Morphology of initials and number of nuclei initiating ... - Science Direct

Trans. Br. mycol. Soc. 90 (3), 36'1-373 (1988)

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MORPHOLOGY OF INITIALS AND NUMBER OF NUCLEI INITIATING CLEISTOTHECIA IN ASPERGILLUS NIDULANS By BEN J. M. ZONNEVELD Cell Biology and Genetics, Kaiserstraat 63, Postbus 9516, NL z300 RA Leiden, The Netherlands The homothallic fungus Aspergillus nidulans initiates fruit bodies or cleistothecia with the formation of short seemingly dikaryotic, strongly branching hyphae. This was shown here for the first time with the help of an aconidial mutant that moreover makes cleistothecia even on dilute media. These branching hyphae formed thick globose, so-called Hiille cells, and subsequently cleistothecia are formed. Differentiated ascogonia or antheridia were not observed. Phenotype and genotype of the ascospores were determined in hybrid and selfed cleistothecia with conidial and ascogonial colour markers. The results indicated indirectly that only two nuclei contribute to the formation of the wall of the fruit bodies and the cleistothecial and ascogonial colours. Fruit bodies or cleistothecia ofthe genus Aspergillus are initiated differently and the teleomorphs are accordingly given different names (Benjamin, 1955). In the Aspergillus glaucus group (teleomorph Eurotium Link) cleistothecia are initiated by a coiled ascogonium often fused with an antheridium. In the Aspergillusfischeri group (teleomorph Sartorya Vuillemin) they are started by a curled ascogonium with no evidence of an antheridium. In Aspergillus quadrilineatus of the A. nidulans group (teleomorph Emericella Berk. & Br.) they are initiated by a hypha forming a wide loop, without a sign of differentiated ascogonia or antheridia. In A. nidulans, one of the genetically best investigated fungal organisms, cleistothecial initiation is notoriously difficult to investigate, due to the heavy layer of conidia preceding cleistothecial development. The formation of the wall of the fruit bodies and the formation of sexual spores are not necessarily correlated as shown by diploid A. nidulans strains producing perfect fruit bodies while meiosis is nearly absent (Elliott, 1960). It is known from genetical data (Pontecorvo et al., 1953) that usually all ascospores of a cleistothecium are derived from a single pair of nuclei. The use here of an aconidial mutant makes it possible to show that cleistothecia are initiated by strongly branching, possibly dikaryotic hyphae. Moreover ascospore colour mutants make it possible to calculate that also the wall of the fruit body is initiated by a single pair of nuclei. MATERIALS AND METHODS

Strains The following strains of Aspergillus nidulans of Glasgow origin were used: biAi, dclAi for the

study of initials (Zonneveld, 1974); biAi, biz with green conidia and blue ascospores and yAz, uiz; sCl2, el3 with white conidia and colourless ascospores for the study of cleistothecia (Apirion, 1963). Yellow (y) and white (w) are conidial colour markers (wild type is green) whereby white is epistatic over yellow; blue (bl) and colourless (cl) are non-autonomous, ascospore colour markers (wild type is red); bi and s designate auxotrophies for biotin and reduced sulphur and del makes many cleistothecia and is nearly aconidial. Media Media used were as described (Zonneveld, 1975) but with 0·8 % glucose + 0'5 % starch for the study of initials and 3 % glucose +0'4 % nitrate for the study of cleistothecia. Further genetical techniques were as described by Clutterbuck (1974). Initials A small inoculum of strain bi Ai, del As was placed in the centre of a plate. Pictures were taken after a few days at 37°C using a binocular microscope. In some cases the plate was stained with 1 % potassium iodide. Scoring of eleistothecia Cleistothecia were collected from 2- to 3-week-old plate cultures, cleaned by rolling over 3 % agar plates and crushed with a needle in small droplets of water on a plastic Petri dish. The phenotypic colour of the ascospores was then scored as red, blue or colourless. The same needle was streaked on a plate to obtain progeny to score for the hybridity of the ascospores, i.e. their genotype with respect to conidial colour, further indicated as the genotype of the ascospores. The

Initiation of cleistothecia in Aspergillus nidulans

370

(b)

(a)

I

J

Fig. 1. Consecutive stages in cleistothecium initiation in the strain biA«, dclAs . (a) Vegetative hyphae; (b) first stage of initiation of cleistothecia; (c) further branching and formation of Hulle cells; (d) ascogenous hyphae start to intertwine; (e-fJ young cleistothecia. Bar = 40 pm.

cleistothecia were classified as selfed, resulting in colonies with only pure green or pure white conidia or as hybrid, resulting in colonies with yellow, green and white conidia. RESUL TS

