rabbit polyclonal anti rdrp  (Eurofins)

Journal: bioRxiv

Article Title: Nanoscale cellular organization of viral RNA and proteins in SARS-CoV-2 replication organelles

doi: 10.1101/2023.11.07.566110

Figure Lengend Snippet: a , Representative confocal images show three types of vgRNA distribution in SARS-CoV-2 infected cells. b , Number of cells assigned to one of the three types at 6 or 24 hpi. c , Cell-integrated vgRNA signal increases significantly from 6 hpi to 24 hpi. p-value = 6⋅10 -8 , two-tailed t-test. d - e , Cell-integrated signal of immunofluorescently detected dsRNA ( d ) and nsp12 ( e ) in SARS-CoV-2 infected cells does not significantly change from 6 hpi to 24 hpi. p-values = 0.13 ( d ) and 0.23 ( e ), two-tailed t-test. f , dsRNA signal correlates with vgRNA signal at 6 hpi (Pearson’s r = 0.76). g , dsRNA signal does not correlate with vgRNA signal at 24 hpi (Pearson’s r = 0.18). Error bars represent mean + SD of the values from individual cells. Scale bars, 10 µm.

Article Snippet: Primary antibodies and the optimal dilutions and concentrations used are as follows: goat polyclonal anti-spike S2 (Novus Biologicals, AF10774-SP, 1:20, 10 µg/mL), mouse monoclonal anti-dsRNA (SCICONS, 10010200, 1:200, 5 µg/mL), rabbit polyclonal anti-RdRp/nsp12 (Sigma-Aldrich, SAB3501287-100UG, 1:500, 2 µg/mL), mouse monoclonal anti-nucleocapsid (Thermo Fisher, MA5-29981, 1:500, 2 µg/mL), rabbit polyclonal anti-nsp3 (Thermo Fisher, PA5-116947, 1:134, 5 µg/mL), sheep polyclonal anti-GFP (Bio-Rad, 4745-1051, 1:1000, 5 µg/mL), and rabbit polyclonal anti-GFP (Novus Biologicals, NB600-308SS, 1:163, 5 µg/mL).

Techniques: Infection, Two Tailed Test

Journal: bioRxiv

Article Title: Nanoscale cellular organization of viral RNA and proteins in SARS-CoV-2 replication organelles

doi: 10.1101/2023.11.07.566110

Figure Lengend Snippet: a - b , Representative confocal images of SARS-CoV-2 infected cells display DL colocalization between nsp12, the catalytic subunit of RdRp (green) and vgRNA (magenta) at both 6 hpi ( a ) and 24 hpi ( b ). c - d , Representative SR images of SARS-CoV-2 infected cells indicate nanoscale association between nsp12 and vgRNA at both 6 hpi ( c ) and 24 hpi ( d ). Insets show magnified images of corresponding regions in colored boxes. e - f , Bivariate pair-correlation functions peak at r = 0 nm indicating association between nsp12 and vgRNA. g , Number of nanoscale puncta of nsp12 per vgRNA cluster. h , SR image of nsp12 with Sec61β suggests encapsulation of nsp12 within ER-derived membranes. Scale bars, 10 µm ( a - b ), 1 µm ( c - d ), 500 nm ( h and insets in c - d ). Dashed lines in c , d and h indicate the edge of the cell nucleus.

Article Snippet: Primary antibodies and the optimal dilutions and concentrations used are as follows: goat polyclonal anti-spike S2 (Novus Biologicals, AF10774-SP, 1:20, 10 µg/mL), mouse monoclonal anti-dsRNA (SCICONS, 10010200, 1:200, 5 µg/mL), rabbit polyclonal anti-RdRp/nsp12 (Sigma-Aldrich, SAB3501287-100UG, 1:500, 2 µg/mL), mouse monoclonal anti-nucleocapsid (Thermo Fisher, MA5-29981, 1:500, 2 µg/mL), rabbit polyclonal anti-nsp3 (Thermo Fisher, PA5-116947, 1:134, 5 µg/mL), sheep polyclonal anti-GFP (Bio-Rad, 4745-1051, 1:1000, 5 µg/mL), and rabbit polyclonal anti-GFP (Novus Biologicals, NB600-308SS, 1:163, 5 µg/mL).

