Supplemental Material

This article contains the following supporting material:

• Supplemental Figure 1 - Gene structure and Southern confirmation of mutation of Neurospora rrg-1. (A) rrg-1 structure and gene replacement. The boxed area indicates the coding region of rrg-1, and the closed triangles indicate the verified intron position. The gene replacement construct using the hph marker under control of the Ashbya TEF gene promoter and terminator is shown below (cassette is bound by thick lines). The insertion points of the hph gene replacement into the rrg-1 gene are indicated by the arrows. The probes used for Southern verification of Δrrg-1 gene replacement mutants (gene replacement), Δrrg-1 rrg-1⁺ bar⁺ rescued strains (bar complementation) or the his-3 targeted rrg-1⁺ and rrg-1^D921N strains (his-3 targeting), are indicated by the solid lines. Letter abbreviations are used to indicate restriction sites: Xh = XhoI, H = HindIII, S = SacI, Xm = XmnI, A = AclI, N = NotI. (B) Southern verification of Δrrg-1 mutants. Genomic DNA from wild-type (74A) and Δrrg-1 homokaryotic mutants 6.2, 11.4, 15.20, 32.15 and 10.10 were digested with HindIII, electrophoresed on an agarose gel, blotted and hybridized with a probe containing the hph cassette and the 3' flanking region of the knockout construct (labeled as "gene replacement" in A). Under these conditions, wild-type yields a 14.9 kb hybridizing fragment and Δrrg-1 mutants exhibit 6.2 and 7.0 kb fragments, due to the presence of a HindIII site in the hph cassette and HindIII sites outside the regions of recombination on both sides of the replacement.
• Supplemental Figure 2 - Alignment of RRG-1 with related response regulator proteins from other fungi. The RRG-1 open reading frame predicted by the Broad Institute (http://www.broad.mit.edu/annotation/fungi/neurospora/) was aligned to other proteins from ascomycete filamentous fungi using ClustalW (Thompson et al., 1994). Species and proteins are N. crassa RRG-1 (NCU01895.2), Chaetomium globosum (CHG06648.1), Fusarium graminearum (FG08948.1) and Magnaporthe grisea (MG02897.4). Boxshade (http://www.ch.embnet.org) was used to highlight identical (black shading) or similar (gray shading) amino acids. The receiver domain is underlined and the conserved aspartate (D921) is indicated with an asterisk (*).