TRANSCRIPTION AND TRANSLATION OF THE SEX MESSAGE IN THE SMUT FUNGUS, USTILAGO VIOL ACE A

Transcription

1 J. Cell Sci. 14, (1974) Printed in Great Britain TRANSCRIPTION AND TRANSLATION OF THE SEX MESSAGE IN THE SMUT FUNGUS, USTILAGO VIOL ACE A I. THE EFFECT OF ULTRAVIOLET LIGHT A. W. DAY AND J. E. CUMMINS Department of Plant Sciences, University of Western Ontario, London, Ontario, Canada SUMMARY Cells of opposite mating type, &x and a 2, of the anther smut, Ustilago violacea assemble a conjugation tube after about 3-4 h of mating on nutrient-free media. Low doses of ultraviolet light (u.v.) delay but do not prevent conjugation in wild-type strains if given in the first 2-3 h of mating. However, irradiation later than this period has little effect and conjugation proceeds normally. The u.v. effect is photoreactivable and we conclude that u.v. induces dinners which affect transcription of specific messenger RNA molecules needed for conjugation (' sex message'). Our evidence suggests that the dimers may cause mistranscription rather than the complete prevention of transcription. The effect of u.v. on conjugation in reciprocal crosses of u.v.- sensitive and wild-type strains indicates clearly that both partners must complete transcription of sex messages in order to conjugate. Inactivation of either partner before transcription prevents conjugation, but conjugation proceeds when either cell is inactivated after transcription of the sex messages has occurred. These results suggest that a mutual and reciprocal exchange of information between the 2 mating-types occurs prior to the assembly of the conjugation tube. INTRODUCTION Conjugation in the anther smut fungus Ustilago violacea provides a useful system for investigating the genetic basis of cellular morphogenesis. In this organism the 2 mating types carry one of two different alleles, a x or a 2, at a single, though possibly complex, locus. We have shown that the morphogenetic events during mating consist of at least 5 stages: (1) intimate pairing of cells and fusion of the glycocalyx; (2) production of bumps on each cell at the site of pairing; (3) elongation of these bumps into 'pegs'; (4) dissolution of opposing walls and plasma membranes of the pegs to form a tube; and (5) elongation of the tube to the mature configuration (Poon, Martin & Day, J 974)- We have shown elsewhere that the 2 alleles are differently regulated during the cell cycle. Allele a x is active only in G x cells (a stringent cell cycle control) while allele a 2 is active during all the phases of the cell cycle (a relaxed cell cycle control) (Cummins & Day, 1973). The cell cycle controls of each allele are maintained in the diploid so that S and G 2 phase a x a 2 cells have a 2 mating ability while G x phase a x a 2 cells are active at both alleles and do not mate with any strain (Day & Cummins, 1973). The studies mentioned above are concerned with the control of when the genes are 30 C EL 14

