Plasmids

Table 5.9: In this work used plasmids

Name Resistance Reference Description

pCR2.1 Amp^R

Kan^R Invitrogen Cloning of PCR and ligated products

p123 Amp^R Aichinger et al. (2003)

Consists of an otef promoter and GFP. For the integration of constructs into the ip locus.

p123-mCherry Amp^R Teichmann et al. (2010) Similar to p123. Consists of mCherry instead of GFP.

pcib1-3xGFP Amp^R

Kan^R Heimel et al. (2010a) For C-terminal fusion of 3xGFP to cib1 in the cib1 locus.

pBS-hhn Amp^R Kämper (2004) Consists of the hygromycin resistance cassette.

Cloning steps of plasmids were checked by restriction analysis and introduced PCR amplicons were verified by sequencing.

Gene deletions

All gene deletions were performed according to Kämper (2004). Flanking gene borders (~1 kb) were PCR amplified from genomic DNA of U. maydis (UM521), which integrated a SfiI restriction site on each border. Both fragments were ligated to a SfiI Hyg^R fragment of the plasmid pBS-hhn (Kämper, 2004). The ligation product integrated into the pCR2.1 TOPO vector (Invitrogen).

pCR2.1-cib1-GFP and pCR2.1-cib1-3xHA

For the cib1-GFP fusion, the 5.5 kb SfiI 3xGFP-Hyg^R fragment of plasmid pcib1-3xGFP (Heimel et al., 2010a) was replaced with the 2.5 kb SfiI GFP-Nat^R fragment from pUMa389 (Becht et al., 2006) to generate the plasmid cib1-GFP. The resulting vector was used to generate plasmid

pCR2.1-98 the PCR fragment followed the procedure as described for pCR2.1-cib1-GFP, yielding the plasmids pCR2.1-cib1^T381A-GFP and pCR2.1-cib1^T381E-GFP.

pCR2.1-cib1^S468A-GFP and pCR2.1-cib1^S468E-GFP

To generate the phospho-null (cib^S468A-GFP) or phospho-mimic (cib1^S468E-GFP) mutation in cib1-GFP, a point mutation was introduced into the ORF of cib1 by standard PCR procedures. Cloning of the PCR fragment followed the procedure as described for cib1-GFP, yielding the plasmids pCR2.1-cib1^S468A-GFP and pCR2.1-cib1^S468E-GFP.

pCR2.1-cib1T381A,S468A-GFP and pCR2.1-cib1T381E,S468E-GFP To generate the double phospho-null (cib1T381A,S468A

-GFP) or double phospho-mimic (cib1T381E,S468E

-GFP) mutations in cib1-GFP, point mutations were introduced into the ORF of cib1 by fusion PCR.

Cloning of the PCR fragments followed the procedure as described for pCR2.1-cib1-GFP, yielding the plasmids pCR2.1-cib1T381A,S468A

-GFP and pCR2.1-cib1T381E,S468E

-GFP.

pCR2.1-Ptef:tTA-tetO:cib1-GFP

For replacement of the cib1 promoter with a tetracycline-regulated promoter, 1 kb upstream of the cib1 start codon and 1 kb of the cib1 open reading frame (ORF) were PCR amplified from genomic DNA, ligated to the SfiI cassette of pUMa707 (Zarnack et al., 2006) and integrated in the pCR2.1 TOPO vector (Invitrogen) generating plasmid pCR2.1-Ptef:tTA-tetO:cib1-GFP

p123-Potef:spp1-mC, p123-Potef:Srspp1-mC, p123-Potef:Uhspp1-mC and p123-Potef:sppA-mC

To generate the spp1-mCherry fusion, the ORF of spp1 (UMAG_02729, UM521) lacking the stop codon was PCR amplified from genomic DNA introducing a BamHI site at the 5’ end and a BspHI site at the 3’ end and integrated into p123-mCherry (Teichmann et al., 2010), to yield p123-Potef:spp1-mC. Cloning of orthologous genes from Sporisorium reilianum Srspp1 (sr13785, strain SRZ1), Ustilago hordei Uhspp1 (UHOR_04354, strain Uh4857-4) and Aspergillus nidulans sppA (ANID_08681, strain AGB551) followed the same procedure, generating plasmids p123-Potef:Srspp1-mC, p123-Potef :Uhspp1-mC and p123-Potef:sppA-mC, respectively.

p123-Potef:YPF1-mC

For cloning of S. cerevisiae YPF1 the ORF (YKL100C, strain sigma 1287) was PCR amplified from genomic DNA introducing BamHI sites at the 5’ and 3’ end removing the stop codon and integrated into p123-mCherry (Teichmann et al., 2010) to yield plasmid p123-Potef:YPF1-mC.

