(JINKS 1952; PONTECORVO

DIRECTIONAL CYTOTOXIC REACTIONS BETWEEN INCOMPATIBLE PLASMODIA OF DZDYMZUM ZRZDZS

JIM CLARK AND O’NEIL RAY COLLINS

Department of Botany, University of California, Berkeley, California 94720

Manuscript received August 2, 1972 Revised copy received October 17, 1972

Transmitted by ROWLAND DAVIS

ABSTRACT

Incompatibility reactions of somatic cells in the myxomycete Didymium iridis are controlled by several loci displaying simple dominance. Pheno- typically dissimilar plasmodia generally undergo a temporary fusion which is quickly terminated by a cytotoxic reaction, whereas phenotypically similar ones undergo fusion which is not followed by such reactions. The size of the killed areas varies from microscopic up to a few square centimeters and is directly correlated with the amount of protoplasmic mixing which occurs. The amount of mixing itself is controlled by the incompatibility loci through regulation of the speed of killing. Each locus allows a characteristic amount of mixing and when two or more loci act together the amount is reduced by a subtractive effect. This results in a smaller killed area because of the more rapid cytotoxic reaction. Such a reaction is also unidirectional for each locus, with killing taking place in the recessive phenotype. Reversion of some heterokaryons to a homokaryotic state within 24 hours is considered evidence for the existence of unidentified weak incompatibility loci which act very slowly.

OMATIC cell fusion is an important means by which heterokaryons are formed, and considerable attention has focused on the significance of hetero-

karyosis in fungal pathogenicity, parasexuality, and in making possible com- plementary gene action studies. Since this has been adequately highlighted (JINKS 1952; PONTECORVO 1956; DAVIS 1966): there is no need for a further review of these subjects in this paper. However, far less emphasis has been placed on how somatic incompatibility systems prevent or impede heterokaryotization. The purpose of this paper is to describe how this is accomplished in the myxomycete Didymium iridis. At the outset it should be pointed out that we interpret heterokaryon incompatibility in this organism as one manifestation of the organism’s general capacity to distinguish between self and non-self, a capacity es- sential to any creature if it is to survive in nature. While this interpretation may not be universally accepted, we conclude that it is the simplest and most plausible one at our present state of knowledge.

The plasmodial stage of D. iridis is well-suited for the study of somatic incompatibility. Its unicellular state, macroscopic size, lack of a cell wall, regenerative and migration capacities make fusion studies relatively routine. Plasmodia can also be kept in the vegetative condition two to six months or longer. Additionally, Genetics 73 : 247-257 February, 1973

248 J. CI.ARK A N D 0. R. COLLINS

I.‘~cu~r: 1 .-l’la~mmlial te’st pairiiig. I X. FIGURE 2.-I’lasmoclial fusion. 15 x dissecting microscope. FIGURE 3.--Plasmodial fusion. 8 0 ~ compound microscope. Marker line equals 0.1 mm. FIGURE 4.-Plasmodial pairing showing no apparent interaction and thus having an incom-

FIGURE 5.-Cytotoxic reaction having an incompatibility rating of 1 . 80x compound micro-

FIGURE 6.-Cytotoxic reaction having an incompatibility rating of 5. 8 0 ~ compound micro-

patibility rating of 0. 80x compound microscope.

scope.

scope.

PLASMODIAL CYTOTOXIC REACTIONS 249

since the haploid myxamoebae, which can also act as gametes, may be main- tained indefinitely in the laboratory, genetically identical plasmodia may be recreated over a span of many years. A combination of such characteristics is lacking in other organisms in which somatic incompatibility has been studied extensively.

A complete review of the literature and detailed life cycle has just been presented (CLARK and COLLINS 1973) and will not be repeated here. A series of pa- pers on the genetics of plasmodial fusion (COLLINS 1966; COLLINS and CLARK, 1968; COLLINS and LING 1972; LING and COLLINS 1970a, b; LING 1972; CLARK and COLLINS 1973) has shown that at least eleven loci, displaying dominant and recessive alleles, control the formation of plasmodial heterokaryons. Up until now, though, little was known of individual function of the various loci and their alleles or of the fate of the heterokaryon after its formation, even though several researchers (COLLINS 1969; CLARK and COLLINS 1968; KERR 1965 ; CARLILE and DEE 1967) have presented either direct or indirect evidence that at least certain heterokaryons may not be stable. In this report data to be presented will deal specifically with the role of individual incompatibility loci in lethal plasmodial interactions, and in heterokaryon survival where no directly observable lethal reaction occurs.

