Supplementary Material

Supplementary Table S1. Oligonucleotide primers used for the assembly of ORF2 and ORF2nat. Target specific sequences are presented in italics; restrictions sites are presented in bold; and deoxyuridine residues are shaded in black. The origin of each group of nucleotides is written beneath them

Name (# of bases) / Sequence
STnat-fwd (50) / AATAAC GAATTC CCATGG CT ATGGAATCAAAGACAACCCAAAACGGATCC
EcoRI NcoI AtST5a (sense)
ST-fwd (68) / AATAAC GAATTC CCATGG CT CACCACCACCACCACCAC ATGGAATCAAAGACAACCCAAAACGGATCC
EcoRI NcoI 6 x His AtST5a (sense)
ST-rev (77) / ACTCCACGUCTCCCGCCAACTTGAGAAGGTCAAAATTCAAAGTCTG GTTATCATGTTGAAGCAAGCCAGTATCTTTG
F2A (5’ fragment, antisense) AtST5a (antisense)
GTnat-fwd (47) / ACGTGGAGUCCAACCCAGGGCCT ATGGCGGAAACAACTCCCAAAGTG
F2A (3’ fragment, sense) UGT74B1 (sense)
GT-fwd (71) / ACGTGGAGUCCAACCCAGGGCCT TGGTCTCATCCTCAATTTGAAAAG ATGGCGGAAACAACTCCCAAAGTG
F2A (3’ fragment, sense) Strep UGT74B1 (sense)
GT-rev (63) / ACCGCAUGTTAGCAGACTTCCTCTGCCCTC CTTCCCTAAACTCTCTATAAACTCGTTAATGCT
T2A (5’ fragment, antisense) UGT74B1 (antisense)
SUR1-fwd (55) / ATGCGGUGACGTCGAGGAGAATCCTGGCCCA ATGAGCGAAGAACAACCACACGCC
T2A (3’ fragment, sense) SUR1 (sense)
SUR1-rev (55) / AATAAC GAGCTC GGTACC TTACATTTCGAGATTATTATCACTCAGTTTCAAAGCT
SacI KpnI SUR1 (antisense)
shortST-fwd (20) / AATAAC GAATTC CCATGG CT
EcoRI NcoI
shortSUR1-rev (22) / AATAAC GAGCTC GGTACC TTAC
SacI KpnI

Supplementary Fig. S1. Southern blot of genomic DNA extracted from different tobacco transgenic lines visualized on a light-sensitive film after detection by chemiluminescence. For each plant, 20 μg of purified DNA were digested with HindIII and subjected to DNA-blot hybridization according to the DIG Application Manual (Roche). The probe used was a DIG-labeled fragment of the nptII (kanR) gene. Lanes 1, 8, and 15: DIG-labeled molecular-weight marker. Lanes 2-6: five different lines transformed with ORF2. Lanes 7 and 9-13: six different lines transformed with ORF2nat. Lane 14: Untransformed (wildtype) tobacco plant

Supplementary note on the synthesis of PATH. As a cautionary note, it is worth mentioning that the synthesis was first attempted by the method of Doszczak and Rachon, which has been reported to give very high yields for other thiohydroxamic acids (Doszczak and Rachon 2002a; Doszczak and Rachon 2002b). This method involves the synthesis of an S-thioacyldithiophosphate to be used as thioacylating agent. However, the production of the corresponding S-thioacyldithiophosphate was not possible (data not shown), most likely because of enolization and further self-thioacylation to produce a thioketene derivative, as has been previously observed for S-thioacyldithiophosphates that contain two α-substituents (Doszczak et al. 2003). Although the desired S-thioacyldithiophosphate was not α,α-disubstituted, the formation of the enolic tautomer might have been favored because of the conjugation of the enolic double bond to the aromatic ring.

References

Doszczak L, Kravtsov VC, Biernat JF, Rachon J (2003) Unexpected thioketene derivative formation during thioacyl dithiophosphate synthesis. Synthetic Commun 33:1797-1808

Doszczak L and Rachon J (2002a) S-thioacyldithiophosphates in the synthesis of thiohydroxamic acids and O-thioacylhydroxylamines. Synthesis 8:1047-1052

Doszczak L and Rachon J (2002b) Synthesis of S-thioacyl dithiophosphates, efficient and chemoselective thioacylating agents. J Chem Soc, Perkin Trans 1 10:1271-1279