p123-Potef:HM13-mC

The cDNA of the human HM13 (BC062595, cDNA clone) was PCR amplified from the vector pCS6(BC062595)-TCH1303-GVO-TRI (BioCat) introducing an XmaI site at the 5’ end, a NcoI site at the 3’ end and removing the stop codon and subsequently ligated into p123-mCherry (Teichmann et al., 2010) to yield p123-Potef:HM13-mC.

p123-Pspp1:spp1-mC

To replace the otef promoter in p123-Potef:spp1-mC with the spp1 promoter, a 1.4 kb spp1 promoter fragment was PCR amplified introducing a NdeI site at the 5’ end and a BamHI site at the 3’ end. The PCR fragment was integrated into p123-Potef:spp1-mC to generate p123-Pspp1:spp1-mC.

p123-Potef:spp1^D279A-mC and p123-Pspp1:spp1^D279A-mC

To generate the catalytically inactive version of spp1 (spp1^D279A), a point mutation was introduced into the ORF of spp1 by standard PCR procedures. Cloning of the PCR fragment followed the procedure as described for p123-Potef:spp1-mC and p123-Pspp1:spp1-mC, yielding the plasmids p123-Potef:spp1^D279A -mC and p123-Pspp1:spp1^D279A-mC, respectively.

p123-Potef:spp1-GFP and p123-Potef:spp1^D279A-GFP

To generate the spp1-GFP fusion as well as a variant with the catalytically inactive spp1 (spp1^D279A -GFP), similar cloning procedures were used as described for p123-Potef:spp1-mC and p123-Potef:spp1^D279A-mC, respectively. PCR fragments were integrated into p123 (Aichinger et al., 2003) instead of p123-mCherry, generating the plasmids p123-Potef:spp1-GFP and p123-Potef:spp1^D279A-GFP.

p123-Potef:pep1-mC

To generate the pep1-mCherry fusion, the ORF of pep1 (UMAG_01987, UM521) lacking the stop codon was PCR amplified from genomic DNA introducing a BamHI site at the 5’ end and a NcoI site at the 3’

end and integrated into p123-mCherry (Teichmann et al., 2010), to yield p123-Potef:pep1-mC.

p123-Potef:tin2-mC

To generate the tin2-mCherry fusion, the ORF of tin2 (UMAG_05302, UM521) lacking the stop codon was PCR amplified from genomic DNA introducing a BamHI site at the 5’ end and a NcoI site at the 3’

end and integrated into p123-mCherry (Teichmann et al., 2010), to yield p123-Potef:tin2-mC.

100 p123-Pspp1[∆UPRE1]:spp1-mC, p123-Pspp1[∆UPRE2]:spp1-mC, p123-Pspp1[∆UPRE1/2]:spp1-mC

To generate the deletion of UPRE1 (Chapter 3.3.10) in the promoter of spp1, the promoter fragments Pspp1-∆UPRE1-LB (Primer: 02729_pro1.4_for and 02729_∆UPRE_rev) and Pspp1-∆UPRE1-RB (Primer: 02729_∆UPRE_for and 02729_pro_rev) were PCR amplified. To generate a full-length promoter lacking the UPRE1, an overlapping PCR (Primer: 02729_pro1.4_for and 02729_pro_rev) was performed on both sub-fragments. The generated Pspp1-∆UPRE1 full-length fragment was cloned as described for p123-Pspp1:spp1-mC, yielding p123-Pspp1[∆UPRE1]:spp1-mC. Deletion of UPRE2 (Chapter 3.3.10) in the spp1 promoter was performed similar as described for deletion of UPRE1, by using different primers for generation of the promoter sub-fragments (02729_pro1.4_for and 02729_∆UPRE2_rev or 02729_∆UPRE2_for and 02729_pro_rev), generating the plasmid p123-Pspp1[∆UPRE2]:spp1-mC. To generate a double deletion of UPRE1 and UPRE2, the promoter sub-fragments were PCR amplified from p123-Pspp1[∆UPRE2]:spp1-mC with the primers 02729_pro1.4_for and 02729_∆UPRE_rev ∆UPRE1/2-LB) or 02729_∆UPRE_for and 02729_pro_rev

(Pspp1-∆UPRE1/2-RB) and cloned as described for p123-P^spp1[∆UPRE1]:spp1-mC, yielding p123-Pspp1[∆UPRE1/2]:spp1-mC.

p123-Potef:02578-GFP

To generate the UMAG_02578-GFP fusion, the ORF of UMAG_02578 lacking the stop codon was PCR amplified from genomic DNA (UM521) introducing a BspHI site at the 5’ and the 3’ end, and integrated into p123 (Aichinger et al., 2003), to yield p123-Potef:02578-GFP.

5.2 Standard methods of microbiology