MATERIALS A N D METHODS

During COLLINS’ and CLARK’S (1968) study it was observed that some incompatible plasmodia underwent a cytoplasmic killing at the point of contact. At first we thought this might be due to an exotoxin, as reported by SEIFRIZ (1944), but closer examination indicated that the cytotoxic reaction was preceded by a transient plasmodial fusion. Therefore, a systematic study was undertaken to determine the s i d i c a n c e of this reaction and the involvement of the individual incompatibility loci. Fusion tests were carried out on water agar in small Petri dishes. Strips of agar containing vigorous pieces of plasmodia were placed opposite each other at a distance of approximately two centimeters. The plasmodia migrate off the agar blocks (Figure 1) and approach each other. When they collide they either fuse and form common veins (Figures 2 and 3), thus becom- ing a single entity, or else an incompatibility reaction occurs. In some combinations this incompatibility takes the form of a cytotoxic reaction after a short period of cytoplasmic mixing following a temporary fusion. The area of intermixed protoplasm loses color, becomes highly vacuolated and is sharply delimited from the rest of the plasmodium (Figures 5 and 6). This area does not recover normal characteristics and will not regenerate a live plasmodium when excised and transplanted to a new culture media. In general the killed protoplasm is invaded by the living portion of the plasmodium after a short period of time and the plasmodium responds to it as if it were unrelated organic matter. The size of this cytotoxic reaction is quite variable even between the same two plasmodia at different point of contact, but the average of all the reactions in several pairings of the same plasmodia is characteristic for that pairing. The small size of the killed area, 0.01 to 0.2 (rarely in special cases up to 2) square millimeters, and the large number of accurate determinations needed, approximately 30, pointed out the need for a simple rating system in the study of plasmodial incompatibility. A high positive linear correlation ( r = 0.98) was found between a series of killed zone size averages for a number of different test pairings and a series of averages, for the same pairings, by use of the following scoring system. Each test plate is scored zero for no observable reaction (Figure 4), one if the largest killed area is greater than zero but less than 0.03 mm2 (Figure 5) and five if at least one killed area is larger than 0.03 mm2 (Figure 6).

RESULTS

Reactions between sixteen known incompatibility classes: Plasmodial fusion

J. CLARK A N D 0. R. COLLINS

TABLE 1

Auerage killed area scores for the cytotoxic reactions between sixteen known phenotypic incompatibility classes tested in all possible pairwise combinations

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

1 =CDEFG 2=CDEfG 3=CDeFG 4=CdEFG 5=cDEFG 6=cdEFG 7=cDEfG 8=CdeFG 9=CDefG 1 O=CDEFg 1 l=CDEfg 12=CdEFg 13=cDEFg 14=cdEFg 15=cDEfg 16=CdeFg