Techniques: Infection, Encapsulation, Derivative Assay

Journal: Molecular Plant-Microbe Interactions®

Article Title: A Necrosis-Inducing Elicitor Domain Encoded by Both Symptomatic and Asymptomatic Plantago asiatica mosaic virus Isolates, Whose Expression Is Modulated by Virus Replication

doi: 10.1094/mpmi-12-10-0279

Figure Lengend Snippet: Fig. 1. Amino acid residue 1,154 of RNA-dependent RNA polymerase (RdRp) alone does not determine necrotic symptoms of Plantago asiatica mosaic virus (PlAMV). A and B, Local symptoms on Nicotiana benthamiana leaves infiltrated with Agrobacterium culture carrying binary plasmid for PlAMV infection. Symptoms induced by Li1, Li6, or their mutants with a single amino acid substitution at amino acid 1,154 of RdRp are shown. Red circles indicate necrosis and the yellow circles indicate no necrosis. C and D, Accumulation of H2O2 in the leaves shown in A and B, respectively, analyzed by diaminobenzedene staining. Photographs were taken 6 days postinfiltration (dpi) (A through D). E, Accumulation of PlAMV RNA in the infiltrated leaves of N. benthamiana. Real-time polymerase chain reaction analysis was performed at 3 dpi using PlAMV coat-protein-specific primers, and the value of PlAMV RNA was normalized relative to the value of EF1A mRNA. Four independent leaves were used for the experiment and mean values (with standard errors) are presented.

Article Snippet: A rabbit polyclonal anti-RdRp antibody (further described in the Results section) diluted to 1/1,000, or rabbit monoclonal anti-c-myc antibody (Cell Signaling Technology, Danvers, MA, U.S.A.) diluted to 1/1,500, was used as the primary antibody.

Techniques: Residue, Virus, Plasmid Preparation, Infection, Staining, Real-time Polymerase Chain Reaction

Journal: Molecular Plant-Microbe Interactions®

Article Title: A Necrosis-Inducing Elicitor Domain Encoded by Both Symptomatic and Asymptomatic Plantago asiatica mosaic virus Isolates, Whose Expression Is Modulated by Virus Replication

doi: 10.1094/mpmi-12-10-0279

Figure Lengend Snippet: Fig. 2. Necrotic symptoms induced by Plantago asiatica mosaic virus (PlAMV) isolates or replicons are correlated with the expression level of RNA-de- pendent RNA polymerase (RdRp). A, Local symptoms on Nicotiana benthamiana leaves infiltrated with Agrobacterium culture expressing each PlAMV-en- coded protein. Each protein is co-expressed with silencing suppressor p19, both under the control of the 35S promoter. An Agrobacterium culture containing pCAMBIA1301, which expresses -glucuronidase (GUS) in the infiltrated area, is used as a negative control. Photographs were taken at 6 days postinfiltra- tion (dpi). Red circles indicate necrosis and yellow circles indicate no necrosis. B, Accumulation of H2O2 in the leaves shown in A analyzed by diaminoben- zedene (DAB) staining. C, Schematic representation of PlAMV cDNA constructs based on the pCAMBIA1301 binary vector. Open boxes represent open reading frames (ORFs). pLi1 contains full-length cDNA of Li1 isolate of PlAMV between the Cauliflower mosaic virus 35S promoter (35S) and the Nos terminator (nos). The boxes labeled with LB and RB represent the left and right border of the T-DNA sequence, respectively. p53U-RdRp1 (designated as replicon in the text) lacks ORF2 to -5 that are not essential for RNA replication but retains the 5 and 3 untranslated region of pLi1. D, Accumulation of RdRp in leaves infiltrated with Agrobacterium culture expressing PlAMV isolates or mutants (left) or each PlAMV replicon (right). A crude membrane (pellet) fraction extracted from the agroinfiltrated leaves was detected by Western blot analysis using anti-RdRp (amino acids 1 to 335) antibody. Coomassie Brilliant Blue staining of the gel is shown as a loading control. E, Local symptoms on N. benthamiana leaves infiltrated with Agrobacterium culture expressing each replicon derived from different PlAMV isolates or mutants. Photographs were taken at 6 dpi. Red circles indicate necrosis and yellow circles indicate no necrosis. F, Accumulation of H2O2 in leaves shown in E analyzed by DAB staining.

