Table 1.
List of primers used for bimolecular fluorescence complementation and pull down assays.
Table 2.
List of primers used for yeast-2-hybrid assays.
Fig 1.
Localization of Iris yellow spot virus (IYSV) nucleocapsid (N) protein in epidermal cells of uninfected transgenic Nicotiana benthamiana plants containing the red nuclear marker histone 2B (RFP-H2B).
Confocal micrographs represent IYSV fusion proteins to the C-terminus of green fluorescent protein (GFP). Columns from left to right show GFP-gene fusion or free GFP (1), RFP-H2B (2), and the overlay of the images (3). (a-c). IYSV N fusion with GFP in RFP-H2B plants; (d-f). Free GFP in RFP-H2B plants. Each micrograph represents minimum 50 cells that were examined for localization. Scale bar = 20μm.
Fig 2.
Localization of Iris yellow spot virus (IYSV) proteins, in epidermal cells of IYSV-infected transgenic Nicotiana benthamiana plants containing the red nuclear marker histone 2B (RFP-H2B), and red endoplasmic reticulum marker (ER-RFP).
Confocal micrographs represent IYSV fusion proteins to the C-terminus of green fluorescent protein (GFP). Columns from left to right show GFP-gene fusion (1), RFP-H2B (2), ER-RFP (3), and the overlay of the images (4). (a-d) GFP-IYSV N co-expression with RFP-H2B and ER-RFP; (e-h) GFP-IYSV NSm co-expression with RFP-H2B and ER-RFP; (i-l) GFP-IYSV NSs co-expression with RFP-H2B and ER-RFP; (m-p) GFP-IYSV GN co-expression with RFP-H2B and ER-RFP; (q-t) GFP-IYSV GC co-expression with RFP-H2B and ER-RFP. Each micrograph represents a minimum of 50 cells that were examined for localization. Scale bar = 20μm.
Fig 3.
In planta interactions of Iris yellow spot virus proteins as examined by bimolecular fluorescence complementation (BiFC) assay.
Interaction assays were performed in leaf epidermal cells of IYSV-infected transgenic Nicotiana benthamiana plants expressing cyan fluorescent protein fused to the nuclear marker histone 2B (CFP-H2B), and cyan endoplasmic reticulum (ER-CFP) marker. Column 1 shows BiFC, column 2 shows localization of CFP-H2B and ER-CFP (nucleus and ER), and column 3 shows a merge of all panels (overlay). The first and second proteins mentioned in each pair of interactors were expressed as C-terminal fusions to the amino-terminal half of YFP and as C-terminal fusions to the carboxy-terminal half of YFP respectively. A set of positive interactions is shown here after testing interactions in all pairwise combinations: (a-c) N/N, (d-f) NSm/ NSm, (g-i) NSm/N. Each micrograph represents a minimum of 50 cells that were examined for interaction. Scale bar = 20μm.
Fig 4.
Maltose binding protein pull-down assays of Iris yellow spot virus (IYSV) proteins.
Self-interaction of nucleocapsid; N (1) and movement; NSm (2) proteins, and cross-interaction of IYSV N and NSm proteins (3). ‘Load’ above the column represents the protein content initially loaded to the MBP column; ‘wash’ above the column comprises of the protein not adhering to the column; ‘elution’ was the material that attached to the column and was desorbed by the addition of maltose. Proteins were subjected to protein gel blot analysis and probed with anti-GST primary antibody. (1.a) MBP-tagged N mixed with GST-tagged N. (1.b) MBP alone mixed with GST-tagged N. (1.c) MBP-tagged N mixed with GST alone. (1.d) GST alone mixed with MBP alone. (1.e) The same combination as 1.a, except that proteins were first subjected to RNase treatment. (2.a) MBP-tagged NSm mixed with GST-tagged NSm. (2.b) MBP alone mixed with GST-tagged NSm. (2.c) MBP-tagged NSm mixed with GST alone. (2.d) The same combination as 2.a, except that proteins were first subjected to RNase treatment. (3.a) MBP-tagged N mixed with GST-tagged NSm.
Fig 5.
Self-interaction of Iris yellow spot virus (IYSV) nucleocapsid (N) protein.
(a) Schematic diagram of the constructs tested for leucine independent growth and β-galactosidase activity. Black box, LexA DBD in pEG202; hatched box, B42 TAD in pJG4-5; blue boxes, IYSV N (N1-full length, amino acids 1–273; N2, amino acids 1–90; N3, amino acids 91–180; N4, amino acids 181–220; N5, amino acids 221–273), white box, full length Cauliflower mosaic virus P6 (amino acids 1–520). Numbers to the left of each pair of constructions correspond to the β-galactosidase assays shown in (b). (b) β-galactosidase activity of yeast transformants expressing constructs as shown in (a).
Fig 6.
Self-interaction of Iris yellow spot virus (IYSV) movement protein.
(a) Schematic diagram of the constructs tested for leucine independent growth and β-galactosidase activity. Black box, LexA DBD in pEG202; hatched box, B42 TAD in pJG4-5; yellow boxes, IYSV NSm (NSm1-full length, amino acids 1–312; NSm2, amino acids 1–160; NSm3, amino acids 100–200; NSm4, amino acids 201–312); white box, full length Cauliflower mosaic virus P6 (amino acids 1–520). Numbers to the left of each pair of constructions correspond to the β-galactosidase assays shown in (b). (b) β-galactosidase activity of yeast transformants expressing constructs as shown in (a).
Fig 7.
Heterotypic interaction of Iris yellow spot virus (IYSV) nucleocapsid (N) and movement (NSm) proteins.
(a) Schematic diagram of the constructs tested for leucine independent growth and β-galactosidase activity. Black box, LexA DBD in pEG202; hatched box, B42 TAD in pJG4-5; blue boxes, IYSV N (N1-full length, amino acids 1–273; N2, amino acids 1–90; N3, amino acids 91–180; N4, amino acids 181–220; N5, amino acids 221–273); yellow boxes, IYSV NSm (NSm1-full length, amino acids 1–312; NSm2, amino acids 1–160; NSm3, amino acids 100–200; NSm4, amino acids 201–312). Numbers to the left of each pair of constructions correspond to the β-galactosidase assays shown in (b). (b) β-galactosidase activity of yeast transformants expressing constructs as shown in (a).