Control of fungal development

DEVELOPMENTAL

BIOLOGY

53,

21-29

(1976)

Control I. The Effects

of Two

Regulatory

MIRIAM University

RICH

of Minnesota, 250 BioScience

of Fungal Genes

Development on Growth

ANDERSON

AND CAROL

in Schizophyllum

commune

S. DEPPE

Department of Genetics and Cell Biology, Center, St. Paul, Minnesota 55108 Accepted

April

26,1976

In Schizophyllum the various events comprising the transition from the homokaryotic to the dikaryotic stage of the life cycle are triggered by two sets of developmental regulatory genes known as the A and B incompatability factors. This paper defines the effects of these genes on growth of surface colonies by establishing the growth curves of dikaryons, comonA heterokaryons, and strains that morphologically mimic dikaryons or heterokaryons because of constitutive mutations in A and/or B. Like homokaryons, all these developmental stages and genetic mimics have triphasic growth curves with definite exponential phases. Growth curves of dikaryons and common-A heterokaryons are indistinguishable from those of their corresponding genetic mimics. However, the growth rate during exponential phase and the timing of the entire curve are dependent upon developmental type, with the consequence that colonies of different developmental stages harvested either at equal times or at equal weights are not necessarily in the same phase of colonial growth. Data presented allow choice of harvesting times such that colonies of the different developmental types will be within the same growth phase. Biochemical differences between homokaryons and dikaryons must be due to differentiation since the two stages have virtually the same growth rate.

tative, filamentous colony called a “homokaryon.” Homokaryons generally have one haploid nucleus in each cell. When compatible homokaryons grow together they give rise to dikaryotic colonies having two nuclei per cell, one derived from each homokaryon. Homokaryons and dikaryons are morphologically distinct. Both stages can grow vegetatively indefinitely. The dikaryon is also capable of inducing fruit bodies (mushrooms), thus engaging in the sexual part of the life cycle that results in production of the haploid spores. In order to be compatible and form a dikaryon, two merging homokaryotic strains must carry unlike A factors and unlike B factors. Each incompatibility factor is responsible for “triggering” a specific subset of the developmental events that occur during the transition from the homokaryon to the dikaryon. There are many different A factors and many different B factors in nature, and these genes are the

INTRODUCTION

The incompatibility factors in Schizophyllum commune and other related fungi are regulatory genes that control the transition from the homokaryotic to the dikaryotic stage of the life cycle. This transition, known as “dikaryosis,” is a developmental transition between two vegetative stages. The physiology and genetic control of this transition have been extensively studied and there are constitutive as well as null mutations of the regulatory genes available to facilitate developmental studies (Raper, 1966; Raper and Raper, 1968; Niederpruem and Wessels, 1969; Koltin, Stamberg, and Lemke, 1972; Kuhn and Parag, 1972; Raper and Hoffman, 1974). This paper describes the effects of these regulatory genes and their constitutive mutants on colonial growth. In Schizophyllum, germination of a haploid spore leads to formation of a vege21 Copyright All rights

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

22

DEVELOPMENTAL

BIOLOGY

mating type genes for the organism. Dikaryosis in Schizophyllum is an ideal system in which to study control of development because of the ease with which the organism is handled for genetic as well as biochemical work and because extensive information is available on the genetics of the control of dikaryosis itself. Dikaryosis entails the following sequence of events: 1. Apparently controlled by neither A nor B. When two colonies grow together some of the intermingling cells of the two merging colonies fuse. These fusions occur whether or not the colonies are compatible and also occur between hyphae within a single colony. 2. Controlled by B factor. Septa of each of the merging homokaryotic colonies break down and nuclei from each colony migrate rapidly throughout the mycelium of the compatible mate. This step occurs only if the two strains have different B factors, and occurs whether or not they have different A factors. Thus septal disruption and reformation, nuclear migration, and a host of associated biochemical changes-alteration in composition of cell walls, increased level of various hydrolytic enzymes, etc. -are said to be “triggered,” “initiated,” or “controlled” by B factor and are referred to as “B-sequence.” That is, Bsequence is that part of the dikaryotic transition that can be triggered by B factor alone, when the mating is between strains carrying identical A factors. (Such matings are symbolized “A= B # .“I 3. Controlled by A factor. Migrant nuclei pair with resident nuclei in the cells of the newly formed dikaryon and septal disruption and nuclear migration cease. Nuclear migration and septal disruption and reformation are a transient stage in fully compatible (A#B#) matings but are constitutive in hemicompatible A=B+ matings. 4. Controlled by A factor. After nuclear pairing, subsequent cellular divisions are “conjugate”; i.e., the two paired nuclei di-

