Journal: Journal of Visualized Experiments : JoVE

Article Title: Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

doi: 10.3791/3791

Figure Lengend Snippet:

Article Snippet: L17 , Biotinylated Solanum Tuberosum (Potato) Lectin , STL , Vector Laboratories , BK-3000.

Techniques: Plasmid Preparation, Positive Control

Journal: Biochimica et biophysica acta. Biomembranes

Article Title: The antifungal plant defensin HsAFP1 induces autophagy, vacuolar dysfunction and cell cycle impairment in yeast

doi: 10.1016/j.bbamem.2020.183255

Figure Lengend Snippet: Ctr_1, Ctr_2, Ctr_3 and HsAFP1_1, HsAFP1_2, HsAFP1_3 represent the control and HsAFP1 treatment of 3 independent experiments, respectively. C. albicans SC5314 cells were treated for 150 minutes with HsAFP1 (2x FC50; 10 μg/mL) in PDB/YPD with 50 mM HEPES pH 7. Colored bars indicate the Z-score, representing the relative expression of a gene compared to the mean of expression for the corresponding gene over all six samples. Up- and down-regulated genes coding for: GPI-anchored proteins, GPI-anchor synthesis, GPI-anchor building block biosynthesis and phosphatidylinositol-specific phospholipase C (PI-specific PLC). Down-regulated iron- and copper-related genes and up-regulation of calcium-related genes. Up- and down-regulated genes involved in macro- or mitophagy, respectively. Up-regulated genes coding for: cell wall biosynthesis and cell cycle enzymes as well as cellular bud located proteins.

Article Snippet: Yeast cells were cultured in the following liquid media: YPD (yeast extract (10 g/L; LabM, UK), peptone (20 g/L; LabM, UK) and glucose (20 g/L; Sigma-Aldrich, USA), YNB (yeast nitrogen base without amino acids; MP Biomedicals, USA) (6.7 g/L), PDB/YPD (potato dextrose broth (19.2 g/L; BD, USA), yeast extract (2 g/L), peptone (4 g/L) and glucose (4 g/L)) adjusted to pH 7 with 50 mM HEPES (Sigma-Aldrich, USA), synthetic complete (SC) medium (CSM (complete amino acid supplement mixture; MP Biomedicals, USA) (0.77 g/L), YNB (6.7 g/L) and glucose (20 g/L)) adjusted to pH 7 with 50 mM HEPES or 1/5 th PDB/YNB; at 30°C and 37°C for S. cerevisiae and C. albicans , respectively.

Techniques: Expressing, Blocking Assay

Journal: Biochimica et biophysica acta. Biomembranes

Article Title: The antifungal plant defensin HsAFP1 induces autophagy, vacuolar dysfunction and cell cycle impairment in yeast

doi: 10.1016/j.bbamem.2020.183255

Figure Lengend Snippet: S. cerevisiae WT and Δbst1 mutant cells were treated for 150 minutes in PDB/YPD with 50 mM HEPES pH 7 at 30°C. BODIPY-HsAFP1 and propidium Iodide (PI; 3 μM) were used as markers for peptide internalization and cell death, respectively. (A) Confocal microscope images of S. cerevisiae cells treated with high HsAFP1 doses (10x FC50; 250 μg/mL). Scale bar: 5 μm. (B) Flow cytometry of WT and Δbst1 mutant cells. WT cells are co-administrated with BODIPY-HsAFP1 (1/2 FC50; 12.5 μg/mL, FC50; 25 μg/mL and 2x FC50; 50 μg/mL)and 2 mM CaSO4, 2 mM CuSO4, 2 mM MgSO4 or 2 mM FeSO4. The % of cells that do have BODIPY-HsAFP1 associated with their surface or internalized (green) and the % of cells that do not have BODIPY-HsAFP1 associated with their surface or internalized (grey) is presented. (C) Flow cytometry of WT and Δbst1 mutant cells. WT cells are co-administrated with BODIPY-HsAFP1 (1/2 FC50; 12.5 μg/mL, FC50; 25 μg/mL and 2x FC50; 50 μg/mL) and 2 mM CaSO4, 2 mM CuSO4, 2 mM MgSO4 or 2 mM FeSO4. The % of cells that both have BODIPY-HsAFP1 associated with their surface/internalized and have compromised membranes is presented in red. All other subpopulations are presented in grey, including the % of cells that do not have BODIPY-HsAFP1 internalized or associated with their surface and do not have compromised membranes, the % of cells that do not have BODIPY-HsAFP1 internalized or associated with their surface and do have compromised membranes, and the % of cells that do have BODIPY-HsAFP1 internalized or associated with their surface and do not have compromised membranes. Data are means ± SD for n ≥ 3 independent experiments. To analyze significant differences in the size of the subpopulations, two-way ANOVA followed by Tukey multiple comparison was performed, with *** and **** representing P < 0.001 and P < 0.0001, respectively.

