Dominance and complementation relationships of conidial longevity mutants of Neurospora crassa

Mechanisms of Ageing and Development, 25 (1984) 79-89 Elsevier Scientific Publishers Ireland Ltd.

79

DOMINANCE AND COMPLEMENTATION RELATIONSHIPS OF CONIDIAL LONGEVITY MUTANTS OF NEUROSPORA CRASSA

KENNETH D. MUNKRES Laboratory of Molecular Biology and Department of Genetics, The University of Wisconsin, Madison, Wisconsin (U.S.A.) (Received May 9th, 1983) (Revision received November 15th, 1983) SUMMARY Previously we reported the occurrence of 16 linked conidial longevity determinant genes in Neurospora. Mutations of those genes are characterized by a reduction of longevity, a pleiotropic morphological defect, and deficiency of five antioxygenic enzymes. On the basis of the linkage and biochemical data, it was proposed that the genes are spatially and functionally redundant. The results of the present investigation support the hypothesis of functional redundancy. All of the mutants examined were dominant to wild type and failed to complement in heterokaryons. These results are discussed in terms of two molecular models of gene function. Key words: Neurospora; Longevity; Mutants; Dominance; Complementation; Conidia

INTRODUCTION In previous studies, 26 conidial longevity mutants (age-) were mapped at 16 genes on the right arm of one of the seven chromosomes, linkage group I [1 ] (Fig. 1). The mutants are characterized by an abnormally short liftspan, [2] a pleiotropic defect in colony morphogenesis, designated aer- [2] and deficiency of five antioxygenic enzymes [3]. There was a marked tendency for the map distance between the genes to be about 5 units. A model was proposed in which spatially repetitive, functionally redundant regulatory genes control the synthesis of the antioxygenic enzymes [1,3]. We speculated that such a situation might provide a mechanism for the amplification of regulatory signals for synthesis of the enzymes in response to changes in flux of free radicals and peroxides [3 ]. The objective of the present investigation was to test the hypothesis of functional genetic redundancy by examining the dominance and complementation properties of the mutant genes. The experimental evidence verifies the theoretical predictions that

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81 the mutant gene should be dominant and non-complementary, and support Fincham's theory [4] that dominance and complementation properties are related manifestations of gene-product interaction. A priori, mutation of any one of a number of redundant genes must be cis dominant to all of the others in a haploid to be expressed and, in a diploid, must also be trans dominant. Neurospora is haploid in the vegetative stage of the life cycle. Classical methodology does not permit direct determination of cis dominance of redundant genes; however, tmns dominance relationships can be determined in two ways; in duplication (partial disomic) strains and in forced heterokaryons [4]. We shall assume that the observed trans dominance indirectly indicates the cis dominance relationship. Viable non-tandem duplications of a chromosomal region are generated in crosses of non-reciprocal translocation strains to strains with normal chromosome arrangement, a fact that has been extensively exploited for establishing the linear order of linked genes by "duplication coverage" which, in effect, is a dominance test [5,6]. The particular strain employed here, T(IR~VIR)NM103, bears a non-reciprocal translocation of the majority of the right arm of linkage group IR to the right tip of linkage group VI [7]. Since the break point in IR is proximal to the age-1 complex [1,7], any one of the mutations of the 16 genes may be tested for dominance in heterozygous duplications generated from crosses to this translocation. Dominance was tested in both heterozygous duplications and forced heterokaryons to determine if the gene product is confined to the nucleus [4]. MATERIALSAND METHODS Stocks

The strains used to prepare forced heterokaryons for dominance and complementation tests are listed in Table I. These strains and the translocation T(IR-~VIR)A NM-103 (FGSC # 2137) were obtained from the Fungal Genetics Stock Center, Humboldt State University, Arcata, CA. The origin and characteristics of the defined wild-types Ela and A.1.9A and the age mutants were previously described [ 1-3 ]. All stocks were stored on culture slants at --20°C. Media and crosses

Vogel's [8] minimal medium.was supplemented with 1.5% sucrose (VM). Slants or plates contained 1.5% Difco Bacto agar. Plates of VM contained 0.8% sorbose and 0.1% sucrose (VSS). Crosses were prepared and viable progeny were selected from random ascospore plating as previously described [1,2]. Scoring the phenotypes o f the age mutants

The age mutants are distinguished from wild type by either their short longevity or

82 TABLE I STRAINS USED TO PREPARE FORCED HETEROKARYONS FOR DOMINANCE AND COMPLEMENTATION TESTS Strain a

trp-l, ylo-1 aur, nic-1, arg-13 aur, are-8 aur, arg-5 pdx-1

FGSC stock no. and mating type

Allele (isolation no.)

