Targeted Proteomics Analysis of Protein Degradation in Plant Signaling on an LTQ-Orbitrap Mass Spectrometer

Targeted proteomics has become increasingly popular recently because of its ability to precisely quantify selected proteins in complex cellular backgrounds. Here, we demonstrated the utility of an LTQ-Orbitrap Velos Pro mass spectrometer in targeted parallel reaction monitoring (PRM) despite its unconventional dual ion trap configuration. We evaluated absolute specificity (>99%) and sensitivity (100 amol on column in 1 μg of total cellular extract) using full and mass range scans as survey scans together with data-dependent (DDA) and targeted MS/MS acquisition. The instrument duty cycle was a critical parameter limiting sensitivity, necessitating peptide retention time scheduling. We assessed synthetic peptide and recombinant peptide standards to predict or experimentally determine target peptide retention times. We applied optimized PRM to protein degradation in signaling regulation, an area that is receiving increased attention in plant physiology. We quantified relative abundance of selected proteins in plants that are mutant for enzymatic components of the N-end rule degradation (NERD) pathway such as the two tRNA-arginyl-transferases ATE1 and ATE2 and the two E3 ubiquitin ligases PROTEOLYSIS1 and 6. We found a number of upregulated proteins, which might represent degradation targets. We also targeted FLAGELLIN SENSITIVE2 (FLS2), a pattern recognition receptor responsible for pathogen sensing, in ubiquitin ligase mutants to assay the attenuation of plant immunity by degradation of the receptor

Targeted Proteomics Analysis of Protein Degradation in Plant Signaling on an LTQ-Orbitrap Mass Spectrometer

Targeted proteomics has become increasingly popular recently because of its ability to precisely quantify selected proteins in complex cellular backgrounds. Here, we demonstrated the utility of an LTQ-Orbitrap Velos Pro mass spectrometer in targeted parallel reaction monitoring (PRM) despite its unconventional dual ion trap configuration. We evaluated absolute specificity (>99%) and sensitivity (100 amol on column in 1 μg of total cellular extract) using full and mass range scans as survey scans together with data-dependent (DDA) and targeted MS/MS acquisition. The instrument duty cycle was a critical parameter limiting sensitivity, necessitating peptide retention time scheduling. We assessed synthetic peptide and recombinant peptide standards to predict or experimentally determine target peptide retention times. We applied optimized PRM to protein degradation in signaling regulation, an area that is receiving increased attention in plant physiology. We quantified relative abundance of selected proteins in plants that are mutant for enzymatic components of the N-end rule degradation (NERD) pathway such as the two tRNA-arginyl-transferases ATE1 and ATE2 and the two E3 ubiquitin ligases PROTEOLYSIS1 and 6. We found a number of upregulated proteins, which might represent degradation targets. We also targeted FLAGELLIN SENSITIVE2 (FLS2), a pattern recognition receptor responsible for pathogen sensing, in ubiquitin ligase mutants to assay the attenuation of plant immunity by degradation of the receptor

Targeted Proteomics Analysis of Protein Degradation in Plant Signaling on an LTQ-Orbitrap Mass Spectrometer

Targeted proteomics has become increasingly popular recently because of its ability to precisely quantify selected proteins in complex cellular backgrounds. Here, we demonstrated the utility of an LTQ-Orbitrap Velos Pro mass spectrometer in targeted parallel reaction monitoring (PRM) despite its unconventional dual ion trap configuration. We evaluated absolute specificity (>99%) and sensitivity (100 amol on column in 1 μg of total cellular extract) using full and mass range scans as survey scans together with data-dependent (DDA) and targeted MS/MS acquisition. The instrument duty cycle was a critical parameter limiting sensitivity, necessitating peptide retention time scheduling. We assessed synthetic peptide and recombinant peptide standards to predict or experimentally determine target peptide retention times. We applied optimized PRM to protein degradation in signaling regulation, an area that is receiving increased attention in plant physiology. We quantified relative abundance of selected proteins in plants that are mutant for enzymatic components of the N-end rule degradation (NERD) pathway such as the two tRNA-arginyl-transferases ATE1 and ATE2 and the two E3 ubiquitin ligases PROTEOLYSIS1 and 6. We found a number of upregulated proteins, which might represent degradation targets. We also targeted FLAGELLIN SENSITIVE2 (FLS2), a pattern recognition receptor responsible for pathogen sensing, in ubiquitin ligase mutants to assay the attenuation of plant immunity by degradation of the receptor

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

PSIG1 interacts with SMG7.

<p><b>a</b>, Schematic structure of PSIG1 and SMG7. <b>b</b>, PSIG1 physically interacts with the C-terminus of SMG7 <i>in vitro</i>. HisMBP-SMG7, HisMBP-SMG7-N or HisMBP-SMG7-C were incubated with GST or GST-PSIG1 and the conjugates were pulled down with Glutathione-Sepharose beads. HisMBP-SMG7, HisMBP-SMG7-N, or HisMBP-SMG7-C were detected by immunoblotting using anti-His antibody. GST-PSIG1 was stained with Coomassie brilliant blue (CBB). Arrowheads indicate the position of each proteins. <b>c</b>, Tyr-575 of PSIG1 is required for interaction with SMG7 <i>in vitro</i>. HisMBP-SMG7 was incubated with GST, GST-PSIG1 ^1–606, GST-PSIG1 ^{1–606 Y575A}, or GST-PSIG1 ^{1–606 W570A/Y575A}, and the conjugates were pulled down with Glutathione-Sepharose beads. HisMBP-SMG7 and GST-PSIG1 were detected by immunoblotting using anti-His antibody or anti-GST antibody. <b>d</b>, PSIG1 and SMG7 co-localize with DCP1, a P-body marker. The images show the CFP signal in cyan, the GFP signal in green, and the mCherry signal in magenta. The merged images indicate the overlay of two signals in yellow or purple and the overlay of three signals in white. White arrowheads indicate overlayed signals. Cellular localization was analyzed at 4 days after inoculation in agroinfiltrated <i>Nicotiana benthamiana</i>. The scale bars represent 10 μm. <b>e</b>, Genomic structure of the <i>SMG7</i> gene. Black boxes indicate the coding region, and white boxes indicate the non-coding region. The PPGF sequence resides downstream of the T-DNA insertion site. <b>f</b>, Induction of RPS4-triggered cell death is pronounced in the <i>smg7-4</i> mutant allele. 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.</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

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