Article Snippet: Yeast cells were cultured in the following liquid media: YPD (yeast extract (10 g/L; LabM, UK), peptone (20 g/L; LabM, UK) and glucose (20 g/L; Sigma-Aldrich, USA), YNB (yeast nitrogen base without amino acids; MP Biomedicals, USA) (6.7 g/L), PDB/YPD (potato dextrose broth (19.2 g/L; BD, USA), yeast extract (2 g/L), peptone (4 g/L) and glucose (4 g/L)) adjusted to pH 7 with 50 mM HEPES (Sigma-Aldrich, USA), synthetic complete (SC) medium (CSM (complete amino acid supplement mixture; MP Biomedicals, USA) (0.77 g/L), YNB (6.7 g/L) and glucose (20 g/L)) adjusted to pH 7 with 50 mM HEPES or 1/5 th PDB/YNB; at 30°C and 37°C for S. cerevisiae and C. albicans , respectively.

Techniques: Mutagenesis, Microscopy, Flow Cytometry

Journal: Biochimica et biophysica acta. Biomembranes

Article Title: The antifungal plant defensin HsAFP1 induces autophagy, vacuolar dysfunction and cell cycle impairment in yeast

doi: 10.1016/j.bbamem.2020.183255

Figure Lengend Snippet: (A) S. cerevisiae WT cells treated 150 min with HsAFP1 (1/2 FC50; 12.5 μg/mL, FC50; 25 μg/mL and 2x FC50; 50 μg/mL) or HsAFP1[H32A][R52A] doses (equivalent to the highest tested HsAFP1 dose) in PDB/YPD with 50 mM HEPES pH 7 at 30°C. Dose-dependent increase of the vacuolar pH and decrease of survival of HsAFP1-treated S. cerevisiae cells as compared to the control (MQ water; black bars), determined via the vacuolar dye BCECF-AM and CFU counting, respectively. Concanamycin A (ConA) was used as positive control for increased vacuolar pH, whereas cell survival was not affected. Equimolar HsAFP1[H32A][R52A] doses (as the highest tested HsAFP1 dose) did not affect vacuolar pH or survival. Means ± SD are presented for n ≥ 3 independent experiments. Significant differences in vacuolar pH or Log10 numbers of CFU between the negative control (MQ water; black bars) and all other treatments were determined via one-way ANOVA followed by Dunnett multiple comparison, with **, *** and **** representing, P < 0.01, P < 0.001 and P < 0.0001, respectively (presented by orange bars). (B) Decrease in replicative lifespan of S. cerevisiae BY4742 cells by HsAFP1 treatment under dietary restriction (DR) conditions, as represented by the dotted arrow. Replicative lifespan of cells on agar plates in the presence or absence of HsAFP1 and containing 2% or 0.05% glucose (= dextrose (D)), with 0.05% glucose representing DR conditions. The HsAFP1 dose used is the maximum dose that does not affect replicative lifespan in 2% glucose. Replicative lifespan results are presented from an experiment containing n = 40 cells tested per condition.

Article Snippet: Yeast cells were cultured in the following liquid media: YPD (yeast extract (10 g/L; LabM, UK), peptone (20 g/L; LabM, UK) and glucose (20 g/L; Sigma-Aldrich, USA), YNB (yeast nitrogen base without amino acids; MP Biomedicals, USA) (6.7 g/L), PDB/YPD (potato dextrose broth (19.2 g/L; BD, USA), yeast extract (2 g/L), peptone (4 g/L) and glucose (4 g/L)) adjusted to pH 7 with 50 mM HEPES (Sigma-Aldrich, USA), synthetic complete (SC) medium (CSM (complete amino acid supplement mixture; MP Biomedicals, USA) (0.77 g/L), YNB (6.7 g/L) and glucose (20 g/L)) adjusted to pH 7 with 50 mM HEPES or 1/5 th PDB/YNB; at 30°C and 37°C for S. cerevisiae and C. albicans , respectively.

