PDF hosted at the Radboud Repository of the Radboud University Nijmegen The following full text is a publisher's version. For additional information about this publication click this link. http://hdl.handle.net/2066/113072 Please be advised that this information was generated on 2021-10-06 and may be subject to change. POST-FUSION INCOMPATIBILITY IN PLASMODIA OF PHYSARUM POLYCEPHALUM J. A. M. Schrauwen POST-FUSION INCOMPATIBILITY IN PLASMODIA OF PHYSARUM POLYCEPHALUM Een onderzoek naar de physiologische veranderingen in een heterokaryon plasmodium van Physarum polycephalum POST-FUSION INCOMPATIBILITY IN PLASMODIA OF PHYSARUM POLYCEPHALUM Een onderzoek naar de physiologische veranderingen in een heterokaryon Plasmodium van Physarum polycephalum PROEFSCHRIFT ter verkrijging van de graad van doctor in de Wiskunde en Natuurwetenschappen aan de Katholieke Universiteit te Nijmegen op gezag van de Rector Magnificus prof.dr J.H.G.I.Giesbers volgens het besluit van het College van Dekanen in het openbaar te verdedigen op woensdag 5 december 1984 des namiddags te 2 uur precies door Johannes Anthonius Maria Schrauwen geboren te Breda И kripj repro meppel PROMOTOR PROF. DR. H.F. LINSKENS Aan: Lia Saskia Ontwerp omslag: onze zoon Bob Bob CONTENTS 1 General introduction 7 2 Cellular interactions in slime moulds 13 3 The lethal reaction as a function of fusion and translation 33 4 DNA as an intermedate and target of the incompatible processes 43 5 Membranes as a site of incompatible reactions 49 6 Changes in properties of the heterokaryon 59 7 General conclusion 71 8 Samenvatting 75 Dankbetuiging Curriculum vitae 1 GENERAL INTRODUCTION 7 General Introduction The investigation of cellular interactions has made In these systems incompatibility is controlled by 1 an extensive growth during the last decades. To­ or 2 genes mostly, with multiple alleles (de Nettan­ pics in this field of research include: court, 1984; Lane, 1981; Linskens and Kroh, 1967; Carlile and Gooday, 1979; Schrauwen, 1984). a) inhibition of pollentube-growth or -development In the animal body a direct contact between cell after self pollination in higher plants (Linskens membranes is common. In plants the stiff cell wall and Kroh, 1967; Linskens 1975 a and b, 1981; de however is a limiting factor for interaction studies Nettancourt, 1984). between cells. Slime moulds provide a good ex­ perimental system for the study of cell fusion and b)rejection of transplanted cells or organs in an­ their consequences. The lack of exgastic cell walls imals (Williamson, 1980) in slime molds make this organism as an excellent model for the investigation of incompatibility reac­ c) allergic reactions (Howlet and Knox, 1984) tions. The slime mould can be cultured under la­ boratory conditions as a microorganism on defined d)effect of non-adhesion after grafting of plants or semi-defined media in the haploid as well as in (Brabec, 1965; Yeoman, 1984) the diploid form (Chaper 2, Fig 1). In our studies the diploid form could be obtained as micro- e) elimination of cells or tissues: -after transporting plasmodia in shake culture and as macro or surface the living material into another environment by plasmodia when cultured in petri dishes (Daniel exudates or toxins already present (bloodtransfu- and Baldwin, 1964; Schiebel, 1973). Several pro­ sion, yeast) -after fusion and mixing of cyto­ perties such as synchronous mitosis and differentia­ plasms of two different cells (fungi,Cadile and tion, of P.polycephalum have evoked an intensive Gooday, 1978; Paramecium ,Tsukii and interest in this organism. This has resulted in well Hiwatashi, 1983; plantprotoplasts, Negrutiu et documentated observations on the physiological al, 1983) processes (Aelen et al, 1983; Hardman et al, 1982; Mohberg and Rusch, 1974; Turnock et al, 1981) AU these interaction phenomena are characterized and genetics (Anderson and Holt, 1981; Dee, by initial "recognition" events, discussed already in 1973) in this slime mould. As in other slime 1877 by Darwin in a botanical context: "It moulds, the cytoplasm of P. polycephalum is sur­ may be said that the two pollens and the two stig­ rounded by a plasma membrane and a slime layer, mas mutually recognize each other" cited according thin at the border of a well growing one day old Heslop-Harrison and Linskens, 1984. Plasmodium cultured on growth medium (Hutter- All recognitions between the participating tissues mann, 1973; Rusch, 1970). This unique envelope or cells are determined genetically. It results in a system of P.polycephalum, together with the non-acceptance of one cell type. This rejection genetical and physiological knowledge described reaction can de effected either by preformed com­ above, makes this slime mould an excellent model pounds in one partner, or produced after the first for both studies on interactions between cells after contact (fungi,Schrauwen, 1984; Carlie and Goo­ fusion and their contact. day, 1978; Lane, 1981;allergy, Howlett and Knox, 1984). This non-acceptance or non-tolerance of cells which in principle should be able to co-exist, is Somatic fusion named incompatibility. The incompatibility When two macroplasmodia of P.polycephalum ap­ phenomena occur in prokaryotes and eukaryotes proach each other they fuse if the plasmodia are of and are widely spread in higher plants, ( de Net- the same age and have identical fus (fusion) genes tancourt, 1984; Heslop-Harrison and Linskens, (Carlile, 1976; Carlile and Dee, 1967; Cooke and 1984) and fungi (Esser and Blaich, 1973; Carlile Dee, 1975; Collins, 1979; Lane, 1981; Schrauwen, and Gooday, 1979). 