Linkage group

A

a

1207 3655

1208 3656

10575,30539 34508, 3416, RU3

11I,V1 IR

1814 1205 2014

1815 1206

34508, DH8 34508, 27947 37803

1R 1, I1 VI

aGenetic symbols: aur, aureseent (allele of albino-I); arg, arginineless;are-8, growth inhibited by a mixture of phenylalanine and tyrosine; nic, nieotinamideless; pdx, pyridoxineless; trp, tryptophanless; and ylo, yellow conidia.

by a pleiotropic defect of the ability of colonies to form aerial hyphae, the conidiophores, designated aer- [2 ]. The rate of aging, or longevity, is estimated by a differential survival test [2]. In this test, the ratio of the percentage survival of conidia after I0 days of aging (Ste) to the percentage viability before aging (So) is measured. The Slo/So value of defined wild types if 90 + 30; whereas that of the mutants is 50 or less. Purification and definition o f translocation genotype After subculturing the translocation stock on VM minimal slants, the differential survival test was performed. The value of Slo/So was 100%; equivalent to that of the defined wild-type Ela. The majority of the colonies failed to form aerial hyphae, resembling the pleiotropic phenotype of the age mutants, aer-. Only six aer ÷ colonies among 768 were observed; they were selected, pooled, subcultured and retested. The differential survival value was 90% and 85% of the colonies were aer ÷. The selected translocation stock was crossed to the wild-type Ela and random ascospores were plated. Among 117 colonies, 102 (87%) were aer +. Since wild-type cultures spontaneously mutate to aer- at a frequency of about 10%, it was concluded that the survival and colony genotype of the selected translocation is equivalent to wild type, age ° aer÷. Selection o f homozygous wild-type duplications In the cross of the translocation to normal sequence, viable progeny are of three types; normal, translocation and duplication sequences [5]. The latter is recognized by

83 female barrenness [5]. Amongst the aer÷ colonies from the cross of the selected translocation to wild type, about 11% were duplications. Such homozygous wild-type duplications were selected as controls for measurement of the phenotype of heterozygous duplications. Selection o f duplication progeny from random isolates Crosses of the duplication progeny as female parent to wild type were either barren or low in ascospore yield, as previously reported [7]. Otherwise, the homozygous wildtype duplications were indistinguishable from normal wild type in growth rate at 35"C or in slant or colony morphology. We observed that the duplications were completely male sterile on corn meal crossing agar. Since this characteristic is technically easier to score than female barrenness, duplication progeny were selected from random isolates by the following procedure. Colonies were isolated to small slants from random ascospore plating and cultured 7 days. Conidia were suspended in 1 ml of sterile distilled water and transferred to a "mating-type" plate with a cotton-tipped applicator (Q-tip) (Solon Mfg. Co., Solon, Maine). The mating-type plates were prepared as follows: Two 9-cm petri dishes, containing 20 ml of 2% corn meal agar (Baltimore Biol. Lab., Baltimore, MD) supplemented with 0.1% sucrose, were centrally inoculated with wild.type conidia of either mating type, wrapped in aluminium foil, and incubated at 250C for 6 - 7 days. (Essentially no aerial hyphae or conidia are formed under these conditions). A grid of 25 squares was drawn on the bottom of the plates with a felt pen. Conidia of various isolates were spotted on the grid and the plates were incubated 3--4 days at 25°C in the dark. Male-fertile strains formed 50-100 black perithecia; whereas the male-sterile duplications exhibited no mating reaction with either mating-type tester. Complementation in non-forced heterokaryons Paired mixtures of age mutant conidia (about 1 X 10 s of each) were made on 1-ml VM slants and incubated 3 days at 35"C in darkness followed by incubation for 4 days at room temperature in continuous white fluorescent light. Conidia of controls or putative heterokaryons were collected in water, diluted, and plated on VSS at a density of 100 or less per plate. The plates were incubated 3 days at 35"C in the dark followed by 5 days at room temperature in continuous fluorescent light. The frequency of colonies with the wild-type phenotype aer÷was observed. Estimate o f nuclear ratios in forced heterokaryons On the assumption that nuclei are distributed at random among the multinucleate conidia, the formulae for estimation of nuclear ratios were derived by Atwood and Mukai [9]. If the proportion of one type of nucleus in a heterokaryon is p and of the other (1 - p) then,

84

p= and

r(1 - - r) + a(n - - 2r)

n(1 - - r) r(1 - - r) + b ( n - - 2r)