Techniques: Positive Control, Negative Control

Journal: Scientific reports

Article Title: Inhibition of potato leafroll virus multiplication and systemic translocation by siRNA constructs against putative ATPase fold of movement protein.

doi: 10.1038/s41598-020-78791-4

Figure Lengend Snippet: Figure 1. (A) Sequence alignments of the ATPase domain of PLRV_MP. Multiple sequence alignments, generated using ClustalW, were manually corrected for domain superimpositions. The number(s) in brackets represent the number of amino acids. (B) Organization of functional motifs on the polypeptide chain of MP of PLRV. Schematics are drawn approximately to the scale and represent the approximate consensus of representative homologs. (C) I-TASSER predicted atomic model of PLRV_MP with all the putative ATP catalysis motifs. [Accession Numbers: Potato leafroll virus isolate PLRV-JF (PLRV JF): ADE93562.1, Potato leafroll virus isolate Anoquia (PLRV Antioquia): QES86451.1, Potato leafroll virus isolate Sta Rosa de Osos-santana (PLRV Santana): AEB78381.1, Potato leafroll virus isolate Turmeque-siguineque (PLRV Tur-sig): AEB78353.1, Potato leafroll virus isolate Sonson-tasajo (PLRV Sonson): AEB78367.1, Potato leafroll virus isolate Siachoque-jurubita (PLRV Jurubita): AEB78379.1, Potato leafroll virus isolate Pasto-el campanero2 (PLRV Pasto-el): AEB78377.1, Potato leafroll virus isolate Pasto-el campanero (PLRV Campa): AEB78375.1, Argentinian Potato leafroll virus isolate PLRV-5 (PLRV Argentina): ADE93558.1, Potato leafroll virus isolate Zipaquira-san Jorge (PLRV Zipaquir): AEB78361.1, Potato leafroll virus isolate Turmeque-siguineque2 (PLRV Turmeque): AEB78373.1, Potato leafroll virus isolate Ipiales-suras (PLRV Suras): AEB78385.1, France Potato leafroll virus strain CIP01 (PLRV CIP01): AAL77949.1, Potato leafroll virus isolate PLRV184 (PLRV 184): AYA73306.1, Potato leafroll virus isolate Villapinzon-bosabita (PLRV Villapinzo): AEB78359.1, Potato leafroll virus isolate Madrid-los arboles2 (PLRV Arboles): AEB78369.1, Potato leafroll virus isolate TZ:LR3M:11 (PLRV TZ:LR3M): AGN48055.1, Potato leafroll virus isolate PLRV171 (PLRV Canada): AYA73300.1, Colombia Potato leafroll virus isolate PLRV165 (PLRV Colombia): AYA73294.1, Tamil nadu Potato leafroll virus isolate OTNI-2 (PLRV Tamilnadu): AFJ11889.1, Jalandgar Potato leafroll virus isolate (PLRV Jalandgar): AFJ11863.1, Karlovce Potato leafroll virus isolate VIRUBRA 1/045 (PLRV VIRUBRA45): ACD93696.1, Australia Potato leafroll virus (PLRV Australia): QBO24572.1, Shimla Potato leafroll virus isolate PBI-6 (PLRV Shimla): AFJ11881.1, Potato leafroll virus (PLRV): QBO24572.1, Prague Potato leafroll virus isolate VIRUBRA 1/046 (PLRV VIRUBRA46): ACD93705.1, Potato leafroll virus isolate Say88 (PLRV Say88): QBO24572.1, Potato leafroll virus isolate Turmeque-siguineque3 (PLRV Siguineque3): AEB78383.1, Potato leafroll virus isolate Facatativa (PLRV Facatat): AEB78357.1, Germany Potato leafroll virus isolate PLRV-DSMZ (PLRV Germany): ADE93559.1, Argentinian Potato leafroll virus isolate PLRV-5 (PLRV Argentinian): ADE93558.1].

