Amplification reactions

2.3.1. Standard PCR

PCR amplifications were performed in a termocycler using the thermostable DNA polymerase of the Roche Expand High Fidelity PCR System. Each reaction contained 300 nM primers, 2.5 mM de MgCl2, 0.8 mM dNTPs mix and 0.05 U/μl of polymerase. Genomic DNA was added at 20 ng/μl and plasmid DNA at 2 ng/μl. PCR cycling conditions were: an initial step of denaturation (5 min, 94ºC) followed by 35 cycles of 35 s at 94ºC, 30 s at the calculated primer annealing temperature and 35 s at 72ºC (or 68ºC for templates larger than 3Kb), and a final extension step at 72ºC (or 68ºC) for 10 minutes. PCR for screening procedures were done with the Velocity DNA Polymerase (Bioline) following the manufacturer´s instructions. For PCR amplification of fragments higher than 7 Kb and/or with high GC, the more robust iProof High-Fidelity DNA Polymerase (BioRad) was used, following the manufacturer´s instructions.

2.3.2. Colony PCR from S. cerevisiae and E. coli cells

To determine the insert after Yeast or Bacterial transformation, with a sterile toothpick a small colony was picked and used directly for a PCR reaction or for more PCR reactions a bigger colony was picked and transfer into a PCR tube containing 10 µl of ddH2O. From this

mix 5µl was used for a PCR with V fin 50µl. The PCR reaction was performed as described in

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2.3.3. Reverse transcriptase PCR

Prior complementary DNA (cDNA) synthesis, the RNA was treated with DNaseI (Fermentas). Therefore 1 μg of total RNA was mixed together with 1 µl DNAse and 1 µl of DNAse buffer (Fermentas) and ddH2O to Vfin: 10µl. After 30 minutes of incubation at 37°C, 1 µl EDTA (25

mM) was added and incubate for 10 minutes at 65°C. For the following first strand cDNA was synthesized different protocols were used. Briefly, 1 µl oligodT primer (100 pmol) and 1 µl dNTPs (0.4 mM) were added and incubate 10 minutes at 65°C. Next, the tube was transferred to ice for 5 minutes. After a short spin 4 µl 1x First Strand Buffer (Invitrogen), 2 µl of 0, 1 M dithiothreitol (DTT) and 1 µl of 4 U/μl of RNAsas RNasin® Plus RNase Inhibitor (Promega) were added and incubated for 2 minutes at 37°C. Then, 1µl retrotransciptase (10 U/μl) was added followed by a 50 minute incubation at 37ºC and a final 15 minute incubation at 70°C to inactivate the enzyme. 30 µl ddH2O were added to a Vfin 50 µl. 5 µl

were used for a quantitative real-time PCR (qPCR) reaction. Optinal distinct qRT-PCR Kits were used. The “Transcriptor Universal cDNA Master” (Roche) or “Transcriptor First Strand cDNA Synthesis Kit” (Roche) following the instructions of the manufacturer.

2.3.4. Real-time quantitative PCR

Quantitative real-time PCR reactions (qPCR) were performed in an iCycler apparatus (BioRad, USA) using iQ SYBR Green Supermix (BioRad, USA), 400 ng cDNA template and 300 nM of each gene-specific primer in a final reaction volume of 15 μl. All primer pairs amplified products of 160 – 200 bp. The following PCR program was used for all reactions: an initial step of denaturation (5 min, 94°C) followed by 40 cycles of 30 s at 94°C, 30 s at 60°C, 30 s at 72°C, and 20 s at 80°C for measurement of fluorescence emission. A melting curve program was run for which measurements were made at 0.5°C temperature increments every 5 s within a range of 55 – 95°C.

Once Ct values were obtained (Ct=number of cycles required for the fluorescent signal to cross the threshold), comparison of multiple samples was performed using relative quantification by the 2-ΔΔCt method (Livak and Schmittgen, 2001; Pfaffl, 2001). For this, the wild type strain was chosen as the calibrator and the expression of the target gene in all

35 other strains was expressed as an increase or decrease relative to the calibrator. To determine the relative expression of a target gene in the test sample and calibrator sample, a reference gene (actin) was used as the normalizer.

