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What Triggers Senescence in Podospora anserina?
Fungal Genetics and Biology 27, 26–35 (1999) Article ID fgbi.1999.1127, available online at http://www.idealibrary.com on
What Triggers Senescence in Podospora anserina?
Corinne Jamet-Vierny,*,1 Miche`le Rossignol,† Vicki Haedens,* and Philippe Silar*,1 *Institut de Ge´ne´tique et Microbiologie, URA 2225, Universite´ de Paris Sud, 91405 Orsay cedex, France; and †Centre de Ge´ne´tique Mole´culaire du C.N.R.S., 91198 Gif sur Yvette cedex, France
Accepted for publication March 16, 1999
recently Griffiths (1992), a particular wild-type fungal isolate is defined as aging-prone when all of the cultures issued from it present a progressive loss of growth potential culminating most often in death. Cases of such reproducible aging have been reported in various fungi (Bo¨ckelmann and Esser, 1986; Caten, 1972; Chevaugeon and Digbeu, 1960; Gagny et al., 1997; Jinks, 1959; Rieck et al., 1982; Rizet, 1953b) and are particularly well documented in Neurospora and Podospora genus (Griffiths, 1992). Most wild-type isolates of Neurospora crassa and N. intermedia are immortal. However, some display true and repetitive aging syndromes. In the first two investigated instances (kalilo and maranhar), the aging-prone phenotype and the senescent state are maternally inherited, and the senescent state has been clearly correlated with the insertion of linear plasmids within the mitochondrial DNA (mtDNA) (Bertrand and Griffiths, 1989; Griffiths, 1992). Unlike Neurospora species, all wild-type isolates of Podospora anserina tested until now are prone to the aging phenomenon called senescence (Rizet, 1953a). It has been clearly established that the senescent state is maternally inherited (Marcou, 1961). Molecular studies have shown that cellular degeneration is correlated with a disorganization of the mtDNA leading to the replacement of the intact mtDNA by circular head-to-tail concatemers originating from particular regions of the mtDNA: the senDNAs (Dujon and Belcour, 1989). Based on their structure and replicative properties, two classes of senDNAs can be defined: (1) senDNA␣ corresponds exactly to the first intron of the mitochondrially encoded cox1 gene or intron ␣ (Osiewacz and Esser, 1984). Intron ␣ is present in all wild-type races (Ku¨ck et al., 1985) and senDNA␣ almost
Jamet-Vierny, C., Rossignol, M., Haedens, V., and Silar, P. 1999. What Triggers Senescence in Podospora anserina? Fungal Genetics and Biology 27, 26–35. Senescence of Podospora anserina is triggered by a cytoplasmic and infectious factor (the determinant of senescence) and is always correlated with mitochondrial DNA modifications, especially with the accumulation of small circular subgenomic DNA molecules, the senDNAs. Several observations have suggested that the senDNAs could be the cytoplasmic and infectious determinant. However, we show here (1) that senDNA molecules can be transferred to a young culture without the cotransmission of the determinant of senescence and (2) that the determinant of senescence does not segregate as a mitochondrial DNA mutation. Overall, our data strongly argue that amplification of senDNA molecules in the mitochondria is not an intrinsic property of these small DNA molecules. They question the nature of the actual determinant of senescence. r 1999 Academic Press
Index Descriptors: Podospora anserina; Senescence; determinant of senescence; mitochondrial DNA; genome stability; senDNA. Whereas all superior organisms seem to display an aging process, lower eukaryotes, such as filamentous fungi, may or may not be mortal. Following Rizet (1953b) and more 1
To whom correspondence and reprint requests should be addressed. Fax.: 01 69 16 70 06. E-mail: [email protected] and [email protected].
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Senescence Determinant in P. anserina
systematically appears during senescence (Dujon and Belcour, 1989; Osiewacz, 1992). It does not accumulate by autoreplication but most likely by a retrotransposition mechanism (Jamet-Vierny et al., 1997a; Sainsard-Chanet et al., 1994). (2) SenDNA and ␥ are highly variable in size but contain common core regions (located in an intergenic region downstream from the cox1 gene for senDNA and near the rRNAs region for senDNA␥) (Cummings et al., 1985; Jamet-Vierny et al., 1997b; JametVierny and Shechter, 1994). These senDNAs are frequently found in senescent cultures but their presence is not systematic. It was recently shown that senDNA molecules are endowed with replicative properties (JametVierny et al., 1997a). Therefore, a common feature of aging in these fungi is that mtDNA is drastically altered as degeneration proceeds. From these data, it is thus usually believed that fungal senescence can be accounted for solely by mitochondrial dysfunction resulting from the presence of altered mtDNA molecules. Abnormal molecules may replicate faster and thus replace the wild-type DNA as it was proposed for the yeast ‘‘petite’’ (Dujon and Belcour, 1989) or for some human diseases involving mtDNA alterations (Wallace, 1992). Alternatively, the presence of modified mtDNA molecules can result in the production of a stress signal inducing the crippled mitochondria that contain them to proliferate (Bertrand, 1995). Disappearance of wild-type mtDNA would result in cellular death since these filamentous fungi are obligate aerobes. Early studies have shown that mycelia of any wild-type strain of P. anserina exist in two physiological states: the nondetermined and the determined states. The transition from the first to the second results from the de novo, random and reproducible appearance of a novel cytoplasmic and infectious factor: the determinant of senescence (Marcou, 1961; Smith and Rubenstein, 1973). This factor seems particulate and amplifies exponentially in growing cells. When a certain threshold of determinant is reached, death of the mycelium ensued. Discovery of mitochondrial dysfunction during senescence pointed toward mitochondria as a possible location for the determinant. Currently, senDNAs, especially senDNA␣, are believed to be this determinant of senescence. This is based on several observations. First, the behavior of the senDNAs is consistent with that of the determinant of senescence since their amount increases during the senescence process and they disappear during rejuvenation of the strains (Koll et al., 1984). Second, deletion of the mtDNA regions generating
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senDNAs results in immortality of the strains (Belcour and Vierny, 1986; Koll et al., 1985). Third, as stated above, the senDNA molecules may be endowed with intrinsic invasive properties, which can explain their deleterious effects. However, none of these arguments is conclusive. On the contrary, several data already suggest that senDNA␣ is not the determinant of senescence. No intron ␣ is present in the close relative P. curvicolla, which does display a very similar senescence syndrome (Bo¨ckelmann and Esser,
FIG. 1. Experimental set up of the anastomosis experiments. Small inoculi of a donor senescent mycelium from the S caps1 mat⫺ wild-type strain, which contained a particular senDNA and senDNA␣, were put onto a set of 18 recipient young thallus (all deriving from the same ascospore germination thallus) of the S capr1 mat⫹ strain, which had grown 1.5 cm on MR petri dishes. After the contaminated cultures had grown for a few centimeters, seven explants of each were taken from a small area (R) located 2 cm upright to the donor inoculum and transferred to independent fresh M2 race tubes. The subcultures were then grown, until the appearance of the dark pigmentation characteristic of the senescent state. Simultaneously, 12 noncontaminated control cultures of the S capr1 strain were similarly processed, except that, on average, only 5 subcultures were grown.
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Jamet-Vierny et al.