Article Snippet: [Accession Numbers: Potato leafroll virus isolate PLRV-JF (PLRV JF): ADE93562.1, Potato leafroll virus isolate Anoquia (PLRV Antioquia): QES86451.1, Potato leafroll virus isolate Sta Rosa de Osos-santana (PLRV Santana): AEB78381.1, Potato leafroll virus isolate Turmeque-siguineque (PLRV Tur-sig): AEB78353.1, Potato leafroll virus isolate Sonson-tasajo (PLRV Sonson): AEB78367.1, Potato leafroll virus isolate Siachoque-jurubita (PLRV Jurubita): AEB78379.1, Potato leafroll virus isolate Pasto-el campanero2 (PLRV Pasto-el): AEB78377.1, Potato leafroll virus isolate Pasto-el campanero (PLRV Campa): AEB78375.1, Argentinian Potato leafroll virus isolate PLRV-5 (PLRV Argentina): ADE93558.1, Potato leafroll virus isolate Zipaquira-san Jorge (PLRV Zipaquir): AEB78361.1, Potato leafroll virus isolate Turmeque-siguineque2 (PLRV Turmeque): AEB78373.1, Potato leafroll virus isolate Ipiales-suras (PLRV Suras): AEB78385.1, France Potato leafroll virus strain CIP01 (PLRV CIP01): AAL77949.1, Potato leafroll virus isolate PLRV184 (PLRV 184): AYA73306.1, Potato leafroll virus isolate Villapinzon-bosabita (PLRV Villapinzo): AEB78359.1, Potato leafroll virus isolate Madrid-los arboles2 (PLRV Arboles): AEB78369.1, Potato leafroll virus isolate TZ:LR3M:11 (PLRV TZ:LR3M): AGN48055.1, Potato leafroll virus isolate PLRV171 (PLRV Canada): AYA73300.1, Colombia Potato leafroll virus isolate PLRV165 (PLRV Colombia): AYA73294.1, Tamil nadu Potato leafroll virus isolate OTNI-2 (PLRV Tamilnadu): AFJ11889.1, Jalandgar Potato leafroll virus isolate (PLRV Jalandgar): AFJ11863.1, Karlovce Potato leafroll virus isolate VIRUBRA 1/045 (PLRV VIRUBRA45): ACD93696.1, Australia Potato leafroll virus (PLRV Australia): QBO24572.1, Shimla Potato leafroll virus isolate PBI-6 (PLRV Shimla): AFJ11881.1, Potato leafroll virus (PLRV): QBO24572.1, Prague Potato leafroll virus isolate VIRUBRA 1/046 (PLRV VIRUBRA46): ACD93705.1, Potato leafroll virus isolate Say88 (PLRV Say88): QBO24572.1, Potato leafroll virus isolate Turmeque-siguineque3 (PLRV Siguineque3): AEB78383.1, Potato leafroll virus isolate Facatativa (PLRV Facatat): AEB78357.1, Germany Potato leafroll virus isolate PLRV-DSMZ (PLRV Germany): ADE93559.1, Argentinian Potato leafroll virus isolate PLRV-5 (PLRV Argentinian): ADE93558.1].

Techniques: Sequencing, Generated, Functional Assay, Virus

Journal: Scientific reports

Article Title: Inhibition of potato leafroll virus multiplication and systemic translocation by siRNA constructs against putative ATPase fold of movement protein.

doi: 10.1038/s41598-020-78791-4

Figure Lengend Snippet: Figure 2. (A) SDS-PAGE analysis of affinity purified his-tagged PLRV_MP expressed in E. coli. Lane 1: Purified protein and lane 2: Marker. (B) Purified MP exhibits basal and DNA-stimulated ATPase activity. (C) Apart from this, MP also exhibits RNA stimulated ATPase activity. Protein concentrations were 2 μM and 10 μM. Only protein is indicated with light grey and protein + dsDNA is indicated with dark grey. The vertical bars represent the mean values of three replicates (n = 3) and the values are highly significant exhibited at 0.001 level. DNA/ RNA concentration was maintained constant at 40 ng for every independent reaction. Negative controls with only buffer were also taken into account while performing the reactions.

