Development of the Amut Bmut Strain of SchizophyUum commune

Transcription

1 Arch. Mikrobiol. 74, (1970) 9 by Springer-Verlag 1970 Development of the Amut Bmut Strain of SchizophyUum commune YIG-AL KOLTIN Department of Food and ]3iotechnology, Technion-lsrael Institute of Technology, H~ifa, Israel Received June 8, 1970 Summary. In search for genetically uniform system for the cytological and biochemical analysis of sexual morphogenesis in Schizophyllum, a dikaryon mimic, the Amut Bmut strain, was studied. The course of nuclear distribution and clamp formation was followed in germlings of the double mutant. It was found that this mutant mimics the interaction between two compatible strains. The developmental sequence leading to dikaryon formation can be precisely timed. Thus, it is suggested that this strain be used for determination of the sequential induction of the enzymatic systems operating in sexual morphogenesis. Sexual morphogenesis in the basidiomycete Schizophyllum commune entails a change from mycelium constituted of uninncleate cells to mycelia constituted of cells containing two compatible nuclei each. The morphogcnetic events of which this transition is a part are regulated by two incompatibility factors, designated A and B (for review see Raper, 1966; Raper and ]~aper, 1968). The events leading to the establishment of the binucleate cells occur only when two mates have different A factor and B factor specificities or when both factors bear a mutation. The resulting binucleate mycelium, termed the dikaryon, is characterized by lateral protrusions which connect the adjacent cells and which serve to maintain the binucleate state. The lateral protrusion is called the clamp connection (Fig. 1). The biochemical and cytological analysis of the morphogenetic progression has been hampered by the Jack of uniformity in the mycelium following an interaction between two compatible strains during the transition from the uninucleate state to the binucleate state. During the transition the mycelinm is a mosaic consisting of homokaryotic and heterokaryotic elements. In search for systems that have genetic uniformity, and are also synchronous, as a means for the study of the time sequence of the biochemical and cytological events of the morphogenetic progression, we found it possible to use a strain bearing a mutation in the B factor (Koltin and Flexer, 1969). Strains carrying mutations

2 124 Y. Koltin: NORMAL Amut Bmut I 9 I 9 I 9 o'-~ F 9 I o 1 9 I 9 I o~ I 9 I 9 I eo ) loot 9 I 9 leol ~ " I 9 I I o~'~~ o ) I 9 I 9 I *el 9 ) Fig. 1. Transition from an uninueleate to binucleate state in a normal interaction between two compatible homokaryons (left) and the Amut Bmut (right) in the incompatibility factors mimic the heterokaryons but have the advantage of being uniform as all the nuclei within each cell are genetically identical and thus operate in synchrony. The current report is a study of a strain bearing mutations in the A and B factors; this strain mimics the dikaryon in its morphology. As will be shown, this strain undergoes a course of development similar to the transition of the uninucleate homokaryons to the binucleate state of the dikaryon and thus may serve as a suitable system for determination of the time sequence of the biochemical and cytological events entailed in this conversion. Materials and Methods The s~rain used was number 1760 of Dr. John R. Raper's collection at Harvard University. The mutation in the A factor was described by Raper, Boyd and Raper (1965) and the mutation in the B factor was described by Parag (1962). Spores obtained from a fruit body of an Amut Bmut strain were spread on cellophane membrane overlaying Schizophyllum complete (Raper and Miles, 1958) semisolid medium. The plates were incubated at 30~ and at various times sections of the cellophane with individual colonies from the germinating spores were fixed and stained as described by Raper (1966). The characteristics described in Tables 1 and 2 were determined by phase contrast microscopy. Results During development of the Amut Bmut strain there is a period in which all cells are uninucleate, followed by a period in which most cells are binucleate (Table 1 and Fig.2). The binueleate state is typical of the fully developed mutant (Raper and Raper, 1966). The latter state is not attained immediately even though each nucleus of an Amut Bmut from the beginning contains all the needed information for the establishment

3 Development of the Amut Bmut Strain of Sehizophyllum commune 125 Table 1. Nuclear distribution in cells o/ an Amut Bmut strain o~ Schizophyllum Number o/ nuclei per cell Time 0 ~ 1 ~ 2 ~ 3 ~ ~ 3 ~ No. (h) Cells G 8G 9 70" -- 6(~ a. O _ 50 A Z v 40 < Z Z oc~ 8r v Z ~ 2e 1C TIME (hrs) Fig. 2. Nuclear distribution in developing mycelium of Amut Bmut strain A uninucleate cells; * binucleate cells of dikaryotic myee]ium. It is only some 60 h after inoculation that the firs~ signs of morphological characteristics typical of the dikaryon (binucleate cells and clamp connections) are visible. Prior to this time the mycelium displays the morphology of the infertile homokaryon e.g.

