Fig 1.
Localisation of Csy4*-GFP fusions in the absence of tagged target RNAs.
(A) C-terminal GFP fusion without a nuclear localisation signal (Csy4*-GFP) shows nuclear and cytoplasmic localisation, including cytoplasmic motile granules (arrow heads). Inset shows enrichment in nucleolus (arrow). (B) Csy4*-GFP motile granules co-localise with periphery of peroxisomes labelled with RFP-SRL (Ser-Arg-Leu) (arrow heads). (C) C-terminal GFP-fusion of Csy4*[mut] (R114/115/118/119A) shows nuclear and cytoplasmic localisation with no motile granules. Inset shows depletion from nucleolus (arrow). All images are maximum intensity z-projections. GFP fluorescence shown green, RFP fluorescence shown magenta and chlorophyll auto-fluorescence shown blue. Scale bars, 10 µm.
Fig 2.
Fluorescent Csy4* fusions label PVX replication sites.
(A) Un-targeted Csy4*-GFP decorates vRNA in the perinuclear ‘X-body’ of PVX tagged with four cognate csy stem-loops. Some of the vRNA is arranged in circular ‘whorls’ with dark centres (arrow heads), as previously described [53]. Faint GFP fluorescence is visible in the nucleoplasm, enriched in the nucleolus (arrow). (B) Nuclear-targeted Csy4*-NLS-GFP similarly labels vRNA with ‘whorls’ in the ‘X-body’ of a four stem-loop tagged PVX. (C) Csy4*-GFP shows a similar ‘X-body’ localisation when the PVX genome is tagged with only two csy stem-loops. (D) When the PVX vRNA is untagged, Csy4*-GFP localisation within the ‘X-body’ is more diffuse, though very weak association with ‘whorls’ can sometimes still be identified (arrow head). By contrast, the nuclear and nucleolar (arrow) signal is comparatively stronger. (RFP channel not shown to more clearly show faint ‘whorls’). GFP fluorescence shown green, RFP fluorescence shown magenta. All images are single z-sections. Scale bars, 10 µm.
Fig 3.
A Csy4*-GFP fusion labels PVX vRNA inside plasmodesmata.
(A, B) Cell wall boundary between two epidermal cells infected with PVX.4xcsy.RFP-2A-CP and expressing Csy4*-GFP. (A) RFP and GFP channel. RFP-2A-CP expressed from the virus labels plasmodesmata. (B) GFP channel only. Csy4*-GFP co-localises with red plasmodesmata and can be seen extending into the inter-cellular wall space (arrow heads). (C, D) Higher magnification image of a cell boundary showing red CP fusion in the dark cell wall space between peripheral cytoplasms of neighbouring cells. (C) RFP and GFP channels. (D) GFP channel only. Csy4*-GFP can clearly be seen to be concentrated in the inter-cellular cell wall space, i.e. the inside of plasmodesmata (arrow heads). (E, F) With an untagged PVX.RFP-2A-CP, co-localisation of Csy4*-GFP with CP in plasmodesmata was rarely observed, and GFP enrichment in PD compared to adjacent cytoplasm was weaker than for a tagged virus (F, arrow heads). GFP fluorescence shown green, RFP fluorescence shown magenta. All images are single z-sections. Scale bars, 10 µm.
Fig 4.
Only Csy4*-GFP detects TMV vRNA containing two binding sites inside plasmodesmata.
(A, B) Csy4*-GFP system. (C, D) PUM-BiFC system. (E, F) MS2CP-GFP system. (G, H) λN22-GFP system. (A, C, E, G) Representative images of TMV VRCs near the nucleus. Note that Csy4*-GFP and λN22-GFP are enriched in the nucleolus, whilst PUM-BiFC is depleted and MS2CP neither enriched nor depleted compared with the nucleoplasm (arrows). (B, D, F, H) Representative images of cell boundaries at the leading edge of viral lesions. Only Csy4*-GFP shows enrichment in punctate structures within the non-fluorescent cell wall layer between adjacent cells, corresponding to plasmodesmata. Each image shows the GFP channel on the left, RFP channel in the middle and merged GFP and RFP channels on the right. GFP fluorescence shown green, RFP fluorescence shown magenta. Panels (A, C, E, G) are maximum intensity z-projections, panels (B, D, F, H) are individual z-sections. Scale bars, 10 µm.
Fig 5.
12xcsy stem-loop tags cause a moderate reduction in PVX infectivity.
(A) Representative (i.e., close to mean lesion size) infection site of PVX.2xcsy.RFP-2A-CP on inoculated leaf, 6 days post inoculation (dpi). (B) Representative (i.e., close to mean lesion size) infection site of PVX.12xcsy.RFP-2A-CP on inoculated leaf, 6 dpi. (C) Box plot of lesion sizes on inoculated leaves, 6 dpi. 2x: PVX.2xcsy.RFP-2A-CP (143 infection sites), 12x: PVX.12xcsy.RFP-2A-CP (85 infection sites). Bold line represents median, box 25% quartile, bars standard deviation (SD) (t-test: n = 3, p = 0.04044, * = significant). (D) Representative (i.e., close to mean fluorescence intensity) systemic infection site of PVX.2xcsy.RFP-2A-CP, 14 dpi. (E) Representative (i.e., close to mean fluorescence intensity) systemic infection site of PVX.12xcsy.RFP-2A-CP, 14 dpi. (F) Box plot of fluorescence intensities on systemically infected leaves, 14 dpi. 2x: PVX.2xcsy.RFP-2A-CP, 12x: PVX.12xcsy.RFP-2A-CP (36 leaves each). Bold line represents median, box 25% quartile, bars SD (t-test: n = 3, p = 0.01957, * = significant). RFP fluorescence shown magenta. All images are maximum intensity z-projections. Scale bars, 1 mm. (G) RT-PCR analysis of systemically infected leaves at 14 dpi. A segment of the PVX genome from the end of the TGB3 ORF to the beginning of the CP ORF was amplified. Arrow heads on right indicate expected product sizes for 2x and 12x tagged viruses, respectively. 1 to 18: three biological replicates with six plants each. +: positive control, plasmids used for inoculation were amplified with the same primers and a mixture of products from 2x and 12x tagged constructs loaded as size standard.
