Uniparental Inheritance and Replacement of Mitochondrial ... - NCBI

Copyright 0 1993 by the Genetics Society of America

Uniparental Inheritance and Replacementof Mitochondrial DNA in Neurospora tetrasperma Steven B. Lee’and John W. Taylor Department of Plant Biology, University of Calfornia, Berkeley, Calfornia 94720 Manuscript received April24, 1991 Accepted for publication April 19, 1993 ABSTRACT This study tested mechanisms proposed for maternal uniparental mitochondrial inheritance in Neurospora: (1) exclusion of conidial mitochondria by the specialized female reproductive structure, trichogyne, due to mating locusheterokaryon incompatibilityand (2) mitochondrial input bias favoring the larger trichogyne over the smaller conidium. These mechanisms were tested by determining the modes of mitochondrial DNA (mtDNA) inheritance and transmission in the absence of mating locus heterokaryon incompatibility following crossesof uninucleate strains of Neurospora tetrasperma with trichogyne (trichogyne inoculated by conidia) and without trichogyne (hyphalfusion).Maternal uniparental mitochondrial inheritance was observed in 136 single ascospore progeny followingboth mating with and without trichogyne using mtDNA restriction fragment length polymorphisms to distinguish parental types. This suggests that maternal mitochondrial inheritance following hyphal fusions is due to some mechanism other than those that implicate the trichogyne. Following hyphal fusion,mututallyexclusive nuclear migration permitted investigation of reciprocal interactions. Regardless of which strain accepted nuclei following sevenreplicate hyphal fusion matings, acceptor mtDNA was the only type detected in 34 hyphal plug and tip samples taken from the contact and acceptor zones.No intracellular mtDNA mixtures were detected. Surprisingly, 3 daysfollowing hyphalfusion, acceptor mtDNAreplaced donor mtDNA throughout the entire colony. To our knowledge,this is the first report of complete mitochondrial replacement during mating in a filamentous fungus.

T

HIS study tests whether two aspects of sexual or

trichogyne-conidial mating a r e responsible for thefailure to transmitthemitochondrialgenome from the male, conidial parent to progeny in ascomycetessuchas Neurospora crassa (MITCHELLa n d MITCHELL 1952; REICHa n d LUCK1966; MANNELLA, PITTENGER and LAMBOWITZ 1979). Twopossible explanations of uniparental mitochondrial inheritance fromthematernal,protoperitheciating,trichogyne parent have been proposed. T h e first is exclusion of conidial cytoplasm (BIRKY1975, 1983), perhaps due tothe vegetative or heterokaryonincompatibility functionassociatedwiththe N . crassa mating type alleles or “idiomorphs” (NEWMEYER 1970;GLASSet al. 1988, METZENBERG1990). The heterokaryon incompatibility function associated with the mating type alleles may immobilize the cytoplasm of the male and allow only the male nucleus washed clean of its cytoplasm through the trichogyne (BIRKY1975, 1978; D. PERKINS,personal communication). This same model, exclusion of male organelles during gamete fusion by cytoplasmic exclusion, has been proposed as a mechanism of maternal uniparental inheritance in plants (BIRKY1978, 1983)and documented for barley(Mo-

’

Present address: Department of Biological Sciences, University of Northem Colorado, Greeley, Colorado 80639. Genetics 134: 1063-1075 (August, 1993)

1988). T h e second possible mechanism is a biased mitochondrial contribution favoring the large trichogyne of the protoperithecial parent over the smallerconidium (JINKS 1964; BIRKY et al. 1978, COSMIDES and TOOBY 1981). These hypothesesweretested by studying mitochondrial inheritance in the pseudohomothallicascomycete, Neurospora tetrasperma in which the heterokaryon incompatibility function associatedwith the mating type locus is inactive (METZENBERG and AHLGREN 1973) due to an inactive toZT allele (JACOBSON 1992),andin whichsexualreproductioncan be achieved by both trichogyne-conidial mating and by fusion of equally sized vegetative hyphae of opposite mating type. Mitochondrial inheritancewas compared in both types ofcrosses of N . tetrasperma using mtDNA restriction fragment length polymorhphisms(RFLPs) as markers. InN. tetrasperma conidial-trichogyne mating, we hypothesized that, due to the inactivityof mating locus heterokaryon incompatibility, male mitochondria might be transmitted through the sexual cycle to offspring. In the hyphal fusion matings, equal amounts ofcytoplasm from equal sized hyphal tips of A and a mating type isolates are brought into contact and there should be n o disparity in cytoplasmic volume or number of mitochondria as must occur in a GENSON

1064

S. B. Lee and J. W. Taylor TABLE 1

Uninucleate strains of N. tetrasperma used in matings Strain

FGSC no.'

Origin

Mating type

Welsh-lbv1 Welsh-lbv2 Perkins-lv2 Perkins-lv1 Lihue-lv1 Lihue-lv2 Hanalei-lbvl Hanalei-lbv2 85A 85a

2503 2504 2505 2506 2508 2509 2510 251 1 1270 1271

Louisiana Louisiana Louisiana Louisiana Kauai, Hawaii Kauai, Hawaii Kauai, Hawaii Kauai, Hawaii Unknown Unknown

A

~

~~

~~

a

A a A a A a

A a ~~

~

These are all single mating type cultures derived by conidial isolation from four-spored pseudohomothallic strains collected in nature, except for 1270 and 1271 which have been inbred (PERKINS, TURNER and BARRY 1976). 'Fungal Genetics Stock Center (Department of Microbiology, University of Kansas Medical Center, Kansas City, Kansas 66103).

