Fig 1.
The NSm protein of tomato spotted wilt tospovirus (TSWV) is physically associated with cellular membranes.
(A) Insertion assay of NSm hydrophobic regions 1 (HR1) and 2 (HR2) into microsomal membranes using the Lep’ construct. Schematic representation of the Lep-derived construct (Lep’) is shown in upper panel. In this Lep’ construct, H1, derived from the glycosylation acceptor site (G2) at the beginning of the P2 domain, will be modified only if the tested HR inserts into the membrane; the G1 site, embedded in an extended N-terminal sequence of 24 amino acids, is always glycosylated. Results of in vitro translation and membrane insertion experiments are shown in the lower panel. Bands of nonglycosylated protein are indicated by a white dot; singly and doubly glycosylated proteins are indicated by one and two black dots, respectively. The protected glycosylated HRs/P2 fragment is indicated by a black triangle. (B) Association of NSm with membrane factions. Total lysate (T) from TSWV-infected or NSm expressing leaves were fractionated into 30,000 × g pellet (P30), 30,000×g supernatant (S30), 100,000×g pellet (P100) and 100,000×g supernatant (S100), and analyzed by immunoblots using antibodies against NSm. The vacuolar H-ATPase (V-H-ATPase) subunit E, phosphoenolpyruvate carboxylase (PEPC) and the luminal binding protein (BiP) were used as a microsomal marker, soluble marker and ER marker, respectively, in the fractionation analysis. (C) Biochemical characterization of NSm associated with membranes. The P30 pellet fraction was treated with original lysis buffer, 0.1 M Na2CO3, 1 M KCl, or 4 M urea, respectively, then separated into supernatant (S30) and pellet (P30) fractions and analyzed by immunoblots using anti-NSm antibodies. (D) Membrane association analysis of TSWV NSm, PVX TGB2 and TGB3 after treatment with 7 M urea. The percentage of proteins eluted in the S30 supernatant or remaining in the P30 pellet, are given at the bottom of the corresponding lanes. (E) Triton X-114 partitioning analysis of TSWV NSm and TMV MP. P30 pellet, aqueous phases (AP) and organic phases (OP) were analyzed by immunoblotting using anti-NSm and anti-HA antibodies, respectively.
Fig 2.
TSWV NSm is localized with the ER and plasmodesmata (PD).
(A-C) Colocalization of NSm-YFP with the ER labeled by mCherry-HDEL at 28 h post infiltration (hpi). Bar, 10 μm. (D-F) Colocalization of NSm-YFP with PD labeled by TMV MP-mRFP at 28 hpi. Bar, 10 μm. Different single planes were used to focus on the ER membrane in panels A-C panels and on the periphery of the cell in panels D-F. (G-I) Plasmolysis assay for PD localization of NSm. N. benthamiana leaves were agroinfiltrated with NSm-YFP, then infiltrated with 10% NaCl at 28 h post agroinfiltration; plasmolyzed cells in the leaf were immediately examined using CLMS. The cell wall (CW) and cytoplasmic membrane (PM) after plasmolysis are marked, respectively, by a purple line and red line. (J) Cofractionation of NSm protein with ER. Extracts of plants transiently expressing NSm were centrifuged on a 20–60% sucrose gradient in the presence or absence of MgCl2. Fractions from top to bottom (1 to 14) were analyzed by immunoblots using anti-NSm, anti-BiP, anti-Arf1 and anti-PEPC antibodies to detect NSm, ER luminal protein, Golgi and soluble protein, respectively.
Fig 3.
Localization of NSm-GFP in the ER membrane network in bombarded cells with the fusion protein and in neighboring cells receiving the fusion protein in leaf epidermis of N. benthamiana.
