Physcomitrella patens Auxin-Resistant Mutants Affect Conserved Elements of an Auxin- Signaling Pathway  Michael J. Prigge, Meirav Lavy, Neil W. Ashton, Mark Estelle  Current Biology  Volume 20, Issue 21, Pages 1907-1912 (November 2010) DOI: 10.1016/j.cub.2010.08.050 Copyright © 2010 Elsevier Ltd Terms and Conditions

Figure 1 Mutations in the Degron motifs of the PpIAA Genes Impair Auxin Response and Developmental Transitions (A) Diagram of the PpIAA proteins showing the positions of domains I, KR, II, III, and IV. The sequences of the degron motif of wild-type and mutant PpIAA proteins are shown. (B–F) Phenotypes of wild-type (pabB4) and ppiaa mutants. The left micrograph in each panel shows the differentiation of green chloronema cells (Ch in panel C) to less pigmented caulonema cells (Ca in panel B) occurs only in wild-type and the weak ppiaa2-250 after eight days of subculture. The photographs to the right in each panel show the phenotypes of wild-type and mutants after 1 month on BCD media (upper) and BCD media supplemented with 1 μM NAA (lower). (G–J) Phenotypes of two representative suppressor mutants with their respective parental strains after 33 days of growth. The black scale bar in (B) represents 300 μm, and the white scale bars in (B) and (J) represent 3 mm. See also Figure S1. Current Biology 2010 20, 1907-1912DOI: (10.1016/j.cub.2010.08.050) Copyright © 2010 Elsevier Ltd Terms and Conditions

Figure 2 Silencing of the PpAFB Genes Results in a Phenotype Similar to that of the ppiaa Mutants (A) NLS4 strain expressing a nuclear-localized GFP-GUS fusion protein. (B–E) Twelve-day-old NLS4 transformants grown on BCD media after tranformation with plasmids targetting the GFP-GUS fusion gene (B), all four PpAFB genes (C), both PpXFB genes (D), and the PpAFB and PpXFB genes (E). (F–G) Twelve-day-old NLS4 transformants grown on BCD media with 1 μM NAA after transformation with the plasmids targeting the GFP-GUS transgene (F) or the PpAFB genes (G). Each image is a merge of chlorophyll autofluorescence (red) and GFP fluorescence (green). The scale bar represents 300 μm. See also Figure S2. Current Biology 2010 20, 1907-1912DOI: (10.1016/j.cub.2010.08.050) Copyright © 2010 Elsevier Ltd Terms and Conditions

Figure 3 TIR1/AFB Proteins Interact with Aux/IAA Proteins in an Auxin-Dependant Manner (A–D) Yeast two-hybrid experiments with lacZ reporter grown for 36–48 hr. The interaction between the BD-AtTIR1 and AD-AtIAA7 fusion proteins was facilitated by the natural auxin IAA (A) and by the synthetic auxin NAA (B). (C) BD-fused TIR1/AFB proteins from both Arabidopsis and P. patens interacted with AD-fused Aux/IAA proteins from both Arabidopsis and P. patens in the presence of 50 μM IAA or 50 μM NAA. (D) Mutations corresponding to the strong ppiaa2 alleles abolished the interaction with PpAFB4, whereas the mutation corresponding to the weak ppiaa2-250 allele reduced the interaction. (E) In-vitro-translated TIR1/AFB proteins from both Arabidopsis and P. patens interacted with purified GST-AtIAA3 (top panels) and GST-PpIAA1B (lower panels). For both experiments, the Ponceau-S-stained GST-IAA protein is shown below the immunoblot for c-Myc-TIR1/AFB detection. Both experiments were carried out at least three times with similar results. + and – indicate the presence and absence, respectively, of 50 μM IAA. See also Figure S3. Current Biology 2010 20, 1907-1912DOI: (10.1016/j.cub.2010.08.050) Copyright © 2010 Elsevier Ltd Terms and Conditions

Figure 4 Expression of Auxin-Induced Genes Is Reduced in ppiaa Mutants qRT-PCR of six genes in protonemal tissue of the wild-type and four mutants treated with 0.06% DMSO (mock), 20 μM NAA, or 10 μM BAP for 13 hr. Each bar reflects the data from three independent RNA extractions with four technical replicates each. Expression of each target gene was normalized to PpACTIN expression and plotted relative to the untreated wild-type samples; error bars represent the SEM. See also Figure S4. Current Biology 2010 20, 1907-1912DOI: (10.1016/j.cub.2010.08.050) Copyright © 2010 Elsevier Ltd Terms and Conditions