trichogyne-conidial mating. We further hypothesized that, in the absence of cytoplasmicinput bias following hyphal fusion mating, mitochondria from either parent would be found in the progeny. We detected only maternal, uniparental mitochondrial inheritance in a total of 136 singleascospore progeny following both hyphal fusion(91) and trichogyne-conidial mating (45) of uninucleate isolates 1271a and 2505Aof N . tetrasperma. This suggests that some mechanism other than either exclusion of mitochondria from the fertilizing conidia by the trichogyne, or bias of input favoring maternal mitochondria is responsible forthe failure of the male to transmit its mitochondria in hyphal fusions. No recombinant mitochondria were detected. The same results were found when either strain acted as the nuclear acceptor and produced perithecia. Surprisingly, 3-4 days following hyphal fusion, nuclear acceptor strain mitochondria replaced nuclear donor strain mitochondria so only one mitochondrial type was present throughout the entire colony. Our results are relevant to models of mtDNAevolution in fungi and to models explaining maternal mitochondrial inheritance and replacement. MATERIALS AND METHODS

Isolates and storage:Isolates of N . tetrasperma were obtained from the Fungal Genetics Stock Center (Department of Microbiology, Universityof Kansas MedicalCenter, Kansas City, Kansas 66 103) as listed in Table 1. The isolates were grown and stored on silica gel as previously described (PERKINS 1977). Media: Different amounts of conidia and perithecia developed ondifferent media as indicated in the lasttwo columns of Table 2. The first letter of the media abbreviation indicates the media used: N for VOGEL'S N medium; W for water. The second symbol indicates the percentage of agar and the third symbol indicates the percentage of sucrose. We chose VOCEL'S N medium (1964), 2% agar, 0.07% sucrose (N2.07) for performing hyphal fusions. This me-

dium supported perithecial formation but suppressed conidiation and minimized the potential for airborne conidia of one colony inoculating the trichogyne of the other. Other mediaused were water, 2% agar (W2.0) for isolationof hyphal tips and N medium, 2% agar, 2.0% sucrose (N2.2) for subculturing hyphal tips. Hyphal fusion: Hyphal fusions were performed by placing cubes (5-7mm on a side) of monokaryotic A and a mycelia, 4-6 cm apart on N2.07 plates. All crosses were made on standard9-cm plasticPetri plates incubated at 30 " . Colonies met after 2-3 days of incubation. Heterokaryon formation and the progress of nuclear migration was indicated by the appearance of perithecia first at the zone of contact and subsequently back along the hyphae leading to one of the inoculum blocks. Repeated matings between 1271aand 2505A showed that nuclear migration could occur in either direction but in only one direction in each mating. The strain which formed perithecia was designated the nuclear acceptor (functionally female) whereas the one which donated its nucleus was designated the nuclear donor (functionally male). Hyphal plug and tip analysis: T o assay mitochondrial type, plugs of mycelium were taken and cultured in 50 ml of N0.2 broth, shaken at 30" for 17-48 hr and harvested. T o permit isolationof hyphal tips,mycelialplugs were cultured on 2% water agar (W2.0) at 25" for 17 hr before tip isolation. T o assay mitochondria in hyphal tips, the tips were cultured on N2.2 agar at 30" for 48 hr. Plugs taken from these colonies were used to start 50 ml N0.2 broth cultures which were shaken at 30" for 17-24 hr and harvested. Trichogyne-conidialmating: Mating was achieved by transferring suspensions of conidia from 2505A to protoperitheciating mycelialmatsof 1271a growing on N2.2 plates as previously described (PERKINS 1986). Nuclear migration and fertilization were indicated by the formation of fertile perithecia at the points of conidial transfer. The reciprocal interaction of 127 l a conidia transferredto 2505A protoperitheciating mycelial mats did not result in fertile perithecia. Mycelial plugs were taken from an areaadjacent to fertile perithecia to assay nuclear type. After subculturing on N2.2 agar at 30°, hyphal plugs taken from these colonies were placed into N0.2 broth at 30" shaking incubation for 1748 hr before harvesting. Isolation and germinationof ascospores: Two to three weeks following mating, ascospores were shot from the perithecia onto the lidsof Petri plates.Ascospores were collected either directly from the lids or by isolating single perithecia and vortexing them in sterile distilled Hz0 to release ascospores. Ascospore suspensions were heated at 60" for 30 min in a microcentrifuge tube, spread on W3.0 plates and single ascospores were isolatedusing a fine needle. Observed ascospore germination was 5 0 4 5 % . Growth of single ascospore cultures:Agar blocks which contained only one germinated ascospore were placed in N2.2 slants at 30" for 2-4 days. Ascospore cultures which continued to grow were subcultured onto N2.2 plates. A hyphal plug of each single ascospore subculture was placed in N2.2 broth at 30" for 3-5 days before harvesting mycelium for DNA isolation. Totalgenomic DNAminipreps: To harvest mycelia, broth cultures were vacuum filtered through cheesecloth, frozen in liquid NP, and lyophilized. Whole-cell DNA was isolated from freeze-dried mycelium by the procedures of ZOLANand PUKKILA (1986) or LEE,MILGROOMand TAYLOR (1988). mtDNA isolation: T o isolate mtDNA from purified mi-

mtDNA Replacement in Neurospora

1065

TABLE 2 Relative amountsof conidia and perithecia producedon different media Type

Agara

Media

% Agar

Name

Crossing (WESTERGAARD and MITCHELL1947)

N (VOGEL1964) N2.07 N2.20 w2.00 3.00 W3.00

Water

Brothb a

N (VOGEL1964)