2 452 A.W. Day andj. E. Cummins transcribed and how their final products are assembled to form the mature copulatory organelle. We report in this paper and in a following one (Cummins & Day, in preparation) studies on the induction, transcription, and translation of the 'sex message' from each allele. In this paper, we report the effect of ultraviolet light on production of the sex message, and the use of u.v.-sensitive mutants to investigate the roles of each mating type allele. In the following paper we report experiments using (i) inhibitors of transcription and translation, and (ii) radioactive tracers to determine when the sex message is synthesized and decoded and the copulatory organelle assembled. MATERIALS AND METHODS Stocks We have used in this study, the wild type haploid stocks 3.^ and a 2 and 2 auxotrophic mutants derived from them, 1. C2 (a lt requiring histidine (hisj) and producing yellow colonies (y)), and (a 2) requiring lysine (/y*i)) (see Day & Jones, 1969). In addition we have used 2 strains, 1.C2u6 and 2.716U6 which both carry the u.v.-sensitive allele, uvs 3 in addition to the other markers in 1. C2 and (Day & Day, 1970). We term strains carrying the uvs a allele as ' S' and strains carrying the wild type uvs + 3 allele as ' R' subsequently in this paper. Culture conditions The stocks were grown in liquid complete medium (Day & Jones, 1968) on a shaker at 20 C. Five millilitres of a culture in or near stationary phase were added to 50 ml of fresh medium and the culture was incubated overnight. Cultures were used the following morning when they were in mid log phase and contained about io 8 cells/ml. Mating conditions Log phase cells of both mating types were washed in sterile distilled water and mixed thoroughly in equal quantities. The cells were centrifuged to concentrate them to at least io 9 cells/ml and about ml was pipetted on water agar (2% agar) and allowed to spread naturally. The water was soon absorbed by the agar leaving a dense mass of cells on the agar surface. The percent conjugation at any time after plating out was estimated by resuspending the cells in water and examining them under the x 40 objective of a Zeiss RA microscope equipped with phase-contrast optics. Conjugated cells were counted as 2 cells (or more if one of the rare multiple conjugations was encountered) in the estimation of the percentage conjugation. Irradiation The cells were irradiated either in water suspension before plating out on water agar (i.e. at zero time for conjugation) or directly on the water agar at various times during conjugation. The u.v. lamp used was described previously and had an intensity during these experiments of 2 J m~ 2 s" 1 (A = 254 nm) at 8 cm distance (Day & Jones, 1968). For low doses the lamp was raised to 14 cm where the intensity was reduced to 1 J m~ 2 s" 1. For photoreactivation treatments the cells were irradiated in closed 100-mm plastic Petri dishes under 2 Westinghouse F15T8CW fluorescent lamps at a distance of 5 cm for 20 min. The total dose was about 1-2 x io 3 J m~ 2 of light of wavelength nm. RNA synthesis Log phase cultures of the 2 mating types were centrifuged, washed and mixed, then [8-14 C]adenine (International Chemical and Nuclear Corporation 55-5 mci/mmol) was added to give 0-5 /ici/ml cell suspension containing approximately 5 x io 8 cells/ml. Following irradiation with different doses,o-i-ml aliquots of cell suspension were deli vered on to o -5 -ml agar pads containing

3 u.v. and conjugation fig chloramphenicol in 16 x 150 mm capped test tubes. Chloramphenicol has no effect on mating at the level employed but effectively inhibits bacterial growth. At the end of each incubation 1 ml of 5 % trichloroacetic acid (TCA) was added and the tubes were frozen. At the end of each experiment the frozen tubes were thawed and the cell suspension was filtered on to a glass fibre filter, washed with 1 % TCA then water, and dried. The radioactivity on each filter was determined using a liquid scintillation detector. Control experiments show that over 90 % of the adenine label was in RNA and the remainder of the label was in DNA. Furthermore, there is evidence that in the auxotrophic strains employed in these experiments the total cellular RNA content remains constant during the course of the experiment. RESULTS The effect of u.v. on the kinetics of conjugation We have established that in an asynchronous population of wild type haploid cells of both mating types, there is an initial burst of synchronous conjugation, between 2-5 and 6 h after plating the cells on water agar (Poon et al. 1974). Conjugations continue to appear at a slower rate between 6 and 18 h as a 1 cells move round the cell cycle and become competent to mate (Cummins & Day, 1973). The auxotrophic strains, 1.C2 and and the uvs 3 mutant stocks derived from them are slower to conjugate and Time, h Fig. 1. The effect of u.v. on the kinetics of conjugation. The cells were irradiated at o h time., no u.v.; A, 10 J nr~ 2 ; #, 20 J nr~

4 454 A.W. Day andj. E. Cummins completed conjugations do not appear until h after plating on water agar. The rate of appearance of conjugations, however, is similar to that of the wild-type strains. Fig. 1 shows the effect on conjugation of 2 u.v. doses given immediately after mixing wild type cells of opposite mating-type. Conjugants were apparent in all 3 cultures at 2-5 h after mating, but the rate of appearance of completed conjugants decreased sharply with increasing u.v. dose. Thus conjugation is delayed but not prevented in an increasing fraction of the population as the u.v. dose is increased (at the low doses used). A practical implication of this is that the time of scoring of per cent conjugation in u.v.-treated cultures is important. In later experiments we scored irradiated cultures n a f ter mixing when there is still a large difference between irradiated cultures and the control. Similarly the age of the culture can affect the percentage conjugation obtained, e.g. the high values in Fig. 1 and the lower values in Fig. 2. We have demonstrated the cell cycle basis of this effect elsewhere (Cummins & Day, 1973). Photoreactivation and conjugation One likely explanation for the delayed conjugation observed after u.v. irradiation is that pyrimidine dimers are induced in a gene or genes concerned with mating and that these dimers either prevent transcription or result in mistranscription, until the lesions are repaired by cellular repair enzymes. Photoreactivating enzymes specifically repair dimers in DNA (Smith & Hanawalt, 1969). Therefore the ability to reverse a u.v. effect with visible light provides strong evidence that the effect is upon DNA. Both o ^ u.v. dose, J m~ 2 Fig. 2. The effect of photoreactivation (PR) on conjugation in u.v.-irradiated cells., u.v.; 0, u.v. + PR.

