Induction and Suppression of Antiviral RNA Silencing by Tomato Spotted Wilt Virus

Authors

Sergio M. Gabriel Peralta, University of Nebraska-LincolnFollow

First Advisor

Hernan Garcia-Ruiz

Date of this Version

7-2017

Document Type

Thesis

Citation

Gabriel Peralta, Sergio M., "Induction and Suppression of Antiviral RNA Silencing by Tomato spotted wilt virus" (2017). Theses, Dissertations, and Student Research in Agronomy and Horticulture.

Comments

A THESIS Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Master of Science Major: Agronomy Under the Supervision of Professor Hernan Garcia-Ruiz Lincoln, Nebraska July, 2017

Copyright (c) 2017 Sergio M. Gabriel Peralta

Abstract

Tomato spotted wilt virus (TSWV) is an emerging pathogen with wide host range and one of the most important viruses of plants. Information regarding processing of negative single stranded RNA viruses such as TSWV in the RNA silencing pathway remains limited. In nature TSWV is only transmitted by thrips as vectors and since infection occurs in both thrips and plants, an experimental system to transmit using thrips and the detection of TSWV were established. In order to understand the processing of TSWV in the RNA silencing pathway, Arabidopsis thaliana as a model plant was used in the genetic analysis against TSWV. Core components of the RNA silencing machinery tested were Dicer-like proteins(DCLs), RNA dependent RNA Polymerases (RDRs), and Argonaute proteins (AGOs). Results suggest DCL4 and DCL2 proteins are involved in the recognition and processing of TSWV. RDR1 is needed for the amplification of siRNAs derived from TSWV. AGOs protein levels remain unaffected after TSWV inoculation compared to mock inoculated plants suggesting that degradation by TSWV does not occur.

It is well known that plant viruses encode suppressors of RNA silencing to facilitate plant infection and TSWV encodes the Non-Structural-small protein (NSs) whose mechanism of suppression has not been elucidated. NSs protein from TSWV was cloned from viral RNA and tagged with a 6HIS-3xFLAG tag at the C terminus for detection (NSs-HF). In transient assays activity of NSs as a suppressor of RNA silencing was corroborated by preventing silencing of green fluorescent protein (GFP) at three days post infiltration (dpi). Infectious clones of TSWV are not available and to determine the mechanism of suppression, NSs-HF was introduced in a previously described inactive model virus Turnip mosaic virus (TuMV-GFP-AS9) which has an inactive suppressor a does not infect wild type plants. The rescue of pathogenicity of this virus was observed after GFP detection under UV light and the presence of the NSs protein by western blotting. These experiments establish a foundation to study TSWV within the RNA silencing pathway and are the basis for future experiments to understand the mechanism of suppression of RNA silencing by TSWV.

Advisor: Hernan Garcia-Ruiz