Plant SNAREs SYP22 and SYP23 interact with Tobacco mosaic virus 126 kDa protein and SYP2s are required for normal local virus accumulation and spread.

Viral proteins often interact with multiple host proteins during virus accumulation and spread. Identities and functions of all interacting host proteins are not known. Through a yeast two-hybrid screen an Arabidopsis thaliana Qa-SNARE protein [syntaxin of plants 23 (AtSYP23)], associated with pre-vacuolar compartment and vacuolar membrane fusion activities, interacted with Tobacco mosaic virus (TMV) 126 kDa protein, associated with virus accumulation and spread. In planta, AtSYP23 and AtSYP22 each fused with mCherry, co-localized with 126 kDa protein-GFP. Additionally, A. thaliana and Nicotiana benthamiana SYP2 proteins and 126 kDa protein interacted during bimolecular fluorescence complementation analysis. Decreased TMV accumulation in Arabidopsis plants lacking SYP23 and in N. benthamiana plants subjected to virus-induced gene silencing (VIGS) of SYP2 orthologs was observed. Diminished TMV accumulation during VIGS correlated with less intercellular virus spread. The inability to eliminate virus accumulation suggests that SYP2 proteins function redundantly for TMV accumulation, as for plant development.

Development of an Expression Vector to Overexpress or Downregulate Genes in Curvularia protuberata.

Curvularia protuberata, an endophytic fungus in the Ascomycota, provides plants with thermotolerance only when it carries a mycovirus known as Curvularia thermotolerance virus (CThTV), and forms a three-way symbiotic relationship among these organisms. Under heat stress, several genes are expressed differently between virus-free C. protuberata (VF) and C. protuberata carrying CThTV (AN). We developed an expression vector, pM2Z-fun, carrying a zeocin resistance gene driven by the ToxA promoter, to study gene functions in C. protuberata to better understand this three-way symbiosis. Using this new 3.7-kb vector, five genes that are differentially expressed in C. protuberata—including genes involved in the trehalose, melanin, and catalase biosynthesis pathways—were successfully overexpressed or downregulated in VF or AN C. protuberata strains, respectively. The VF overexpression lines showed higher metabolite and enzyme activity than in the control VF strain. Furthermore, downregulation of expression of the same genes in the AN strain resulted in lower metabolite and enzyme activity than in the control AN strain. The newly generated expression vector, pM2Z-fun, has been successfully used to express target genes in C. protuberata and will be useful in further functional expression studies in other Ascomycota fungi.

MicroRNA-625 inhibits the proliferation and increases the chemosensitivity of glioma by directly targeting AKT2.

Glioma is a malignant tumor for which new therapies are needed. Growing evidence has demonstrated that microRNAs (miRNAs) have a major effect on glioma development. Here, we aimed to characterize a novel anti-cancer miRNA, miR-625, by investigating its expression, function, and mechanism of action in glioma progression. The expression of miR-625 and its target mRNA in human glioma tissues and cell lines was assessed by real-time PCR, western blotting, and immunohistochemistry. Functional significance was assessed by examining cell cycle progression, proliferation, apoptosis, and chemosensitivity to temozolomide in vitro, and by examining growth of subcutaneous glioblastoma in a mouse model in vivo. We found that miR-625 expression was significantly lower in human glioma samples and cell lines than in normal brain tissue and human astrocytes. Furthermore, miR-625 overexpression not only suppressed glioma cell proliferation in culture and in the tumor xenograft model but also induced cell cycle arrest and apoptosis. AKT2 was identified as a direct miR-625 target in glioma cell lines, and AKT2 overexpression reversed the suppressive effects of miR-625 in the cell lines and the tumor xenograft model. Finally, we found that the sensitivity of glioma cells to temozolomide was increased by miR-625 overexpression, and this was reversed by concomitant AKT2 expression. In conclusion, our findings suggest that the miR-625-AKT2 axis could be a new prognostic marker and diagnostic target for gliomas.

Mutual association of Broad bean wilt virus 2 VP37-derived tubules and plasmodesmata obtained from cytological observation.

The movement protein VP37 of broad bean wilt virus 2 (BBWV 2) forms tubules in the plasmodesmata (PD) for the transport of virions between cells. This paper reports a mutual association between the BBWV 2 VP37-tubule complex and PD at the cytological level as determined by transmission electron microscopy. The generation of VP37-tubules within different PD leads to a different occurrence frequency as well as different morphology lines of virus-like particles. In addition, the frequency of VP37-tubules was different between PD found at different cellular interfaces, as well as between single-lined PD and branched PD. VP37-tubule generation also induced structural alterations of PD as well as modifications to the cell wall (CW) in the vicinity of the PD. A structural comparison using three-dimensional (3D) electron tomography (ET), determined that desmotubule structures found in the center of normal PD were absent in PD containing VP37-tubules. Using gold labeling, modification of the CW by callose deposition and cellulose reduction was observable on PD containing VP37-tubule. These cytological observations provide evidence of a mutual association of MP-derived tubules and PD in a natural host, improving our fundamental understanding of interactions between viral MP and PD that result in intercellular movement of virus particles.

