Journal: Microbiology Spectrum

Article Title: Citrus psorosis virus 24K protein inhibits the processing of miRNA precursors by interacting with components of the biogenesis machinery

doi: 10.1128/spectrum.03513-23

Figure Lengend Snippet: Subcellular colocalization of 24K and miRNA biogenesis proteins. ( A ) (i) Colocalization of 24K:mRFP with CFP:AtDCL1 in nuclear aggregates. ( B ) (i) Colocalization of 24K:mRFP with AtHYL1:YFP in nuclear aggregates. ( C ) (i) Colocalization of 24K:mRFP with AtSE:YFP in nuclear aggregates. (ii) The mRFP control does not colocalize in nuclear aggregates with any of the tested proteins. ( D ) Western blot analyses of extracts from N. benthamiana leaves in colocalization assays at 3 dpa. Anti-RFP (@RFP) or anti-GFP (@GFP) monoclonal antibodies were used for 24K or biogenesis proteins, respectively. Coomassie blue stain is shown as loading control. The numbers below correspond to normalized band density. Ratios between AtDCL and AtDCL1/24K (240 kDa); AtHYL1 and AtHYL1/24K (75 kDa); AtSE and AtSE/24K (110 kDa) are shown under the lines. Scale bar: 10 µm.

Article Snippet: GFP (or its variants, CFP or YFP) and RFP fusion proteins were detected with anti-GFP (3H9) monoclonal antibody (Chromotek, Germany) and anti-RFP (6G6) monoclonal antibody (Chromotek, Germany), respectively.

Techniques: Control, Western Blot, Bioprocessing, Staining

Journal: Microbiology Spectrum

Article Title: Citrus psorosis virus 24K protein inhibits the processing of miRNA precursors by interacting with components of the biogenesis machinery

doi: 10.1128/spectrum.03513-23

Figure Lengend Snippet: Interaction between 24K protein and miRNA biogenesis proteins. ( A ) In vivo analysis of BiFC assays in epidermal cells of N. benthamiana plants. The merge panels show positive interaction between N-mCitrine:24K and C-mCitrine:AtHYL1 (i) and N-mCitrine:24K and C-mCitrine:AtSE (ii) and no interaction between N-mCitrine:24K with C-mCitrine:AtDCL1 (iii). (iv). Negative control: N-mCitrine:24K + C-mCitrine (empty vector). Chloroplasts were marked in blue in the case of the negative interactions. In all cases, Fib:mRFP was used as an expression control of infiltrated samples. Scale bar: 10 µm. ( B ) Co-IP assay between 24K:mRFP and AtHYL1. INPUT and UNBOUND controls correspond to input and output fractions without binding to bead system, respectively. The IP fraction corresponds to immunoprecipitated proteins. (−) corresponds to the negative control using beads without @HYL1 antibody. (+) corresponds to the immunoprecipitated samples with @HYL1 attached to the beads. The anti-RFP (@RFP) and anti-HYL1 (@HYL1) monoclonal antibodies were used in each case to develop the blots. The presence of both proteins in the IP fraction indicates a positive interaction. ( C ) Co-IP assay between the 24K:mRFP and AtSE:YFP protein. The INPUT and UNBOUND controls correspond to input and output fractions without binding to bead system, respectively. The IP fraction corresponds to immunoprecipitated proteins with @GFP attached to the beads. Free mRFP was used as a negative control. The anti-RFP (@RFP) and anti-GFP (@GFP) antibodies were used in each case to develop the blots. The presence of both proteins in IP fraction indicates a positive interaction.

Article Snippet: GFP (or its variants, CFP or YFP) and RFP fusion proteins were detected with anti-GFP (3H9) monoclonal antibody (Chromotek, Germany) and anti-RFP (6G6) monoclonal antibody (Chromotek, Germany), respectively.

Techniques: In Vivo, Negative Control, Plasmid Preparation, Expressing, Control, Co-Immunoprecipitation Assay, Binding Assay, Immunoprecipitation, Bioprocessing

Journal: Journal of Virology

Article Title: A Lysine Residue Essential for Geminivirus Replication Also Controls Nuclear Localization of the Tomato Yellow Leaf Curl Virus Rep Protein

doi: 10.1128/JVI.01910-18

Figure Lengend Snippet: Nuclear localization of Rep from two TYLCV strains is controlled by the conserved lysines. (A) Subcellular localization of RFP-tagged RepTYLCV (TYLCV-Alb13) variants in N. benthamiana upon transient expression with Agrobacterium. Arrowheads indicate fluorescence in the cytoplasm, and asterisks mark nonfluorescent or weakly fluorescent nuclei. Scale bars represent 5 μm. (B) Immunoblot of the Rep-RFP fusion proteins upon transient expression in N. benthamiana. Proteins were detected with an anti-RFP antibody. To demonstrate equal protein loading, the membranes were stained with Ponceau S. (C) Micrographs of epidermal leaf cells transiently expressing RepTYLCV K-to-A triple mutant-RFP and stained with ER-Tracker Green. From the left in order: RFP channel, ER-Tracker, merge of RFP and ER-Tracker, bright field, and graph representing the normalized fluorescence intensity of RFP and ER-Tracker (GFP) along the lines in the micrographs. Note that the two signals are shifted and do not overlap, indicating that they localize in different positions. Scale bars represent 50 μm. (D) Subcellular localization of RFP-tagged Rep TYLCV-Almeria WT and K-to-A variants in N. benthamiana. For each sample, one representative epidermal cell is shown with a 4× zoom of its nucleus; arrowheads indicate fluorescence in the cytoplasm, and asterisks mark nonfluorescent or weakly fluorescent nuclei. The scale bars represent 5 μm. (E) Box plot showing the RFP fluorescence intensity ratio in the cytoplasm versus nucleus for the images shown in panel D; a total of 16 cells were analyzed. (F) Box plot depicting the RFP fluorescence intensity ratio in the cytoplasm versus nucleus for the images shown in panel A; a total of 8 cells per sample were analyzed. The statistical analysis used is described in the legend to Fig. 2.

Article Snippet: Immunodetection of the proteins was performed according to standard protocols using anti-RFP antibody (Chromotek 6G6; 1:1,000) to detect the Rep-RFP fusion proteins and antihemagglutinin (anti-HA) antibody (Roche 3F10; 1:2,000) for Rep-SCFP C fusions as primary antibodies and anti-rat (Pierce 31470; 1:10,000) or anti-mouse (Pierce 31430; 1:10,000) as secondary antibodies.

Techniques: Expressing, Fluorescence, Western Blot, Staining, Mutagenesis