N0.20

% Sucrose Perithecia

2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00

0.00 0.05 2.00 0.00 0.05 0.07 1.oo 2.00

0.00

2.00

Conidia

+ + + ++

-

+ +++

+++

-

+++ ++++ ++++

+ +++ ++++

+ + +++

+++

100 X 15 mm Petri plates. 50-100 ml of liquid in a 250-ml flask.

tochondria, mycelia were grown in 2 liters of N0.2 broth for 36-48 hr at30 O and thenharvested by filtration through cheesecloth. Hyphae were disrupted using a glass bead homogenizer (Bead Beater, Biospec Products, Bartelsville, Oklahoma). Mitochondria were isolated by sucrose gradient centrifugation and lysed, and mtDNA was purified as deand LAMBOWITZ (1 978),and TAYLOR scribed by MANNELLA and NATVIC(1989). Restriction endonuclease analysis:Samples of purified mtDNA or total DNA were digested with EcoRI or BglII under reaction conditions recommended by the supplier (New England BioLabs, Beverly, Massachusetts). DNAfragments were separated by electrophoresis in horizontal submerged 0.8% agarose gels (4-mmthick) in TAE buffer (100 mM Tris-HC1, pH 8.3; 125 mM Na-acetate; 10 mM EDTA) for 8-12 hr at 1 V/cm. Gels were stained using ethidium bromide (0.5 Pg/ml) for 30 min and destained in distilled water for 15 min. Generally, one mini-prep provided enough DNA for 10- 15 digests. DNA filter hybridization:Electrophoretically separated DNA fragments in agarose gels were depurinated for 15 min in 0.25 M HCI, denatured in 1.5 M NaCl, 0.5 M NaOH for 30 min, and neutralized in 1.5 M NaCl, 0.5 M Tris-HC1, pH 7.2, for 30 min. DNA in agarose gels was transferred onto Hybond N nylon membranes (Amersham, Arlington Heights, Illinois) by the general method of SOUTHERN (1975) using capillary flowas described by SCHLEIFand WENSINK(1981). mtDNA probes (0.5-1.0 rg) were prepared by nick-translation with ['*P]dATP (RIGBYet al. 1977).

Nylon membranes were shaken for 4-8 hr at 65" in prehybridization solution [0.9 M NaCl; 0.05 M sodium phosphate; 5 mM EDTA; 0.2% sodium dodecyl sulfate (SDS); 100 rg/ml calf thymus or salmon sperm DNA; 5 X Denhardt's solution (1% Ficoll, 1% polyvinylpyrrolidone, 1% BSA-Pentax Fraction V)]. A heat-denatured, radioactive probe was added directly to this solution and allowed to hybridize at 65" for 8-1 2 hr. Blots were washed three times for 30 min with agitation in 85-95" 1 X SSC (MANIATIS, FRITSCHand SAMBROOK 1982) plus 0.1% SDS. Hybridized filters were used to expose X-ray film (Kodak X-AR) for 4 hr to4 days at -80 O . RESULTS

To study mtDNA inheritance in N . tetrasperma we needed monokaryotic strains of opposite mating type

NUCLEARMIGRATION

I

2503A 2505A

1270A I

I

I

2510A

25088 1

I

Key JNILATERAI

4~-

A

L o -

31LATERAL

4 0 - A W

A+a

MUTUALLY EXCLUSIVE

A O +

1

) ' AZc

FIGURE1.-Distribution patterns of perithecial formation following hyphal fusion between uninucleate strains of N. tetrusperma. Arrows point to the strain($ which formed perithecia in each interaction. Size of the arrows indicate relative amounts of perithecial formation. The key indicates the potential modes of nuclear migration which may have resulted in the distribution patterns.

exhibiting interfertility by hyphal fusions, interfertilityby trichogyne-conidia interactions and different mtDNA restriction fragment patterns. To assess interfertility by hyphal fusion, monokaryotic N . tetrasperma strains of A and a mating types were crossed in the combinations shown in Figure 1. In all interactions,fertileperitheciaformed at the zone of contact. However, three different patternsof perithecial distribution were observed (Figure 1). These patterns were indicative of three modes of nuclearmigration: (1) unilateralnuclearmigration resulting in one parent, and always the same parent, formingperithecia, (2) bilateralnuclearmigration resulting in both parents forming perithecia simultaneously and (3) mutually exclusive unilateral nuclear migration resulting in either parent forming perithe-

S. B. Lee and J. W. Taylor

1066 B

A

1

2

1

2

3

4

4:::

- 11.5 - 9.4 -

7.4

FIGURE2,”mtDNA of uninucleate strains of N. tetruspemu. mtDNA was purified, restriction digested, separated by 0.8% agarose gel electrophoresis, and visualized after staining in EtBr with UV irradiation. (A) 1270 and 1271, strains recovered from the same dikaryon, exhibit identical Haelll mtDNA restriction fragment patterns (lanes 1 and 2). (B) Strains recovered from different dikaryons, 2505A and 1271a exhibit differences in both EcoRI (lanes 1 and 2) and EglIl mtDNA restriction fragmentpatterns (lanes 3 and 4).

cia butnot simultaneously. We used the mutually to follow mitochondrial exclusive matingpattern transmission in reciprocal interactions. T w o pairs of isolates, 2505A X 2509a and 2505A X 1271a, exhibited this pattern. In hyphal fusion mating, 2505A X 1271a produced more viable (black) ascospores than did 2505A X 2509a, and therefore was chosen for further analysis. T o assess interfertility by trichogyne-conidial interactions, 2505A conidia were used to fertilize trichogynes from protoperithecia of 1271a and resulted in fertile ascospores. T h e reciprocalmatingbetween 1271a conidia and 2505A perithecia produced false perithecia (DODGE1935, 1936; DODGEand SEAVER 1938) which lacked asci and ascospores. Purified mtDNA from each isolate listed in Table 1 was analyzed for RFLPs as described in MATERIALS AND METHODS. Haploid monokaryotic strains of o p posite mating type recovered from a single natural 1271a, had identical dikaryon, e.g., 1270A and mtDNA restriction fragment patterns (data not shown) butstrainsrecoveredfromdifferentdikaryons, e.g., 2505A and 1271a, had different mtDNAs (Figure 2). T h e monokaryotic isolates we selected, 2505A and

