MUTATIONS MODIFYING SEXUAL MORPHOGENESIS IN

MUTATIONS MODIFYING SEXUAL MORPHOGENESIS IN SCHIZOPHYLLUMl

CARLENE A. RAPER AND JOHN R. RAPER

The Biological Laboratories, Haruard Uniuersity, Cambridge, Massachusetts

Received June 13, 1966

HE morphogenetic sequence leading to the establishment of the fertile dikaryon from two self-sterile homokaryons in the tetrapolar Hymenomycete

Schizophyllum commune consists of a number of distinct stages. After hyphal fusion between compatible homokaryons, (a) reciprocal nuclear exchange and migration convert both homokaryons into a dikaryon by (b) pairing of the compatible nuclei, ( c ) conjugate nuclear division, (d) the formation of hook- cells, lateral hyphal outgrowths associated with conjugate division, (e) hook-cell and hyphal septation, and ( f ) hook-cell fusion. The dikaryon, capable of in- definite propagation, normally bears fruiting bodies, within which occur karyo- gamy, meiosis, and the formation of haploid spores to complete the life cycle. This process is common to all tetrapolar fungi; its nature and regulation appears to be identical in Schizophyllum, Coprinus, Collybia, Pleurotus, etc. Relevant information from species other than S. commune is accordingly included (for compre- hensive review see RAPER 1966).

All of the events from nuclear migration through hook-cell fusion are regulated jointly by the A and B incompatibility factors. The two factors assort independently and are each constituted of two linked loci, (Y and ,8, each with multiple alleles (RAPER, BAXTER, and MIDDLETON 1958; RAPER, BAXTER, and ELLINGBOE 1960). The entire developmental sequence can occur only when the paired homokaryons carry different alleles in at least one of the loci of the A factor and in one of the loci of the B factor. The role of each factor is evident from the sequences of events that occur in the two hemicompatible heterokaryons. In the establishment of the heterokaryon in which the B factors are different but the A factor is identical, A= BZ, only nuclear migration occurs. This is the major event of the B sequence. In the establishment of the heterokaryon in which the A factors are different but the B factor is identical, A# B=, nuclear migration either does not occur or is severely restricted (SNIDER 1965), but the events beginning with nuclear pairing and ending with hook-cell septation proceed normally. This is termed the A sequence. Pairing and conjugate division of the two parental nuclei occur in growing apical cells as part of the A sequence; but the subsequent failure of hook-cell fusion in each apical cell prevents the entrapped daughter nucleus from passing from the hook-cell to the newly formed subapical cell, which then contains a daughter nucleus of the other parental type. The establishment of a

' This woib was suppxted by Pub l~c IIealth Service Grant AI OGlE4

Genet:rh 5.1: 1151-l l ( iX November 1966.

1152 C. A. RAPER AND J. R. RAPER

dikaryotic subapical cell is thus prevented. Only the new apical cell, which contains the other daughter nuclei of both parental types, remains binucleate (Fig- ture IC). Allelic differences must exist in both factors, A# BZ, for completion of the AB sequence, beginning with nuclear migration and ending with hook-cell fusion and the establishment of the fertile dikaryon (Figure 1, d and D).

The control imposed by the incompatibility factors on this entire process is completely disrupted by mutations at the incompatibility loci. All of several known mutations of A and of B factors (located in the AP and BP loci) have resulted in the loss of the normal discriminatory functions of self-recognition and self-incompatibility of the affected factors (PARAG 1962; DAY 1963; RAPER, BOYD, and RAPER 1965). The effect of one mutated factor in the homokaryon is thus equivalent to the effect of two different wild-type factors of the same series in a heterokaryon. In a homokaryon carrying a mutated A factor, Amut Bx (where x is any wild factor), the A sequence operates continually, and the homokaryon therefore mimics the A# B= heterokaryon; in an Ax Bmut homokaryon, the B sequence operates continually, and the homokaryon therefore mimics the A= B# heterokaryon; in an Amut Bmut homokaryon, the entire AB sequence is expressed, and the homokaryon therefore mimics the A# B# heterokaryon, the dikaryon.

Knowledge of the AB sequence is incomplete at best. While a characterization of the two sequences as expressed in the hemicompatible heterokaryons and their homokaryotic mimics is desirable, a more detailed understanding should be possible through a study of mutations, other than at the incompatibility loci, that disrupt specific stages in the two sequences.

The present paper describes and analyzes several such mutations. These modifier mutations are expressed only when part or all of the sequence is operating, i.e., in all mycelia except normal homokaryons and A= B= heterokaryons. Nine mutations representing two phenotypes have been described (RAPER and RAPER 1964). More recently, seven modifier mutations of three additional phenotypes were identified, and these, together with representatives of the first two types, are compared. The disruptive effects of the different modifiers are compared by a quantitative analysis of nuclear distribution and septal characteristics.

MATERIALS A N D METHODS

Most of the strains of S. commune examined in this study-normal as well as those that carried known auxotrophic mutations, modifier mutations, and primary mutations at the Ai3 incompatibility locus-originated in this laboratory. One homokaryon with a primary mutation at the BP locus was kindly supplied by DR. YAIR PARAG, Hebrew University, Jerusalem.

Mycelia of A= B# phenotype constitutes a selective system for the modifier mutations; they are expressed as sectors of more vigorous and morphologically altered growth. The specific origins of mutations MI-M9 are given elsewhere (RAPER and RAPER 1964). Mutations MIO-MI6 originated as follows: MIO, spontaneous in Ax Bmut; M i l , induced in Ax Bmut by ethyl methane- sulfonate (RAPER, BOYD, and RAPER 1965); M12-Mi6, induced in Ax Bmut by unfiltered 100 kv X-radiation. M12-Mi6 were isolated and partially characterized by MR. YIGAL KOLTIN of these laboratories.