TABLE 1 Longevity and mtDNA Content of Subcultures Derived from the 18 Contaminated (A) and 12 Control (B) Cultures (A)
Contaminated culture E1
E2
E3
E4
Longevity of the subcultures (cm)
Donor senDNAa
Contaminated culture
18.7 19.5 20.0 20.5 21.6 25.4 29.3
⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹
E7
14.6 15.0 15.2 17.0 17.5 17.9 22.5 7.8 7.8 8.0 9.2 11.4 13.7 17.2 15.3 16.3 17.1 17.1 20.0 20.7 27.1
E5
7.5 11.5 12.8 13.5 16.3 28.2
E6
9.4 11.0 12.5 13.7 19.4 21.1
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Longevity of the subcultures (cm)
Donor senDNAa
Contaminated culture
16.5 17.5 17.8 19.5 19.9 22.5 29.5
⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫹
E 13
E8
12.2 19.8 21.7 22.8 30.2 31.1 32.8
⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹
E 14
9.7 11.5 14.1 16.2 18.5 20.9 22.3
E9
10.8 12.5 12.8 12.8 15.0 16.7 18.5
E 15
16.1 16.9 18.0 20.0 20.5 23.1 27.2
E 10
12.3 12.5 15.0 15.5 18.5 19.9 20.6
E 16
15.7 16.2 16.7 17.5 19.0 22.8 32.0
⫹ ⫺ ⫹ ⫹ ⫹
⫹
⫹ ⫹
⫹ ⫹ ⫹ ⫹
⫹ ⫹ ⫹ ⫹ ⫹
Longevity of the subcultures (cm) 10.1 13.2 14.8 16.6 17.5 18.5 18.7
E 11
7.8 8.9 9.5 10.0 10.5 11.0 15.4
E 17
12.7 14.5 15.0 16.0 16.2 17.7 20.0
E 12
12.0 12.8 13.3 14.2 14.3 16.0 17.2
E 18
11.8 13.0 13.7 14.1 15.7 16.0 19.7 20.6
⫹ ⫹
Donor senDNAa ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹
⫹ ⫹ ⫹ ⫹
⫹ ⫹
⫹ ⫹ ⫹
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Senescence Determinant in P. anserina
Table 1—Continued (B)
Control culture C1
Longevity of the subcultures (cm) 14.4 17.0 18.7 20.1 26.5
Donor senDNAa
Control culture
Longevity of the subcultures (cm)
C7
16.9 22.3 23.3 21.2 21.2 13.0 16.5 22.2 23.0
⫺ ⫺
15.4 17.1 18.5 19.6
⫺ ⫺
C8
C3
15.0 21.7 23.2 25.5
⫺
C9
15.8 17.5 18.0 19.0 19.0
C4
16.0 19.0 21.7 22.0 22.5
C 10
17.3 17.5 19.5 20.0 21.2
C5
16.5 19.0 20.7 22.7 23.5
C 11
18.4 20.5 21.4 22.5
C6
17.0 17.2 19.0 20.0 23.5
C 12
12.0 12.0 12.8 13.1 13.5 14.0 16.6 22.0 22.5
C2
Donor senDNAa ⫺
⫺
a A plus indicates that donor senDNA was detectable in the senescent subcultures, after hybridization of the blot with the appropriate probe. A minus indicates that this senDNA was not detectable by hybridization under the same conditions. No indication is reported when the analysis of the mtDNA content has not been performed.
1986). Intact intron ␣ is present in some immortal mex strains (Koll et al., 1985). SenDNA␣ is present in the permanently nonsenescent double mutant strain i viv (Tudzynski et al., 1982). Senescence can occur sporadically in the absence of senDNA␣ (Dujon and Belcour, 1989). More recently, nuclear mutations that strongly impair senDNA␣ amplification without preventing senescence were obtained (Borghouts et al., 1997; Silar et al., 1997) and mex strains were induced to senescence by the sole
transfer of senDNA molecules through anastomosis (Jamet-Vierny et al., 1997a). In this paper, we show that senDNAs can be efficiently transmitted via anastomoses (cytoduction) to recipient mycelia, without the cotransfer of the determinant of senescence. Meiotic analysis shows that a mitochondrial mutation and the determinant of senescence do not segregate in a similar fashion. These data question the actual nature of the determinant of senescence.
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MATERIALS AND METHODS Media, Fungal Strains, and Lifespan Measurements Culture conditions and genetics methods are described in Esser (1974). The strains used derived from the S strain of Podospora anserina (Rizet, 1952). capr1 is a mitochondrial mutation that promotes a resistance to chloramphenicol and an increased longevity (Belcour and Begel 1977, 1980). Longevity of a subculture is defined as the distance covered on M2 medium by the mycelium between the point where the spore germinates (or where the explant is reinoculated) and the point where the apical cells die. The lifespan of a strain is defined as the mean longevity of parallel cultures. On M2 medium and under our race tube conditions, the S capr 1 strain has a lifespan of 15.1 ⫾ 2.1 cm, and the S caps1 wild-type strain has a lifespan of 9.5 ⫾ 1.0 cm.
Nucleic Acid Manipulation DNA analysis experiments were performed by standard methods (Ausubel et al., 1987). MtDNA was extracted using the miniprep procedure (Lecellier and Silar, 1994). The cloned monomer of senDNA and of senDNA␣ used as hybridization probes have been described (ViernyJamet, 1988).
Anastomosis Experiments The protocol, called contamination, was designed by Belcour (Sellem et al., 1996) to introduce mitochondrial mobile sequences from a donor mycelium into a recipient. In our case, a small inoculum of the donor senescent mycelium was put onto the recipient young thallus that had grown a few centimeters on an MR petri dish. This inoculum was taken from a senescent S caps1 mat⫺ culture about 1 to 0.5 cm before its arrest of growth. It contained large amounts of a senDNA, whose monomer was well characterized, and large amounts of senDNA␣. It was placed on the growing front of the recipient S capr1 mat⫹ thallus. Under these conditions, local anastomoses occur. They permit the exchange of cytoplasm but do not allow the propagation of the nuclei from the donor into the recipient. Indeed, to ensure that the mycelium, which grew after contamination, was not colonized by donor nuclei, we checked that it did exclusively conserve nuclei with the mat⫹ mating type of the S capR1 mat⫹ recipient strain. Contamination plates contained the MR medium
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Jamet-Vierny et al.
since the frequence of spontaneous appearance of the determinant of senescence is lower on this medium than on M2, as the longevity of wild-type is much increased on MR (Coppin et al., 1993) when compared to M2 (Silar and Picard, 1994). Longevity of the contaminated cultures was subsequently performed on M2 medium.
RESULTS The Mere Presence of senDNAs Is Not Sufficient to Commit the Mycelium for Senescence During previous anastomosis experiments between young and senescent strains, which were performed to follow the modalities of senDNAs transmission (Jamet-Vierny et al., 1997a), we observed a provocative phenomenon. While in most cases, the determinant of senescence was transmitted with the senDNAs, in some other cases we noticed that transmission of senDNAs was not associated with transmission of the determinant of senescence. To confirm these data, a specially designed anastomosis experiment was carried out as summarized in Fig. 1. Briefly, a senescent S caps 1 mat⫺ donor culture was used to contaminate an undetermined S capr1 mat⫹ recipient. Afterward, we investigated (1) the presence or absence of the determinant of senescence by measuring the longevity of the contaminated mycelia with explants taken 2 cm downstream of the contamination point (in area R) and (2) the senDNA content of the contaminated recipient mycelium. Control plates were processed in a similar way, except that no contaminating explant was added. The results are presented in Table 1. They were used to establish two survival curves according to Marcou (1961), one corresponding to the contaminated cultures and the other to the control cultures (Fig. 2). In this representation, the logarithm of the percentages of surviving subcultures is plotted as a function of growth length. The curves have a characteristic shape: a plateau, which corresponds to the shortest distance grown by all subcultures before exhibiting the senescent phenotype, and thereafter an exponential decrease of the percentage of surviving subcultures. Such a profile signifies that senescence results from the random but reproducible appearance of a factor called the determinant of senescence (Marcou, 1961). The plateau represents the distance of growth, which is required to reach the final phase of senescence after the determinant appeared (or incubation distance). The slope of the
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Senescence Determinant in P. anserina
FIG. 2. Lifespan curves of the contaminated and control cultures. The data are reported according to Marcou (1961) as the logarithm of the percentage of living subcultures with respect to growth length. Survivors are plotted every centimeter. Extensive data are reported in Table 1.