1 --p =

n(1 - - r )

where r = proportion o f heterokaryotic conidia; a, b = proportion o f conidia homokaryotic for type 1 and type 2 nuclei; and n = average number o f nuclei per conidium. "In N . crassa, n does not differ between homo- and heterokaryotic conidia, it is usually safe to assume a figure o f 2.5" [8]. In a "forced h e t e r o k a r y o n " bearing different nutritional markers, the value o f r is defined by the proportion o f conidia that grow on minimal medium. The proportion o f homocaryotic conidia is determined b y plating on media supplemented with one or the other nutritional requirement [8]. RESULTS AND DISCUSSION Both the age and a e r phenotypes o f mutations o f four loci o f the age-1 complex were dominant to the wild-type allele in heterozygous duplication strains (Table II). In fact, TABLE II T R A N S DOMINANCE OF MUTANT PHENOTYPES OF FOUR LOCI OF THE age-1 COMPLEX IN HETEROZYGOUS DUPLICATION STRAINS (age-/age o) Duplication strain no.

age locus in duplication

Survival phenotype So

D-3-11 -15 -16 D4-17 D~-12 -24 D-31~ Controls: Mutant no. 3-10a 4.9A 6-18A 31 Wild-type (age °) Wild-type duplications: D-18-6 D-6-25-1 D-4-17-1

St.

No. colonies St./S o

aer ÷

Total

1.7 1.7 1,7 1.5 1.6 1.6 1.3

100 40 40 67 35 63 80

1 11 1 1 9.5 7.5 11

1 28 2 1 27 12 14

0 0 0 17a 0 25 a 0

174 162 120 312 144 260 260

1.7 1.5 1.6 1.3

73 100 100 100 100

38 52 28 40 90

52 52 28 40 90

0 1 0 0 90

1130 430 4640 1000 100

108 78 104

85 92 102

79 118 98

536 382 182

653 477 246

aSomatic recombinants(?), see text.

85

the survival of the duplications was significantly less than that of the haploid mutants alone. This effect cannot be attributed a non-specific consequence of chromosomal duplication because the survival of the homozygous wild-type duplications was equivalent to that of typical haploid wild types. One of two sister duplications containing a mutation of the a g e - l . 6 locus, and the duplication containing a mutation of the a g e - l . 5 locus yielded a small frequency of a e r ÷ colonies (Table II). Since the frequency was much greater than the frequency of such colonies in the haploid mutant controls, they apparently did not arise by reverse mutation of the a g e - locus in the duplication. Many of the a e r ÷ colonies in these two duplications were not morphologically equivalent to wild type. The formation of aerial conidiophores on wild-type colonies is radially symmetrical over the entire surface, whereas many of the a e r ÷ colonies in these duplications were asymmetrical, i.e. sectored for the two phenotypes. Turner [7l noted that euploid derivatives arise in vegetative culture of duplications prepared from the same translocation employed here, and concluded that they arise by mitotic recombination and haploidization. Six a e r ÷ colonies, selected from each of the two duplications, were found to remain duplications. In view of this result and the sectoring phenomenon, it appears that the present instability of these duplications may be a consequence of mitotic recombination without haploidization. Despite this complication, dominance of the mutations is established by the observations that the m a j o r i t y of the colonies of these exceptional duplications were a e r - , and that five other duplications yielded no a e r ÷ colonies among several hundred. Dominance was clearly indicated by the observation that the spontaneous mutation frequency in three homozygous wild-type duplications was about twice that of the haploid wild type (Table III). The experimental procedure and the interpretation of the dominance test are more TABLE III SPONTANEOUS MUTATION FREQUENCIES IN HOMOZYGOUS WILD-TYPE DUPLICATIONS aer+/aer÷ AND HAPLOIDWILD-TYPEaer+ Strain

Mutation f r e q u e n c y a (%) (aer--,aer-)

Duplications: D18 D4-17-1 D6-25-1 average

Wild-type Ela

X

$

n

18 26 20

5 2 8

6 3 6

21 10

5

6

aThe average (x) and standard deviation (s) of n measurements. For each measurement, about 100 colonies were examined.

