Expression of dikaryon-specific mRNAs of Schizophyllum commune in relation to incompatibility genes, light, and fruiting

EXPERIMENTAL MYCOLOGY 12, 60-69 (1988)

Expression of Dikaryon-Specific mRNAs of Schizophyllum commune in Relation to Incompatibility Genes, Light, and Fruiting MARCEL H.J. RUITERS,JOHANNES H. SIETSMA, AND JOSEPH G. H. WESSELS Department

of Plant

Physiology, Biological Centre, University 9751 NN Haven, The Netherlands

Accepted for publication

of Groningen,

Kerklaan

30,

October 21, 1987

RUITERS, M. H. .I., SIETSMA, J. H., AND WESSELS, .I. G. H. 1988. Expression of dikaryonspecific mRNAs of Schizophyllum commune in relation to incompatibility genes, light, and fruiting. Experimental Mycology 12,60-69. The concentrations of eight specific mRNAs abundantly present in a fruiting dikaryon but not in the progenitor monokaryons of Schizophyllum commune were measured in various heterokaryons and in homokaryons that carry constitutive mutations in the incompatibility genes. Only a heterokaryon with different alleles for both incompatibility genes and a homokaryon with mutations in both genes, both fruiting dikaryons, produced large amounts of these mRNAs. In dark-grown colonies of the dikaryon these dikaryon-specific mRNAs were virtually absent. After transfer of colonies to light these mRNAs and fruit bodies were concomitantly formed in approximately the same region of the colony. Some of these sequences were also formed in a fruiting monokaryon. These results suggest that these abundant mRNAs that are normally regulated by the concerted activities of the incompatibility genes and by light have no role in vegetative growth but are possibly required for fruiting. Q 1988Academic PEW 1~. INDEX DESCRIPTORS: gene expression, in S. commune development; incompatibility genes, control of mRNAs; light regulation of fruiting and mRNAs; Schizophyllum commune; mushroom formation, regulation of mRNAs; dikaryon-specific mRNA.

lead to dikaryosis. These heterokaryons never fruit. Similar morphogenetic processes can be switched on in homokaryons, without mating, by so-called constitutive mutations in the incompatibility genes. When present in both the A and B incompatibility genes of a homokaryon, these mutations switch on all processes leading to dikaryosis and a mycelium that completely mimics the heterokaryotic dikaryon, including the formation of fruit bodies (Raper, 1983). A similar genetic system regulating cellular and multicellular morphogenesis has been shown to occur in Coprinus cinereus (Swamy et al., 1984). In previous studies we described the isolation of a number of cDNA clones derived from abundant mRNAs present in the heterokaryotic dikaryon at the time of fruiting, particularly in the fruiting structures, but absent in the monokaryon (Dons et al., 1984; Mulder and Wessels, 1986). It was

Matings in Schizophyllum commune are controlled by two incompatibility genes, A and B, for which extensive series of alleles exist (Raper, 1966, 1983). Each of the A and B genes actually consists of two loci, a and j3. Two monokaryons mate to form a dikaryon when they have allelic differences in at least one of the two loci of each of the incompatibility genes. The dikaryon formed is a stable heterokaryon with clamp connections at the septa and two nuclei, one from each of the mates, per hyphal compartment. Unlike the monokaryon the dikaryon readily forms fruit bodies in which terminally differentiating basidia produce basidiospores after fusion and meiosis of the paired nuclei. Matings between monokaryons carrying allelic differences at only one of the incompatibility genes result in common-A and common-B heterokaryons, each of which express parts of the morphogenetic processes which together 60 0147-5975188 $3.00 Copyright 0 1988 by Academic Press, Inc. AU rights of reproduction in any form reserved.