Fig 6.
PVX VRCs are recruited adjacent to plasmodesmata when intercellular movement is complemented by TMV 30k protein.
(A) Fluorescent infection site of PVX.ΔTGB.30k.4xcsy.RFP-2A-CP. (B) Localisation of a PVX replicase (165k)-GFP fusion to the VRC in a PVX.ΔTGB.30k.4xcsy.RFP-2A-CP infected cell. Circle indicates location of nucleus. (C) Localisation of vRNA labeled by Csy4*-GFP within the VRC in a PVX.ΔTGB.30k.4xcsy.RFP-2A-CP infected cell. No circular arrangement of vRNA in ‘whorls’ observed. Circle indicates location of nucleus. (D) Csy4*-GFP labels vRNA of PVX.ΔTGB.30k.4xcsy.RFP-2A-CP inside plasmodesmata (arrow heads), but unlike during native PVX transport, RFP-2A-CP is not co-enriched within plasmodesmata. (E) Csy4*-GFP vRNA imaging at the leading edge of a PVX.ΔTGB.30k.4xcsy.RFP-2A-CP lesion. Yellow arrow indicates approximate direction of viral spread. The inset is a separate higher magnification image acquired at the boxed region. vRNA can be observed in ‘caps’ at plasmodesmata entrances, as well as inside the channels labelled with 30k-CFP fusion expressed in trans (arrow heads). (A full maximum projection of the same image is shown in S4A and S4B Fig to confirm the presence of virus-expressed RFP in the cell on the left.) (F) 165k-GFP replicase marker located in ‘caps’ at the entrances of plasmodesmata labelled with aniline blue-staining of callose. (The full image from which panel F is derived is presented in S4C and S4D Fig and shows multiple similar ‘caps’ containing 165k and CP.) GFP fluorescence shown green, RFP fluorescence shown magenta, CFP and aniline blue fluorescence shown cyan. Panels (A-C) are maximum intensity z-projections, (E) is a projection of two z-sections and (D, E inset and F) are single z-sections. Scar bars, 100 µm (A), 10 µm (B-C, E-F), 5 µm (D).
Fig 7.
‘Self-tracking’ PVX constructs demonstrate preferential access of Csy4*-GFP expressed from the viral genome in cis.
(A) Representative Western blot of total protein from leaf systemically infected with untagged PVX.Csy4*-GFP-T2A-CP and probed with antibody against GFP. Black arrow heads indicate expected sizes of free GFP, GFP-T2A and Csy4*-GFP-T2A fusion proteins. White arrow head indicates expected size of Csy4*-GFP-T2A-CP fusion. (B-E) Untagged PVX.Csy4*-GFP-T2A-CP. (B) Representative infection site; (C) VRC; (D) vRNA inside plasmodesmata (arrow heads). (E) Leading edge of an infection site, yellow arrow indicates approximate direction of viral spread. vRNA can be observed in plasmodesmata projecting from the most recently infected cell (arrow heads). (F-H) PVX.2xcsy.Csy4*-GFP-T2A-CP tagged directly upstream of the Csy4* ORF. (F) Representative single-celled infection site; (G) VRC; (H) vRNA inside plasmodesmata (arrow heads). (I-K) PVX.Csy4*-GFP-T2A-CP.2xcsy tagged immediately downstream of the CP ORF. (I) Representative small multi-cellular infection site; (J) VRC; (K) vRNA inside plasmodesmata (arrow heads). (L) Schematic representation of ‘self-tracking’ PVX constructs (not to scale). (M) Comparison of lesion sizes of untagged PVX.Csy4*-GFP-T2A-CP (0x; 118 infection sites) and upstream-tagged PVX.2xcsy.Csy4*-GFP-T2A-CP (2x [u.s.]; 25 infection sites). Bold line represents median, box 25% quartile, bars SD (t-test: n = 3, p = 0.0009156, *** = significant). (N) Comparison of lesion sizes of untagged PVX.Csy4*-GFP-T2A-CP (0x; 134 infection sites) and downstream-tagged PVX.Csy4*-GFP-T2A-CP.2xcsy (2x [d.s.]; 24 infection sites). (t-test: n = 3, p = 0.02117, * = significant). Experiments shown in panels M and N were conducted separately with identical conditions (inoculating first two true leaves of four week-old plants, leaves imaged at 6 dpi), but at different times of the year, and are therefore presented and analysed separately. Note that the upstream-tagged virus produced mostly (17/25 = 68%) single-celled infection sites when the untagged control had larger lesions (F, M), whilst the downstream-tagged virus produced mostly (18/24 = 75%) small multicellular infection sites even though the control had smaller lesions at this time (I, N). All images are maximum intensity z-projections with the exception of (D, G, H, K) which are individual z-sections. Scale bars, 100 µm (B, F, I), 10 µm (C-E, G-H, J-K).