The strains were cultured and mated on semisolid medium (SNIDER and RAPER 1958) in

SEXUAL MORPHOGENESIS I N S C H I Z O P H Y L L U M 1153

standard plastic petri dishes at 22°C. All A= B=, A# B=, and certain modified heterokaryons were established by forced heterokaryosis between complementary auxotrophic strains. Micro- scopic observations of septal characteristics and nuclear distribution were made on stained, intact mycelia. The preparation of these materials are described elsewhere (RAPER 1966).

A survey of the characteristics of each mycelial type was made by examining apical segments of intact hyphal systems about 20 cells in length, branches included. Each survey was made by determining the number of nuclei, the type of septation, and the morphology of hook cells in each of 90 to 460 cells in a dozen or more hyphae chosen at random from mycelia of several independent cultures of the same basic genotype.

Observations by phase microscopy were made on live mycelia grown on membranes overlying nutrient medium. A strip of membrane with an adherent young mycelium was placed on a slide and overlaid with a drop of warm, 14% gelatin and a cover glass. In such preparations, nuclei and septa were plainly visible, and the mycelia survived for 3 to 4 days.

RESULTS

The role of the modifier mutations is only understandable in terms of departures from the normal sequence that the mutations effect. The following account accordingly presents experimental results in the order: (a) the normal sequence and its control by the incompatibility factors, (b) the genetics of the modifier mutations, and (c) aberrations caused by the modifier mutations.

Sexual morphogenesis: Sexual morphogenesis, in part or in whole and normal or modified, is discernible in mycelial morphology (Figure 1) . The specific correlations of incompatibility genotypes and morphology are given in the first line of Table 1.

Two parameters, the number of nuclei per cell and the frequencies of septa of various types, strikingly reveal quantitative differences among the various mycelia. Uninucleate cells with simple septa in the homokaryon and binucleate cells with clamp connections in the dikaryon represent two uniform and anti- thetical conditions. The two hemicompatible heterokaryons represent two distinct intergrades between the homokaryotic and dikaryotic states. Nonuniform nuclear distribution, 0 and > 2 nuclei per cell, and pseudoclamps characterize the A= B# and A# B= heterokaryons, respectively. These features are quanti- fied in Table 2 and graphically presented in Figure 2. The phenotypic mimicry of heterokaryons by homokaryons with mutated incompatibility loci is supported by similarities in nuclear distribution and septal characteristics (Table 2, lines 4 us. 14,20 us. 27, and 28 us. 34).

Nuclear distribution bears a distinct relationship to septal character in mycelia of the various incompatibility genotypes. Beyond the quantitative analysis presented in Table 2 and Figure 2, additional observations on stained and live mycelia suggest some bases for this relationship. Most mycelia appear to have the regular pattern of nuclear division in apical cells only and septal formation per- pendicular to the plane of nuclear division immediately following division, and the septa remain intact indefinitely. Nuclear exchange from cell to cell is effec- tively prevented by the septa, which have been shown to be complex structures in S. commune (SNIDER 1965) and other Hymenomycetes (GIRBARDT 1961; MOORE and MCALEAR 1962; GIESY and DAY 1965).

1154 C. A. RAPER A N D J. R. RAPER

I;rc;urtr: I .-Ihsic types of morphology relevant to incompatihility genotypes antl modifier mutation,. A-I) i i r i t l a-tl-Grass antl hyphal morphology of normal mycelia. Aa-hornokaryon. Rh-A= n+ hrtrrokaryon. Cc-A# n= hrtcrokaryon. Dd-A+ n# hetrtmkaryon, the tlikary- on. E-F and e-f-Gross and hyphal morphology of reprcsenfativr niodilircl mycelia. See Table f for spccific correlations. Arrows drsignatr apical crlls. thr tips of which lir to the right. a-f: 600 x m’rgnificiition.

SEXUAL MORPHOGENESIS IN SCHIZOPHYLLUM 1155

TABLE 1

Mycelial and hyphal morphology correlated with incompatibility genotypes and modifier mutations in full dose

Incompatibility genotypes

Modifier mutations' Ax Bx A = B= A= B# Az Bmut A# B= Amut Bx A# B# ilmut Bmul

None A.a+ A.a B.b B.b C.c C c,e D.d D d[c]

I 1 (M2-M9) A.a A.a F.e[c] F.f E.e F.c[e] F.e[c] 1 ( M I ) A.a A.a F.e F.f E.e,a D.d

I11 (MIO) A.a A.a F.e,f F.f C.c D.d I V ( M I 1 , M15. M I 6 ) A.a A.a A.a A.a C.c D.d

V (M12-MI 4) A.a A.a D.e D.e E.e D.e

* I-V designate phenotypic classes. + Mycelial morphology, capital letters; hyphal morphology, lower-case letters (illustrated in Figure 1). Brackets indicate relative rarity.