curve is indicative of the frequency of the determinant appearance. These curves give two important informations. First, under the conditions of the experiment, the incubation distance for the S capr 1 mat⫹ control cultures is estimated to about 12.5 cm. Therefore, we can conclude that all the contaminated cultures that have grown a distance higher than 12.5 cm (79% of the cultures) did not receive the determinant of senescence at the time of contamination. Second, the slopes of the curves are nearly identical, but incubation distance is slightly shorter for the experimental curve. These slopes indicate that the frequency of appearance of the determinant is roughly the same in the contaminated and control cultures. The shortened incubation distance indicates that the time necessary between determination and the expression of senescence is slightly reduced in the experimental cultures compared to that in the control cultures. MtDNA of some contaminated cultures was extracted at two stages of growth, first from explants taken on the petri dishes in area R located close to the contamination point and second from explants taken in the race tubes, 1 cm before the arrested edge of the senescent culture (see Fig. 1). The restriction and hybridization pattern of this mtDNA was then compared with that of the donor mtDNA. The mtDNA obtained from area R was in all cases very similar
to that present in a young control culture. On the other hand, the mtDNA recovered from senescent subcultures in race tubes displayed a high quantity of senDNA molecules identical to the ones found in the senescent donor (Table 1 and Fig. 3A). SenDNA are highly variable and it is unusual to recover identical monomers from independent cultures. It is therefore most likely that the observed senDNA derived from the donor. Confirming this, the analyzed senescent control cultures display variable senDNAs, all different from the donor senDNA (Fig. 3B). In addition to senDNA, the senescent donor contained a high quantity of senDNA␣. The HaeIII restriction pattern of the mtDNA reveals variable quantities of this senDNA in the senescent contaminated cultures (Fig. 3A). These molecules could result either from transmission from the donor of minute amounts of senDNA␣ during contamination and/or from de novo generation in the recipient as we previously showed (Jamet-Vierny et al., 1997a). Noteworthy, most of the contaminated senescent subcultures analyzed had grown more than 12.5 cm and therefore were not determined for senescence at the point of contamination.
The Determinant of Senescence and the capr1 Mitochondrial Mutation Display Different Meiotic Segregation Modalities In previous reports, it was observed that the determinant of senescence and the capr1 mitochondrial mutation display different meiotic behaviors. Indeed, in crosses between the capr1 and caps1 strains, the ascospores produced by a single perithecium are either all resistant to chloramphenicol or all sensitive; no ascospores that carry both alleles are observed (Belcour and Begel, 1977). On the contrary, it is possible to find ascospores determined and nondetermined for senescence within the same perithecium in a cross between a senescent and a young culture (Marcou, 1961). We thus decided to repeat these two experiments in a single step by crossing a young S capr1 mat⫹ culture with a senescent S caps1 mat⫺ culture. As a control, the young S capr1 mat⫹ culture was also crossed with the young S caps1 mat⫺ culture from which the senescent culture was obtained. To this end, hyphae from both partners were loosely ground together and inoculated on fresh medium. Self-fertile cultures were obtained consisting of an heterocaryotic heteroplasmic capr1 mat⫹/caps1 mat⫺ that contain the determinant of senescence in the experimental cross and that do not contain the determinant in the control cross. From each culture, eight perithecia were randomly picked up and for
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Jamet-Vierny et al.
FIG. 3. Restriction and hybridization patterns of some of the contaminated (A) and control (B) subcultures. (Left) Ethidium-stained gels of the HaeIII-digested mtDNA from senescent subcultures issued from contamination E14 (reported in Table 1A) (A) and from some of the control cultures (C1, C2, C3, C7, and C8, reported in Table 1B) (B). (Right) Results of the hybridization with a probe that specifically hydridizes with the region that is common to all senDNA and that is included in fragment HaeIII n° 16 (2.1 kb). The monomer of the particular senDNA present in the senescent culture used as donor contains this common region and a large part of the adjacent HaeIII fragment n°1. This monomer has only one HaeIII site and after digestion of the mtDNA, it appears as a single fragment of about 8.8 kb. Consequently, the probe hybridizes specifically with this 8.8-kb fragment (and, as seen in Fig. 3B, lane C7: 16.9 cm, with any senDNA that possesses a similar organization). CC, mtDNA extracted from area R (see Fig. 1). Donor, mtDNA from the donor senescent culture. YC, mtDNA extracted from a young control culture. HaeIII 16, position of fragment HaeIII n° 16. ␣, monomer of senDNA␣ (2.5 kb). The longevity of each culture is indicated. In (A) two identical mtDNA samples of the E14 subculture with a longevity of 20.9 cm are present on the gel.
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Senescence Determinant in P. anserina
appearance and spreading of a cytoplasmic and infectious factor (Marcou, 1961; Smith and Rubenstein, 1973). Death is always correlated with the presence of particular defective mtDNA molecules (Dujon and Belcour, 1989). Because these molecules are constantly present in senescent mycelia and may have invasive properties (see the Introduction), they are potential candidates to be the infectious factor. We show here that senDNA␣ or  can be transmitted to nondetermined mycelia without the transfer of the determinant of senescence (in 49 of the 52 tested cultures), confirming our previous observations (Jamet-Vierny et al., 1997a). Noteworthy, during the experiment, we observed that contamination with the senDNAs did not seem to significantly affect the probability of the appearance of the determinant, suggesting that presence of a low amount of senDNAs is not sufficient to commit the mycelium to senescence. However, the incubation distance is slightly diminished. This indicates, as already postulated (JametVierny et al., 1997a), that senDNA amplification, at the expense of intact mtDNA, participates in cellular death. The contamination clearly supplies the cells with senDNAs, and this likely decreases the time required to reach a lethal level of senDNA. We also show that the determinant of senescence and the capr1 mitochondrial mutation display different meiotic segregation modalities. Whereas all the ascospores of a given perithecium are homogenous for either capr1 or caps1, these are heterogenous for the presence of the determinant of senescence. These data confirm the fact that very few molecules of mtDNA (possibly one), and thus very few mitochondria, are used during perithecium formation (Belcour and Begel, 1977). In view of all these observations and the previous uncertainties about the true molecular nature of the
each, 13 to 24 ascopores were checked (1) for the presence of the capr1 or caps1 allele and (2) for their determined versus nondetermined status. Due to the large amount of cultures generated in this experiment, the presence or absence of the determinant of senescence was ascertained by inoculating a single culture per ascospore in M2 medium race tubes. Because this method is not very accurate, very stringent criteria were used to differentiate between nondetermined and determined ascospores. Cultures with longevity inferior to 80% of the incubation distance (10.0 cm for capr strains and 4.5 cm for caps strains) were considered issued from a determined ascospore. Those with a longevity superior to 130% of the incubation distance (16.0 cm for capr strains and 6.0 cm for capS strains) were considered issued from a nondetermined ascospore. Ascopores that generate cultures whose longevity fell between these two values were considered not ascertained. The results of this experiment are summarized in Table 2. As previously reported, perithecia were homogenous with respect to the capr1 mutation since all ascopores from one perithecium were either all resistant or all sensitive to chloramphenicol. On the contrary, the determinant of senescence was transmitted in a very heterogeneous fashion in the experimental cross since seven of the eight tested perithecia contained a mixture of determined and nondetermined ascospores.
