PCR amplification of the partial and full-length coding regions of NtMTHFRs and functional complementation of a yeast mthfr mutant by NtMTHFR1. A, A 300-bp fragment was amplified from tobacco cDNAs using degenerate primers targeting MTHFR. B, The NtMTHFR1 genomic DNA contains 11 exons (blue rectangles) and 10 introns (black solid lines). Regions used for the RNAi sense and antisense arms, as well as the spacer, are shown in orange and red, respectively. C, Full-length coding regions of MTHFRs amplified from cDNAs from W38, N. sylvestris (Ns), and N. tomentosiformis (Nto) using a pair of primers targeting the MTHFR. D, Functional complementation of a yeast mthfr mutant by NtMTHFR1. Yeast strains DAY4 (wild type; 1), RRY3 (null mutant met12met13; 2), RRY3 with the pVT103-U vector alone (3), and RRY3 with pVT103-U expressing NtMTHFR1 (4) were grown in minimal medium in the presence (+) or absence (−) of Met. [See online article for color version of this figure.]