The outstanding characteristic of the A== B# heterokaryon and of its Ax Bmut mimic is the erratic distribution of nuclei. Certain features frequently seen in stained preparations of these mycelia, but rarely in other mycelia, possibly ex- plain the nonuniform nuclear distribution: (a) an occasional nucleus lying across a septum; (b) occasional half septa; (c) numerous hyphal bridges between adjacent hyphae; (d) synchronous division of aggregated nuclei in inter- calary as well as in apical cells; and (e) an excess of nuclei to cells in the ratio of 7:5. The nuclear movement and septal alterations that underlie the features seen in stained material have been observed in living Ax Bmut homokaryons by phase-contrast microscopy. Within a period of ten minutes, a septum that initially blocked completely the passage of nuclei and visible mitochondria became altered to permit the passage of a constricted nucleus. One and a half hours later, the septum extended only half way across the hypha, and nuclei were observed to pass through it freely. The constant disruption of septa, the frequent formation of hyphal bridges, and the movement of nuclei across cells and hyphae to from aggregates suggest that the continual operation of the B sequence, in the absence of the A sequence, involves nuclear aggregation as well as the initial disruption of septa (LEHFELDT 1923; NOBLE 1937; GIESY and DAY 1965) and rapid migration of nuclei (BULLER 1931; SNIDER 1965). There is no evidence for any regular association of the two constituent nuclei in the A= B# heterokaryon (SNIDER and RAPER 1965; RAPER and SAN ANTONIO 1954; RAPER and RAPER 1964). In fact, one type of nucleus appears to predominate locally and nuclear aggregates, as frequently seen in apical cells as in subapical cells, are almost always of one constituent type.

By contrast, nuclear distribution in the A# B= heterokaryon is quite regular. Whereas the cells of the A# B= heterokaryon and of its mimic, the Amut Bx homokaryon, are mostly uninucleate, there is an important distinction between the apical cells of main hyphae and all subapical cells, apical cells of branches included. The apical cells of main hyphae in the A# B= heterokaryon and in its mutant mimic are mostly binucleate (Table 3). Conjugate division of the nuclei is seen frequently in these apical cells, the paired nuclei representing the two


TABLE 2

Nuclear and septal characteristics correlated with incompatibility genotypes and modifier mutations

Genotypes Sample Nuclei per cell-% Septal typ8-X no.

Incompatibility Modifier. of cells 0 1 2 5-25 S PC CC

1 2 3$ 4$ 5 63 73 8 9

10 11 1 22 13 14 15 16 17 18 19 20% 21 $ 223 23$ 242 252 263 27 28 29 303

32 33 34 35

313

Ax Bx

A= B= A= B#

Ax Bmut

A# B=

Amut Bx A# B#

I1 ( M 2 )

I(MI) + I(MI)

II(M2) + II(M.2)

III(MI0) + III(MI0)

II(M2) + III(MI0) IV(MII) + IV(M1I)

I1 ( M 2 )

II(M2) + II(M3)

I(MI) + III(MI0)

V(MI4) + V(MI4)

I1 ( M 2 ) III(MI0) IV(MI1)

I(MI) + I(MI) II(M2) + II(M2)

III(Mf0) + III(MI0)

V(MI4)

II(M2) + II(M3)

IV(MIf) + IV(MIf) V(Mi4) + V(Mf4)

I(M1) + I(MI) II(M2) + II(M2)

III(MI0) + III(Mf0) II(M2) + II(M3)

V(Mf4) t V ( M f 4 ) Amut Bmut . . . .

II(M2)

308 97

200 284 193 246 151 180 226 181 199 253 121 21 7 214 273 222 23 1 126 341 435 159 72

242 91

232 227 484 106 114 166 110 130 181 21 0

10 81 7 2 11 84 4 1 5 9 2 3 0

28 M 16 12 13 77 8 2 26 48 16 10 10 74 13 3 2 6 6 7 7 0 24 51 13 12 9 8 3 8 0

20 62 12 6 6 8 9 3 2 7 9 0 3 0

53 23 10 14 27 22 20 31 12 48 27 13 18 30 24 28 4 9 0 6 0

IQ 83 5 2 15 66 16 3 14 77 8 1 7 9 1 2 0

10 85 5 0 5 8 7 6 2

18 61 17 4 22 75 3 0 19 68 11 2 1 7 9 2 0 4 5 9 0 1

10 70 18 2 5 80 14 1 4 2 9 4 0 8 8 3 8 1

12 19 67 2 14 80 6 0

100 0 0 100 0 0 100 0 0 98 1 1 31 69 0 41 53 6 10 90 0 37 63 0 24 65 11 7 92 1

47 53 0 100 0 0 29 70 1 98 2 0 57 15 28 30 51 19 15 54 31

loo 0 0 35 65 0 12 88 0 75 25 0 21 79 0 4 96 0

17 83 0 3 97 0

25 75 0 8 92 0 0 4 96 3 3 94 8 92 0

10 90 0 3 3 94

31 69 0 5 24 71

13 87 0

I-V designate phenotypic classes. t S = simple septa, PC = septa with pseudoclamps, CC = septa with clamp connections f Forced heterokaryosis.

constituent types (RAPER and RAPER 1964). Conversely, most subterminal cells of the two types of mycelia are uninucleate, and the remainder are either anucleate or multinucleate. Branch hyphae, which form only on subapical cells, have predominantly uninucleate apical cells in both mycelia (Table 3) . Most septa of hyphae with binucleate apical cells have septate, nucleate pseudoclamps, whereas most septa of hyphae with uninucleate apical cells (branch hyphae) have anucleate pseudoclamps, either septate or nonseptate (Table 4). It is thus clear both


TABLE 3

Effect of modifier mutations on nuclear pairing: nuclear pairing in apical cells of main and branch hyphae of pseudoclumped mycelia with uninucleate subapical cells

Apical cells ~~

Main hyphae Branch hyphae Types of

Inrompatibility modifier Uninucleate Binucleate Uninucleate Binucleate genotypes mutations No. No. % No. No. %

A# B= None 7 50 88 63 4 6 I S 1 21 0 0 29 0 0 I1 + I1 47 3 6 46 0 0 I11 + I11 0 38 100 49 4 8 IV + IV 3 17 85 26 2 7 v + v 20 1 5 36 0 0

Amut Bx None 15 21 58 72 0 0

A= B# I + I 27 3 10 19 0 0 I1 + I1 27 7 21 56 1 2 v + v 23 0 0 42 2 5

A# B# I1 + I1 13 28 68 57 0 0 v + v 26 0 0 53 0 0

Amut Bmut I1 17 3 15 37 2 5

from previous and present observations that the A sequence involves fairly regular pairing of the two constituent nuclei, hook-cell formation, conjugate nuclear division, and hook-cell septation.