2.5 4.5 2.3 2.5 1.7 3.3 1.7 1.8 0.4 0 0 0.2 0 0.1 0

2.9 3.8 1.9 1.8 2.3 1.3 0 0 0 0 1 0 0

1.3 2.5 2 1.9 0 0 0 0 0 0 0 0 5 0 . 6 0 0 0 0 0 0 0

0 . 5 0 0 0 0 0 0 0 0 0 0 0 0 0 . 2 0

2.8 0.4 3.8 0.7 2.1 1.3 5 0 0 0 0 0 0 0.2

1.4 4.4 0.4 5 0.6 0 0 0 0.2 0 0 0.3

0.9 1.7 0 0.5 0 0.6 0 0 0.5

1.6 3.1 4.6 1.1 1.5 2.3 1.3 2.8 0 2.7 0.7

0.4 2.3 3.7 0.5 1.5

1.3

1.2 5 0.5 5

within the Honduran isolate of Didymium iridis was shown to be controlled by five loci with alternative dominant or recessive alleles (COLLINS and CLARK 1968). However, since only a few plasmodial genotypes were known, it was necessary to expand what was known before the present research could proceed. Sixteen incompatibility classes were determined (CLARK and COLLINS 1973) and one to three different plasmodia of each class were paired in all possible combinations with six replications of each pairing. An average of these six scores was then calculated to produce a rating for each pairing. In this form it became evi- dent that the different pairings having the same plasmodial phenotypes had approximately the same size ratings. An analysis of variance indicated that the

TABLE 2

Auerage killed area of the cytotoxic reaction between plasmodia differing at specific loci

Loci Average Loci Average Loci Average

C 3.6 DF 0.5 CFG 0.0 D 2.3 DG 0.0 DEI? 0.7 E 4.9 EF 2.3 DEG 0.0 F 2.0 EG 0.1 DFG 0.0 G 0.1 FG 0.2 EFG 0.0 CD 1.4 CDE 2.6 CDEF 0.8 CE 3.0 CDF 0.4 CDEG 0.3 CF 2.9 CDG 0.0 CDFG 0.0 CG 0.1 CEF 1.9 CEFG 0.0 DE 2.3 CEG 0.1 DEFG 0.1

CDEFG 0.3

PLASMODIAL CYTOTOXIC REACTIONS 25 1

differences between the phenotype pairing means was significant and therefore it was decided that a phenotypic average would be a valid measurement. An examination of the results (Table 1) immediately indicates that plasmodia with common G phenotypes (either both plasmodia dominant or both recessive) produce the majority of the cytotoxic reactions while plasmodia with dissimilar G phenotypes seldom produce a killed zone. This suggests a direct link between the size of the cytotoxic reaction and the incompatibility phenotypes. With this in mind Table 1 was used to compare the various possiblc combinations of tests in which the phenotypes were dissimilar at any one locus or different combinations of loci. As indicated in Table 2, each locus produced a killed zone of characteristic size (C=3.6 ,D=2.3 ,E=4.9 ,F=2,G=O. l ) . Whentheplasmodiadifferattwoor more loci the effect is generally subtractive, e.g., CD = 1.4, which is smaller than either C or D alone. This combined effect is independent of the way the two plasmodia differ; the dominant alleles may all be in one plasmodium or divided between the two.

Close examination of the point of plasmodial contact indicates that the killed zone corresponds to the area of protoplasmic mixing that occurs during the transient fusion. The size of the mixed protoplast area, and therefore of the killed area, depends upon the speed of the killing reaction. A slow reaction allows for more mixing and thus a larger killed zone is formed, while a rapid reaction allows for little mixing and thus a small killed zone is formed. The occasional small killed zone observed in repeated tests of pairings which generally give no notice- able reactions indicates that a very rapid reaction may be taking place. These results are interpreted to mean that the size of the killed zone is inverse to thc strength of the loci. Thus, G is the strongest locus since it allows the smallest zone while E is the weakest since it allows the largest. As the plasmodia differ at more and more loci the strength of the reaction increases and a smaller area of killing results.

Second generation plasmodia (CLARK and COLLINS 1973) gave comparable results when they were used to check the previous scores. Directional eflect of the cytotoxic reaction: During the course of the previous tests it was observed that the cytotoxic reaction was often directional, with the killed area occurring in only one of the paired plasmodia. In order to explore this phenomenon further, a series of tests was conducted to determine if there was a correlation between specific alleles and the directional effect (Table 3 ) . Pairings were first made between plasmodia which differed at only one locus and the location of the killed area recorded for twenty replications of each pairing. Although the cytotoxic reaction occurs in either plasmodium or is shared between them, there is a definite bias toward its occurrence in plasmodia carrying the recessive trait. This trend was most pronounced in combinations which produce a large cytotoxic reaction area. Pairings were then made between plasmodia which differed at two loci with both dominant traits occurring in one plasmodium. Similar results were obtained, but with the cytotoxic reaction being shared more frequently and with a lessening bias towards its occurrence in the recessive plasmodium. This is expected since two loci acting together would tend to produce

