CYTOPLASMIC INCOMPATIBILITY STUDIES IN THE MYXOMYCETE DIDYMIUM IRIDIS: RECOVERY AND NUCLEAR SURVIVAL IN HETEROKARYONS'

At least eleven somatic compatibility loci exist in the myxomycete Didymium iridis. Cell fusion is controlled by at least seven fusion loci (Fusl-Fus7). Cytoplasmic compatibility is controlled by at least four clear-zone loci (Czl-Cz4). Plasmodia with identical phenotypes at all seven fusion loci, but different phenotypes at the clear-zone loci, will fuse temporarily, but fusion is soon blocked by cytoplasmic reactions which prevent complete mixing. Areas which contain cytoplasm from two incompatible plasmodia become clearly delineated from healthy cytoplasm. Such areas, termed clear zones, have been isolated and found to recover. If clear zones are sectioned into several small pieces, not all pieces will recover, indicating that toxic cytoplasmic reactions have occurred. Plasmodial fusion studies and F1 studies of recovered clear zones indicate that the clear-zone loci may also control nuclear survival in heterokaryons. CYTOPLASMIC INCOMPATIBILITY has been studied in the true slime molds (Clark and Collins, 1973a, b), in the higher fungi (Esser and Kuenen, 1967), and in Protozoa (Hildemann, 1972). The relatively small size of most protists makes it difficult to observe cytoplasmic interactions in these organisms. Slime mold plasmodia, however, are giant cells and cytoplasmic interactions upon cell fusion are easily observable at 45 x with a dissecting microscope. In addition, cytoplasmic interactions occur within 1-5 min after cell contact. Somatic cell compatibility in plasmodia of the true slime mold Didynium iridis (Ditmar) Fries is controlled by a system of at least eleven loci each with dominant and recessive alleles (Ling and Collins, 1970a, b; Ling, 1971; Collins and Ling, 1972). Two plasmodia must be phenotypically identical at all these somatic compatibility loci in order to fuse freely (Fig. 1). These loci can be categorized into two classes, depending on whether they allow a temporary fusion or no fusion at all (Ling and Ling, 1974). Fusion and heterokaryon production do not occur if two plasmodia differ phenotypically at any of the seven fusion loci Fusl-Fus7 (Fig. 2). Temporary fusion is controlled by four clearzone loci (Czl-Cz4). Fusion between two plasmodia with phenotypic differences at any of the clear-zone loci is terminated within a few minutes by coagulation reactions (Clark and Collins, 1 Received for publication 13 July 1973. Research supported by PHS Grant GM19165 and a University of Delaware Research Foundation Grant to the first author. The competent assistance of Andrew Liu is gratefullv acknowledged. 1973a, b; Upadhyaya and Ling, 1972; Ling and Upadhyaya, 1973). Areas formed by intermixing of two incompatible plasmodia lose pigment and appear quite different from the normal surrounding protoplasm (Fig. 3). These affected areas have been termed "clear zones" (Collins and Clark, 1968). Clear-zone formation has often been found to be directional. When plasmodia of two different phenotypes are repeatedly tested, most of the clear zones are formed in one member of the pair (Clark and Collins, 1973a; Ling and Upadhyaya, 1973). Clark and Collins (1973a) assumed that toxic reactions occurred within the clear zones. It was observed by previous workers that veins of living protoplasm often appeared within clear zones, but these were interpreted as invasion by healthy plasmodia. However, in the current study it was determined that if clear zones were excised from the surrounding plasmodia, recovery still took place and thus heterokaryons were produced. It is still quite possible that both invasion by healthy plasmodium and recovery normally take place within clear zones. This communication reports for the first time the reco,very pattern of heterokaryons formed between plasmodia differing at one or more clearzone loci. The fusion properties and survival of marker alleles in recovered clear zones are discussed in detail. MATERIALS AND METHODS-Plasmodia chosen for these studies were phenotypically identical at all fusion loci Fusl-Fus7. Thus fusions were possible, but extensive mixing of protoplasm was pre-