Supplemental Figure Captions
Figure S1. Schematic drawings of the conceivable siRNA signal spread into a flower and
the effect of DMC1 silencing on meiosis. (a) Drawing of a closed flower indicating the
allocation of DMC1 silencing signal (red arrows). Grey: wild type; blue: DMC1 hpRNAi
plants producing DMC1 siRNA; green: scion tissue not effected. (b) Scheme of the
potential effects of DMC1 silencing on meiosis and tetrad formation redrawn after
(McGarry and Kragler 2013).
Supplemental Figure S2. (a) Schematic drawing and sequence alignment of the used
inverted repeat hairpin construct producing DMC1 siRNA in Nicotiana tabacum and
Arabidopsis thaliana. The Brassica carinata DMC1 (GenBank: DM473019.1) and the
Arabidopsis thaliana DMC1 cDNA (TAIR: At3g22880) cDNA sequences were used to
express via a CaMV 35S promoter an inverted repeat transcript producing siRNA
targeting DMC1 in Nicotiana tabacum and Arabidopsis thaliana, respectively. BLAST
alignment of the used Brassica carinata cDNA stretch and the Nictotiana tabacum
DMC1 CDS (NtDMC1) sequences shows that they have identical nucleotide regions
longer than 21 nucleotides (red) and two regions with only one nucleotide difference
within 21 nucleotides (blue). (b) Seed production in the first and third flowers on nongrafted and grafted Nicotiana tabacum plants crossed with wild-type pollen. A significant
difference in seed production of grafted plants and rescue of seed production after
fertilization (blue columns) was observed. This suggests that the root-stock produced
DMC1 silencing signal preferably penetrates anthers formed in the first flowers of an
inflorescence. Error bars: Standard deviation of the means.
Supplemental Figure S3. NtDMC1 RT-PCR of Nicotiana tabacum transgenic lines and
wild-type plants, and CLSM images of pollen samples from grafted plants. (a) Semiquantitative RT-PCR of DMC1 transcripts present in transgenic lines and wild type.
Specific Nicotiana tabacum DMC1 RT-PCR products were detected in total RNA
isolated from mature wild-type Nicotiana tabacum leaves. Note that only a faint PCR
band was detected in RNA isolated from leaves in Brassica carinata DMC1 hpRNAi
lines. RNase control shows that the amplified PCR product did not result from genomic
DNA contamination. (b) CLSM microscopy images of propidium iodide stained pollen
samples used in the FACS analysis. Red: stained pollen coat and nuclei. vN: vegetative
nuclei; gN: generative nuclei. Arrowheads indicate misshaped pollen.
Figure S4. Additional CLSM images of YFP fluorescence in sectioned floral tissues
formed on grafted 35S:YFP-∆N DMC1 Nicotiana tabacum plants. Green: YFP
fluorescence and auto-fluorescence of developing spores within anthers. Blue:
chloroplast/plastid auto-fluorescence. Arrows indicate cells showing no or weak decrease
of YFP fluorescence. Arrowheads indicate tissues and cells with decreased or no
detectable YFP fluorescence.
Figure S5. Additional CLSM images of erGFP fluorescence present in leaves and in
sectioned floral tissues formed on agro-infiltrated Nicotiana benthamiana plants.
Indicated 35S driven mock control (RFP) or invGFP constructs were expressed in agroinfiltrated sources leaves. Green: GFP fluorescence and weak auto-fluorescence of
forming spores within anthers. Blue: chloroplast/plastid auto-fluorescence. Arrowheads
indicate lack or decreased and arrows indicate unchanged presences of erGFP
fluorescence in specified tissues.
Figure S6. Binocular and CLSM images of YFP-AtDMC1 fluorescence detected in
Arabidopsis thaliana tissues 35S:YFP-AtDMC1 plants grafted on AtDMC1 hpRNAi rootstock plants. Green: YFP-AtDMC1 fluorescence. Blue: chloroplast/plastid autofluorescence. Arrowheads indicate lack or decreased and arrows indicate unchanged
presences of YFP fluorescence in specified plant tissues.
Figure S7. Image of 35S:AtDMC1 hpRNAi, 35S:YFP-AtDMC1 and grafted transgenic
plants showing empty siliques. The used AtDMC1 silencing line is nearly sterile
(arrowheads indicate first sterile flowers) whereas transgenic 35S:YFP-DMC1 plants
grown under the same conditions produce siliques producing normal seed numbers.
Grafted plants supported by 35S:AtDMC1 hpRNAi root-stocks show aberrant seed
production (arrowheads) suggesting spread of the DMC1 siRNA signal into sporogenic
tissues. Note that within an inflorescence of the grafted plants the first 3 to 4 flowers are
often less impaired in their seed production (arrows).
Figure S8. Additional CLSM images showing the distribution of symplastic-loaded
fluorescein and HTPS in flower tissues of Arabidopsis thaliana and Nicotiana tabacum.
Upper panels: Additional images showing dye loading into petioles of Arabidopsis
thaliana and CLSM images suggesting the unloading in flowers/anthers and presence of
HPTS in vacuoles of cells in cauline leaves. Middle panel: Presence of fluorescein in
DAPI stained (red) cross-sectioned flower tissue (arrowheads) after 24 h of dye loading.
Lower panel. Z-stack merged images (fluorescein loaded and control) of the crosssectioned Nicotiana tabacum flower presented in Fig. 9B. Green: fluorescein/HPTS;
Blue: chloroplastic/plastid auto-fluorescence; Red: DAPI stained nuclei.
Figure S9. Pollen shape and size features of the training datasets and an example
showing image processing and SVM classification for pollen from wild-type and
Brassica carinata DMC1 hpRNAi transgenic (for details of the analysis see supplemental
methods).