Article Snippet: A rabbit polyclonal anti-RdRp antibody (further described in the Results section) diluted to 1/1,000, or rabbit monoclonal anti-c-myc antibody (Cell Signaling Technology, Danvers, MA, U.S.A.) diluted to 1/1,500, was used as the primary antibody.

Techniques: Virus, Expressing, Control, Negative Control, Staining, Construct, Plasmid Preparation, Labeling, Sequencing, Membrane, Western Blot, Derivative Assay

Journal: Molecular Plant-Microbe Interactions®

Article Title: A Necrosis-Inducing Elicitor Domain Encoded by Both Symptomatic and Asymptomatic Plantago asiatica mosaic virus Isolates, Whose Expression Is Modulated by Virus Replication

doi: 10.1094/mpmi-12-10-0279

Figure Lengend Snippet: Fig. 3. Expression of RNA-dependent RNA polymerase (RdRp) protein but not its RNA sequence induces necrotic symptoms on Nicotiana benthamiana. A, Northern blot analysis of RNAs accumulated in the leaves infiltrated with Agrobacterium culture expressing either Li1, 53U-RdRp1, or a mixture of 5U- RdRp1 + p19. Northern blot analyses were performed at the days indicated on the top of the figure, using strand-specific probes to detect positive- or nega- tive-strand Plantago asiatica mosaic virus (PlAMV) genomic RNAs as indicated on the left. Ethidium bromide staining of ribosomal RNA (rRNA) was used as the loading control. B, Local symptoms on N. benthamiana leaves infiltrated with Agrobacterium culture containing T-DNA constructs used for transient expression of RdRp. An Agrobacterium culture containing pCAMBIA1301, which expresses -glucuronidase (GUS) in the infiltrated area, is used as a nega- tive control and that containing p53U-RdRp as a positive control. Photographs were taken at 6 days postinfiltration (dpi). Red circles indicate necrosis and yellow circles indicate no necrosis. C, Accumulation of H2O2 in leaves shown in B analyzed by diaminobenzedene staining. D, Accumulation of RdRp in leaves infiltrated with Agrobacterium culture containing each RdRp expression vector. Western blot analysis was performed using anti-RdRp antibody, and Coomassie Brilliant Blue staining of the gel is shown as a loading control.

Article Snippet: A rabbit polyclonal anti-RdRp antibody (further described in the Results section) diluted to 1/1,000, or rabbit monoclonal anti-c-myc antibody (Cell Signaling Technology, Danvers, MA, U.S.A.) diluted to 1/1,500, was used as the primary antibody.

