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M. A. Mayo - One of the best experts on this subject based on the ideXlab platform.

  • Nucleolar localization of Potato Leafroll Virus capsid proteins.
    Journal of General Virology, 2005
    Co-Authors: Sophie Haupt, Tanya Stroganova, Gill Fraser, George H. Duncan, Hugh Barker, M. A. Mayo, Eugene V Ryabov, Michael E Taliansky
    Abstract:

    Potato Leafroll Virus (PLRV) encodes two capsid proteins, major protein (CP) and minor protein (P5), an extended version of the CP produced by occasional translational ‘readthrough’ of the CP gene. Immunogold electron microscopy showed that PLRV CP is located in the cytoplasm and also localized in the nucleus, preferentially targeting the nucleolus. The nucleolar localization of PLRV CP was also confirmed when it was expressed as a fusion with green fluorescent protein (GFP) via an Agrobacterium vector. Mutational analysis identified a particular sequence within PLRV CP involved in nucleolar targeting [the nucleolar localization signal (NoLS)]. Minor protein P5 also contains the same NoLS, and was targeted to the nucleolus when it was expressed as a fusion with GFP from Agrobacterium. However, P5–GFP lost its nucleolar localization in the presence of replicating PLRV.

  • evidence for rna mediated defence effects on the accumulation of Potato Leafroll Virus
    Journal of General Virology, 2001
    Co-Authors: H. Barker, M. A. Mayo, Eugene V Ryabov, Kara D Mcgeachy, Uli Commandeur, Michael E Taliansky
    Abstract:

    In plants infected with Potato Leafroll Virus (PLRV), or other luteoViruses, infection is very largely confined to cells in the vascular system. Even in tobacco plants transformed with PLRV full-length cDNA, in which all mesophyll cells should synthesize infectious PLRV RNA transcripts, only a minority of the mesophyll cells accumulate detectable amounts of Virus. We have explored this phenomenon further by transforming a better PLRV host, Nicotiana benthamiana, with the same transgene, by superinfecting transformed plants with Potato Virus Y and by producing tobacco plants in which cells contained both PLRV cDNA and DNA encoding the P1/HC-Pro genes of the potyVirus Tobacco etch Virus. A greater proportion of cells in superinfected plants or in doubly transgenic plants accumulated PLRV than did in singly transgenic tobacco plants. However, most cells in these plants did not accumulate Virus. To investigate restriction of the multiplication of Viruses containing PLRV sequences, transgenic plants were infected with a chimeric Virus that consisted of Tobacco mosaic Virus (TMV) containing genes for either the coat protein (CP) of PLRV or jellyfish green fluorescent protein (GFP) in place of the TMV coat protein. The Virus that encoded PLRV CP spread more slowly and accumulated less extensively than did the Virus that expressed GFP. The results support the suggestion that an RNA-mediated form of resistance that resembles post-transcriptional gene silencing operates in non-vascular cells and may be part of the mechanism that restricts PLRV to vascular tissue in conventionally infected plants.

  • umbraVirus gene expression helps Potato Leafroll Virus to invade mesophyll tissues and to be transmitted mechanically between plants
    Virology, 2001
    Co-Authors: Eugene V Ryabov, M. A. Mayo, H. Barker, Gillian Fraser, Michael E Taliansky
    Abstract:

    Potato Leafroll Virus (PLRV) was mechanically transmissible when inocula also contained the umbraVirus Pea enation mosaic Virus-2 (PEMV-2). In plants infected with PLRV and PEMV-2, PLRV accumulated in clusters of mesophyll cells in both inoculated and systemically infected leaves. No transmissions were obtained by coinoculation with Potato Virus Y, Potato Virus X (PVX), Tobacco mosaic Virus, or Cucumber mosaic Virus (CMV), although PLRV was transmissible from mixtures with CMV(ORF4) (a recombinant that contained the movement protein (MP) gene of the umbraVirus Groundnut rosette Virus (GRV) in place of the CMV MP gene). In contrast, neither a recombinant PVX that expressed GRV MP nor a mutant of CMV(ORF4), in which the CMV 2b gene was untranslatable, was able to help PLRV transmission. Possibly both a cell-to-cell movement function and counterdefense mechanisms such as those that block posttranscriptional gene silencing are involved in movement of PLRV within plants and its mechanical transmission between plants.