DIKARYON-SPECIFIC

mRNAs IN Schirophy&n

subsequently shown that suppression of fruit-body formation by growth of the dikaryon in darkness, in the presence of elevated carbon dioxide or in shaken suspen-r sion culture, did not abolish the presence of, these mRNAs, but only lowered their concentrations (Wessels et al., 1987). Apparently, these dikaryon-specific mRNAs can also appear in the absence of fruit bodies. In the present study we measured the concentrations of the dikaryon-specific mRNAs in coisogenic mycelia in which the various morphogenetic processes were switched on by allelic differences in the incompatibility genes or by mutations in these genes. High concentrations of these mRNAs were found only in dikaryons irrespective of their homokaryotic or heterokaryotic nature. In addition, when grown as a colony from a single inoculum instead of as a confluent lawn from a mycelial macerate, the dikaryon-specific mRNAs were clearly elevated by illumination and appeared in the region of fruit-body initiation. These results add to the evidence that these dikaryon-specific mRNAs have no role in the primary morphological transition from

Constitution

of Incompatibility

Incompatibility genes”

Strains and crosses

6%

monokaryon to dikaryon but role in secondary processes cuning in the dikaryon, inclu MATERIALS

AND METHODS

Strains. The coisogenic homokar~~~s of S. commune used are listed in Table 1. The two strains 4-39 (A41B41, CBS 341.81) and 4-40 (A43B43, CBS 340.81), originally obtained from the culture collection of Professor J. R. Raper, served as the source of the wild-type incompatibility genes. Strain 4-40 was regularly backcrossed to stram 4-39 to maintain isogenicity and, among then- progenies, strains containing A43B43, and A43B41 were recovered. The homokaryon 1758 (Amu coisogenic to strain 4-39 and originally also obtained from the culture collection of Professor J. R. Raper, was used as a source of the mutant incompatibility alleles, Jt was backcrossed several times to strain (A4lB41) and, from the progenies, AmutBmut, A4lBmut, and Amu were isolated. The monokaryon HF-2 (A43

TABLE 1 Genes and Expressed Morphogenetic

Nuclear constitution

A

B

A4lB41 A43B43 ‘441A43 A43B41

al-$1 a&p6 d-p1 a4-/%

f&/32 al-/?1 al-#1 a%/32

Homokaryon

A41B41 X A41B43

al-/U

d-~2/al-/?l

Heterokaryon

AllBmut

commune

Morphogenetic pathways

Pathways

HYPbal morphology

Fnliting --__ No

A-off, B-off

Monokaryon

A-off, B-on

Constitutive nuclear migration

No

A-on, B-off

Pseudoclamped hyphae

NO

Homokaryon Homokaryon

al-j?1

a3-/w)

Homokaryon

al-/Ula4-j36

al-/D

Heterokaryon

AmutB41

al-/31(l)

a?-/32

Homokaryon

A41B#l X A43B43 Arm&Smut

al+‘lla4-P6 al+l(l)

a?-/?2/al-/31

Heterokaryon Homokaryon

A-on, B-on

Dikaryon

Yes

(7-wxl)

A43826 ffF2

a4+6

03-p1

Homokaryon

A-off, B-off

Monokaryon

Yes -

A41B43

X

A43B43

a Constitutive

mutations are indicated by a number in parentheses behind the affected locus.

62

RUITERS,

SIETSMA,

isogenic to 4-39, was selected by Dr. M. Raudaskoski (University of Helsinki) for its capability to readily produce fruit bodies after induction by light and was kindly provided by her. Culture conditions. To compare mRNAs in wild-type homokaryons, heterokaryons, and homokaryons carrying primary mutations, mycelia were grown from homogenates evenly spread over 0.7% (w/v) minimal agar medium as described (Dons et al., 1984). The heterokaryons were synthesized by inoculation with 1: 1 mixtures of the homogenates of the proper wild-type homokaryons (Wessels and Niederpruem, 1967). This resulted in a uniform expression of the heterokaryotic phenotypes, even in the case of the common-B heterokaryon in which no nuclear migration occurs. Incubations were done under continuous light (about 1000 ix) at 24°C and duplicate cultures were harvested 96 h after inoculation. This was done by separating mycelial mats from the setisolid medium with a spatula after which the mats were frozen in liquid nitrogen and stored at -70°C. To estimate the abundance of mRNAs in various regions of mycelial colonies, petri dishes containing complete medium (Raper and Hofman, 1974) with 1.5% (w/v) agar were inoculated in the center with a plug of mycelium (1 mm in diameter) and incubated for 4 days at 24°C in complete darkness. The colonies (about 3 cm in diameter) were then transferred to light (1000 lx) or left in darkness. The advance of the periphery of the colonies was marked each day at the bottom of the petri dish as concentric rings (dark-grown cultures were marked under a red safety light). The dikaryon and the monokaryotic fruiter HF2 produced fruit bodies 2 days after transfer to light in a ring about 5 mm wide, with the inner boundary approximately 3 mm behind the margin of the colony at the moment of transfer to light (see Fig. 1). At various times areas corresponding to the marked rings were cut from the colonies and the mycelial rings were

