Fig. S1 (A) Replacement strategy for deletion of bcatf1. The flanking regions of bcatf1 were amplified via polymerase chain reaction (PCR). Modified primer (9/10 and 11/12; see Table S1 in Supporting Information) integrated specific restriction sites (SacI/SacII and Apa/XhoI, respectively) for endonucleases into the flanking regions, allowing for directed cloning of these fragments into the pNR1 replacement vector. OliC, promoter from Aspergillus nidulans; nat1, nourseothricin acetyltransferase (Streptomyces noursei); T-tub, tubulin terminator from Botrytis cinerea. A linear fragment obtained by digestion of pNR1_Δbcatf1 with SacI and ApaI was used for the transformation of B. cinerea wild-type strain B05.10. Primers used for amplification of the flanking regions: 3, BcAtf1_F1_for/9; 4, BcAtf1_F1_rev/10; 5, BcAtf1_F2_for/11; 6, BcAtf1_F2_rev/12. (B) Verification of homologous integration in the bcatf1 locus and control of homokaryotic deletion strains (B.9, N3, N.6, L.11). Diagnostic PCR was performed using primers 1 (BcAtf1_probe_for/3) and 2 (BcAtf1_probe_rev/4) for amplification of the wild-type fragment (0.5 kb). Primers 7 (BcAtf1_for control 2/5) and 9 (pOliR/7) (1.8 kb) were used to amplify the DNA fragment F1 consisting of the flanking region 1 upstream of bcatf1 and the first part of the resistance cassette promoter region. Primers 8 (BcAtf1_F2_rev_control/6) and 10 (T-tub2/8) (0.9 kb) were used to amplify a fragment F2 with the end of the resistance cassette terminator and the flanking region 2 downstream of bcatf1. Amplification of these DNA fragments should only be possible in cases of homologous integration of the replacement fragment into the bcatf1 gene locus. (C) Southern blot analysis of Δbcatf1 strains. Genomic DNA was digested with HindIII and separated via agarose gel electrophoresis. Flanking region 2 used for the replacement cassette served as probe. The F2 probe hybridized with the wild-type 1.8-kb HindIII fragment, as well as with the 1.1-kb fragment of the Δbcatf1 mutants. Marker: DNA ladder mix (Fermentas, St. Leon-Roth, Germany).

Fig. S2 Hyphal morphology of Δbcatf1 in comparison with the wild-type B05.10. The mutant's hyphae are elongated in comparison with B05.10 hyphae, but show regular normal septation. Scale bar, 100 μm. The strains were grown for 2 days on CM-overlaid microscope slides and stained with calcofluor white. After 2–4 days of incubation in a humid chamber at 20 °C, the colonies were incubated for 5 min in 1% (w/v) calcofluor white solution and then washed with water.

Fig. S3 Quantification of conidiospore production of Δbcatf1. Test tubes contain the quantified conidiospore solutions. Conidiospores were harvested from agar plates and spore suspensions of each strain were diluted in H2O to 5 × 105 spores/mL; 4 × 104 spores were plated on CM (three plates/strain) and incubated at 18 °C for 2 weeks under permanent near-UV light to enhance conidiospore formation. After 2 weeks, conidiospores were floated off the plates in a defined volume of 15 mL H2Obidest, filtered over a Nytex membrane and washed twice with H2Obidest. Spores were resuspended in 20 mL of H2Obidest. For spore quantification, dilution series were counted.

Fig. S4 Growth in the presence of oxidative stressors. Growth of the Δbcatf1 strain, Δbcsak1 strain and wild-type B05.10 on different stress-inducing media after 3 days. CM was supplemented with H2O2 (20 mM), menadione (500 μM) and NaCl (1 M).

Fig. S5 Expression of botrydial cluster genes in Δbcatf1 strain and B05.10. Activation of bcbot1/CND5 and bcbot2/CND15, respectively, is dependent on calcineurin, as its inhibitor, cyclosporin A (CsA), inhibits their expression. A strikingly enhanced expression of these cluster genes can be observed in the bcatf1 deletion strain, which is reduced with the addition of CsA under day/night growth conditions (A). There is no expression of the cluster genes visible in the wild-type and only a slight CsA-independent expression in Δbcatf1 when cultures are grown in the dark (B). Loading control: rRNA.

Fig. S6 Isolated toxins from parental and mutant strains.

Fig. S7 Physical interaction between DNA-binding domain of transcription factors and promoters revealed by yeast one-hybrid assay. Haploid yeast strains harbouring the Botrytis cinerea DNA-binding domains of the transcription factors ATF1 or CreA in frame with the Gal4 activator domain were grown on horizontal lines, whereas haploid strains harbouring the promoters of the regulated genes upstream of a HIS3 reporter gene were grown on vertical lines. One medium without histidine, with the growth of diploids (large colonies) at the intersections, indicates positive one-hybrid interactants. The positive control CreA binds to all five promoters, whereas ATF1 does not bind to any of the tested promoters.

Fig. S8 Differentiation of Δbcatf1, the wild-type B05.10 and the complementation strain cΔbcatf1 under standard cultivation conditions. Growth test. (A) After 3 days on complete medium (B5 + 2% glucose + 1 g/L yeast extract), the wild-type (WT) and complementation strain cΔbcatf1 grow flat on top of the medium, whereas the mutant produces aerial hyphae. (B) Under permanent light on complex medium [potato dextrose agar (PDA) + bean], the WT and complementation strain cΔbcatf1 produce large amounts of conidiospores causing the black colour of the mycelium. The Δbcatf1 strain hardly produces any spores. (C) After 2 weeks in permanent darkness (B5 + 2% glucose + 1 g/L yeast extract), the WT and complementation strain cΔbcatf1 form sclerotia, whereas there is no formation of sclerotia, but only of conidiospores, in the Δbcatf1 strain.

Fig. S9 Pathogenicity assay of Δbcatf1, the wild-type B05.10 and complementation strain cΔbcatf1. Agar plaques were placed on leaves of Phaseolus vulgaris. Photographs were taken 2 days post-infection.

Table S1 Primers used in this work (sequence: 5′ → 3′).

Table S2 Gene expression profile of Δbcatf1. +/−, up-/down-regulated genes in Δbcatf1.

mpp778_sm_Figs-Tabs.doc25533KSupporting info item
mpp778_sm_FS7.doc4132KSupporting info item

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