(A) Scheme of cell-to-cell transport of NSm in leaf epidermis of N. benthamiana by bombardment. (B and C) Cell-to-cell movement of GFP-GFP (B) and NSm-GFP (C) in leaf epidermis of N. benthamiana. Bar, 50 μm. (D-Q) NSm-GFP was localized in the interconnected ER network in cells bombarded with the fusion protein and in neighboring cells that subsequently received the protein. A low magnification image to show that NSm moved intercellularly after bombardment (D). Bar, 20 μm. A region with three cells showing NSm movement (Cell 1 to Cell 3) in image D was magnified (boxed region) to show colocalization of NSm-GFP with the ER labeled by mCherry-HDEL (E). Bar, 10 μm. The boxed region in image E corresponding to the respective initially bombarded cell (Cell 1) and the second (Cell 2) and third layer (Cell 3) of cells into which NSm moved was further split into three channels to show colocalization of NSm-GFP with the ER labeled by mCherry-HDEL (Cell 1, F-H; Cell 2, J-L; Cell 3, N-P). Colocalization of NSm and ER in the respective cells was further analyzed by overlapping fluorescence spectra (I, M and Q). Bar, 10 μm.
Table 1.
Cell-to-cell movement of TSWV NSm in leaf epidermis of Nicotiana benthamiana after bombardment.
Fig 4.
Impairment of membrane integration of NSm inhibits its cell-to-cell movement in leaf epidermis of N. benthamiana.
(A) Diagram of amino acid residues for hydrophobic regions (HR1 to HR2) and two aspartate substitution mutants at sites of amino acids 133–135 and 177–179 in NSm. (B) Triton X-114 partitioning analysis of NSm133-135D and NSm177-179D aspartate substitution mutants. P30 pellet, aqueous phase (AP) and organic phase (OP) were analyzed by immunoblots using anti-NSm antibodies, respectively. (C-E) Cell-to-cell movement analysis of NSm133-135D (D) and NSm177-179D (E) mutant in N. benthamiana after bombardment. NSmWT (C) was used as a positive control. Bar, 50 μm.
Fig 5.
Mutations in NSm that blocked correct ER sorting or altered sorting to other subcellular localization inhibits NSm cell-to-cell movement in leaf epidermis of N. benthamiana.
(A-C) Subcellular localization of NSm4A/5A mutant in epidermal cell. ER marker mCherry-HDEL was used for colocalization analysis. Bar, 10 μm. (D-F) Colocalization analysis of NSm230A/232A mutant with ER labeled by mCherry-HDEL. Bar, 10 μm. (G-I) Cell-to-cell movement analysis of NSm4A/5A (H) and NSm230A/232A (I) mutants in N. benthamiana after bombardment. NSmWT (G) was used as a positive control. Bar, 50 μm.
Table 2.
BFA disruption of the interconnected ER network inhibited cell-to-cell movement of TSWV NSm in Nicotiana benthamiana.
Fig 6.
Cell-to-cell trafficking of NSm is significantly reduced in the nonbranched ER network of the rhd3 mutant of A. thaliana compared with the Col-0 wild-type (WT).
(A and B) Morphology of the ER network labeled by mCherry-HDEL in WT (A) and rhd3-8 (B), respectively. Bar, 10 μm. (C and D) Comparison of intercellular movement of NSm-GFP in WT and rhd3-8 plants after bombardment. Bar, 20 μm.
Table 3.
Cell-to-cell movement of TSWV NSm in WT (Col-0) and rhd3 mutant of Arabidopsis thaliana after bombardment.
Fig 7.
Viral systemic infection is significantly delayed in the nonbranched ER network rhd3 mutant of A. thaliana compared with the Col-0 wild-type (WT).
(A and B) Symptoms of WT and rhd3-8 plants inoculated with mock (A) or TSWV (B) at 15 dpi. (C) Disease development was delayed in rhd3-8 mutant compared with WT after inoculation with TSWV. (D) Immunoblots of extracts from leaves of WT and rhd3-8 plants after systemic infection with TSWV and probing with monoclonal antibodies against TSWV nucleocapsid at 15 dpi. Protein loading is indicated by Ponceau S staining. The accumulation of TSWV in WT and rhd3-8 plants was quantified.