Induction and genetic characterization of ultraviolet-sensitive mutants in the elm tree pathogen Ophiostoma ulmi (sensu lato)

Mycol. Res.

98 (8), 943-953 (1994)

943

Printed in Grellt Britain

Induction and genetic characterization of ultraviolet-sensitive mutants in the elm tree pathogen Ophiostoma ulmi (sensu lato)

LOUIS BERNIER! AND MARTIN HUBBES 2 1 2

Centre de recherche en biologie forestiere, Faculte de foresterie et de geomatique, Universite Laval, Cite universitaire, Quebec, Canada, GlK 7P4 Faculty of Forestry, University of Toronto, Toronto, Ontario, Canada, M55 383

The lethal and mutagenic effects of ultraviolet (uv) irradiation on Ophiostoma ulmi sensu lato were tested. Exposure to uv rays increased the &equency at which benomyl-resistant mutants were recovered in five wild-type strains representing the non-aggressive species 0. ulmi and both Eurasian and North American races of the aggressive O. novo-ulmi. Treatment of yeast-like cells hom wildtype strain MH 75 and laboratory strain LB 44-R-2 with the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine allowed the recovery of four uv-sensitive mutants. All mutants were photoreactivable, whereas one strain was resistant to caffeine. Meiotic analysis provided evidence that the four strains carried non-allelic mutations. The uvs1 and uvs3 loci were linked and found to occur on linkage group I. The uvs2 and uvs4 loci were assigned to linkage groups III and IV, respectively. Two of the uv-sensitive strains also appeared to be hyperrnutable and might therefore be useful for the induction of additional types of mutants in O. ulmi sensu lata.

The ascomycetous fungus Ophiosloma ulmi sensu lalo (s.1.) is responsible for the two Dutch elm disease pandemics which have occurred during this century, and is composed of three distinct entities: the non-aggressive (NAG) subgroup, also known as 0. ulmi, and the two races, Eurasian (EAN) and North American (NAN), of the aggressive subgroup also referred to as O. novo-ulmi (Brasier, 1991). Studies on 0. ulmi s.1. have understandably focused on its pathogenicity to elm trees, and have led to the formulation of several hypotheses on the mechanisms by which the pathogen kills its host So far, however, no gene product has been conclusively identified as a major contributor to pathogenicity. Genetic analysis of many species of tree pathogenic fungi is impeded by problems such as the difficulty of culturing the pathogen in the laboratory, the absence of a sexual stage in vilro or even in vivo, or the lack of reliable bioassays for pathogenicity testing. Ophiostoma ulmi s.l. represents an exception, as it is well suited for both classical and molecular genetic studies (Brasier, 1988; Bernier, 1993). It is a heterothallic species with two mating types, A and B, determined by two alleles at a Single locus (Brasier, 1984). Perithecia will form as a result of a successful cross between sexually compatible individuals. Although they are rarely found in nature, perithecia are obtained relatively easily in vitro, either by pairing of mycelia with different mating types, or by controlled fertilization of protoperithecia of a recipient strain with conidia or yeast-like cells of a donor parent (Brasier, 1984). Mendelian analysis of naturally occurring variants has shown that pathogenicity in 0. ulmi s.1. is controlled by a predominantly additive polygenic nuclear gene system (Brasier & Gibbs, 1976; Brasier, 1987, 1988; Kile

& Brasier, 1990), and has allowed the identification of one

major pathogenicity gene in the EAN race (Brasier, 1987). Meiotic analysis of laboratory strains carrying spontaneous and induced mutations is proViding information on linkage groups (Webber, Mitchell & Smith, 1986; Mitchell, 1987; Bernier & Hubbes, 1990a, b), whereas pulse field gel electrophoresis has allowed the separation of chromosomes and the comparison of molecular karyotypes (Royer et al., 1991; Dewar & Bernier, 1993). The development of an efficient genetic transformation system based on hygromycin resistance (Royer el al., 1991) has added another tool for the investigation and manipulation of the pathogen. Finally, naturally occurring markers such as vegetative incompatibilitytype loci and restriction fragment-length polymorphisms of nuclear and mitochondrial DNA have proved to be useful for population studies (Brasier, 1984; Hintz et al., 1991, 1993; Jeng et al., 1991; Bates, Buck & Brasier, 1993 a, b; Brasier el al., 1993). We have previously reported the use of an alkylating agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), to induce mutations in yeast-like cells of O. ulmi 5.1. (Bernier & Hubbes, 1990 a). Although treatment with MNNG provided several useful markers, some phenotypes such as adenine- or lysine-deficient mutants were consistently recovered at higher frequencies, whereas others could not be recovered. In addition, the use of MNNG has its drawbacks: exposure to this chemical often results in comutation, the simultaneous induction of mutations in closely linked genes (Cerda-Olmedo & Ruiz-Vazquez, 1979). Moreover, MNNG is a potent carcinogen, which must be handled and disposed of with great care. Therefore, we chose to test another mutagen, ultraviolet

