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Supplementary methods
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Disruption of the ligD gene
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The upstream (2.0 kb) and downstream (2.0 kb) flanking regions of the ligD ORF were
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amplified by using the primers (attB4-L4-F and attB1-L4-R for the upstream,
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attB2-L4-F and attB3-L4-R for the downstream) and the A. oryzae genomic DNA as
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template. The fragments were cloned as 5’ and 3’ entry clones (pg5'L4 and pg3'L4), and
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then connected with pgCArgB containing the argB marker (Jin et al. 2007) and
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pDESTR4-R3 (Invitrogen), generating pgLDA. The gene disruption fragment for ligD
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was amplified by the primers (attB4-L4-F and attB3-L4-R) and pgLDA as template, and
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introduced in the NSAR1 strain (Jin et al. 2004). M+Met medium supplemented with
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0.01% adenine was used for selection of transformants. In order to verify disruption of
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the ligD gene, genomic DNAs were analyzed by PCR using the primers (L4p-2056_F
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and L4t+52_R).
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Disruption of the pyrG gene
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The upstream (1.5 kb) and downstream (1.5 kb) flanking regions of the pyrG ORF were
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amplified by using the primers (attB4-PYRG-F and attB1-PYRG-R for the upstream,
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attB2-PYRG-F and attB3-PYRG-R for the downstream) and the A. oryzae genomic
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DNA as template. The fragments were cloned as 5’ and 3’ entry clones (pg5'PG and
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pg3'PG), and then connected with pgEaA containing the adeA marker (Jin et al. 2007)
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and pDESTR4-R3, generating pgPGaA. The gene disruption fragment for pyrG was
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amplified by the primers (attB4-PYRG-F and attB3-PYRG-R) and pgPGaA as template,
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and introduced in the NSR-ΔlD2 strain (ΔligD). M+Met medium supplemented with 20
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mM uridine was used for selection of transformants. In order to confirm disruption of
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the pyrG gene, genomic DNAs of the transformants were subjected to PCR analysis
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using the primers (pGp-1548_F and pGt+104_R).
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Disruption of the tppA gene
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The 0.3 kb upstream and 1.3 kb downstream flanking regions of the tppA ORF was
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amplified with the primers (aB2-tp+p_F and f-tp+p_R for the upstream, f-tp+p_F and
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aB3-tp+p_R for the downstream) and the A. oryzae genomic DNA as template. The two
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fragments were fused with the primers (aB2-tp+p_F and aB3-tp+p_R) and cloned,
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generating the 3’ entry clone (pg3'tp+p). The pyrG gene was amplified with the primers
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(aB1-pG-F and aB2-pG-R) and the A, oryzae genomic DNA and cloned as the center
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entry clone, pgEpG. The entry clones (the 5’ entry clone (pg5'tp) containing the 1.3 kb
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tppA upstream region (Jin et al. 2007), pgEpG, pg3'tp+p and the destination vector
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(pDESTR4-R3) were connected, generating pgtpPG. The gene disruption fragment for
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tppA was amplified by the primers (tppA-F’ (Jin et al. 2007) and aB3-tp+p_R) and
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pgtpPG as template, and introduced in the NSPlD1 strain (ΔligD ΔpyrG). M+Met
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medium was used for selection of transformants. In order to verify disruption of the
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tppA gene, PCR analysis was performed using the primers (tpp-1340_F and tpp+237_R)
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and genomic DNAs of the transformants as template.
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Disruption of the pepE gene
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The 0.3 kb upstream and 1.9 kb downstream flanking regions of the pepE ORF were
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amplified with the primers (aB2-pE+p_F and f-pE+p_R for the upstream, f-pE+p_F and
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attB3R-TpepE3’-AS for the downstream) and the genomic DNA as template. The two
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fragments were fused with the primers (aB2-pE+p_F and attB3R-TpepE3’-AS) and
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cloned, generating the 3’ entry clone, pg3'pE+p. The 5’ entry clone (pPepEP4-P1R)
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containing the 2.0 kb tppA upstream region (Jin et al. 2007), pgEpG, pg3'pE+p and the
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destination vector, pDESTR4-R3, were connected, generating pgpEpG. The gene
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disruption fragment for pepE was amplified by the primers (aB2-pE+p_F and
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attB3R-TpepE’-AS) and pgPGaA as template, and introduced in the NSPlD-tA3 strain
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(ΔligD ΔpyrG ΔtppA). M+Met medium was used for selection of transformants.
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Genomic DNAs were used as template for PCR analysis using the primers (pEp-488_F
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and pEt+162_R) for verification of pepE gene disruption.
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