127 la, had easily distinguishedmtDNA RFLPs. Either parent had the potential to accept nuclei but not simultaneously and this permitted investigation of mitochondrial transmission in separate reciprocal interactions, one for 2505A as “female” and one for 127 1 as “female.” Comparingresults from reciprocal interactionscontrolledfor effects of strain-specific mitochondrial replication bias. If one strain had mitochondria with a replicative advantage, those mitochondria would be theone type detected in both interactions regardless of which parent peritheciated. In addition, if one straincontainedmtDNA with sequences which exhibited a sequenceconversion bias (polar recombination) such as yeast omega (ZIN and BUTOW1985) and var 1 regions (BUTOW,PERLMAN and GROSSMAN 1985), this would also be observed as the dominanttype regardlessof which parent peritheciated. Maternalmitochondrialinheritanceandtransmission followed trichogyne-conidialinteractions: 2505A conidia were used to fertilize 127 l a mycelium grown on N2.07for 1 week by placing a drop of conidial suspension on the centerof the culture. Perithecia began to darken and enlarge1 week following fertilization. After perithecia formed but before ascospores were shot, 10 hyphal plugs were taken 0.5 cm from perithecia and mtDNA from these subcultures were analyzed. Hyphae from all plugs contained only resident 127 l a mtDNA (data not shown). These 10 hyphal plug subcultures never formed perithecia, even 3 months after fertilization. Approximately3 weeks after fertilization, ascospores were shot and harvested. Forty-five single ascospores were germinated, and mtDNA from these cultures were analyzed. All ascospore cultures exhibited the maternal 127 l a mtDNA restriction fragment pattern (Figure 3). T h e reciprocal interaction using 127 l a conidia inoculating 2505A trichogynes didnot result in fertile matings and was not investigated further. Presence of both A and a nuclei was indicated by the formationof fertile perithecia following hyphal fusion: Inoculation of both2505A and 1271aon N2.07 resulted in mating by hyphal fusion. T h e onset of fusion was determined by microscopic observation of the contact betweenthe two colonies. T h e presence of both A and a nuclei in the zone of contact and the nuclear acceptor strainwas indicated by the formation of fertileperithecia in the matedculturesand in cultures taken from these zones. In one interaction shown (Figure 4), 2505A is the nuclear acceptor and 1271a is the nuclear donor whereas in the reciprocal interaction (see Figure 6), 127l a is the nuclear acceptor and 2505A is the nuclear donor. Mitochondrial transmission and replacement was observed by hyphal plug analysis: T o study mtDNA

Neurospora

1

2

3

4

5

6

7

8

in

Replacement mtDNA

1067

910111213141516171819u)

FIGURE3.-mtDNA of single ascospore progeny from trichogyne-conidial mating between 2505A conidia and 1271a mycelia. Patterns represent Southern hybridization of radiolabeled 127 l a mtDNA to nylon membranebound, Bglll digested, total DNA isolated from ascospore cultures. Lane 1, 1271a maternal mtDNA; lane 2, 2505A paternal mtDNA; lanes 3-20, single ascospore progeny mtDNA exhibiting 127l a maternal mtDNA pattern.

FIGURE4.-Hyphal fusionmating of N. tetraspenna isolates is the nuclear 127 la and 2505A. 127 is thela nuclear donor, 2505A acceptor. Maturing peritheciaa r e shown after fusion.T h e perithecia became darker and all contained fertile ascospores. T h e t h r e e zones sampled for mtDNA are indicated: I, nuclear donor, 1271a; 2, zone of contact; 3, nuclear acceptor, 2505A.

transmission, hyphal plugs (shown in Figures 4 and 6) were taken from three zones: the nuclear donor zone, the zone of contact and the nuclear acceptor zone. These zones were sampledat 2 and 7 days after fusion and mtDNA type was assayed. Results of mtDNA

FIGURE5.-mtDNA of hyphal plugs takenfrom the zones shown in Figure 4a t 2 and 7 days following hyphal fusion matings between nuclear acceptor, 2505A and nuclear donor, 1271a. Patterns r e p resent Southern hybridization of radiolabeled 1271a mtDNA to membrane-bound, EcoRI-digested, total DNA isolated from hyphal plug subcultures. Parental mtDNAs are shown: lane 1, 1271a nuclear donor; lane 2,2505A nuclear acceptor. Hyphal plug mtDNAs taken at 2 days after hyphal fusion exhibit resident mtDNA patterns: lane 3, donor zone showing 127 la mtDNA; lane4, contact zone showing mtDNA mixture of both 127 la and 2505A; lane5, acceptor zone showing 2505A mtDNA. Hyphal plug mtDNAs taken seven days after hyphal fusion exhibit only nuclear acceptor 2505A mtDNA: lane 6, donor zone now showing 2505A mtDNA instead of 1271a mtDNA; lane 7, contact zone now showing only 2505A mtDNA instead of a mixture; lane 8, acceptor zone still showing 2505A mtDNA.

analyses for reciprocal hyphal plug experiments are shown in Figures 5 and 7. Radiolabeled purifiedmtDNAfrom1271a selectively hybridized to mtDNA restriction fragments of bothparents,1271aand2505A(Figure 5, lanes 1 and 2; Figure 7, lanes 1 and 2) and these restriction fragment patterns were used to distinguish parental mtDNAs indicated in the figures with the following symbols: 8 for nuclear donor and9 for nuclear acceptor. Two days after fusion, we detected only resident mtDNA in donor and acceptorzones (Figure 5 , lanes 3-5; Figure 7, lanes 3-5). For both interactions, mycelium from the nuclear acceptor zone exhibited only nuclearacceptormtDNArestrictionfragment patterns and mycelium from nuclear donor zone exhibited only nuclear donor mtDNA specific restriction fragments. In thezone of contacttwo days after fusion we observed an apparent mixture of mtDNA fragments from both parental genomes (Figure5, lane 4). Seven days after fusion, we detected nuclear accep tor mtDNA in the samples analyzed from the nuclear acceptor zone (Figure5, lane 8). Unexpectedly, seven days after fusion,mtDNA of the nuclearacceptor

