Current Genetics
Current Genetics (1983) 7:405- 407
© Springer-Verlag 1983
Short Communication Mitochondrial Genetics of Coprinus: Recombination of Mitochondrial Genomes J. L. C. Baptista-Ferreira*, Androulla Economou, and Lorna A. Casselton School of Biological Sciences, Queen Mary College, Mile End Road, London E1 4NS, England
Summary. The formation of the sexual mycelium or dikaryon in the basidiomycete Coprinus cinereus involves exchange and migration of nuclei without accompanying exchange of mitochondria. The dikaryotic growth which appears around the periphery of mated monokaryons has exclusively the mitochondrial genome of the recipient cells. Recombination of mitochondrial genomes is not, however, precluded during dikaryosis. Using monokaryons with different mitochondrial gene mutations, [aeu-lO] causing cytochrome aa3 deficiency and [cap-l.1] conferring resistance to chloramphenicol, it was shown that recombinant mitochondria arise in the zone of contact of mated monokaryons. Key words: Coprinus -
Mitochondrial recombination
migration of donor nuclei through the established cells of each recipient monokaryon. This establishes tip cells with the dikaryotic pair of nuclei but because there is no accompanying exchange of organelles, the dikaryon which grows out has exclusively the cytoplasm of the recipient mycelium. Since dikaryotisation is a reciprocal event, in any one mating two discrete dikaryons can be formed which have identical nuclei but different cytoplasms. To determine whether there is any opportunity for recombination of organelle genomes during dikaryosis, matings were made between two mutants having different mitochondrial gene mutations. Recombinant genomes were detected in the region where hyphal anastomoses occur.
Introduction
Materials and Methods
Despite the developmental complexity of the higher basidiomycetes there is no differentiation of specialised sex cells and the interaction which leads to sexual reproduction is between purely vegetative hyphae (see Casselton 1978). Surprisingly this interaction can lead to the complete exclusion of mitochondria from one parent (Casselton and Condit 1972) analogous to the maternal inheritance seen in ascomycete fungi such as Neurospora crassa (Mitchell and Mitchell 1952). The sexual interaction in Coprinus cinereus is between hyphae of two monokaryons, which, if compatible, gives rise to the dikaryon on which fruit bodies develop. Uniparental inheritance of mitochondria derives from events following hyphal anastomosis. There is first a reciprocal exchange of nuclei followed by
Strains. CC9 A6B6 [aeu-lO] derived from H9 wild type by mutagenesis with N-methyl Nlnitro-N nitrosoguanidine (Casselton and Condit 1972). CDR1 A6B6 [cap-l.1] derived from TC10 wild type by selection for spontaneous mutation to chloramphenicol resistance.
Mitochondrial Gene Mutations
Offprint requests to." L. A. Casselton * Present Address: Faculty of Science, Lisbon, Portugal
The [acu-lO] mutation causes loss of cytochrome a a but with the appearance of a modified cytochrome
Media and Growth Conditions. The glucose and acetate media were those used by Scaly-Lewisand Casselton (1978). Chloramphenicol base was added to molten medium after autoclaving to give a final concentration of 2 mg/ml. Cultures were grown at 37 °C. Chlamydospore isolation was as described by Lewis (1961).
