Figure 1
From: The transcription factor Spores Absent A is a PKA dependent inducer of Dictyostelium sporulation
Identification of SpaA by REMI mutagenesis and validation by gene knock-out. (A) REMI mutant phenotype. Wild-type Ax2 cells, transformed with cotC-mRFP were subjected to REMI mutagenesis and a clone, b39, with reduced red fluorescence in slugs and fruiting bodies was isolated. Bar: 50 µm. (B) Individual cells. Prespore cells and spores harvested from Ax2 and b39 slugs and fruiting bodies, respectively, were photographed under epifluorescence with longer exposure for the b39 clone. Bar: 3 µm. (C) Phylogeny of mutant gene. B39 was defective in a CudA-like nuclear factor, SpaA. SpaA sequence was aligned with all D.discoideum CudA-like proteins (DDB_G prefix), an Entamoeba histolytica CudA (AAC41578) and the closest homologs to SpaA in the Dictyostelids D. purpureum (DPU1258757), D. lacteum (DLA_07383), Polysphondylium pallidum (PPL_00580) and D. fasciculatum (DFA_08790). A well conserved segment of the alignment (AA184-AA589 of SpaA) was subjected to Bayesian phylogenetic inference51. The tree is rooted at midpoint and posterior probabilities of the nodes are indicated. Colour coding of species names: red, green, blue violet: major groups 1, 2, 3 and 4 of Dictyostelia, respectively, amber: non-dictyostelid Amoebozoa. (D,E) Recapitulated spaA- mutant. A spaA knock-out was generated by homologous recombination (see Supplementary Fig. S2). Wild type Ax2 and spaA- cells were distributed on non-nutrient agar and photographed after slugs (D) and fruiting bodies (E) had formed. Bar: 100 µm. (F). Spore morphology. Ax2 and spaA- spores were fixed and stained with anti-spore antibodies (left) or Calcofluor (right), and photographed under phase contrast (bottom) or epifluorescence (top). Bar: 5 µm. (G) Sporulation efficiency. Fruiting bodies were developed from 3 × 106 cells plated on 1 cm2 filters. Filters were vortexed with 0.1% Triton-X100 after 21 h when fruiting bodies had formed and at two later time points. After spore counting, the percentage of spores relative to the number of plated cells was calculated. Mean and SD of three independent experiments.