A precise relationship between the A and B sequences is suggested by the following characteristics of the fully compatible heterokaryon, A# B#: (a) establishment of the heterokaryon by nuclear migration; (b) occurrence of nuclear pairing and conjugate division in all apical cells: (c) uniform condition of clamp connections and binucleate cells; (d) strict 1: 1 ratio of the two constituent nuclear types in all cells; and (e) absence of any evidence for disruption of septa, formation of hyphal bridges, and nuclear movement between cells and hyphae. Apparently the part of the B sequence responsible for the disruption of septa ceases to function once the invading nuclei reach terminal cells and pair

TABLE 4

Relationship between nuclear pairing and type of septation in mycelia with pseudoclamped hyphae and uninucleate subapicu2 cells

Type of septation' (percent) No. of No. of hyphae cells S .%PC ASPC NSPC

Main hyphae with binucleate apical cells 49 502 9 10 11 70 Branch hyphae with uninucleate apical cells 348 707 17 49 24 10 Main hyphae with uninucleate apical cells 83 1,086 26 45 23 6

* S =simple septa, -4PC =anucleate pseudoclamps, ASPC=anucleate, septate pseudoclamps, NSPC=nucleate, septate pseudoclamps.


I N C O M PAT I B I 1 I T Y G E NO T Y P E S A x B x A=Bf & B = A # B #

2 I I - - 0)

U

L

0)

0

.- al

U

x

- 2

z 0

I- 3

-

m

b o

- U

I? 0

U U W A U 3 z ..... ... :;:;: gi ::A :I::: 1

...

.e...

UNMOD I F I ED

M O D I F I E D ( F u l l D o s e )

I (M1)

II (M2)

y [M14), S I M I L A R

m(Ml0)

m(MI1)


with resident nuclei. The A sequence then begins to operate and the further function of the B sequence involves only the fusion of hook cells.

Genetics of modifier mutations: Sixteen independent mutations have been found to modify sexual morphogenesis in several basic ways. These mutations are designated Ml-M16, and each was identified initially by its effect upon the morphology of the A= B Z heterokaryon or its mutant mimic, the Ax Bmut homokaryon. With the exception of M9, each mutation has been shown to segregate normally with its wild-type allele. The data on segregation indicate no linkage of any of the modifiers to either the A or the B incompatibility factor. The 16 modifier mutations are assigned to five phenotypes as follows: type I, M I ; type 11, M2-M9; type 111, MIO; type IV, M I 1 , M15, M I & ; and type V, M12-MI4. Modifier mutations belonging to the various types will henceforth be designated specifically by number, i.e., M l - M I 6 and generally or collectively by type, i.e., I-V. Although types I and 11, MI-M9, have been characterized previously (RAPER and RAPER 1964), they will be discussed here insofar as new information about them has become available and also in their relationship to the more recently characterized types 111, IV, and V.

A determination of the genetic relationships of the mutations is seriously hampered by two features of types I1 and V. First, heterokaryons formed by the mating of compatible strains, each carrying a modifier of either type, rarely if ever form heterokaryotic fruiting bodies; second, modifiers of both types are dominant to their wild alleles, and complementation tests between independent mutations are therefore impossible. Nevertheless, it has been possible to make an analysis of successful crosses in the pairing in all combinations of six selected modifiers, representative of all five types (Table 5 ) . The expected ratio of 3 M: 1 f for two unlinked mutations obtains with a probability by chi-square test of > 0.05 in all fertile combinations of M I , M2, M6, MlO,M11, and M14, except for M 2 X M6. The high ratio of M:+ in this pairing suggests that M 2 and M6 are loosely linked.

Beyond this, four crosses, each involving two independent mutations of the same type, provide additional information. M6 x M7, both of type 11, yielded no wild progeny and are either allelic or closely linked (RAPER and RAPER 1964). Crosses between all three representatives of type IV yielded mutant/wild progeny as follows (with P values for expected segregation for two unlinked loci) : M11 x

FIGURE 2.-Graphic comparisons of nuclear content of cells and septal characteristics in mycelia of different incompatibility genotypes with and without full doses of representatives of each of the phenotypic classes of modifier mutations. The top graph compares unmodified incompatibility phenotypes. The four remaining graphs detail the phenotypes resulting from incompatibility genotypes in association with modifier mutations. In all graphs, the bars in each vertical column add to 95-100% (<5% values omitted). The population of cells in respect to nuclear content, indicated by light shading in the left column for each mycelium, is divided into thre: categories: uninucleate (bottom) ; binucleate (top) ; anucleate and multinucleate, i.e., 0 and >2 (middle). Septal type, indicated by dark shading in the right column, is also divided into three categories: simple septa (bottom) ; septa with clamp connections (top) ; and septa with pseudoclamps (middle) (Data from Table 2.) “No effect” signifies no recognized effect of modifier and ns dztailed analysis.