AND

WESSELS

frozen in liquid nitrogen and stored at - 70°C. Isolation of total RNA. Frozen mycelium with adhering agar was ground to a powder under liquid nitrogen in a mortar and the RNA was extracted by the hot-phenol procedure as described by Hoge et al. (1982). Denaturing gel electrophoresis indicated intact RNA in all cases. The yield from the cultures grown from mycelial homogenates was 40-60 pg RNA/g wet wt of mycelium and the yield from the different rings from a colony varied from 40 to 750 kg RNA/ring. Estimation of the concentrations of specific mRNAs. Glyoxalated samples of total RNA (3.0-3.5 kg) were dotted onto Genescreen-plus membrane and hybridized to [a-32P]dCTP-labeled probes as described (Mulder and Wessels, 1986). Hybridization signals were monitored by scintillation counting and/or by scanning autoradiograms with a Joyce-Loebel microdensitometer. The probes used were cDNA sequences originally cloned in pBR327 and represented mRNAs specifically present in the fruiting dikaryon (pSc-1, 2,4, 5, 6, 7, 9, and 14) and mRNAs present in both monokaryotic and dikaryotic mycelia (pSc3 and 10) (Dons et al., 1984; Mulder and Wessels, 1986). For the experiment described here the cDNA inserts were actually recloned into pGEM-1 vectors (Promega, Biotec). For quantification of the concentrations of the probed RNAs, 150 pg of the insert of pSc-1 was mixed with 3.4 pg of yeast RNA and dotted in the same way. Variations in the total amount of RNA dotted were corrected for by hybridizing with a clone containing part of the 18s ribosomalRNA gene of S. commune. RESULTS

Concentration of specific RNAs in relation to expressed morphogenetic pathways. The various homokaryons and heterokaryons of S. commune (Table 1) were grown from mycelial homogenates for 4 days on

DIKARYON-SPECIFIC

mRNAs

IN

Schizophyllum

commune

93’ 4 5 6 7

//--

Q

10

(.> 0

FIG. 1. Schematic drawing of growth zones of Schizophyllum commune colonies grown in continuous darkness (A) or transferred to light after growth for 4 days in darkness (B). Numbers indicate the ages of the growth zones in days at the time of harvest for measurements of mRNA concentrations, 6 and 10 days after inoculation. The narrow outer growth zone is caused by contact of the advancing mycelial front with the edge of the petri dish. The sites where fruit bodies arise in the light are schematically indicated.

the surface of agar plates. The homokaryons with wild-type incompatibility genes displayed abundant aerial mycelium and the hyphae all contained simple septa and one nucleus per hyphal compartment (monokaryon; A-off, B-off). The presence of different alleles for the B genes in the heterokaryon (common-A heterokaryon) or a primary mutation in the B gene of the homokaryon (Bmut) both switched on the B morphogenetic sequence (A-off, B-on). In conjunction with the A sequence this B sequence is responsible for transient nuclear migration during formation of the dikaryon and for the fusion of the hook cells to form clamp connections. Operation of the B sequence in the absence of the A sequence causes constitutive nuclear migration through dissolved septa and an irregular nuclear distribution. In addition, the hyphae

are irregularly shaped and no aerial mycelium is formed (flat mycelium). The formation of a common-B heterokaryon (Aon, B-off) is normally difficult to achieve, without nutritional forcing, because in the absence of the B sequence nuclear migration is blocked. However, mixing the appropriate homokaryons at inoculation apparently provided for so many contact sites where fusions could occur that even in the absence of nuclear migration the A-on phenotype, that is, the abundant presence of pseudoclamped hyphae (hyphae with hook cells that fail to fuse), could be observed throughout the culture. A similar phenotype was observed for the homokaryon with a primary mutation in the A gene. These A-on B-off mycelia also grew with little aerial mycelium. The presen.ce allelic differences in both the A and the

