Model system-guided protein interaction mapping for virus isolated from phloem tissue

Authors: Deblasio, Stacy; JOHNSON, RICHARD - University Of Washington; MACCOSS, MICHAEL - University Of Washington; Gray, Stewart; Heck, Michelle

Submitted to: Journal of General Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/30/2016
Publication Date: 10/20/2016
Citation: Deblasio, S.L., Johnson, R., Maccoss, M., Gray, S.M., Cilia, M. 2016. Model system-guided protein interaction mapping for virus isolated from phloem tissue. Journal of General Virology. 15:4601-4611.

Interpretive Summary: Many plant pathogens that are transmitted by insects strictly infect the plant's vascular system. The strict requirement for vascular infection renders vascular-localized pathogens extraordinarily difficult to study because the pathogens are difficult to isolate. They are low in overall abundance relative to other molecules in the plant leaf. Here, we report the first successful application of a high throughput method for virus-plant protein interaction identification to a plant virus that is localized to the plant's vascular tissue during the course of a virus infection. We used Potato leafroll virus (PLRV) infecting potato, a natural host of the virus. Information about plant-PLRV protein interactions is important because it enables a deeper understanding of how PLRV infects and is transmitted from plant to plant. When we compared the PLRV-plant protein interactions in potato to the protein interactions we discovered using a model solanaceous host, we found that the overlap was strikingly high. The confidence of weak interactions from the potato dataset was boosted by their identification in the model plant-PLRV interaction network. Our method will be useful for other scientists seeking to apply protein interaction technologies to other pathogens infecting the phloem vascular system and lend support to using model pathosystems for studying recalcitrant plant pathogens where feasible.

Technical Abstract: Potato leafroll virus (PLRV) is an agriculturally important phloem-limited pathogen that causes significant yield loss in potato (Solanum tuberosum) and a model virus in the Luteoviridae. Encoding only a small repertoire of viral proteins, PLRV relies on carefully orchestrated protein-protein interactions with its host and vector to ensure survival in the plant and further its transmission. Localization of virions strictly to phloem cells, is advantageous for virus acquisition by sap-sucking aphid vectors but renders protein interaction studies with PLRV and other related viruses extremely challenging. Characterization of these interactions at the proteomic level may offer new disease control opportunities. Previously, we reported the identification of over 1000 host and 3 viral proteins in complex with PLRV using co-immunoprecipitation coupled to high-resolution mass spectrometry (co-IP-MS). In these experiments, virus was over-expressed in N. benthamiana leaf cells using an infectious cDNA clone. Although N. benthamiana is a model host of PLRV, the model plant is not suitable to study the host-specific effects on PLRV movement and tissue tropism. In this study, we extend our co-IP-MS approach to include systemically infected Solanum tuberosum, a natural host of the virus. Comparing two different co-immunoprecipitation support matrices, we identified 39 high confidence S. tuberosum proteins and one viral protein (P1 polyprotein) specifically associated with virus isolated from infected phloem tissue. An additional 145 proteins were identified to interact directly or in complex with virus at varying degrees of confidence but were found to be high confidence interactions in the N. benthamiana network. Bioinformatics analysis revealed that these host proteins regulate diverse cellular processes but are enriched for functions involved in organelle membrane transport and protein translation. Interestingly, 88% overlapped in identity with proteins we identified in complex with virus in N. benthamiana with 51 percent sharing homology with proteins identified in the plasmodesamal proteome of Arabidopsis thaliana suggesting a role in regulating viral movement. Our results demonstrate that model system proteomics is extremely valuable for understanding protein interactions regulating infection in recalcitrant pathogens such as phloem-limited viruses.