Journal: The New Phytologist

Article Title: Receptor‐like kinases BIR1 and BIR3 modulate antiviral resistance by different mechanisms

doi: 10.1111/nph.70216

Figure Lengend Snippet: Hormone regulation of BRASSINOSTEROID INSENSITIVE1‐ASSOCIATED RECEPTOR KINASE 1 ( BAK1 ) ‐INTERACTING RECEPTOR‐LIKE KINASE ( BIR ) gene expression in Arabidopsis. (a) Reverse transcription quantitative polymerase chain reaction (RT‐qPCR) analysis of BIR1 , BIR2 , and BIR3 transcript levels in plants infected with tobacco rattle virus (TRV) 5 d after inoculation (left) or treated with 1 mM of salicylic acid (SA) (right) compared with mock‐treated controls. (b) Time‐course analysis of (JA) and abscisic acid (ABA) levels in leaves of noninoculated, mock‐inoculated, and TRV‐infected Arabidopsis, determined by gas chromatography coupled to time‐of‐flight mass spectrometry (GC‐TOF‐MS). dpi, days postinoculation. Error bars represent SD from five independent biological replicates. (c) RT‐qPCR analysis of BIR1 , BIR2 , and BIR3 transcript levels in wild‐type (WT; Columbia‐0 (Col‐0)) plants treated with H 2 O (mock), 1 mM of methyl Jasmonate (MeJA), or 100 μM of ABA. (d) RT‐qPCR analysis of the hormone‐responsive marker genes: PATHOGENESIS‐RELATED 1 ( PR1 ) (SA), PLANT‐DEFENSIN 1.2 ( PDF1.2 ) (JA), and RESPONSIVE TO DESICCATION 29A ( RD29A ) (ABA) in response to SA, MeJA, and ABA treatments. Measures were taken using the same set of replicates for each condition. All data were normalized to the CBP20 internal control and compared with mock‐treated plants (set to 1). Data are mean ± SD analyzed by unpaired t‐test (a, d) or one‐way ANOVA and Tukey's multiple comparison test (b, c) ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05. Experiments were repeated with similar results.

Article Snippet: Immunoprecipitation of mCherry‐tagged BIR1 protein from Arabidopsis leaves was performed using RFP‐Trap (Chromotek, Planegg, Germany) according to the manufacturer's protocol.

Techniques: Gene Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Infection, Virus, Gas Chromatography, Mass Spectrometry, Marker, Control, Comparison

Journal: The New Phytologist

Article Title: Receptor‐like kinases BIR1 and BIR3 modulate antiviral resistance by different mechanisms

doi: 10.1111/nph.70216

Figure Lengend Snippet: Genetic dissection of the antiviral response in bir1‐1 Arabidopsis mutants. (a, b) Reverse transcription quantitative polymerase chain reaction (RT‐qPCR) analysis of tobacco rattle virus (TRV) genomic RNA levels in TRV‐infected Arabidopsis wild‐type (WT; Columbia‐0 (Col‐0)) and various mutant combinations, including bir1‐1 sobir1‐1 , sobir1‐13 , bir2‐1 , bak1‐4 pad4‐1 sobir7‐1 (a), and bir1‐1 sobir1‐1 pad4‐1 , bir1‐1 pad4‐1 sobir7‐1 (b), at 5 d postinoculation (dpi). Morphological phenotypes of each genotype at 5 dpi with TRV are also shown. A noninoculated bir1‐1 pad4‐1 mutant is included for size comparison. Relative TRV RNA levels were normalized to the CBP20 internal control and compared with WT plants (set to 1). Data are mean ± SD analyzed by one‐way ANOVA followed by Tukey's multiple comparison test; ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05. Experiments were repeated with similar results. BIR, BAK1‐INTERACTING RECEPTOR‐LIKE KINASE . BAK1 , BRASSINOSTEROID INSENSITIVE1‐ASSOCIATED RECEPTOR KINASE 1 , PAD4 , PHYTOALEXIN DEFICIENT 4 . SOBIR, SUPPRESSOR OF BIR1‐1.

Article Snippet: Immunoprecipitation of mCherry‐tagged BIR1 protein from Arabidopsis leaves was performed using RFP‐Trap (Chromotek, Planegg, Germany) according to the manufacturer's protocol.