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difficult with heterokaryons than with duplications. First, it is desirable to employ selective markers to be confident that heterokaryosis has occurred. Second, because mycelia and conidia are multinucleate, it is desirable to estimate the ratio of the two nuclear types to determine if the heterokaryon is "balanced"; a skewed nuclear ratio yields cells whose phenotype resembles that of the majority. Finally, because of random distribution, some cells will have skewed nuclear ratios even when the average proportions of the two types are equal. A justification for dominance tests with heterokaryons when the dominance relationship is known in a heterozygous duplication is to determine if the gene products are confined to the nucleus. Conldia of the heterokaryon (a; age-l.5, arg-S) (a; age°; trp-l) were plated on minimal and tryptophan-supplemented media (Table IV). The frequency of heterokaryotic conidia was 0.49. The frequency of the two nuclear types, computed as described in Methods, was 0.52 and 0.48; hence, the heterokaryon was balanced. The phenotype of the auxotrophs alone was aer+; whereas only 8 of 116 colonies of the heterokaryon were clearly aer ÷. About 20% of the 116 colonies were sectored tor the aer phenotype. The small proportion of wild-type and sectored colonies probably reflects a skewness of the nuclear ratio in favor of wild type, either present initially in the conidia or arising during germin. ation and growth. Despite these complications, it is reasonably clear that mutant no. 4 of the age-l.5 locus is dominant. Since this mutant also proved to be dominant in a duplication (Table II), it appears that the mutant gene product is not confined to the nucleus. The results with another forced heterokaryon of mutant no. 8 of the age-l.2 locus with wild type revealed that the majority of heterokaryotic conidia gave rise to pure aer- colonies (Table IV), indicating dominance of the mutant phenotype. The results of one set of complementation tests of pairwise mixed cultures of mutants representing seven of the genes of the age-1 complex are summarized in Table V. Since no aer ÷ colonies were observed among several hundred in any of the mixtures, it appears that the genes are non-complementary, Le. functionally redundant. These experiments were performed with mutants of the a mating type. The experiments were repeated with the same mutants of the A mating type; again no complementation was observed. TABLE IV DOMINANCE OF MUTATIONSOF TWO GENES OF THE age-1 COMPLEX IN FORCED HETEROKARYON$ WITH WILD TYPEa age locus in heterokaryon

Mutant no. in heterokaryon

Frequency of heterokaryotic conidia

No. colonies Total

aer÷

age-l.5 age.l.2

4 8

0.49 0.11

116 103

8 11

aThe heteroksxyon was (a, age-l, arg-5) (a, age °, trp.1).

Differential survival (S,o/SJ

0

87 TABLE V NON-COMPLEMENTATION OF MUTANTS OF THE age-1 COMPLEX The experimental procedure is described in Methods. The data are arranged in a "complementation matrix" [8]. Numbers along the diagonal are homokaryotic controls. A dash indicates the test was not performed. age locus

Mutant

no.

no.

No. aer+ colonies/total observed Mutant no.

1.3 1.7 1.5 1.4 1.6 1.6

2 3 4 5 6 13

2

3

4

5

6

13

1/463

0/116 0/481

0/130 0/81 1/162

0/113 0/117 0/142 0/311

0/206 0/54 0/87 0/147 0/601

0/14 0/15 0/11 0/93

Heterokaryosis may not occur if strains are heterogeneous for heterokaryon incomparability genes [911 this was not expected because the mutants were all induced and backcrossed into the same wild type. Furthermore, the formation o f heterokaryons with either wild type or other age mutants by the use o f forcing nutritional markers[Tables IV and VI] indicates that heterokaryon incomparability does not account for the failure to complement. TABLE VI NON-COMPLEMENTATION OF aer PHENOTYPE OF age-1 MUTANTS IN FORCED HETEROKARYONS A loop of conidia of two strains was mixed on a 1-ml VM agar slant. Slants receiving a strain beating the are-8 marker contained a mixture of phenylalanine and tyrosine to inhibit its growth. Cultures were incubated for 3 days at 35°C in the dark followed by 4 days at room temperature in continuous cool white fluorescent light. Heterokaryon formation in the mixtures of 1.3 + 1.8 and 1.2 + 1.3 was verified by dominance of the pigment marker aur ÷. Conidia were collected in water, diluted and spread on VSS agar plates containing phenylalanine and tyrosine. Three plates of each heterokaryon were prepared with about 100 conidia per plate. The plates were incubated 3 days at 350C followed by 4 days at room temperature in continuous light. Each culture formed many colonies, indicating heterokaryotic conidia; however, no wild-type aer ÷ colonies were observed, indicating non-complementation of the age loci with respect to the pleiotropic aer phenotype. age-1 loci in heterokaryon

Mutant no. in het erokaryon

Heterokaryon component markers

nic-1, arg-13, + aur, are-8 aur, arg-5 + aur, are-8 aur, aro-8 +aur, arg.5 aur, arg-5 + aur, are-8 aur, arg-5 + nit-l, arg-13