64

RUITERS,

SIETSMA,

genes in the homokaryons mixed at the time of inoculation or the presence of primary mutations in both the A and the B genes of a singly inoculated homokaryon resulted in full expression of the dikaryotic phenotype (A-on, B-on). In the heterokaryotic dikaryon only hyphae with clamp connections were seen, while in the homokaryotic dikaryon also hyphae with pseudoclamps were observed. In contrast to all the other cultures, these A-on B-on cultures fruited abundantly. As expected, the dikaryon-specific mRNAs were all very low or undetectable in the homokaryons with wild-type incompatibility alleles (Table 2). Similarly low concentrations were found in the A-off Bon mycelia and A-on B-off mycelia, regardless of their homokaryotic or heterokaryotic nature. However, in both the homokaryotic and the heterokaryotic dikaryons elevated concentrations of these dikaryon-specific mRNAs were found, their abundancies resembling those found previously in the heterokaryotic dikaryon (Mulder and Wessels, 1986). Clearly, both the A- and B-morphogenetic sequences must operate for these mRNA concentrations to rise. Abundance

AND

WESSELS

The nonspecific mRNAs were present at somewhat varying concentrations with no apparent correlation to the operation of the A- or B-morphogenetic sequence. Concentrations of specific mRNAs during light-induced fruiting. When a dikaryon of S. commune (4-39 X 4-40) was grown from a mycelial homogenate in surface culture under continuous light, fruiting occurred throughout the culture. However, when grown for 4 days in the dark from a plug inoculum on complete medium, transfer of the colony to light induced fruiting at a ring approximately 5 mm wide with the inner boundary 3 mm within the original margin of the dark-grown colony. Cultures remaining in the dark never fruited. Growth was monitored daily by marking the margin of the colony at the bottom of the petri dish (Fig. 1). Linear growth rates in the dark and in the light were 5.6 and 5.3 mm day-‘, respectively. This inhibition of linear growth rate by light has been observed previously (Raudaskoski and Viitanen, 1982). From duplicate colonies, rings corresponding to the growth zones were harvested after growth of the colonies for a total of 6 and 10 days, respectively, and RNA was extracted. The concentrations of

TABLE 2 of Dikaryon-Specific and Nonspecific mRNAs in 4-Day Surface Cultures of Monokaryons Heterokaryons with Wild-Type and Mutated Incompatibility Genes

and

Percentage of total RNA x 103” Dikaryon-specific

A41 B41 A43B43 A41 B43

Nonspecific tnRNAs

mRNAs

SC-1

SC-2

SC-4

SC-S

SC-~

SC-7

SC-9

SC-14

SC-3

SC-10

co.9 co.9 <0.9

co.9 1.5 2.4

3.6 5.1 10.8

co.9 co.9 <0.9

co.9 co.9 co.9

<0.9 <0.9 1.5

co.9 1.8 1.8

co.9 co.9 co.9

49.5 63.3 60.9

1.5 <0.9 <0.9

1.8 0.9

40.9 1.2

57.0 2.4

2.4 4.8

A41B41 AIlBmut

x A41B43

1.5 1.8

1.8 co.9

<0.9 4.2

1.2 co.9

1.8 co.9

<0.9 <0.9

A41B43 AmutB41

x A43B43

1.2 1.2

1.2 1.5

<0.9 2.4

co.9 co.9

co.9 CO.9

co.9 2.4

co.9 1.8

0.9 2.4

41.4 38.1

<0.9 2.7

A41B41 x A43B43 AmutBmut

30.6 34.2

2.7 6.0

98.4 96.0

7.5 4.5

23.4 20.4

5.1 3.6

2.4 3.0

41.4 36.6

0.9 3.9

2.4 1.8

a Each value represents the mean of two cultures. Maximum variation between mRNAs concentrations independent cultures was 1.2% of the values indicated.

in the

mRNAs IN Schizophyllum commune

DIKARYON-SPECIFIC

the specific mRNAs are shown in Fig. 2. In dark-grown cultures the dikaryon-specific mRNAs, and also the two monitored nonspecific mRNAs, were absent or at very low concentrations except for the SC-~ mRNA, which appeared in the oldest part

DARK

LIGHT

I 1 2 t

6 10

4 8

2 6

4 2 0 0 4 2 0 8 6 4 age of colony region (days)

2

0

FIG. 2. Concentrations of dikaryon-specific mRNAs (SC-~, 2,4, 5, 6,7: 9, and 14) and nonspecific mRNAs (SC-~ and 10) in the various growth zones indicated in Fig. 1. mRNA SC-~ is omitted from the figure because it was too low to be detected in any of the zones. Shaded bars represent concentrations as present 6 days after inoculation. Open bars represent concentrations as present 10 days after inoculation. Cuhures were grown continuously in the dark (A) or transferred to light after 4 days (B).