944

Mutants of Ophiostoma ulmi sensitive to uv (uv) light, which has been widely used in genetic studies and manipulations of micro-organisms, is safe and reproducible in its effects (Normansell, 1982). Studies on prokaryotes and eukaryotes have shown that the effectiveness of uv mutagenesis is reduced by the ability of living organisms to repair uv-Iight-induced damage to their DNA. This is done by a variety of mechanisms operating either at the pre-replicative or post-replicative level. While some mechanisms, such as photoreactivation and excision repair, have been described as error-proof, there is also evidence for the occurrence of error-prone repair (or damage tolerance) as part of the more general SOS response (Saunders, Allsop & Holt, 1982; Lewin, 1985; Bernstein & Bernstein, 1991). Enhanced mutation ratios can thus be obtained by blocking the error-proof pathways while leaving the errorprone systems intact. Here we describe protocols for the inductions of mutants in O. ulmi 5.1. by uv irradiation. The sensitivity and mutability of wild-type strains representing different biotypes were first evaluated and compared. Attempts were then made to obtain strains which would be hypersensitive and hypermutable, following chemical inhibition of the excision-repair pathway or by mutation of DNA repair genes.

MATERIALS AND METHODS Strains and culture media The lethal and mutagenic effects of uv radiation were first tested against five wild-type strains: 0. novo-ulmi strains MH 75 (NAN), VA (NAN) and T 88 (EAN); and O. ulmi strains Q 412 and S II6. These strains had been previously characterized with respect to pathogenicity, cultural characters, and electrophoretic protein and isoenzyme patterns (Takai, 1980; Bernier, ]eng & Hubbes, 1983; ]eng & Hubbes, 1983; ]eng, Bernier & Brasier, 1988). Ultraviolet-hypersensitive mutants were recovered from strains MH 75 and LB 44-R-2. The latter is a laboratory strain derived from a series of crosses between strain VA and mutants derived from strain MH 75 following treatment with MNNG (Bernier & Hubbes, 1990a). Strain LB 44-R-2 has an NAN-background but carries two mutant alleles, BENIR-1 and nic1-I, conferring increased tolerance to benomyl and requirement for nicotinamide, respectively. Additional wild-type and laboratory strains, all with an NAN background, were used in heterokaryon tests and sexual crosses. Formulations for complete (CM), minimal (MM) and crossing media were as described previously (Bernier & Hubbes, 1990 a). Strains, genotypes and phenotypes are deSignated according to Yoder, Valent & Chumley (1986). Mating-type alleles are designated Amt and Bmt (Brasier, 1988). The gene symbols ade, arg, Iys, met, nic and ura represent requirements for adenine, arginine, lysine, methionine, nicotinamide and uracil in auxotrophic mutants; mt and BENR indicate mating type and tolerance to benomyL respectively.

Sensitivity to uv of wild-type strains In a preliminary experiment, pieces of mycelium (ca 5 mm 2 ) were taken from the periphery of a culture of strain MH 75

and transferred on to solid CM supplemented with 0'01 % (w Iv) sodium deoxycholate (SD) to induce colonial growth (Bernier & Hubbes, 1990a). Six plates were prepared, each one containing 14 samples. After 3 d of incubation at 25°C in darkness, the colonies were replica-plated on to two sets of plates of CM + SD. One set of replicas was placed under a Philips 30 W germicidal uv lamp (2'4] m- 2 S-l at 254 nm), at a distance of 57 cm from the lamp, which had been prewarmed for 15 min. The lids were carefully removed and the conidia were exposed for periods from 10 to 180 s. The lids were put back and the plates returned to the incubator. Irradiation and subsequent manipulations were carried out in the absence of visible light to avoid photoreactivation. The remaining set of replicas was kept as unirradiated controls. The effects of photoreactivation and caffeine on the sensitivity and mutability of strain MH 75 were also tested. Yeast-like cells from a 7-d-old culture in liquid MM were transferred into fresh liquid MM at a concentration of 4 x 10 6 cells ml- 1 . After 40 h incubation at 25° on a rotary shaker (100 rpm), equal amounts of exponentially growing cultures were aseptically dispensed into two empty Erlenmeyer flasks. Caffeine, which inhibits light-independent excision repair, was then added to one of the cultures at a final concentration of 0'5 mg ml- 1 and both samples were incubated for an additional period of 30 min (Fong & Bockrath, 1979). The cultures were harvested separately, filtered through eight layers of cheesecloth, and cells were washed twice by centrifugation at 4800 rpm and resuspended in sterile distilled water. Survival rates were measured by plating cells at concentrations ranging from 200 to 2000 cells plate-Ion CM agar or on CM supplemented with caffeine at 0'5 mg ml- 1 (for caffeine-treated cells). Forward mutation frequencies were examined by seeding cells at high concentrations (1-10 x 10 7 cells ml- 1 ) on CM agar (or CM + caffeine) supplemented with benomyl at 1'2 I-lg ml- 1 (Bernier & Hubbes, 1990a). In all treatments, plates were exposed to uv rays for 5-100 s, with three replicates per treatment. The effect of photoreactivation on survival was tested by incubating culture plates under fluorescent light for 50 min, immediately after uv irradiation (Chang & Tuveson, 1967). Dose-response curves were established for other strains from the NAG, EAN and NAN biotypes of O. ulmi 5.1. Liquid cultures in CM were prepared as described earlier. Irradiation was carried out in the dark and without caffeine, with three replicates per treatment.