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S . B. Lee and J. W. Taylor

1 2 3 4 5 6 7 8

FIGURE6.-Hyphalfusion mating of N . tetrasperma isolates 127 la and2505A. 2505A is the nuclear donor, 127 la is the nuclear acceptor. Maturing perithecia are shown after fusion. T h e perithecia formed near the inoculum of 1271a and in the contact zone became darker after oneweek and all contained fertile ascospores. The three zones sampled for mtDNA are indicated: 1,nuclear acceptor, 1271a; 2, zone of contact; 3, nuclear donor, 2505A.

strain had replaced that of the nuclear donor so that only acceptor mtDNA was present in all samples includingthecontactzoneandnucleardonor zone (Figure 5, lanes 6 and 7; Figure 7,lanes 7 and 8). We were unable to detect any mtDNA mixtures in the contact zones seven days after fusion. Similarresults were observed for seven replicate experiments, three when 2505A was the nuclear acceptor and four when 127 la was the nuclear acceptor. A total of 70 hyphal plug samples were analyzed: 26 from the nuclear donor zone, 18 from the zone of contact, and 26 from the nuclear acceptor zone. In replicate matings, the replacement of male mtDNA by female mtDNA occured regardless ofwhich strain acted female and this phenomenon was observed as early as 3 days after fusion. Testing of alternative explanationsby analysis of colony hyphal tips, plugs and backcrosses: T h e replacement of mtDNA on the nuclear donor side of the Petri plate by mtDNA of the nuclear acceptor may also be explained by the growth of the nuclear acceptor mycelium over, underor through that of the nuclear donor mycelium. If the acceptor had grown into the donor zone, then hyphal plug cultures and subcultures from the nuclear donor zone should initially includeamixtureof mycelia. If the nuclear acceptor grew faster than the nuclear donor, eventually onlynuclearacceptormitochondria would be detected in these cultures taken fromthe donorzone. If this explanation were true, plugs taken from the

FIGURE7.-mtDNA of hyphal plugs taken from the zones shown in Figure 6at 2 and 7 days following hyphal fusion matings between nuclear acceptor, 1271a and nuclear donor, 2505A. Patterns rep resent Southern hybridization of radiolabeled 1271a mtDNA to membrane-bound, Egllldigested, total DNA isolated from hyphal plug subcultures. Parental mtDNAs are shown: lane 1, 2505A nuclear donor; lane 2, 1271a nuclear acceptor. Hyphal plug mtDNAs taken at 2 days after hyphal fusion exhibit resident mtDNA patterns: lane 3, acceptor zone showing 1271a mtDNA; lane 4, contact zone showing mtDNA of 127la; lane 5, donor zone showing 2505A mtDNA. Hyphal plug mtDNAs taken 7 days after hyphal fusion exhibit only nuclear acceptor 1271a mtDNA: lane 6, accep tor zone still showing 1271a mtDNA; lane 7, contact zone now showing only 1271a mtDNA instead of a mixture; lane 8, donor zone now showing 127 la mtDNA instead of 2505A mtDNA.

nuclear donor after 7 days should contain hyphal tips of both parents and subcultures of these plugs should lead to mating and productionof fertile perithecia. To test this alternative explanation and to examine the timing of mitochondrialtransmission more closely, hyphal plugs from the nuclear acceptor, contact and nuclear donor zones were taken at 2, 5.5, 7.5, 14.5 and 82.5 hr after fusion. T h e plugs were grown 24 hr onH z 0 agar, and fromeach plug three hyphal tips were subcultured and mtDNAwas analyzed as above. Between 2 and 14.5 hr after fusion, mtDNA ofonly the nuclear acceptor strain was detected in all 4 hyphal plug and 12 hyphal tip subcultures taken from the nuclear acceptor zone and all 4 hyphal plug and 12 hyphal tip subcultures from the zone of contact (Table 3). Three days after fusion, mtDNA of only the nuclear acceptor strainwas detected in all 3 tip (and 1 1hyphal plug) subcultures taken fromthe zone of contact and all 3 tip(and17 plug) subculturesofthenuclear acceptor zone. The entire colony, including samples from the nuclear donor zone (3 tip and 17 hyphal plug subcultures) were homogeneous for nuclear acceptor mtDNA.

mtDNA Replacement in Neurospora

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TABLE 3 mtDNA type in hyphal plugs and tips sampled from three zones at different times following fusion

1 2 3 4 5 6

7 8 9 10 1 1 1 2 1 3 1 4 1 5 1 6 171819 2 0 2 1 2 2 2 3 2 4 2 5 2 6

Zone of hyphal fusion cross sampledn mtDNA type detected

Nuclear acceptor Nuclear donor Mixtures

Days after fusion

Nuclear Contact donor or fusion zone zone

Nuclear acceptor zone

Total all zones

53 >3

(21) 2 I 7 (19) 20 (20) 14 (14)

21 (21) 20 (20)

54 (54)

53 >3

19 (21) 0 (20)

0 (19) 0 (14)

0 (21) 0 (20)

0 (54)

53 >3

0 (21) 0 (20)

2(19) 0 (14)

O(21) 0 (20)

0 (54)

I

Data of reciprocal interactions from seven separate matings of 2505A and 127 la monokaryotic strains of N. tetrasperma. Number of hyphal plug and tip samples with mtDNA type followed by the total number of samples taken in parentheses.

Colonies formed from hyphal plug and tip subcultures of the nuclear donor zone taken from 3 to 21 days after fusion never formed perithecia, indicating they were monokaryotic. Hyphal plug subcultures of the nuclear donor strain were backcrossed against the nuclear accepting strainand resulted in the formation of perithecia indicating the presence of donor strain nuclei. However, no perithecia formed when the donor subculture was backcrossed to the donor parent which is further evidence that mycelium of the nuclear acceptor did not grow over, under or through the nuclear donor and that the donor mycelium remained monokaryotic. Two days after fusion, two plugs exhibited mixed mtDNA types from the zone of contact. These mixtures were rare, were never observed in single hyphal tips and are probably due to sampling mixtures of donor and acceptor mycelia in the contact zone. Single ascospore analysis demonstrated maternal uniparental mtDNA inheritance following hyphal fusion mating: Single ascospores were harvested following hyphal fusion and analyzedasdescribed in methods. All 91 single-ascospore cultureshadthe nuclear accepting strain mitochondrial DNA (Figure 8). In total, 136 single ascospore cultures exhibited maternal uniparental inheritance (91 from hyphal fusion and 45 fromtrichogyne-conidial mating).