5

of the colony between the 6th and In 6-day-old cultures transferred t the 4th day the mRNAs were present approximately at the position where the body primordia were seen (cf. Fig. 1). mRNAs decreased again in this region when growth was allowed to continue in the light for 4 more days, but some of them (mRNAs SC-~, 4,7, and 9) were also detectable in a second area behind the margin of the lo-day-old colony. This correlated with the position in these colonies where a few new primordia arose that did not deve further (Fig. 1). In the period between 6th and IOth day a few of the primordia in the inner ring developed into full-grown fruit bodies. This experiment suggested that the measured mRNAs play no role in the vegetative growth of the dikaryon because they were very low in dark-grown cultures. They appeared as a response to light not so much at the growing edge of the colonies but at positions which approximated the positions where fruit bodies arose. Time course of appearance of specific mRNAs after transfer to light. TF fruiting monokaryon 4-39, the dikaryo~ 439 x 4-40, and the monokaryotic fruiter HF2 were grown from plug-inocula on complete medium in the dark for 4 days, Rings, 5 mm wide with the inner borders 3 mm within the margins of the colonies, were then marked on the bottoms of the dishes and the cultures were transfer-r light. Rings corresponding to the markings were then cut from the colonies at various times after transfer to light and processed for measurements of the mRNA &o~~c~~t~~tions . Figure 3 shows the abundances of the specific mRNAs in the colonial rings from the moment the cultures were shifte light (time zero). In the dikaryon and the monokaryon HF2 distinct fruit-body primordia were visible on the rings after 2 days. The monokaryon 4-39 remained vegetative. None of the cultures fruited when

RUITERS,

SIETSMA,

AND WESSELS

80 t 60-

F 8 f

time

.

(days)

FIG. 3. Appearance of dikaryon-specific mRNAs and nonspecific mRNAs after transfer of darkgrown colonies (4 days) to light. The concentrations were measured in an isolated ring of 5-mm width including 3 mm of the outer border of the dark-grown mycelium. (A) Dikaryon 4-39 X 4-40; (B) monokaryon 4-39; (C) monokaryon HFZ.

cultivation was continued in the dark. After illumination of the dikaryon all the dikaryon-specific mRNAs except SC-~ rose concomitantly with the appearance of fruit bodies. Significantly, the dikaryon-specific mRNAs SC-~, SC-~, and SC-~ also rose in the monokaryon HF2 concomitant with the appearance of the monokaryotic fruit bodies. The other dikaryon-specific mRNAs were not detectable in HF2, which was particularly noteworthy since mRNA SC-~ reached rather high concentrations in the fruiting dikaryon. The low concentration of the nonspecific mRNA SC-~ in all these cultures was also significant because this n-RNA could reach extremely high concentrations in cultures grown from spread mycelial homogenates (Mulder and Wessels, 1986; compare Table I). On the other

hand, the nonspecific mRNA SC-10 reached unusually high concentrations in the fi-uiting ring of dikaryotic mycelium. Figure 3 also shows that some of the dikaryonspecific mRNAs had a tendency to peak a second time after 4 to 5 days of illumination. This corresponded to the time when a few of the fruit-body promordia started to expand their pilei. DISCUSSION

In previous studies (Dons et al., 1984; Mulder and Wessels, 1986) a number of cDNA clones were isolated that represented the abundant mRNAs formed in the dikaryon at the time of fruiting but which remained very low in the monokaryotic mates from which the dikaryon was formed.