DISCUSSION All wild-type strains of Podospora anserina examined so far present a degenerative process that is controlled in a constant way. This senescence process is triggered by the
TABLE 2 Meiotic Behavior of the Determinant of Senescence and the capr1 Mitochondrial Mutation Young S capr1 mat⫹ ⫻ young S capS1 mat⫺
Young S capr1 mat⫹ ⫻ senescent S capS1 mat⫺
Perithecium
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
capS capr
21 0
23 0
20 0
23 0
24 0
19 0
24 0
23 0
0 13
0 19
0 18
0 21
0 18
0 20
0 23
22 0
Det⫹ Det⫺ ?
0 21 0
0 23 0
0 20 0
0 23 0
0 24 0
0 19 0
0 23 1
0 23 0
2 8 3
15 0 4
5 10 3
5 12 4
2 15 1
2 15 3
2 17 4
10 10 2
Note. For the eight perithecia from each cross, the table gives the number of ascospores found to be capS or capr and their nondetermined (Det⫺) or determined (Det⫹) status. ? Not ascertainable (see text).
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determinant of senescence (Griffiths, 1992), two alternative hypotheses can be proposed. The determinant of senescence could be a sufficient level of senDNAs. Once a threshold of senDNA is reached, a physiological cellular change would trigger amplification as proposed by Bertrand (1995). However, this hypothesis is not very consistent with the heterogenous segregation of the determinant of senescence observed in meiosis. Indeed, we would expect that the determinant of senescence, if it is associated with senDNA that are located within the mitochondria, would segregate in a fashion similar to a mitochondrial mutation. We thus favor the hypothesis that an additional cytoplasmic and infectious factor is required to permit the senDNAs amplification. According to this proposition and contrary to the previous models, amplification of mtDNA deleted molecules would not be the etiology of senescence but a mere symptom. Nonetheless, it would be an important symptom since it is likely to be the actual reason for cellular death. In an overlooked report, Raynal (1980) related experiments that were based on a different rational but which led to the same conclusion. To date, we have no ideas about the molecular nature of the determinant. Recently, we have proposed that the determinant of senescence acts on the amplification of senDNAs through modifications in the expression of nuclear encoded proteins, possibly at the translational level (Silar et al., 1997). These mediator proteins would be imported in mitochondria and alter the maintenance of the mitochondrial genome. Involvement of the mitochondrial import machinery in modulating the mitochondrial DNA maintenance of P. anserina was recently shown (JametVierny et al., 1997c). Our proposition is not unconventional. Indeed, Griffiths and Yang (1993) have recently described several cases of aging in geographical isolates of Neurospora intermedia. In some instances, nuclear determination is suspected but tetrad analysis does not support any simple model. In addition, in some of these cases, aging does not seem to involve clear mitochondrial dysfunction. Similarly, we have recently reported a new degenerative syndrome in Podospora anserina. This syndrome is also triggered by a cytoplasmic and infectious element, which is not connected with mitochondrial dysfunction (Silar et al., 1999).
ACKNOWLEDGMENTS We thank M. J. Daboussi, M. Picard, and H. Lalucque for useful discussions. This work was supported by grants from Universite´ de Paris
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Jamet-Vierny et al.
XI, Centre National de la Recherche Scientifique, Association Francaise contre les Myopathies, and Fondation pour la Recherche Me´dicale.
REFERENCES Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Strul, K. 1987. Current Protocoles in Molecular Biology. Wiley Interscience, New York. Belcour, L., and Begel, O. 1977. Mitochondrial genes in Podospora anserina: Recombination and linkage. Mol. Gen. Genet. 153: 11–21. Belcour, L., and Begel, O. 1980. Life-span and senescence in Podospora anserina: Effect of mitochondrial genes and functions. J. Gen. Microbiol. 119: 505–515. Belcour, L., and Vierny, C. 1986. Variable DNA-splicing sites of a mitochondrial intron: Relationship to the senescence process in Podospora. EMBO J. 5: 609–614. Bertrand, H. 1995. Senescence is coupled to induction of an oxidative phosphorylation stress response by mitochondrial DNA mutations in Neurospora. Can. J. Bot. (Suppl. 1): S198–S204. Bertrand, H., and Griffiths, A. J. F. 1989. Linear plasmids that integrate into mitochondrial DNA in Neurospora. Genome 31: 155–159. Bo¨ckelmann, B., and Esser, K. 1986. Plasmids of mitochondrial origin in senescent mycelium of Podospora curvicolla. Curr. Genet. 10: 803– 810. Borghouts, C., Kimpel, E., and Osiewacz, H. D. 1997. Mitochondrial DNA rearrangements of Podospora anserina are under the control of the nuclear gene grisea. Proc. Natl. Acad. Sci. USA 94: 10768–10773. Caten, C. E. 1972. Vegetative incompatibility and cytoplasmic infection in fungi. J. Gen. Microbiol. 72: 221–229. Chevaugeon, J., and Digbeu, S. 1960. Un second facteur cytoplasmique infectant chez le Pestallozzia annulata. C. R. Acad. Sci. Paris 251: 3043–3045. Coppin, E., Arnaise, S., Contamine, V., and Picard, M. 1993. Deletion of the mating type sequences in Podospora anserina abolishes mating without affecting vegetative function and sexual differentiation. Mol. Gen. Genet. 241: 409–414. Cummings, D. J., MacNeil, I. A., Domenico, I., and Matsuura, E. T. 1985. Excision-amplification of mitochondrial DNA during senescence in Podospora anserina. DNA sequence analysis of three unique plasmids. J. Mol. Biol. 185: 659–680. Dujon, B., and Belcour, L. 1989. Mitochondrial DNA instabilities and rearrangements in yeasts and fungi. In Mobile DNA. (D. E. Berg and M. M. Howe, Eds.), pp. 861–878. American Society for Microbiology, Washington, DC. Esser, K. 1974. Podospora anserina. In Handbook of Genetics (R. C. King, Ed.), pp. 531–551, Vol. 1., Plenum, New York. Gagny, B., Rossignol, M., and Silar, P. 1997. Cloning, sequencing, and transgenic expression of Podospora curvicolla and Sordaria macrospora eEF1A genes: Relationship between cytosolic translation and longevity in Filamentous fungi. Fung. Genet. Biol. 22: 191–198. Griffiths, A. J. F. 1992. Fungal senescence. Annu. Rev. Genet. 26: 351–372. Griffiths, A. J. F., and Yang, X. 1993. Senescence in natural population of Neurospora intermedia. Mycol. Res. 97: 1379–1387.
