Figure S3.
Molecular characterization of the tagged CSLD loci. (A) Diagram illustrates the result of HDR mediated insertion of mScarlet-i (red) sequence in a generic CSLD genomic locus. The position of the protospacer (PS) sequences is indicated with an orange arrow. The dashed vertical lines indicate the junction between the knock-in construct and upstream and downstream genomic sequences. Small arrows above the diagrams represent primers used for genotyping. PCR products obtained with the indicated primer pairs are shown below the diagram. Expected sizes for wild type (WT) and edited loci are shown for each CSLD locus. Molecular weight is indicated in kb. (B) Diagram illustrates the result of HDR mediated insertion of mEGFP (green) sequences in the CSLD6 genomic locus. Coding exons are indicated by thick boxes and untranslated exons are indicated by thin boxes. Thin lines indicate intronic regions. The dashed vertical lines indicate the junction between the knock-in construct and upstream and downstream genomic sequences. Small arrows above the diagrams represent primers used for genotyping. Scale bar is 0.5 kb. PCR products obtained with the indicated primer pairs are shown below the diagram. Predicted sizes for correct products are indicated below each gel. Molecular weight is indicated in kb. (C) Gametophore phyllids form normally in 3–4-wk-old plants regenerated from ground tissue, with no phyllid patterning defects visible as seen in csld2/6KO plants (see Fig. 2, G and H), demonstrating that the tagged CSLD6 is functional. Scale bar, 500 µm. (D) Similar to mScarlet-CSLD6, mEGFP-CSLD6 is enriched in cytosolic punctae and at the apical plasma membrane of tip growing cells. Images are from a time lapse acquisition of confocal images of the medial plane of a growing protonemal cell. Time is indicated by min:sec. Yellow and orange dotted lines indicate the shape of the cell at 00:00, and 05:50 times, respectively. Scale bar, 10 µm. (E) Maximum projection of a confocal Z-stack of a dividing protonemal cell shows that mEGFP-CSLD6 accumulates at the cell division plane. Large globular structures are chloroplasts which are more concentrated near the division plane and auto-fluoresce in the GFP channel. Source data are available for this figure: SourceData FS3.

Molecular characterization of the tagged CSLD loci. (A) Diagram illustrates the result of HDR mediated insertion of mScarlet-i (red) sequence in a generic CSLD genomic locus. The position of the protospacer (PS) sequences is indicated with an orange arrow. The dashed vertical lines indicate the junction between the knock-in construct and upstream and downstream genomic sequences. Small arrows above the diagrams represent primers used for genotyping. PCR products obtained with the indicated primer pairs are shown below the diagram. Expected sizes for wild type (WT) and edited loci are shown for each CSLD locus. Molecular weight is indicated in kb. (B) Diagram illustrates the result of HDR mediated insertion of mEGFP (green) sequences in the CSLD6 genomic locus. Coding exons are indicated by thick boxes and untranslated exons are indicated by thin boxes. Thin lines indicate intronic regions. The dashed vertical lines indicate the junction between the knock-in construct and upstream and downstream genomic sequences. Small arrows above the diagrams represent primers used for genotyping. Scale bar is 0.5 kb. PCR products obtained with the indicated primer pairs are shown below the diagram. Predicted sizes for correct products are indicated below each gel. Molecular weight is indicated in kb. (C) Gametophore phyllids form normally in 3–4-wk-old plants regenerated from ground tissue, with no phyllid patterning defects visible as seen in csld2/6KO plants (see Fig. 2, G and H), demonstrating that the tagged CSLD6 is functional. Scale bar, 500 µm. (D) Similar to mScarlet-CSLD6, mEGFP-CSLD6 is enriched in cytosolic punctae and at the apical plasma membrane of tip growing cells. Images are from a time lapse acquisition of confocal images of the medial plane of a growing protonemal cell. Time is indicated by min:sec. Yellow and orange dotted lines indicate the shape of the cell at 00:00, and 05:50 times, respectively. Scale bar, 10 µm. (E) Maximum projection of a confocal Z-stack of a dividing protonemal cell shows that mEGFP-CSLD6 accumulates at the cell division plane. Large globular structures are chloroplasts which are more concentrated near the division plane and auto-fluoresce in the GFP channel. Source data are available for this figure: SourceData FS3.

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