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Genetics, Vol. 175, 785-794, February 2007, Copyright © 2007
doi:10.1534/genetics.106.063081
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,1
* Centre de Génétique Moléculaire, CNRS, 91198 Gif-sur-Yvette Cedex, France and Laboratorium voor Erfelijkheidsleer, Wageningen Universiteit, 6703 BD Wageningen, The Netherlands
1 Corresponding author: Centre de Génétique Moléculaire, Centre National de la Recherche Scientifique, 1 Ave. de la Terrasse, 91198 Gif-sur-Yvette Cedex, France.
E-mail: maas{at}cgm.cnrs-gif.fr
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Senescence in Neurospora can almost invariably be ascribed to the presence of a single mitochondrial plasmid, such as one of the linear invertron-type mitochondrial plasmids pKalilo (BERTRAND et al. 1985, 1986; MYERS et al. 1989) and pMaranhar (COURT et al. 1991) or one of the circular mitochondrial retroplasmids pVarkud and pMauriceville (AKINS et al. 1989). These plasmids are all able to integrate into the mitochondrial genome of their hosts and interfere with mitochondrial function, which is allegedly the cause of death of the cultures that carry them. Often they are found close to or within the genes encoding the mitochondrial rRNAs (BERTRAND et al. 1985; BERTRAND and GRIFFITHS 1989; MYERS et al. 1989; CHIANG et al. 1994; M. F. P. M. MAAS, unpublished results). The invertron-type plasmids allegedly integrate via short DNA sequence homology (SSH) with the mitochondrial target site, whereas the retroplasmids integrate via a reverse transcription step. pVarkud and pMauricevile both contain an open reading frame (ORF) encoding a reverse transcriptase (RT) that can generate hybrid mitochondrial cDNA via RNA template switching. Following hybrid cDNA formation, the latter plasmids may integrate via homologous recombination (CHIANG et al. 1994).
Little is known about suppressors of "plasmid-based" senescence in Neurospora. On the basis of the analogy with senescence in Podospora anserina, one might expect that modifications of the respiratory chain would act as suppressors of senescence (DUFOUR and LARSSON 2004). Thus far, however, there is no evidence to support this. In the case of pKalilo, there are reports of nuclear-encoded suppressors: In octads collected from crosses between senescing and nonsenescing laboratory strains of Neurospora, several cases were found in which senescence and "immortality" segregated in a four-to-four ratio (GRIFFITHS et al. 1992). In one case the titer of pKalilo was reduced to barely detectable levels, whereas in another case the titer of this plasmid was high but stable and associated with copies of the plasmid integrated into the mitochondrial chromosome. These nuclear-encoded suppressors of pKalilo-based senescence were not characterized any further. A search for suppressors among natural Neurospora isolates was purportedly unsuccessful (DEBETS et al. 1995). In the latter search, however, not every pKalilo-carrying isolate senesced, and suppressors may hence have been present.
Recently, we resampled the natural Hawaiian population of Neurospora intermedia (MAAS et al. 2005). Again several pKalilo-carrying isolates did not senesce. We analyzed these isolates. The ability to tolerate the plasmid was maternally inherited, indicating a cytoplasmic origin of this trait. Long-lived strains were characterized by a stable plasmid titer and lacked "de novo" integration events. In at least two of them, stably "integrated" or recombinant plasmid copies were present. Using a "plasmid-tagging" method based on the semirandom two-step (ST–PCR) procedure described by CHUN et al. (1997), we analyzed the sequences flanking these copies. As opposed to those from short-lived isolates, integrated or recombinant copies from long-lived isolates were complete and showed no evidence of a recombination process involving short DNA sequence homology. Additional analysis of the flanking sequences revealed the presence of a cryptic mitochondrial retroplasmid. This element was present in all five suppressor isolates. In at least two of the five, it had recombined with the pKalilo plasmid and the mitochondrial genome, apparently involving a template-switching reverse transcriptase. We discuss a mechanism by which different types of mitochondrial plasmids may interact and their detrimental effect on the host can be suppressed.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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DNA extraction and Southern analysis:
For DNA extraction, cultures were grown in liquid VMM for 
24 hr at 25°. Mycelium was harvested, dried between filter paper, and ground using liquid nitrogen, followed by a standard phenol/chloroform-based DNA extraction (SAMBROOK et al. 1989). Prior to the addition of phenol and chloroform, samples were incubated for 1 hr at 37° with proteinase-K (100 µg/ml final concentration), except those to be used in a ST–PCR (see further on).