.

_._.ah.$

FIGURE8.-mtDNA of single ascospore progeny from hyphal fusion matings between nuclear donor 127 la and nuclear acceptor 2505A. Patterns represent Southern hybridization of radiolabeled 1271amtDNA to nylon membrane-bound, BglIIdigested, total DNA isolated from ascospore cultures. Lanes 25 and 26, parental mtDNA of 2505A (acceptor) and 1271a (donor) respectively. Lanes 1-24, ascospore progeny mtDNA which allshow the nuclear accep tor, 2505A. mtDNA pattern.

Maternal mitochondrial inheritance resulting from fusion of equal sized hyphae suggests that a mechanism other than a contribution bias favoring the female gamete (trichogyne) or an exclusion of conidial mitochondria from the trichogyne is responsible for the failure of the male to transmit its mitochondria. Unexpectedly, following hyphal fusion, nuclear acceptor mtDNA replaced nuclear donor mtDNAeven in nuclear donor mycelium. In addition to information on mitochondrial inheritance in N . tetrasperma, this study has provided information on patterns of nuclear and mitochondrial migration. Beginning with trichogyne-conidial mating, we will discuss these results in relation to studies of other fungi, and in light of models proposed to explain organellemigration,inheritance,andreplacement, and mitochondrial evolution. DISCUSSION Maternal uniparental mtDNA inheritance following trichogyne-conidial mating:Conidia from 2505A Our results show that mitochondrialinheritance fertilized trichogynes of 127 la andresulted in viable was identical in both trichogyne-conidial and hyphal ascospores but the reciprocal combination produced fusion matings. In all cases, only mitochondria of the nuclearaccepting parent were found in the ascoonly protoperithecia or false perithecia (DODGE1935; spores. T h e hypothesis that the trichogyne is responPERKINS1986). T h e failure to produce viableascosible for preventing inheritance of mitochondria from spores in this combination may be due to poor conithe conidial parent was not supported by our results: diation of 127 l a or female sterility of 2505A (personal mitochondrial inheritance was the same whether or communications from B. T U R N E R , N. RAJU and D. not trichogynes were involved in nuclear exchange. JACOBSON).

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When 2505A conidiafertilized 127 la protoperithecia, production of fertile perithecia was limited to the inoculated area. Nuclei, which moved from the conidia through trichogynes to the ascogonia, wereapparently unable to move from the ascogonia into surrounding monokaryotic hyphae. Our results show that these nuclei are capable of migrating through at least 4 cm of hyphae of the opposite mating type in hyphalfusion matings. Apparently, the trichogyne and ascogonium can restrict nuclear migration. This restriction is probably not due to the heterokaryon incompatibility function of mating type, as this function is inactive in N. tetrasperma (METZENBERG and AHLGREN 1973;JACOBSON 1992). Mitochondrial DNA ofthe protoperitheciating parent was detected in all45 ascospore progeny examined from trichogyne-conidium matings. Mitochondria of the male acting parent were neither detected in ascospores nor hyphae sampled next to fertile perithecia. This result of uniparental, maternal mtDNA inheritance is consistent with previous studies ofmtDNA inheritance in N. crassa using genetic (MITCHELLand MITCHELL 1952)and molecular approaches (MANNELLA,PITTENGER and LAMBOWITZ 1979; REICHand LUCK1966). The inactivity of the heterokaryon incompatibility function associated with mating type in N. tetrasperma (JACOBSON 1992) had no effect on the outcome of mitochondrial inheritance. Different patternsof nuclear migrationfollowing hyphal-fusion mating: Nuclear migration following hyphal fusion is a well documented phenomenon in many fungi (DOWDING and BULLER1940; DOWDING and HORand BAKERSPIEL1954; HINTZ,ANDERSON GEN 1988; MAY and TAYLOR 1988), including N. tetrasperma (DODGE1935). As described in the Introduction, inactivity of the vegetative-incompatibility function of the mating type locus in N. tetrasperma permits hyphal fusion, in contrast to N. crassa in which the same locus is active (JACOBSON 1992) and hyphae fuse and protoplasmic killing follows fusion thereby preventing the formation of stable heterokaryons between incompatible combinations of opposite mating type strains. In hyphal fusions between three different pairs of uninucleate strains of N. tetrasperma, we observed four different patterns of nuclear migration as inferred from patterns of perithecial formation: (1) no migration, (2) migration from one strain into the other,i.e., unilateral, (3) migration of each into the other at the same time, i.e., bilateral, and (4) migration of each into the otherbut not at the same time, i.e., mutually exclusive. Nuclear migration patterns were alsoinferred from a perithecial formation by DODGE(1 935), and general model to explain nuclear migration has been proposed by KEMP (1976). This model involves the production