Article Snippet: [Accession Numbers: Potato leafroll virus isolate PLRV-JF (PLRV JF): ADE93562.1, Potato leafroll virus isolate Anoquia (PLRV Antioquia): QES86451.1, Potato leafroll virus isolate Sta Rosa de Osos-santana (PLRV Santana): AEB78381.1, Potato leafroll virus isolate Turmeque-siguineque (PLRV Tur-sig): AEB78353.1, Potato leafroll virus isolate Sonson-tasajo (PLRV Sonson): AEB78367.1, Potato leafroll virus isolate Siachoque-jurubita (PLRV Jurubita): AEB78379.1, Potato leafroll virus isolate Pasto-el campanero2 (PLRV Pasto-el): AEB78377.1, Potato leafroll virus isolate Pasto-el campanero (PLRV Campa): AEB78375.1, Argentinian Potato leafroll virus isolate PLRV-5 (PLRV Argentina): ADE93558.1, Potato leafroll virus isolate Zipaquira-san Jorge (PLRV Zipaquir): AEB78361.1, Potato leafroll virus isolate Turmeque-siguineque2 (PLRV Turmeque): AEB78373.1, Potato leafroll virus isolate Ipiales-suras (PLRV Suras): AEB78385.1, France Potato leafroll virus strain CIP01 (PLRV CIP01): AAL77949.1, Potato leafroll virus isolate PLRV184 (PLRV 184): AYA73306.1, Potato leafroll virus isolate Villapinzon-bosabita (PLRV Villapinzo): AEB78359.1, Potato leafroll virus isolate Madrid-los arboles2 (PLRV Arboles): AEB78369.1, Potato leafroll virus isolate TZ:LR3M:11 (PLRV TZ:LR3M): AGN48055.1, Potato leafroll virus isolate PLRV171 (PLRV Canada): AYA73300.1, Colombia Potato leafroll virus isolate PLRV165 (PLRV Colombia): AYA73294.1, Tamil nadu Potato leafroll virus isolate OTNI-2 (PLRV Tamilnadu): AFJ11889.1, Jalandgar Potato leafroll virus isolate (PLRV Jalandgar): AFJ11863.1, Karlovce Potato leafroll virus isolate VIRUBRA 1/045 (PLRV VIRUBRA45): ACD93696.1, Australia Potato leafroll virus (PLRV Australia): QBO24572.1, Shimla Potato leafroll virus isolate PBI-6 (PLRV Shimla): AFJ11881.1, Potato leafroll virus (PLRV): QBO24572.1, Prague Potato leafroll virus isolate VIRUBRA 1/046 (PLRV VIRUBRA46): ACD93705.1, Potato leafroll virus isolate Say88 (PLRV Say88): QBO24572.1, Potato leafroll virus isolate Turmeque-siguineque3 (PLRV Siguineque3): AEB78383.1, Potato leafroll virus isolate Facatativa (PLRV Facatat): AEB78357.1, Germany Potato leafroll virus isolate PLRV-DSMZ (PLRV Germany): ADE93559.1, Argentinian Potato leafroll virus isolate PLRV-5 (PLRV Argentinian): ADE93558.1].

Techniques: SDS Page, Affinity Purification, Purification, Marker, Activity Assay, Concentration Assay

Journal: Scientific reports

Article Title: Inhibition of potato leafroll virus multiplication and systemic translocation by siRNA constructs against putative ATPase fold of movement protein.

doi: 10.1038/s41598-020-78791-4

Figure Lengend Snippet: Figure 3. Reverse-transcription polymerase chain reaction (RT-PCR) was performed to amplify sense and antisense PLRV_MP sequences from the leaves of infected potato plants. (A) Amplified cDNA fragments were analyzed by electrophoresis on 0.8% agarose gel. M: 100 bp DNA ladder; lanes 1 and 2: PCR products of sense and antisense MP (471 bp). (B) The pHANNIBAL plasmids were purified from E. coli DH5a cultures and analyzed by electrophoresis on 0.8% agarose gel. M: 1 kb DNA ladder; lanes 1: pHANNIBAL plasmid (5.83 kb). (C) The pART27 binary plasmid was purified from E. coli DH5a culture and analyzed by electrophoresis on 0.8% agarose gel. M: 1 kb DNA ladder; lane 1: pART27 plasmid (11.6 kb). (D) Confirmation of siRNA constructs in pHANNIBAL and pART27 binary vector through restriction analysis. M: 1 kb DNA ladder; lane 1: undigested pHANNIBAL; lane 2: antisense MP ligated with pHANNIBAL (pHANNIBAL-antisense MP construct); lane 3: both antisense and sense MP ligated with pHANNIBAL (pHANNIBAL-MP-siRNA construct); lane 4: HindIII and BamHI digestion of pHANNIBAL- antisense MP construct shows the release of a 471 bp fragment; lane 5: XhoI and KpnI digestion of pHANNIBAL-MP siRNA construct also shows the release of 471 bp fragments; lane 6: XhoI and BamHI digestion of pHANNIBAL-MP siRNA construct shows the release of a ~ 1500 bp fragment; and lane 7: NotI digestion of pHANNIBAL-MP siRNA construct shows the release of two fragments of ~ 4 kb (including sense MP, intron, and antisense MP sequence) and 3.5 kb. (E) The whole siRNA cassette (~ 4 kb) was transferred from pHANNIBAL to pART27, and this was further confirmed by NotI restriction analysis. (F) Schematic representation of pART27-MP siRNA constructs used for transient expression by agroinfiltration.