5 u.v. and conjugation 455 dark repair and photoreactivation enzymes have been shown to be active in U. violacea (Day & Day, 1970). In the next experiment we report the effect of visible light on mating in u.v.-irradiated cells. Samples of cells from a mixed log phase population of the auxotrophic strains 1. C2 and were irradiated with different doses of ultraviolet. Some samples were further irradiated for 20 min with visible light. The cells were plated on water agar and the percent conjugation was measured at 18 h (Fig. 2). In several experiments, the u.v. dose response curve plotted on a log scale has been more or less linear at low doses with only a small shoulder and an extrapolation number of about 2 (Smith & Hanawalt, 1969). The effect of ultraviolet was strongly photoreactivable so that conjugation in photoreactivated cultures was as high as in the unirradiated control culture, except after the highest u.v. dose. Photoreactivating light alone, however, only slightly affected conjugation. This result implies that the effect of ultraviolet on conjugation is through dimer formation in DNA. We suggest that such dimers interfere with transcription of mrna (sex message) from the gene or genes concerned with sexual morphogenesis until they are repaired by one of the cellular repair processes. The doses needed to inhibit total transcription are generally high, of the same order of magnitude as the doses needed to prevent colony formation (Smith & Hanawalt, 1969). However, in this study, the dose needed to inhibit conjugation is low and causes very little lethality in wild type cells. Thus a dose of 20 J m~ 2 causes about a 10% decrease in colony-forming ability when the cells are plated on complete medium, and only a 1 % decrease in this ability when the cells are plated on mating medium (water agar). The greater survival on water agar is thought to be related to the extended cell cycle on this medium, which might permit more repair of the ultraviolet damage (Day & Day, 1969). The low dose of u.v. used makes it unlikely that the total amount of RNA synthesis would be affected. It is probable that the u.v. causes either mistranscription of RNA, or inhibition of a small specific fraction of RNA species including the sex message. The next experiment confirms that the total amount of RNA synthesized is not greatly affected by these doses of u.v. RNA synthesis in irradiated mating cells The auxotrophic strains 1.C2 and which have wild-type u.v. resistance (R) were mixed in equal quantities, and so were the u.v.-sensitive strains 1. C2U6 and 2.716U6 carrying the recessive allele (S). [ 14 C]adenine was added to each culture and samples from each were immediately irradiated for different times. Aliquots were plated on water agar and left to mate. Samples were removed at intervals for 6 h after plating on water agar, and assayed for 14 C-labelled RNA (Fig. 3). Total transcription in the resistant populations (Fig. 3 A) is interesting in that the highest dose of u.v. used (100 J m~ 2 ) slightly enhanced RNA synthesis in the first 4 h of mating and caused RNA degradation in the 4-6 h period. This dose corresponds to about 20 % survival on complete medium, and about 50% survival on water agar, as judged by colony-

