bbwv 1  (DSMZ)

Journal: Molecular Plant Pathology

Article Title: RNA2‐encoded VP37 protein of Broad bean wilt virus 1 is a determinant of pathogenicity, host susceptibility, and a suppressor of post‐transcriptional gene silencing

doi: 10.1111/mpp.12979

Figure Lengend Snippet: Schematic representation of cDNA constructs of broad bean wilt virus 1 (BBWV‐1). (a) Combination of RNA1 (pBBWV1‐R1) and RNA2 (pBBWV1‐R2) cDNA constructs of BBWV‐1 based on nucleotide sequence of isolate Ben used for the wild‐type infectious clone (pBBWV1‐Wt). The 35S promoter of cauliflower mosaic virus is upstream of the first nucleotide of each BBWV‐1 cDNA. The hepatitis delta virus ribozyme (Rbz) and the nopaline synthase terminator (t‐Nos) are engineered in tandem after a polyA tail. (b) Combination of RNA1 (pBBWV1‐R1) and RNA2 (pBBWV1‐R2:G492C) cDNA constructs with a single point substitution in the VP37 start codon at nucleotide position 492 (AUG > AUC, Met > Ile) used to obtain a BBWV‐1 infectious clone not expressing VP37 (pBBWV1‐G492C)

Article Snippet: To quantify the viral titre of plants agroinfiltrated with pBBVW1‐R1/pBBWV1‐R2 (pBBWV1‐Wt) and pBBWV1‐R1/pBBWV1‐R2:G492C (pBBWV1‐G492C) two TaqMan (Sigma) probes specific for BBWV‐1 RNA1 (positions 1,123 to 1,148) and RNA2 (positions 1,791 to 1,820) were designed using Primer Express (Applied Biosystems) software.

Techniques: Construct, Sequencing, Expressing

Journal: Molecular Plant Pathology

Article Title: RNA2‐encoded VP37 protein of Broad bean wilt virus 1 is a determinant of pathogenicity, host susceptibility, and a suppressor of post‐transcriptional gene silencing

doi: 10.1111/mpp.12979

Figure Lengend Snippet: Biological characterization of pBBWV1‐Wt and pBBWV1‐G492C cDNA infectious clones in different herbaceous host species. (a) Symptoms induced by pBBWV1‐Wt and pBBWV1‐G492C in Nicotiana benthamiana , broad bean, and pepper plants in comparison with those induced by BBWV‐1 isolate Ben at 21 days postagroinfiltration (dpa). (b) Northern blot analysis of plants agroinfiltrated with pBBWV1‐Wt (lane 1), pBBWV1‐G492C (lane 2), and mock‐infiltrated used as negative control (lane 3) (at the top). Ribosomal RNAs (rRNAs) used as loading controls are shown at the bottom. Each lane corresponds to a pool of three total RNA samples

Article Snippet: To quantify the viral titre of plants agroinfiltrated with pBBVW1‐R1/pBBWV1‐R2 (pBBWV1‐Wt) and pBBWV1‐R1/pBBWV1‐R2:G492C (pBBWV1‐G492C) two TaqMan (Sigma) probes specific for BBWV‐1 RNA1 (positions 1,123 to 1,148) and RNA2 (positions 1,791 to 1,820) were designed using Primer Express (Applied Biosystems) software.

Techniques: Clone Assay, Northern Blot, Negative Control

Journal: Molecular Plant Pathology

Article Title: RNA2‐encoded VP37 protein of Broad bean wilt virus 1 is a determinant of pathogenicity, host susceptibility, and a suppressor of post‐transcriptional gene silencing

doi: 10.1111/mpp.12979

Figure Lengend Snippet: Quantitative reverse transcription PCR amplification of both (+) BBWV‐1 genomic and (−) replication‐associated RNA1 strands of total RNA samples obtained from agroinfiltrated leaf areas of plants from different host species agroinfiltrated with pBBWV1‐Wt or pBBWV1‐G492C

Article Snippet: To quantify the viral titre of plants agroinfiltrated with pBBVW1‐R1/pBBWV1‐R2 (pBBWV1‐Wt) and pBBWV1‐R1/pBBWV1‐R2:G492C (pBBWV1‐G492C) two TaqMan (Sigma) probes specific for BBWV‐1 RNA1 (positions 1,123 to 1,148) and RNA2 (positions 1,791 to 1,820) were designed using Primer Express (Applied Biosystems) software.

