2013 Annual Report
1a.Objectives (from AD-416):
The two luteovirid structural proteins, capsid protein (CP) and the readthrough protein (RTP), and the nonstructural P17 movement protein each contain multiple functional domains that interact either in cis or in trans with various plant, aphid, and/or aphid symbiont proteins to direct the overall virus transmission process. Our objective is to identify how these three proteins orchestrate the movement of virus within the plant and the aphid to facilitate the transmission of virus between hosts. Specific to this subcontract of the larger NIFA grant are experiments on the P17 protein and on the discovery of siRNAs in aphids that are viral in origin.
1b.Approach (from AD-416):
P17 mutants of Potato leafroll virus are generated and used in co-immunoprecipitation experiments to identify plant and aphid proteins that can interact with P17. These protein samples are forwarded to Ithaca for MS analysis. siRNA populations are characterized in extracts from whole aphids and specific aphid tissues after they have fed on purified virus, infected plants or healthy plants.
The P17 movement protein is known to be a virus-encoded movement protein that facilitates the cell-to-cell movement of poleroviruses by modifying plasmodesmata and targeting virus to these sites. The homologous protein in luteoviruses appears to direct viral RNA to the nuclear envelope and may contain a novel nuclear envelope targeting domain. To further map the functional sites in P17, mutants were constructed, one knock-out and several point substitutions, which did not affect the coding region of the overlapping CP gene. When agro-inoculated into N. benthamiana plants, the wild-type virus and the mutants accumulated to similar levels in agroinfiltrated tissues, but the mutants accumulated to lower levels than WT in systemically infected tissues. All mutations were retained in progeny virus extracted from systemically infected tissues. The P17 mutants were all detected by immunolocalization in fewer phloem cells than WT virus and were distributed mainly in outer phloem cells, whereas WT virus was observed in both inner and outer phloem tissues. All point mutations and the P17 knock-out mutant (null) accumulated and were distributed similarly in N. benthamiana tissues. As expected, these mutants did not move systemically in natural hosts of the virus such as potato, Physallis floridana or hairy nightshade. Aphids (M. persicae) were fed on systemically infected N. benthamiana leaves for 48 hr and transferred in groups of 5 to healthy recipient plants to determine transmission efficiency. WT virus was efficiently transmitted and several of the P17 mutants were transmitted somewhat less efficiently, while the null mutant and other P17 mutants were not transmitted. Given that the aphids fed equally well on plants infected by all P17 mutants and WT and the distribution and accumulation of P17 mutants were similar, it was surprising that the lack of virus transmission was not likely a function of virus availability. Additional point mutations are being constructed to look for different phenotypes, i.e. increased accumulation with reduced aphid transmission, or efficient transmission and altered distribution/accumulation.
To facilitate future studies, the full-length P17 gene was cloned into the pMAL-c2 vector and expressed as a fusion protein with the maltose-binding protein (MBP) tag in bacterial cells. This fusion protein was purified using affinity chromatography on amylose resin, subjected to LC-MS-MS/MS analysis for sequence confirmation, and used as an antigen for production of antibodies. The antibodies did not react with purified virus in direct ELISA, but a positive reaction was obtained in TAS-ELISA when anti-P17 antibody was used as capturing antibody and anti-CP antibody was used as detecting antibody if infected sap was the sample. The anti-P17 antibodies reacted with a single 17-kDa band when purified virions were analyzed on Western blots. These preliminary studies show that P17 is associated with virions and that the association survives the relatively harsh treatments of virus purification.