|JOHNSON, RICHARD - University Of Washington|
|MAHONEY, JACLYN - Boyce Thompson Institute|
|KARASEV, ALEXANDER - University Of Idaho|
|MACCOSS, MICHAEL - University Of Washington|
Submitted to: Molecular Plant-Microbe Interactions
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/3/2014
Publication Date: 12/11/2014
Citation: Deblasio, S.L., Johnson, R., Mahoney, J., Karasev, A., Gray, S.M., Maccoss, M., Cilia, M. 2014. Insights into the polerovirus-plant interactome revealed by co-immunoprecipitation and mass spectrometry. Molecular Plant-Microbe Interactions. DOI: 10.1094/MPMI-11-14-0363-R.
Interpretive Summary: Protein interactions regulate the majority of cellular processes during plant development and viral infection. Developing an understanding of these proteins interactions, such as determining the identity of the plant and virus proteins involved, figuring out where the interactions are occurring within a cell, and identifying which proteins are interacting with each other will provide us with critical insights into their function. This paper presents a method that uses an antibody recognizing a plant virus to capture the virus and the proteins interacting with that virus during plant infection. The proteins are identified using mass spectrometry and bioinformatics. The strategy enables us to confidently identify the composition of the protein complexes and minimizes the time necessary to go from a living, infected plant to an isolated protein complex. The virus we used for these experiments, Potato leafroll virus, belongs to an economically important family of viruses called the Luteoviridae that infect nearly every cultivatable crop grown around the world. Viruses in the Luteoviridae are transmitted solely by herbivorous, sap-sucking aphid vectors. It is indisputable that the development and application of new technologies for studying proteins and protein interactions will enable us to decipher how viruses in the Luteoviridae infect plants and are transmitted by insects to provide novel inroads for the development of precision disease management strategies.
Technical Abstract: The identification of host proteins that interact with virus proteins is a major challenge for the field of virology. Phloem-limited viruses pose extraordinary challenges for in vivo protein interaction experiments because these viruses are localized in very few and highly specialized host cells. We present a method that uses co-immunoprecipitation and mass spectrometry to identify plant-virus protein complexes with Potato leafroll virus (PLRV) from the genus Polerovirus, family Luteoviridae. We describe optimization of conditions for tissue lysis, protein extraction, co-immunoprecipitation, and peptide analysis using tandem mass spectrometry. The technique enables us to capture the full-length form of the readthrough protein (RTP), the virus protein that regulates PLRV phloem tropism. Our analysis revealed RTP isoforms that are posttranslationally modified and allowed for enrichment of the RTP from systemically infected tissue where virus is low in abundance. The approach minimizes the time it takes to isolate virus protein complexes from plants for characterization by mass spectrometry.