  • mechanical transmission of Potato Leafroll Virus
    Journal of General Virology, 2000
    Co-Authors: M. A. Mayo, Eugene V Ryabov, Gillian Fraser, Michael E Taliansky
    Abstract:

    Like typical luteoViruses, Potato Leafroll Virus (PLRV) cannot be transmitted mechanically by rubbing plants with solutions containing Virus particles. However, PLRV was found to be mechanically transmissible from extracts of plants that had been inoculated by viruliferous aphids and then post-inoculated with Pea enation mosaic Virus-2 (PEMV-2). Unlike the asymptomatic infections induced by either Virus alone, double infections in Nicotiana benthamiana induced necrotic symptoms with some line patterning and vein yellowing. Infective PLRV was recovered from a purified Virus preparation by inoculating plants mechanically with purified Virus particles mixed with PEMV-2. Similarly, Beet mild yellowing Virus was readily transmitted mechanically from mixtures containing PEMV-2. PLRV was also transmissible from mixtures made with extracts of plants infected with Groundnut rosette Virus, although less efficiently than from mixtures containing PEMV-2. This novel means of transmitting PLRV, and perhaps other poleroViruses, should prove very useful in a number of fields of luteoVirus research.

  • aphid acquisition and cellular transport of Potato Leafroll Virus like particles lacking p5 readthrough protein
    Phytopathology, 2000
    Co-Authors: F E Gildow, M. A. Mayo, J W Lamb, B Reavy, G H Duncan, J A T Woodford, Ronald T Hay
    Abstract:

    ABSTRACT Lepidopteran cells (Spodoptera frugiperda) produced isometric Virus-like particles (VLP) when infected with a recombinant baculoVirus Ac61 that contained the Potato Leafroll Virus (PLRV) coat protein gene modified with an N-terminal histidine tag (P3-6H). Cells infected with AcFL, a recombinant baculoVirus that expressed cDNA copies of the PLRV genome RNA, did not produce Virus-like particles (VLP). In cell lines doubly infected with Ac61 and AcFL, VLP were formed that contained PLRV-RNA packaged in P3-6H coat protein (FL). Both the P3-6H and the FL particles were morphologically indistinguishable from particles of PLRV despite the fact that they lacked the P5 readthrough protein present in wild-type PLRV. When aphids (Myzus persicae) were fed on, or injected with, purified PLRV, or VLP of either type (FL or P3-6H) and examined by electron microscopy, no differences were observed among treatments for particle endocytosis, transcellular transport, or exocytosis at the aphid midgut or accessory salivary glands. Particles were observed in the salivary canals and in the salivary duct leading out of the aphid. These results suggest that P5 readthrough protein of PLRV may not be essential for cellular transport of Virus through aphid vectors.

Stewart M Gray - One of the best experts on this subject based on the ideXlab platform.

  • the innate immunity protein c1qbp functions as a negative regulator of circulative transmission of Potato Leafroll Virus by aphids
    bioRxiv, 2020
    Co-Authors: Stacy L Deblasio, Michael J Maccoss, Richard S Johnson, Jennifer R Wilson, Cecilia Tamborindeguy, Patricia V Pinheiro, Stewart M Gray
    Abstract:

    ABSTRACT The vast majority of plant Viruses are transmitted by insect vectors with many crucial aspects of the transmission process being mediated by key protein-protein interactions. Yet, very few vector proteins interacting with Virus have been identified and functionally characterized. Potato Leafroll Virus (PLRV) is transmitted most effectively by Myzus persicae, the green peach aphid, in a circulative, non-propagative manner. Using an affinity purification strategy coupled to high-resolution mass spectrometry (AP-MS), we identified 11 proteins from M. persicae displaying high probability of interaction with PLRV and an additional 23 vector proteins with medium confidence interaction scores. Two of these proteins were confirmed to directly interact with the structural proteins of PLRV and other luteovirid species via yeast two-hybrid with an additional vector protein displaying binding specificity. Immunolocalization of one of these direct PLRV-interacting proteins, an orthologue of the human innate immunity protein complement component 1 Q subcomponent-binding protein (C1QBP), shows that MpC1QBP partially co-localizes with PLRV within cytoplasmic puncta and along the periphery of aphid gut epithelial cells. Chemical inhibition of C1QBP in the aphid leads to increased PLRV acquisition and subsequently increased titer in inoculated plants, supporting the role of C1QBP as a negative regulator of PLRV accumulation in M. persicae. We hypothesize that the innate immune function of C1QBP is conserved in aphids and represents the first instance of aphids mounting an immune response to a non-propagative plant Virus. This study presents the first use of AP-MS for the in vivo isolation of functionally relevant insect vector-Virus protein complexes.

  • the interaction dynamics of two Potato Leafroll Virus movement proteins affects their localization to the outer membranes of mitochondria and plastids
    Viruses, 2018
    Co-Authors: Stacy L Deblasio, Michael J Maccoss, Richard S Johnson, Ana Rita Rebelo, Stewart M Gray, Michelle Heck
    Abstract:

    The Luteoviridae is an agriculturally important family of Viruses whose replication and transport are restricted to plant phloem. Their genomes encode for four proteins that regulate viral movement. These include two structural proteins that make up the capsid and two non-structural proteins known as P3a and P17. Little is known about how these proteins interact with each other and the host to coordinate Virus movement within and between cells. We used quantitative, affinity purification-mass spectrometry to show that the P3a protein of Potato Leafroll Virus complexes with Virus and that this interaction is partially dependent on P17. Bimolecular complementation assays (BiFC) were used to validate that P3a and P17 self-interact as well as directly interact with each other. Co-localization with fluorescent-based organelle markers demonstrates that P3a directs P17 to the mitochondrial outer membrane while P17 regulates the localization of the P3a-P17 heterodimer to plastids. Residues in the C-terminus of P3a were shown to regulate P3a association with host mitochondria by using mutational analysis and also varying BiFC tag orientation. Collectively, our work reveals that the PLRV movement proteins play a game of intracellular hopscotch along host organelles to transport the Virus to the cell periphery.

  • Potato Leafroll Virus structural proteins manipulate overlapping yet distinct protein interaction networks during infection
    Proteomics, 2015
    Co-Authors: Stacy L Deblasio, Michael J Maccoss, Stewart M Gray, Michelle Cilia, Richard J Johnson, Michelle Sweeney, Alexander V Karasev
    Abstract:

    Potato Leafroll Virus (PLRV) produces a readthrough protein (RTP) via translational readthrough of the coat protein amber stop codon. The RTP functions as a structural component of the virion and as a nonincorporated protein in concert with numerous insect and plant proteins to regulate Virus movement/transmission and tissue tropism. Affinity purification coupled to quantitative MS was used to generate protein interaction networks for a PLRV mutant that is unable to produce the read through domain (RTD) and compared to the known wild-type PLRV protein interaction network. By quantifying differences in the protein interaction networks, we identified four distinct classes of PLRV-plant interactions: those plant and nonstructural viral proteins interacting with assembled coat protein (category I); plant proteins in complex with both coat protein and RTD (category II); plant proteins in complex with the RTD (category III); and plant proteins that had higher affinity for virions lacking the RTD (category IV). Proteins identified as interacting with the RTD are potential candidates for regulating viral processes that are mediated by the RTP such as phloem retention and systemic movement and can potentially be useful targets for the development of strategies to prevent infection and/or viral transmission of Luteoviridae species that infect important crop species.

