The ER-membrane transport system is critical for intercellular trafficking of the NSm movement protein and Tomato Spotted Wilt Tospovirus

Plant viruses move through plasmodesmata to infect new cells. The plant endoplasmic reticulum (ER) is interconnected among cells via the ER desmotubule in the plasmodesma across the cell wall, forming a continuous ER network throughout the entire plant. This ER continuity is unique to plants and has been postulated to serve as a platform for the intercellular trafficking of macromolecules. In the present study, the contribution of the plant ER membrane transport system to the intercellular trafficking of the NSm movement protein and Tomato spotted wilt tospovirus (TSWV) is investigated. We showed that TSWV NSm is physically associated with the ER membrane in Nicotiana benthamiana plants. An NSm-GFP fusion protein transiently expressed in single leaf cells was trafficked into neighboring cells. Mutations in NSm that impaired its association with the ER or caused its mis-localization to other subcellular sites inhibited cell-to-cell trafficking. Pharmacological disruption of the ER network severely inhibited NSm-GFP trafficking but not GFP diffusion. In the Arabidopsis thaliana mutant rhd3 with an impaired ER network, NSm-GFP trafficking was significantly reduced, whereas GFP diffusion was not affected. We also showed that the ER-to-Golgi secretion pathway and the cytoskeleton transport systems were not involved in the intercellular trafficking of TSWV NSm. Importantly, TSWV cell-to-cell spread was delayed in the ER-defective rhd3 mutant, and this reduced viral infection was not due to reduced replication. On the basis of robust biochemical, cellular and genetic analysis, we established that the ER membrane transport system serves as an important direct route for intercellular trafficking of NSm and TSWV

Characterization of the inner membrane protein BB0173 from Borrelia burgdorferi

Abstract Background The bacterial spirochete Borrelia burgdorferi is the causative agent of the most commonly reported arthropod-borne illness in the United States, Lyme disease. A family of proteins containing von Willebrand Factor A (VWFA) domains adjacent to a MoxR AAA+ ATPase have been found to be highly conserved in the genus Borrelia. Previously, a VWFA domain containing protein of B. burgdorferi, BB0172, was determined to be an outer membrane protein capable of binding integrin α3β1. In this study, the characterization of a new VWFA domain containing membrane protein, BB0173, is evaluated in order to define the location and topology of this multi-spanning membrane protein. In addition, functional predictions are made. Results Our results show that BB0173, in contrast to BB0172, is an inner membrane protein, in which the VWFA domain is exposed to the periplasmic space. Further, BB0173 was predicted to have an aerotolerance regulator domain, and expression of BB0173 and the surrounding genes was evaluated under aerobic and microaerophilic conditions, revealing that these genes are downregulated under aerobic conditions. Since the VWFA domain containing proteins of B. burgdorferi are highly conserved, they are likely required for survival of the pathogen through sensing diverse environmental oxygen conditions. Conclusions Presently, the complex mechanisms that B. burgdorferi uses to detect and respond to environmental changes are not completely understood. However, studying the mechanisms that allow B. burgdorferi to survive in the highly disparate environments of the tick vector and mammalian host could allow for the development of novel methods of preventing acquisition, survival, or transmission of the spirochete. In this regard, a putative membrane protein, BB0173, was characterized. BB0173 was found to be highly conserved across pathogenic Borrelia, and additionally contains several truly transmembrane domains, and a Bacteroides aerotolerance-like domain. The presence of these functional domains and the highly conserved nature of this protein, strongly suggests a required function of BB0173 in the survival of B. burgdorferi

Viral systemic infection is significantly delayed in the nonbranched ER network <i>rhd3</i> mutant of <i>A</i>. <i>thaliana</i> compared with the Col-0 wild-type (WT).

<p>(A and B) Symptoms of WT and <i>rhd3-8</i> plants inoculated with mock (A) or TSWV (B) at 15 dpi. (C) Disease development was delayed in <i>rhd3-8</i> mutant compared with WT after inoculation with TSWV. (D) Immunoblots of extracts from leaves of WT and <i>rhd3-8</i> 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 <i>rhd3-8</i> plants was quantified.</p

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 <i>N</i>. <i>benthamiana</i>.

<p>(A-C) Subcellular localization of NSm^4A/5A mutant in epidermal cell. ER marker mCherry-HDEL was used for colocalization analysis. Bar, 10 μm. (D-F) Colocalization analysis of NSm^230A/232A mutant with ER labeled by mCherry-HDEL. Bar, 10 μm. (G-I) Cell-to-cell movement analysis of NSm^4A/5A (H) and NSm^230A/232A (I) mutants in <i>N</i>. <i>benthamiana</i> after bombardment. NSm^WT (G) was used as a positive control. Bar, 50 μm.</p

Impairment of membrane integration of NSm inhibits its cell-to-cell movement in leaf epidermis of <i>N</i>. <i>benthamiana</i>.

<p>(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 NSm^133-135D and NSm^177-179D 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 NSm^133-135D (D) and NSm^177-179D (E) mutant in <i>N</i>. <i>benthamiana</i> after bombardment. NSm^WT (C) was used as a positive control. Bar, 50 μm.</p

The NSm protein of tomato spotted wilt tospovirus (TSWV) is physically associated with cellular membranes.

<p>(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 <i>in vitro</i> 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 × <i>g</i> pellet (P30), 30,000×<i>g</i> supernatant (S30), 100,000×<i>g</i> pellet (P100) and 100,000×<i>g</i> 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.</p

Cell-to-cell trafficking of NSm is significantly reduced in the nonbranched ER network of the <i>rhd3</i> mutant of <i>A</i>. <i>thaliana</i> compared with the Col-0 wild-type (WT).

<p>(A and B) Morphology of the ER network labeled by mCherry-HDEL in WT (A) and <i>rhd3-8</i> (B), respectively. Bar, 10 μm. (C and D) Comparison of intercellular movement of NSm-GFP in WT and <i>rhd3-8</i> plants after bombardment. Bar, 20 μm.</p

TSWV NSm is localized with the ER and plasmodesmata (PD).

<p>(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. <i>N</i>. <i>benthamiana</i> 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.</p