Techniques: Amplification

Journal: Molecular Plant Pathology

Article Title: RNA2‐encoded VP37 protein of Broad bean wilt virus 1 is a determinant of pathogenicity, host susceptibility, and a suppressor of post‐transcriptional gene silencing

doi: 10.1111/mpp.12979

Figure Lengend Snippet: Transient expression of BBWV‐1 VP37 in Nicotiana benthamiana plants using the pPVX202 viral vector. (a) Schematic representation of pPVX‐derived cDNA constructs. (b) Symptoms induced by PVX‐derived constructs in upper noninoculated leaves of N. benthamiana plants at 7 days postinoculation (dpi): mild mosaic in plants inoculated with pPVX‐Ø and severe mosaic and small necrotic lesions (arrows) in those inoculated with pPVX‐VP37. Mock‐inoculated N. benthamiana plants were used as negative controls. (c) Northern blot analysis of upper noninoculated leaves of N. benthamiana plants inoculated with pPVX‐Ø and pPVX‐VP37 using a PVX‐specific riboprobe. Ribosomal RNAs (rRNAs) used as loading controls are showed at the bottom. Each lane corresponds to a pool of three total RNA samples

Article Snippet: To quantify the viral titre of plants agroinfiltrated with pBBVW1‐R1/pBBWV1‐R2 (pBBWV1‐Wt) and pBBWV1‐R1/pBBWV1‐R2:G492C (pBBWV1‐G492C) two TaqMan (Sigma) probes specific for BBWV‐1 RNA1 (positions 1,123 to 1,148) and RNA2 (positions 1,791 to 1,820) were designed using Primer Express (Applied Biosystems) software.

Techniques: Expressing, Plasmid Preparation, Derivative Assay, Construct, Northern Blot

Journal: Molecular Plant Pathology

Article Title: RNA2‐encoded VP37 protein of Broad bean wilt virus 1 is a determinant of pathogenicity, host susceptibility, and a suppressor of post‐transcriptional gene silencing

doi: 10.1111/mpp.12979

Figure Lengend Snippet: Systemic necrosis as consequence of programmed cell dead (PCD) induced by transient expression of BBWV‐1 VP37 using the pPVX202 viral vector in upper noninoculated leaves of Nicotiana benthamiana plants. (a) Staining of superoxide ion ( O 2 ‐ ) deposits with nitroblue tetrazolium (NBT) staining method (arrow). (b) Visualization of groups of dead cells using the trypan blue staining method (arrows)

Article Snippet: To quantify the viral titre of plants agroinfiltrated with pBBVW1‐R1/pBBWV1‐R2 (pBBWV1‐Wt) and pBBWV1‐R1/pBBWV1‐R2:G492C (pBBWV1‐G492C) two TaqMan (Sigma) probes specific for BBWV‐1 RNA1 (positions 1,123 to 1,148) and RNA2 (positions 1,791 to 1,820) were designed using Primer Express (Applied Biosystems) software.

Techniques: Expressing, Plasmid Preparation, Staining

Journal: Molecular Plant Pathology

Article Title: RNA2‐encoded VP37 protein of Broad bean wilt virus 1 is a determinant of pathogenicity, host susceptibility, and a suppressor of post‐transcriptional gene silencing

doi: 10.1111/mpp.12979

Figure Lengend Snippet: RNA silencing suppression activity of BBWV‐1 VP37. (a) Leaves of Nicotiana benthamiana 16c plants lit with a handheld UV lamp at 3 days postagroinfiltration (dpa). Plants were agroinfiltrated with p35S‐GFP in combination with p35S‐VP37, p35S‐p19, or p35S‐Ø. (b) Northern blot analysis of green fluorescent protein (GFP) mRNAs and siRNAs extracted from agroinfiltrated tissue patches at 3 dpa. Ribosomal RNAs (rRNAs) used as loading controls are shown at the bottom. Each lane corresponds to a pool of three total RNA samples. (c) Visualization using a fluorescence stereomicroscope of N. benthamiana leaves agroinfiltrated with p35S‐VP37, p35S‐Ø, or p35S‐p19 at 4 dpa. Agroinfiltrated leaves were mechanically inoculated with pTCV‐GFP RNA transcripts 1 day later. Bars represent 50 µm

Article Snippet: To quantify the viral titre of plants agroinfiltrated with pBBVW1‐R1/pBBWV1‐R2 (pBBWV1‐Wt) and pBBWV1‐R1/pBBWV1‐R2:G492C (pBBWV1‐G492C) two TaqMan (Sigma) probes specific for BBWV‐1 RNA1 (positions 1,123 to 1,148) and RNA2 (positions 1,791 to 1,820) were designed using Primer Express (Applied Biosystems) software.