The cell biology of Tobacco mosaic virus replication and movement.

Successful systemic infection of a plant by Tobacco mosaic virus (TMV) requires three processes that repeat over time: initial establishment and accumulation in invaded cells, intercellular movement, and systemic transport. Accumulation and intercellular movement of TMV necessarily involves intracellular transport by complexes containing virus and host proteins and virus RNA during a dynamic process that can be visualized. Multiple membranes appear to assist TMV accumulation, while membranes, microfilaments and microtubules appear to assist TMV movement. Here we review cell biological studies that describe TMV-membrane, -cytoskeleton, and -other host protein interactions which influence virus accumulation and movement in leaves and callus tissue. The importance of understanding the developmental phase of the infection in relationship to the observed virus-membrane or -host protein interaction is emphasized. Utilizing the latest observations of TMV-membrane and -host protein interactions within our evolving understanding of the infection ontogeny, a model for TMV accumulation and intracellular spread in a cell biological context is provided.

The VP37 protein of Broad bean wilt virus 2 induces tubule-like structures in both plant and insect cells.

VP37 protein of Broad bean wilt virus 2 (BBWV-2) is a multifunctional protein that binds single-strand nucleic acids, interacts with viral coat protein (CP) and potentiates the virus cell-to-cell movement in its host plant. In this study, tubule-like structures filled with virus-like particles were observed by Electron Microscopy in plasmodesmata in walls of Chenopodium quinoa leaf cells infected with BBWV-2. Immunogold labeling using VP37 protein specific antibody demonstrates that the VP37 is a component of the tubular structures. When VP37 was fused with the green fluorescent protein (VP37-GFP) and expressed in BY-2 protoplasts or in insect Tn cells, green fluorescent tubules of various lengths were produced, protruding from the surface of the expressing cells. These findings suggest that the movement of BBWV-2 between cells is mediated by the tubular structures that contain the VP37 protein, and the VP37 protein itself is capable of inducing these tubule-like structures in cells. Our results also suggest that the plant and insect cell factors involved in the tubule formation have conserved features.

Cell-to-cell trafficking, subcellular distribution, and binding to coat protein of Broad bean wilt virus 2 VP37 protein.

Broad bean wilt virus 2 (BBWV 2) is a member of the genus Fabavirus of the family Comoviridae. To date, a movement protein (MP) of BBWV 2 has not been described. Here we demonstrate that the green fluorescent protein (GFP)-VP37 fusion protein can move from initial bombarded cells to neighboring cells in Nicotiana benthamiana epidermal leaves. In addition, the GFP-VP37 fusion protein localizes as a halo around the nucleus and as punctate spots on the cell periphery in N. benthamiana epidermal leaf cells and BY-2 suspension cells. Fluorescence near the nucleus also was co-localized with the endoplasmic reticulum in BY-2 cells. Fibrous networks were found in GFP-VP37 agro-infiltrated N. benthamiana epidermal leaf cells. Deletion analyses indicated that the C-terminal region of the VP37 protein is essential for localization at the cell periphery. Using a blot overlay assay and bimolecular fluorescence complementation assay, the purified 6xHis-tagged VP37 protein was shown to bind specifically to the small coat protein of BBWV 2. The above results indicate that VP37 is a movement protein.

[Production of monoclonal antibodies to Lily symptomless virus and application in lily virus detection].

Four hybridoma cell lines, 2A2, 5H9, 5H2 and 5E12, secreting monoclonal antibodies (MAbs) against Lily symptomless virus (LSV) were produced by fusing mouse myeloma cells (SP2/0) with spleen cells from BALB/C immunized by the LSV particles. The four MAbs could specifically react with LSV. The titres of ascitic fluids of the four MAbs are up to 10(-6) in ELISA. Isotypes and subclasses of 5H9 and 5E12 belong to IgG1 while those of 2A2 and 5H2 belong to IgG3. Isotypes of light strains of the four MAbs all belong to kappa. The four MAbs were used in antigen-coated plate (ACP)-ELISA for LSV detection, and ACP-ELISA could successfully detect 1.8 ng of purified LSV or virus in plant sap diluted 1:300. The presence of LSV in field lily tissues was investigated with ACP-ELISA.