Abstract
During a plant viral infection, host–pathogen interactions are critical for successful replication and propagation of the virus through the plant. RNA silencing suppressors (RSSs) are key players of this interplay, and they often interact with different host proteins, developing multiple functions. In the Potyviridae family, viruses produce two main RSSs, HCPro and type B P1 proteins. We focused our efforts on the less known P1b of cucumber vein yellowing virus (CVYV), a type B P1 protein, to try to identify possible factors that could play a relevant role during viral infection. We used a chimeric expression system based on plum pox virus (PPV) encoding a tagged CVYV P1b in place of the canonical HCPro. We used that tag to purify P1b in Nicotiana-benthamiana-infected plants and identified by mass spectrometry an importin-β-like protein similar to importin 7 of Arabidopsis thaliana. We further confirmed the interaction by bimolecular fluorescence complementation assays and defined its nuclear localization in the cell. Further analyses showed a possible role of this N. benthamiana homolog of Importin 7 as a modulator of the RNA silencing suppression activity of P1b.
Highlights
Plant viral infections are based on a myriad of protein interactions that represent the fight between the host, trying to stop pathogen replication and spread, and the virus, searching to exploit plant resources for its own benefit [1,2,3]
We engineered a plum pox virus (PPV) construct in which HCPro was replaced by cucumber vein yellowing virus (CVYV) P1b with an Strep tag II in tandem (SIII) N-terminal tag to facilitate purification of the protein during viral infection (Figure 1a)
Following the protocol described [52], we purified SIIIP1b from the pPPV-SIIIP1b-inoculated tissue and performed in parallel the same purification process using the tissue inoculated with pPPV (Figure 1b)
Summary
Plant viral infections are based on a myriad of protein interactions that represent the fight between the host, trying to stop pathogen replication and spread, and the virus, searching to exploit plant resources for its own benefit [1,2,3]. HCPro is a well-known multifunctional protease that helps in aphid transmission and viral particle formation, acting as an RSS at least in potyviruses and rymoviruses [24] It is a protein extensively studied, and the list of identified interacting host factors continuously grows [4,24,25,26]. Cucumber vein yellowing virus (CVYV) is an ipomovirus that infects cucurbitaceae species, such as cucumber, melon, and watermelon [30] It does not encode HCPro and presents at the beginning of the polyprotein two P1 endopeptidases of type A and type B (named P1a and P1b, respectively) [31]. Our characterization of the interaction between P1b and the N. benthamiana homolog of Importin 7 and its effect in the plant identified a new possible role of this host protein as a regulator of the action of viral RSSs
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