For Southern analysis, total DNA was digested using PstI, MvnI, or both, separated, and blotted onto positively charged nylon membranes (SAMBROOK et al. 1989). Blots were probed with the 8.2-kb KpnI fragment of the prototypic pKalilo plasmid (VICKERY and GRIFFITHS 1993), labeled using Digoxigenin-11-dUTP (DIG–dUTP). Detection was done using the chemiluminescent substrate CSPD according to the manufacturer's instructions (Boehringer Mannheim, Indianapolis).
ST–PCR-based amplification of plasmid flanking sequences:
To amplify pKalilo flanking sequences from senescent and long-lived isolates, a plasmid-tagging approach was used on the basis of the ST–PCR procedure given by CHUN et al. (1997). ST–PCR consists of a touchdown PCR to generate fragments incorporating a certain adaptor sequence, followed by a regular PCR to specifically amplify the fragments containing the plasmid flanking sequences. The first step was done using a primer located within the terminal inverted repeat (TIR) region of the plasmid (primer 1.1: 5'-GAAATGATAAAAAGATCACAAAGGG-3'), in combination with the partially degenerate primer given in the original protocol (primer 1.2: 5'-GGCCACGCGTCGGACTAGTAC-N(10)-GATAT-3'). The latter anneals at sites throughout the mitochondrial genome, 300 bp apart. It is distributed uniformly throughout the mitochondrial genome, and thus the method provides an unbiased way of amplifying plasmid flanking sequences. The following step was done using a second primer located within the TIR region of the plasmid (primer 2.1: 5'-GGGTTAAAATAGGAACAAAAGGGG-3'), in combination with a primer comprising the specific part of the partially degenerate primer (primer 2.2: 5'-GGCCACGCGTCGGACTAGTAC-3'). The method is illustrated in Figure 1. Reaction conditions are given in Table 1.
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using the pGEM-T Easy Vector system (Promega, Madison, WI). Plasmid DNA was recovered using a High Pure plasmid isolation kit (Roche) and sequencing was done according to the methods described by SANGER (1977), using Big Dye terminators version 3 (Applied Biosystems, Foster City, CA). We compared the sequences obtained with sequences from the GenBank database using BLAST version 2.2.9 (ALTSCHUL et al. 1997).
Test for the retroelement in other isolates:
To test other N. intermedia wild types for the presence of the novel mitochondrial retroelement, PCRs were done using primers located within the putative RT gene of the element: 5'-TAACTTAACCGCCAACGTAT-3' and 5'-GACCCCTTTCTTCCAGTTTAT-3'. These were based on the sequence derived from isolate NI28.01 and yield a PCR product size of 341 bp.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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The long-lived isolates were reciprocally crossed with short-lived ones from the same collection. Three of five long-lived natural isolates (NI15.02, NI28.01, and NI30.04) produced viable progeny when crossed with a wild-type isolate of the opposite mating type. The other two (NI05.01 and NI31.02) produced viable ascospores only when used as a male. Infertility also commonly occurred in crosses using random combinations of short-lived isolates. It is hence not necessarily associated with the longevity trait, but perhaps due to a general form of sexual incompatibility. Growth rates of short- and long-lived isolates did not significantly differ from one another (ANOVA: F = 1.83, d.f. = 1, P = 0.190).
