Subcellular localization of IAA7, IAA8, and IAA17.

<p>GFP fusions of IAA7, IAA8, and IAA17 were transiently expressed in <i>Arabidopsis</i> mesophyll protoplasts. NLS-tdTomato was co-introduced both as a nuclear marker and as a control for transformation. GFP, NLS, and BF (top) represent GFP and tdTomato fluorescence and bright field images, respectively. n: nucleus, c: cytosol. Bars  = 10 μm.</p

Auxin-dependent interaction of IAA8 with the TIR1 auxin receptor.

<p>(A) Yeast (EGY48::pJK103) cells were co-transformed with bait plasmid, which contained a LexA DNA-binding domain (BD)-TIR1 fusion (BD-TIR1) in pEG202, and prey plasmid, which contained activation domain (AD)-Aux/IAA fusions (AD-Aux/IAA) in pJG4-5. Auxin-dependent interactions in each transformant were assayed by plating on SD(Gal)/-Ura/-His/-Trp/-Leu medium with 100 μM IAA (+IAA). Auxin-independent interactions were examined using the same medium without IAA (−IAA). (B) The BiFC assay was used to detect protein-protein interactions between Aux/IAA repressors and TIR1. <i>nYFP-Aux/IAA</i> fusions or <i>nYFP-GUS</i> and <i>cYFP-TIR1</i> were transiently co-expressed in <i>Arabidopsis</i> mesophyll protoplasts with a nucleus marker plasmid, NLS-tdTomato. To monitor the auxin-dependent interaction, 10 μM IAA was added before microscopic observation (+IAA). BiFC, NLS and BF (top) represent YFP, tdTomato fluorescence, and bright field images, respectively. n: nucleus. Bars  = 10 μm.</p

Detection of the interaction between IAA8 and ARFs by yeast two-hybrid and BiFC assays.

<p>(A) Yeast (EGY48::pJK103) cells were co-transformed with BD-IAA8 bait plasmid and AD-ARFCTD prey plasmid as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043414#pone-0043414-g004" target="_blank">Figure 4</a>. The strength of each interaction was evaluated by measuring β-gal activity using the o-nitrophenyl β-D-galactopyranoside (ONPG) method. Vector indicates empty vector (negative control experiment). (B) A BiFC assay was carried out to monitor the protein-protein interaction between IAA8 and ARFs. <i>ARF-nYFP</i> fusions or <i>nYFP-GUS</i> and <i>IAA8-cYFP</i> were transiently co-expressed in <i>Arabidopsis</i> mesophyll protoplasts with a nuclear marker plasmid, NLS-tdTomato. BiFC, NLS, and BF (top) represent YFP, tdTomato fluorescence, and bright field images, respectively. n: nucleus. Bars  = 10 μm.</p

<i>IAA8</i> overexpression suppresses the auxin-inducible expression of early auxin-responsive genes in roots.

<p>Wild-type (Col-0) and XVE::IAA8 (line #8, #11) roots treated first with 10 μM β-estradiol for 24 h and then with 1 μM IAA for 0 to 60 min were subjected to semi-quantitative RT-PCR analysis of the auxin marker genes, <i>IAA1</i>, <i>IAA19</i> and <i>GH3.3</i> together with endogenous <i>IAA8</i> gene expression (end) and of both endogenous and exogenous (end + exo) <i>IAA8</i> gene expression. EF1α was used as an internal control. All semi-quantitative RT-PCR figures are representative of three biological replicates.</p