Genetics of rheumatoid arthritis contributes to biology and drug discovery

A major challenge in human genetics is to devise a systematic strategy to integrate disease-associated variants with diverse genomic and biological datasets to provide insight into disease pathogenesis and guide drug discovery for complex traits such as rheumatoid arthritis (RA)1. Here, we performed a genome-wide association study (GWAS) meta-analysis in a total of >100,000 subjects of European and Asian ancestries (29,880 RA cases and 73,758 controls), by evaluating ~10 million single nucleotide polymorphisms (SNPs). We discovered 42 novel RA risk loci at a genome-wide level of significance, bringing the total to 1012–4. We devised an in-silico pipeline using established bioinformatics methods based on functional annotation5, cis-acting expression quantitative trait loci (cis-eQTL)6, and pathway analyses7–9 – as well as novel methods based on genetic overlap with human primary immunodeficiency (PID), hematological cancer somatic mutations and knock-out mouse phenotypes – to identify 98 biological candidate genes at these 101 risk loci. We demonstrate that these genes are the targets of approved therapies for RA, and further suggest that drugs approved for other indications may be repurposed for the treatment of RA. Together, this comprehensive genetic study sheds light on fundamental genes, pathways and cell types that contribute to RA pathogenesis, and provides empirical evidence that the genetics of RA can provide important information for drug discovery

Isolation of Natural Fungal Pathogens from Marchantia polymorpha Reveals Antagonism between Salicylic Acid and Jasmonate during Liverwort-Fungus Interactions

The evolution of adaptive interactions with beneficial, neutral and detrimental microbes was one of the key features enabling plant terrestrialization. Extensive studies have revealed conserved and unique molecular mechanisms underlying plant–microbe interactions across different plant species; however, most insights gleaned to date have been limited to seed plants. The liverwort Marchantia polymorpha, a descendant of early diverging land plants, is gaining in popularity as an advantageous model system to understand land plant evolution. However, studying evolutionary molecular plant–microbe interactions in this model is hampered by the small number of pathogens known to infect M. polymorpha. Here, we describe four pathogenic fungal strains, Irpex lacteus Marchantia-infectious (MI)1, Phaeophlebiopsis peniophoroides MI2, Bjerkandera adusta MI3 and B. adusta MI4, isolated from diseased M. polymorpha. We demonstrate that salicylic acid (SA) treatment of M. polymorpha promotes infection of the I. lacteus MI1 that is likely to adopt a necrotrophic lifestyle, while this effect is suppressed by co-treatment with the bioactive jasmonate in M. polymorpha, dinor-cis-12-oxo-phytodienoic acid (dn-OPDA), suggesting that antagonistic interactions between SA and oxylipin pathways during plant–fungus interactions are ancient and were established already in liverworts.The Max-Planck-Gesellschaft to H.N.; Japan Society for the Promotion of Science KAKENHI [17K07665 to S.K.]; and Spanish Ministry for Science and Innovation grant [BIO2016-77216-R (MINECO/FEDER) to R.S.]

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

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 PLANT SMY2-TYPE ILE-GYF DOMAIN-CONTAINING PROTEIN 1 (PSIG1) and show that PSIG1 helps to restrict cell death induction during pathogen infection. Inactivation of PSIG1 does not result in spontaneous lesions, and enhanced cell death in psig1 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 smg7-4 mutant allele mimics the cell death phenotype of the psig1 mutants. Intriguingly, the psig1 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

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

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

<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

PTI responses in the <i>psig1</i> mutants.

<p><b>a</b>, Flg22-induced ROS production in the <i>psig1</i> mutants. Data are shown as the mean ± SE. <b>b</b>, Flg22-induced MAPK activation in the <i>psig1</i> mutants. <b>c</b>, Flg22-induced callose deposition in the <i>psig1</i> mutants. Callose deposition was quantified with Image J software. 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). The scale bar represents 200 μm. <b>d</b>, The <i>psig1-1</i> mutant has a slight dwarf phenotype. Photograph of 6-week-old plants grown under short day conditions. <b>e</b>, <i>PR1</i> gene expression in 10-day-old seedlings. 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> < 0.01). <b>f</b>, Flg22-induced ROS production in the <i>psig1-1 sid2-2</i> mutants. Data are shown as the mean ± SE. <b>g</b>, Flg22-induced ROS production in the <i>psig1-1 rbohD</i> mutants. Data are shown as the mean ± SE.</p