Cleistothecia of Aspergillus nidulans develop to full size after exhaustion of the medium (Zonneveld, 1972). The initiation of the cleistothecia however takes place earlier. Wild-type A. nidulans produces first an abundance of asexual spores (conidia) followed by the formation of initials and the development of the cleistothecia. This thick layer of conidia makes it nearly impossible to observe the initiation of the cleistothecium. Moreover, even if few conidia are produced the number of cleisto-

thecial initials is so high that they will be hidden completely. The mutant bi Ai, delAt used here was isolated as producing few conidia but an abundance of cleistothecia (Zonneveld, 1974). More important, low numbers of cleistothecia are produced on media with low amounts of glucose (0'8 %), a situation in which the wild type rarely produces any cleistothecia. The first sign of the initiation of the cleistothecium is the formation of a short, wavy hypha contrasting with the long, straight, vegetative hyphae (Fig. 1 a-c). This hypha then branches profusely, followed by the formation of thick globose Hulle cells, subsequently solid bodies appear increasing in size and developing to fullsize fruit bodies (Fig. 1 d-fJ. It was established by genetical data (Pontecorvo et al., 1953) that only two nuclei are responsible for the formation of all

Ben J. M. Zonneveld ( 10-1 0 0 000) ascospores in a single cleistothecium. This results in three types of cleistothecium from a heterokar yon , selfed of one or of the oth er parent and hybrid ones . Occasionally twin fruiting bodie s also occur. These three types can easily be checked by the use of different conidial spore colour markers. Here we crossed wild type (green) and y,w (wh ite because w is epistatic). This resu lts in (1) selfed cleistothecia with ascospores leading to colonies with green conidia; (2) se1fed cleistothecia with ascospore s leading to colonies with white conidia, and (3) hybrid cleistothecia with ascospores leading to colonies with mixed white, green and yellow conidia. Apirion (1963) has isolated from the wild type with red ascospores, mutants with blue and colourless ascospores . These colours are non-autonomous, i.e . are not determined by the genotype of the ascospores themselves but by th e genotype of nuclei that form the wall of the fruit body and consequently determine th e colour of the walls of the clcist othecia and ascospores. T o determine the number of nuclei contributing to the formation of the wall of the fruit bodies a cro ss between two strains differing in ascospore and conidiospore colours was made . With three genotypes of ascosp ores for conidial colour (white, gre en and mixed yellow- white-green) and three phenotypic classes of ascospores red, blue and colourless, nine different classes are possible. In three independent

Table

1.

37 1

experiments cleistothecia were placed in one of these classes as described in Materials and Methods and this resulted in the num bers given in Table 1. The ratio of the two different nuclei in a heterokaryon can be strongly unequ al depending on the genotypes used. From Table 1 one can calcu late how man y nuclei of each parent have contributed to the different genotypes of the ascospores (shown by the colour of the conidia) (T able 2). The green parent con tributes four times more nuclei than the white parent. Starting from thi s, it is possible to test for random mixing of the nuclei by calculating the expected nuclei with the three genotypes of ascospores (T able 3). The numbers found and calculated are reasonably close except that the number of found selfed white cleistothecia is higher. So the random mixing in th e hyphae is not perfect, but sufficient to calculate from it the number of nuclei forming the initial population of th e wall of the fruit bodies (T able 4). It is here assumed that with small numbers of nuclei any mixture of nucle i coding for blue and colourless nuclei will result in a wild type (red) colour. If we compare the numbers calculated with 1, 2, 3 or 4 starting nuclei with the actual found numbers it is clear that the numbers calculated with two nuclei come most close and that with 3,4 (or more) nuclei the calculated numbers are more and more deviating from the actually scored numbers.

N umber of cleistothecia in nine diffe rent classes f rom the cross biAt b12 x yA2 W2 Se12 cl3 Phenot ype (ascospore colour).. . Genotype (conidial colour).. ,

Red

Blue

Colo urless

Hyb

Whi

Gre

Hyb

Whi

Gre

Hyb

Whi

Gre

54

13 26 11 60

69 121 67 257

32 40 54 126

9 7 4 20

368 191 290

32 19 23

32 13 23

849

74

77

38 28 27 113

Experiment

1 2 3 Total

109

61

224

H yb = hybrid (green , yellow and white); Whi = white ; Gre = green,

Table 2. Number of nuclei of each par ent cont ributing to genotypes of ascospores Nuclei from green parent

Nuclei from white parent

424 1219 157

424 2438

424

1800

2862 = 79'S %

Number Hybrid cleistothecia Selfed green cleistothecia Selfed white cleisto thecia Total 14

738

314 20'S %

=

M Y C 90

Initiat ion of cleistothecia in Aspergillus nidulans

37 2

Table 3. N umber of cleistothecia expected differing in gen otype with respect co conidial colour based 0 11 numb er of nu clei calculate d in T able 2 Found Number Hybr id cleistothecia Selfed green cleistothecia Selfed white cleistothecia

4 24 1219 157

0

"

23 68 9

Calculated 2 x 79'5 x 20'5 79' 5 x 79'5 20'5 x 20'5

Number

""

32'6 63'2 4'2

58 7 1138 75

Table 4. Ca lcula tion of numb er of ph enotypically different cleistot hecia If I, 2, 3 or 4 nuclei cont ribute co th e wall of f rui t bodies compa red with actua l f ound numb ers of cleistothecia

Cleistothecial colour Red Blue Colourless

Found Number 54 1 99 5 264

Expected with : 3 nuclei

1 nucleus

"" 3° 55'3 14'7

Numb er ° 143 1 69 3

0

2 nuclei

"

° 79'5 20'5

Numb er 587 1138 75

"

"

32'6 63'2 4'2

Nu mber 880 903 16

4 nuclei

""

4 8'9 5°' 2 0'9

Number 1078 7 19 3

""

59'9 39'9 0'2

Figures are calculated assuming 79'5 " 0 nuclei from the green parent and 20' 5 0 0 nuclei from the white parent. (See Tables 2 and 3.)

