Extended Data Fig. 9: BASS11 aggregate formation in P. anserina and Bab/BASS11 incompatibility.

a, Western-blot of P. anserina crude extracts expressing GFP-BASS11^Bab (GFP-Bab(86-109)) in the [b*] and [b] state or GFP-BASS11^Bab N100P fractionated by centrifugation at 17,000g probed with an anti-GFP antibody (T, total extract, S, supernanant, P, pellet). b, Representative example of a micrograph of a P. anserina strain co-expressing GFP-BASS11^Bab (GFP-Bab(86-109)) and BASS11^Agp-RFP (Agp(2-25)-RFP). Scale bar is 5 µm. c, Representative example of a barrage reaction between a strain expressing Bab-GFP (Bab) and strains expressing GFP-BASS11 in the soluble [b*] and aggregated [b] state assayed on corn meal agar medium. The red arrowhead points to the barrage reaction. On the right an interpretative model of Bab/[b] incompatibility is given. In this model, in [b*] strains BASS11 is soluble, upon cell fusion with a strain expressing Bab, no activation of Bab occurs and the fusion cell is viable. In [b] strains, BASS11 is aggregated into an amyloid state and upon fusion with a strain expressing Bab, Bab is converted and activated and causes death of the fusion cell. d, Cell death in fusion cells of strains expressing Bab (Bab(86-109)-RFP) and BASS11 (GFP-Bab(86-109)). The upper panels correspond to methylene blue staining for dead cells, the lower panel to a GFP/RFP overlay. Arrowheads point to the presumed fusion site. Scale bar is 5 µm. Three representative examples are shown. e, GFP-BASS11 [b] prion propagation assay based on acquisition of the barrage reaction to a tester strain expressing Bab. Two rows of six [b*] state strains expressing GFP-BASS11 were inoculated on the plate (grey bracket) together with a row of strains expressing Bab (center row). The left and right rows differ by the strain inoculated at the bottom of the plate, either [b*] (bottom right) or [b] strain (bottom left). Note the barrage formation in the confrontation zone in the left row, indicating that [b*] strains have been converted to the [b] state at the time of contact with the Bab-tester row. f, Sexual incompatibility associated to the Bab/BASS11 interaction. Counts correspond to the number of fertilized sexual organs (perithecia) formed in the following crosses : ΔhellpΔhet-sΔhellf::Bab x ΔhellpΔhet-sΔhellf (ΔΔΔ::Bab x ΔΔΔ) (shades of grey, two independent crosses used as control), ΔΔΔ::GFP-BASS11 x ΔΔΔ (blue, one cross) and ΔΔΔ::Bab x ΔΔΔ::GFP-BASS11 (red and pink, two independent crosses). Counts are means +/− s.d. of numbers of perithecia per cm in the confrontation zone (8 technical replicates). P-values are calculated from one-way ANOVA and are Tukey’s multiple comparison tests comparing the values to the first control cross. Images of the confrontation zone showing individual perithecia (white arrowheads) are given on the right of the histogram. The upper image is ΔΔΔ::Bab x ΔΔΔ (fertile control) and the lower image, ΔΔΔ::Bab x ΔΔΔ::GFP-BASS11. Scale bar is 2 mm. g, Mutations in the BASS11-motif and the N-terminal region of Bab abolish sexual incompatibility. Strains expressing Bab and Bab mutants as marked were crossed with strains expressing aggregated GFP-BASS11 (ΔΔΔ::Bab x ΔΔΔ::GFP-BASS11). All tested mutations restore fertility and increase formation of fertilized sexual organs (perithecia). h, Mutations in the BASS11-motif and the N-terminal region of Bab abolish vegetative incompatibility. Mutations in Bab abolish the barrage reaction in confrontations with strains expressing aggregated BASS11. The confrontations are shown on synthetic medium (left) and corn meal agar (right). The red arrowhead points to the barrage reaction between the strain expressing Bab and GFP-BASS11.