1160 C. A. RAPER AND J. R. RAPER

TABLE 5

Genetic relationships of modifier mutations: segregation of modified to unmodified progeny

Modifier mutations. I(M1) II(M2) II(M6) III(Mf0) IV(M11) V ( M f 4 )

I ( M 2 ) 58/0 55/14 38/14 43/22 72/22 42/10 I I ( M 2 ) - 50/7+ 64/20 M/28 -

III(M2O) 54/0 75/25 42/9 IV ( M I 2 ) 28/0 79/25

I 1 ( M 6 ) - 61/11 46/22 -

V ( M I 4 ) -

* I-V designate phenotypic classes. t By x 2 test for 3:l segregation, P=0.01-0.02; for all other fertile heteroallelic crosses, P>0.05.

M15, 35/15 ( P = 0.5); MI1 X M16, 55/8 ( P 1 0.03); M15 x M16, 48/18 ( P = 0.7). The three type IV mutations are clearly located in three distinct loci.

The samples of 50 to 100 progeny analyzed in all of the crosses are not adequate to determine precise relationships between the mutations; the analysis of nine modifiers, however, has demonstrated with certainty the separate location of five: M I , M2, M6, MZO, and MIZ. Furthermore, phenotypic differences between the four other types of mutations and the three type IV mutations (M11, M15, and M I & ) suggest that these latter represent two more loci, although their independ- ence from mutations of other types has not been demonstrated. The three type V mutations (M12, M13, and M14) probably represent at least one additional locus. Little attention has been paid to the location of the modifiers in respect to known loci other than the A and B factors, with which all assort independently. M11 is the sole exception; it is linked to thin, a morphological mutation, at 16 crossover units and to uracilless-2 at 3 crossover units. The order of these loci was not established.

Expression of modifiers: The several mutations are assigned to the five different phenotypes, I to V, according to their characteristic patterns of effects upon the A, B, and AB sequencies of sexual morphogenesis. The effects of the modifiers, however, must be considered in three different levels of dosage: full dose, homoallelic double dose in heterokaryons and single dose in homokaryotic mimics of heterokaryons; half-dose, a single dose in one of the constituents of heterokaryons, in which dominance or recessiveness of the mutations to their wild alleles becomes relevant to their phenotypic effects; and doubly heteroallelic dose, single doses of different mutations of the same type or of different types in the two constituents of heterokaryons, in which dominance, recessiveness, epis- tasis, and additivity contribute to the determination of phenotypic effects.

The effects of the mutations relate to (a) alterations in gross and microscopic morphology (Figure 1; Table l ) , (b) quantitative changes in nuclear distribution and septal characteristics (Table 2; Figure 2), and (c) the relationship between nuclear pairing in apical cells and the mode of septation (Tables 3 and 4).

1. Put1 dose: The effects of the modifier mutations upon the morphology of


Mated __ homokaryons

or

Homokaryot ic equivalents -

Controlling factor(s)

Ax Bx Ax Bx Ax Bx - x X X

Ay Bx AY BY Ax By - x X X

Ay Bx AY BY Ax By

HYPHAL FUSION a I

I I I

I I Amut Bmut AxBmut

B - A A and B

I I I

I I HYPHAL FUSION

a I Amut Bmut AxBmut

B - A A and B

I B f

I I Sequence operates when - A # A# and B #

FIGURE 3.-Modification of sexual morphogenesis by modifier mutations of the five phenotypic classes. The normal developmental sequences controlled by the two incompatibility factors, separately and together, follow the solid vertical lines connecting the events comprising dikaryosis. Specific effects of full doses of the modifier mutations of phenotypic classes I to V are indicated by arrows. A cross bar represents a blocking of all subsequent events in the series; a dotted line around an event indicates the bypassing of that event; and a dotted line to an event indicates the induction of that event. Parentheses denote a partial effect.

the heterokaryons and their mimics are specified in Table 1 and illustrated in Figure 1.

Figure 3 presents a diagram of the morphogenetic progression, with the effects that are caused by mutations of the five types in the A, AB, and B sequences (exemplified in the A# B=, A# B#, and A= BZ heterokaryons, respectively) indicated either as interruptions of normal processes or as the induction of stages or structures that are normally absent.

Note: (a) Types I, 11, and V have generally opposite effects in the A sequence, in which they block the normal process, and in the B sequence, in which they block one normal process but induce abnormal processes. (b) Type I affects both A and B sequencies and type I11 affects the B sequence, but neither is expressed in the AB sequence. (c) Type IV renders any strain carrying it incap- able of accepting nuclei in interaction with any mate, i.e., unilaterally. IV completely blocks the B sequence when it alone is operating but has no effect


upon the AB sequence when the basic handicap of unilateral mating is bypassed by forced heterokaryosis. An A= B# heterokaryon with a full dose of IV can also be established by forced heterokaryosis, and such a heterokaryon is a faith- ful mimic of the A= B= heterokaryon. Similarly, the phenotype of IV Ax Bmut is a mimic of the normal homokaryon. IV thus blocks nuclear aggregation as well as nuclear migration. (d) Although type I1 causes the fusion of a small portion of the induced hook cells in the B sequence, it completely blocks hook-cell fusion in the AB sequence.

