Meiotic chromosome spreads in wild type, <i>spo11-1rec8</i>, <i>TDM1-P17</i> and <i>spo11-1rec8 TDM1-P17</i> plants.

<p>(A to D) Meiosis in wild type. (A) Five bivalents align at metaphase I and (B) pairs of homologous chromosome are distributed into two nuclei at telophase I. (C) Five pairs of sister chromatids align on the two metaphase plates. (D) Four balanced nuclei are formed at telophase II. (E to H) Meiosis in <i>spo11-1rec8</i>. The first division resembles a mitotic division with (E) alignment of 10 pairs of chromatids on the metaphase plates and (F) segregation into two groups of 10 chromatids. (G) Single chromatids fail to align properly at metaphase II, resulting in (H) a variable number of unbalanced nuclei at telophase II. (I to J) Meiosis in wild type plants transformed with <i>TDM1-P17</i>. A single, meiosis I-like division is observed. (K to L) Meiosis in <i>spo11-1 rec8</i> plants transformed with <i>TDM1-P17</i>. A single, mitotic-like division is observed. Scale bar = 10μM.</p

The <i>spo11-1rec8</i> (s)-40 mutant produces dyads and is tetraploid.

<p>(A to C). Male meiotic products stained by toluidine blue. (A) Wild type produces tetrads of spores. (B) <i>spo11-1 rec8</i> double mutant produces polyads (aberrant meiotic products with more than four spores). (C) <i>spo11-1 rec8 (s)-40</i> produces dyads of spores. (D to F) Mitotic karyotype. (D) Wild type is diploid, having ten chromosomes aligned on mitotic metaphase plates. (E) <i>spo11-1 rec8</i> is diploid. (F) The three <i>spo11-1 rec8</i> (s)-40 M1 plants were tetraploid, having 20 chromosomes aligned on mitotic metaphase plates. Scale bar = 10μM.</p

TDM1 localization.

<p>(A,B). GUS histochemical assays on plants transformed by <i>TDM1</i>:<i>GUS</i>. (A) The signal (blue) is detected in anthers of young buds, corresponding to the meiotic stage. (B) A close up of a group of anthers showing that the signal is detected in meiocytes. (C-L). Immunolocalization of TDM1::Myc (green). The DNA appears in red. TDM1::Myc is detected from mid-prophase to the tetrad stage. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005856#pgen.1005856.s005" target="_blank">S5 Fig</a> for specificity of the signal.</p

Summary of <i>TAM-TDM1-OSD1-SMG7</i> epistasis interactions.

<p>Summary of <i>TAM-TDM1-OSD1-SMG7</i> epistasis interactions.</p

Ploidy analysis of <i>spo11-1 rec8 TDM1-P17 and TDM1-P17</i> offspring.

<p>Ploidy analysis of <i>spo11-1 rec8 TDM1-P17 and TDM1-P17</i> offspring.</p

Tubulin immuno-localisation at meiosis <i>TAMΔD</i>, <i>TDM1-P17L</i> and <i>TAMΔD TDM1-P17L</i>.

<p>Tubulin appears in green and DNA in red. (A-D) <i>TAMΔD</i>. (A) Metaphase I. (B) Telophase I. (C) Metaphase II. (D) Metaphase III with the formation of four spindles. (E-F) <i>TDM1-P17L</i>. (E) Metaphase I. (F) Telophase I (G) Dyad of spores. No second division is observed. (H-K) <i>TAMΔD TDM1-P17L</i>. (H) Metaphase I. (I) Telophase I. (J) Aberrant stage with apparent uncoupling of spindle formation and chromosome condensation. (K) Aberrant stage with chromosomes scattered in the cell. No figures of meiosis II or meiosis III were observed. Scale bar = 10μM.</p

Analysis of the effect of expression of mutated <i>TDM1</i> in wild type and a series of mutants.

<p>For each experiment, the total number of analysed primary transformants is indicated. A minimum of 50 cells were observed per primary transformant.</p

TDM1 interacts with itself, CDC27a and CDC20.1.

<p>(A) Yeast two-hybrid. Interaction between the bait fused with the GAL4 DNA binding domain (pDEST32) and the pray fused with the GAL4 activation domain (pDEST22) is revealed by growth on media depleted of histidine (-his, weak interaction) or media depleted of both histidine and adenine (-His—Ade, strong interaction). Three replicates are shown for each test. (B to I) Bimolecular fluorescent complementation (BiFC). Interaction between two proteins is revealed by reconstituting the fluorescence of split YFP, in pavement cells that are shaped like the pieces of a jigsaw puzzle. (A) Positive control: YFP^N::DEF and YFP^C::GLO, two interacting components of the <i>Anthirrinum majus</i> MADS box transcription factors DEFICIENS and GLOBOSA. (B) YFP^N::TDM1 and YFP^C::TDM1. (C) YFP^N::TDM1 and YFP^C::TDM1. (D) YFP^N::TDM1 and YFP^C::CDC20.1. (E) YFP^N::DEF and YFP^C::CDC20.1. (F) YFP^N::DEF and YFP^C::TDM1. (G) YFP^N::TDM1 and YFP^C::GLO. (H) YFP^N::CDC20.1 and YFP^C::GLO. (I) YFP^N::DEF and YFP^C::CDC20.1. Scale bar = 100μm.</p

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

<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