DEV035048 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Verification of the proper integration of PpFIE constructs at 5′ and 3′ genomic loci, as detected by PCR analysis. ΔPpFIE (A,D). Gene replacement of PpFIE with AtFIE (B,E) and PpFIE-GUS fusion (C,F). The arrows indicate primers used to detect the integration of the recombinant DNA. uidA, uidA coding region; nos-ter, nopaline synthase polyadenylation signal; nptII, nptII expression cassette. 5′ and 3′ indicate the site of integration at the respective genomic loci.

• Supplemental Figure S2 -

  Fig. S2. Expression analysis of ΔPpFIE and AtFIE-co lines. (A) RT-PCR analysis of PpFIE expression in wild-type and ΔPpFIE lines. (B) PpFIE and AtFIE expression in the wild type and AtFIE-co lines. rRNA was used as a control for equal amounts of templates in all experiments.

• Supplemental Figure S3 -

  Fig. S3. Verification of FIE/fie,PpFIE/∼ genotype and transgene expression by genomic PCR and RT-PCR. (A) AtFIE expression in the wild type and in FIE/fie,PpFIE/∼. (B) PpFIE expression in FIE/fie,PpFIE/∼. (C) Detection of the fie allele by RFLP analysis. In panels A and B the RNA was purified from mature siliques.

• Supplemental Figure S4 -

  Fig. S4. Transmission of the fie mutant allele via the female gametophyte in the presence of the PpFIE transgene. (A) Detection of the fie allele by RFLP analysis. The DraI restriction site marks the mutant allele, whereas the amplified product from the wild-type allele remains uncut. (B) Genomic PCR amplifying the PpFIE transgene. (C-E) Siliques obtained by self pollination of FIE/fie,PpFIE/∼ female (C), wild-type (D) and F1 (E) plants. F1 plants resulted from a cross between FIE/fie,PpFIE/∼ as female and wild-type parents. Brown seeds contain aborted embryos and green seeds bear viable embryos.

• Supplemental Figure S5 -

  Fig. S5. Alignment of the SET domain. At, Arabidopsis thaliana; Pp, Physcomitrella patens; Dm, Drosophila melanogaster; Hs, Homo sapiens; V, Paramecium bursaria chlorella virus. The SET domain active site is marked with *, cofactor binding site is marked with ** and histone multiplicity site marked with *** (Qian et al., 2006).

  Qian, C., Wang, X., Manzur, K., Sachchidanand Farooq, A., Zeng, L., Wang, R. and Zhou, M. M. (2006). Structural insights of the specificity and catalysis of a viral histone H3 lysine 27 methyltransferase. J. Mol. Biol. 359, 86-96.

• Supplemental Figure S6 -

  Fig. S6. Genomic structure of the ΔPpFIE mutant and confirmation of targeting by Southern analyses. Upper panel, Southern analyses for the ΔPpFIE loci in three independent lines. Genomic DNA from lines 1 to 3 were digested with EcoRI and hybridized with a labeled nptII probe. A separate probe was used to detect the Lambda DNA size marker, digested with BstEII. A single insert was detected at the expected size in all lines. Lower panel, schematic illustration of the ΔPpFIE genomic locus. The expected fragment of 7914 bp is released upon digestion with EcoRI bearing the disrupting cassette carrying the nptII gene.