Potyviruses are non-persistently transmitted by aphid vectors with the assistance of a viral accessory factor known as helper component (HC-Pro), a multifunctional protein that is also involved in many other essential processes during the virus infection cycle. A transient Agrobacterium-mediated expression system was used to produce Plum pox virus (PPV) HC-Pro in Nicotiana benthamiana leaves from constructs that incorporated the 59 region of the genome, yielding high levels of HC-Pro in agroinfiltrated leaves. The expressed PPV HC-Pro was able to assist aphid transmission of purified virus particles in a sequential feeding assay, and to complement transmission-defective variants of the virus. Also, HC-Pro of a second potyvirus, Tobacco etch virus (TEV), was expressed and found to be functional for aphid transmission. These results show that this transient system can be useful for production of functionally active HC-Pro in potyviruses, and the possible uses of this approach to study the mechanism of transmission are discussed.
Potyviruses are plant pathogens transmitted by aphids in a non-persistent manner. During transmission, the virus-encoded factor helper-component protease (HCPro) is presumed to act as a molecular bridge, mediating the reversible retention of virions to uncharacterized binding sites in the vector mouthparts. Whilst the predicted interaction between HCPro and the coat protein (CP) of virions has been confirmed experimentally, the characterization of putative HCPro-specific receptors in aphids has remained elusive, with the exception of a report that described binding of HCPro of zucchini yellow mosaic virus to several cuticle proteins. To identify other aphid components that could play a role during transmission, this study used purified HCPro of tobacco etch virus (TEV) in far-Western blotting assays as bait to select interactors among proteins extracted from aphid heads. With this approach, new HCPro-interacting proteins were found, and several were identified after mass spectrometry analysis and searches in databases dedicated to aphid sequences. Among these interactors, a ribosomal protein S2 (RPS2) was chosen for further investigation due to its homology with the laminin receptor precursor, known to act as the receptor of several viruses. The specific interaction between RPS2 and TEV HCPro was confirmed after cloning and heterologous expression of the corresponding Myzus persicae gene. The possible involvement of RPS2 in the transmission process was further suggested by testing a variant of HCPro that was non-functional for transmission due to a mutation in the conserved KITC motif (EITC variant). This variant retained its ability to bind CP but failed to interact with RPS2.
INTRODUCTIONNatural dissemination of most plant viruses is mediated by insect vectors, in either circulative or non-circulative processes, with aphids (Hemiptera, Aphididae) being the most common vectors (Pirone & Perry, 2002;Ng & Falk, 2006;Hogenhout et al., 2008). Whilst the viral components implicated in transmission are frequently well known, there is substantially less knowledge about the insect counterparts, and only a few published studies have identified insect products with the capacity to interact with virions or viral components that might play a role in plant virus transmission. Using far-Western blotting or similar methodologies, studies with circulative plant viruses have found proteins interacting with virions at key points of the pathways followed by these viruses inside their insect vectors. Examples include a thrips-transmitted tospovirus (Medeiros et al., 2000) and several aphid-transmitted members of the family Luteoviridae (van den Heuvel et al., 1997;Li et al., 2001;Seddas et al., 2004). A promising approach for further advances in this field is represented by the recent adoption of combined genetic and proteomic tools in the analysis of vector specificity in luteovirids (Yang et al., 2008).In the case of non-circulative plant viruses, the insect vector factors implicated in transmission remain largely unknown. The ...
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