Induction and recovery of uv-hypersensitive mutants Exponentially growing cultures of strains MH 75 and LB 44R-2 were prepared in liquid MM (MH 75) or nicotinamidesupplemented MM (LB 44-R-2) and mutagenized as described by Bernier & Hubbes (1990a). Briefly, yeast-like cells were filtered through eight layers of cheesecloth, washed twice by centrifugation, resuspended in 0'067 M phosphate buffer (pH 7'5) at 2 x 10 7 cells ml- 1 and treated with MNNG for 90 min, in the dark and with constant agitation. The reaction was stopped and the mutagen washed off by two rounds of centrifugation in cold modified Fries' medium (pH 8'0) supplemented with sodium thiosulphate. The cells were

L. Bernier and M. Hubbes

resuspended in sterile distilled water and plated on to solid CM+SD. After 4-7 d of incubation, survivors were hand-transferred on to fresh CM + SO, with up to 45 samples plate-I. A total of 1584 survivors was recovered from MH 75, whereas 264 colonies were obtained from strain LB 44-R-2. The colonies were incubated at 25° for 3 d before they were replicatransferred on to two sets of plates containing CM + SO. One set was kept as control, whereas the second set was uvirradiated for 60 s. After 2-4 d of incubation, the control and irradiated plates were compared and putative uv-sensitive (uvs-) mutants were hand-picked, subcultured, and re-tested in the same way. Characterization of uvs- mutants Dominance and allelism of putative uvs- phenotypes were investigated on CM and MM agar by the streak method of Cox & Barry (1968). Putative heterokaryons were replicatransferred on to two sets of plates. One set was irradiated for 30-120 s. Dose-response curves were also established for putative mutants, with three replicates per treatment. Survival was measured on CM agar, whereas mutation frequencies were estimated by monitoring the emergence of benomylresistant mutants or of prototrophic colonies (for uvsphenotypes from LB 44-R-2). The response of uvs- strains to photoreactivation and caffeine was investigated as described earlier. The sensitivity of wild-type and mutant strains to ethyl methane sulphonate (EMS) was also determined. Exponentially growing cultures in liquid CM were filtered through eight layers of cheesecloth, washed by centrifugation and resuspended in 4 ml 0'05 M phosphate buffer (pH 7'0), at 4 x 10 7 cells ml- I and treated with 0'1 M EMS for 30 min. One ml of 10% sodium thiosulphate was then added, the cells were centrifuged once, resuspended in sterile distilled water and plated on to solid CM + SO for the determination of survival rates. Three replicates were used for each treatment. The meiotic segregation of uvs+ and uvs- phenotypes was studied by crossing uvs- mutants with sexually compatible uvs+ strains. Additional crosses were carried out with laboratory strains carrying well-characterized genetic markers in order to map the UV5 loci on linkage groups. The procedure for meiotic analysis has been described before (Bernier & Hubbes, 1990a, b). Induction of additional mutants with uv light The uvs- strains derived from MH 75 and LB 44-R-2 were uvtreated in an attempt to induce and recover auxotrophs. Exponentially growing cultures were resuspended in sterile distilled water at a concentration of 1 x 10 7 cells ml- I . Ten ml of each suspension was aseptically transferred into a plastic Petri dish and the samples were irradiated for up to 60 s. An aliquot of the irradiated suspensions was kept for survival counts, whereas 5 ml was transferred into 125 ml Erlenmeyer flasks covered with aluminium foil and containing 50 ml of sterile liquid MM supplemented with chloramphenicol, streptomycin sulphate and cycloheximide at 30, 100 and 100 J..lg ml-l, respectively (Brasier, 1981; Miller, Sands & Strobel, 1981). The flasks were incubated on a rotary shaker 60

945 (100 rpm) for 5 d and auxotrophic mutants were recovered

after nystatin enrichment (Snow, 1966), following the modified procedure of Bernier & Hubbes (1990a) for O. ulmi. Specific types of auxotrophs were obtained by plating nystatin-treated cells on to MM supplemented with individual bases or amino acids. Colonies growing on these media were replica-plated on to MM agar and screened for auxotrophs. Auxotrophs with mutations at either adel or ade710cus could be recovered directly from CM agar due to the occurrence of a pink coloration in colonies carrying these mutations (Bernier & Hubbes, 1990a, b; Bernier, unpub!. results). Statistical analysis Unless indicated otherwise, means were subjected to an analysis of variance (ANOVA) using the Statistical Analysis System (SAS Institute Inc., 1989). Standard mathematical transformations were applied to variates which did not meet the requirements for ANOVA: survival and mutation rates were transformed to arcsin and log, respectively. Following ANOVA means were compared and ranked by the WallerDuncan k-ratio t test.

