Strains

N. crassa strains used in this work are summarized in Table 3. As indicated, several strains used in this study were obtained from the Fungal Genetics Stock Center (FGSC) at the University of Missouri, USA.

The single deletion strains used had been generated within the framework of the Neurospora genome project hosted at Dartmouth Medical School, Great Britain, following the procedure outlined in (Dunlap et al., 2007). A detailed description is available at http://www.dartmouth.edu/~neurosporagenome/protocols.html. In all cases, genes had been disrupted through targeted replacement by a hygromycin resistance cassette containing the selectable marker hph^R; strains had been verified by Southern blotting. For rho-1 and NCU00668, only heterokaryotic deletion strains were available, in which both nuclei harbouring the deletion of the gene (marked by the presence of the hph^R gene) and nuclei of wild type are present, the latter providing shelter from the (lethal) effects of the deletion.

Conditional mutants of cdc-24 had been generated by UV mutagenesis and identified as described in (Seiler and Plamann, 2003); cdc-42, rac and rho-1 loss-of-function and temperature-sensitive strains had been created by S. Seiler (Germany) applying RIP (repeat induced point mutation) mutagenesis (Selker et al., 1989).

The majority of N. crassa strains generated in this study were obtained by transformation applying vectors targeted to the his-3 locus (cp. Table 2 and descriptions below). These plasmids contain the respective expression cassette flanked by a region allowing homologous recombination to the 3 locus and concomitant restoration of a functional his-3 allele (and thus, histidine prototrophy) in transformants obtained from his-3 strains possessing a mutant his-3 allele (Aramayo and Metzenberg, 1996; Margolin et al., 1997).

For all strains, expression of the desired fusion protein was routinely verified by Western blotting (see section 4.7.3) using antibodies for detection of the respective epitope tag.

Generally, strains for expression of proteins fused to fluorescent proteins were generated by transforming strain his-3 (FGSC #6103) with expression vectors targeted to the his-3 locus (see Table 2 and section 4.4.6) and selecting histidine-prototrophic transformants.

Functionality of the expressed proteins was tested by crossing the resulting strains (which still contain the endogenous wild type copy of the gene) with the respective hygromycin-resistant (heterokaryotic or homokaryotic) deletion mutant; offspring carrying the deletion nucleus were identified by their hygromycin resistance. Suppression of phenotypic defects associated with the deletion was used as evidence for functionality.

Deviating from this procedure, strain mus52::bar his-3 (FGSC #9720) was cotransformed with a fragment of ppRho1_N_GFP_Rho1 targeted to the rho-1 locus by homologous recombination and pCSN44 (see Table 2 and section 4.4.6) to create strain sgfp-rho-1 (endog.), in which rho-1 is sgfp-tagged at the endogenous locus. Cotransformants were selected by their resistance towards hygromycin (based on ectopic integration of the hygromycin resistance cassette delivered by pCSN44) and expression of GFP-RHO1, as verified by Western blotting. Crossing with wild type to remove the mus52 mutation and verification by Southern blotting are still pending.

Strains for overexpression of myc-tagged CDC42 and RAC in the cdc-42(18-4) background were obtained by transforming the corresponding temperature-sensitive strain with plasmids pBScpcmyc3hyg_Cdc42 or pBScpcmyc3hyg_Rac (see Table 2 and section 4.4.7), which are thought to integrate ectopically into the genome; transformants were selected on hygromycin.

As a control, a strain harbouring the empty expression vector ectopically integrated was prepared in parallel.

For expression of HA-, myc- and FLAG-tagged fusion proteins for interaction analysis by coimmunoprecipitation and pulldown experiments, strains were generated by transforming auxotrophic strains trp-3; his-3 or nic-3; his-3, respectively, with the corresponding vectors (see Table 2 and section 4.4.7) targeting expression cassettes to the his-3 locus. Resulting histidine-prototrophic transformants were isolated and fusion protein expression checked.