Article Snippet: [Accession Numbers: Potato leafroll virus isolate PLRV-JF (PLRV JF): ADE93562.1, Potato leafroll virus isolate Anoquia (PLRV Antioquia): QES86451.1, Potato leafroll virus isolate Sta Rosa de Osos-santana (PLRV Santana): AEB78381.1, Potato leafroll virus isolate Turmeque-siguineque (PLRV Tur-sig): AEB78353.1, Potato leafroll virus isolate Sonson-tasajo (PLRV Sonson): AEB78367.1, Potato leafroll virus isolate Siachoque-jurubita (PLRV Jurubita): AEB78379.1, Potato leafroll virus isolate Pasto-el campanero2 (PLRV Pasto-el): AEB78377.1, Potato leafroll virus isolate Pasto-el campanero (PLRV Campa): AEB78375.1, Argentinian Potato leafroll virus isolate PLRV-5 (PLRV Argentina): ADE93558.1, Potato leafroll virus isolate Zipaquira-san Jorge (PLRV Zipaquir): AEB78361.1, Potato leafroll virus isolate Turmeque-siguineque2 (PLRV Turmeque): AEB78373.1, Potato leafroll virus isolate Ipiales-suras (PLRV Suras): AEB78385.1, France Potato leafroll virus strain CIP01 (PLRV CIP01): AAL77949.1, Potato leafroll virus isolate PLRV184 (PLRV 184): AYA73306.1, Potato leafroll virus isolate Villapinzon-bosabita (PLRV Villapinzo): AEB78359.1, Potato leafroll virus isolate Madrid-los arboles2 (PLRV Arboles): AEB78369.1, Potato leafroll virus isolate TZ:LR3M:11 (PLRV TZ:LR3M): AGN48055.1, Potato leafroll virus isolate PLRV171 (PLRV Canada): AYA73300.1, Colombia Potato leafroll virus isolate PLRV165 (PLRV Colombia): AYA73294.1, Tamil nadu Potato leafroll virus isolate OTNI-2 (PLRV Tamilnadu): AFJ11889.1, Jalandgar Potato leafroll virus isolate (PLRV Jalandgar): AFJ11863.1, Karlovce Potato leafroll virus isolate VIRUBRA 1/045 (PLRV VIRUBRA45): ACD93696.1, Australia Potato leafroll virus (PLRV Australia): QBO24572.1, Shimla Potato leafroll virus isolate PBI-6 (PLRV Shimla): AFJ11881.1, Potato leafroll virus (PLRV): QBO24572.1, Prague Potato leafroll virus isolate VIRUBRA 1/046 (PLRV VIRUBRA46): ACD93705.1, Potato leafroll virus isolate Say88 (PLRV Say88): QBO24572.1, Potato leafroll virus isolate Turmeque-siguineque3 (PLRV Siguineque3): AEB78383.1, Potato leafroll virus isolate Facatativa (PLRV Facatat): AEB78357.1, Germany Potato leafroll virus isolate PLRV-DSMZ (PLRV Germany): ADE93559.1, Argentinian Potato leafroll virus isolate PLRV-5 (PLRV Argentinian): ADE93558.1].

Techniques: Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Infection, Amplification, Electrophoresis, Agarose Gel Electrophoresis, Purification, Plasmid Preparation, Construct, Sequencing, Expressing

Journal: Scientific reports

Article Title: Inhibition of potato leafroll virus multiplication and systemic translocation by siRNA constructs against putative ATPase fold of movement protein.