1.3 + 1.8

10 + 16

1.2 + 1.5

8 + 4

1.1 + 1.2 1.2 + 1.8 1.2 + 1.3

12 + 8 8 + 16 8 + 10

88

Heterokaryons of five combinations of the age genes were prepared with enforcing nutritional markers (Table VI). No complementation of the aer phenotype was observed among several hundred colonies of each of the heterokaryons. The mutants grow normally in VM liquid medium. The growth of 15 paired mixtures of six mutants in liquid medium was tested (Table VII); 'six of the mixtures failed to grow. One interpretation of these results is that heterokaryons of these mutants undergo "negative complementation". Alternatively, the cells may excrete metabolites that are mutually inhibitory. Whatever the interpretation, the results indicate that not all of the mutations are functionally identical, at least with respect to this phenomenon. A molecular model of dominance was suggested by Fincham and Day [4]. If a functional gene product is a dimer or higher multimer of one polypeptide, and the hybrid protein formed in the heterozygote is non-functional or nearly so, then insufficient wild-type protein may be formed and the phenotype of the heterozygote is mutant. This same model was used by Fincham to explain the phenomenon of negative complementation in which the phenotype of a heterokaryon, consisting of one complete and one partial ("leaky") mutant or two leaky mutants, is completely mutant. In view of the fact that the age mutations are dominant, such a model might also explain the failure of the age mutants to complement. In view of the functional redundancy hypothesis, any one mutation of the 16 genes of the age-1 complex must be a priori be cis dominant to its neighbors to be expressed. The observation that all of the four mutant loci tested are trans dominant in duplication heterozygotes supports that view. If the functionally redundant genes are all expressed under the experimental conditions, then Fincham's model of dominance would require not only both cis and trans formation of non-functional hybrid protein, but also excessive production of the mutant polypeptide in order to titrate out all of the wild-type polypeptides. A plausible working hypothesis is that the age genes encode proteins whose function is

TABLE VII NEGATIVE COMPLEMENTATIONOF GROWTHOF CELL MIXTURES OF age-1 MUTANTS Paired mixtures of mutants nos. 2, 3, 4, 5, 6, 8 and 12 were made with a loop of conidia in 1 ml of VM liquid medium. The cultures were incubated at 35°C and observed after 18 and 36 h. The mixed cultures in the table failed to grow, whereas other mixtures and each mutant alone grew vigorously. Mutant no. in mixture exhibiting negative complementation

4+2 4+5 4+6 4+8 12+5 12+6

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the regulation of the synthesis of the five antioxygenic enzymes [3], If that, is the case, another molecular model o f dominance m a y be considered. If the genes encode positiveacting regulatory proteins which bind to promoter sites adjacent to cistrons encoding the enzymes, then either overproduction or increased affinity o f the non-functional regulatory protein for the promoter sites could block transcription o f the enzyme cis-

trons. ACKNOWLEDGEMENTS This research was supported by the College o f Agriculture and Life Sciences, the Graduate School, and the National Institute on Aging (AG 00930). Contribution no. 2655 from the Department o f Genetics. REFERENCES 1 K.D. Munkres and C.A. Furtek, Linkage of conidial longevity determinant genes in Neurospora crassa. Mech. Ageing Dev., in press. 2 K.D. Munkres and C.A. Furtek, Selection of conidial longevity mutants of Neurospora crassa. Mech. Ageing Dev., in press. 3 K.D. Munkres, R.S. Rana and E. Goldstein, Genetically determined conidial longevity is positively correlated with superoxide dismutase, catalase, glutathione peroxidase, cytochrome c peroxidase, and ascorbate free radical reductase in Neurospora. Mech. Ageing Dev., 24 (1984) 83-100. 4 J.R.S. Fincham and P.R. Day, Fungal Genetics, 3rd edn., Blaekwell Scientific Publications, Oxford, 1971, pp. 258-259. 5 D.D. Perkins and E.G. Barry, The cytogenetics of Neurospora. Adv. Genet., 19 (1977) 133-285. 6 R.L. Metzenberg, M.K. Gleason and B.S. Littlewood, Genetic control of alkaline phosphatase in Neurospora: the use of partial diploids in dominance studies. Genetics, 77 (1974) 25-43. 7 B.C. Turner, Euploid derivatives of duplications from a translocation in Neurospora. Genetics, 85 (1977) 439-460. 8 R.H. Davis and F.J. deSerres, Genetic and microbiological techniques for Neurospora crassa. Methods Enzymol., 17A (1970) 79-143. 9 K.C. Atwood and F. Mukai, Nuclear distribution in conidia of Neurospora heterokaryons. Genetics, 40 (1955) 438-443.