Trikaryon formation and nuclear selection in pairings between heterokaryons and homokaryons of the root rot pathogen Heterobasidion parviporum

Pairings between heterokaryons and homokaryons of Agaricomycete fungi (he-ho pairings) can lead to either heterokaryotization of the homokaryon or displacement of the homokaryotic nucleus through migration of nuclei from the heterokaryon into the homokaryon. In species of Agaricomycetes with multinucleate cells (>2 nuclei per cell), he-ho pairings could result in the stable or transient formation of a hypha with three genetically different nuclei (trikaryons). In this study, he-ho pairings were conducted using the multinucleate Agaricomycete Heterobasidion parviporum to determine whether trikaryons can be formed in the laboratory and whether nuclear genotype affects migration and heterokaryon formation. Nuclei were tracked by genotyping the heterokaryotic mycelium using nucleus-specific microsatellite markers. The data indicated that certain nuclear combinations were favored, and that nuclei from some strains had a higher rate of migration. A high percentage of trikaryons (19 %) displaying three microsatellite alleles per locus were identified among subcultures of the he-ho pairings. Using hyphal tip and conidial isolation, we verified that nuclei of three different mating types can inhabit the same mycelium, and one of the trikaryotic strains was judged to be semi-stable over multiple sub-culturing steps, with some hyphal tips that retained three alleles and others that reduced to two alleles per locus. These results demonstrate that nuclear competition and selection are possible outcomes of heterokaryon-homokaryon interactions in H. parviporum and confirm that ratios of component nuclei in heterokaryons are not strictly 1:1. The high rate of trikaryon formation in this study suggests that fungi with multinucleate cells may have the potential for greater genetic diversity and recombination relative to dikaryotic fungi.