Results and Discussion
406
Mitochondrial Genetics of Coprinus
Table 1. Somatic segregation of mitochondrial phenotypes during dikaryosis in the cross [acu-lO cap-1+] x [aculO+ cap-1.1 ] Cytoplasm
Dikaryon
Somatic segregation
of dikaryon
phenotype
Parental ~cu-i 0 cap-1+
Recombinant acu-lO+ cap-1.1
acu-lO + cap-1+
acu-lO cap-l.1
[A] mycelial sample (a) [acu-lOcap-1+]
[acu-lO cap-1+]
(b) [acu-lO+ cap-1.1]
60 60
0 0
0 0
0 0
[acu-10 cap-1.1 ] (1) (2)
0 0
60 60
0 0
0 0
(1) (2)
0 0
14 2O
106 100
0 0
0 0 0
98 28 0
73 132 180
0 0 0
(c) Junction
(1) a (2)
[B] chlamydospore samples Junction
b(i) [acu-lO+ cap-1.1] (ii) [acu-lO+ cap-l.l] (iii) [acu-lO+ cap-1+]
a (1) and (2) indicate separate crosses b (i) (ii) (iii) indicate separate isolates
oxidase (Casselton and Condit 1972). We can detect no differences in the mitochondrial DNAs of mutant and wild type by restriction enzyme analysis (unpublished data) and a relatively high reversion frequency o f 10 4 indicates that [acu-lO] is a point mutation. The respiratory deficiency caused by [acu-lO] results in weak growth and inability to utilise acetate as sole carbon source for growth. The [cap-l.1] mutation confers resistance to the antibiotic chloramphenicol. Analogous mutations in Saccharomyces cerevisiae result in alteration to the mitochondrial ribosomes and map in the mitochondrial gene encoding the large (21S)ribosomal RNA (Borst and Grivell 1978; Dujon 1980). It is assumed that similar mutations in other fungi, Schizosaccharomyces pombe (Seiz et al. 1977), Aspergillus nidulans (Gunatilleke et al. 1975; Lazarus and Turner 1977), and Podospora anserina (Belcour and Begel 1977) all have a similar functional basis. [cap-l.1 ] causes some 50% reduction of normal growth on drug-free medium but does not affect the cytochrome complement and hence growth on acetate is unimpaired. Matings and Sampling Procedure Matings were made by placing inocula of the two mutant monokaryons lcm apart on an agar plate and allowing the two mycelia to grow together. After 4 - 5 days the two discrete reciprocal dikaryons had developed at the periphery of the two mated cultures. Samples were removed from three areas:
1. The [acu-lO] recipient dikaryon. This should have only [acu- 10 cap- 1+ ] parental type mitochondria. 2. The [eap-l.1] recipient dikaryon. This should have only [acu-10 + cap-1.1 ] parental type mitochondria. 3. The area adjacent to the junctions where mated monokaryons had anastomosed and nuclear migration was initiated. This region was not dikaryotic but inocula developed into dikaryon on subculture. In the first instance three inocula were taken from each reciprocal dikaryon and six (three from each side) from the junction area. These samples were grown for a further 5 days to allow development of colonies and to promote somatic segregation. Twenty samples were then taken from each colony and tested for phenotype. This gave a total of 60 samples for each reciprocal dikaryon and 120 samples from the junction regions. The results of sampling two matings are given in Table 1 [A]. It will be seen that, as expected, only the parental mitochondrial types were derived from the two reciprocal dikaryons (a) and (b), either [acu-lO cap-1 +] or [acu-lO + cap-l.1]. From the junction regions (c) samples showed somatic segregation for the parental [acu-lO + cap-l.1] phenotype and a new [acu-lO + cap1+ ] phenotype.
Non-recovery o f [acu- 10] from the Junction Region Non-recovery of [acu-lO], either the parental or a recombination double mutant from the junction region is not unexpected. [acu-lO] causes such a gross respiratory
Mitochondrial Genetics of Coprinus defect that [acu-lO ÷] mitochondria would rapidly be selected in a mixed mitochondrial population. Moreover, although [cap-l] mutations are readily selected in wild strains, they cannot be selected in [acu-lO] mutants suggesting that the doubly mutant genome is inviable.
