Journal: The New Phytologist

Article Title: A lysin motif effector subverts chitin‐triggered immunity to facilitate arbuscular mycorrhizal symbiosis

doi: 10.1111/nph.16245

Figure Lengend Snippet: RiSLM ) is highly induced in intraradical mycelium (IRM) in a wide range of host plants. (a) RiSLM is highly induced in five hosts Medicago truncatula (Medicago), Allium schoenoprasum (Chives), Nicotiana benthamiana (Nicotiana), Solanum lycopersicum (Tomato) and Lotus japonicus (Lotus) compared with germinating spores (GS). Transcripts per million (TPM) was used to represent expression levels. Error bars represent SE from either three replicates (for Medicago, Chives, and Nicotiana) or two replicates (for Lotus and Tomato), collected from independent RNA sequencing (RNAseq) experiments. (b) Quantitative PCR analysis, showing that RiSLM (relative to Rhizophagus irregularis elongation factor 1α , RiEF ) is induced during arbuscular mychorrizal colonization and arbuscule (ARB) development as represented by relative expression of RiEF and the ARB‐specific phosphate transporter ( MtPT4 ) normalized by Medicago elongation factor 1α ( MtEF ). Error bars represent SE from three biological replicates. (c) RiSLM expression based on RNAseq analyses of laser microdissected ARBs and IRM compared with extraradical mycelium (ERM) and GS. Error bars represent SE from three biological replicates. For all figures, different characters indicate significant differences (least significant difference P < 0.05)

Article Snippet: A time‐course experiment in Medicago revealed that RiSLM expression correlates with fungal colonization and abundance, as reflected by the expression of R. irregularis Elongation Factor 1α ( RiEF ) and the ARB‐specific phosphate transporter ( MtPT4 ; Javot et al. , ) (Fig. b).

Techniques: Expressing, RNA Sequencing, Real-time Polymerase Chain Reaction

Journal: The New Phytologist

Article Title: A lysin motif effector subverts chitin‐triggered immunity to facilitate arbuscular mycorrhizal symbiosis

doi: 10.1111/nph.16245

Figure Lengend Snippet: Lysin motif (LysM) effectors are a conserved feature of arbuscular mychorrizal (AM) fungi. LysM effectors from different AM fungal species/isolates, including six Rhizophagus irregularis isolates (DAOM197198/RiSLM, A1, A4, A5, B3, and C2), Rhizophagus clarus , Gigaspora rosea , and Gigaspora margarita , were aligned using M afft and grouped using G eneious tree builder ( https://www.geneious.com ). Signal peptide and LysM domain are marked.

Article Snippet: A time‐course experiment in Medicago revealed that RiSLM expression correlates with fungal colonization and abundance, as reflected by the expression of R. irregularis Elongation Factor 1α ( RiEF ) and the ARB‐specific phosphate transporter ( MtPT4 ; Javot et al. , ) (Fig. b).

Techniques:

Journal: The New Phytologist

Article Title: A lysin motif effector subverts chitin‐triggered immunity to facilitate arbuscular mycorrhizal symbiosis

doi: 10.1111/nph.16245

Figure Lengend Snippet: RiSLM) does not block symbiotic responses. (a) Binding of RiSLM to lipo‐chitooligosaccharides (LCOs) LCO‐IV(C18:1, sulphated LCO (sLCO)) and LCO‐IV(C18:1, nonsulphated LCO (nsLCO)) (black and blue circles, respectively) as determined by microscale thermophoresis and corresponding to the same binding event. Inset: two binding events occurring at low (0.1–100 nM) and high concentrations (100 nM to 10 µM without reaching saturation) of LCO‐IV(C18:1, S). Error bars represent SD from two independent measurements using two batches of independently purified proteins. (b, c) Quantitative PCR (qPCR) analysis of Medicago truncatula R108 roots treated with (b) 10 nM nonsulphated myc‐LCOs or (c) sulphated myc‐LCOs in combination with either 10 or 50 nM RiSLM. Three symbiotic marker genes, PUB1 , TUBB1 , or Vapyrin , were used to monitor symbiotic responses induced by LCOs, using Med ic ago elongation factor 1α ( MtEF ) as reference gene. Error bars represent SE from three biological replicates. (d) qPCR analysis of Medicago R108 roots treated with water ( n = 3) or 100 nM chitotetraose (CO4, n = 3) in combination with either 100 nM ( n = 4) or 500 nM RiSLM ( n = 3), showing that RiSLM does not strongly suppress CO4‐triggered symbiotic marker gene induction. Different letters indicate significant difference (least significant difference P < 0.05) between different treatments.

Article Snippet: A time‐course experiment in Medicago revealed that RiSLM expression correlates with fungal colonization and abundance, as reflected by the expression of R. irregularis Elongation Factor 1α ( RiEF ) and the ARB‐specific phosphate transporter ( MtPT4 ; Javot et al. , ) (Fig. b).