2.3.5. Fusion PCR

Fusion PCR or overlap extension represents a new approach to genetic engineering (Ho et al., 1989; Yang et al., 2004) and is schematically represented in Figure x. Complementary oligodeoxyribonucleotide (oligo) primers and the polymerase chain reaction are used to generate two DNA fragments with overlapping ends. In this PCR (PCRI) reaction a ~1, 5 kp fragment upstream (prom) of the target gene and a ~1, 5 kp fragment downstream (term) of the target gen is amplified. For the prom region the primer pair X_prom_F/X_term_R and for the term region the primer pair X_term_F/X_term_R were used, where X stands for the target gene (table x). The primer X_term_R and X_term_F contain a tail which is homologue to the hygromycin resistance cassette which was amplified in parallel using the M13F/M13R primer (A). These fragments are combined in a two subsequent 'fusion' reaction (Fusion PCRI, prom+hyg and term+hyg) in which the overlapping ends anneal, allowing the 3' overlap of each strand to serve as a primer for the 3' extension of the complementary strand (B). The resulting fusion product was used as a template for the next PCR reaction (Fusion PCRII). Primers were added to amplify the fusion product produced in the first fusion PCR (C). In this work, this technique was used for the generation of gene knockout constructs, where part of the ORF of the gene was replaced with the hygromycin resistance cassette. The fragments were either purified with the commercial GENECLEAN Turbo Nucleic Acid Purification kit or the “glass milk method” or precipitated before used for transformation. For PCR fusion of final fusion fragments larger than 5 Kb, the more robust iProof High-Fidelity DNA Polymerase (BioRad, Madrid, Spain) was used, following the manufacturer´s instructions. Gene knockout was performed by homologous recombination (E) after replacing the target gene with the two fusion PCR products (D). All primers used to make the construct and confirm positive knockout mutants are indicated in figure 1.

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prom Fusion PCR I

(w/o primer) T trpC Hyg

R P gpdA term T trpC HygR _{P gpdA} B X prom_R X term_R prom term X term_F T trpC HygR _{P gpdA} PCR I X prom_F M13For M13Rev A X term_R

T trpC HygR _{P gpdA} term

T trpC HygR _{P gpdA} HygG HygG prom X prom_F Fusion PCR II (with primer) C term HygR T trpC HygR prom D Fusion PCR Fragments for Transformation Homologous Recombination T trpC HygR P gpdA prom term HygR term prom ORF X_inEx_F X_inEx_R term E T trpC HygR _{P gpdA} prom term X_prom_F_ver X_prom_F X_term_R_ver X_term_R HygY HygG F Verification PCR´s of knockout mutans P gpdA

Figure 1. Schematic representation of the Fusion PCR technique used to generate split-maker for gene knockout in F. oxysporum via homologous recombination. Amplifiaction of the upstream (prom region) and downstream region (term region) of the target gene with primer which contain homolgoues tails to the Hygromycin resistance cassette. The PCR products obtained are used as templates for a PCR reaction with no oligos (PCR2), resulting in annealing of complementary template sequences and extension by the polymerase. The final reaction (PCR3) uses the PCR2 product as a template for amplification with the external primer and the primer set inside the Hyg cassette. Transformation with the two PCR fragments resulting in homologous recombination and reconstruction of the complete Hyg cassette inside the target gene locus.

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2.3.6. Generation of a F. oxysporum cDNA library

The cDNA for the library was generated by using the SMARTTM technology (MatchmarkerTM Library Construction& Screening Kits User Manual, Clontech) following the manufacturer´s instructions with some modeifications. Complete RNA were extracted (as described in section x) of F. oxysporum growing in liquid MM media. The first strand synthesis was performed using CDS III primer (hybridize to the 3`-end of poly A+RNAs) following the manufacture protocols for the SMARTTM technology but with 20 reactions (total volume 320 µl first strand synthesis). From the first-strand cDNA 160 µl were used for the Long-Distance PCR with a total volume of 3200 µl aliquoted in 64 PCR tubes with Vfin: 50 µl. The Long-

Distance PCR was performed by using the Expant High Fidelity Polymerase (Roche) and 22 PCR cycles where the number of thermal cycles used based on the amount of RNA used in the first-strand synthesis and fewer cycles are better to avoid nonspecific PCR products. The cDNA was precipitated to Vfin: 200 µl. After agarosegel confirmation Nanodrop measurement

reveal a concentration of 4700 ng/µl cDNA. A total volume of 100 µl was loaded through CHROMA SPINTM TE-400 Columns to fractionate and select for cDNA > 200 pb and resulted in Vfin: 200 µl cDNA with a concentration of 260 ng/µl. This cDNA was diluted for test transformation before used for the yeast library construction.