Degree Type

Dissertation

Date of Award

2013

Degree Name

Doctor of Philosophy

Department

Entomology

Major

Microbiology

First Advisor

Bryony C. Bonning

Abstract

Insect transmission of plant viruses results in tremendous economic loss within the agricultural sector worldwide. Aphids account for nearly half of insect-borne plant virus transmission. Viruses in the family Luteoviridae are transmitted by aphids in a persistent-circulative manner that requires specific molecular interactions between the aphid and virus. Ingested virions cross the aphid gut and salivary gland epithelial barriers using receptors that have not been identified. We assessed the binding of a model luteovirid, Pea enation mosaic virus (PEMV), to brush border membrane vesicles (BBMV) of the pea aphid, Acyrthosiphon pisum using a two-dimensional far-western blot method. Pea aphid membrane alanyl aminopeptidase N (APN) was identified by mass spectrometry following specific binding to PEMV virions and to a PEMV coat protein-eGFP fusion peptide (CP-P-eGFP). The binding of PEMV to APN was confirmed by multiple methods including a pull-down assay, surface plasmon resonance (SPR) analysis, and by increased binding of CP-P-eGFP to baculovirus-expressed pea aphid APN in Sf9 cells. We also show that a peptide (GBP3.1) that was previously shown to impede uptake of PEMV into the pea aphid also binds to APN. Based on these results, we conclude that APN is a putative gut receptor for PEMV in the pea aphid and if confirmed would be the first insect receptor identified for a plant virus. Interestingly, PEMV appears to bind to a different, as yet unidentified, receptor in a second vector, Myzus persicae, suggesting that different gut receptors may be used by luteoviruses in different vector species.

Luteoviruses are acquired when aphids ingest the phloem sap of an infected plant. Phloem proteins have been shown to associate with luteovirus particles and facilitate aphid transmission in in vitro feeding assays. We showed an increase of virus in the hemocoel of aphids fed on artificial diet containing purified PEMV with bovine serum albumin (BSA) compared to aphids fed on virus in the absence of BSA. Interestingly, BSA reduced the amount of a mutant virus lacking the minor structural protein readthrough domain (RTD) detected in the aphid hemocoel. We also demonstrated that the PEMV RTD binds to multiple aphid proteins. SPR analysis indicated that the CP and RTD both bind to BSA. Based on these data, models are presented to account for the role of the RTD and mechanism by which BSA and plant proteins facilitate virus entry into the aphid hemocoel.

Little is known about the role of glycans in mediating luteovirus-aphid interactions. We used the lectins Concanavalin A (ConA) and Galanthus nivalis agglutinin (GNA) for lectin blot analysis of BBMV and confirmed that pea aphid proteins are glycosylated with mannose and glucose moieties. APN, the PEMV gut receptor, is glycosylated with mannose residues. However, we did not detect any binding of PEMV to a synthesized tri-mannose glycan that is common in insects using both isothermal titration calorimetry or a carbohydrate microarray. These results suggest that mannose by itself is not involved in PEMV-APN binding. ConA bound to PEMV indicating that viral structural proteins are glycosylated. The potential role of virus glycosylation in aphid transmission of luteoviruses is discussed. Taken together, our increased understanding of luteovirus-aphid vector interaction will facilitate research into other plant virus-insect vector systems, and the development of mitigation strategies.

Copyright Owner

Lucas Benjamin Linz

Language

en

File Format

application/pdf

File Size

173 pages

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