Presentation Title

Using the BioID Approach to Identify Proteins Interacting with the P0 Protein from Turnip Yellows Virus

Faculty Mentor

Melanie Sacco

Start Date

23-11-2019 8:45 AM

End Date

23-11-2019 9:30 AM

Location

112

Session

poster 2

Type of Presentation

Poster

Subject Area

biological_agricultural_sciences

Abstract

RNA silencing is a conserved process used as an antiviral mechanism in plants and invertebrates. Dicer-like enzymes cleave viral double-stranded RNA replication intermediates into virus-derived small RNA (vsiRNA) fragments that are incorporated into the RNA-induced silencing complex (RISC) composed of argonaute (AGO) proteins. The vsiRNAs guide the endonuclease activity of AGO1 in the RISC to cleave viral RNAs by complementary base-pairing target RNAs. All plant RNA viruses have evolved viral suppressors of RNA silencing (VSR) proteins to interfere with RNA silencing. Turnip yellows virus (TuYV) is an economically important virus in the genus Polerovirus that infects a wide host range of plants, such as lettuce, turnip, and canola. The TuYV VSR protein P0 targets AGO1 for autophagic degradation and prevents RISC formation. The exact mechanism of how P0 targets AGO1 for autophagic degradation remains unknown. The purpose of this project is to identify potential protein-protein interactions involving P0 to identify cellular targets of P0 function and to define components of the AGO1 degradation pathway with which P0 interacts using the BioID proximity-labeling technique to covalently tag proteins interacting with P0 with biotin in the plant cell. Beta-glucuronidase (GUS), wild-type P0, and four mutant P0 genes were each ligated into pBin61 to create six fusion proteins with the promiscuous biotin ligase BirA* and a hemagglutinin (HA) epitope. The wild-type P0 fusion was functionally validated by agroinfiltration into Nicotiana plants for VSR and hypersensitive response assays, showing that BirA* fusion does not impair P0 activities. Protein extracts were analyzed via pull-down assays and blotting for validation of BioID construct expression and detection of biotin-tagged proteins. Tandem mass spectrometry will be done to identify potential P0 target proteins. This work will advance research our understanding of TuYV infection in plants, which is important for developing virus-resistant crops and understanding similar mammalian viruses.

This document is currently not available here.

Share

COinS
 
Nov 23rd, 8:45 AM Nov 23rd, 9:30 AM

Using the BioID Approach to Identify Proteins Interacting with the P0 Protein from Turnip Yellows Virus

112

RNA silencing is a conserved process used as an antiviral mechanism in plants and invertebrates. Dicer-like enzymes cleave viral double-stranded RNA replication intermediates into virus-derived small RNA (vsiRNA) fragments that are incorporated into the RNA-induced silencing complex (RISC) composed of argonaute (AGO) proteins. The vsiRNAs guide the endonuclease activity of AGO1 in the RISC to cleave viral RNAs by complementary base-pairing target RNAs. All plant RNA viruses have evolved viral suppressors of RNA silencing (VSR) proteins to interfere with RNA silencing. Turnip yellows virus (TuYV) is an economically important virus in the genus Polerovirus that infects a wide host range of plants, such as lettuce, turnip, and canola. The TuYV VSR protein P0 targets AGO1 for autophagic degradation and prevents RISC formation. The exact mechanism of how P0 targets AGO1 for autophagic degradation remains unknown. The purpose of this project is to identify potential protein-protein interactions involving P0 to identify cellular targets of P0 function and to define components of the AGO1 degradation pathway with which P0 interacts using the BioID proximity-labeling technique to covalently tag proteins interacting with P0 with biotin in the plant cell. Beta-glucuronidase (GUS), wild-type P0, and four mutant P0 genes were each ligated into pBin61 to create six fusion proteins with the promiscuous biotin ligase BirA* and a hemagglutinin (HA) epitope. The wild-type P0 fusion was functionally validated by agroinfiltration into Nicotiana plants for VSR and hypersensitive response assays, showing that BirA* fusion does not impair P0 activities. Protein extracts were analyzed via pull-down assays and blotting for validation of BioID construct expression and detection of biotin-tagged proteins. Tandem mass spectrometry will be done to identify potential P0 target proteins. This work will advance research our understanding of TuYV infection in plants, which is important for developing virus-resistant crops and understanding similar mammalian viruses.