Senescence Determinant in P. anserina
Jamet-Vierny, C., Boulay, J., Begel, O., and Silar, P. 1997a. Contribution of various classes of defective mitochondrial DNA molecules to senescence in Podospora anserina. Curr. Genet. 31: 171–178. Jamet-Vierny, C., Boulay, J., and Briand, J. F. 1997b. Intamolecular crossing-overs generate deleted mitochondrial DNA molecules in Podospora anserina. Curr. Genet. 31: 162–170. Jamet-Vierny, C., Contamine, V., Boulay, J., Zickler, D., and Picard, M. 1997c. Mutations in genes encoding the mitochondrial outer membrane proteins Tom70 and Mdm10 of Podospora anserina modify the spectrum of mitochondrial DNA rearrangements associated with cellular death. Mol. Cell. Biol. 17: 6359–6366. Jamet-Vierny, C., and Shechter, E. 1994. Senescence-specific mitochondrial DNA molecules in P. anserina: Evidence for transcription and normal processing of the introns. Curr. Genet. 25: 538–544. Jinks, J. L. 1959. Lethal suppressive cytoplasms in aged clones of Aspergillus glaucus. J. Gen. Microbiol. 21: 397–409. Koll, F., Begel, O., Keller, A. M., Vierny, C., and Belcour, L. 1984. Ethidium bromide rejuvenation of senescent cultures of Podospora anserina: Loss of senescence-specific DNA and recovery of normal mitochondrial DNA. Curr. Genet. 8: 127–134. Koll, F., Belcour, L., and Vierny, C. 1985. A 1100-bp sequence of mitochondrial DNA is involved in senescence process in Podospora: Study of senescent and mutant cultures. Plasmid 14: 106–117. Ku¨ck, U., Kappelhoff, B., and Esser, K. 1985. Despite mtDNA polymorphism the mobile intron (plDNA) of the CO1 gene is present in ten different races of Podospora anserina. Curr. Genet. 10: 59–67. Lecellier, G., and Silar, P. 1994. Rapid methods for nucleic acids extraction from Petri dish-grown mycelia. Curr. Genet. 25: 122–123. Marcou, D. 1961. Notion de longe´vite´ et nature cytoplasmique du de´terminant de se´nescence chez quelques champignons. Ann. Sci. Natur. Bot. 11: 653–764. Osiewacz, H. D. 1992. Genetic control of aging in the ascomycete Podospora anserina. In Biology of Aging (Zwilling and Balduini, Eds.), pp. 153–164. Springer-Verlag, Berlin, Heidelberg. Osiewacz, H. D., and Esser, K. 1984. The mitochondrial plasmid of Podospora anserina: A mobile intron of a mitochondrial gene. Curr. Genet. 8: 299–305. Raynal, A. 1980. Relationships between mitochondria and longevity determinants in the ascomycete Podospora anserina. Exp. Mycol. 4: 48–55.
35 Rieck, A., Griffiths, A. J. F., and Bertrand, H. 1982. Mitochondrial variants of Neurospora intermedia from nature. Can. J. Genet. Cytol. 24: 741–759. Rizet, G. 1952. Les phe´nome`nes de barrages chez Podospora anserina. I Analyse ge´ne´tique des barrages entre souches S et s. Rev. Cytol. Biol. Veget. 13: 51–92. Rizet, G. 1953a. Sur la longe´vite´ des souches de Podospora anserina. C. R. Acad. Sci. Paris 237: 1106–1109. Rizet, G. 1953b. Sur l’impossibilite´ d’obtenir la multiplication ve´ge´tative ininterrompue et illimite´e de l’ascomyce`te Podospora anserina. C. R. Acad. Sci. Paris 237: 838–840. Sainsard-Chanet, A., Begel, O., and Belcour, L. 1994. DNA doublestrand break in vivo at the 38 extremity of exons located upstream of group II introns. Senescence and circular DNA introns in Podospora mitochondria. J. Mol. Biol. 242: 630–643. Sellem, C. H., d’Aubenton-Carafa, Y., Rossignol, M., and Belcour, L. 1996. Mitochondrial intronic ORFs in Podospora: Mobility and consecutive exonic sequence variations. Genetics 143: 777–788. Silar, P., Haedens, V., Rossignol, M., and Lalucque, H. 1999. Propagation of a novel cytoplasmic infectious and deleterious determinant is controlled by translational accuracy in Podospora anserina. Genetics 151: 87–95. Silar, P., Koll, F., and Rossignol, M. 1997. Cytosolic ribosomal mutations that abolish accumulation of circular intron in the mitochondria without preventing Senescence of Podospora anserina. Genetics 145: 697–705. Silar, P., and Picard, M. 1994. Increased longevity of EF-1␣ high fidelity mutants in Podospora anserina. J. Mol. Biol. 235: 231–236. Smith, J. R., and Rubenstein, I. 1973. The development of ‘‘Senescence’’ in Podospora anserina. J. Gen. Microbiol. 76: 283–296. Tudzynski, P., Stahl, U., and Esser, K. 1982. Development of a eukaryotic cloning system in Podospora anserina. I. Long-lived mutants as potential recipients. Curr. Genet. 6: 219–222. Vierny-Jamet, C. 1988. Senescence in Podospora anserina: A possible role for nucleic acid interacting proteins suggested by the sequence analysis of a mitochondrial DNA region specifically amplified in senescent cultures. Gene 74: 387–398. Wallace, D. C. 1992. Mitochondrial genetics: A paradigm for aging and degenerative diseases. Science 256: 628–632.
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What Triggers Senescence in Podospora anserina?
Corinne Jamet-Vierny,*,1 Miche`le Rossignol,† Vicki Haedens,* and Philippe Silar*,1 *Institut de Ge´ne´tique et Microbiologie, URA 2225, Universite´ de Paris Sud, 91405 Orsay cedex, France; and †Centre de Ge´ne´tique Mole´culaire du C.N.R.S., 91198 Gif sur Yvette cedex, France
Accepted for publication March 16, 1999
recently Griffiths (1992), a particular wild-type fungal isolate is defined as aging-prone when all of the cultures issued from it present a progressive loss of growth potential culminating most often in death. Cases of such reproducible aging have been reported in various fungi (Bo¨ckelmann and Esser, 1986; Caten, 1972; Chevaugeon and Digbeu, 1960; Gagny et al., 1997; Jinks, 1959; Rieck et al., 1982; Rizet, 1953b) and are particularly well documented in Neurospora and Podospora genus (Griffiths, 1992). Most wild-type isolates of Neurospora crassa and N. intermedia are immortal. However, some display true and repetitive aging syndromes. In the first two investigated instances (kalilo and maranhar), the aging-prone phenotype and the senescent state are maternally inherited, and the senescent state has been clearly correlated with the insertion of linear plasmids within the mitochondrial DNA (mtDNA) (Bertrand and Griffiths, 1989; Griffiths, 1992). Unlike Neurospora species, all wild-type isolates of Podospora anserina tested until now are prone to the aging phenomenon called senescence (Rizet, 1953a). It has been clearly established that the senescent state is maternally inherited (Marcou, 1961). Molecular studies have shown that cellular degeneration is correlated with a disorganization of the mtDNA leading to the replacement of the intact mtDNA by circular head-to-tail concatemers originating from particular regions of the mtDNA: the senDNAs (Dujon and Belcour, 1989). Based on their structure and replicative properties, two classes of senDNAs can be defined: (1) senDNA␣ corresponds exactly to the first intron of the mitochondrially encoded cox1 gene or intron ␣ (Osiewacz and Esser, 1984). Intron ␣ is present in all wild-type races (Ku¨ck et al., 1985) and senDNA␣ almost
Jamet-Vierny, C., Rossignol, M., Haedens, V., and Silar, P. 1999. What Triggers Senescence in Podospora anserina? Fungal Genetics and Biology 27, 26–35. Senescence of Podospora anserina is triggered by a cytoplasmic and infectious factor (the determinant of senescence) and is always correlated with mitochondrial DNA modifications, especially with the accumulation of small circular subgenomic DNA molecules, the senDNAs. Several observations have suggested that the senDNAs could be the cytoplasmic and infectious determinant. However, we show here (1) that senDNA molecules can be transferred to a young culture without the cotransmission of the determinant of senescence and (2) that the determinant of senescence does not segregate as a mitochondrial DNA mutation. Overall, our data strongly argue that amplification of senDNA molecules in the mitochondria is not an intrinsic property of these small DNA molecules. They question the nature of the actual determinant of senescence. r 1999 Academic Press
Index Descriptors: Podospora anserina; Senescence; determinant of senescence; mitochondrial DNA; genome stability; senDNA. Whereas all superior organisms seem to display an aging process, lower eukaryotes, such as filamentous fungi, may or may not be mortal. Following Rizet (1953b) and more 1
To whom correspondence and reprint requests should be addressed. Fax.: 01 69 16 70 06. E-mail: [email protected] and [email protected].