DI S C US SI O N

The figures show for th e first time the earliest stages of th e ini tiation of clei stothecia in A spergillu s nidulans. N o sign of differ entiated ascogon ia or antheridia was foun d . The initiating hyphae differ fro m th e veg etative hyphae in their shape and frequency of branching. The wavy appearance could be due to frequent sep ta tion, suggesting that th ese hyphae are dikaryotic. T h is cou ld be a way to ens ure th at bo th for the formation of th e wall of the fr uit bodies and the sex ual spore formation a pair of n uclei is set apart. This may also explain more easily the uniparental inheritance of mitochondria, found by Rowland & Turner (1976) . The classes of cleistothecia in Table 1 devi ate from th e re sults of Ap irion (1963) in that he did not find b lue or colou rl ess h ybrid cleistoth ecia, although h e stated th at th ey could have been found . T h is may be due to the fact that he selected on ly large cleistothecia whereas here they are ch osen at random . This implies that the size of the clei sto thecium is not determined by the pair of nuclei forming the ascospores, but b y the pa ir of nuclei forming th e cleistothecial wa ll. The re sults of Table 2 show that the green parent contributed four tim es more n uclei to the cleistothecia than the white parent . This is most like ly caused by the fact that the strain biA i biz is on ly bi otin deficient, a deficiency th at n eeds a very sma ll supply of bio tin onl y. Table 3, ass uming random mixing of nuclei , pred icted more

selfed white clei stothecia . This indicates that th ere is not a perfect homogeneous mixing of nuclei. Scored numbers seem clo se enough to allow the calculations of Table 4. Here we see that th e num bers calc u lated match those fo und best if it is assu m ed that onl y tw o n uclei form th e star ting point for the in itiation of th e wall of the fru it body . This deviates from the resu lts found in the heterothallic N eurospora crassa (Johnson , 1976) in which by ana lysis of mosaics 200-300 nuclei were found to form the starting population. T h e surplus of colourless cleistothecia in Table 4 may be caused by the inadvertent classification of too young cleist othecia as colourless. The results presented here may help in expl aining mo re precisely several aspects of ini tiation and dev elopment of cleistoth ecia. RE FERENCES

APIRION, D . ( 1963). Formal and physiological genetics of ascospore colour in A spergillu s nidulans. Genetical R esearch, Cambridge 4, 276-283 .

BENJAMIN, C. R. ( 1955). Ascocarps of A spergillu s and Penicillium . My cologia 47, 669-68 7. CLUTTERBUCK, A. J. (1974) . A spergillu s nidulans . In Handbook of Genetics vol. I (ed . R . C. King), pp. 447-510. New York, London: Plenum Press. ELLIOTT, C. G. (1960 ). The cytology of Aspe rgillus nidulans. Genetical R esearch 1 , 462- 477. JOHNSON, T . E. (1976). Analysis of pattern formation in

Ben J. M. Zonneveld Neurospora perithecial development using genetic mosaics. Developmental Biology 54, 23-36. PONTECORVO, G., ROPER, J. A., HEMMONS, L. M. , MACDoNALD, K. D. & BUFTON, A. W . J. (1953). The genetics of Aspergillus nidulans. Advan ces in Genetics 5, 14 1-238 . ROWLAND , R. T. & TURNER, G . (1976). Maternal inheritance of extranuclear mit ochondrial markers in Asp ergillus nidulan s. Genetical R esearch, Cambridge 28 , 281-29° · ZONNEVELD, B. J. M . (1972). Morphogenesis in A spergillus nidulans. The significance of alpha-s ,3-glucan of

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the cell wall and alph a-l ,3-glucanase for c1eistothecium development. Biochimica B iophysica Acta 273, 174187. ZO"'NEVELD, B. J. M . (1974). Alpha-r .j-glucan synth esis correlated with alpha-v .j-glucanase synt hesis, conidiation and fructification in morphogenetic mutants of A spergillu s nidulans. J ournal of General M icrobiology 8 1,445-451. ZONNEVELD, B. J. M. (1975). Sexual differentiation in Asp ergillus nidulans. The requirement for manganese and its effect on alpha- r .j -glucan synth esis and degradation. Archives of M icrobiology 105, 101-104.

(Received f or publication 6 July 1987)

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