Table 1.
Plasmids used for this study.
Fig 1.
Sequence alignment between part of the genes encoding wild-type repB and repB from the pTF::Ri binary vector.
The T to C changes are highlighted. The repB stop codon is marked in red. Upper strand: wild-type repB from GenBank X04833; lower strand: pTF::Ri sequence.
Fig 2.
Alignment of the oriRi RepB C-terminus with other RepB C-termini.
Arrow indicates the Y299H point mutation. The number at right indicates the last amino acid of each RepB protein. (GenBank accession: oriRi, # X04833; pRL7, Rhizobium leguminosarum bv. viciae plasmid #AM236081; C58linear, Agrobacterium tumefaciens str. C58 linear chromosome # AE007870.2; pRL12, Rhizobium leguminosarum bv. viciae plasmid pRL12 # AM236086; pRtTA1d, Rhizobium leguminosarum bv. trifolii strain TA1 plasmid # FJ592234; pNGR234b, Sinorhizobium fredii NGR234 plasmid pNGR234b # CP000874; p42b, Rhizobium etli CFN 42 plasmid p42b # NC_007763; pRLG204, Rhizobium leguminosarum bv. trifolii WSM2304 plasmid pRLG204 # NC_011371; pR132504, Rhizobium leguminosarum bv. trifolii WSM1325 plasmid pR132504; pRL9, Rhizobium leguminosarum bv. viciae plasmid pRL9 # NC_008379; pRLG201, Rhizobium leguminosarum bv. trifolii WSM2304 plasmid pRLG201; pNGR234a; Sinorhizobium fredii NGR234 plasmid pNGR234a # U00090; pR132502, Rhizobium leguminosarum bv. trifolii WSM1325 plasmid pR132502; pRtTA1b, Rhizobium leguminosarum bv. trifolii strain TA1 plasmid pRtTA1b # FJ592235; pRLG202, Rhizobium leguminosarum bv. trifolii WSM2304 plasmid pRLG202 3 # NC_011366; p42e, Rhizobium etli CFN 42 plasmid p42e # NC_007765; pRL8, Rhizobium leguminosarum bv. viciae plasmid pRL8 # NC_008383; pTiBo542, Agrobacterium tumefaciens Ti plasmid pTiBo542 # DQ058764; pTiC58, Agrobacterium tumefaciens str. C58 Ti plasmid # NC_003065; pTiA6, Agrobacterium tumefaciens octopine-type Ti plasmid # AF242881; p42a, Rhizobium etli CFN 42 plasmid p42a # CP000134; pRtTA1a, Rhizobium leguminosarum bv. trifolii TA1 plasmid pRtTA1a # HM032068; pRi1724, Agrobacterium rhizogenes plasmid pRi1724 # NC_002575; pRtTA1c, Rhizobium leguminosarum bv. trifolii TA1 plasmid pRtTA1c # EU555187; pRL10, Rhizobium leguminosarum bv. viciae plasmid pRL10 # NC_008381; pRLG203, Rhizobium leguminosarum bv. trifolii WSM2304 plasmid pRLG203 # NC_011370). Yellow, red, and blue shading indicate generally conserved amino acids.
Fig 3.
Growth of A. tumefaciens with a) pMON83937, WT-RepB; b) pMON83937-RepBY299H, and c) pMON83937-repBT486C silent mutation. Panel A) Colony size on solidified medium. Panel B) Growth rate in liquid medium (shown as OD at A600 on the Y axis): a1, a2; two colonies from pMON83937, b1, b2; two colonies from pMON83937-RepBY299H, c1, c2, two colonies from pMON83937-repBT486C.
Fig 4.
Agrobacterium plasmid copy number determination by ddPCR.
A) A. tumefaciens no binary plasmid control; B) pMON67438 oriV replicon; C) pMON83937 oriRi native repB; D) pMON83937-RepBY299H; E) pMON83937-repBT486C; F) pMON83937-RepBY299H + repBT486C double mutations. Error bars are shown in the diagram. * B, D and F are significant higher copies than C (native oriRi) and E (T486C silent mutation) at P = 0.05. B (RK2 oriV) is also significantly higher than D and F (the oriRi mutants). Small letters a, b, c and d above the bars indicate statistically significantly different groups (data and statistical analysis in S1 Table).
Fig 5.
Effect of the oriRi with RepBY299H mutation on quality transgenic canola plant production.
Total oriV (pMON67438) events, n = 421; total oriRi (pMON83937) events, n = 379; total oriRi RepBY299H (pMON138207) events n = 462. Error bars are shown in the diagram. *significant difference from the RK2 oriV and the oriRi RepBY299H at P = 0.01. TF: transformation frequency (transgenic events/explant number); 1 copy CP4: single copy CP4 positive frequency; 1 copy CP4 BKB-free: single copy CP4 positive, backbone-free transgenic plant frequency; BKB+: backbone positive transgenic plant frequency. Raw data and statistical analyses in S2 Table for the canola transformation.