26
1087-1845/99 $30.00 Copyright r 1999 by Academic Press All rights of reproduction in any form reserved.
Senescence Determinant in P. anserina
systematically appears during senescence (Dujon and Belcour, 1989; Osiewacz, 1992). It does not accumulate by autoreplication but most likely by a retrotransposition mechanism (Jamet-Vierny et al., 1997a; Sainsard-Chanet et al., 1994). (2) SenDNA and ␥ are highly variable in size but contain common core regions (located in an intergenic region downstream from the cox1 gene for senDNA and near the rRNAs region for senDNA␥) (Cummings et al., 1985; Jamet-Vierny et al., 1997b; JametVierny and Shechter, 1994). These senDNAs are frequently found in senescent cultures but their presence is not systematic. It was recently shown that senDNA molecules are endowed with replicative properties (JametVierny et al., 1997a). Therefore, a common feature of aging in these fungi is that mtDNA is drastically altered as degeneration proceeds. From these data, it is thus usually believed that fungal senescence can be accounted for solely by mitochondrial dysfunction resulting from the presence of altered mtDNA molecules. Abnormal molecules may replicate faster and thus replace the wild-type DNA as it was proposed for the yeast ‘‘petite’’ (Dujon and Belcour, 1989) or for some human diseases involving mtDNA alterations (Wallace, 1992). Alternatively, the presence of modified mtDNA molecules can result in the production of a stress signal inducing the crippled mitochondria that contain them to proliferate (Bertrand, 1995). Disappearance of wild-type mtDNA would result in cellular death since these filamentous fungi are obligate aerobes. Early studies have shown that mycelia of any wild-type strain of P. anserina exist in two physiological states: the nondetermined and the determined states. The transition from the first to the second results from the de novo, random and reproducible appearance of a novel cytoplasmic and infectious factor: the determinant of senescence (Marcou, 1961; Smith and Rubenstein, 1973). This factor seems particulate and amplifies exponentially in growing cells. When a certain threshold of determinant is reached, death of the mycelium ensued. Discovery of mitochondrial dysfunction during senescence pointed toward mitochondria as a possible location for the determinant. Currently, senDNAs, especially senDNA␣, are believed to be this determinant of senescence. This is based on several observations. First, the behavior of the senDNAs is consistent with that of the determinant of senescence since their amount increases during the senescence process and they disappear during rejuvenation of the strains (Koll et al., 1984). Second, deletion of the mtDNA regions generating
27
senDNAs results in immortality of the strains (Belcour and Vierny, 1986; Koll et al., 1985). Third, as stated above, the senDNA molecules may be endowed with intrinsic invasive properties, which can explain their deleterious effects. However, none of these arguments is conclusive. On the contrary, several data already suggest that senDNA␣ is not the determinant of senescence. No intron ␣ is present in the close relative P. curvicolla, which does display a very similar senescence syndrome (Bo¨ckelmann and Esser,
FIG. 1. Experimental set up of the anastomosis experiments. Small inoculi of a donor senescent mycelium from the S caps1 mat⫺ wild-type strain, which contained a particular senDNA and senDNA␣, were put onto a set of 18 recipient young thallus (all deriving from the same ascospore germination thallus) of the S capr1 mat⫹ strain, which had grown 1.5 cm on MR petri dishes. After the contaminated cultures had grown for a few centimeters, seven explants of each were taken from a small area (R) located 2 cm upright to the donor inoculum and transferred to independent fresh M2 race tubes. The subcultures were then grown, until the appearance of the dark pigmentation characteristic of the senescent state. Simultaneously, 12 noncontaminated control cultures of the S capr1 strain were similarly processed, except that, on average, only 5 subcultures were grown.
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28
Jamet-Vierny et al.
TABLE 1 Longevity and mtDNA Content of Subcultures Derived from the 18 Contaminated (A) and 12 Control (B) Cultures (A)
Contaminated culture E1
E2
E3
E4
Longevity of the subcultures (cm)
Donor senDNAa
Contaminated culture
18.7 19.5 20.0 20.5 21.6 25.4 29.3
⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹
E7
14.6 15.0 15.2 17.0 17.5 17.9 22.5 7.8 7.8 8.0 9.2 11.4 13.7 17.2 15.3 16.3 17.1 17.1 20.0 20.7 27.1
E5
7.5 11.5 12.8 13.5 16.3 28.2
E6
9.4 11.0 12.5 13.7 19.4 21.1
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Longevity of the subcultures (cm)
Donor senDNAa
Contaminated culture
16.5 17.5 17.8 19.5 19.9 22.5 29.5
⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫹
E 13
E8
12.2 19.8 21.7 22.8 30.2 31.1 32.8
⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹
E 14
9.7 11.5 14.1 16.2 18.5 20.9 22.3
E9
10.8 12.5 12.8 12.8 15.0 16.7 18.5
E 15
16.1 16.9 18.0 20.0 20.5 23.1 27.2
E 10
12.3 12.5 15.0 15.5 18.5 19.9 20.6
E 16
15.7 16.2 16.7 17.5 19.0 22.8 32.0
⫹ ⫺ ⫹ ⫹ ⫹
⫹
⫹ ⫹
⫹ ⫹ ⫹ ⫹
⫹ ⫹ ⫹ ⫹ ⫹
Longevity of the subcultures (cm) 10.1 13.2 14.8 16.6 17.5 18.5 18.7
E 11
7.8 8.9 9.5 10.0 10.5 11.0 15.4
E 17
12.7 14.5 15.0 16.0 16.2 17.7 20.0
E 12
12.0 12.8 13.3 14.2 14.3 16.0 17.2
E 18
11.8 13.0 13.7 14.1 15.7 16.0 19.7 20.6
⫹ ⫹
Donor senDNAa ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹
⫹ ⫹ ⫹ ⫹
⫹ ⫹
⫹ ⫹ ⫹
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Senescence Determinant in P. anserina
Table 1—Continued (B)
Control culture C1
Longevity of the subcultures (cm) 14.4 17.0 18.7 20.1 26.5
Donor senDNAa
Control culture
Longevity of the subcultures (cm)
C7
16.9 22.3 23.3 21.2 21.2 13.0 16.5 22.2 23.0
⫺ ⫺
15.4 17.1 18.5 19.6
⫺ ⫺
C8
C3
15.0 21.7 23.2 25.5
⫺
C9
15.8 17.5 18.0 19.0 19.0
C4
16.0 19.0 21.7 22.0 22.5
C 10
17.3 17.5 19.5 20.0 21.2
C5
16.5 19.0 20.7 22.7 23.5
C 11
18.4 20.5 21.4 22.5
C6
17.0 17.2 19.0 20.0 23.5
C 12
12.0 12.0 12.8 13.1 13.5 14.0 16.6 22.0 22.5
C2
Donor senDNAa ⫺
⫺
a A plus indicates that donor senDNA was detectable in the senescent subcultures, after hybridization of the blot with the appropriate probe. A minus indicates that this senDNA was not detectable by hybridization under the same conditions. No indication is reported when the analysis of the mtDNA content has not been performed.