VOLUME

53, 19’76

vide simultaneously making use of a special morphological feature, the hook cell. The conjugate division mechanism permits the maintenance of two different haploid nuclei per cell as a stable state during subsequent vegetative growth. All the processes involved in conjugate division except the last one (hook cell fusion) depend only on the A factor. Under suitable conditions, vegetatively growing dikaryons induce mushrooms and reproduce sexually. Fusion of the two haploid nuclei takes place in basidial cells of the mushroom and is followed immediately by meiosis and spore production. In the heterokaryon that results from A=B # matings (the common-A heterokaryon) nuclear migration and septal disruption and reformation are constitutive. The hyphal filaments are gnarled and irregularly branched, and the cell wall is imperfect, as evidenced by extrusions of cytoplasm. Growth is slow and the overall colony morphology is sickly looking and nearly lacking in growth above the surface of the medium. There are constitutive mutations in the B factor, which we refer to as “Bcon” (Bconstitutive). The mutants resemble the common-A heterokaryon when they carry a normal A factor. This is symbolized “Ax Bcon .” Crosses between strains with different A factors but common-B factors (A#B = matings) do not produce heterokaryons unless special genetic tricks are used to bypass the B-controlled migration stage. When such tricks are used, the resulting common-B heterokaryons resemble Acon Br strains (a constitutive mutant A factor and a normal B factor). The nuclei in the apical cells are paired and go through all the processes of conjugate division except the last, hook cell fusion. Strains carrying both Acon and Bcon are morphologically indistinguishable from normal dikaryons. They produce mushrooms and normal spores. These spores (all of Acon Bcon genotype) germi-

Control

nate to give rise to transient homokaryons that spontaneously dikaryotize, produce mushrooms, and complete the life cycle in the absence of any mating.

of Fungal

Development

23

was autoclaved for 20 min in l-liter batches, and poured at a temperature of about 60°C to reduce condensation. A layer of medium a minimum of &mm thick (in the center) was used in each petri plate. METHODS Thinner medium shortens the exponential It is possible to Very briefly, the technique employed in phase of colony growth. this study involves growing a fungal col- lengthen exponential phase somewhat by ony on a water-permeable membrane overmoving the colony to a new plate of melying agar medium and repeatedly removdium occasionally. With the slow-growing ing the membrane with colony from the strains (those carrying Bcon and commonmedium for weighing without disturbing A heterokaryons), the change of medium is necessary in order to prolong the expothe colony’s growth. Weighings are carried out by placing the membrane with colony nential phase enough to measure it. on sterile aluminum foil on the pan of an The cellophane membrane material used was obtained as a free sample from analytical balance. Each weighing must DuPont (no. CNC50367, type 150 PD 52). be finished before water loss due to evapoMembranes were cut to 6- x 6-cm squares ration becomes significant. This technique for fast-growing strains or 4-cm circles for is described in detail elsewhere (Deppe, slow-growing strains. The membranes 1976). The strains used in these experiments should be no larger than needed because are descendants of the Raper 699 series the extra surface area allows greater wawhich have been backcrossed more than 30 ter loss and weighing is then less accurate. were sterilized briefly in generations. Thus they are largely iso- The membranes water, drained, laid on the genie to 699 and to each other except for distilled the A and B factors. The wild-type homohardened medium, and allowed to stand karyons used included Raper 1736 (A41 until they stopped losing water into the B43), Raper 1737 (A43 B41), M4 (A41 medium (equilibration) - about 5 days at B41), M5 (A43 B43), M6 (A41 B43), and 20°C. All growth experiments were done at 20°C. Slower growth rates are obtained M7 (A43 B41). The wild-type dikaryons and common-A heterokaryons were estabwith old medium, so plates should be used have equililished by mating appropriate pairs of the as soon as the membranes homokaryons listed above: 1736 + 1737, brated. M4 + M5, M4 + M6, M5 + M7, and M6 + Inocula were taken from membrane culM7. The homokaryons with constitutive tures grown at 20°C. A sharp knife was mutations in A and/or B were descendants used to cut the colony and membrane siof Raper 2394 f&on Bcon) backcrossed to multaneously. A standard 3- x 3-mm square of colony measured from and invarious of the homokaryons listed above. These constitutive mutations are those cluding the advancing hyphal front was used by Wang and Raper (1969, 1970) and used (Deppe, 1976). Inocula were cut under derived originally by Parag (1962) and 17x magnification to allow accuracy in Raper, Boyd, and Raper (1965). measuring size and to make certain the The colonies were grown on a standard fungus was cut cleanly and not torn. minimal medium containing 20 g of gluMost strains can be inoculated from colcose, 1 g of L-asparagine, 0.46 g of KHxP04, onies of any age as long as the hyphal front 1 g of K2HP04, 0.5 g of MgS04, 0.12 mg of is available. However, the inocula for Ax thiamin HCI, 1 ml of trace elements soluBcon’s and common-A heterokaryons tion (Hunter, 1950), and 20 g of agar should be from colonies which are as young as possible for two reasons. These (Sigma Type IV) per liter. The medium