RESULTS Sensitivity of wild-type strains to uv radiations The sensitivity of strain MH 75 to uv radiation was first assessed qualitatively by exposing replica-plated conidia. No visible effect was detected for exposure periods of 60 s or less. Longer irradiation times, however, reduced the germination of conidia on CM (Fig. 1). The response of strain MH 75 was also analysed quantitatively by exposing known concentrations of yeast-like cells and monitoring both survival and forward mutation frequencies. The survival curve exhibited a broad shoulder when both light-dependent- and dark-repair mechanisms were allowed to operate (Fig. 2). A shorter shoulder was observed in the absence of photoreactivation. Pre-incubation and exposure in the presence of caffeine resulted in a significant decline in survival at low doses. The negative effect of caffeine was only partly compensated by photoreactivation. In the absence of photoreactivation, caffeine induced a slight but not significant increase in the frequency at which benomyl-resistant phenotypes were recovered after exposure times of up to 30 s. At increased uv doses, mutants were recovered at a higher frequency in the absence of caffeine. Comparison of dose-effect curves and mutation rates to benomyl resistance among the three biotypes of 0. ulmi 5.1. showed that EAN strain T 88 was the most resistant and least mutable, whereas NAG strain Q 412 was the most sensitive and mutable (Fig. 3 a, b). Induction, recovery and characterization of uvs- mutants Fifteen putative uvs- phenotypes were recovered after MNNG mutagenesis of strain MH 75, at a dose that killed 90% of the original cell population. One additional mutant was derived from strain LB 44-R-2. Mutant colonies growing on CM agar were detected by their higher degree of sensitivity to a 60 s irradiation treatment (Fig. 4). Putative uvs- mutants were subcultured and re-tested: four strains still expressed a mutant MYC 98

Mutants of Ophiostoma ulmi sensitive to uv

946

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Fig. 1. Survival of O. ulmi 5.1. wild-type strain MH 75 (uvs+) to uv irradiation. Colonies growing on CM agar (0) were replicatransferred on to two sets of CM plates. One set of replicas (I) was irradiated for periods ranging from 10 to 180 s. Growth of colonies after exposure to uv light was compared with growth of control (C) replicas.

phenotype. Three of these strains, designated LB 04870l, LB 048709 and LB 048712, were derived from strain MH 75, whereas mutant LB 048714 originated from strain LB 44-R-2. The mutants were morphologically indistinguishable from their progenitors. Irradiation of colonies on CM agar indicated the occurrence of different degrees of sensitivity to uv and EMS among the mutants (Table I). Mutant strain LB 048709 was highly sensitive to EMS; a significant different was also observed between strain LB 048714 and its wild-type progenitor, LB 44-R-2. Comparison of uv dose-response

curves (Fig. 5) showed that, in the absence of photoreactivation, strain LB 048701 was slightly more sensitive than strain MH 75, whereas the survival of strains LB 048709 and LB 048714 decreased exponentially at low doses. Strain LB 048712 displayed an intermediate level of sensitivity. Most complementation tests on CM agar yielded negative results. A heterokaryon formed on MM agar between uvsmutant LB 048714 (nic1-1 BENIR-1) and uvs+ strain LB 108511 (adel-1 benls-1) had a uvs+ phenotype. PrototrophiC heterokaryons formed on MM agar between strain

L. Bernier and M. Hubbes

947

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Fig. 2. Lethal and mutagenic effects of uv radiations on 0. ulmi s.l. wild-type strain MH 75. Survival (--) was estimated under conditions that either stimulated or inhibited error-proof mechanisms. ./::" Photoreactivation; 0, photoreactivation + caffeine; 0, no photoreactivation, no caffeine; D, no photoreactivation, caffeine. Mutability (---) was assessed by monitoring the frequency of forward mutations to benomy\, in the absence of photoreactivation. . , Caffeine; e, no caffeine. Values are shown±s.E. where these exceed the dimensions of the symbols. For a given exposure time, survival (small letters) or mutability (capital letters) values were not significantly different if followed by the same letters, according to Waller-Duncan k-ratio t test (P = 0'05) following ANOVA. LB 048714 and ade- phenotypes derived from uvs- strain LB 048701 could not be replica-plated satisfactorily on MM. Prototrophic heterokaryons were also obtained in liquid MM, but the cultures grew slowly and failed to germinate after they were seeded on MM agar. The effect of caffeine and of photoreactivation was tested on uvs· strains in order to identify the repair mechanisms affected by the mutations (Fig. 6). The four strains tested were photoreactivable. Strain LB 048701 showed no sensitivity to caffeine. This strain was also slightly more resistant to benomyl than wild-type MH 75; the mutability of both strains.was thus assessed and compared on medium in which the concentration of fungicide was increased to 1'8 I-lg ml- I . Strain LB 049701 was not Significantly more mutable than MH 75 (Table 2). The other uvs- strains were moderately to highly sensitive to caffeine. The mutability of strains LB 048709 and LB 048712 appeared comparable or slightly lower than that of the wild type. Strain LB 048714 was highly mutable: the recovery frequency of nic+ revertants increased nearly 200-fold after only 10 s of exposure to uv rays and was significantly higher than the value obtained for the wild-type progenitor, LB 44-R-2, at a comparable level of survival (Table 2). The spontaneous mutation rate also differed significantly between the two strains.