1986). Intact intron ␣ is present in some immortal mex strains (Koll et al., 1985). SenDNA␣ is present in the permanently nonsenescent double mutant strain i viv (Tudzynski et al., 1982). Senescence can occur sporadically in the absence of senDNA␣ (Dujon and Belcour, 1989). More recently, nuclear mutations that strongly impair senDNA␣ amplification without preventing senescence were obtained (Borghouts et al., 1997; Silar et al., 1997) and mex strains were induced to senescence by the sole
transfer of senDNA molecules through anastomosis (Jamet-Vierny et al., 1997a). In this paper, we show that senDNAs can be efficiently transmitted via anastomoses (cytoduction) to recipient mycelia, without the cotransfer of the determinant of senescence. Meiotic analysis shows that a mitochondrial mutation and the determinant of senescence do not segregate in a similar fashion. These data question the actual nature of the determinant of senescence.
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30
MATERIALS AND METHODS Media, Fungal Strains, and Lifespan Measurements Culture conditions and genetics methods are described in Esser (1974). The strains used derived from the S strain of Podospora anserina (Rizet, 1952). capr1 is a mitochondrial mutation that promotes a resistance to chloramphenicol and an increased longevity (Belcour and Begel 1977, 1980). Longevity of a subculture is defined as the distance covered on M2 medium by the mycelium between the point where the spore germinates (or where the explant is reinoculated) and the point where the apical cells die. The lifespan of a strain is defined as the mean longevity of parallel cultures. On M2 medium and under our race tube conditions, the S capr 1 strain has a lifespan of 15.1 ⫾ 2.1 cm, and the S caps1 wild-type strain has a lifespan of 9.5 ⫾ 1.0 cm.
Nucleic Acid Manipulation DNA analysis experiments were performed by standard methods (Ausubel et al., 1987). MtDNA was extracted using the miniprep procedure (Lecellier and Silar, 1994). The cloned monomer of senDNA and of senDNA␣ used as hybridization probes have been described (ViernyJamet, 1988).
Anastomosis Experiments The protocol, called contamination, was designed by Belcour (Sellem et al., 1996) to introduce mitochondrial mobile sequences from a donor mycelium into a recipient. In our case, a small inoculum of the donor senescent mycelium was put onto the recipient young thallus that had grown a few centimeters on an MR petri dish. This inoculum was taken from a senescent S caps1 mat⫺ culture about 1 to 0.5 cm before its arrest of growth. It contained large amounts of a senDNA, whose monomer was well characterized, and large amounts of senDNA␣. It was placed on the growing front of the recipient S capr1 mat⫹ thallus. Under these conditions, local anastomoses occur. They permit the exchange of cytoplasm but do not allow the propagation of the nuclei from the donor into the recipient. Indeed, to ensure that the mycelium, which grew after contamination, was not colonized by donor nuclei, we checked that it did exclusively conserve nuclei with the mat⫹ mating type of the S capR1 mat⫹ recipient strain. Contamination plates contained the MR medium
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Jamet-Vierny et al.
since the frequence of spontaneous appearance of the determinant of senescence is lower on this medium than on M2, as the longevity of wild-type is much increased on MR (Coppin et al., 1993) when compared to M2 (Silar and Picard, 1994). Longevity of the contaminated cultures was subsequently performed on M2 medium.
RESULTS The Mere Presence of senDNAs Is Not Sufficient to Commit the Mycelium for Senescence During previous anastomosis experiments between young and senescent strains, which were performed to follow the modalities of senDNAs transmission (Jamet-Vierny et al., 1997a), we observed a provocative phenomenon. While in most cases, the determinant of senescence was transmitted with the senDNAs, in some other cases we noticed that transmission of senDNAs was not associated with transmission of the determinant of senescence. To confirm these data, a specially designed anastomosis experiment was carried out as summarized in Fig. 1. Briefly, a senescent S caps 1 mat⫺ donor culture was used to contaminate an undetermined S capr1 mat⫹ recipient. Afterward, we investigated (1) the presence or absence of the determinant of senescence by measuring the longevity of the contaminated mycelia with explants taken 2 cm downstream of the contamination point (in area R) and (2) the senDNA content of the contaminated recipient mycelium. Control plates were processed in a similar way, except that no contaminating explant was added. The results are presented in Table 1. They were used to establish two survival curves according to Marcou (1961), one corresponding to the contaminated cultures and the other to the control cultures (Fig. 2). In this representation, the logarithm of the percentages of surviving subcultures is plotted as a function of growth length. The curves have a characteristic shape: a plateau, which corresponds to the shortest distance grown by all subcultures before exhibiting the senescent phenotype, and thereafter an exponential decrease of the percentage of surviving subcultures. Such a profile signifies that senescence results from the random but reproducible appearance of a factor called the determinant of senescence (Marcou, 1961). The plateau represents the distance of growth, which is required to reach the final phase of senescence after the determinant appeared (or incubation distance). The slope of the
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Senescence Determinant in P. anserina
FIG. 2. Lifespan curves of the contaminated and control cultures. The data are reported according to Marcou (1961) as the logarithm of the percentage of living subcultures with respect to growth length. Survivors are plotted every centimeter. Extensive data are reported in Table 1.
curve is indicative of the frequency of the determinant appearance. These curves give two important informations. First, under the conditions of the experiment, the incubation distance for the S capr 1 mat⫹ control cultures is estimated to about 12.5 cm. Therefore, we can conclude that all the contaminated cultures that have grown a distance higher than 12.5 cm (79% of the cultures) did not receive the determinant of senescence at the time of contamination. Second, the slopes of the curves are nearly identical, but incubation distance is slightly shorter for the experimental curve. These slopes indicate that the frequency of appearance of the determinant is roughly the same in the contaminated and control cultures. The shortened incubation distance indicates that the time necessary between determination and the expression of senescence is slightly reduced in the experimental cultures compared to that in the control cultures. MtDNA of some contaminated cultures was extracted at two stages of growth, first from explants taken on the petri dishes in area R located close to the contamination point and second from explants taken in the race tubes, 1 cm before the arrested edge of the senescent culture (see Fig. 1). The restriction and hybridization pattern of this mtDNA was then compared with that of the donor mtDNA. The mtDNA obtained from area R was in all cases very similar
to that present in a young control culture. On the other hand, the mtDNA recovered from senescent subcultures in race tubes displayed a high quantity of senDNA molecules identical to the ones found in the senescent donor (Table 1 and Fig. 3A). SenDNA are highly variable and it is unusual to recover identical monomers from independent cultures. It is therefore most likely that the observed senDNA derived from the donor. Confirming this, the analyzed senescent control cultures display variable senDNAs, all different from the donor senDNA (Fig. 3B). In addition to senDNA, the senescent donor contained a high quantity of senDNA␣. The HaeIII restriction pattern of the mtDNA reveals variable quantities of this senDNA in the senescent contaminated cultures (Fig. 3A). These molecules could result either from transmission from the donor of minute amounts of senDNA␣ during contamination and/or from de novo generation in the recipient as we previously showed (Jamet-Vierny et al., 1997a). Noteworthy, most of the contaminated senescent subcultures analyzed had grown more than 12.5 cm and therefore were not determined for senescence at the point of contamination.
The Determinant of Senescence and the capr1 Mitochondrial Mutation Display Different Meiotic Segregation Modalities In previous reports, it was observed that the determinant of senescence and the capr1 mitochondrial mutation display different meiotic behaviors. Indeed, in crosses between the capr1 and caps1 strains, the ascospores produced by a single perithecium are either all resistant to chloramphenicol or all sensitive; no ascospores that carry both alleles are observed (Belcour and Begel, 1977). On the contrary, it is possible to find ascospores determined and nondetermined for senescence within the same perithecium in a cross between a senescent and a young culture (Marcou, 1961). We thus decided to repeat these two experiments in a single step by crossing a young S capr1 mat⫹ culture with a senescent S caps1 mat⫺ culture. As a control, the young S capr1 mat⫹ culture was also crossed with the young S caps1 mat⫺ culture from which the senescent culture was obtained. To this end, hyphae from both partners were loosely ground together and inoculated on fresh medium. Self-fertile cultures were obtained consisting of an heterocaryotic heteroplasmic capr1 mat⫹/caps1 mat⫺ that contain the determinant of senescence in the experimental cross and that do not contain the determinant in the control cross. From each culture, eight perithecia were randomly picked up and for
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Jamet-Vierny et al.