252 J. CLARK AND 0. R. COLLINS

TABLE 3

Location of the cytotoxic reaction in tests between plasmodia of different phenotypes

Set 1 Number of times killed area occurred in:

Phenotype Rating of total Dominant Recessive Both Pairing killed area plasmodium plasnodium plasmodia

C c D-d E-e F-f

CD-cd CF-cf DE-de EF-ef

3.6 2.3 4.9 2.0 1.4 2.9 2.3 2.3

10 5

15 11 9

10 6 7

7 9 5 6 8

10 12 11

Set 2 Number of times killed area occurred in:

Phenotype Differential Strongest Weakest Both pairing rating' plasmodium+ plasmodium plamodia

Cd-cD 1.3 Cf-cF 1.6 Df-dF 0.3 cF-Cf 1.3 dE-De 2.6 eF-Ef 2.9

2,3$ 396 15,11 492 797 9,Il 3,4 8,6 9,IO 494 576 11,lO 5 6 9 2 6 12

* A differential rating is calculated by subtracting the smallest cytotoxic rating of the opposing loci from the largest. This represents the excess killing capacity of the plasmodium having the dominant trait causing the greatest amount of killing. + The strongest plasmodium is the plasmodium which has the dominant trait causing the greatest amount of killing.

$ Results of two separate pairings.

smaller killed zones and as demonstrated in the first pairings the smaller reactions have a less definite directional effect. Plasmodia differing at two loci with each plasmodium having one of the dominant traits were then tested (set #2) . Since the previous tests predict that the killing will occur in the recessive plasmodium and since both plasmodia have a recessive trait, one would expect that the killed area would be shared between the two plasmodia. This was found to be the case in the predominant number of tests, but a non-random directional effect was also displayed. If two plasmodia, for example phenotypes Cf and cF, are tested one can calculate the directional effect by considering each locus separately. The C locus can cause a 3.6 rated killed area in the cF plasmodium while the F locus can cause a 2 rated killed area in the Cf plasmodia. Subtracting the smallest (2) from the largest (3.6) gives a difference of 1.6 in favor of the Cf plasmodium's killing capacity. And in most tests the directional effect was away from the plasmodium whose killing potential was greater, as predicted in this manner.

Tests for heterokaryon surviual: Although heterokaryon formation by the fusion of plasmodia is easily accomplished, little is known about the survival of these


heterokaryons. The multiple allelic mating system of Didymium iridis offers a convenient system for testing heterokaryon survival. This system operates in such a way that different isolates typically carry unlike pairs of alleles. Vegetative fusions which bring together non-identical pairs of mating factors are apparently rare, probably because of dissimilarity in the plasmodial compatibility systems of the various isolates (COLLINS and LING 1972). In the laboratory, appropriate interisolate crosses sometimes yield hybrids which are similar enough genetically to allow for occasional production of mating type heterokaryons.

A clone, 66, with the A4 mating type from a Panamanian isolate was crossed with a Honduran clone, 2, of mating type Al. The resulting F, plasmodium was induced to fruit and a number of F, clones were isolated. All clones of mating type Al were crossed with clone 20 (mating type A2) of the Honduran isolates and the resulting plasmodia, carrying mating types A1 and A2, were then tested for fusion against plasmodium 66.20 which carried mating types A4 and A2. One plasmodium (66.2-14.20) was found which fused and thus formed a heterokaryon composed of A1A2 and A2A4 nuclei. Plasmodial designations which indicate heri- tage are used in this report. For example an F, plasmodium derived from crossing parent clones 66 and 2 is designated 66.2 and F, clones derived from it are labeled 66.2-1, 66.2-2, etc. If one of these F, clones (66.2-1) is crossed with parent clone 20 the resulting plasmodium is given a 66.2-1.20 designation. Although the designations become fairly complicated after a series of crosses they are very useful in assigning pedigrees.