1984). 9 Contact of two plasmodia of different strains not The lethal reaction as a function of fusion and followed by fusion may result in the growth of the plasmodia along or even over each other. In many systems secrations are responsible for a Fusion of two plasmodia of different strains may killer-like reaction. Experiments to check the pos­ be followed by a post-fusion reaction that destroys sible presence of comparable exudates in plasmo­ the fused Plasmodium partially (gedeeeltelijk) and dia of P. polycephalum are described in Chapter 3. sometimes totally (Carlile and Dee, 1967; Collins, A destructive reaction can be obtained only after 1979; this thesis ). The failure to fuse is indicated the fusion of a sensitive and killer plasmodium in as fusion-incompatibility and the destruction with the heterokaryon. Therefore, special attention has post-fusion incompatibility. Both reactions are been paid to events occuring during the 6 hour genetically determined and are associated to period between the moment of fusion and the lysis heterogenetic incompatibility (Esser and Blaich, reaction. This is the time period during which 1973; Carlile and Gooday, 1978; Lane, 1981; non-t.olcrance comes to expression. The genetical Schrauwen, 1984). The number of loci that govern control of the post fusion-incompatibility reaction fusion are about 13 (Collins, 1982; Schrauwen, suggests the involvement of transcription and 1984). Interactions after fusion are controlled by translation of genes in the post-fusion interaction multiple pairs of alleles, "dominant" and "recessive" processes. To obtain insight into these metabolic (Carlile, 1976; Carlie and Gooday, 1978). This processes, ihibitors of RNA (Fouquet et al, 1975) means two plasmodia of P.polycephalum fuse and and protein (Nations et al, 1974) synthesis are ex­ form a heterokaryon if their fusion loci are identi­ cellent tools with which to test the involvement of cal. This fusion may be followed by the destruc­ these macromoleculcs. Results of such experi­ tion of a part or of the total heterokaryon, called ments and their consequences are discussed in lysis or the lethal reaction, if the composition of Chapter 3. the let (lethal) alleles is different (Schrauwen, 1982). In the absence of sufficient nutrients or DNA as an inetermediair and target of the incom­ with great differences in plasmodial size of the two patibility processes. strains at the moment of fusion no lethal reaction appears. However, the fused heterokaryon obtains In addition to translation (Chapter 3), transcription the properties of one strain within 48 hours after may also be a part of the series of reactions that fusion irrespective of the occurrence of a partial enters into the lethal reaction. Minor (small) lysis reaction (Carlile, 1972). The plasmodium modifications at one or both DNA strands of the that survives after the fusion of two different incompatible plasmodia prior to fusion may show Plasmodia, with a partial or without a self- the influence of DNA at the lethal reaction (Fun- destruction reaction, is described as the killer derud et al, 1978). Results of experiments that strain; the other is called the sensitive strain. describe the possible effect of DNA from both The properties of the killer or sensitive strain is plasmodia on the lethal reaction are shown in dependent on the set of let alleles of the two mat­ Chapter 4. ing types. The strain with major dominant let al­ Fusion of sensitive and killer plasmodia having leles will be the killer. This means that a strain great differences in size causes no lethal reaction can be the killer type in one fusion reaction and (Carlile, 1972). However, the heterokaryon of the sensitive type after fusion with another strain. killer and sensitive changed in a plasmodium with The lethal reaction is expressed in P.polycephalum the properties of killer within 48 hours. There is a 4-6 hours after fusion. change of phenotype as well as the capacity to The properties of P.polycephalum summerized summon a lethal reaction after fusion with a sensi­ above make the slime mould an excellent tive plasmodium (Chapter 3). This suggests that in model-system for incompatibility studies at the the heterokaryon the DNA of the sensitive strain molecular level. has disappeared. To check this suggestion DNA This thesis describes a number of studies on somat­ strands of sensitive and killer plasmodium were ic incompatibility with this model organism.