Origin of [acu- 10 + cap- 1+] Mitochondria This phenotype could arise by recombination, reversion o f [acu-lO] or [cap-l.1] or result from a mixed mitochondrial DNA population. Mutation can be ruled out. None of the samples taken from the two reciprocal dikaryons formed in the same matings showed evidence of reversion of either mutation (Table I[A] (a) and (b)). More conclusively, matings set up between homoallelic [acu-lO] mutants and homoalMic [cap-l.1] mutants were analysed similarly and from 480 samples from each no revertants of either mutation were obtained. Mixed mitochondrial genomes can also be ruled out. Results presented in Table I[A] derive from testing multicellular inocula and these are more likely to have mixed mitochondrial populations than single cells. Single cells of the dikaryon can be sampled by taking the chlamydospores which form on the undersurface of the mycelium. Three junction derived dikaryons were sampled in this way and the segregation data given in Table I[B]. All possible phenotypes were again tested for but even from single cells the [acu-lO] mitochondria could not be recovered. The important observation is that from two dikaryons (i) and (ii) having the parental [acu-lO + cap-l.1] phenotype there was segregation in chlamydospores for both this and the recombinant [acu- 10 + cap- 1 ÷ ] phenotype whereas from the dikaryon already expressing the recombinant phenotype (iii) no further segregation occurred. [cap-l.1 ] is therefore dominant to [cap-l+], thus the [acu-lO + cap-1 +] phenotype
407 cannot derive from a mixed mitochondrial DNA population but must result from recombination. The data presented show that recombination of mitochondrial genomes can occur in the basidiomycete Coprinus cinereus but it is confined to the region of hyphal anastomoses. Because nuclear migration is independent of cytoplasmic movement, it leads to dikaryosis with uniparental inheritance of mitochondria. This has the important implication in natural populations that sexual reproduction will be unaffected by any mitochondrially determined incompatibility. In terms of mitochondrial genetics this basidiomycete system is particularly useful. On the one hand, uniparental inheritance in reciprocal dikaryons allows rapid identification of a cytoplasmic mutation and on the other, crosses are easily effected by taking cells from the junction region.
References Belcour L, Begel O (1977) Mol Gen Genet 153:11-21 Borst P, Grivell LA (1978) Cell 15:705-723 Casselton LA (1978) Dikaryon formation in higher basidiomycetes. In: Smith JE, Berry DR (eds) The Filamentous Fungi, vol. III. Edward Arnold Casselton LA, Condit A (1972) J Gen Microbiol 72:521-527 Dujon B (1980) Cell 20:185-197 GunatiUeke IAUN, Scazzocchio C, Arst HN (1975) Mol Gen Genet 137:269-276 Lazarus CM, Turner G (1977) Mol Gen Genet 156:303-311 Lewis D (1961) Genet Res 2:141-155 Mitchell MB, Mitchell HK (1952) Proc Natl Acad Sci USA 38: 442-449 Sealy-Lewis HM, Casselton LA (1978) Mol Gen Genet 164:211215 Seiz G, Wolf K, Kaudewitz F (1977) Mol Gen Genet 155:339- 346 Communicated by K. Esser Received June 27, 1983
Current Genetics (1983) 7:405- 407
© Springer-Verlag 1983
Short Communication Mitochondrial Genetics of Coprinus: Recombination of Mitochondrial Genomes J. L. C. Baptista-Ferreira*, Androulla Economou, and Lorna A. Casselton School of Biological Sciences, Queen Mary College, Mile End Road, London E1 4NS, England
Summary. The formation of the sexual mycelium or dikaryon in the basidiomycete Coprinus cinereus involves exchange and migration of nuclei without accompanying exchange of mitochondria. The dikaryotic growth which appears around the periphery of mated monokaryons has exclusively the mitochondrial genome of the recipient cells. Recombination of mitochondrial genomes is not, however, precluded during dikaryosis. Using monokaryons with different mitochondrial gene mutations, [aeu-lO] causing cytochrome aa3 deficiency and [cap-l.1] conferring resistance to chloramphenicol, it was shown that recombinant mitochondria arise in the zone of contact of mated monokaryons. Key words: Coprinus -
Mitochondrial recombination
migration of donor nuclei through the established cells of each recipient monokaryon. This establishes tip cells with the dikaryotic pair of nuclei but because there is no accompanying exchange of organelles, the dikaryon which grows out has exclusively the cytoplasm of the recipient mycelium. Since dikaryotisation is a reciprocal event, in any one mating two discrete dikaryons can be formed which have identical nuclei but different cytoplasms. To determine whether there is any opportunity for recombination of organelle genomes during dikaryosis, matings were made between two mutants having different mitochondrial gene mutations. Recombinant genomes were detected in the region where hyphal anastomoses occur.