Four such heterokaryons were produced with varying proportions of the original homokaryotic plasmodia. The heterokaryons were then fed and allowed to grow for seven days before they were induced to sporulate. Clones isolated from these fruitings were then tested for mating type by appropriate crosses with known mating types. In the first heterokaryon, derived from plasmodia of equal size, only small numbers of spores were isolated from two sporangia to get a quick indication of mating type survival. Retrieval of A2 and A4 mating types only (Table 4, heterokaryon 1) suggested that the A1A2 nuclei did not survive to become incorporated into spores. In view of this, heterokaryons were produced in which there was a 4 to 1, 10 to 1 and 20 to 1 size ratio in favor of the A1A2 plasmodium. Even under these conditions only AZ and A4 mating types were recovered (Table 4, heterokaryons 2, 3 and 4). In the 4 to 1 and 10 to 1 sporangial samples, approximately equal numbers of A2 and A4 mating types were recovered in large enough numbers to indicate that the A2 mating type was derived from the A2A4 nuclei only.

Differential nuclear division rates is often suggested as the cause of heterokaryon reversion to the homokaryotic state ( PONTECORVO 1946). This could explain the loss of A1A2 nuclei after seven days of growth, but if that were the case fruitings produced just after fusion should contain all three mating types. IIomokaryotic plasmodia were transferred to water agar and exposed to light in order to induce fruitings; when some of the transfers started to fruit the rest were used to produce heterokaryons which fruited within 24 hours at fusion. In the first two of these heterokaryons (Table 4, heterokaryons 5 and 6) only A2 and

254 J. CLARK A N D 0. R. COLLINS

TABLE 4

Mating types recovered from heterokaryons composed of 66.20 (A4A*) and 66.2-14.20 (AlAZ) plasmodia

Seven days of growth

Heterokaryon and A‘AZ to Mating types recovered sporangml number A1A2 ratio A’ A2 A4

1-1 1-2 1-total 2-1 2-2 ,%total 3-1 3-2 3-3 3-4 3-total &total

1:l 1:l 1:l 1:4 1:4 1:4 1:io 1:lO 1:lO 1:lO 1:lO 1 :20

0 0 0 0 0 0 0 0 0 0 0 0

5 6

11 11 12 23 10 16 10 10 46 8

3 2 5

11 10 21 12 13 12 15 52 2

One day of growth 5-1 1:l 0 12 8 5-2 1:l 0 9 6 5-total 1:l 0 21 14 &total 1:l 0 4 8 7-1 1:l 6 0 4 7-2 1:l 30 0 24 7-total 1:l 36 0 223

Microplasmodia 8-A 1:lO 0 13 7 8-B 1:lO 0 5 6 8-C 1:lO 0 18 18

A4 mating types were again found, but in the third (Table 4, heterokaryon 7) A’ and Az mating types were recovered. This represented the missing nuclei of the heterokaryon but now the A2A4 nuclei were missing. It is suggested that the rapid fruiting immediately after fusion somehow reversed the normal loss of nuclei. One possibility is that the A1A2 plasmodium was so close to fruiting prior to fusion that its nuclei could not be eliminated. And since a considerable number of nuclei are normally lost during fruiting the “less ready to fruit” A2A4 plasmodium nuclei could have been eliminated in this manner.

The possibility that the missing nuclei were present in the plasmodium until fruiting, during which they were lost, was then considered. KERR (1965), using mutant markers of clonal plaques in a homothallic myxomycete, Didymium nigripes, reported one marker which was present in reduced proportion within the heterokaryon until fruiting, at which time it completely disappeared. TO test for this type of phenomenon a 10 to 1 ratio heterokaryon was fragmented in a Waring blender until no macroscopic pieces remained. After 24 hours the smallest microplasmodia observed under a dissecting microscope at 9OX were iso-


lated. Phase contrast microscopy indicated an original microplasmodium containing five nuclei. Therefore, if the nuclei had remained in the original IO: 1 ratio each microplasmodium would have had approximately a 50% chance of containing all A1A2 nuclei (the A2A4 nuclei not present because of chance). Three microplasmodia which had grown to large size and fruited were tested for mating type. Again only A2 and A4 mating types were recovered (Table 4, heterokaryon 8) which suggests that the heterokaryons lose their A1A2 nuclei before fruiting.

