Location: Emerging Pests and Pathogens Research2013 Annual Report
1a. Objectives (from AD-416):
This proposal focuses on developing a platform and pipeline to integrate biomarker technology into IPM strategies for the control of multiple hemipteran insect species that transmit circulative plant viruses. The Overall objective is to dissect the molecular and cellular functions of several virus proteins that together regulate the phloem specific movement of virus in host plants and the tissue specific movement of virus in aphid vectors. Specifically we plan to: 1. Identify the active domains of the RTP and its interacting plant proteins that operate to limit the virus to phloem tissues where it is available to aphids. 2. Identify aphid and plant proteins that interact with the CP and RTP, and the mechanisms that allow these complexes to orchestrate the transport of virus across aphid gut and salivary tissues and the survival of virus in the aphid hemolymph. 3. Identify the mechanisms by which the P17 influences the uptake of virus by aphids and influences the transmission efficiency to other plant hosts.
1b. Approach (from AD-416):
We have shown the CP without RTP can assemble virions, but they are movement impaired in both plants and aphids. CP and RTP act together to regulate virus movement and survival in the aphid. RTP also functions as a soluble protein to confine virus to phloem tissues. P17, a nonstructural virus movement protein, acts in a host dependent manner and affects virus uptake during aphid feeding. Recently, we have identified a number of aphid and aphid symbiont proteins that are linked to virus transmission. Also we have identified a number of plant proteins that co-purify with transmissible virus, but are absent from virus that cannot move through the aphid. The logical extension of this work is to identify which of the aphid and plant candidate proteins are truly interacting with which virus protein and to determine how these various proteins orchestrate virus movement in the plant and aphid to ensure maximum transmission and continued virus existence. Our approach will focus on the use of the latest techniques in targeted proteomics and cell biology to identify aphid, symbiont and plant proteins that interact with each of the virus proteins. Mutations in the virus and plant proteins that disrupt these interactions will be used to identify how and where each protein functions independent of, and in concert with, other virus proteins in both plants and aphids. In vitro feeding and microinjection techniques allow us to mix and match various virions and proteins for study in various aphid tissues. GFP tagged proteins will be used to localize interactions to specific tissues and cell organelles.
3. Progress Report:
Optimization of methods to discover virus-plant protein interactions. Plant proteins interacting with the virus (PLRV) capsid structural proteins (CP and RTP) were initially identified by nanoflow liquid chromatography coupled to tandem mass spectrometry (nLC-MS/MS) analysis of virus purified from Nicotiana benthamiana. Using this approach, we were able to detect 31 plant proteins enriched in purified virus fractions or not found in the analysis of proteins purified from non-infected tissue using the same workflow. Peptides derived from the C-terminal domain of the RTP were not detected due to a proteolytic cleavage occurring during virus purification. In contrast, co-immunoprecipitation (co-IP) of PLRV and associated plant proteins directly from infected plant tissue enabled us to detect peptides spanning the entire RTP and preserve the plant protein interactions with the C-terminal domain previously shown to be important for retaining PLRV within the phloem. Interestingly, virus purified using sodium sulfite was not acquired into the aphid gut and therefore not transmissible. Using nLC-MS/MS, we determined that the plant-virus interactome was significantly altered by sodium sulfite. We hypothesize that the virus-interacting plant proteins are mediating virus uptake into the gut by stimulating endocytosis. This study was published in PlosOne. Work is ongoing to determine how these plant proteins may regulate the intimate interaction between virus, plant and aphid vector. Potato leafroll virus-plant protein interactions vary between hosts of the virus. Using a 96-well plate Co-IP format we developed and coupled to nLC-MS/MS on an Orbitrap-Velos mass spectrometer, we identified PLRV-interacting plant proteins from potato and N. benthamiana. Using protein databases now available for both hosts, together with label-free quantitation based on spectral counts and MS1 peak integration, we identified 76 and 24 proteins exclusively in the co-IP reactions from potato and N. benthamiana, respectively. Fifteen proteins were shared in both species. An additional 71 and 30 proteins were found enriched = 3 fold in PLRV co-IP from infected S. tuberosum and N. benthamiana, respectively, compared to healthy tissue control co-IP reactions. Most of these plant proteins did not interact with purified virus (described above), indicating they may interact with the soluble form of RTP or transiently with assembled virions. GO analysis of these putative viral binding partners revealed an abundance of proteins involved in plant defense, carbohydrate metabolism and vesicle transport, as well as an enrichment of proteins localizing to the membranes of organelles PLRV associates with during infection, i.e. mitochondria, chloroplasts, vacuoles and plasmodesmata. Optimization of methods to discover virus-aphid protein interactions. A co-IP approach was developed to identify aphid proteins interacting with luteovirids. Using the aphid vector Schizaphis graminum and the RPV strain of the Cereal yellow dwarf virus, we identified 31 aphid proteins that interact with RPV virions. To help achieve this objective, we completed a comprehensive proteomic analysis of gut and salivary tissues in S. graminum. Using these data, we can pinpoint certain RPV-interacting proteins to the gut or the accessory salivary glands. We had previously shown that one of these RPV-interacting proteins, sucrase, is up-regulated in vector genotypes of S. graminum and is exclusively found in the gut proteome. A second RPV-interacting protein, cyclophilin B, was previously characterized to have two isoforms in the S. graminum population. Both isoforms of cyclophilin B interact with RPV and cyclophilin A also interacts with RPV indicating that cyclophilin may play a major role in the translocation of RPV through aphid tissues. Interestingly, the plant homolog of cyclophilin was shown to associate with PLRV, suggesting that luteovirids may exploit similar trafficking pathways in both host and vector to facilitate its movement and transmission. This work was published in PlosOne Couple molecular virology and targeted proteomics to define functional roles for host- virus-vector protein-protein interactions. To define the functional roles of the plant and aphid proteins we identify in complex with wild-type PLRV, we are using our collection of PLRV capsid and readthrough protein mutants to monitor perturbations in the plant/aphid-virus complexes and to identify specific interaction sites within the virus. For experiments using structural protein mutants we optimized a co-IP workflow using antibody-conjugated magnetic beads to isolate plant-virus complexes within 1 hour, a time frame shown by a cooperator to effectively capture transient viral interactions and lower binding of unspecific proteins. Preliminary experiments identified 24 N. benthamiana proteins that interact with wild-type PLRV in the co-IP, but do not interact or interact less efficiently with a PLRV mutant that does not translate the minor capsid protein (¿RTD). These data provide proof-of-concept that coupling molecular virology with co-IP-nLC-MS/MS can map protein interactions onto the functional domains of the virus capsid. Additionally, 19 P17 mutants have been constructed and are being tested for effects on virus replication, movement and aphid transmission.