24

DEVELOPMENTAL

BIOLOGY

slow-growing strains can easily become genetically modified because there is a selective advantage for additional mutations that increase the growth rate. Also, in this material the hyphal front becomes more uneven as the colony grows older, so uniform 3- x 3-mm inocula are more readily obtainable from young colonies. Weighings were normally read to the nearest 0.5 mg. However, the weight of the slow-growing material was estimated to the nearest 0.1 mg even though the sensitivity of the technique is about ~~0.5 mg. The initial weight is subtracted from all subsequent ones, the weights graphed on semi-log paper with time on the linear scale, and the doubling time during exponential phase is extrapolated to determine the specific growth rate.

VOLUME

TABLE

1

APPROXIMATE EXTENT OF THE EXPONENTIAL IN INDIVIDUAL COLONIES OF VARIOUS DEVELOPMENTAL TYPES”

Homokaryons S-60 4-50 S-50 5-65 7-55 4-55 3-60 3-50 10-55 l-55 10-100 5-80 8-85 9-55 5-55 4-60

RESULTS

The growth of approximately 225 colonies was followed in this study. Data from three representative colonies of each developmental stage or genetic mimic of a developmental stage are presented in the graphs that follow and are discussed below. The three colonies shown are always from at least two different experiments. In addition, a summary of various parameters for a subset of the colonies is presented (cf. Tables 1 and 2) to enable the reader’s evaluation of how the colonies graphed compare to those not shown. The tables do not include the earlier experiments; many of these did not include the entire growth curve. Nor do they include colonies lost before the end of the experiment because of contamination, genetic modification, or technical mistakes, or some occasional colonies whose curves, for unknown reasons, showed too much scatter to permit accurate drawing of the lines. In general, it was colonies of the more slow-growing types that generated the greatest problems of all sorts-contamination during the longer experiments, genetic modification, etc. -as well as showing greater scatter within curves and greater variation from

53, 1976

Acon

Bx’s

PHASES

Dikaryons A’s

Z-50 3-45 4-50 5-45 2-40 1.5-40 2-35 2.5-40 2-40 1.5-30 4.5-45 l-30 I-30 2-35 2.5-40 1-35 3-45 1.5-35 3-35 2-35 3-40 1.5-30 l-30

3-60 5-60 5-70 2-45 7-60 5-60 5-55 3-45 3-40 3-50

-

1.5-22 2.5-20 3-28 3-45 3-18 2.5-16 2.5-22 3.5-20 1-17 l-18

Acon &on’s

Ax Bcon’s

2.5-35 4-45 2.5-40 4-45 2-45 1.5-50 2.5-50 1.5-45 Z-50 2-45 1.5-40 3-45 3-40 5-75

1.5-17 3-20 2.5-19 3-22 3-18 2.5-17 1-22 1-17 3-25 3-30 4-45 2.5-30

-

a In milligrams

colony to colony within and among experiments. A variety of strains was used to show the degree of variation from homokaryon to homokaryon and from dikaryon to dikaryon, etc. We found that there was no more variation among different homokaryons than among different colonies of a single homokaryotic strain. The same holds true for each of the other developmental types studied. (The strains used all belong to a highly coisogenic series.) Colonies of all de,velopmental stages and mimics of developmental stages exhibited three-stage growth curves including extended exponential phases. However, the doubling times during the exponential