Techniques: Expressing, Sequencing, Northern Blot, Virus, Staining, Control, Construct, Positive Control, Plasmid Preparation, Western Blot

Journal: Molecular Plant-Microbe Interactions®

Article Title: A Necrosis-Inducing Elicitor Domain Encoded by Both Symptomatic and Asymptomatic Plantago asiatica mosaic virus Isolates, Whose Expression Is Modulated by Virus Replication

doi: 10.1094/mpmi-12-10-0279

Figure Lengend Snippet: Fig. 4. Necrotic symptoms in Nicotiana benthamiana are induced in an RNA-dependent RNA polymerase (RdRp) dose-dependent but not amino acid 1154- dependent manner. N. benthamiana leaves were infiltrated with a mixture of two Agrobacterium cultures containing estradiol-inducible RdRp constructs (pER8-RdRp) together with p19. Three days later, leaves were treated with estradiol at the indicated concentrations. A, Local symptoms on N. benthamiana leaves transiently expressing RdRp of four different Plantago asiatica mosaic virus (PlAMV) isolates or mutants by estradiol-inducible system. At 3 days postinfiltration (dpi) of a mixture of Agrobacterium cultures containing p19 and either four pER8-RdRp variants or pER8-sGFP (synthetic green fluorescent protein), estradiol was treated on the same leaves at a concentration of 50 µM. Red circles indicate necrosis and yellow circle indicates no necrosis. B, Accumulation of H2O2 of the leaves shown in A analyzed by diaminobenzedene (DAB) staining. C, Accumulation of RdRp and its mRNA in the infiltrated area shown in A. Western blot analysis was conducted using anti-c-myc antibody. Coomassie Brilliant Blue staining as a loading control of Western blot analysis and ethidium bromide staining of ribosomal RNA (rRNA) as the loading control of Northern blot analysis are also shown. D, Local symptoms on N. benthamiana leaves expressing different levels of RdRp. N. benthamiana leaves were infiltrated with a mixture of Agrobacterium cultures containing p19 and pER8-RdRp1. Three days later, different levels of estradiol whose concentrations are indicated (µM) were infiltrated in the leaves. Photographs were taken 3 days after estradiol treatment. Red circles indicate necrosis and yellow circle indicates no necrosis. E, Accumulation of H2O2 in leaves shown in D analyzed by DAB staining. F, Estradiol dose-dependent RdRp expression assessed by Western blot analysis. Estradiol concentrations (µM) are indicated at the top of the figure. Protein samples were extracted 2 days after estradiol treatment and Western blot analysis was performed as described above.

Article Snippet: A rabbit polyclonal anti-RdRp antibody (further described in the Results section) diluted to 1/1,000, or rabbit monoclonal anti-c-myc antibody (Cell Signaling Technology, Danvers, MA, U.S.A.) diluted to 1/1,500, was used as the primary antibody.

Techniques: Construct, Expressing, Virus, Concentration Assay, Staining, Western Blot, Control, Northern Blot

Journal: Molecular Plant-Microbe Interactions®

Article Title: A Necrosis-Inducing Elicitor Domain Encoded by Both Symptomatic and Asymptomatic Plantago asiatica mosaic virus Isolates, Whose Expression Is Modulated by Virus Replication

doi: 10.1094/mpmi-12-10-0279

Figure Lengend Snippet: Fig. 5. Necrotic symptom-triggering domain resides in the putative RNA helicase domain (HEL) of Plantago asiatica mosaic virus (PlAMV) RNA-depend- ent RNA polymerase (RdRp). A, Schematic representation of the constructs used for the transient expression of each domain of PlAMV RdRp and typical symptoms they induced. For simplicity, the prefix p53U was omitted from the plasmid names. PlAMV-Li1 and Li6-derived regions are indicated by blank and hatched boxes, respectively. Local symptoms and H2O2 accumulation in leaves 6 days postinfiltration with each construct are shown on the right. Red circles indicate necrosis. B, Quantification of cell death in the leaves expressing RdRp domains were derived from Li1 by electrolyte leakage. Error bars rep- resent standard error (n = 5).

Article Snippet: A rabbit polyclonal anti-RdRp antibody (further described in the Results section) diluted to 1/1,000, or rabbit monoclonal anti-c-myc antibody (Cell Signaling Technology, Danvers, MA, U.S.A.) diluted to 1/1,500, was used as the primary antibody.

Techniques: Virus, Construct, Expressing, Plasmid Preparation, Derivative Assay