Figure 6. The TPL CRA repression domain behaves similarly in yeast and plants.

(A) Bimolecular fluorescence complementation assay performed in tobacco. Each image is an epi-fluorescent micrograph taken at identical magnification from tobacco epidermal cells at 2 days post injection. The YFP image is colored green (left panel). p35S:H2B-RFP was used as a control and is false-colored magenta (right panel). (B) Co-immunoprecipitation of MED21 and TPL from tobacco leaves. MED21-YFP-HA was immunoprecipitated using anti-HA, and YFP-TPL was detected using the YFP fusion. Actin was used to demonstrate that the purification had removed non-specific proteins. Numbers on the left of blots indicate sizes of protein standards in kilodaltons. (C) Design of UAS-TPL-IAA14^mED and UAS-MED21 constructs. Mutation of the conserved lysine residues in the EAR domain disrupted potential interactions with endogenous TPL/TPR proteins. The IAA14 degron has been mutated (P306S) to render it auxin insensitive. UAS: upstream activating sequence; ttRBCS: Rubisco terminator sequence. (D) Auxin-induced degradation of IAA14 is absolutely required for initiation of lateral root development (cartoon, left). An enhancer trap line (J0121) expresses GAL4-VP16 and UAS-GFP in in xylem pole pericycle cells. (E) N-terminal domains of TPL were sufficient to repress the development of lateral roots in Arabidopsis seedlings. The density of emerged lateral roots was measured in T1 seedlings at 14 days after germination. (F) N-terminal deletions in AtMED21 were sufficient to dominantly increase the development of lateral roots in Arabidopsis seedlings. The density of emerged lateral roots was measured in T1 seedlings at 14 days after germination. (E, F) Lowercase letters indicate significant difference (ANOVA and Tukey HSD multiple comparison test; p<0.001).

Figure 6—figure supplement 1. The TPL-MED21 interaction is required for repression in plants.

(A) MED21 and TPL co-immunoprecipitated with AtMED10B from tobacco extracts. Each construct was expressed under the viral 35S promoter, and tissues were harvested after 2 days of injection. MED10B was purified by incubation with IgG sepharose beads (see Materials and methods), and the presence of interacting proteins was determined by western blotting. (B, C) Engineering and prototyping a variant of TPLN-IAA14^mED, which carries mutations in the EAR domain (EAR^AAA) and in the degron (P306S) in yeast. (B) Cartoon schematic of the mutations tested during prototyping of the TPLN188-IAA14mED construct. In each case, the identical glycine-serine linker (GS) was used as the flexible linker between the 2xHA-TPLN188 protein and the portion of IAA14 retained in the construct. (C) Time-course flow cytometry of TPLN-IAA14^mED strains following auxin addition. Strains containing the TPLN-IAA14^mEDwere tested in both haploid and diploid strains and demonstrated similar repression profiles. Every point represents the average fluorescence of 5–10,000 individually measured yeast cells (a.u.: arbitrary units). Auxin (IAA-10 µM) was added at the indicated time (gray bar, +Aux). Two independent experiments are shown for each construct. (D) Transient expression of indicated TPL constructs in tobacco. DR5:Venus: the synthetic DR5 auxin promoter (Ulmasov et al., 1997) driving Venus; ARF19: p35S:AtARF19-1xFLAG; GAL4:VP16: pUBQ10:GAL4-VP16, TPLN-X-UAS-TPL-IAA14^mED with various TPL truncations or mutations. (E, F) The TPL-N terminus functions as a repressor independently of the MED13 component of the Mediator CDK8 kinase module in Arabidopsis. The GAL4:UAS-driven dominant lateral root repression constructs (UAS-TPLN188-IAA14, UAS-Monomer-IAA14) and the dominant slr mutant were crossed to the med13 mutant (gct-5, a.k.a. mab2-2, SAIL_1169 H11). We performed time-course lateral root density assays and genotyped the progeny in the F1 and F2 generations. We observed an ~1% transmission rate of gct-5 homozygotes. (E) Time course of the mean of lateral root density over days 10–14 post germination ± standard error. (F) Boxplots of lateral root density for day 14 (E). Lowercase letters indicate significant difference (ANOVA and Tukey HSD multiple comparison test; p<0.001).

Figure 6—figure supplement 2. The essential gene MED21 is required for normal lateral root development in plants.

(A) Identification and characterization of a novel CAS9-based insertional mutation in MED21. The MED21 genomic locus (AT4G04780) is shown as a cartoon, with a zoom-in on the beginning of the coding sequence highlighted with the amino acid sequence. The location of the med21/MED21 mutant (WiscDsLox461-464K13, see triangle) and the sgRNA we employed (see green annotation and NGG PAM site) is highlighted. The insertion of a G at nucleotide position +214 after the transcriptional start site abrogates the sgRNA site (red annotation above with i214G). A representative sequencing trace demonstrates the position where the heterozygote carries i215G, and the predicted effect to the coding sequence is shown at the top right – a red arrow indicates the first codon affected by the i214G mutation. The inset pictures at the top left demonstrate the embryo lethality phenotype in med21^i214G/MED21 heterozygote siliques. White asterisks indicate the embryos that have begun to degenerate. These aborted seeds are visibly brown, indicating that fertilization took place allowing the seed coat to form before development failed. (B) Med21/MED21 heterozygotes are haplo-sufficient for lateral root development. Lateral root density (number of lateral roots/primary root length) was calculated at 10 days post germination. Lowercase letters indicate significant difference (ANOVA and Tukey HSD multiple comparison test; p<0.005). (C) MED21-CAS9 repressors targeted to MED21 display increased lateral root densities. Lateral root density (number of lateral roots/primary root length) was calculated at 10 days post germination. Numbers below boxplots are p-values for pairwise comparisons with control using a Wilcoxon rank-sum test. (D) Ratio of lateral root lengths to total root lengths (lateral root lengths + primary root length) in dCAS9 repressor lines targeting MED21 calculated at 10 days post germination. Statistical tests (ANOVA and Wilcox test) are reported above the graph. (E) Representative root traces of dCAS9 repressor lines targeting MED21 calculated at 10 days post germination.