The following points are not evident in Figure 3. (a) Types I1 and V render the A, AB, and B sequences subsequent to nuclear migration essentially identical, but they differ strikingly in one important feature: whereas type I1 in full dose prevents fruiting in the A# B# heterokaryon, type V permits copious fruiting in the A# B# heterokaryon and induces fruiting in the A= B# heterokaryon. The progeny of the fruiting bodies borne on heterokaryons, however, are mono- typic, i.e., all identical, and like one parent only. (b) Binucleate apical cells occur in frequencies of 10% and 21%, respectively, in A= B# heterokaryons that carry I and 11. A previous analysis showed comparable incidences of heterokaryotic hyphal tips in mycelia carrying mutations of these types (RAPER and RAPER 1964). (c) A curious relationship exists in the effects of I and I1 on the B sequence: mutations of both types block nuclear aggregation in the A= B# heterokaryon but not in its Ax Bmut mimic, and both types induce some hook-cell fusion only in the latter. This is the one exception noted to the rule that the modifiers have identical effects upon heterokaryons and upon their respective homokaryotic mimics.

TABLE 6

Expression of modifier mutations in single dose and in homoallelic and heteroallelic double dose in A= B# and A# B# heterokaryons

Modifier mutations None I ( M 1 ) II(M2) II(M6) III(M1O) I V ( M f 1 ) i V ( M 1 4 )

a . A=B#: I ( M 1 ) I 1 ( M 2 ) I I ( M 6 ) I I I ( M l 0 ) IV( M 1 1 ) t V ( M 1 4 )

b. A#B#:

I I ( M 2 ) I 1 ( M 6 )

IV (Mil)+ V ( M 1 4 )

1 ( M I )

I I I ( M l 0 )

+* ++ ++ ++ ++ + ++ ++ + + ++ + + - -

+

++ ++ ++ ++ ++ ++ + ++ ++ + ++ + w 4 1

' f =partially modified phenotype. -k f =fully modified phenotype. Number in brackets indicates modifier mutation

+ Unilateral mating reaction. expressed.


2. Single dose: Further differentiation among the five types of modifiers is possible from a comparison of their effects when present in single dosage in A= B# and A# B# heterokaryons (Table 6, left column). Single dose effects in A# B= heterokaryons have not been studied.

Mutations of types I11 and IV are recessive to their wild alleles in the B sequence and are not expressed in the AB sequence. Although IV strains interact unilaterally, the heterokaryotic products of their interaction with fully compatible wild mates are normal in all respects. Mutations of two other types, I1 and V, are partially dominant in both B and AB sequences. The single mutation of type I is partially dominant in the B sequence and is not expressed in the AB sequence (Table 6, left colum, a and b) .

Partial dominance signifies an expression of the mutant phenotype that is intermediate between the unmodified and full-dose phenotypes. For example, an A= B# heterokaryon with a single dose of I1 ( M 2 ) has pseudoclamps at only 53% of its septa (90% in full dose) (Table 2). An A# B# heterokaryon with single dose of I1 displays a partial suppression of hook-cell fusion, evident as frequent pseudoclamps interspersed with true clamp connections. and a reduction in fruiting competence, whereas a full dose of I1 completely suppresses hook-cell fusion and fruiting.

3. Double hetermllelic dose: In every case that has been examined, the effects of two single doses of different mutations of the same phenotype can be rationalized from their individual dominance or recessiveness in single dose and the effects that they cause in full dose. Double heteroallelic doses of type I1 (Table 6) and of type V are thus additive and give the same result as full dose in either case. Two different mutations of type IV, each recessive to its wild allele and the cause of unilateral interaction, have no effect in A= B# heterokaryons established by nutritional forcing on minimal medium,

The situation with double heteroallelic doses of mutations of different phenotypes, however, is less simple and cannot be rationalized in detail from previously known characteristics of the mutations involved (Table 6) . The more significant departures from the interactions that might have been expected are the following:

I and I11 in the A= B# heterokaryon are additive, although I11 alone is recessive. I and I1 in the A# B# heterokaryon result in the suppression of 11, which is partially

dominant and expressed in single dose; I is epistatic to 11. I and I11 in the A# B# heterokaryon elicit a response comparable to that of a single dose

of 11, although neither I nor I11 has any effect in the A# B# heterokaryon even in full dose. IV and V in the A= B# heterokaryon interact to produce a mixed mycelial type that

appears to be partially homokaryotic and partially pseudoclamped with fruiting-body initials. Since IV is recessive, only the effect of V could have been expected.

One further possibility for genic interactions among the different modifiers is provided by homokaryotic mimics of heterokaryons, Ax Bmut, Amut Bx, and Amut Bmut, that carry full doses of two different mutations. Such interactions have been examined only to the extent of combining a IV mutation with mutations of the other four types in the Ax Bmut genotype. The effect is the same in


all cases: IV is epistatic to all other modifier mutations, and only the phenotype of IV, indistinguishable from the normal homokaryon, is expressed.

Pseudochmps: The formation of pseudoclamps at most septa is a feature that many modified mycelia share with the unmodified A# B= heterokaryon (Figure 2). The careful examination of any pseudoclamped mycelium, however, reveals three major types of pseudoclamps: (a) anucleate nonseptate (Figure le) ; (b) anucleate septate (Figure le ) ; and (c) nucleate septate (Fig- ure IC). Less frequently, the pseudoclamps may be nucleate nonseptate (1 to 2%), and a few of any of these types may be reversed, i.e., curved toward the apical, rather than subapical, cell. Furthermore, as many as 10 to 20% of the cells may bear any of the above types of pseudoclamps between septa (Figure le, f).

