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     

Structure of a Gelasinospora linear plasmid closely related to the kalilo plasmid of Neurospora intermedia

We have determined the complete nucleotide sequence of a linear mitochondrial plasmid from a natural isolate of a homothallic species ofGelasinospora. The plasmid genome is 8231 by long. It carries terminal inverted repeats of 1137 bp. Extending inwards from the terminal repeats are two long open reading frames coding for putative proteins with similarity to DNA and RNA polymerases. These are separated by a short intergenic region. The plasmid sequence shows remarkable similarity to that of theNeurospora intermedia senescence-plasmid kalilo. Overall the two plasmids have a similar genetic organization and are clearly homologous at the sequence level. The main differences are in the intergenic region and in the terminal repeats.     

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.     

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.     

Heterokaryotic transmission of senescence plasmid DNA in Neurospora

Summary 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.     

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.     

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.     

Distribution of seven homology groups of mitochondrial plasmids in Neurospora: evidence for widespread mobility between species in nature

A survey of mitochondrial DNAs from over 225 Neurospora and related fungal isolates from around the world uncovered three new homology groups of mitochondrial plasmids, two divergent subgroups of the Fiji plasmid family, and extended previous data about plasmid distribution patterns. Newly-discovered circular plasmids, Java and MB1, and the linear Moorea plasmids, were found in relatively-few isolates. A large proportion of isolates (51%) were found to have these or previously-discovered plasmids in the Varkud, kalilo, LaBelle, or Fiji families. Plasmids in most families were found in isolates world-wide and distributed nearly randomly with respect to species. As many as three types of plasmids were found in single isolates, and plasmids typically were found alone or in pairs in a random, independent pattern. The regional clustering of some plasmids was independent of species. providing a strong argument that horizontal transfer of plasmids occurs frequently in nature. Some plasmid families were much more diverse than others. The Fiji plasmids are a superfamily composed of distinct subgroups defined by degrees of cross-hybridization. Between some subgroups there were large regions of non-homology.     

Characterization of inl + transformants of Neurospora crassa obtained with a recombinant cosmid-pool

Summary We constructed a Neurospora crassa gene library in a cosmid vector and used the cosmid-pool DNA to transform an inl, rg Neurospora crassa strain to inositol prototrophy. The inl ⁺ colonies obtained in this experiment proved to be integrative type transformants. Genetic analysis revealed that the integration event occurred at or near the inl locus. In one of the transformants the inl ⁺ trait exhibited mitotic and meiotic instability. In hybridization experiments free plasmids were detected in the F₁ progeny of the transformants. We were able to recover eleven different plasmids from the F₁ progeny of the transformants. None of these plasmids proved to carry a functional copy of the inl ⁺ gene as judged by its transforming ability. Possible explanations for the observed phenomena are discussed.     

Characterization of the termini of linear plasmids from Nectria haematococca and their use in construction of an autonomously replicating transformation vector

Summary The mitochondria of isolate FS37 from Nectria haematococca mating population I (Fusarium solani f. sp. cucurbitae) contain two linear plasmids, pFSCI and pFSC2, of 9.2 and 8.3 kbp, respectively. Evidence for a protein blocking the 5 termini of these plasmids was obtained from exonuclease digestion experiments. A single protein band with an apparent M_r of 80 K was labeled when the DNA-protein complex of either plasmid was reacted with [¹²⁵I] Bolton-Hunter reagent and then digested with DNase I. DNA sequence analysis of the termini of both plasmids revealed long inverted repeats of 1,211 by (pFSC1) and 1,027bp (pFSC2). No sequence similarity was found between the terminal inverted repeats (TIRs) of pFSC1 and pFSC2, nor was any similarity identified between the TIRs of the these plasmids and sequences of TIRs from other linear DNAs. A restriction fragment containing the TIR of pFSCI conferred autonomous replication when incorporated into an integrative transformation vector of Ustilago maydis.     

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.     

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.     

Double or triple sets of replication functions as inverted and direct repeats on in vitro reconstructed streptococcal MLS resistance plasmids

In vitro rearrangement of plasmid pDB102 together with comparative studies of other streptococcal plasmids allowed the localization of replication and copy control functions on sequences which were present on pDB102 and its naturally occurring ancestor pSM19035 as duplicates in inverted orientation. Evidence is presented that neither the presence of duplicate replication regions nor their arrangement in inverted orientation was essential for plasmid survival. Among the in vitro reconstructed plasmids were several that stably carried two or three sets of replication and copy control functions either as inverted or direct repeats or both. A copy control mutation is described which led to a tenfold increase of copy number over that of the naturally occurring plasmid pSM19035.     