Techniques: Activity Assay, Northern Blot, Fluorescence

Journal: Molecular Plant Pathology

Article Title: RNA2‐encoded VP37 protein of Broad bean wilt virus 1 is a determinant of pathogenicity, host susceptibility, and a suppressor of post‐transcriptional gene silencing

doi: 10.1111/mpp.12979

Figure Lengend Snippet: Schematic representation of cDNA constructs of broad bean wilt virus 1 (BBWV‐1). (a) Combination of RNA1 (pBBWV1‐R1) and RNA2 (pBBWV1‐R2) cDNA constructs of BBWV‐1 based on nucleotide sequence of isolate Ben used for the wild‐type infectious clone (pBBWV1‐Wt). The 35S promoter of cauliflower mosaic virus is upstream of the first nucleotide of each BBWV‐1 cDNA. The hepatitis delta virus ribozyme (Rbz) and the nopaline synthase terminator (t‐Nos) are engineered in tandem after a polyA tail. (b) Combination of RNA1 (pBBWV1‐R1) and RNA2 (pBBWV1‐R2:G492C) cDNA constructs with a single point substitution in the VP37 start codon at nucleotide position 492 (AUG > AUC, Met > Ile) used to obtain a BBWV‐1 infectious clone not expressing VP37 (pBBWV1‐G492C)

Article Snippet: To quantify the viral titre of plants agroinfiltrated with pBBVW1‐R1/pBBWV1‐R2 (pBBWV1‐Wt) and pBBWV1‐R1/pBBWV1‐R2:G492C (pBBWV1‐G492C) two TaqMan (Sigma) probes specific for BBWV‐1 RNA1 (positions 1,123 to 1,148) and RNA2 (positions 1,791 to 1,820) were designed using Primer Express (Applied Biosystems) software.

Techniques: Construct, Sequencing, Expressing

Journal: Molecular Plant Pathology

Article Title: RNA2‐encoded VP37 protein of Broad bean wilt virus 1 is a determinant of pathogenicity, host susceptibility, and a suppressor of post‐transcriptional gene silencing

doi: 10.1111/mpp.12979

Figure Lengend Snippet: Biological characterization of pBBWV1‐Wt and pBBWV1‐G492C cDNA infectious clones in different herbaceous host species. (a) Symptoms induced by pBBWV1‐Wt and pBBWV1‐G492C in Nicotiana benthamiana , broad bean, and pepper plants in comparison with those induced by BBWV‐1 isolate Ben at 21 days postagroinfiltration (dpa). (b) Northern blot analysis of plants agroinfiltrated with pBBWV1‐Wt (lane 1), pBBWV1‐G492C (lane 2), and mock‐infiltrated used as negative control (lane 3) (at the top). Ribosomal RNAs (rRNAs) used as loading controls are shown at the bottom. Each lane corresponds to a pool of three total RNA samples

Article Snippet: To quantify the viral titre of plants agroinfiltrated with pBBVW1‐R1/pBBWV1‐R2 (pBBWV1‐Wt) and pBBWV1‐R1/pBBWV1‐R2:G492C (pBBWV1‐G492C) two TaqMan (Sigma) probes specific for BBWV‐1 RNA1 (positions 1,123 to 1,148) and RNA2 (positions 1,791 to 1,820) were designed using Primer Express (Applied Biosystems) software.

Techniques: Clone Assay, Northern Blot, Negative Control

Journal: Molecular Plant Pathology

Article Title: RNA2‐encoded VP37 protein of Broad bean wilt virus 1 is a determinant of pathogenicity, host susceptibility, and a suppressor of post‐transcriptional gene silencing

doi: 10.1111/mpp.12979

Figure Lengend Snippet: Quantitative reverse transcription PCR amplification of both (+) BBWV‐1 genomic and (−) replication‐associated RNA1 strands of total RNA samples obtained from agroinfiltrated leaf areas of plants from different host species agroinfiltrated with pBBWV1‐Wt or pBBWV1‐G492C

Article Snippet: To quantify the viral titre of plants agroinfiltrated with pBBVW1‐R1/pBBWV1‐R2 (pBBWV1‐Wt) and pBBWV1‐R1/pBBWV1‐R2:G492C (pBBWV1‐G492C) two TaqMan (Sigma) probes specific for BBWV‐1 RNA1 (positions 1,123 to 1,148) and RNA2 (positions 1,791 to 1,820) were designed using Primer Express (Applied Biosystems) software.