RUITERS,

SIETSMA,

Because the only genetic difference between these mycelia was the presence of single alleles for the two incompatibility genes in the monokaryons and the presence of two different alleles of both genes in the dikaryon, the presence of different alleles for one or both incompatibility genes must have been responsible for the appearance of these mRNAs. In this study we show that allelic differences at both incompatibility genes must exist in a heterokaryon to effect a rise in these mRNAs. In common-A and common-B heterokaryons these mRNAs are as low as those in the monokaryons. Additional evidence for the regulation of the dikaryon-specific mRNAs by the concerted action of the incompatibility genes was obtained by showing that in homokaryons that morphologically mimic the heterokaryons due to constitutive mutations in the incompatibility genes, these mRNAs only appear in the double mutant which mimics the heterokaryotic dikaryon both in hyphal morphology and fruiting capacity. The genetic regulation of these dikaryon-specific mRNAs therefore resembles the regulation of diploid- and sporulation-specific mRNAs by different alleles of the mating-type genes in Saccharomyces cervisia (Clancy et al., 1983; PercivalSmith and Segall, 1984). Although the precise function of these alleles in S. cerevisiae is still largely unknown (Garber and Segall, 1986) the molecular regulation of these mRNAs by the interaction of the products of the two mating-type genes is rapidly being uncovered (Mitchell and Herskowitz, 1986). However, the system of S. commune is much more complex because two different mating-type genes exist, each with a large number of alleles (Raper, 1966, 1983). Also, the interactions between the alleles entail the differentiation of the dikaryon and the development of multicellular fruit bodies in which meiosis and sporulation eventually occur. A relationship between the appearance of the dikaryon-specific mRNAs and fruiting

AND

WESSELS

6-J

was previously surmised (Mulder and sels, 1986) because these mRNAs very low in vegetatively growing dikaryon, rose steeply in concentration during fruitbody formation, and were higher in concentration in the developing fruit bodies than in the supporting vegetative mycelium. The cultures used in those studies wer from mycelial homogenates spre minimal medium. When fruit-bod opment in such cultures was inhibited wit 5% carbon dioxide or absolute darkness, or by growing the dikaryon in shaken suspension cultures, the dikaryon-specific mRNAs nevertheless appeared at the time that fruiting would have occurred under noninhibiting conditions, although at somewhat lower concentrations (Wessels et al., 1987). Also the three nonspecific examined rose with culture age an bly were selected originally because ~ftbe~r abundance at the time of harvest for preparing the cDNA bank (Dons et al., 1984), i.e., the time of maximal expression o fruiting in the dikaryon before the glucose was depleted. The associ the dikaryon-specific mRNAs with fruiting could therefore be a coincidence or their production may be necessary but not sufficient for fruiting to occur. To reexamine the relationship the dikaryon-specitic mRNAs an in this study, we measured their concentrations in colonies grown from a plug inoculum. We used this method because it avoids possible artifacts induced by hornQ~e~i~~~g the mycelium and enables measurements of RNAs in regions representing hyphae of different ages. To induce fruit bodies in a predictable area we used dark-light transfers as employed by Raudaskoski and tanen (1982) and Yii-Mattila (1985) to st the effects of light on fruiting. It turn that the dikaryon-specific mRNAs, a nonspecific mRNAs examined, ap only after transfer of the colonies to light and only in the region where fruit bodies arose. As in cultures grown from a ~y~cl~~~

68

RUITERS,

SIETSMA,

homogenate (Mulder and Wessels, 1986) the mRNAs peaked in the fruiting ring of mycelium. The decline was followed by the appearance of a second wave of increased concentration for some of the dikaryonspecific mRNAs (notably mRNAs SC-1 and SC-~) at the advancing front of the colony where new fruit bodies arose. Although the dikaryon-specific mRNAs have been shown to be abundant in full-grown fruit bodies (Mulder and Wessels, 1986) it should be noted that such fruit-bodies were probably too few in number compared to the many abortive fruit bodies to prevent the decline in the specific mRNAs. Light did not induce any of the measured mRNAs in the nonfruiting monokaryon but at least mRNAs SC-~, SC-~, and SC-~ were induced in a monokaryon (HF2) carrying alleles that permit monokaryotic fruiting. However, these fruit bodies were abnormally shaped and produced no spores. Preliminary experiments have indicated that the presence of other haploid-fruiting alleles also results in the formation of “dikaryon-specific mRNAs.” This tends to strengthen the suggestion that these mRNAs are actually involved in fruiting. Formally, such haploidfruiting alleles in S. commune (Leslie and Leonard, 1979; Esser et al., 1979) can be thought of as representing alleles of secondary regulatory genes for the dikaryonspecific mRNAs, with the incompatibility genes exerting the primary control. They would thus be analogous to the RMEZ gene of yeast (Mitchell and Herskowitz, 1986). Although a causal relationship of the dikaryon-specific mRNAs with fruiting remains to be proven, they are probably not involved in the vegetative growth of dikaryotic hyphae nor in the monokaryondikaryon transition. They appear absent from colonies of the dikaryon growing in complete darkness and only appear in the light behind the growing front of the colony. This agrees with the late appearance of these sequences in cultures grown from mycelial homogenates as previously shown