  • cross linking measurements of the Potato Leafroll Virus reveal protein interaction topologies required for virion stability aphid transmission and Virus plant interactions
    Journal of Proteome Research, 2012
    Co-Authors: Juan D Chavez, Stewart M Gray, Michelle Cilia, Chad R Weisbrod, Jimmy K Eng, James E Bruce
    Abstract:

    Protein interactions are critical determinants of insect transmission for Viruses in the family Luteoviridae. Two luteovirid structural proteins, the capsid protein (CP) and the readthrough protein (RTP), contain multiple functional domains that regulate Virus transmission. There is no structural information available for these economically important Viruses. We used Protein Interaction Reporter (PIR) technology, a strategy that uses chemical cross-linking and high resolution mass spectrometry, to discover topological features of the Potato Leafroll Virus (PLRV) CP and RTP that are required for the diverse biological functions of PLRV virions. Four cross-linked sites were repeatedly detected, one linking CP monomers, two within the RTP, and one linking the RTP and CP. Virus mutants with triple amino acid deletions immediately adjacent to or encompassing the cross-linked sites were defective in virion stability, RTP incorporation into the capsid, and aphid transmission. Plants infected with a new, infectio...

  • small deletions in the Potato Leafroll Virus readthrough protein affect particle morphology aphid transmission Virus movement and accumulation
    Journal of General Virology, 2008
    Co-Authors: Peter Palukaitis, Stewart M Gray, Kari A Peter, Delin Liang
    Abstract:

    Potato Leafroll Virus (PLRV) capsid comprises 180 coat protein (CP) subunits, with some percentage containing a readthrough domain (RTD) extension located on the particle's surface. The RTD N terminus is highly conserved in luteovirids and this study sought to identify biologically active sites within this region of the PLRV RTD. Fourteen three-amino-acid-deletion mutants were generated from a cloned infectious PLRV cDNA and delivered to plants by Agrobacterium inoculations. All mutant Viruses accumulated locally in infiltrated tissues and expressed the readthrough protein (RTP) containing the CP and RTD sequences in plant tissues; however, when purified, only three mutant Viruses incorporated the RTP into the virion. None of the mutant Viruses were aphid transmissible, but the Viruses persisted in aphids for a period sufficient to allow for Virus transmission. Several mutant Viruses were examined further for systemic infection in four host species. All mutant Viruses, regardless of RTP incorporation, moved systemically in each host, although they accumulated at different rates in systemically infected tissues. The biological properties of the RTP are sensitive to modifications in both the RTD conserved and variable regions.

Qiaochun Wang - One of the best experts on this subject based on the ideXlab platform.

  • long term preservation of Potato Leafroll Virus Potato Virus s and Potato spindle tuber viroid in cryopreserved shoot tips
    Applied Microbiology and Biotechnology, 2018
    Co-Authors: Minrui Wang, Qiaochun Wang, Haiyan Chen, Lei Zhao, Zhenhua Cui, Zhibo Zhang, Dagragnar Blystad
    Abstract:

    Availability of and easy access to diverse plant Viruses and viroids is a prerequisite in applied and basic studies related to Viruses and viroids. Long-term preservation of Viruses and viroids is difficult. A protocol was described for long-term preservation of Potato Leafroll Virus (PLRV), Potato Virus S (PVS), and Potato spindle tuber viroid (PSTVd) in cryopreserved shoot tips of Potato cv. Zihuabai. Shoot regrowth levels following cryopreservation were higher in 1.5 mm-shoot tips (58–60%) than in 0.5-mm-ones (30–38%). All shoots recovered from 0.5-mm-shoot tips were PVS- and PSTVd-preserved, but none of them were PLRV-preserved. Cryopreservation of 1.5-mm-shoot tips resulted in 35% and 100% of PLRV- and PVS- and PSTVd-preserved shoots. Studies on cell survival patterns and Virus localization provided explanations to the varying PLRV-preservation frequencies produced by cryopreservation of the two sizes of shoot tips. Although micropropagation efficiencies were low after 12 weeks of subculture following cryopreservation, similar efficiencies were obtained after 16 weeks of subculture in pathogen-preserved shoots recovered from cryopreservation, compared with the diseased in vitro stock shoots (the control). Pathogen concentrations in the three pathogens-preserved shoots analyzed by qRT-PCR were similar to those in micropropagated shoots. The three pathogens cryopreserved in shoot tips were readily transmitted by grafting and mechanical inoculation to Potato plants. PLRV, PVS, and PSTVd represent a diverse range of plant Viruses and viroid in terms of taxonomy and infectious ability. Therefore, shoot tip cryopreservation opens a new avenue for long-term preservation of the Virus and viroid.