4 126 Y. Koltin: nninucleate cells devoid of clamp connections. Binucleate cells and clamp connections appear gradually from 69 h after inoculation of the spores. At 74 h the ratio of uninucleate cells to binueleate cells is ca. 1 : 1 ; at 90 h the ratio is ca. 1 : 5, which is typical of the fully developed Amut Bmut and slightly lower than the dikaryon resulting from the interaction between 2 compatible homokaryons. Thus, our system, comprised of only one type of nucleus, mimics the entire transition from the uninueleate state to the binueleate state typical of the course of development following the interaction between compatible strains. An indication of the synehrony in nuclear action is seen from the course of clamp development (Table 2). Normally, clamp formation is Table 2. Septal characteristics during the development o/ the Amut ]3mut strain Type o~ septa a Time SS ~ UC ~ TC ~ No. ~ (h) Cells Clamped b SS, simple septa (a septum defined by a cross wall only), UC, unfused clamp connection, TC, true clamp connection (a clamp connection fused with the adjacent cell). b Percent clamped of total septa observed (including unfused clamp connection and true clamp connection). initiated following the pairing of two compatible nuclei in the terminal cell. It is the clamp which henceforth maintains the binucleate state in each cell. When clamp formation is initiated, however, in a normal interaction only the terminal cells arc heterokaryotic whereas the rest of ~he cells are homokaryotic. In the Amut Bmut strain, 60 h after inoculation clamp formation is initiated but is not confined to ~he terminal cells. At 69 h it is seen clearly that along a single hypha clamp formation is initiated in about one of every two septa. However, simple septa (septa in which clamps have not been formed) are found between cells which are already clamped. Between h more than 75~ of the septa bear clamp connections although many of the clamps are unfused to the adjacent cell. At 90 h, less than 100/o of the septa are undamped. The presence of unclamped septa between clamped septa indicates that

5 Development of the Amut Bmut Strain of Schizophyllum commune L27 the system operative in clamp formation is initiated at a specific time in many nuclei. Since clamp initiation is not limited to the terminal cells we avoid to a great extent the formation of the mosaic typical of the transition found in a normal mating interaction. The timing of the course of clamp initiation and development provides points of reference which can be correlated with specific enzymatic activity and cytological features associated with clamp formation. A similar situation occurs with respect to the formation of binueleate cells. Such cells are dispersed between uninucleate cells 69 h after inoculation. The frequency of binueleate cells, however, increases sharply from this time to 90 h (Table 1, Fig.2). The formation of fused clamps does not increase at the same rate. Thus, it appears that initial formation of binueleate cells is not associated with clamp formation but rather with the placement of the cross walls. Only at 90 h a good correspondence between clamp formation and binucleate cells is found (790/0 binucleate cells and 77~ fused clamp connections). Discussion This study demonstrates that the sequential initiation of various functions in the Amut Bmut strain mimics the morphogenetic development of the dikaryon. The use of the Amut Bmut strain enables the performance of these studies in a system in which the genetic constitution of each cell is identical. The results may serve as points of reference in the design of experiments to detect the activation of enzyme systems during various stages of development and the correlation of various cytological features associated with the morphogentic sequence. Such studies are currently in progress. In addition, the use of the Amut Bmut strain demonstrates that the initial establishment of binucleate cells in the dikaryon is not a random event. The fact that binucleate cells are formed at a specific time and precede clamp formation, suggest that two mechanisms must operate in the establishment of binucleate cells (a) septation in a way to secure pairing of nuclei (b) the clamp formation which normally functions after the nuclei have paired. The mechanisms suggested are not specific for the Amut Bmut strain but can be distinguished clearly in this system due to the genetic uniformity of the nuclei within the hyphae. Pairing of the two compatible nuclei in a mating interaction between two compatible strains precedes the formation of clamp connections. Pairing could occur only if the mechanism which maintains the uninucleate distribution of nuclei in the homokaryons was altered so as to form the septa beyond the paired nuclei.

6 128 Koltin: Development of the Amut Bmut Strain of Schizophyllum commune References Koltin, Y., Flexer, A. S. : Alteration of nuclear distribution in B-mutant strains of Schizophyllum commune. J. Cell Sci. 4, (1969). Parag, Y. : Mutations in the B incompatibility factor of Schizophyllum commune. Proc. nat. Acad. Sei. (Wash.) 48, (1962). Raper, C. A., Raper, J. R, : Mutations modifying sexual morphogenesis in Schizophyllum. Genetics 54, (1966). Raper, J. R. : Genetics of sexuality in higher fungi. New York: Ronald Press Boyd, D. It., Raper, C. A. : Primary and secondary mutations at the incompatibility loci in Schizophyllum. Proc. nat. Acad. Sci. (Wash.) 53, (1965). -- Miles, P. G.: The genetics of Schizophyllum commune. Genetics 43, (1958). -- Raper, C. A. : Genetic regulation of sexual morphogenesis in Schizophyllum commune. J. Elisha Mitchell Sei. Soe. 84, (1968). Dr. Yigal Koltin Department of Botany Genetics Unig University of Tel-Aviv Tel-Aviv, Israel