Meiotic analysis of uvs mutants Random FI progeny were extracted from perithecia resulting from matings between uvs- mutants and sexually compatible

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Fig. 3. Comparison of the lethal (a) and mutagenic (b) effects of uv radiations on different biotypes of O. ulmi 5.1. Both survival and rates of mutation to ben R phenotype were assessed in the absence of photoreactivation and caffeine. and e, NAN O. novo-ulmi strains MH 75 and VA, respectively; ./::" EAN O. novo-ulmi strain T 88; D and . , NAG O. ulmi strains Q 412 and S 116, respectively. Values are shown ± S.E. where these exceed the dimensions of the symbols. For a given exposure time, values were not significantly different if followed by the same letters, according to Waller-Duncan k-ratio t test (P = 0'05) following ANOV A.

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uvs+ strains. With the exception of crosses involving LB 048701, the frequency of uvs+ and uvs· phenotypes among the progeny fitted a 1: 1 ratio (Table 3). The four uvsmutations appeared to be non-allelic, as indicated by the recovery of uvs+ recombinants in crosses between uvsparents (Table 4). Both uvsI and uvs310ci showed linkage with markers on linkage group I, whereas uvs2 was tightly linked to the ade7 locus on linkage group III. Locus uvs4 was not linked to any of the markers tested and was aSSigned to a new linkage group, no. IV (Fig. 7). 60-2

Mutants of Ophiosfoma ulmi sensitive to uv

948

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Fig. 4. Screening of colonies from 0. ulmi 5.1. laboratory strain LB 44-R-2 (uvs+) for MNNG-induced uvs- phenotypes. Colonies were replica-plated on to CM agar. One set of replicas (I) was irradiated for 60 s. Growth of colonies following exposure to uv light was compared with growth of control (C) replicas. The arrow indicates a putative uvs- mutant. Table 1. Tolerance of wild-type and uv-sensitive strains of 0. ulmi 5.1. to uv radiation and to EMS

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Uv irradiation Length of exposure (s) Strain

Phenotype Origin

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20 40 60 100 Survival (%t ~

uvs+ MH 75 LB 048701 uvs LB 048709 uvs LB 048712 uvs LB 44-R-2 uvs+ LB 048714 uvs

MH 75 MH75 MH 75 MH 75 Laboratory strain LB 44-R-2

+t+ + ± + + ± + + + ±

90'0 86'6±5'8 a 6'1±1'2 b 79'9±5'6 a 99'9±0'2 A 78'6±8'7 B

• With the exception of MH 75, entries show mean ±SE, Means were not significantly different if followed by the same letters according to Waller-Duncan k-ratio t test following ANaYA (P = 0'05). Small letters refer to comparisons among uvs- strains derived from MH 75, whereas capital letters indicate comparison between LB 44-R-2 and LB 048714. t Growth of colonies from irradiated plates as compared with growth of colonies from non-irradiated control replica places, +, No detectable difference; ±, slightly less vigorous than control; -, no or very little growth,

Induction and recovery of auxotrophs from uvs- mutants Yeast-like cell suspensions from wild-type strain MH 75 and from the four uvs- mutants were subjected to uv irradiation followed by nystatin enrichment, and examined for the presence of auxotrophs. The length of the exposure period to uv rays was adjusted for each individual strain based on its susceptibility and mutability (Fig. 6). The proportion of pink ade- mutants among survivors from LB 048701 and LB 048714 was twice as high as in MH 75, whereas no mutant was detected in populations from LB 048709 and LB 048712 (Table 5), Other types of auxotroph were also recovered from all uvs- mutants except strain LB 048709,

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Exposure time (s) Fig. 5. Sensitivity of O. ulmi 5.1. uvs- strains to uv radiations, Experiments were carried out in the absence of photoreactivation and caffeine. 0, Wild-type MH 75; D, uvs- LB 048701; . , uvsLB 048709; e, uvs- LB 049712; /'::,., uvs- LB 048714. Values are shown ± S,E. where these exceed the dimensions of the symbols. For a given exposure time, values were not significantly different if followed by the same letters, according to Waller-Duncan k-ratio t test (P = 0'05) following ANOVA.