FIG. 3. Restriction and hybridization patterns of some of the contaminated (A) and control (B) subcultures. (Left) Ethidium-stained gels of the HaeIII-digested mtDNA from senescent subcultures issued from contamination E14 (reported in Table 1A) (A) and from some of the control cultures (C1, C2, C3, C7, and C8, reported in Table 1B) (B). (Right) Results of the hybridization with a probe that specifically hydridizes with the region that is common to all senDNA and that is included in fragment HaeIII n° 16 (2.1 kb). The monomer of the particular senDNA present in the senescent culture used as donor contains this common region and a large part of the adjacent HaeIII fragment n°1. This monomer has only one HaeIII site and after digestion of the mtDNA, it appears as a single fragment of about 8.8 kb. Consequently, the probe hybridizes specifically with this 8.8-kb fragment (and, as seen in Fig. 3B, lane C7: 16.9 cm, with any senDNA that possesses a similar organization). CC, mtDNA extracted from area R (see Fig. 1). Donor, mtDNA from the donor senescent culture. YC, mtDNA extracted from a young control culture. HaeIII 16, position of fragment HaeIII n° 16. ␣, monomer of senDNA␣ (2.5 kb). The longevity of each culture is indicated. In (A) two identical mtDNA samples of the E14 subculture with a longevity of 20.9 cm are present on the gel.
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33
Senescence Determinant in P. anserina
appearance and spreading of a cytoplasmic and infectious factor (Marcou, 1961; Smith and Rubenstein, 1973). Death is always correlated with the presence of particular defective mtDNA molecules (Dujon and Belcour, 1989). Because these molecules are constantly present in senescent mycelia and may have invasive properties (see the Introduction), they are potential candidates to be the infectious factor. We show here that senDNA␣ or  can be transmitted to nondetermined mycelia without the transfer of the determinant of senescence (in 49 of the 52 tested cultures), confirming our previous observations (Jamet-Vierny et al., 1997a). Noteworthy, during the experiment, we observed that contamination with the senDNAs did not seem to significantly affect the probability of the appearance of the determinant, suggesting that presence of a low amount of senDNAs is not sufficient to commit the mycelium to senescence. However, the incubation distance is slightly diminished. This indicates, as already postulated (JametVierny et al., 1997a), that senDNA amplification, at the expense of intact mtDNA, participates in cellular death. The contamination clearly supplies the cells with senDNAs, and this likely decreases the time required to reach a lethal level of senDNA. We also show that the determinant of senescence and the capr1 mitochondrial mutation display different meiotic segregation modalities. Whereas all the ascospores of a given perithecium are homogenous for either capr1 or caps1, these are heterogenous for the presence of the determinant of senescence. These data confirm the fact that very few molecules of mtDNA (possibly one), and thus very few mitochondria, are used during perithecium formation (Belcour and Begel, 1977). In view of all these observations and the previous uncertainties about the true molecular nature of the
each, 13 to 24 ascopores were checked (1) for the presence of the capr1 or caps1 allele and (2) for their determined versus nondetermined status. Due to the large amount of cultures generated in this experiment, the presence or absence of the determinant of senescence was ascertained by inoculating a single culture per ascospore in M2 medium race tubes. Because this method is not very accurate, very stringent criteria were used to differentiate between nondetermined and determined ascospores. Cultures with longevity inferior to 80% of the incubation distance (10.0 cm for capr strains and 4.5 cm for caps strains) were considered issued from a determined ascospore. Those with a longevity superior to 130% of the incubation distance (16.0 cm for capr strains and 6.0 cm for capS strains) were considered issued from a nondetermined ascospore. Ascopores that generate cultures whose longevity fell between these two values were considered not ascertained. The results of this experiment are summarized in Table 2. As previously reported, perithecia were homogenous with respect to the capr1 mutation since all ascopores from one perithecium were either all resistant or all sensitive to chloramphenicol. On the contrary, the determinant of senescence was transmitted in a very heterogeneous fashion in the experimental cross since seven of the eight tested perithecia contained a mixture of determined and nondetermined ascospores.
DISCUSSION All wild-type strains of Podospora anserina examined so far present a degenerative process that is controlled in a constant way. This senescence process is triggered by the
TABLE 2 Meiotic Behavior of the Determinant of Senescence and the capr1 Mitochondrial Mutation Young S capr1 mat⫹ ⫻ young S capS1 mat⫺
Young S capr1 mat⫹ ⫻ senescent S capS1 mat⫺
Perithecium
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
capS capr
21 0
23 0
20 0
23 0
24 0
19 0
24 0
23 0
0 13
0 19
0 18
0 21
0 18
0 20
0 23
22 0
Det⫹ Det⫺ ?
0 21 0
0 23 0
0 20 0
0 23 0
0 24 0
0 19 0
0 23 1
0 23 0
2 8 3
15 0 4
5 10 3
5 12 4
2 15 1
2 15 3
2 17 4
10 10 2
Note. For the eight perithecia from each cross, the table gives the number of ascospores found to be capS or capr and their nondetermined (Det⫺) or determined (Det⫹) status. ? Not ascertainable (see text).
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34
determinant of senescence (Griffiths, 1992), two alternative hypotheses can be proposed. The determinant of senescence could be a sufficient level of senDNAs. Once a threshold of senDNA is reached, a physiological cellular change would trigger amplification as proposed by Bertrand (1995). However, this hypothesis is not very consistent with the heterogenous segregation of the determinant of senescence observed in meiosis. Indeed, we would expect that the determinant of senescence, if it is associated with senDNA that are located within the mitochondria, would segregate in a fashion similar to a mitochondrial mutation. We thus favor the hypothesis that an additional cytoplasmic and infectious factor is required to permit the senDNAs amplification. According to this proposition and contrary to the previous models, amplification of mtDNA deleted molecules would not be the etiology of senescence but a mere symptom. Nonetheless, it would be an important symptom since it is likely to be the actual reason for cellular death. In an overlooked report, Raynal (1980) related experiments that were based on a different rational but which led to the same conclusion. To date, we have no ideas about the molecular nature of the determinant. Recently, we have proposed that the determinant of senescence acts on the amplification of senDNAs through modifications in the expression of nuclear encoded proteins, possibly at the translational level (Silar et al., 1997). These mediator proteins would be imported in mitochondria and alter the maintenance of the mitochondrial genome. Involvement of the mitochondrial import machinery in modulating the mitochondrial DNA maintenance of P. anserina was recently shown (JametVierny et al., 1997c). Our proposition is not unconventional. Indeed, Griffiths and Yang (1993) have recently described several cases of aging in geographical isolates of Neurospora intermedia. In some instances, nuclear determination is suspected but tetrad analysis does not support any simple model. In addition, in some of these cases, aging does not seem to involve clear mitochondrial dysfunction. Similarly, we have recently reported a new degenerative syndrome in Podospora anserina. This syndrome is also triggered by a cytoplasmic and infectious element, which is not connected with mitochondrial dysfunction (Silar et al., 1999).
ACKNOWLEDGMENTS We thank M. J. Daboussi, M. Picard, and H. Lalucque for useful discussions. This work was supported by grants from Universite´ de Paris
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Jamet-Vierny et al.
XI, Centre National de la Recherche Scientifique, Association Francaise contre les Myopathies, and Fondation pour la Recherche Me´dicale.
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