From each fertile cross two complete, eight-spored asci were collected. Cultures derived from the eight spores of each of these asci were individually tested for senescence by means of serial subculturing. For practical reasons the experiment was terminated after 30 vegetative subcultures (equivalent to 10 weeks) and longevity is here henceforth defined as having a "life span" of >30 subcultures. Results are given in Table 2. Although life span had a clear Mendelian component (e.g., as observed in the second ascus from the cross using NI16.04 as a female and NI30.04 as a male; Table 2), the longevity trait itself (i.e., having a life span of >30 subcultures) showed a clear pattern of maternal inheritance: When a long-lived isolate was used as a female, all eight cultures derived from an ascus were long lived; when the same isolate was used as a male, all eight were short lived. Although isolates NI05.01 and NI31.02 were female sterile, the longevity trait of these two isolates was not inherited paternally. We therefore concluded that the longevity trait was of a cytoplasmic origin.
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Senescence suppressor isolates have stable plasmid titers and are not subject to de novo integration events:
In senescing isolates, pKalilo typically increases in titer and integrates into the mitochondrial genome. In long-lived isolates, however, this was not the case. In long-lived isolates the plasmids neither increased in titer nor integrated de novo into the mitochondrial genome (a typical example is given in Figure 2). In strain NI05.01 we were able to detect, by Southern analysis, also a stably integrated or recombinant copy of the plasmid. This copy was already present in the first subculture and remained stable for at least 30 subcultures. It was also stable in the sexual cycle (and thus cosegregated with the senescence suppression trait). In the four other long-lived strains we were unable to detect, at least by Southern analysis, stably integrated copies. As discussed below, however, this was possible by PCR-based methods, at least in isolate NI28.01. In contrast to senescing isolates, senescence suppressor isolates hence have stable plasmid titers and are not subject to de novo integration events.
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Senescence suppressor isolates NI05.01 and NI28.01 contain hybrid mitochondrial plasmids:
The RFLP data indicated inverted repeats adjacent to the integrated plasmid from NI05.01. ST–PCRs yielded a single product (see Table 3), which is consistent with this. Sequencing revealed that the plasmid from isolate NI05.01 was directly flanked by a short (65 bp) region of mtDNA corresponding to a region between the nd5 and cob genes (corresponding to 32,734–32,798 of the mitochondrial genome sequence of N. crassa strain 74-OR23-1A; Whitehead Institute/MIT Center for Genome Research), directly followed by an unknown sequence containing a large ORF (orf1) encoding a putative reverse transcriptase (Figure 2).
We found similarly organized sequences in isolate NI28.01. Because these were not visible on Southern blots and could be found only using PCR experiments, it must mean that they were present at a substoichiometric level. Like that from NI05.01, the recombinant or integrated pKalilo homolog from isolate NI28.01 was directly flanked by a short region of mtDNA, this time corresponding to a cDNA copy of trna-arg (including the post-transcriptionally added 3'-terminal CCA sequence) followed by the 5' terminus of the rns gene (encoding the small subunit mitochondrial rRNA). These sequences in turn were followed by a sequence as in isolate NI05.01 (see Figure 2). The latter sequence again contained a large ORF encoding a putative reverse transcriptase. Additional PCRs and sequencing showed that the entire molecule mapped as a circular element composed of pKalilo, mtDNA, and a novel element. Derivatives hereof also existed, with the pKalilo sequence nearly entirely deleted (Figure 3).
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The novel element is a cryptic mitochondrial retroplasmid:
The novel element has an AT-biased (60% AT) nucleotide content and is maternally inherited (hence cosegregating with the senescence suppression trait), consistent with its mitochondrial location. Although it does not share significant DNA sequence homology with any currently known element, at the protein level it does: It contains a single large ORF (orf1) putatively encoding a protein with similarity to the RTs of mitochondrial retroplasmids and group II introns. It contains all the conserved domains of the RNA-dependent DNA polymerases (POCH et al. 1989; XIONG and EICKBUSH 1990). The predicted 663-aa-long protein is similar to the RT of the retroplasmid pTHR1 from Trichoderma harzianum (GenBank accession no. AAF89327; ANTAL et al. 2002), with 48% similarity and 35% identity over a length of 395 amino acids (E-value = 4 x 10–44), closely followed by the RTs of the retroplasmids pVarkud from N. intermedia and pMauriceville from N. crassa (GenBank accession nos. AAA70286 and AAA70287, respectively; NARGANG 1986), with 46% similarity and 29% identity over a length of 354 aa for both RTs (E-value = 1 x 10–26). A protein sequence alignment is given in Figure 4.