DISCUSSION Exposure of yeast-like cell suspensions of O. ulmi 5.1, to the alkylating agent MNNG led to the recovery of four stable uvsensitive mutants, Complementation tests showed that at least one of the mutations, at the uvs4 locus, was recessive. The

L. Bernier and M. Hubbes

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Exposure time (s) Fig. 6. Comparative response of O. uIml s.I. wild-type (+ ) and uv-sensitive (-) phenotypes to the lethal (--) and mutagenic (---) effects of uv radiations. Survival was first assessed in the absence of both photoreactivation and caffeine (0). Effects of photoreactivation (,6.) and caffeine (0) were also measured. Mutability was evaluated by recording frequencies of forward mutations conferring resistance to benomyl at 1'2 Ilg (e) or 1'8 Ilg (~.l ml- 1 , or reverse mutation to prototrophy (.) in the absence of photoreactivation and caffeine. Values are shown±s.E. where these exceed the dimensions of the symbols. For a given exposure time, survival values were not significantly different if followed by the same letters, according to Waller-Duncan k-ratio t test (P = 0'05) following ANOV A.

Mutants of Ophiostoma ulmi sensitive to uv

950

Table 2. Paired comparisons of rates of mutation in uv-tolerant (uvs+) and uv-sensitive (UVS") strains of O. ulmi 5.1. after uv irradiation Length of exposure (s)

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(uvs+) (UVS")

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Table 3. Segregation of mutant (UVS") and wild-type (uvs+) phenotypes in random F1 ascospore progeny of 0. ulmi s.l.

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Progeny Cross (uvs+, <;2) x (UVS", 6)

Mutant/ 2 Mutant Wild wild X

FC 245 (Ami) x LB 048701 (Bml) LB 98-R-2 (Ami) x LB 048701 (Bmf) FC 245 (Ami) x LB 048709 (Bml) FG 245 (Ami) x LB 048712 (Bml) W 2 (Bml) x LB 048714 (Ami) W 2 (Bml) x LB 048714 (Ami)

29 53 17 21 49 64

57 78 18 23 61 75

0'51 0'68 0'94 0'91 0'80 0-85

9'12' 4'77' 0'03

0'09 1-31 0'87

• Indicates that observed ratios differed significantly from the expected 1: 1 ratio at P = 0'05.

other mutations could not be characterized due to the failure to produce stable heterokaryons among strains carrying these mutations. Meiotic analysis indicated that each of the four mutant phenotypes most likely resulted from a mutation at a single locus. The predominance of uvs+ phenotypes in the progeny from the crosses involving strain LB 048701 is thought to reflect a lower level of viability associated with the mutant phenotype, as has been observed for other 0. ulmi s.1. mutant phenotypes (Bernier & Hubbes, 1990a, b). Linkage analysis provided direct evidence that the mutations in strains

Table 4. Recombination frequencies among auxotrophic, drug-tolerance and uvs" markers in 0. ulmi s.l. Progeny Cross (<;2x6) LB 98-R-2 (Ami) x LB 048701 (Bml)

Loci

P

R

T

%R

benlR x uvsl

91 63 60 55 58 58 32 41 164 86 46 91 69 39 40 46 66 51

40 68 70 75 76 64 30 41 3 58 32 41 75 37 45 69 73 59

131 131 130 130 134 122 62 82 167 144 78 132 144 76 85 115 139 110

30-5 51'9 53.8 57'7 56-7 52-3 48-4 50-0 1-8 40-3 41-0 31'1 52-1 48-7 52'9 60-0 52-5 53-6

argl x uvsl

LB LB LB LB LB LB

100-R-2 (Ami) x LB 048701 (Bml) 100-R-2 (Ami) x LB 048712 (Bml) 100-R-2 (Ami) x LB 241-R-99 (Bml) 100-R-2 (Ami) x LB 018614 (Bml) 99-R-2 (Ami) x LB 048709 (Bmf) 1I8-R-2 (Ami) x LB 048709 (Bmt)

uvs2 x uvsl uvs2 x uvs3 uvs2 x uvs4 uvsl x mell benlR x uvs2 benlR x uvs2 ade7 x uvs2

LB 1I8-R-2 (Amt) x LB 048712 (Bmt)

benlR x uvs3

LB 98-R-2 (Ami) x LB 048712 (Bml) LB 1I8-R-2 (Ami) x LB 048712 (Bmf)

argl x uvs3

LB 381-R-l (Bml) x LB 384-R-6 (Ami) LB 316-R-7 (Amt) x LB 319-R-8 (Bml) W 2 (Bmt) x LB 048714 (Ami)

ade7 x uvs3 uvs4 x uvs3 uvs4 x met! nicl x uvs4

2

X

19'9" 0'2 0'8 3'1 2'4 0'3 0'0 0'0 155'2" 5'4' 2'5 13'1" 0'3 0'1 0'3 4'6' 0'4 0'6

P, parental types; R, recombinant types; T, total number of progeny; %R. recombination frequency between markers_ In crosses between two uvs" parents, R was estimated as 2 x no. of uvs+ colonies. X' 'values for goodness of fit to a 1 : I recombinant: parental ratio, or to a I: 3 uvs+ recombinant: remaining progeny ratio in crosses between uvs" parents.• and" indicate that the recombination frequency differed significantly from the expected ratio at P = 0'05 and 0'0 I, respectively.

