Table 7: Plasmids constructed and used in this study
pME Description Organism Marker Source
pJET1.2 E. coli AmpR Fermentas
2.4.1 Construction of plasmids for V. dahliae knock-out strains
The primers and plasmids for deleting THI4 and PA14_2 in V. dahliae are listed in Table 5, Table 6 and Table 7.
For deleting thiamine synthase THI4, the nourseothricin (nat) resistance was amplified from the pME3793 vector with primers CH93fw/rev. The 5'-prime and 3'-prime flanking regions of around 1.5 kb were amplified from V. dahliae JR2 genomic DNA by CH123fw/rev and CH124fw/rev. A constitutive gpdA promoter was used for the resistance cassette and amplified by CH127fw/94rev from vector pKO2. Fusion PCR was performed with the primer pair CH123fw/124rev. The knockout cassette was cloned blunt end into pJET1.2 and into the pKO2 vector via 5'-prime EcoRI and 3'-prime XbaI restriction sites (resulting in the vectors pME4105 and pME4106). The knockout cassette was cloned into V. dahliae JR2 by Agrobacterium tumefaciens mediated transformation (ATMT).
Deletion of the membrane protein PA14_2 was performed by insertion of nourseothricin (natR) resistance into the locus. Amplification of the resistance cassette from the pME3793 vector with the primers CH93fw/rev was performed. The 1.5 kb 5'-prime and 3'-prime flanking regions were amplified from genomic V. dahliae JR2 DNA by using CH119fw/rev and CH120fw/rev primers. Fusion PCR was then performed together with the constitutive gpdA promotor (CH127fw/94rev from vector pKO2) by using the primers CH119fw/CH120rev. Then the knockout cassette was cloned into pJET1.2 and into pKO2 vector via 5'-prime EcoRV and 3'-prime SbfI (SdaI) restriction sites (pME4107 and pME4108 were the resulting plasmids). The knockout cassette was transformed linear into V. dahliae JR2 via protoplastation.
2.4.2 Construction of plasmids for complementation of Vd∆THI4 and Vd∆PA14_2 Complementation was on the one hand performed by using the original Vd JR2 open reading frame (resulting in the plasmids pME4109 and pME4111) and on the other hand by using the Vl 43 dahliae-like allele (resulting in the plasmids pME4110 and pME4112) for heterologous complementation. For both, flanking regions were derived of the JR2 genome.
For construction the GeneArt® Seamless Cloning & Assembly Kit (life technologiesTM, Darmstadt, Germany) was used to fuse three parts of the complementation cassette and the pBluescript®II KS+ vector.
For construction of the ∆THI4 complementation plasmid THI4 open reading frame was amplified from genomic DNA using the primers CH150fw/134rev like the 1.5 kb up- and
downstream flanking regions (using the primers CH175fw/149rev and CH162fw/175rev). The constitutive gpdA promoter was amplified from pME4105 by using CH169fw/159rev and finally the hygromycin resistance gene (hph) was amplified with the primers CH160fw/161rev from the pGS1 vector. The trpC terminator at least was amplified from the pAN7-1 vector using CH176fw/rev as primers. Before using the GeneArt® Seamless Cloning
& Assembly Kit the upstream flanking region and open reading frame were fused. The promoter was fused with the resistance and the downstream flanking region by PCR. The trpC terminator was used as a single part. The three parts of the complementation cassette were ligated into pBluescript®II KS+ vector via PmeI restriction sites.
The complementation cassette of ∆PA14_2 was constructed like the one of thiamine synthase.
The 1.5 kb up- and downstream flanking regions (CH173fw/149rev, CH162fw/173rev) and the PA14_2 open reading frame (CH153fw/84rev) were amplified from genomic DNA. The constitutive gpdA promotor was amplified from the plasmid pME4107 by using the primers CH169fw/159rev and finally the hygromycin resistance gene (hph) was amplified with CH160fw/161rev from the pGS1 vector. At least trpC terminator was amplified by CH176fw/rev from the pAN7-1 vector. Before using GeneArt® Seamless Cloning &
Assembly Kit the upstream flanking region and open reading frame were fused and the promoter was connected with the resistance and the downstream flanking region by PCR. The trpC terminator was used as a single part. The three parts of the complementation cassette were fused blunt end into pBluescript®II KS+ vector via PmeI restriction sites. Finally all cassettes were transformed linear into Vd∆THI4 and Vd∆PA14_2 deletion strains via protoplastation.
2.4.3 Construction of GFP-tagged plasmids
Single GFP (single green fluorescing protein) was cloned downstream to VdTHI4 and VdPA14_2 genes (resulting in the vectors pME4116 and pME4117). Fusing the cassette was done by GeneArt® Seamless Cloning & Assembly Kit (life technologiesTM, Darmstadt, Germany) with two cassette parts and the pBluescript®II KS+ vector.
For the VdTHI4-GFP cassette the plasmid pME4109 was used as template. Upstream flanking region and open reading frame were amplified with the primers CH175fw/141rev from the plasmid pME4109. The GFP gene was amplified by CH142fw/177rev from pFA6a-GFP plasmid and the two parts were fused (CH175fw/177rev) by PCR. As a second part the terminator, promoter, resistance and downstream flanking region were amplified as one fragment with the primers 178fw/175rev from the plasmid pME4109. The GFP cassette was
cloned by fusing the two fragments blunt end into pBluescript®II KS+ vector via PmeI restriction sites.
For generating the cassette of VdPA14_2-GFP pME4111 was used as template and upstream flanking region together with VdPA14_2 open reading frame were amplified using primers CH173fw/146rev. The GFP was generated by PCR from the pFA6a-GFP plasmid using the primes CH147fw/177rev. The two parts were fused with 173fw/177rev by fusion PCR. As a second part the terminator, promoter, resistance and downstream flanking region were amplified as one fragment by the primers 178fw/173rev from the plasmid pME4111. The gfp cassette was cloned by fusing the two fragments blunt end into pBluescript®II KS+ vector via PmeI restriction sites.
In order to create the positive control, GFP-gpdA-natR was amplified from the plasmid pME3929 by using the primers CH183fw/rev. For generating the constitutive promotor gpdA pME 4109 was used as the template and the primers CH182fw/rev and CH184fw/rev. All three fragments were cloned into pBluescript®II KS+ vector blunt end via PmeI restriction sites and named pME4118. All cassettes were transformed linear into V. dahliae JR2 via protoplastation.