hawaiian strains of the heterothallic fungus Neurospora intermedia. Its properties include terminal inverted repeats, two ORFs coding for a presumptive DNA and an RNA polymerase, and the ability to cause senescence in its original host and in the closely related species Neurospora crassa. We have examined natural isolates alleged to contain plasmids homologous to kalilo. Most of these isolates do in fact contain plasmids with so close an identity to kalilo as to be certain relatives. We found a new case of kalilo in Neurospora tetrasperma from Moorea-Tahiti, and a new case of LA-kalilo (previously found only in N. tetrasperma) in N. crassa from Haiti. A previously unreported, substantially shorter, kalilo variant has been found in three geographically separate isolates of the heterothallic species Neurospora discreta. Therefore, if the previously reported kalilo variant from the genus Gelasinospora is included, in all there are four members of the kalilo plasmid family. The main differences between these plasmids are in the terminal inverted repeats (TIRs). The phylogeny of the TIR sequences is largely congruent with that of nuclear DNA in the species in which they are found, suggesting that the plasmids are related by vertical descent throughout the evolution of these species. However, there are two cases of a plasmid found in a heterothallic and a pseudohomothallic species in the same global area; these cases might have arisen from more recent horizontal transmission or introgression. Received: 14 July / 17 September 1999     

Inversions and recombinations in mitochondrial DNA of the (SG-1) cytoplasmic mutant in two Neurospora species

Summary The mitochondrial DNAs of [SG-1] cytoplasmically-mutant and wild-type strains of Neurospora crassa and Neurospora sitophila were examined by comparative restriction endonuclease analyses. The mtDNA of N. sitophila wild type of Whitehouse differs from type II mtDNA of N. crassa by insertions of 3.3 kb in EcoRI-9, and 1.2 kb in EcoRI-3, and a deletion of 1.1 kb in EcoRI-5. These DNA heteromorphisms provided convenient markers for tracing N. crassa [SG-1] mtDNA during and after its transfer into N. sitophila. The [SG-1] cytoplasmic mutant in both N. crassa and N. sitophila has a distinctive inversion that connects the fragment EcoRI-4 with HindIII-10a. The [SG-1] mtDNA from N. crassa remained essentially intact after it was transferred by crosses into N. sitophila. In each species, a unique second inversion occured in the [SG-1] mtDNA after the transfer was made. In N. sitophila, polar recombination in heteroplasmons between [SG-1] and wild-type preferentially yields strains with mtDNAs that contain the maximum possible number of insertions in the cob and co-1 loci of the EcoRI-3 region of the mitochondrial chromosome.     

Unstable cytoplasms in Hawaiian strains of Neurospora intermedia

Summary By subjecting a large sample of natural isolates of N. intermedia to prolonged serial subculturing, 26 cytoplasmic variants have been identified. These variants show senescence, and finally death at some strain-specific point in the subculture series. All senescent strains are from the Hawaiian archipelago, where their incidence in natural populations is high. Senescent cultures can be female-fertile. Random ascospore analyses show that (i) senescence is maternally inherited; (ii) different stages of senescence give different proportions of senescent progeny; and (iii) ascospores from one cross show different degrees of senescence. These results indicate that senescence is determined by a genetic factor which re sides in the cytoplasm. This factor promotes instability of the cytoplasm, resulting initially in cytoplasmic heterogeneity shown by ascus and conidium sampling, and finally in death. Molecular studies to be published elsewhere show that the progression through senescence to death is correlated with the occurence of abnormalities in cytochrome content and mitochondrial DNA. The Hawaiian word kalilo (dying), symbolised [kal], is proposed to denote these cytoplasms.     

A recombinant plasmid carrying the mitochondrial plasmid sequence of Neurospora intermedia LaBelle yields new plasmid derivatives in Neurospora crassa transformants

Summary We have studied the efficacy of transformation of Neurospora crassa with a chimaeric plasmid. We constructed a recombinant plasmid, pMK2, consisting of the mitochondrial plasmid of N. intermedia LaBelle, a part of the qa gene cluster of Neurospora and the Escherichia coli plasmid pBR322. Compared to plasmid pVK88, not containing the LaBelle sequence, the pMK2 plasmid gives a five-fold increase in transformation of the qa2⁺ gene. Analysis of the DNA from Neurospora transformants revealed that the pMK2 plasmid is not stable. The qa insert as well as the LaBelle part of pMK2 are rapidly lost from the plasmid. In most cases the qa insert integrates into the nuclear DNA of the host. Plasmids recovered from Neurospora transformants are rearranged and show insertions or deletions. Some of these plasmids are described here. In most cases the qa insert and the LaBelle sequence of plasmid pMK2 have been deleted. Frequently plasmid dimers, carrying an insertion of mitochondrial DNA, are recovered.     

