BACKGROUND: Localization and interaction studies of viral proteins provide important information about their replication in their host plants. Tospoviruses (Family Bunyaviridae) are economically important viruses affecting numerous field and horticultural crops. Iris yellow spot virus (IYSV), one of the tospoviruses, has recently emerged as an important viral pathogen of Allium spp. in many parts of the world. We studied the in vivo localization and interaction patterns of the IYSV proteins in uninfected and infected Nicotiana benthamiana and identified the interacting partners.
PRINCIPAL FINDINGS: Bimolecular fluorescence complementation (BiFC) analysis demonstrated homotypic and heterotypic interactions between IYSV nucleocapsid (N) and movement (NSm) proteins. These interactions were further confirmed by pull-down assays. Additionally, interacting regions of IYSV N and NSm were identified by the yeast-2-hybrid system and β-galactosidase assay. The N protein self-association was found to be mediated through the N- and C-terminal regions making head to tail interaction. Self-interaction of IYSV NSm was shown to occur through multiple interacting regions. In yeast-2-hybrid assay, the N- and C-terminal regions of IYSV N protein interacted with an N-terminal region of IYSV NSm protein.
CONCLUSION/SIGNIFICANCE: Our studies provide new insights into localization and interactions of IYSV N and NSm proteins. Molecular basis of these interactions was studied and is discussed in the context of tospovirus assembly, replication, and infection processes.
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The work was supported by PPNS No. 0684, Department of Plant Pathology, College of Agricultural, Human and Natural Resource Sciences, Agricultural Research Center, Hatch Project # WNPO 0545, Washington State University, Pullman, WA 99164-6430, USA. The authors also acknowledge the support from the Franceschi Microscopy and Imaging Center (FMIC) at the Washington State University, Pullman.
Tripathi, Diwaker; Raikhy, Gaurav; Goodin, Michael M.; Dietzgen, Ralf G.; and Pappu, Hanu R., "In Vivo Localization of Iris yellow spot Tospovirus (Bunyaviridae)-Encoded Proteins and Identification of Interacting Regions of Nucleocapsid and Movement Proteins" (2015). Plant Pathology Faculty Publications. 49.
S1 Fig. High-resolution confocal micrographs showing the localization of Iris yellow spot virus (IYSV) proteins in epidermal cells of IYSV-infected transgenic Nicotiana benthamiana plants containing red endoplasmic reticulum marker (ER-RFP).
journal.pone.0118973.s002.TIF (2150 kB)
S2 Fig. Confocal micrographs to show the interaction of Iris yellow spot virus (IYSV) nucleocapsid (N) and movement (NSm) proteins in epidermal leaf cells of transgenic Nicotiana benthamiana (CFP-H2B) plants by bimolecular fluorescence complementation (BiFC) assay.
journal.pone.0118973.s003.TIF (2363 kB)
S3 Fig. In planta interactions of Tomato spotted wilt virus proteins as examined by bimolecular fluorescence complementation (BiFC) assay.