Techniques: Dissection, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Virus, Infection, Mutagenesis, Comparison, Control

Journal: The New Phytologist

Article Title: Receptor‐like kinases BIR1 and BIR3 modulate antiviral resistance by different mechanisms

doi: 10.1111/nph.70216

Figure Lengend Snippet: Effects of BRASSINOSTEROID INSENSITIVE1‐ASSOCIATED RECEPTOR KINASE 1 (BAK1)‐INTERACTING RECEPTOR‐LIKE KINASE1 (BIR1) induction on flg22‐ and nlp20‐elicited pattern‐triggered immune (PTI) responses in Arabidopsis. (a) Accumulation of reactive oxygen species (ROS) in dexamethasone (DEX)‐treated wild‐type (WT), BIR1 L6, and BIR1 L9 inducible plants after elicitation with 1 μM flg22, 1 μM nlp20, or H 2 O (mock). Data represent mean ± SD of relative fluorescence units. (b) Reverse transcription quantitative polymerase chain reaction (RT‐qPCR) analysis of RESPIRATORY BURST OXIDASE HOMOLOG D ( RBOHD ) transcript levels after treatment with H 2 O (mock) or 1 μM flg22 for 45 min in DEX‐treated (DEX+) and nontreated (DEX‐) samples. Values were normalized to the CBP20 internal control and compared with mock‐treated plants (set to 1). Data are mean ± SD analyzed by unpaired t ‐test. **, P < 0.01; *, P < 0.05. (c) Ethylene accumulation in WT, BIR1 L6, and BIR1 L9 transgenic plants after treatment with H 2 O (mock), flg22, and nlp20 at 1 μM or 100 nM. Data represent average values ± SD. (d) Immunoprecipitation of mCherry‐BIR1 fusion proteins (arrowhead) from DEX‐treated and mock‐treated samples of BIR1 L6 and BIR1 L9 plants used in (a) and (b). Experiments were repeated with similar results. NLP, NECROSIS AND ETHYLENE‐INDUCING PEPTIDE.

Article Snippet: Immunoprecipitation of mCherry‐tagged BIR1 protein from Arabidopsis leaves was performed using RFP‐Trap (Chromotek, Planegg, Germany) according to the manufacturer's protocol.

Techniques: Fluorescence, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Control, Transgenic Assay, Immunoprecipitation

Journal: The New Phytologist

Article Title: Receptor‐like kinases BIR1 and BIR3 modulate antiviral resistance by different mechanisms

doi: 10.1111/nph.70216

Figure Lengend Snippet: Transcriptional changes associated with BRASSINOSTEROID INSENSITIVE1‐ASSOCIATED RECEPTOR KINASE 1 (BAK1)‐INTERACTING RECEPTOR‐LIKE KINASE1 (BIR1) induction in Arabidopsis. (a) Morphology of representative dexamethasone (DEX)‐inducible BIR1‐mCherry transgenic Arabidopsis plants (line 9, L9) after 4 d of 30 μM of DEX or mock treatment (H 2 O). (b) Western blot analysis using anti‐mCherry antibody to detect BIR1‐mCherry protein accumulation after 4 d of DEX treatment. BIR1 transcript levels in wild‐type (WT) and BIR1 L9 plants were quantified using RNA‐Seq (Fragments Per Kilobase of transcript sequence per Millions of base‐pairs sequenced (FPKM)) and reverse transcription quantitative polymerase chain reaction (RT‐qPCR). RT‐qPCR values were normalized to the CBP20 internal control and compared with mock‐treated (set to 1). Data are mean ± SD analyzed by unpaired t ‐test. ***, P < 0.001. (c) Principal component analysis (PCA) of RNA‐Seq data from mock‐treated WT (Mock‐WT), mock‐treated BIR1 L9 (Mock‐L9), DEX‐treated WT (DEX‐WT), and DEX‐treated BIR1 L9 (DEX‐L9) plants. (d) Number of up‐ or downregulated transcripts (|log 2 (fold change)| ≥ 1 and adjusted P ‐value ≤ 0.01) in each pairwise comparison. (e) Scatterplot of selected Kyoto Encyclopedia of Genes and Genomes (KEGG) terms of differentially expressed genes (DEGs) between DEX‐treated and mock‐treated BIR1 L9 plants. Dot size represents the gene count, and color indicates the significance of term enrichment. Gene Ontology (GO) terms with adjusted P ‐value < 0.05 are significantly enriched.

Article Snippet: Immunoprecipitation of mCherry‐tagged BIR1 protein from Arabidopsis leaves was performed using RFP‐Trap (Chromotek, Planegg, Germany) according to the manufacturer's protocol.