Cloning and preliminary characterization of a molybdenum cofactor gene of Neurospora crassa

Summary A Neurospora crassa library, constructed in a derivative of the plasmid pBR322 (pRK9), was used to transform two E. coli ch1D molybdenum cofactor mutants (ch1D, ch1D::Mu). Subsequently, one transformant from each of three independent transformation experiments was restriction mapped. All three transformants had an identical N. crassa DNA insert (4.2 kb). Southern Blot analysis with one of the plasmids (pMoCo, 1:4) showed hybridization to a single band of N. crassa genomic DNA. When pMoCo plasmid (1:4) was used to transform various E. coli nitrate reductase mutants (ch1A, ch1B, ch1C, ch1D, ch1E, ch1G and ch1M), the pMoCo plasmid was capable of restoring E. coli nitrate reductase activity to only the ch1D mutant. In vitro reconstitution experiments using wild-type, ch1D and ch1D; pMoCo cell-free extracts as a source of molybdenum cofactor (MoCo) were performed with the N. crassa MoCo mutants nit-1, nit-7 and nit-8. MoCo from wild-type E. coli cell-free extracts was capable of reconstituting NADPH : nitrate reductase activity to all three N. crassa mutants. MoCo from ch1D; pMoCo cell-free extracts was capable of reconstituting more NADPH : nitrate reductase activity to the N. crassa mutants than cell-free extracts from the original ch1D mutant.     

Plasmids of mitochondrial origin in senescent mycelia of Podospora curvicolla

Summary Podospora curvicolla displays symptoms of senescence similar but not quite identical to those reported for Podospora anserina. In Podospora curvicolla single hyphae may escape from death leading to a new growth front and consequently to a mode of growth characterized by alternating phases of growth and non-growth. Restriction analyses and hybridization experiments have revealed that the Podospora curvicolla type of senescence is correlated with plasmids originating from amplification of a single distinct region of the mitochondrial DNA containing the IrRNA gene. In the yeast transformation system sequences of this region may function as autonomously replicating sequences (ARS). Plasmids (pl1, pl2 and pl3) isolated from different, independently aged mycelia are largely homologous to each other but differ in their excision/junction sites and have different sizes: 10.85 kb (pl1), 9.01 kb (p12) and 10.50 kb (pl3). The sequence of the most frequently occurring plasmid in ageing strains of Podospora anserina is absent in Podospora curvicolla either as free plasmid DNA or as an integrated part of the mtDNA. Possibly there is a correlation between the absence of this particular sequence in Podospora curvicolla and the type of senescence displayed in this organism.     

The mitochondrial plasmid of the true slime mold <Emphasis Type="Italic">Physarum polycephalum</Emphasis> bypasses uniparental inheritance by promoting mitochondrial fusion

Mitochondrial DNA (mtDNA) is inherited maternally in most eukaryotes. Linear mitochondrial plasmids in higher plants and fungi are also transmitted from the maternal parent to the progeny. However, mF, which is a mitochondrial linear plasmid of Physarum polycephalum, evades uniparental mitochondrial inheritance. We examined 36 myxamoebal strains of Physarum and isolated three novel mF⁺ strains (JE8, TU111, NG111) that harbored free mF plasmids. These strains were mated with the mF^– strain KM88. Of the three mF^– × mF⁺ crosses, only KM88 × JE8 displayed complete uniparental inheritance. However, in KM88 × TU111 and KM88 × NG111, the mtDNA of KM88 and mF of TU111 and NG111 were inherited by the plasmodia and showed recombination. For example, although the mtDNA of TU111 was eliminated, the mF of TU111 persisted and became inserted into the mtDNA of KM88, such that recombinant mtDNA represented 80% of the total mtDNA. The parental mitochondria fused to yield giant mitochondria with two or more mitochondrial nucleoids. The mF appears to exchange mitochondria from the recipient (paternal) to the donor (maternal) by promoting mitochondrial fusion.The first two authors have equally contributed to this work     