of a nuclear migration inhibitor substance and response to theinhibitor, encoded by each nuclear genotype. Nuclear migration inonly one direction between strains can be due to only one partner producing a nuclear migration inhibitor, or only one being sensitive to the inhibitor produced by the other. Bilateral nuclear migration could be explained by either a lackofsensitivity to the inhibitor or the lackof inhibitor production by both partners or combinations of both. This model can account for lack of migration, unilateral migration and bilateral migration, but is not sufficient for explaining mutuallyexclusive nuclear migration. Additional factors that couldeffect the direction of migration include temperature and light (light affects nuclear migration inGelasinospora, DOWDING and BULLER1940), or even relative age or rates of development in the parental cultures, although parental cultures were ofapproximately equal age in all our fusion experiments. Mechanisms accounting for unilateral nuclear migration couldinvolve microtubule function asoband MORRIS served in Aspergillus nidulans (OAKLEY 1980; RAUDASKOSKI 1972) or nuclear destruction as observed following plasmodial fusion of sensitiveand and killer strains of Physarum polycephalum (BORDER CARLILE 1974; LANE and CARLILE 1979; SCHRAUWEN 1979). One nuclear type may escape destruction by having different microtubule associated proteins or patterns of (WEIL,OAKLEY and OAKLEY 1986), DNA methylation (MONK 1990). If one nuclear type escapes destruction by methylation, then it should be possible to use a methylation-sensitive restriction endonuclease to distinguishnucleiwith and without et al. 1990; STEIGERWALD, methylated DNA (PFEIFER PFEIFER and RIGGS 1990). Irrespective of the mechanism of nuclear migration, the process by which one nucleus is selected to migrate and the otherto remain stationary is unknown. Hyphal fusion mating resultsin rapid segregation of mtDNAtypeanduniparentalmtDNAinheritance: After hyphal fusion, the nuclear acceptor strain received nuclei from the nuclear donor but not mitochondria (Table 3). Migration of nuclei into one partner without concomitant mitochondrial migration hasalso been observed inbasidiomycetes, Coprinus cinereus (MAY and TAYLOR 1988), Agaricus bitorquis (HINTZ,ANDERSON and HORGEN1988), Armillaria bulbosa (SMITHet al. 1990), andStereum hirsuitum and S. complicatum (AINSWORTH et al. 1992). We found no support for ourhypothesis that fusion of equally sized hyphal tips, presumably with equal amounts of parental mtDNA, should lead to inheritance of both mtDNAs or their recombination. This differs from other laboratory experiments of other ascomycetes and basidiomycetes in which recombina-

mtDNA Replacement in Neurospora

tion of mtDNA or mitochondrial mosaics have been reported (BORSTand GRIVELL 1978; ZIN and BUTOW 1985; BAPTISTA-FERREIRA, CASSELTON and ECONOMOU 1985; ECONOMOU et ai. 1987; EARLet al. 1981; HINTZ,ANDERSON and HORGEN 1988; MAYand TAYLOR 1988; SMITH et al. 1990). Although all of our evidence is that one mtDNA type quickly prevails in the zone of fusion in N . tetrasperma, both mitochondrial typesmayhave existed togetherforshort periods of time or in unequal stoichiometries and have gone undetected by our methods. However, similar laboratory methods have detected mtDNA heterogeneity in other fungi (MAY and TAYLOR 1988; HINTZ,ANDERSONand HORGEN 1988; SMITHet al. 1990). Segregation of mitochondrial types usually occurs after several successive generations following laboratory crosses of many fungi (MANNELLAand LAMBOWITZ 1978; COSMIDIES and TOOBY 198 1; SRIPRAKASH and BATUM198 1;SILLIKER and YANand COLLINS1988; MIRFAKHRAI,TANAKA AGISAWA 1990; TREAT-CLEMMONS BIRKY and 1983); however, in nature, recent evidence suggests that rapid segregation of mitochondrial types occurs in the basidiomycete genus, Armillaria (SMITHet al. 1990). Small contributions of paternal mtDNA maybe undetected duetoeither insensitivityof detection methods (LANSMAN, AVISEand HUETTEL1983; MARTIN 1989) or small sample size (only 136 single ascospores were analysed for mtDNA type). Detection of minority mtDNA populations, represented by as little as one cell, inas little material as one spore isnow possibleusing the polymerase chain reaction (PCR, 1990). By using SAIKIet al. 1985; LEEand TAYLOR PCRLARSSON et al. (1992) were able exclude the presence of maternal deleted mtDNA from daughter cells and tisssues at a fractional levelofless than 1:100,000. PCR has been also applied to detecting paternal mtDNAfollowingcrossesofDrosophila (KONDO, MATSUURAand CHIGUSA 1992). and facilitates sensitive detection of DNA type in very large numbers (VIGILANTet al. 1989). Replacement of nuclear donor mtDNA: All of our evidence indicates that nuclei moved from the donor into theacceptor while mitochondria moved from the acceptor into the donor and replaced donor mtDNA (Figure 9). Our results provide further evidence which support genetic and biochemical results for Coprinus 1988) and Aspergillus nicinereus (MAYand TAYLOR dulans (OAKLEY and RINEHART1985) indicating that nuclei and mitochondria have different mechanisms for movement. Based on observation in other organisms, replacement can be due to (1) migration of one mitochondrionthroughoutthe recipient mycelium coupled with destruction of the other type, (2) an organelle replication advantage in one strain or (3) recombina-

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tion between mitochondria with the identifying features of one genome always being incorporated in the recipient. Uniparental mitochondrial inheritance observed in Physarum polycephalum (KAWANOet al. 1987; KAWANO and KURIOWA1989) may be due to elimination of one parental mitochondrial genotype in the zygotes and young developing plasmodia and a genomic restriction system hasbeen implicated (MELANDet al. 199 1). Destruction of paternal organelles has been observed in the zygotesofsomemammals and algae (BIRKY 1978). One mechanism proposed involves nucleases that cause the extensive destruction of one parent’s chloroplast (ct) genome in Chlamydomonas (SAGER and RAMANIS1973; SAGER,SANOand GRABOWY 1984; KUROIWA 1985). Similarly, uniparental ctinheritance in Chlamydomonas may also be due to destruction of one parent’s unmethylated ctDNA by a 1983; methylation sensitive nuclease(CHARLESWORTH BOYNTON et al. 1987) similar to epigenetic inheritance (MONK 1990). It is also possible that organelle DNA from both parents is destroyed but at different rates (CHIANG1976). However, extensivemethylation of ctDNA did not affect ct gene transmission (BOLENet al. 1982) and no evidence for methylation of mtDNA was found following uniparental mtDNA inheritance in Chylamydomonas(BECKERS et al. 199 1) supporting previous reports that themechanism of ctDNAinheritance must occur through some other mechanism probably due to alleles at or near the nuclear mating et al. 1987). locus (BOYNTON Recombination between mitochondrial molecules with the identifying features of one genome always being incorporated in the recipient has been previously observed in yeast (COENet al. 1970; DUJONand 1976) and Coprinus (CASSELTON and CONSLONIMSKI DIT 1972). Unidirectional movementofsequences from one mitochondrion to another in N . crassa has been referred to as polar recombination (MANNELLA and LAMBOWITZ 1979). However, our results cannot be explained by a type of “polar recombination,” gene conversion or unidirectional recombination seen in other fungi (STRAUSBERG and BUTOW 198 1 ; BUTOW, PERLMAN and GROSSMAN 1985; JACQUIER and DUJON1985; ZIN and BuTOW 1985; SKELLYand CLARK-WALKER 1990) because mtDNA sequences of either strain were able to replace the other depending on which strain peritheciated. Mixtures of mitochondrial DNA restriction fragment patterns were rarely found in hyphal plugs, and never detected in tips or ascospores which would be expected if some recombination event was responsible for replacement. Our logic, that the reciprocal nature of mitochondrial migration precludes some intrinsic feature of one mitochondrial molecule favoring it over the