ANDERSON TABLE

-

Control

AND DEPPE

2 Doubling time during exponential phase (hr)

Number of colonies with this doubling time

Homokaryon

10 11 12

6 9 1

Dikaryons

11 12 13

4 5 1

Acon

11 12

5 10

11 12 13 14 15

1 5 5 13 1

20 21 23 24 27

5 2 1 1 1

20 21 22 24 26

3 1 7 2 2

Bcon’s

Acon’s

Common-A karyons

hetero-

Bcon’s

-

phases as well as the times and weights at which colonies begin and cease exponential growth depend critically upon the developmental stage involved. Thus colonies of different developmental stages that are harvested at uniform times or at uniform masses are not necessarily in comparable phases of colonial growth. The characteristics of each developmental stage are described below. The differences found and their implications for those who do biochemical work on this aspect of Schizophyllum development are dealt with in the discussion. Homokaryons Three typical growth curves of homokaryons are shown in Fig. 1. Colonies generally grow exponentially at least from the

of Fungal

25

Development

time they weigh 8-10 mg until they weigh 50-55 mg, and many colonies enter exponential earlier or leave it later (cf. Table 1). Their doubling times during exponential are about lo-11 hr (cf. Table 2). The exponential phase can be lengthened somewhat by transferring a colony to fresh medium after each weighing, but the doubling time remains the same. The homokaryon has the fastest growth rate of any stage studied, and it enters and leaves the exponential phase at higher weights than the other developmental stages. Dikaryons and Strains Both A and B

Constitutive

for

Growth curves of dikaryotic colonies are not distinguishable from those of the Aeon Bcon strains that are their genetic mimics. Three typical growth curves of dikaryons and three of Acon Bcon’s are shown in Figs. 2 and 3, respectively. Colonies from this developmental stage generally grow exponentially at least from the time they weigh 4-5 mg until they weigh 40-50 mg (cf. Table 1). Some enter exponential much earlier or leave much later, and it is possible to extend the exponential phase by transferring the colony to fresh medium occasionally. The doubling time during exponential is about 11-12 hr (cf. Table 2). Thus the growth rate of dikaryons and Aeon Bcon’s is statistically indistinguishable from that of homokaryons. They may grow slightly more slowly than homokaryons, but the difference, if it is real, is small. A-Constitutive

Strains

Figure 4 shows three typical growth curves ofAeon Bx colonies. They generally grow exponentially at least from the time they weigh 3-5 mg until they weigh 30-35 mg (cf. Table 1). Some colonies are in exponential much earlier or later than this and it is possible to extend the exponential phase somewhat by placing the colony on fresh medium occasionally. This does not alter the growth rate. The doubling time

DEVELOPMENTAL

BIOLOGY

VOLUME

53, 1976

FIGS. 1-6. In each figure, the three curves are on different time scales. The abscissa is marked in lo-hr intervals, and the age (in hours) of each colony at the beginning of the curve is given immediately below the first point in the curve. FIG. 1. Homokaryons: 1736 (O), 5.17 (A), and M4 (W). FIG. 2. Dikaryons: 1736 + 1737 (O), M6 + M7 (A), and M4 + M5 (B). FIG. 3. Acon &on’s: F140 (O), F131 (A), and F132 (B). FIG. 4. Acon Bx’s: F133 (O), 7 (A), and F133 (m). FIG. 5. A= heterokaryons: M4 + M6 (O), M5 + M7 (A), and M4 + M6 (m). All three colonies were transferred to fresh medium every 12 hr from the age of 3 days until the end of the experiment. FIG. 6. Ax &on’s: 2547 (O), Ml2 (A), and Ml1 (m). The latter two were transferred to fresh medium every 12 hr from the age of 3 days until the end of the experiment. The first one was not.

during exponential Table 2).

