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

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

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

<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

Additional file 7: of Quantitative evaluation of protocorm growth and fungal colonization in Bletilla striata (Orchidaceae) reveals less-productive symbiosis with a non-native symbiotic fungus

Quantitative evaluation of symbiotic cells in a protocorm under conditions with high concentrations of oatmeal. (a) The number of symbiotic cells per one protocorm under 2×- and 4×-strength oatmeal conditions for four weeks after seeding. (b) Ratio of the number of symbiotic cells at each stage in a symbiotic protocorm under 2×- and 4×-strength oatmeal conditions. Error bars represents the standard error of the mean in ten protocorms. The experiments were repeated three times with similar results. (PDF 140 kb

The GYF domain protein PSIG1 dampens the induction of cell death during plant-pathogen interactions

<div><p>The induction of rapid cell death is an effective strategy for plants to restrict biotrophic and hemi-biotrophic pathogens at the infection site. However, activation of cell death comes at a high cost, as dead cells will no longer be available for defense responses nor general metabolic processes. In addition, necrotrophic pathogens that thrive on dead tissue, take advantage of cell death-triggering mechanisms. Mechanisms by which plants solve this conundrum remain described. Here, we identify <i>PLANT SMY2-TYPE ILE-GYF DOMAIN-CONTAINING PROTEIN 1 (PSIG1)</i> and show that <i>PSIG1</i> helps to restrict cell death induction during pathogen infection. Inactivation of PSIG1 does not result in spontaneous lesions, and enhanced cell death in <i>psig1</i> mutants is independent of salicylic acid (SA) biosynthesis or reactive oxygen species (ROS) production. Moreover, PSIG1 interacts with SMG7, which plays a role in nonsense-mediated RNA decay (NMD), and the <i>smg7-4</i> mutant allele mimics the cell death phenotype of the <i>psig1</i> mutants. Intriguingly, the <i>psig1</i> mutants display enhanced susceptibility to the hemi-biotrophic bacterial pathogen. These findings point to the existence and importance of the SA- and ROS-independent cell death constraining mechanism as a part of the plant immune system.</p></div

The GYF domain is required for the cell death but not growth regulation.

<p><b>a</b>, Expression of <i>PSIG1</i>^<i>Y575A</i> complements the <i>psig1-1</i> growth phenotype. Photograph of 5-week-old plants grown under long day conditions (12 h light/ 12 h dark). <b>b</b>, <i>PSIG1</i> gene expression in 5-week-old plants. Data are shown as the mean ± SE. Statistical groups were determined using the Tukey HSD test. Statistically significant differences are indicated by different letters (<i>p</i> < 0.05). <b>c</b>, Plants were spray inoculated with 1 x 10⁸ c.f.u. ml^-1 of <i>Pto AvrRPS4</i> under long day condition (12 h light/ 12 h dark), and dead cells were visualized by trypan blue staining 1 day after inoculation. The scale bar represents 200 μm. <b>d</b>, Trypan blue stained area. Plants were spray inoculated with 1 x 10⁸ c.f.u. ml^-1 of <i>Pto AvrRPS4</i>, and dead cells were visualized by trypan blue staining 1 day after inoculation. The stained area was measured using an imaging software. Two leaves were taken from each of 4 individual plants. The box plot indicates the area of trypan blue stained cells. Boxes show upper and lower quartiles of the data, and black lines represent the medians. Statistical groups were determined using the Tukey HSD test. Statistically significant differences are indicated by different letters (<i>p</i> < 0.05).</p

<i>PSIG1</i> is required for flg22-induced cell death suppression.