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300 bp from the proposed 3' transcript terminus and not conserved between the element from NI05.01 and that from NI28.01. Also pTHR1 did not have a clear cloverleaf structure at its 3' terminus (ANTAL et al. 2002). The retroelement thus probably corresponds to a retroplasmid that is similar in structure to the 2.6-kb pTHR1 plasmid. We tested all of our other isolates for the presence of the novel retroelement using PCR. It was detected in all five long-lived isolates, but not in the other pKalilo-carrying isolates. This suggests a meaningful correlation between the presence of the novel element and the suppression of pKalilo. The element was not found in isolates that lacked pKalilo.
Template switching was involved in the formation of the hybrid mitochondrial plasmids:
Retroplasmids are able to incorporate mitochondrial sequences via the formation of hybrid cDNAs. This is also known from retroviruses and certain group II introns (SELLEM et al. 2000) and probably occurs by an erroneous template switching activity of the RT they encode (AKINS et al. 1989; CHIANG et al. 1994; CHIANG and LAMBOWITZ 1997). The incorporated mitochondrial sequences often correspond to mitochondrial tRNAs. This is allegedly so because of the structural similarity between the tRNAs and the 3' end of the retroplasmids' transcripts.
An incorporated copy of the trna-arg gene was found in the element from isolate NI28.01 (see Figure 3). At the 3' border of this copy a CCA trinucleotide sequence was present. This trinucleotide is added post-transcriptionally, and the sequence must hence reflect an in vivo cDNA. Furthermore, the sequence flanking the 3' end of the trna-arg copy corresponded to the exact 5' terminus of the rns gene. This must also reflect an in vivo cDNA. Similarly, in NI05.01 a sequence was found that corresponded to the putative 5' terminus of the cob transcript. The sequence directly bordering the copy of pKalilo (Table 3, case 4) resembles the consensus sequence of mitochondrial 5' transcript termini (KENNEL and LAMBOWITZ 1989; KUBELIK et al. 1990; ARGANOZA and AKINS 1995). Although it is located 431 bp upstream of the 5' terminus of the cob transcript that was originally described by KUBELIK et al. (1990), it may reflect an alternative transcription initiation site.
Altogether, we show that the element found in strains NI05.01 and NI28.01 is a cryptic mitochondrial retroelement similar to the plasmids of the pVarkud group. Its putative template-switching activity probably led to the formation of the hybrid molecules observed in strains NI05.01 and NI28.01.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Although previous work did suggest pleiotropic effects of pKalilo on the reproductive fitness of its host (BOK and GRIFFITHS 2000), we have not been able to reproduce these results using isogenic combinations of isolates with and without pKalilo (M. F. P. M. MAAS, unpublished results). Moreover, also the invertron-type plasmid pAL2-1 from P. anserina shortens life span without any notable beneficial effects on reproduction (MAAS et al. 2004). Thus there appears to be no trade-off between life span and reproduction with regard to plasmid-based senescence, contrary to expectations from evolutionary explanations of aging. Furthermore, plasmid-based senescence does not appear to respond to calorie restriction in a life-span-extending manner as it does in all other aging organisms (MAAS et al. 2004). It is thus probably best seen as a lethal disease. In any case, it is fundamentally different from the senescence phenomenon originally described by RIZET (1953). Therefore also a different mode of suppression may be expected.