The significance of the differing proportions in which these types of pseudoclamps occur in the various mycelia, particularly with respect to septation, is obscure, but there is a strong correlation between nucleate pseudoclamps and hyphae with binucleate apical cells. The combined data from all mycelia composed predominantly of pseudoclamped and uninucleate cells show that 70% of all septa along the axes of main hyphae with binucleate apical cells have nucleate pseudoclamps, while only 6% of the septa along the axes of main hyphae with uninucleate apical cells have nucleate pseudoclamps (Table 4). The apical cells of branch hyphae of both types of main hyphae are predominantly uninucleate (97%), and only 10% of their septa carry nucleate pseudoclamps (Table 4). The correlation between anucleate and nucleate pseudoclamps us. uni- and binucleate apical cells reflects the observed mode of nuclear division in the two cases. When only a single nucleus is present, the division is along the axis of the cell (Figure le) , whereas, when two nuclei divide conjugately, one divides axially and the other divides obliquely to deposit one daughter in the hook cell (Figure IC). The specific nature of the pseudoclamps is thus an accurate indication of the Occurrence of nuclear pairing. This correlation is not meaningful for pseudoclamped mycelia composed of cells with highly varied numbers of nuclei, e.g., some of the modified A z Bmut mycelia (Table 2, lines 15 to 17). The septa in such mycelia appear to become disrupted to permit the free exchange of nuclei from cell to cell.

DISCUSSION A N D CONCLUSIONS

The effects of the modifier mutations upon the partial and combined morphogenetic sequences regulated by the A and B incompatibility factors indicate an intricate relationship among the events comprising dikaryosis. Certain conclusions, however, may be drawn from the study about each of the events and thus provide some additional insight into the morphogenetic progression. These conclusions follow, each with its supporting evidence. I. Nuclear migration is not a necessary antecedent to subsequent events: Mu-

tations of type IV prevent nuclear migration, but when nuclear migration is circumvented by forced heterokaryosis, all of the subsequent events in the AB sequence occur normally.

2. Nuclear migration must inuolue cytoplasmic factors: Migration of wild-type nuclei into cytoplasm preformed under the influence of type IV mutations is rigidly prohibited, while nuclei carrying these mutations migrate rapidly through preformed normal cytoplasm.

3. Nuclear aggregation in the B sequence is blocked by the operation of the A sequence: Nuclear aggregation, a normal event in the B sequence, does not occur when the A sequence is also operative in the dikaryon. 4. Nuclear aggregation is independent of nuclear migration: Types I, 11, and

V block nuclear aggregation but have no effect upon nuclear migration.


5. Hook-cell formation is not dependent upon nuclear pairing: Types I, 11, and V block nuclear pairing in the A sequence but do not affect hook-cell formation; types I, 11, 111, and V induce hook-cell formation in the B sequence, but only I and I1 also induce nuclear pairing in a small fraction of the apical cells of the A= B# heterokaryon.

6. Nuclear pairing, a normal event in the A sequence, may be influenced by the operation of the B sequence: Type I blocks nuclear pairing in the A sequence but not in the AB sequence, and I1 blocks nuclear pairing in the A sequence but only partially in the AB sequence.

7. Conjugate division is probably a consequence of nuclear pairing: All observed divisions of paired nuclei in the A# B= and A# B# heterokaryons and in their mutant mimics and of grouped nuclei in the A= BZ heterokaryon and its mutant mimic have been synchronous; no evidence has been found of genetic disruptions to synchronous nuclear division in the absence of disruption to nuclear pairing.

8. Hook-cell septation is probably a consequence of conjugate division of paired nuclei, but it can also occur independently of conjugate nuclear division: Hook- cell septation virtually always occurs following conjugate nuclear division, and no mutative disruption to hook-cell septation has been found save for a mutation, described by DANA H. BOYD (personal communication), that blocks all septation both in the absence and in the presence of sexual morphogenesis. Hook-cell septation occurs about 30% of the time following axial divisions of single nuclei in apical cells.

9. Hook-cell fusion is an integral component of both the A and B sequences: Hook-cell fusion does not occur in the A sequence and occurs only sporadically, often abnormally, in hook cells induced by types I, 11, and I11 in the B sequence.

IO. The control of hook-cell fusion is independent of the controls for preceding events: Type I1 blocks hook-cell fusion just after the preceding event, hook-cell septation, in the combined AB sequence.

11. A n extensive complex of genes controls sexual morphogenesis: Of nine modifier mutations studied intensively, at least five and probably eight are at distinct loci, and these have at least five distinct phenotypes.

A more precise understanding of dikaryosis might emerge from the study of additional modifier mutations, especially recessive mutations, with simpler and more distinct disruptive effects than most of the mutations now available. A variety of available selective systems, some of which have not yet been exploited, would be required in the search for such mutations. Modifier mutations are expressed in great abundance in the numerous mycelial systems available, and greater care in the selection of cases for study should provide mutations that would, for example, lead in the B sequence to (a) disruption of nuclear migration but not of nuclear aggregation and (b) prevention of nuclear aggregation but not the induction of hook-cell formation. Similarly, mutations should be found that, in the A sequence, (a) prevent hook-cell formation but do not impair nuclear pairing, (b) prevent nuclear pairing but have no effect upon events in the B


sequence and, in the AB sequence, (c) prevent hook-cell fusion without affecting prior events.

Physiological and biochemical approaches perhaps offer the best hope for elucidating the disruptive effects of modifier mutations. The determination, by serological techniques and by acrylamide-gel electrophoresis, of significant differences in the protein spectra of the dikaryon versus its two component homokaryons (RAPER and ESSER 1961; DICK 1965; C . S. WANG and J. R. RAPER, unpub- lished) suggests that specific protein differences may be correlated with specific events in dikaryosis. A study of the possible effects of extracts from mutant mycelia upon wild-type mycelia and vice versa must await, at least, the development of effective means of getting the extracts into the cells.

