IN VIVO LOCALIZATION AND INTERACTION STUDIES OF TOSPOVIRUS (GENUS: TOSPOVIRUS; FAMILY: BUNYAVIRIDAE) PROTEINS

Diwaker Tripathi

IN VIVO LOCALIZATION AND INTERACTION STUDIES OF TOSPOVIRUS (GENUS: TOSPOVIRUS; FAMILY: BUNYAVIRIDAE) PROTEINS Tospoviruses (family: Bunyaviridae) are one of the most agronomically important viruses that affect numerous agriculture crops. Two distinct tospoviruses, Tomato spotted wilt virus (TSWV) and Iris yellow spot virus (IYSV), are important pathogens of a wide variety of crops. Localization and interaction studies of IYSV proteins were performed in the host plant, Nicotiana benthamiana. Our localization studies in infected plants suggested a change in localization of the nucleocapsid (N) protein from the cytoplasm to cell periphery. The interaction analyses of tospovirus proteins were performed using Bimolecular Fluorescence Complementation (BiFC), pull down, and Yeast-2-Hybrid (Y2H) assays. Interaction studies of IYSV proteins demonstrated homotypic and heterotypic interactions between IYSV N and NSm proteins. The observations were discussed in the context of tospovirus assembly, replication, and infection processes. To understand the molecular basis of mixed infections caused by tospoviruses, interaction between N and NSm proteins of TSWV and IYSV was also studied. Results showed that the N and NSm proteins of IYSV interact with their counterparts coded by TSWV in a doubly infected plant. Interacting regions of these proteins were also identified by the yeast-2-hybrid systems. Molecular interactions between cognate proteins of these viruses may be of crucial significance for the understanding of pathogenesis, evolution, and development of efficient and durable control strategies for these viruses. Acibenzolar-S-Methyl (ASM) is a functional analog of salicylic acid (SA) that activates plant's local and systemic acquired resistance (SAR) responses against a wide variety of pathogens. Two different hosts of IYSV-Datura stramonium and Nicotiana benthamiana were used as model plants to develop a set of descriptors to study the effect of SAR inducers on IYSV infection. ASM- and buffer-treated D. stramonium and N. benthamiana plants were mechanically inoculated with IYSV. Symptom development and virus levels were monitored by the enzyme linked immunosorbent assay (ELISA), reverse-transcriptase polymerase chain reaction (RT-PCR), and real-time- RT-PCR (q-RT-PCR). A significant reduction in IYSV levels in ASM-treated plants was noticed. In addition, ASM-treated plants showed reduced viral symptoms (lesion number and sizes), compared to buffer-treated plants. The knowledge gained from this study could be used in developing SAR based chemical options for virus management.

IN VIVO LOCALIZATION AND INTERACTION STUDIES OF TOSPOVIRUS (GENUS: TOSPOVIRUS; FAMILY: BUNYAVIRIDAE) PROTEINS

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