6 456 A. W. Day andj. E. Cummins forming ability (Day & Day, io,7o)athe lower dose of 20 J m~ 2 did not significantly affect RNA synthesis or degradation. Again, in the sensitive cells there were no large differences between unirradiated cells and cells which had been treated with 10 or 100 J m~ 2 (Fig. 3B). There was a slight decrease in RNA synthesis of about 5-10 % in the cells treated with 100 J m~ 2 compared to the control cells. This slight reduction has been observed in another experiment, and may indicate that a small fraction of RNA synthesized during 25 r 20.= Time, h Fig. 3. The effect of u.v. on RNA synthesis during conjugation. Duplicate points were taken for each time and the curves are derived from averages of these points. A, using strains (1.C2 x 2.716) with the wild type allele for u.v. resistance (uvs 3 + ):, no u.v.; A, 20 J m~ 2 ;, 100 J m~ 2. B, strains (1.C2U6 x 2.716U6) with the u.v.-sensitive allele (uvs 3 ): 9, no u.v.; A, 10 J m~ 2 ;, 100 J m~ 2. mating may be inhibited by u.v. irradiation. This u.v. dose is relatively high for sensitive cells and would cause complete loss in colony-forming ability on water agar. Even the dose of 10 J m~ 2 would decrease survival to as little as o-i% on water agar (Day & Day, 1970). These experiments indicate that u.v. doses high enough to reduce or eliminate colony-forming ability do not greatly inhibit either the initial or the total RNA synthesized in irradiated cultures and may even induce extra RNA synthesis for a few hours. We report in a subsequent paper (Cummins & Day, in preparation) that the bulk of RNA synthesized on water agar by auxotrophic strains is probably mrna and/or

7 u.v. and conjugation 457 transfer RNA as the level of stable RNA remains constant. If the bulk of the RNA synthesized under mating conditions is mrna then it is likely that the main influence of u.v. on mating is to cause mistranscription of the sex-message. The effect of irradiation at different stages of conjugation Fig. 4 illustrates the effect of u.v. irradiation of 20 J m~ 2 at different times during conjugation in the resistant strains, 1.C2 and 2.716, and in the sensitive strains, 1.C2u6 and 2.716U A Time, h Fig. 4. The effect of u.v. at different times during conjugation in wild type and in sensitive strains. The percent conjugation at 24 h (solid line) in uvs uvs 3 + (R + R, A) and uvs 3 + uvs 3 (S + S, #) was determined following irradiation (20 J m~ 2 ) at different times during conjugation. The kinetics of conjugation in control, unirradiated cultures are shown as dotted lines (R + R, A; S + S, O). The dashed lines represent the percent conjugation at 24 h in R x R (upper) and S x S (lower) in the absence of u.v. It is clear that there is a period of 2-3 h after mating is initiated in which the mating ability of cells is reduced by ultraviolet. Irradiation after this period has relatively little effect on mating ability. The dose used, 20 J m~ 2, prevents about 50 % of resistant cells from mating, and is sufficient to eliminate mating in the sensitive strains, if given in the

8 458 A. W. Day andj. E. Cummins critical period. This critical period precedes the assembly of the copulatory organelle. Thus in the sensitive strains, u.v. given at 3-75 h when there are only 2 % completed conjugations, does not prevent 38 % of the remaining cells from completing conjugation. Only about 23 % fail to conjugate. This result is even more striking in the resistant strains, which mated more synchronously in this experiment, as at 3-75 h there are only 3 % completed conjugants, but 74 % go on to complete conjugation after irradiation and only 2 % fail. Experiments which we describe in a subsequent paper, using radioactive tracers, and also inhibitors of transcription and translation, indicate that transcription of the sex message is completed in most cells after 3 h of mating, and that translation of these messages is largely completed by about h (Cummins & Day, in preparation). The results shown in Fig. 4 are therefore consistent with an hypothesis that u.v. inhibits conjugation by influencing transcription of the sex message. The contribution of each allele to conjugation The effect of low doses of u.v. on matings between uvs + 3 (R) and uvs 3 (S) cells can be used to indicate whether both alleles are active in contributing sex message, or one merely' triggers' the other. A dose of 20 J m~ 2 inhibits conjugation far more efficiently in a sensitive strain than in a resistant one (Fig. 4). Thus in a mating of a sensitive cell with a resistant one, ultraviolet can almost completely disrupt transcription in the sensitive partner, while the resistant partner will continue to transcribe functional sex message. If the reciprocal combinations S(a x ) x R(a 2 ) and R(aj) x S(a 2 ) show different abilities to conjugate following irradiation at different stages, then the contribution of the 2 mating types must be different in quantity and/or in temporal aspects. However, as shown in Fig. 5 the two combinations have similar curves for sensitivity to u.v. and both are very close to the curve for S^) x S(a 2 ) cells, especially at earlier times. We conclude that each allele is transcribing essential sex messages right from initiation of sex message transcription to the end of this specific transcription period (0-2-5 n )- Disruption of transcription of either allele is sufficient to inhibit mating. Recovery of ability to mate in irradiated cells The curves shown in Figs. 4 and 5 were scored after n on mating medium. Scorings at about 48 h indicate that many of the competent R x R cells do eventually conjugate, regardless of when the u.v. dose has been given. Thus the irradiated resistant cells eventually repair the damage, transcribe functional sex messages and conjugate. However, S x S, S x R, and R x S curves are essentially the same at 48 as at 24 h. This is not surprising as 20 J m~ 2 is sufficient to kill % of uvs 3 cells. Thus uvs 3 cells that complete the transcription of sex message before irradiation can complete conjugation even though they are 'dead' as judged by the criterion of colony-forming ability. However, sensitive cells that are irradiated before or during transcription cannot repair the damage and neither conjugate nor form colonies.