Previous studies on the mutative disruption of sexual development in homothallic fungi, such as the Ascomycete Sordaria (ESSER and STRAUB 1958; OLIVE 1956, 1958; CARR and OLIVE 1959), have illustrated the complexity of sexual morphogenesis in forms lacking incompatibility control of sexuality. Of 18 mutations to self-sterility in Sordaria macrospora, for example, 14 were found to be nonallelic and could be assigned to at least four distinct phenotypes-it is the enviable good fortune of the investigators, ESSER and STRAUB, that all of the mutations were recessive and hence subject to complementation tests. Similar studies, recently begun by DR. PAUL A. LEMKE of these laboratories, have indicated comparable complexity in the sexual progression, i.e., dikaryosis, in a homothallic species related to Schizophyllum (LEMKE 1966). In the Basidiomycete Sistotrema brinkmanni, 40 of 41 mutations to sterility were recessive to their wild alleles and represented at least four phenotypes. The specific mutative disruptions observed involved: (a) hook-cell formation, (b) hook-cell fusion, (c) fruiting-body formation, and (d) sporulation. Although complementation tests for all combinations of the mutations have not been performed, numerous tests that have been completed indicate a high degree of nonallelism among the mutations.

The regulation of sexual morphogenesis by incompatibility genes necessarily complicates the study of dikaryosis in heterothallic Basidiomycetes, but the dual regulation of sexuality by two incompatibility factors in tetrapolar forms such as Schizophyllum permits examination of the two partial sequences in isolation and of their relationship to one another in dikaryosis. Perhaps the most intriguing aspect of incompatibility control in this system is the unique advantage it offers for the study of a genetically regulated process of differentiation in a eukaryotic organism.

S U M M A R Y

Sexual morphogenesis, the process of dikaryosis in Schizophyllum commune, is the summation of two separable sequences of events that are regulated by two distinct pairs of linked loci, the A and B incompatibility factors, respectively. Severe disruption to this morphogenetic progression is caused by numerous modifier mutations, 16 of which are here characterized and analyzed. These 16 modifier mutations (a) have no expression in the absence of sexual morpho-


genesis, (b) segregate normally and assort independently of both A and B incompatibility factors, (c) occupy a minimum of five (and more probably eight) loci, and (d) belong to five distinct phenotypes with respect to their specific effects upon the developmental sequences controlled by the incompatibility factors. Reprecentatives of the five types have been compared in a quantitative analysis of their effects on two phenotypic variables in the morphogenetic progression: nuclear distribution and septal characteristics. The study leads to several conclusions about the nature and interdependence of the specific stages in the progression.

LITERATURE CITED

BULLER, A. H. R., 1931

CARR, A. J. H., and L. S. OLIVE, 1959

Researches on Fungi. IV. Longman’s Green, & Co., London

Genetics of Sordaria fimicola. 111. Cross-compatibility among self-fertile and self-sterile cultures. Am. J. Botany 46: 81-91.

DAY, P. R., 1963 Mutations affecting the A mating type locus in Coprinus Zagopus. Genet. Res.

Physiological aspects of tetrapolar incompatibility. Pp. 72-80. Incompatibility

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DICK, S., 1965 in Fungi. Edited by K. ESSER and J. R. RAPER. Springer, Berlin.

Genetische Untersuchungen an Sordaria macrospora Auersw., Kompensation und Induktion bei genbedingten Entwicklungsdefekten. Z. Vererb. 89 : 729-746.

The septal pores of Coprinus Zagopus (Fr.) sensu Buller in relation to nuclear migration. Am. J. Botany 52: 287-294.

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ESSER, K., and J. STRAUB, 1958

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GIRBARDT, M., 1961

LEHFELDT, W., 1923 Uber die Entstehung des Paarkernmyzels bei heterothallischen Basidio- myzeten. Hedwigia 64: 13-51.

LEMKE, P. A., 1966 The genetics of dikaryosis in a homothallic Basidiomycete Sistotrema

Fine structure of Mycota. 7. Observations on septa of

The morphology and cytology of Typhula trifolii Rostr. Ann. Botany, N. S.

brinkmanni. Thesis. Harvard University, Cambridge.

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OLIVE, L. S., 1956 Genetics of Sordaria fimicola. I. Ascospore color mutants. Am. J. Botany 43: 97-107. __ 1958 On the evolution of heterothallism in fungi. Am. Naturalist 92: 233-25 1.

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RAPER, J. R., M. G. BAXTER, and A. H. ELLINGBOE, 1960

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RAPER, J. R., D. H. BOYD, and C. A. RAPER, 1965 Primary and secondary mutations at the incompatibility loci in Schizophyllum. Proc. Natl. Acad. Sci. U.S. 53: 132.4-1332.

RAPER, J. R., and K. ESSER, 1961 Antigenic differences due to the incompatibility factors in Schizophyllum commune. Z. Vererb. 92: 439-444.

RAPER, J. R., and J. P. SAN ANTONIO, 1954 Heterokaryotic mutagenesis in Hymenomycetes. 1. Heterokaryosis in Schizophyllum commune. Am. J. Botany 41 : 69-86.

SNIDER, P. J., 1965 Incompatibility and nuclear migration. Pp. 52-70. Incompatibility in Fungi. Edited by K. ESSER and J. R. RAPER. Springer, Berlin.

SNIDER, P. J., and J. R. RAPER, 1958 Nuclear migration in the Basidiomycete Schizophyllum commune. Am. J. Botany 45: 538-546. - 1965 Nuclear ratios and genetic complementation in common-A heterokaryans of Schizophyllum commune. Am. J. Botany 52 : 547-552.

C . A. RAPER A N D J. R. RAPER

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MUTATIONS MODIFYING SEXUAL MORPHOGENESIS IN