<p><b>a</b>, Phosphoregulation of PSIG1 in the PAMP-signaling mutants. Relative abundance of the ‘DIQGSDNAIPLpSPQWLLSKPGENK’ phosphopeptide upon flg22 treatment. Arabidopsis seedlings were treated with 1 μM flg22 for 10 min or received a mock treatment (dH₂O) prior to phosphoproteome analysis. Data are shown as the mean ± SD from three independent experiments. <b>b</b>, The <i>bak1-5</i> and <i>bik1 pbl1</i> mutants induce cell death upon <i>Hpa</i> Noco2 infection. Plants were inoculated with spores of <i>Hpa</i> Noco2, and dead cells on true leaves were visualized by trypan blue staining 5 days after inoculation. The scale bar represents 200 μm. <b>c</b>, Induction of RPS4-triggered cell death is pronounced in the <i>bak1-5</i> and <i>bik1 pbl1</i> mutants. Plants were spray inoculated with 1 x 10⁸ c.f.u. ml^-1 of <i>Pto AvrRPS4</i>, and dead cells were visualized by trypan blue staining 2 days after inoculation. The scale bar represents 200 μm. <b>d</b>, Flg22-induced restriction of effector injection by <i>Pto</i> is intact in the <i>psig1-1</i> mutant. Leaves were infiltrated with 100 nM flg22 or received a mock treatment (dH₂O). Twenty-four h after the pretreatments, plants were spray inoculated with 1 x 10⁸ c.f.u. ml^-1 of <i>Pto AvrRPM1</i>, and dead cells were visualized by trypan blue staining 24 h after inoculation. The scale bar represents 200 μm. <b>e</b>, Suppression of flg22-induced FB1-triggered cell death is compromised in the <i>psig1-1</i> mutant. Leaves were infiltrated with FB1 after mock (dH₂O) or flg22 pretreatments. Control leaves were infiltrated with dH₂O (mock) after mock (dH₂O) or flg22 pretreatments. Photographs were taken 4 days after FB1 infiltration. Dead cells were visualized by trypan blue staining. The scale bar represents 200μm.</p

GYF domain proteins.

<p><b>a</b>, Schematic structure and the phosphorylation site of PSIG1. Ser-39 was found to be the phosphorylation site. The red box indicates the GYF domain. <b>b</b>, Relative abundance of the ‘DIQGSDNAIPLpSPQWLLSKPGENK’ phosphopeptide upon flg22 treatment. Arabidopsis seedlings were treated with 1 μM flg22 or received a mock treatment (dH₂O) prior to phosphoproteome analysis. Data are shown as the mean ± SD from three independent experiments. <b>c</b>, Aligned amino acid sequences of the GYF domains from diverse eukaryotic species. Key residues for the GYF domain are delineated as white text on a black background. At, Os, Smo, Phpat, Cre, Kfl, Hs and Sc stand for following species: <i>Arabidopsis thaliana</i>, <i>Oryza sativa</i>, <i>Selaginella moellendorffii</i>, <i>Physcomitrella patens</i>, <i>Chlamydomonas reinhardtii</i>, <i>Klebsormidium flaccidum</i>, <i>Homo sapiens</i> and <i>Saccharomyces cerevisiae</i>, respectively. <b>d</b>, Phylogenetic tree and schematic structures of GYF-domain proteins from diverse eukaryotes. Species abbreviations are defined in Fig 1C. Numbers on the phylogenetic tree indicate the bootstrap values. Red boxes indicate the GYF domain.</p

<i>PSIG1</i> negatively regulates the induction of cell death during pathogen infection.

<p><b>a</b> and <b>c</b>, Induction of RPS4-triggered cell death was pronounced in the <i>psig1-1</i> mutant in an SA and ROS-independent manner. Plants were spray inoculated with 1 x 10⁸ c.f.u. ml^-1 of <i>Pto AvrRPS4</i>, and dead cells were visualized by trypan blue staining 2 days after inoculation. The scale bar represents 200 μm. <b>b</b> and <b>d</b>, Trypan blue stained area. Plants were spray inoculated with 1 x 10⁸ c.f.u. ml^-1 of <i>Pto AvrRPS4</i>, and dead cells were visualized by trypan blue staining 2 days after inoculation. The stained area was measured using an imaging software. Two to 3 leaves were taken from each of at least 5 individual plants for <b>b</b>. Three leaves were taken from each of 3 individual plants for <b>d</b>. The box plot indicates the area of trypan blue stained cells. Boxes show upper and lower quartiles of the data, and black lines represent the medians. Statistical groups were determined using the Tukey HSD test. Statistically significant differences are indicated by different letters (<i>p</i> < 0.05). <b>e</b>, The <i>psig1-1</i> mutant induces cell death upon <i>Hpa</i> Noco2 infection. Plants were inoculated with spores of <i>Hpa</i> Noco2, and dead cells on true leaves were visualized by trypan blue staining 5 days after inoculation. White arrowheads indicate infection hyphae of <i>Hpa</i> Noco2 and red arrowheads indicate dead cells. Scale bars in the upper and lower panels indicate 200 μm and 100 μm, respectively.</p