L. Bernier and M. Hubbes

951

Table 5. Recovery of auxotrophs from uvs+ and uvs- strains of 0. ulmi 5.1. after uv irradiation and nystatin enrichment Auxotrophs'

Strain

Genotype

Irradiation time (s)

MH 75 LB 048701 LB 048709 LB 048712 LB 048714

wild type uvsluvs2uvs3uvs4-

60 60 25 60 30

From CM

From selective media

ade- /T

lys- /T

met- /T

ura- /T

7/1100 (0'6) 3/232 (1-3) 0/222 (0'0) 0/1200 (0'0) 1/80 (I'2)

NTt

NT

NT

12/173

9/148

NT

NT

2/87 0/21

2/88 1/28

5/172 0/47 5/37 1/44

• ade- phenotypes represent pink colonies on complete medium (CM) carrying mutations at the adel or ade7locus. Lys-. met- and ura- phenotypes were identified by plating uv-irradiated- and nystatin-treated cells on minimal medium (MM + nicotinamide for LB 048714) supplemented with either lysine. methionine or uraciL respectively. Colonies growing on the selective media were replica-plated on to MM for the identification of auxotrophs. Data are shown as number of auxotrophs/total number of colonies tested on each type of medium. The percentage of pink mutants which were recovered is indicated in parentheses. t Not tested.

LB 048709 (uvs2-1), LB 048712 (uvs3-1) and LB 048714 (uvs4I) were non-allelic and located on three different linkage groups. Indirect evidence suggested that the mutation in LB 048701 (uvsI-1) was also non-allelic and that the uvsI and uvs3 loci were linked. Both uvs 1 and uvs3 were found to be linked to the benIR locus, but uvs3 also showed linkage to argI, whereas no linkage was apparent between argI and uvsI. Repeated failure to cross LB 048701 with laboratory strains carrying the uvs3- I mutation or other mutations affecting loci on linkage group I prevented a more precise localization of the uvsI locus. We had previously reported the occurrence of at least three linkage groups in O. ulmi 5.1. (Bernier & Hubbes, 1990b). Linkage group III was identified by a single locus, metI, but we have recently obtained evidence that this locus belongs to linkage group II (Bernier, unpub!. results). Meiotic analysis of uvs- mutants, however, suggested the occurrence of at least four linkage groups in O. ulmi 5.1. (Fig. 7). The uvs2 and ade7 loci were tightly linked to each other but unlinked to the other markers tested: they were therefore assigned to linkage group III. The uvs4 locus segregated independently from all other markers tested and is thus presumed to represent a fourth linkage group. It is likely that more linkage groups occur in 0. ulmi 5.1., as suggested by electrophoretic karyotypes reported elsewhere (Royer et al., 1991; Dewar & Bernier, 1993). Exposure to uv rays is a safe, reliable and convenient way to induce mutations in a wide variety of organisms, particularly in bacteria and fungi (NormanselL 1982; Saunders et al., 1982; Beckerich et al., 1984). Ultraviolet light offers the advantage, when compared with chemical mutagens such as MNNG and EMS, of producing a broad range of DNA alterations resulting from base-pair substitutions (including both transitions and transversions), frameshifts and non-revertible deletions, in order of decreasing frequency (Kilbey, DeSerres & MaIling, 1971; Lawrence, 1991). Several studies, however, have demonstrated the occurrence of efficient uv-damage repair mechanisms in living organisms, particularly in the bacterium E. coli and in the yeast Saccharomyces cerevisiae. These mechanisms involve both error-proof and error-prone repair pathways. By inhibiting the activity of the former while allowing the latter to operate, it is theoretically possible to obtain higher yields of mutants.