Introduction
Materials and Methods
Despite the developmental complexity of the higher basidiomycetes there is no differentiation of specialised sex cells and the interaction which leads to sexual reproduction is between purely vegetative hyphae (see Casselton 1978). Surprisingly this interaction can lead to the complete exclusion of mitochondria from one parent (Casselton and Condit 1972) analogous to the maternal inheritance seen in ascomycete fungi such as Neurospora crassa (Mitchell and Mitchell 1952). The sexual interaction in Coprinus cinereus is between hyphae of two monokaryons, which, if compatible, gives rise to the dikaryon on which fruit bodies develop. Uniparental inheritance of mitochondria derives from events following hyphal anastomosis. There is first a reciprocal exchange of nuclei followed by
Strains. CC9 A6B6 [aeu-lO] derived from H9 wild type by mutagenesis with N-methyl Nlnitro-N nitrosoguanidine (Casselton and Condit 1972). CDR1 A6B6 [cap-l.1] derived from TC10 wild type by selection for spontaneous mutation to chloramphenicol resistance.
Mitochondrial Gene Mutations
Offprint requests to." L. A. Casselton * Present Address: Faculty of Science, Lisbon, Portugal
The [acu-lO] mutation causes loss of cytochrome a a but with the appearance of a modified cytochrome
Media and Growth Conditions. The glucose and acetate media were those used by Scaly-Lewisand Casselton (1978). Chloramphenicol base was added to molten medium after autoclaving to give a final concentration of 2 mg/ml. Cultures were grown at 37 °C. Chlamydospore isolation was as described by Lewis (1961).
Results and Discussion
406
Mitochondrial Genetics of Coprinus
Table 1. Somatic segregation of mitochondrial phenotypes during dikaryosis in the cross [acu-lO cap-1+] x [aculO+ cap-1.1 ] Cytoplasm
Dikaryon
Somatic segregation
of dikaryon
phenotype
Parental ~cu-i 0 cap-1+
Recombinant acu-lO+ cap-1.1
acu-lO + cap-1+
acu-lO cap-l.1
[A] mycelial sample (a) [acu-lOcap-1+]
[acu-lO cap-1+]
(b) [acu-lO+ cap-1.1]
60 60
0 0
0 0
0 0
[acu-10 cap-1.1 ] (1) (2)
0 0
60 60
0 0
0 0
(1) (2)
0 0
14 2O
106 100
0 0
0 0 0
98 28 0
73 132 180
0 0 0
(c) Junction
(1) a (2)
[B] chlamydospore samples Junction
b(i) [acu-lO+ cap-1.1] (ii) [acu-lO+ cap-l.l] (iii) [acu-lO+ cap-1+]
a (1) and (2) indicate separate crosses b (i) (ii) (iii) indicate separate isolates
oxidase (Casselton and Condit 1972). We can detect no differences in the mitochondrial DNAs of mutant and wild type by restriction enzyme analysis (unpublished data) and a relatively high reversion frequency o f 10 4 indicates that [acu-lO] is a point mutation. The respiratory deficiency caused by [acu-lO] results in weak growth and inability to utilise acetate as sole carbon source for growth. The [cap-l.1] mutation confers resistance to the antibiotic chloramphenicol. Analogous mutations in Saccharomyces cerevisiae result in alteration to the mitochondrial ribosomes and map in the mitochondrial gene encoding the large (21S)ribosomal RNA (Borst and Grivell 1978; Dujon 1980). It is assumed that similar mutations in other fungi, Schizosaccharomyces pombe (Seiz et al. 1977), Aspergillus nidulans (Gunatilleke et al. 1975; Lazarus and Turner 1977), and Podospora anserina (Belcour and Begel 1977) all have a similar functional basis. [cap-l.1 ] causes some 50% reduction of normal growth on drug-free medium but does not affect the cytochrome complement and hence growth on acetate is unimpaired. Matings and Sampling Procedure Matings were made by placing inocula of the two mutant monokaryons lcm apart on an agar plate and allowing the two mycelia to grow together. After 4 - 5 days the two discrete reciprocal dikaryons had developed at the periphery of the two mated cultures. Samples were removed from three areas:
1. The [acu-lO] recipient dikaryon. This should have only [acu- 10 cap- 1+ ] parental type mitochondria. 2. The [eap-l.1] recipient dikaryon. This should have only [acu-10 + cap-1.1 ] parental type mitochondria. 3. The area adjacent to the junctions where mated monokaryons had anastomosed and nuclear migration was initiated. This region was not dikaryotic but inocula developed into dikaryon on subculture. In the first instance three inocula were taken from each reciprocal dikaryon and six (three from each side) from the junction area. These samples were grown for a further 5 days to allow development of colonies and to promote somatic segregation. Twenty samples were then taken from each colony and tested for phenotype. This gave a total of 60 samples for each reciprocal dikaryon and 120 samples from the junction regions. The results of sampling two matings are given in Table 1 [A]. It will be seen that, as expected, only the parental mitochondrial types were derived from the two reciprocal dikaryons (a) and (b), either [acu-lO cap-1 +] or [acu-lO + cap-l.1]. From the junction regions (c) samples showed somatic segregation for the parental [acu-lO + cap-l.1] phenotype and a new [acu-lO + cap1+ ] phenotype.