9 u.v. and conjugation r 4-5 Fig. 5. The effect of u.v. at different times during conjugation in crosses of wild type (R) with sensitive (S) strains. The percent conjugation following irradiation (20 J m~ 2 ) is plotted against time at which irradiation was applied., R(a!) + R(a 2 ); V, S(a x ) + R(a 2 ); A, R(a,) + S(a 2 );, S(a 1 ) + S(a 2 ). DISCUSSION The results presented above indicate that small doses of ultraviolet can disrupt transcription of the sex message in U. violacea. As total RNA synthesis is not significantly affected by such low doses, we propose that the accuracy of transcription of the sex message is diminished by the presence of u.v.-induced dimers in the gene or genes concerned with conjugation. It is possible that the mrna synthesized will have altered bases opposite the dimers, or more likely perhaps that it will be prematured, terminated, or will result in a 'frame-shift'. We are currently devising experiments to determine the extent and nature of u.v.-induced alterations in the sex message. We will discuss in the following paper the timing of transcription and translation of the sex message relative to the assembly of the copulatory organelle. Here we concentrate on the most interesting aspect of the experiments with ultraviolet light - the ability to manipulate the contributions of each mating type allele separately during conjugation, by using reciprocal combinations of u.v.-sensitive and resistant cells. These experiments indicate that there is a mutual exchange of information between

10 460 A. W. Day andj. E. Cummins the mating cells during the first 3 h of mating and prior to the major morphological changes - a period which may appropriately be called courtship. The information exchange precedes assembly at a time when the membranes of the individual cells are intact and the sole cell-to-cell contact is by means of the hairs on their glycocalyxes. During courtship conjugation is prevented if either partner is inactivated with ultraviolet. Thus transcription of sex messages from both cells is necessary to complete conjugation. It is not yet clear whether each cell, once induced by the other, continues to transcribe sex messages until the message is complete, even when its partner is inactivated, or whether there is a mutual and sequential induction of genes which is halted if one partner is inactivated. What our experiments do show, however, is that both partners must complete the transcription of the sex message to complete conjugation. We wish to thank Mrs Vera Marnot and Miss Kristina Nielsen for excellent technical help. The work was supported by grant number A5062 of National Research Council of Canada to A. W. Day. REFERENCES CUMMINS, J. E. & DAY, A. W. (1973). The cell cycle regulation of mating-type alleles in a smut, Ustilago violacea. Nature, Lond. 245, DAY, A. W. & CUMMINS, J. E. (1973). Temporal allelic interactions: A new kind of dominance. Nature, Lond. 245, DAY, A. W. & DAY, L. L. (1970). Ultraviolet light sensitive mutants of Ustilago violacea. Can. Jf. Genet. Cytol. 12, DAY, A. W. & JONES, J. K. (1968). The production and characteristics of diploids in Ustilago violacea. Genet. Res. n, DAY, A. W. & JONES, J. K. (1969). Sexual and parasexual analysis of Ustilago violacea. Genet. Res. 14, POON, N. H., MARTIN, J. & DAY, A. W. (1974). Conjugation in Ustilago violacea. I. Morphology. Can.jf. Microbiol. (in Press). SMITH, K. C. & HANAWALT, P. C. (1969). Molecular Photobiology. Inactivation and Recovery. New York and London: Academic Press. (Received 9 August 1973)