is about

Common-A Heterokaryons tutive Strains

12-14 hr (cf. and B-Consti-

Three typical growth curves of commonA heterokaryons and three of Ax Bcon’s are shown in Figs. 5 and 6, respectively. Growth curves for Ax Bcon strains are indistinguishable from those of the hetero-

karyons they mimic. The colonies generally grow exponentially at least from the time they weigh about 3 mg until they weigh about 17-18 mg. Somewhat longer exponentials are commonly found, but colonies seldom remain in exponential over a tenfold increase in colony weight (cf. Table 1). It is usually necessary to place these colonies on fresh medium at least once per day in order to obtain exponential phases

ANDERSON

AND DEPPE

even this long. The doubling time during exponential phase is usually 20-22 hr with slower times up to about 27 hr occurring less frequently (cf. Table 2). This stage is by far the slowest growing and gives by far the most variation in growth curves. The exponential phases start earlier and end earlier than those of colonies from the other three stages. SUMMARY

AND

DISCUSSION

This study shows the following: (1) Dikaryons, A- and B-constitutives, A-constitutives, common-A heterokaryons, and Bconstitutives, like homokaryons, have triphasic colonial growth curves with definite exponential phases. (2) The timing of the beginning and ending of the exponential phase as well as the growth rate during exponential phase depends upon the developmental type. The latter result has two serious implications for those who wish to do biochemical work on dikaryosis in Schizophyllum. The rest of this section is devoted to discussing these two implications. First of all, it is commonly accepted that biochemical comparisons of two different strains are meaningful only if the two strains are (1) grown in the same manner and (2) harvested in the same phase of growth. The initial motivation behind the work described in this paper lay in our need to satisfy the latter requirement. One possible means of obtaining comparable material from different developmental types is to harvest the material at equal ages regardless of weight. However, this study shows that such material is often in different phases of growth. For example, at the age of 125 hr a homokaryon commonly weighs about 60 mg and is in postexponential phase or in transition to postexponential. But an Ax Bcon commonly weighs less than 2 mg and is in preexponential or early exponential phase. To attribute biochemical differences between the two to differentiation would be extremely naive.

Control

of Fungal

Development

27

Given that colonies of different developmental types are not comparable if harvested at equal ages, another possibility would be to harvest colonies at equal weights regardless of age. This study shows that such colonies are not necessarily comparable either. For example, a 40mg homokaryon is in exponential phase. A dikaryon or Acon Bcon of 40 mg would be at the transition from exponential to postexponential and an Acon Bx strain would be in early postexponential. Common-A heterokaryons and Ax Bcon’s would have been in postexponential for some time. This paper provides the information that allows one to harvest any of these developmental types during their exponential phases. Further, all developmental types are in exponential over the weight range of lo-17 mg, so experiments can be designed so that all the material is harvested at equivalent weights as well. Whether this latter is necessary is unknown. Data have been presented (Deppe, 1976) showing that the ratio of dry weight to fresh weight of Schizophyllum colonies is constant throughout the exponential phase but rises sharply thereafter. In addition, we have preliminary data indicating that the cyclic AMP and cyclic GMP levels are constant throughout the exponential phase. Thus it may be that the colonial exponential phase is pragmatically equivalent to the exponential phase of bacterial cultures, where all biochemical amounts, ratios, and processes per unit mass are constant throughout the exponential phase, and one may harvest different cultures anywhere within that phase and be assured of comparability. Biochemical studies on Schizophyllum are frequently done on material grown in liquid shake cultures. The cultures are inoculated with vegetative material macerated with a blender so that the fragment size ranges from less than one cell to more than a hundred cells. One can extrapolate from work with individual membrane grown colonies that such flasks would con-