Techniques: Transgenic Assay, Western Blot, RNA Sequencing, Sequencing, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Control, Comparison

Journal: The New Phytologist

Article Title: Receptor‐like kinases BIR1 and BIR3 modulate antiviral resistance by different mechanisms

doi: 10.1111/nph.70216

Figure Lengend Snippet: BRASSINOSTEROID INSENSITIVE1‐ASSOCIATED RECEPTOR KINASE 1 (BAK1)‐INTERACTING RECEPTOR‐LIKE KINASE1 (BIR1) induction interferes with flg22‐elicited pattern‐triggered immune (PTI) gene expression in Arabidopsis. (a) Reverse transcription quantitative polymerase chain reaction (RT‐qPCR) analysis of the PTI marker genes FLG22‐INDUCED RECEPTOR‐LIKE KINASE 1 ( FRK1 ) and PATHOGENESIS‐RELATED 1 ( PR1 ) in wild‐type (WT; Columbia‐0 (Col‐0)), BIR1 L6, and BIR1 L9 inducible plants after treatment with H 2 O (mock) or 1 μM flg22 for 45 min. Plants were pretreated with or without dexamethasone (DEX) as indicated. (b) RT‐qPCR analysis of BIR1 transcript levels in DEX‐treated samples before flg22 treatment. All data were normalized to the CBP20 internal control and compared with mock‐treated plants (set to 1). Data are mean ± SD analyzed by unpaired t ‐test. **, P < 0.01; *, P < 0.05. All experiments were repeated with similar results.

Article Snippet: Immunoprecipitation of mCherry‐tagged BIR1 protein from Arabidopsis leaves was performed using RFP‐Trap (Chromotek, Planegg, Germany) according to the manufacturer's protocol.

Techniques: Gene Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Marker, Control

Journal: The New Phytologist

Article Title: Receptor‐like kinases BIR1 and BIR3 modulate antiviral resistance by different mechanisms

doi: 10.1111/nph.70216

Figure Lengend Snippet: Transient BRASSINOSTEROID INSENSITIVE1‐ASSOCIATED RECEPTOR KINASE 1 (BAK1)‐INTERACTING RECEPTOR‐LIKE KINASE1 (BIR1) expression partially inhibits poly(I:C)‐triggered callose deposition at plasmodesmata (PD) in Nicotiana benthamiana . (a) Callose deposition at PDs was visualized by aniline blue staining of epidermal cells in N. benthamiana leaves transiently expressing a 35S‐driven BIR1‐HA construct or an empty vector (EV). Callose deposition was observed after treatment with H 2 O (control) or 50 ng μl −1 poly(I:C). Bars, 10 μm. (b) Quantification of PD callose content in leaves infiltrated with EV or BIR1‐HA in response to H 2 O, poly(I:C), or flg22 (10 μM). Each dot represents the mean callose intensity at individual PDs (> 100) measured in three to four leaf disks from at least three independent biological replicates per treatment and analyzed by one‐way ANOVA followed by Tukey's multiple comparison test; ****, adjusted P ‐value < 0.0001; ***, P < 0.001. (c) Western blot analysis using anti‐HA and anti‐mCherry antibodies to detect BIR1‐HA and BIR1‐mCherry proteins, respectively, in agroinfiltrated areas at 2 d postinoculation (dpi). (d) Western blot analysis using anti‐HA antibodies to detect HA‐tagged BIR1 and GFP proteins in areas co‐infiltrated with TRV‐GFP and EV (−) or BIR1‐HA (+) at 3 and 5 dpi. Red Ponceau is shown as a loading control. (e) Transient expression of BIR1 promotes viral spreading in N. benthamiana leaves. Whole leaves were first infiltrated with Agrobacterium containing BIR1‐HA under the 35S promoter, BIR1‐mCherry under a dexamethasone (DEX)‐inducible promoter, or the corresponding EV, and then spot‐infiltrated (indicated by dash circles) into the same area with Agrobacterium carrying TRV‐GFP. For DEX‐inducible BIR1‐mCherry expression, 30 μM DEX was added to the infiltration solution. Photographs were taken under ultraviolet light 2 dpi to visualize GFP fluorescence. Bars, 1 cm. All experiments were repeated with similar results. TRV, tobacco rattle virus; GFP, green fluorescent protein.

Article Snippet: Immunoprecipitation of mCherry‐tagged BIR1 protein from Arabidopsis leaves was performed using RFP‐Trap (Chromotek, Planegg, Germany) according to the manufacturer's protocol.

Techniques: Expressing, Staining, Construct, Plasmid Preparation, Control, Comparison, Western Blot, Fluorescence, Virus