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Time ofter contoct

other, can be applied equally well to replication advantage (PISKUR 1989) or organelle destruction. However, we cannot rule out some extrinsic feature, perhapsassociatedwith nuclear acceptance or female behavior, which confers a replication advantage or destructive ability onthe maternal mitochondrial DNA. It is also conceivablethat the state of differentiation of the two parental colonies may play an important role in determining which mitochondrial DNA is ultimately retained. The state of differentiation appears to correlate to an unexpected nonrandom mitochondrial DNA segregation in human cell hybrids (SHAY and ISHII1990). Nuclear gene products such as MGTl in yeast may play an important role in the transmission of mtDNA as mutations for MGTl have beenfound to reverse biased transmission(ZWEIFEL and FANCMAN 199 1). foreign Replacement of resident mtDNAwith mtDNA produces a novel combination of mitochondrial and nuclear genomes. The reciprocal combinationalso produced fertile ascospores and indicates that, at least in some combinations, exchange of organelles is possible without immediate consequence to viability in N. tetrasperma. This study is the first report of complete mitochondrial replacement following intraspecific mating of a filamentous fungus. Complete replacement of resident mtDNA by foreign mtDNA has been observed in heteroplasmic flies initially possessing endogenous and foreign mtDNA by intra-and interspecific transplantation of germ plasm in Drosophila melanogaster (NIKI, CHICUSAand MATSUURA1990) and may involved the nuclear genomes (MATSUURA,NIKI and

FIGURE9.-Cartoon of nuclear and mitochondrial movement and replacement following hyphal fusion mating in N. tetrasperma. Nuclear movement is indicated by - - - - - . Mitochondrial movement is indicated by o o o o 0.

CHICUSA1991, TSUJIMOTO, NIKI, and MATSUURA 1991; MATSUURA1991). These flies are fertile and the transplanted mtDNA is stably maintained in their offspring. Replacement of bovinemitochondrial DNA was demonstrated following pedigree analysis in 13 of 35 maternal lineages investigatedand occurred within one generation (KOEHLERet al. 199 1). Evolution and mitochondrial inheritance: Our experiments show that in trichogyne-conidialmatings only mitochondria of 127 l a would be found in ascospores but in hyphal fusion matings, mitochondria of both parents could be incorporated into ascospores, albeit not in the same interaction. This result is essentially the same for N . crassa, and supports the assump tion of mtDNA uniparental inheritance used for mtDNA evolutionary inference of Neurospora sf$. (TAYLOR and NATVIC 1989). Although we know that both mtDNAs can be transmitted to the next generation without hybridization, we do not know if there is a bias inthe proportionof each type that is transmitted in nature. Our understanding of mitochondrial inheritance in nature is clouded by our ignorance of fungal life history. We do not know how common each type of mating is in nature, or even the diversity of matings that are possible in nature. By analogy withN. crassa, it seems likely that the first, or primary, colonies of N . tetrasperma arising following fire come from heatactivated ascospores. Dikaryoticspores would produce colonies with perithecia whose ascospores would perpetuatethe mitochondria of theparent. The rare monokaryotic ascosporewould produce a potential mating partner that would be subject to a rain of

Replacement mtDNA

conidia from both dikaryotic and monokaryotic mycelia. It would seem unlikely that two primary, monokaryotic colonies could mate by hyphal fusion. However, if conidia formed by primary colonies could start secondary colonies,then many ofthese colonies would be haploid and have the opportunity to mate by hyphal fusion with other secondary colonies or with primary colonies. Furthermore, even dikaryotic primary colonies could act as partners in di-mon matings (DODGE and SEAVER 1938) similar to those in basidiomycetes (MAY and TAYLOR 1988). Of course, these haploid colonies would also be subjected to a rain of conidia from all colonies inthe vicinity. Unstable vegetative fusion can lead to nonparental mitochondrial-nuclear combinations in N . intermedia (COLLINSand SAVILLE1990) and to transmission of plasmids (GRIFFITHSet al. 1990) and the transfer of mitochondria between isolates of different vegetative incompatibility groups of Fusarium oxysporum (GoRDON and OKAMOTO 1992) indicates the importance of hyphal fusion interactions in the nuclear and extranuclear genetic structure of natural populations of fungi. The importance of the different mating strategies of N . tetrasperma to genetic exchange in nature are not well understood and should be tested in nature using a population oriented approach (e.g., SPIETH 1975). We would like to thank DAVIDPERKINS,BARBARA TURNER and DAVIDJACOBSON fortheir advice and discussions. We are also grateful to JOE HANCOCK, GEORGIANA MAY,TOMWHITE,and the late ALLAN WIISON for their careful reading of this manuscript and helpful suggestions. This project was supported in part by a U.S. Department of Agriculture Fellowship to S.B.L. and National Science Foundation BSR85-16513 to J.W.T.

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