Experiments with various micro-organisms have shown that error-proof systems can be inhibited physically and chemically. Therefore, it is common practice to carry out uvmutagenic treatments in the dark to prevent photoreactivation, and in the presence of caffeine or acriflavine, two inhibitors of light-independent excision repair (Saunders et al., 1982). Inhibition of either of these pathways was found to increase the sensitivity of uvs+ cells of 0. ulmi 5.1. to uv radiation. Excision repair may be more important than photoreactivation for repairing uv damage in uvs+ individuals, since the survival of both strains MH 75 and LB 44-R-2 was more affected by caffeine then by photoreactivation (Figs 2 and 6). Preincubation and exposure in the presence of caffeine, however, did not significantly increase the frequency at which benR mutants were recovered from strain MH 75. An alternative strategy, based on the induction and recovery of uv-sensitive strains with blocks in error-proof pathways, was therefore investigated in order to increase the rate at which mutants could be recovered after exposure to uv light. Four uv-hypersensitive mutants were recovered and characterized. In the presence of visible light, an enzyme, encoded by the phr gene in E. coli, uncouples the thymine dimers induced in the DNA by uv irradiation. In S. cerevisiae, at least two nuclear loci control photoreactivation (James & Nasim, 1987). This pathway was not affected in the four 0. ulmi uvs- mutants induced by MNNG, since all were found to be photoreactivable (Fig. 6). In the absence of light, enzymes encoded by the uvrA, Band C genes in E. coli will remove the dimer and some adjacent bases, and repair the Single-strand gap thus created. Most (99%) excision-repair events are believed to involve 'short-patch' repair (Lewin, 1985). In S. cerevisiae, at least five rad genes have been shown to be important for the incision step of excision repair (Bailly et al., 1991). Excision repair, like photoreactivation, can proceed without DNA replication. Our data suggest that the slightly higher uv sensitivity of strain LB 048701 resulted from a block in the excision-repair pathway, since this mutant was resistant to the effect of caffeine. Strain LB 048701 showed no significant increase in mutability to benomyl tolerance; however, Ade- auxotrophs were recovered from uv-treated LB 048701 cells at a higher frequency than from MH 75 cells. A third system, post-replicative repair, involves DNA

952

Mutants of Ophiostoma ulmi sensitive to uv replication and recombination, is thought to include both error-proof and error-prone pathways, and is responsible for the cell's ability to repair damage induced by various stress agents, including physical and chemical mutagens, as well as heat shock (Bernstein & Bernstein, 1991; Suzuki et al., 1991). While post-replicative repair is well documented in E. coli (Walker, 1985), evidence that it also occurs in fungi and other eukaryotes is accumulating (Fincham, Day & Radford, 1979; Taylor & Holliday, 1984). In S. cerevisiae, for example, one group of rad genes controls recombination repair of doublestrand breaks induced by ionizing radiation, whereas another group is involved in repair of damage induced by both ionizing and non-ionizing radiation (James & Nasim, 1987). Our data suggest that blocks in post-replicative repair, possibly in error-proof pathways, were responsible for the phenotypes of uvs- strains LB 048709, LB 048712 and LB 048714. These strains were photoreactivable, responded to caffeine and were moderately to highly sensitive to EMS. They could also recombine meiotically and were still mutable. With the exception of strain LB 048714, however, there was no evidence for a hypermutable phenotype. Exposure of strain LB 048714 and of its progenitor, LB 44-R-2, to uv rays for 10 sand 100 s, respectively, resulted in similar levels of survival and to a significantly higher mutation rate in the former. Strain LB 048714 thus appears to be hypermutable, and its phenotype may result from the constitutive enhancement of an error-prone repair mechanism, since the rate of spontaneous mutations in LB 048714 was Significantly higher than in its wild-type progenitor. Identification of the genes controlling pathogenicity, and understanding of the biochemical and physiological processes involved in pathogenesis, remain at the forefront of Dutch elm disease research. The analysis of field isolates of O. ulmi 5.1. and of their sexual progeny has already provided valuable information on the genetic control of pathogenicity, by exploiting the natural occurrence of variability for this trait (Brasier, 1987). This approach is, however, limited by factors such as the complexity of the polygenic control system (Brasier, 1988), and the negative interactions which complicate the analysis of progeny from 0. novo-ulmi x O. ulmi crosses (Brasier & Gibbs, 1976; Brasier, 1987; Kile & Brasier, 1990). Other approaches, based on the use of well-characterized laboratory strains, are thus needed to dissect pathogenicity into its individual, simpler components. We have already described a procedure for the chemical induction of strains carrying various types of mutation (Bernier & Hubbes, 1990a). We have now presented evidence that uv irradiation provides a physical alternative to MNNG. Although most of the work reported here was carried out on a few laboratory strains, the response of the wild-type NAG, EAN and NAN strains, which were also analysed, suggests that these protocols could be used successfully in other laboratories and with different strains. The recovery of uvs- mutants LB 048701 and LB 048714 is significant, as these strains also appear to be hypermutable and could thus be very useful in future mutagenesis work. In addition, the transformation system described by Royer et aI. (1991), based on the random integration of vector DNA into the nuclear genes of 0. ulmi 5.1., provides a third method for mutagenesis. Hence, the

induction of additional genetic variation in O. ulmi 5.1. appears relatively straightforward. However, the recovery of strains carrying detectable mutations in genes controlling or regulating pathogenicity remains a major challenge. The authors wish to thank Josee Dufour and Cynthia Honhart for their technical assistance, Sylvain Boisclair for helping with the statistical analyses, and Clive M. Brasier and Joan F. Webber for sending 0. ulmi 5.1. strains. This work was made pOSSible by grants from the Natural Sciences and Engineering Research Council (Canada) to both authors and by a grant from Fonds FCAR (Quebec) to L. Bernier.

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(Accepted 8 February 1994)

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