For growth, the strains thus generated still depended on supplementation of the medium with tryptophane or nicotinamide, respectively. To prepare coimmunoprecipitation analyses, desired combinations of these strains were fused to produce prototrophic heterokaryotic strains simultaneously expressing two fusion proteins; to this end, conidia of the respective pair of strains (one Nic^-, the other Trp^-) were combined on VMM (see (Kawabata and Inoue, 2007) for details).

Table 3: N. crassa strains used in this work. (Note that heterokaryotic fusion strains used in coimmunoprecipitation analysis are not listed in the table; see text for details on their generation.) Genetic features are denoted as (EC) if ectopically integrated.

Strain Genotype or description Reference/source

cdc-24(10-19) cdc-24(10-19) A (Seiler and Plamann, 2003) cdc-24(19-3) cdc-24(19-3) A (Seiler and Plamann, 2003) cdc-24(24-21) cdc-24(24-21) A (Seiler and Plamann, 2003) cdc-42(18-4) cdc-42(18-4) a S. Seiler, unpublished

cdc-42(18-7) cdc-42(18-7) S. Seiler, unpublished

rac(7-1) rac(7-1) A S. Seiler, unpublished

rac(11-21) rac(11-21) S. Seiler, unpublished

rac(11-23) rac(11-23) S. Seiler, unpublished

44 4. Materials and Methods

Strain Genotype or description Reference/source

rac(7-1);cdc-42(18-4) rac(7-1) cdc-42(18-4) S. Seiler, unpublished

rho-1(9-1) rho-1(9-1) a S. Seiler, unpublished

rho-1(10-1) rho-1(10-1) a S. Seiler, unpublished

Δrho-2; rho-1(9-1) Δrho-2::hph^R rho-1(9-1) S. Seiler (Δrho-2 x rho-1(9-1))

00668-sgfp his-3⁺::Pccg-1-NCU00668⁺-sgfp A this study 00668-sgfp (compl.) ΔNCU00668::hph^R his-3⁺

::Pccg-1-NCU00668⁺-sgfp (Δmus51::bar^R ?)

this study (FGSC #11488 x 00668-sgfp)

00668-yfp his-3⁺::Pccg-1-NCU00668-yfp A this study mCherry-rho-1 his-3⁺::Pccg-1-mCherry-rho-1⁺ A this study

sgfp his-3⁺::Pccg-1-sgfp A this study

sgfp-rho-1 his-3⁺::Pccg-1-sgfp-rho-1⁺ A this study sgfp-rho-1 (endog.) Δmus52::bar^R his-3^-

Prho-1-sgfp-rho-1⁺ hyg^R(EC) A yfp-rac (compl.) Δrac::hph^R his-3⁺::PgpdA-yfp-rac⁺

(Δmus51::bar^R ?)

HA-rho-1 trp-1^- his-3⁺::Pccg-1-ha-rho-1⁺ Y. Heilig, unpublished HA-rho-2 trp-1^- his-3⁺::Pccg-1-ha-rho-2⁺ this study

3xmyc-rho-1 trp-1^- his-3⁺::Pccg-1-3xmyc-rho-1⁺ this study 3xmyc-rho-2 trp-1^- his-3⁺::Pccg-1-3xmyc-rho-2⁺ this study 12xmyc-rho-1 trp-1^- his-3⁺::Pccg-1-12xmyc-rho-1⁺ this study 3xFLAG-00668 nic-3^- his-3⁺ was used for amplification of DNA. For expression of MBP- and GST-tagged fusion proteins, E. coli strain se ™ 2(DE3) [F^-, ompT, hsdS_B (r_B- m_B-), gal, dcm (DE3), pRARE2 (Cam^R)]

(Merck KGaA, Germany) was used.

S. cerevisiae strain AH109 [MATa, trp1-901, leu2-3, 112, ura3-52, his3-200, gal4Δ, gal80Δ, LYS2::GAL1_UAS-GAL1_TATA-HIS3, GAL2_UAS-GAL2_TATA-ADE2, URA3::MEL1_UAS-MEL1_TATA-lacZ]

((James et al., 1996) and Clontech, USA) was employed for yeast two-hybrid analyses.