Non-recovery o f [acu- 10] from the Junction Region Non-recovery of [acu-lO], either the parental or a recombination double mutant from the junction region is not unexpected. [acu-lO] causes such a gross respiratory
Mitochondrial Genetics of Coprinus defect that [acu-lO ÷] mitochondria would rapidly be selected in a mixed mitochondrial population. Moreover, although [cap-l] mutations are readily selected in wild strains, they cannot be selected in [acu-lO] mutants suggesting that the doubly mutant genome is inviable.
Origin of [acu- 10 + cap- 1+] Mitochondria This phenotype could arise by recombination, reversion o f [acu-lO] or [cap-l.1] or result from a mixed mitochondrial DNA population. Mutation can be ruled out. None of the samples taken from the two reciprocal dikaryons formed in the same matings showed evidence of reversion of either mutation (Table I[A] (a) and (b)). More conclusively, matings set up between homoallelic [acu-lO] mutants and homoalMic [cap-l.1] mutants were analysed similarly and from 480 samples from each no revertants of either mutation were obtained. Mixed mitochondrial genomes can also be ruled out. Results presented in Table I[A] derive from testing multicellular inocula and these are more likely to have mixed mitochondrial populations than single cells. Single cells of the dikaryon can be sampled by taking the chlamydospores which form on the undersurface of the mycelium. Three junction derived dikaryons were sampled in this way and the segregation data given in Table I[B]. All possible phenotypes were again tested for but even from single cells the [acu-lO] mitochondria could not be recovered. The important observation is that from two dikaryons (i) and (ii) having the parental [acu-lO + cap-l.1] phenotype there was segregation in chlamydospores for both this and the recombinant [acu- 10 + cap- 1 ÷ ] phenotype whereas from the dikaryon already expressing the recombinant phenotype (iii) no further segregation occurred. [cap-l.1 ] is therefore dominant to [cap-l+], thus the [acu-lO + cap-1 +] phenotype
407 cannot derive from a mixed mitochondrial DNA population but must result from recombination. The data presented show that recombination of mitochondrial genomes can occur in the basidiomycete Coprinus cinereus but it is confined to the region of hyphal anastomoses. Because nuclear migration is independent of cytoplasmic movement, it leads to dikaryosis with uniparental inheritance of mitochondria. This has the important implication in natural populations that sexual reproduction will be unaffected by any mitochondrially determined incompatibility. In terms of mitochondrial genetics this basidiomycete system is particularly useful. On the one hand, uniparental inheritance in reciprocal dikaryons allows rapid identification of a cytoplasmic mutation and on the other, crosses are easily effected by taking cells from the junction region.
References Belcour L, Begel O (1977) Mol Gen Genet 153:11-21 Borst P, Grivell LA (1978) Cell 15:705-723 Casselton LA (1978) Dikaryon formation in higher basidiomycetes. In: Smith JE, Berry DR (eds) The Filamentous Fungi, vol. III. Edward Arnold Casselton LA, Condit A (1972) J Gen Microbiol 72:521-527 Dujon B (1980) Cell 20:185-197 GunatiUeke IAUN, Scazzocchio C, Arst HN (1975) Mol Gen Genet 137:269-276 Lazarus CM, Turner G (1977) Mol Gen Genet 156:303-311 Lewis D (1961) Genet Res 2:141-155 Mitchell MB, Mitchell HK (1952) Proc Natl Acad Sci USA 38: 442-449 Sealy-Lewis HM, Casselton LA (1978) Mol Gen Genet 164:211215 Seiz G, Wolf K, Kaudewitz F (1977) Mol Gen Genet 155:339- 346 Communicated by K. Esser Received June 27, 1983