28

DEVELOPMENTAL

BIOLOGY

tain colonies whose growth curves would not be synchronous because larger inocula give rise to colonies that enter exponential much earlier than colonies generated by the smaller inocula (Deppe, 1976). In addition, the culture reinoculates itself when fragments break off larger colonies. The reinoculation probably occurs to varying extents and with various timings in material of different developmental stages since the stages vary in growth rate, branching pattern, and possibly also friability. At harvest time such cultures are probably heterogeneous mixtures of preexponential, exponential, and postexponential colonies as well as of dead material, and the fraction of the material that is in these phases at any standard harvesting time probably varies with the developmental type. Even if one were willing to use heterogeneous cultures, there is no obvious way to determine the age, if any, at which cultures of the different developmental types would be comparable. Biochemical comparisons of the different stages are hardly meaningful when any differences discovered could be due to differences in phase of growth rather than stage of development. The second implication of this work concerns a problem that classical developmental biologists rarely forget-the dilemma of distinguishing between processes and parameters related to growth and those related to differentiation, given that most systems that differentiate usually undergo dramatic alterations in growth rate simultaneously. Developing sea urchin embryos and differentiating Dictyostelium discoideum are systems that are attractive to developmental biologists because they undergo differentiation in the absence of significant growth, and thus without alteration of growth rate. Dikaryosis in Schizophyllum should be added to the list of developmental systems in which one can do studies that avoid the problem of confounding growth and development; it is a developmental transition

VOLUME

53. 1976

between two vegetative stages that have virtually the same growth rate. This system offers the additional advantage that one can get ready incorporation of radioactive labels, etc., into both the “before” and “after” stages of development because of the fact that both are very actively growing vegetative stages. However, the common-A heterokaryons and their Ax Bcon genetic mimics grow very slowly compared to homokaryons and dikaryons. Hence, any difference between, for example, a homokaryon and an Ax Bcon strain may not be caused by the difference in developmental stage, but rather may merely be a correlate of slow growth. A possible solution to this problem might be to use, as additional controls, homokaryotic colonies grown in a manner in which they have a gro_wth rate comparable to those of the Ax Bcon strain. We have preliminary data indicating that one can obtain different doubling times during the exponential phase by altering the nitrogen source in the medium. If control homokaryons with slow growth rates resemble the homokaryon and not the Ax Bcon strain with respect to the parameter being studied, one could probably safely conclude that the difference between the two developmental stages is, in fact, a function of differentiation. This work was supported by grants from the University of Minnesota Graduate School and the Research Corporation to the junior author, and by Grant No. IN-13 from the American Cancer Society. REFERENCES DEPPE, C. (1976). Growth kinetics of surface colonies of tilamentous fungi. J. Gen. Micro. (in press). HUNTER, S. H., PROVASOLI, L., SCHATZ, A., and HASKINS, Z. P. (1950). Some approaches to the study of the role of metals in the metabolism of microorganisms. Proc. Amer. Phil. Sot. 94,152. KOLTIN, Y., STAMBERG, J., and LEMKE, P. A. (1972). Genetic structure and evolution of the incompatibility factors in higher fungi. Bact. Reu. 36,156. KUHN, J., and PARAG, Y. (1972). Protein-subunit aggregation model for self-incompatibility in higher fungi. J. Thor. Biol. 35, 77. NIEDERPRUEM, D. J., and WESSELS, J. G. H. (1969).

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AND DEPPE

Cytodifferentiation and morphogenesis in Schizophyllum commune. Bact. Rev. 33, 505. PARAG, Y. (1962). Mutations in the B incompatibility factor in Schizophyllum commune. Proc. Nat. Acad. Sci. USA 48, 743. RAPER, J. R. (1966). “Genetics of Sexuality in Higher Fungi.” Ronald, New York. RAPER, J. R., BOYD, D. H., and RAPER, C. A. (1965). Primary and secondary mutations at the incompatibility loci in Schizophyllum. Proc. Nat. Acad. Sci. USA 53, 1324. RAPER, J. R., and HOFFMAN, R. M. (1974). Schizo-

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phyllum commune. “Handbook of Genetics,” Vol. I, p. 597. Plenum Press, New York. RAPER, J. R., and RAPER, C. A. (1968). Genetic regulation of sexual morphogenesis in Schizophyllum commune. J. Elisha Mitchell Scientific Sot. 84, 267. WANG, C.-S., and RAPER, J. R. (1969). Protein specificity and sexual morphogenesis in Schizophyllum commune. J. Bacterial. 99, 291. WANG, C.-S., and RAPER, J. R. (1970). Isozyme patterns and sexual morphogenesis in Schizophyllum commune. Proc. Nat. Acad. Sci. USA 66, 882.