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United States Department of Agriculture

Agricultural Research Service

Research Project: DETECTION, IDENTIFICATION, AND CHARACTERIZATION OF NEW AND EMERGING VIRAL AND BACTERIAL DISEASES OF ORNAMENTAL PLANTS

Location: Floral and Nursery Plants Research Unit

Title: Subcellular localization of the barley stripe mosaic virsus triple gene block proteins

Authors
item Lim, Hyoun
item Bragg, Jennifer
item Ganesan, Uma -
item Ruzin, Steven -
item Schichnes, Denise -
item Lee, Mi Yeon -
item Vaira, Anna Maria -
item Ryu, Ki Hyun -
item Hammond, John
item Jackson, Andrew -

Submitted to: Journal of Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 22, 2009
Publication Date: July 1, 2009
Repository URL: http://hdl.handle.net/10113.45115
Citation: Lim, H.S., Bragg, J.N., Ganesan, U., Ruzin, S.E., Schichnes, D., Lee, M., Vaira, A., Ryu, K., Hammond, J., Jackson, A.O. 2009. Subcellular localization of the barley stripe mosaic virsus triple gene block proteins. Journal of Virology 83(18):9432-9448. http://dx.doi.org/10.1128/JVI.00739-09.

Interpretive Summary: An increased understanding of the ways in which virus infection spreads throughout the host plant may lead to new ways of interfering in the infection process and thus conferring protection to plants. Different kinds of plant virus utilize different types of movement proteins to aid in the infection process; Barley stripe mosaic virus (BSMV), which causes an important disease of barley, has a “triple gene block” (TGB) of proteins that interact to fulfill this function. Previous studies demonstrated that each of the three TGB proteins is required for efficient viral movement in both monocotyledonous and dicotyledous plant hosts, and that self-interactions of TGB1, and interactions of TGB3 with both TGB1 and TGB2 are necessary. In the current studies, fluorescently-labeled individual proteins and specifically mutated proteins were expressed in leaf cells by means of Agrobacterium-mediated infiltration in order to examine the effects of protein interactions on subcellular localization and virus spread. Confocal microscopy revealed that the TGB3 protein localizes at the cell wall in close proximity to plasmodesmata (cytoplasmic connections between adjoining cells). Deletion or mutagenesis of a single carboxy-terminal amino acid residue was sufficient to disrupt localization of TGB3 to the cell wall, and also prevented TGB3-dependent localization of TGB2 to the cell wall. Mutations in TGB1 showed that self-interactions of TGB1 were not required for interactions with TGB2 and TGB3. These results may lead to increased understanding of the way in which other viruses spread in infected plants, and to new means of conferring virus resistance.

Technical Abstract: Barley stripe mosaic virus (BSMV) spreads from cell-to-cell through the coordinated actions of three triple gene block proteins (TGB1, TGB2, and TGB3) arranged in overlapping open reading frames (ORFs). Our previous studies (Lawrence and Jackson, 2001a,b) have shown that each of these proteins is required for cell-to-cell movement in monocot and dicot hosts. We (Lim et al 2008) recently have found that TGB1 engages in homologous interactions leading to formation of a ribonucleoprotein (RNP) complex containing viral genomic and messenger RNAs, and also have demonstrated that TGB3 functions in heterologous interactions with TGB1 and TGB2. We have now used Agrobacterium-mediated protein expression in Nicotiana benthamiana leaf cells and site-specific mutagenesis to determine how TGB protein interactions influence their subcellular localization and virus spread. Confocal microscopy revealed that the TGB3 protein localizes at the cell wall (CW) in close association with plasmodesmata (PD), and that deletion or mutagenesis of a single amino acid at the immediate C-terminus can disrupt CW targeting. TGB3 also directed localization of TGB2 from the endoplasmic reticulum (ER) to the CW, and this targeting was shown to be dependent on TGB2:TGB3 protein interactions. Optimal localization of the TGB1 protein at the CW also required TGB2 and TGB3 interactions, but in this context, site-specific TGB1 helicase motif mutants varied in their localization patterns. The results suggest that the ability of TGB1 to engage in homologous binding interactions is not essential for targeting to the CW. However, the relative expression levels of TGB2:TGB3 influenced the membrane and CW distributions of TGB1 and TGB2. Moreover, in both cases localization of TGB1 at the CW was optimal at the 10:1 TGB2:TGB3 ratios occurring in virus infections, and mutations reducing CW localization had corresponding effects on BSMV movement phenotypes. These results support a model whereby TGB protein interactions function in subcellular targeting of movement protein complexes and movement processes of BSMV.

Last Modified: 9/22/2014
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