Journal: The New Phytologist

Article Title: Receptor‐like kinases BIR1 and BIR3 modulate antiviral resistance by different mechanisms

doi: 10.1111/nph.70216

Figure Lengend Snippet: BRASSINOSTEROID INSENSITIVE1‐ASSOCIATED RECEPTOR KINASE 1 (BAK1)‐INTERACTING RECEPTOR‐LIKE KINASE1 (BIR1) induction inhibits flg22‐ and poly(I:C)‐triggered callose deposition at plasmodesmata (PD) in Arabidopsis. (a) Quantification of PD callose content in wild‐type (WT) plants pretreated with or without 30 μM dexamethasone (DEX) for 3 d in response to H 2 O (control), 50 ng/μl poly(I:C), or 1 μM flg22. (b) Aniline blue staining to visualize callose deposition at PDs in epidermal cells of WT plants in response to poly(I:C) or flg22. (c) Quantification of PD callose content in BIR1 L9 plants pretreated with or without DEX in response to H 2 O, poly(I:C), or flg22. (d) Aniline blue staining of BIR1 L9 transgenic plants pretreated with or without DEX in response to H 2 O, poly(I:C), or flg22. Photographs were taken 30 min after treatments. BIR1‐mCherry fluorescence was confirmed in DEX‐treated BIR1 L9 plants within the same images. Inlays show magnified views of the boxed areas. Bars, 10 μm. (e) Quantification of PD callose content in WT and sobir1‐12 mutant plants in response to H 2 O or poly(I:C). (a, c, e) Each dot represents the mean callose intensity at individual PDs (> 100) measured in three to four leaf disks from at least three independent biological replicates per treatment and analyzed by one‐way ANOVA followed by Tukey's multiple comparison test; ****, adjusted P ‐value < 0.0001; ***, P < 0.001; **, P < 0.01. SOBIR1, SUPPRESSOR OF BIR1‐1 1 . All experiments were repeated with similar results.

Article Snippet: Immunoprecipitation of mCherry‐tagged BIR1 protein from Arabidopsis leaves was performed using RFP‐Trap (Chromotek, Planegg, Germany) according to the manufacturer's protocol.

Techniques: Control, Staining, Transgenic Assay, Fluorescence, Mutagenesis, Comparison

Journal: The New Phytologist

Article Title: Receptor‐like kinases BIR1 and BIR3 modulate antiviral resistance by different mechanisms

doi: 10.1111/nph.70216

Figure Lengend Snippet: Genetic dissection of the antiviral response in bir3‐2 Arabidopsis mutants. (a) Reverse transcription quantitative polymerase chain reaction (RT‐qPCR) analysis of tobacco rattle virus (TRV) genomic RNA levels in TRV‐infected Arabidopsis wild‐type (WT, Columbia‐0 (Col‐0)) and various mutant combinations, including bir3‐2 , csa1‐2 , bir3‐2 bak1‐4 csa1‐2 , and bir3‐2 bak1‐4 eds1‐12 , at 5 d postinoculation (dpi). (b) RT‐qPCR analysis of TRV genomic RNA levels in TRV‐infected WT (Col‐0), bak1‐4 , pad4‐1 , eds1‐2 , and pad4‐1 eds1‐2 at 5 dpi. Morphological phenotypes of each genotype at 5 dpi with TRV are also shown. A noninoculated bir3‐2 bak1‐4 mutant is included for size comparison. Relative TRV RNA levels were normalized to the CBP20 internal control and compared with WT plants (set to 1). Data are mean ± SD analyzed by one‐way ANOVA followed by Tukey's multiple comparison test; ****, P < 0.0001; **, P < 0.01; *, P < 0.05. Experiments were repeated with similar results. (c) Model for BAK1‐INTERACTING RECEPTOR‐LIKE KINASE (BIR)‐mediated regulation of antiviral defense. The BIR1 and BIR3 proteins are induced during virus infection, influenced by antagonistic interactions between salicylic acid (SA) and jasmonic acid (JA) hormone signaling. Induction of BIR2 during infection is affected by SA, but not JA. BIR1 negatively regulates antiviral defense through mechanisms that may include pattern‐triggered immunity (PTI) gene expression and plasmodesmata (PD) callose deposition as well as yet unidentified pathways independent of reactive oxygen species (ROS) or the BAK1, SOBIR1, or PAD4 signaling components. BIR3 represses an antiviral response that relies on the activation of BAK1‐ and EDS1/PAD4‐dependent effector‐triggered immunity (ETI), likely involving intracellular nucleotide‐binding leucine‐rich repeat (NLR) receptors, leading to asymptomatic resistance. Solid arrows indicate activation, blunt‐ended arrows denote repression, and dashed arrows represent potential effects on antiviral defenses. BAK1 , BRASSINOSTEROID INSENSITIVE1‐ASSOCIATED RECEPTOR KINASE 1 ; CSA1 , CONSTITUTIVE SHADE AVOIDANCE 1; EDS1 , ENHANCED DISEASE SUSCEPTIBILITY 1 ; PAD4 , PHYTOALEXIN DEFICIENT 4 .

Article Snippet: Immunoprecipitation of mCherry‐tagged BIR1 protein from Arabidopsis leaves was performed using RFP‐Trap (Chromotek, Planegg, Germany) according to the manufacturer's protocol.

Techniques: Dissection, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Virus, Infection, Mutagenesis, Comparison, Control, Gene Expression, Activation Assay, Binding Assay