1.8.15
COMPARISON OF HETEROKARYON FORMATION BY HYPHAL ANASTOMOSIS AND PROTOPLAST FUSION IN FUSARIUM

BR CORTES, R BLANCA, DN KUHN and R REJANE

Department of Biological Sciences, Florida International University, Miami, Florida 33199, USA

Background and objectives
Parasexuality requires the initial formation of heterokaryons followed by karyogamy and haploidization. Previously, we have observed karyogamy within 2 weeks in heterokaryons formed by hyphal anastomosis. Heterokaryons can be formed in Fusarium by hyphal anastomosis between vegetatively compatible strains. Infrequently, we have also observed heterokaryon formation by hyphal anastomosis between strains in different vegetative compatibility groups (VCG) [1]. Such heterokaryons are morphologically indistinguishable from those formed within a VCG. Heterokaryons can theoretically be formed between any two strains by fusion of protoplasts [2]. We have used a battery of genetic and molecular markers including auxotrophy, allozymes, electrophoretic karyotyping and amplified fragment length polymorphisms (AFLP) to investigate the formation of intra- and inter-VCG heterokaryons by both hyphal anastomosis and protoplast fusion. In addition, we have attempted to establish when karyogamy occurs in heterokaryons formed by protoplast fusion.

Materials and methods
UV generation of auxotrophic mutants, auxanography and methods to force heterokaryon formation were as described [1]. Protoplast generation and fusion protocols used followed the guidelines outlined in [2]. Allozymes were done on cellulose acetate gels (Helena Laboratories, Beaumont, Texas). The electrophoretic karyotype was done using the Contour-clamped homogeneous field (CHEF-DRII) apparatus (Biorad) and for the AFLPs we used the protocols outlined by Perkin-Elmer. Analysis was done on an Applied Biosystems 377 automated sequencer.

Results and conclusions
Heterokaryon formation between auxotrophic mutants via hyphal anastomosis was observed in intra-VCG pairings. Inter-VCG and inter-forma speciales heterokaryon formation were also observed via hyphal anastomosis. We noticed a correlation between the isolates that had undergone multiple rounds of mutagenesis and their ability to overcome incompatibility barriers. Furthermore, when these isolates were paired with unmutagenized strains they were still able to overcome incompatibility and form heterokaryons between VCGs and formae speciales. Thus for heterokaryons formed by hyphal anastomosis, the incompatibility barrier is not as stringent as previously thought, and the contribution to incompatibility is not equally shared by both parents. Because removal of the cell wall by protoplasting should also remove incompatibility barriers, we are comparing heterokaryon formation between hyphal anastomosis and protoplast fusion in inter-VCG and inter-formae speciales heterokaryons. In heterokaryons formed by hyphal anastomosis, karyogamy can be detected within 2 weeks of formation. We are investigating the time course of karyogamy in heterokaryons formed by protoplast fusion, through allozyme and AFLP analysis of the uninuclear microconidia of the heterokaryons.

Funded by NIGMS/NIH grant number GM08205.

References
1. Cortes BR, 1996. MSc thesis, Florida International University
2. Adams G, Johnson N, Leslie JF, Hart LP, 1987. Experimental Mycology 11, 339-353