A second similar heterokaryon was produced in which the A4 mating type clone was a 66.2-F, instead of the 66 clone as in the first heterokaryon. Hetero- karyons with 1: 1, 10: 1 and 20: 1 size ratio in favor of the A1A2 plasmodium were formed and induced to fruit after seven days’ growth. Again only A2 and A4 mating types were recovered in all three heterokaryons except for a 1 : 1 ratio heterokaryon in which one Al mating type clone was recovered. This one excep- tion may have been due to the survival of a few A1A2 nuclei in the heterokaryon or it could also be due to the incorporation of an encysted amoebae into the sporangium. (The amoebae which are crossed to produce the plasmodium are still in the culture at the time of fruiting.)

Since these heterokaryons, in which the homokaryons were identical at 85% of the loci, were unstable, further inbreeding was undertaken. However, the same behavior (exclusion of A1A2 nuclei) was seen after two more generations of inbreeding (98% identical) and attempts to further inbreed the plasmodia to an even greater extent failed. The non-survival of the mating type heterokaryon even after this extensive inbreeding is thus believed to be due to a heterokaryon incompatibility system. Therefore, it is tentatively suggested that the mating locus functions directly as a heterokaryon incompatibility locus or is closely linked to one or more unidentified compatibility loci.

DISCUSSION

Heterokaryon incompatibility in the true fungi which also deals with the fusion of coenocytes probably represents the most comparable situation to that described in this paper for the myxomycete D. iridis. In the Ascomycete Neuro- spora crassa heterokaryon survival is determined by three major loci ( GARNJOBST 1955; WILSON and GARNJOBST 1966) and several minor loci (PITTENGER and BRAWNER 1961; DE SERRES 1962). When hyphae come into contact they fuse, but the fusion cells will die shortly after mixing of the protoplasm if they are not genotypically identical for the major incompatibility loci. The mating locus, as in Didymium iridis, seems to be closely linked to, or is itself, a major incompatibility factor (NEWMEYER 1970). Neurospora sitophila (MISHRA 1971) , Rhizoc- tonia solani (FLENTJE and STRETTON 1964), Aspergillus glaucus (JONES 1965) , Aspergillus nidulans ( JINKS et al. 1966) , and Podospora anserina ( ESSER 1956; BERNET 1967) are other fungi in which heterokaryon incompatibility has been reported. The occurrence of both sexual and asexual incompatibility in the same haploid hyphae can lead to more than a single interpretation of results. In fact, the formation of a dikaryon as a normal part of the sexual cycle in the Basidiomy- cetes greatly reduces the possibility of finding separate heterokaryon incompati-

25 6 J. CLARK A N D 0. R. COLLINS

bility systems in this group. But the work in the Ascomycetes especially seems to indicate the widespread occurrence of multilocus somatic cell incompatibility systems. These are often manifested by cytotoxic reactions.

We believe that the primitive slime mold, like its more complex counterparts in the living world, must cope with the problems of recognition of potentially foreign matter, whether represented by non-living matter, other species, genetically non-identical members of the same species. or even portions of itself which become foreign through genetic change. In this context, the elimination of different nuclei in a myxomycete or fungal heterokaryon would be functionally equi- valent to the cell surveillance system proposed for vertebrates (BURNET 1970). If these systems are in fact functionally comparable then the unique genetic and cultural characteristics of the Myxomycetes will make them very good models for testing for homostatic control through cytotoxicity. But before this can be decided it will be necessary to study and characterize specific gene products via extraction and micro-injection experiments to determine the nature of the unidirectional killing reaction.

The authors gratefully acknowledge financial support to the first author from an NDEA fellowship and NSF traineeship, and from XSF grants GB-5275 and GB-15770 and an NIH Biomedical Sciences Support Grant through the University of California, Berkeley, to the second author. This report, in part, is condensed from a portion of a dissertation submitted to the Uni- versity of California, Berkeley, in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

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(JINKS 1952; PONTECORVO

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