Techniques: Amplification

Journal: Molecular Plant Pathology

Article Title: RNA2‐encoded VP37 protein of Broad bean wilt virus 1 is a determinant of pathogenicity, host susceptibility, and a suppressor of post‐transcriptional gene silencing

doi: 10.1111/mpp.12979

Figure Lengend Snippet: Transient expression of BBWV‐1 VP37 in Nicotiana benthamiana plants using the pPVX202 viral vector. (a) Schematic representation of pPVX‐derived cDNA constructs. (b) Symptoms induced by PVX‐derived constructs in upper noninoculated leaves of N. benthamiana plants at 7 days postinoculation (dpi): mild mosaic in plants inoculated with pPVX‐Ø and severe mosaic and small necrotic lesions (arrows) in those inoculated with pPVX‐VP37. Mock‐inoculated N. benthamiana plants were used as negative controls. (c) Northern blot analysis of upper noninoculated leaves of N. benthamiana plants inoculated with pPVX‐Ø and pPVX‐VP37 using a PVX‐specific riboprobe. Ribosomal RNAs (rRNAs) used as loading controls are showed at the bottom. Each lane corresponds to a pool of three total RNA samples

Article Snippet: To quantify the viral titre of plants agroinfiltrated with pBBVW1‐R1/pBBWV1‐R2 (pBBWV1‐Wt) and pBBWV1‐R1/pBBWV1‐R2:G492C (pBBWV1‐G492C) two TaqMan (Sigma) probes specific for BBWV‐1 RNA1 (positions 1,123 to 1,148) and RNA2 (positions 1,791 to 1,820) were designed using Primer Express (Applied Biosystems) software.

Techniques: Expressing, Plasmid Preparation, Derivative Assay, Construct, Northern Blot

Journal: Molecular Plant Pathology

Article Title: RNA2‐encoded VP37 protein of Broad bean wilt virus 1 is a determinant of pathogenicity, host susceptibility, and a suppressor of post‐transcriptional gene silencing

doi: 10.1111/mpp.12979

Figure Lengend Snippet: Systemic necrosis as consequence of programmed cell dead (PCD) induced by transient expression of BBWV‐1 VP37 using the pPVX202 viral vector in upper noninoculated leaves of Nicotiana benthamiana plants. (a) Staining of superoxide ion ( O 2 ‐ ) deposits with nitroblue tetrazolium (NBT) staining method (arrow). (b) Visualization of groups of dead cells using the trypan blue staining method (arrows)

Article Snippet: To quantify the viral titre of plants agroinfiltrated with pBBVW1‐R1/pBBWV1‐R2 (pBBWV1‐Wt) and pBBWV1‐R1/pBBWV1‐R2:G492C (pBBWV1‐G492C) two TaqMan (Sigma) probes specific for BBWV‐1 RNA1 (positions 1,123 to 1,148) and RNA2 (positions 1,791 to 1,820) were designed using Primer Express (Applied Biosystems) software.

Techniques: Expressing, Plasmid Preparation, Staining

Journal: Molecular Plant Pathology

Article Title: RNA2‐encoded VP37 protein of Broad bean wilt virus 1 is a determinant of pathogenicity, host susceptibility, and a suppressor of post‐transcriptional gene silencing

doi: 10.1111/mpp.12979

Figure Lengend Snippet: RNA silencing suppression activity of BBWV‐1 VP37. (a) Leaves of Nicotiana benthamiana 16c plants lit with a handheld UV lamp at 3 days postagroinfiltration (dpa). Plants were agroinfiltrated with p35S‐GFP in combination with p35S‐VP37, p35S‐p19, or p35S‐Ø. (b) Northern blot analysis of green fluorescent protein (GFP) mRNAs and siRNAs extracted from agroinfiltrated tissue patches at 3 dpa. Ribosomal RNAs (rRNAs) used as loading controls are shown at the bottom. Each lane corresponds to a pool of three total RNA samples. (c) Visualization using a fluorescence stereomicroscope of N. benthamiana leaves agroinfiltrated with p35S‐VP37, p35S‐Ø, or p35S‐p19 at 4 dpa. Agroinfiltrated leaves were mechanically inoculated with pTCV‐GFP RNA transcripts 1 day later. Bars represent 50 µm

Article Snippet: To quantify the viral titre of plants agroinfiltrated with pBBVW1‐R1/pBBWV1‐R2 (pBBWV1‐Wt) and pBBWV1‐R1/pBBWV1‐R2:G492C (pBBWV1‐G492C) two TaqMan (Sigma) probes specific for BBWV‐1 RNA1 (positions 1,123 to 1,148) and RNA2 (positions 1,791 to 1,820) were designed using Primer Express (Applied Biosystems) software.

Techniques: Activity Assay, Northern Blot, Fluorescence