AND WESSELS

(Mulder and Wessels, 1986; Wessels et al., 1987). Why darkness completely suppresses the appearance of these mRNAs in colonies but not in cultures grown from a mycelial homogenate is now under investigation. ACKNOWLEDGMENT The skillful technical assistance of Jan Springer is gratefully acknowledged. REFERENCES CLANCY, M. J., BUTEN-MAGGEE, B., STRAIGHT, D. J., KENNEDY, A. L., PARTRIDGE, R. M., AND MAGEE, P. T. 1983. Isolation of genes expressed preferentially during sporulation in the yeast Saccharomyces cerevisiae. Proc. Nati. Acad Sci USA 80: 3mo--3004.

DONS, J. J. M., MULDER, G. H., ROUWENDAL, G. J. A., SPRINGER, J., BREMER, W., AND WESSELS, J. G. H. 1984. Sequence analysis of a split gene involved in fruiting from the fungus Schizophyllum comune. EMBO .I. 3: 2101-2106. ESSER, K., SALEH, F., AND MEINHARDT, F. 1979. Genetics of fruit body production in higher basidiomycetes. Curr. Genet. 1: 85-88. GARBER, A. T., AND SEGALL, J. 1986. The SPS4 gene of Saccharomyces cerevisiae a major sporulationspecific mRNA. Mol. Cell. Biol. 6: 447%4485. HOGE, J. H. C., SPRINGER, J., AND WESSELS, J. G. H. 1982. Changes in complex RNA during fruit body initiation in the fungus Schizophyllum commune.

Exp.

Mycol.

6: 233-243.

LESLIE, J. F., AND LEONARD, T. J. 1979. Monokaryotic fruiting in Schizophyllum commune: genetic control of the response to mechanical injury. Mol. Gen. Genet. 175: 5-12. MITCHELL, A. P., AND HERSKOWITZ, I. 1986. Activation of meiosis and sporulation by repression of the RMEl product of yeast. Nature (London) 319: 738142.

MULDER, G. H., AND WESSELS, J. G. H. 1986. Molecular cloning of RNAs differentially expressed in monokaryons and dikaryons of Schizophyllum commune in relation to fruiting. Exp. Mycol. 10: 214227.

PERCIVAL-SMITH, A., AND SEGALL, J. 1984. Isolation of DNA sequences preferentially expressed during sporulation in Saccharomyces cerevisiae. Mol. Cell. Biol. 4: 142-150. RAPER, C. A. 1983. Controls for development and differentiation in basidiomycetes. In Secondary Metabolism and Differentiation (J. M. Bennett and A. Ciegler, Eds.), pp. 195-238. Dekker, New York.

DIKARYON-SPECIFIC

mRNAs IN Schizophyllum

RAPER, J. A. 1966. Genetics of Sexuality in Higher Fungi. Ronald Press, New York. RAPER, J. R., AND HOFFMAN, R. M. 1974. Schizophyllum. In Handbook of Genetics (R. C. King, Ed.), Vol. 1, pp. 597-626. Plenum, New York. RAUDASKOSKI, M., AND VIITANEN, H. 1982. Effect of aeration and light on fruit body induction in Schizophyllum

commune.

Trans.

Brit.

Mycol.

Sot.

78: 89-

96. SWAMY, S., UNO, I., AND ISHIKAWA, T. 1984. Morphogenetic effects of mutations at the A and B incompatibility factors in Coprinus cinereus. J. Gen. Microbial. 130: 3219-3224.

commune

69

WYESSELS,J. G. H., AND NIEDERPREUM, D. J. 1967. Role of cell-wall glucan degrading enzyme in mating of Schizophyllum commune. J. Bacterial. 94: 1.5941602. WESSEL~, J. G. H., SPRINGER, I., AND MULDER, G. H. 1987. Expression of dikaryon-specific and non-specific mRNAs of Schizophylium commune in relation to environmental conditions and fruiting. S. Gen. Microbial. 133: 2557-2561. YLI-MATTILA, T. 1985. Action spectrum for fruiting in the basidiomycete Schizophyllum commune. Physiol. Plant.

65: 286-293.