Plasmid recovery from transformants and the isolation of chromosomal DNA segments improving plasmid replication in Neurospora crassa

Summary The efficient recovery of plasmid DNA from Neurospora crassa transformants is described. Lithium acetate-treated spores were transformed with plasmid DNA and grown in mass in liquid culture. The resulting mycelial growth was harvested and plasmid DNA was extracted and used to transform E. coli to ampicillin resistance. Although at low frequency, routine recovery of plasmid pSD3 which carries the Neurospora qa-2 ⁺ gene and pBR322 sequences has been demonstrated. About 10% of the recovered plasmids carried deletions and transformed Neurospora at a higher frequency. The liquid culture procedure was also used in attempts to isolate autonomously replicating sequences (ars). In order to select for a stable vector which contains an ars sequence, a clone bank containing a selectable marker (qa-2 ⁺) and Neurospora chromosomal BamHI fragments was constructed and used to transform Neurospora. Several plasmid isolates resulting from a screening of the clone bank showed an improvement in the efficiency of recovery from Neurospora transformants. The properties of one such isolated plasmid, pJP102, suggest that it may contain an ars sequence. Some potential applications of these results for cloning in Neurospora and other filamentous fungi are discussed.     

FK506-binding protein of Neurospora crassa (NcFKBP) mediates sensitivity to the immunosuppressant FK506; resistant mutants identify two loci

Summary Growth of Neurospora crassa wild-type is inhibited by micromolar concentrations of the immuno-suppressive macrolide FK506. Spontaneous and induced mutations that confer resistance to FK506 identified two loci, fkr-1 and fkr-2. They map on the right arm of linkage group V on either side of inl with fkr-1 being centromere proximal. Allele fb (fkr-2) lacks immunodetectable N. crassa FK506-binding protein (NcFKBP). This demonstrates that the sensitivity of N. crassa towards FK506 is mediated by NcFKBP. FK506-binding proteins have been shown to be highly conserved, i.e., found in all eukaryotic cells tested, and to exhibit peptidyl-prolyl cis-trans isomerase (PPIase) activity in vitro. Possible functions for the loci are discussed. Apart from the resistance to FK506 no other mutant phenotype was detected not even in double mutants that lacked NcFKBP as well as cyclophilin. Cyclophilin mediates the cytotoxic effect of the immunosuppressive drug Cyclosporin A and is also characterized by PPIase activity in vitro. Both FK506-resistant alleles studied exhibit incomplete dominance in forced heterokaryons. A mechanism is proposed to explain this dominance especially in view of the NcFKBP-deficient allele, fb.     

Homologous linear plasmids in mitochondria of three species of wheat bunt fungi,Tilletia caries,T. laevis and T. controversa

Summary All isolates of Tilletia spp. investigated (five isolates of T. caries, including one from Japan, two isolates of T. laevis, and five isolates of T. controversa) contained a linear DNA plasmid ranging in size from 7.2 to 7.6 kb. All plasmids were highly homologous to each other as shown by DNA-DNA hybridization and comparison of restriction enzyme sites. Variability in the size of the plasmid was found to be due to differences within a central region of the plasmid. No homology between the plasmid and mitochondrial or nuclear DNA was found, but the mitochondrial origin of the plasmid was confirmed.     

Mitochondrial DNA of the filamentous ascomycete Cochliobolus heterostrophus

Summary The mitochondrial chromosome of Cochliobolus heterostrophus is a circle approximately 115 kb in circumference, among the largest known from fungi. A physical map of C. heterostrophus mtDNA was constructed using the restriction enzymes BamHI, EcoRI, and PvulI by DNA-DNA hybridizations with cloned or purified mtDNA BamHI fragments. The following sequences were located on the mtDNA map: (1) the large and small mitochondrial ribosomal RNA genes (identified by heterologous hybridization to cloned Neurospora crassa rRNA genes); (2) the sequence homologous to a mitochondrial plasmid present in one field isolate of C. heterostrophus; and (3) a 1.05 kb EcoRI fragment that functions as an autonomously replicating sequence in Saccharomyces cerevisiae. An examination of mtDNA from 23 isolates of C. heterostrophus collected worldwide revealed polymorphisms in restriction enzyme sites. One such polymorphism, coupled with data on a polymorphism in nuclear rDNA, suggests that there are two genetically distinct but geographically overlapping mating populations of C. heterostrophus in the world.