Figure S5.
Analysis of TPL and TPL-interacting protein activity in Arabidopsis lateral root development. (A) Protein expression analysis by western blot for UAS-driven TPL-IAA14 constructs. For all samples, four 14-day grown root tissues were pooled from two representative T2 lines from Fig. 6 D. The middle panel is comprised of LisH mutants in the context of an intact H8, and the right panel is comprised of LisH mutants in the context of a mutant H8. Proteins were extracted and run by SDS-PAGE and western blotting for the HA-epitope tag. Equal volume was run on each lane. (B) Protein expression analysis by western blot for UAS-driven AtSPT4.1 constructs. For all samples, four 14-day grown root tissues were pooled from three representative T2 lines from Fig. 6 E. Proteins were extracted and run by SDS-PAGE and western blotting for the MYC-epitope tag. Equal volume was run on each lane. (C) Protein expression analysis by western blot for UAS-driven AtSPT5.2 constructs. For all samples, four 14-day grown root tissues were pooled from three representative T2 lines from Fig. 6 F. Proteins were extracted and run by SDS-PAGE and western blotting for the MYC-epitope tag. Equal volume was run on each lane. This construct was lowly expressed and was at the limits of detection. (D) Schematic of the integrase switch approach to generate a cell-type specific loss of function. In this approach a T-DNA in an essential gene that normally can only be carried as a heterozygote is rescued by a ubiquitously expressed version of the essential gene. This rescue transgene also carries Integrase binding sites (triangles) flanking the ubiquitous (pUBQ10) promoter. Upon expression of the integrase (PhiC31) from a separate transgene driven by a cell type–specific promoter (pGATA23), the PhiC31 will bind to these binding sites, catalyze an irreversible inversion of the promoter sequence, and therefore active a reporter gene that is facing in the opposite direction on the same strand of DNA (here this is a fluorescent protein reporter, FP). This generates loss of function mutations in the lateral root primordial cells, as cartooned in the seedling in the middle of the panel. We chose MED21, SPT6L and TAF5 as targets to destabilize TPL activity in lateral root primordium cells, to test the ability to modulate development of lateral roots initiation as diagrammed by the cartoon at the right. (E–G) Integrase switched essential lines demonstrate developmental root phenotypes. Scanned images of seedlings grown on plates for 14 days. (E) MED21 integrase switch lines exhibit short stubby roots that grow out from the primary root. (F and G) (F) SPT6L and (G) TAF5 integrase switch lines exhibit very short roots that do not exit the primary root and are not visible in plate scans. (E and F) Scale bars represent 10 mm. (H–K) Epifluorescence micrographs of roots of T2 plants from representative integrase switch lines. All images were taken 12 days after germination at 20× magnification. Arrowheads indicate initiating lateral roots based upon induction of pGATA23:PhiC31-triggered switching. (H) Wild-type mTurq to mScarlet control switch, mScarlet shown alone. (I)med21/med21, MED21:6xHA to mScarlet experimental switch, mScarlet shown alone. (J)taf5/taf5, RFP:TAF5 to Venus experimental switch, Venus shown alone. (K)spt6l/spt6l, SPT6L:GFP to mScarlet experimental switch, both channels are shown. For H–K, the same scale bar at top right applies to all images and represents 50 µm. Source data are available for this figure: SourceData FS5 Refer to the image caption for details.

Analysis of TPL and TPL-interacting protein activity in Arabidopsis lateral root development. (A) Protein expression analysis by western blot for UAS-driven TPL-IAA14 constructs. For all samples, four 14-day grown root tissues were pooled from two representative T2 lines from Fig. 6 D. The middle panel is comprised of LisH mutants in the context of an intact H8, and the right panel is comprised of LisH mutants in the context of a mutant H8. Proteins were extracted and run by SDS-PAGE and western blotting for the HA-epitope tag. Equal volume was run on each lane. (B) Protein expression analysis by western blot for UAS-driven AtSPT4.1 constructs. For all samples, four 14-day grown root tissues were pooled from three representative T2 lines from Fig. 6 E. Proteins were extracted and run by SDS-PAGE and western blotting for the MYC-epitope tag. Equal volume was run on each lane. (C) Protein expression analysis by western blot for UAS-driven AtSPT5.2 constructs. For all samples, four 14-day grown root tissues were pooled from three representative T2 lines from Fig. 6 F. Proteins were extracted and run by SDS-PAGE and western blotting for the MYC-epitope tag. Equal volume was run on each lane. This construct was lowly expressed and was at the limits of detection. (D) Schematic of the integrase switch approach to generate a cell-type specific loss of function. In this approach a T-DNA in an essential gene that normally can only be carried as a heterozygote is rescued by a ubiquitously expressed version of the essential gene. This rescue transgene also carries Integrase binding sites (triangles) flanking the ubiquitous (pUBQ10) promoter. Upon expression of the integrase (PhiC31) from a separate transgene driven by a cell type–specific promoter (pGATA23), the PhiC31 will bind to these binding sites, catalyze an irreversible inversion of the promoter sequence, and therefore active a reporter gene that is facing in the opposite direction on the same strand of DNA (here this is a fluorescent protein reporter, FP). This generates loss of function mutations in the lateral root primordial cells, as cartooned in the seedling in the middle of the panel. We chose MED21, SPT6L and TAF5 as targets to destabilize TPL activity in lateral root primordium cells, to test the ability to modulate development of lateral roots initiation as diagrammed by the cartoon at the right. (E–G) Integrase switched essential lines demonstrate developmental root phenotypes. Scanned images of seedlings grown on plates for 14 days. (E) MED21 integrase switch lines exhibit short stubby roots that grow out from the primary root. (F and G) (F) SPT6L and (G) TAF5 integrase switch lines exhibit very short roots that do not exit the primary root and are not visible in plate scans. (E and F) Scale bars represent 10 mm. (H–K) Epifluorescence micrographs of roots of T2 plants from representative integrase switch lines. All images were taken 12 days after germination at 20× magnification. Arrowheads indicate initiating lateral roots based upon induction of pGATA23:PhiC31-triggered switching. (H) Wild-type mTurq to mScarlet control switch, mScarlet shown alone. (I)med21/med21, MED21:6xHA to mScarlet experimental switch, mScarlet shown alone. (J)taf5/taf5, RFP:TAF5 to Venus experimental switch, Venus shown alone. (K)spt6l/spt6l, SPT6L:GFP to mScarlet experimental switch, both channels are shown. For H–K, the same scale bar at top right applies to all images and represents 50 µm. Source data are available for this figure: SourceData FS5

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