Neurospora crassa nuclear genome contains analogy of Saccharomyces cerevisiae genes for ribosomal RNA processing

Summary Neurospora crassa wild type genome shows DNA sequences which are homologous to the sequences present in the rRNA processing genes of the yeast Saccharomyces cerevisiae. Five such processing genes from yeast, viz., RNA1 through RNA5, cloned in plasmid pBR322 were transformed in Escherichia coli strain LE392. Southern blots containing DNAs from these clones were restricted with several restriction endonucleases along with DNAs from lambda phage, rice (plant) and neuroblastoma (animal), were hybridized with ³²P-labelled nick-translated N. crassa nuclear DNA under very stringent conditions. Autoradiograms of these blots revealed that four yeast rRNA processing genes (RNA1, RNA2, RNA3, and RNA4) showed homology with N. crassa nuclear DNA but such analogs were not found in DNAs representing prokaryotes, phages, higher plants and animals.     

Characterization of mutations in the two-component histidine kinase gene AbNIK1 from Alternaria brassicicola that confer high dicarboximide and phenylpyrrole resistance

Highly iprodione- and fludioxonil-resistant field and laboratory isolates of A. brassicicola were found to be either moderately sensitive or tolerant to osmotic stress. AbNIK1, a two-component histidine kinase gene, was isolated from a fungicide-sensitive strain. The predicted protein possessed the six tandem amino acid repeats at the N-terminal end, which is a landmark of osmosensor histidine kinases from filamentous fungi. A comparison of the nucleic acid sequences of the AbNIK1 gene from fungicide-sensitive and fungicide-resistant isolates revealed the presence of mutations in six of the seven resistant strains analyzed. Null mutants were all found to be moderately sensitive to osmotic stress, indicating that they are similar to Neurospora crassa Type I os-1 mutants. Only one mutation, corresponding to a single amino acid change within the H-box of the kinase domain, was found in an osmotolerant strain. These results suggest that AbNIK1p participates in osmoregulation and that expression of the fully functional enzyme is essential for dicarboximide and phenylpyrrole antifungal activities.     

Transformation of Phanerochaete chrysosporium and Neurospora crassa with adenine biosynthetic genes from Schizophyllum commune

Summary Protoplasted basidiospores of two different adenine auxotrophs of the lignin-degrading basidiomycete Phanerochaete chrysosporium were transformed to prototrophy using plasmids containing genes encoding adenine biosynthetic enzymes from Schizophyllum commune. Fragments containing these genes were subcloned into pUC18 and P. chrysosporium transformants obtained with these subclones were analyzed. The subclones were mapped for restriction sites and the approximate locations of the complementing genes were determined. One of these plasmids was used to transform the Neurospora crassa auxotrophic strain ade2, thereby identifying the S. commune ade5 biosynthetic gene as encoding phosphoribosylaminoimidazole synthetase.     

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     

Restricted distribution of the Tad transposon in strains of Neurospora

Summary A simple colony blot procedure was used to screen 336 Neurospora strains for the presence of the transposable element Tad. These strains included the standard laboratory wild types, all of the available Neurospora isolates collected from the Ivory Coast, and all wild-collected Neurospora crassa isolates available from the Fungal Genetics Stock Center. Tad was found only in the strain of origin from Adiopodumé, Ivory Coast, where it is present in multiple copies. Three other strains of African origin were found to have single copy sequences that are related to Tad.     

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.     

Linear extrachromosomal DNA in the morel Morchella conica

Summary Altogether 18 different strains of the genus Morchella were assayed for the presence of extrachromosomal genetic elements. It was shown that 8 out of 13 strains of the Morchella conica group contain plasmids of comparable size (6 kb and 8 kb respectively). The 5 representatives of Morchella esculenta were not found to contain extrachromosomal DNA. The plasmid of one strain (nr. 3) was further analysed. By restriction analyses and electron microscopy it was confirmed that the plasmid is linear having a molecular weight of 6 kb. It was further shown that it carries at both ends inverted repeats of 0.75 kb.