Comparison of meiosis in GFP-containing and corresponding non-GFP-containing strains.
In Sordaria (homothallic Ascomycete) a single ascospore and thus a single haploid nucleus can complete the entire life cycle from germination to meiotic sporogenesis. Vegetatively growing mycelium is plated on suitable medium and after 6 days perithecia that develop in a synchronous wave contain mainly asci in meiotic prophase. Prophase nuclear stages have been accurately defined by ultrastructural analysis (Zickler 1977). These studies and DAPI staining revealed that asci and nuclear sizes as well as nucleolus morphology are tightly coupled to prophase stages. In particular ascus growth that increases from 10 m just after karyogamy to 60 m at zygotene, 100 m at mid-pachytene and 150 m at diplotene, is a powerful tool for staging. On the assumption that ascus development is unaffected, ascus lengths and nuclear diameters can thus be used to determine prophase stages in mutants altered in the chromosomal program (e.g. Storlazzi et al. 2003; Tesse et al. 2003).
Sordaria meiotic mutants (like higher plant mutants) do not arrest when either recombination or synapsis is defective. Also, all GFP tags tested so far are well tolerated in Sordaria and all the tagged proteins analyzed in this and previous studies are fully functional based on in vivo genetic complementation criteria (below). All the GFP tags are inserted ectopically because homologous recombination is a rare event. Tagged constructs were first introduced by transformation in a WT strain and then crossed to the relative mutant strain to evaluate the complementation phenotype.
Ascus sizes, synapsis and ascospore formation of each strain containing a GFP-tagged protein in a WT or a mutant background are compared to an isogenic strain lacking the GFP tag. The overall progression of meiosis is sensitively revealed by scanning rosettes of asci from multiple perithecia, which implies scanning of hundreds of meiotic nuclei (100-150 per perithecium) at all stages from prekaryogamy to sporulation. Even minor deviations from the normal program are immediately apparent. An abnormality in the fraction of asci of different sizes is also readily detectable without quantification. Given this situation, a delay in progression from one chromosomal stage to another, or in chromosome status at any particular stage(s) is detectable as a disparity in chromosomal state versus ascus size. No differences in ascus sizes and meiotic progression were found when the GFP-tagged strains used for this study (see table) were compared to the corresponding isogenic strains (WT and mutants) lacking the GFP-tag. A slight defect in sporulation (90 to 95% of viable spores instead of 100% in WT) was detected in the WT strain carrying Rad51-GFP. However, meiosis was normal and no supplementary defect was observed in the different mutant backgrounds (see table). As we have no rad51 mutant to test the complementation of Rad51-GFP, we used an anti-Rad51 antibody to compare the number of foci in all strains (see table).