doi: 10.1038/s41598-020-78791-4

Figure Lengend Snippet: Figure 4. (A) Symptoms observed in the tertiary leaves of the PLRV infected potato plants. (1) PLRV infected control without agroinfiltration; (2) agroinfiltrated with the empty vector pART27; (3) agroinfiltrated with the plasmid containing only antisense sequence (pART27-antisense MP); (4) agroinfiltrated with the plasmid containing only sense sequence (pART27-sense MP); (5) agroinfiltrated with the pART27-MP siRNA construct; (B) Confirmation of expression of siRNA using Northern blotting analysis. Higher expression of siRNA in the leaves at 10 dpi (lane 1) was observed as compared to 5 dpi (lane 2), whereas empty vector (mock) agro-infected leaves did not show any siRNA (lane 3). (C) Detection of PLRV RNA by RT-PCR. Amplified cDNA fragments (627 bp) were analyzed by electrophoresis on 0.8% agarose gel. M: 100 bp marker; lane 1: PLRV was observed in control plant; lane 2: PLRV from tertiary leaves of the plant containing the empty vector pART26; lane 3: PLRV from tertiary leaves of the plant containing the antisense construct (pART27-antisense MP); lane 4: PLRV from tertiary leaves of the plant containing the sense construct (pART27-sense MP); lane 5: no PLRV in tertiary leaves of the plant containing MP siRNA (pART27-MP). (D) Actin PCR was performed as an internal control.

Article Snippet: [Accession Numbers: Potato leafroll virus isolate PLRV-JF (PLRV JF): ADE93562.1, Potato leafroll virus isolate Anoquia (PLRV Antioquia): QES86451.1, Potato leafroll virus isolate Sta Rosa de Osos-santana (PLRV Santana): AEB78381.1, Potato leafroll virus isolate Turmeque-siguineque (PLRV Tur-sig): AEB78353.1, Potato leafroll virus isolate Sonson-tasajo (PLRV Sonson): AEB78367.1, Potato leafroll virus isolate Siachoque-jurubita (PLRV Jurubita): AEB78379.1, Potato leafroll virus isolate Pasto-el campanero2 (PLRV Pasto-el): AEB78377.1, Potato leafroll virus isolate Pasto-el campanero (PLRV Campa): AEB78375.1, Argentinian Potato leafroll virus isolate PLRV-5 (PLRV Argentina): ADE93558.1, Potato leafroll virus isolate Zipaquira-san Jorge (PLRV Zipaquir): AEB78361.1, Potato leafroll virus isolate Turmeque-siguineque2 (PLRV Turmeque): AEB78373.1, Potato leafroll virus isolate Ipiales-suras (PLRV Suras): AEB78385.1, France Potato leafroll virus strain CIP01 (PLRV CIP01): AAL77949.1, Potato leafroll virus isolate PLRV184 (PLRV 184): AYA73306.1, Potato leafroll virus isolate Villapinzon-bosabita (PLRV Villapinzo): AEB78359.1, Potato leafroll virus isolate Madrid-los arboles2 (PLRV Arboles): AEB78369.1, Potato leafroll virus isolate TZ:LR3M:11 (PLRV TZ:LR3M): AGN48055.1, Potato leafroll virus isolate PLRV171 (PLRV Canada): AYA73300.1, Colombia Potato leafroll virus isolate PLRV165 (PLRV Colombia): AYA73294.1, Tamil nadu Potato leafroll virus isolate OTNI-2 (PLRV Tamilnadu): AFJ11889.1, Jalandgar Potato leafroll virus isolate (PLRV Jalandgar): AFJ11863.1, Karlovce Potato leafroll virus isolate VIRUBRA 1/045 (PLRV VIRUBRA45): ACD93696.1, Australia Potato leafroll virus (PLRV Australia): QBO24572.1, Shimla Potato leafroll virus isolate PBI-6 (PLRV Shimla): AFJ11881.1, Potato leafroll virus (PLRV): QBO24572.1, Prague Potato leafroll virus isolate VIRUBRA 1/046 (PLRV VIRUBRA46): ACD93705.1, Potato leafroll virus isolate Say88 (PLRV Say88): QBO24572.1, Potato leafroll virus isolate Turmeque-siguineque3 (PLRV Siguineque3): AEB78383.1, Potato leafroll virus isolate Facatativa (PLRV Facatat): AEB78357.1, Germany Potato leafroll virus isolate PLRV-DSMZ (PLRV Germany): ADE93559.1, Argentinian Potato leafroll virus isolate PLRV-5 (PLRV Argentinian): ADE93558.1].

Techniques: Infection, Control, Plasmid Preparation, Sequencing, Construct, Expressing, Northern Blot, Reverse Transcription Polymerase Chain Reaction, Amplification, Electrophoresis, Agarose Gel Electrophoresis, Marker