Techniques: Blocking Assay, Binding Assay, Microscale Thermophoresis, Purification, Real-time Polymerase Chain Reaction, Marker

Journal: The New Phytologist

Article Title: A lysin motif effector subverts chitin‐triggered immunity to facilitate arbuscular mycorrhizal symbiosis

doi: 10.1111/nph.16245

Figure Lengend Snippet: Host‐induced gene silencing of RiSLM ) reduces mycorrhization in Medicago truncatula . (a) Frequency (F%) remains the same, whereas mycorrhization intensity in the root (M%) and arbuscule abundance in the root (A%) are reduced in RiSLM ‐silenced roots. For the empty vector control (EV), 12 biological replicates were used. For RiSLM RNA interference (RNAi), 10 biological replicates were used. (b) Quantitative PCR analysis of control and RNAi roots showing RiSLM expression level relative to Rhizophagus irregularis elongation factor 1α ( RiEF ) and MtPT4 expression levels relative to Medicago elongation factor 1α ( MtEF ). Successful silencing of RiSLM (relative to RiEF ) reduces RiEF and MtPT4 expression (relative to MtEF ), indicating reduced mycorrhization and arbuscule abundance. Twelve replicates (individual transgenic roots) were used for EV. Eleven replicates were used for RNAi. Student’s t ‐test P ‐values are indicated for (a) and (b). For all box plots, boxes represent interquartile range (IQR) and whiskers represent 1.5IQR. (c, d) Wheat germ agglutinin‐Alexa Fluor 488 staining of mycorrhization in (d) RiSLM ‐silenced roots and (c) control roots (c). Bars, 200 µm.

Article Snippet: A time‐course experiment in Medicago revealed that RiSLM expression correlates with fungal colonization and abundance, as reflected by the expression of R. irregularis Elongation Factor 1α ( RiEF ) and the ARB‐specific phosphate transporter ( MtPT4 ; Javot et al. , ) (Fig. b).

Techniques: Plasmid Preparation, Control, Real-time Polymerase Chain Reaction, Expressing, Transgenic Assay, Staining

Journal: The Journal of Biological Chemistry

Article Title: Chemical Proteomics Identifies Heterogeneous Nuclear Ribonucleoprotein (hnRNP) A1 as the Molecular Target of Quercetin in Its Anti-cancer Effects in PC-3 Cells *

doi: 10.1074/jbc.M114.553248

Figure Lengend Snippet: Identification and validation of quercetin binding targets using immunoblotting and surface plasmon resonance binding assays. A, silver-stained gel showing proteins bound to the affinity column in the presence or absence of quercetin. T, total cell lysates; Un, unbound fraction; W, proteins that did not bind quercetin; E, bound proteins eluted. The indicated protein bands were excised from the gel and digested with trypsin, and the peptides were analyzed by MS. The identified quercetin-binding proteins are listed in Table 1. B, both unbound and bound proteins were separated by SDS-PAGE on 10% gels and immunoblotted with antibodies against vinculin, nucleolin, EF-1α, and hnRNPA1. C and E, recombinant full-length hnRNPA1 (aa 1–320) protein and the C-terminal region of hnRNPA1 (aa 268–320) were individually covalently coupled to a Biacore CM5 chip following the manufacturer's instructions. Serial dilutions of quercetin (final concentrations, 10 to 0.15 μm) were perfused over the immobilized proteins to allow association to occur, and dissociation was then monitored. Solvent correction was performed by subtracting vehicle-alone signals and those of quercetin perfused over uncoupled dextran matrix. D and F, saturation curve fitting of quercetin-full-length hnRNPA1 and quercetin-C-terminal region hnRNPA1 (aa 268–320) was plotted using GraphPad Prism software. The Kd values were calculated to be about 8.9 μm for full-length hnRNPA1 and about 1.7 μm for the C-terminal region of hnRNPA1. QCT, quercetin. RU, response units. Error bars, S.E.

Article Snippet: The rabbit anti-vinculin polyclonal antibody, anti-α-tubulin, and rabbit anti-EF-1α polyclonal antibody were from GeneTex Inc.

Techniques: Biomarker Discovery, Binding Assay, Western Blot, SPR Assay, Staining, Affinity Column, SDS Page, Recombinant, Solvent, Software

Journal: The Journal of Biological Chemistry

Article Title: Chemical Proteomics Identifies Heterogeneous Nuclear Ribonucleoprotein (hnRNP) A1 as the Molecular Target of Quercetin in Its Anti-cancer Effects in PC-3 Cells *

doi: 10.1074/jbc.M114.553248

Figure Lengend Snippet: Identification of quercetin-binding proteins by LC-MS/MS An individual ion score of >43 indicates identity or extensive homology ( p < 0.05). The protein score is derived from ion scores as a non-probabilistic basis for ranking protein hits.

Article Snippet: The rabbit anti-vinculin polyclonal antibody, anti-α-tubulin, and rabbit anti-EF-1α polyclonal antibody were from GeneTex Inc.

Techniques: Derivative Assay, Membrane