We have shown that suppression of pKalilo-based senescence, as it is found in the natural population of N. intermedia from Hawaii, is maternally inherited. Although the suppressors described by GRIFFITHS et al. (1992) showed a clear Mendelian pattern of inheritance, the latter suppressors were derived from laboratory stocks. It is not self-evident that they would be similar to suppressors from natural populations.
We show that the senescence suppression trait is associated with a stable titer of the autonomously replicating plasmid and lack of de novo integration events. This was also the case in the suppressors described by GRIFFITHS et al. (1992). Short-lived isolates typically accumulated variant mtDNA molecules with pKalilo integrated into or close to one of the mitochondrial rRNA genes as was also previously observed (BERTRAND et al. 1985; BERTRAND and GRIFFITHS 1989; MYERS et al. 1989; CHIANG et al. 1994). This was, however, not the case in long-lived isolates. The five isolates that were able to tolerate pKalilo were all coinfected with a novel mitochondrial retroelement. In at least two of them, stable recombinants of the two heterologous plasmids were found, including short intervening stretches of sequence derived from the mitochondrial genome. The latter molecules were probably generated via hybrid cDNA formation by an erroneous template-switching activity of the RT encoded by the novel element: The intervening stretches of mtDNA in the recombinant molecules clearly reflected in vivo cDNA. Recombination via hybrid cDNA formation is also known from retroplasmids of the Varkud group (AKINS et al. 1989; CHIANG et al. 1994; CHIANG and LAMBOWITZ 1997).
Although similar effects of the pTHR1 plasmid from T. harzianum have not been reported (ANTAL et al. 2002), pVarkud and pMauriceville are both associated with a senescence phenotype (AKINS et al. 1989): They integrate into the mitochondrial genome, generating suppressive mtDNA variants that gradually replace the wild-type mitochondrial genome. This presumably causes senescence by interfering with mitochondrial function. The newly discovered retroelement may similarly be an unknown senescence plasmid. Testing this would require an isolate containing the retroelement but devoid of pKalilo. Unfortunately we have not yet obtained such an isolate; of the 64 natural isolates tested only the long-lived pKalilo-carrying isolates contained the element. Since the novel retroelement may be a senescence plasmid itself, our observation is puzzling. The simplest explanation would be that the element is not associated with a virulent phenotype and that this is dominant over the effect of pKalilo. Alternatively, pKalilo may be suppressed by means of interference competition with the novel element. Although parasitic virulence is generally driven by resource competition within hosts (EBERT 1998), interference competition is believed to attenuate virulence rather than enforce it (MASSEY et al. 2004). Competition for resources will generally select for a more rapid exploitation of the host. It will select for higher within-host growth rates and higher virulence. The increase in titer of pKalilo in senescing isolates seems to evidence this. Interference competition on the other hand (e.g., by integrating into the competing plasmid, thus forming a so-called "defective interfering particle"), will lead to slower within-host growth rates and reduced virulence.
It would be interesting to experimentally test this hypothesis, by combining cytoplasms from different isolates carrying structurally similar (homologous) or different (heterologous) types of senescence plasmids (i.e., circular retroplasmids and linear invertron-type plasmids). If virulence suppression by means of interference competition is a naturally important phenomenon, the effects of most potential senescence plasmids would frequently go undetected. Many Neurospora strains contain a mix of mitochondrial plasmids and interference competition between plasmids might be at work in these strains. Heterologous types of plasmids have been found together in a single strain more than once. Interestingly, a strain of N. intermedia has been described by GRIFFITHS and YANG (1995), in which two heterologous types of plasmids had recombined. This strain contained five prominent mitochondrial plasmids: three of the "zhisi" and two of the "harbin" homology group. In one culture of this strain, the levels of the original plasmids dropped to undetectable levels and novel, recombinant plasmids appeared.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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FOOTNOTES |
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Communicating editor: M. S. SACHS
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pKAL, in gray). Note the terminal trinucleotide sequence repeat of the novel element (underlined).