  • cryotherapy of Potato shoot tips for efficient elimination of Potato Leafroll Virus plrv and Potato Virus y pvy
    Potato Research, 2007
    Co-Authors: Qiaochun Wang, Yong Liu, Yonghong Xie, Misa You
    Abstract:

    Viral diseases constitute a major constraint to high yield and high quality production of Potato. Potato Leafroll Virus (PLRV) and Potato Virus Y (PVY) are among the most damaging Potato Viruses and are prevalent in most Potato growing areas. In the present study, attempts were made to eliminate PLRV and PVY by three cryogenic protocols, i.e., encapsulation-dehydration, encapsulation-vitrification and droplet. Results showed that both PLRV and PVY could be efficiently eliminated by cryogenic treatments with 83–86% and 91–95% of frequencies of Virus-free plantlets obtained for the former and latter, respectively. Frequencies of Virus-free plantlets produced by cryogenic treatments were higher than those by meristem culture (56% for PLRV and 62% for PVY) and thermotherapy (50% for PLRV and 65% for PVY), and similar to those by thermotherapy followed by meristem culture (90% for PLRV and 93% for PVY). Survival (75–85%) and regrowth (83–89%) from cryo-treated shoot tips were higher than those from meristem culture (50–55%) and thermotherapy followed by meristem culture (40–50%), but similar to those from thermotherapy (80–87%). The morphology of the plantlets regenerated from cryo-treated shoot tips was similar to that of non-treated plantlets. Thus, cryotherapy would provide an alternative method for efficient elimination of Potato Viruses, and can be simultaneously used for long-term storage of Potato germplasm and for production of Virus-free plants.

L Torrance - One of the best experts on this subject based on the ideXlab platform.

  • a fully recombinant elisa using in vivo biotinylated antibody fragments for the detection of Potato Leafroll Virus
    Journal of Virological Methods, 2009
    Co-Authors: Ahmad Almrabeh, Angelika Ziegler, Graham H Cowan, L Torrance
    Abstract:

    A recombinant antibody fusion protein, V3HCL, which was shown previously to have specific reactivity for Potato Leafroll Virus (PLRV), was labeled with biotin using standard chemical coupling procedures and by an in vivo method. The in vivo method proved superior giving reproducible V3HCL-biotin preparations. A fully recombinant ELISA was devised incorporating V3HCL, V3HCL-biotin and streptavidin alkaline phosphatase conjugate. This assay gave comparable results for PLRV detection in Potato to an assay based on immunoglobulins. The V3HCL-biotin preparations were stable and retained specific activity for more than 1 year when stored at 4 degrees C or -20 degrees C. The results demonstrate that scFv reagents derived from synthetic phage display platforms can provide effective alternatives to assays incorporating immune reagents.

  • fusion proteins of single chain variable fragments derived from phage display libraries are effective reagents for routine diagnosis of Potato Leafroll Virus infection in Potato
    Phytopathology, 1999
    Co-Authors: R L Toth, M. A. Mayo, K Harper, L Torrance
    Abstract:

    ABSTRACT A panel of 11 different single-chain variable fragment antibodies (scFv) that bind to Potato Leafroll Virus (PLRV) has been studied to assess each one's suitability as practical diagnostic tools. The scFv, previously obtained from naive phage display libraries, were expressed in Escherichia coli as fusion proteins. The fusion proteins comprised scFv joined to either the human light chain kappa constant domain (CL), an amphipathic helix (Zip), a combination of CL and Zip, or alkaline phosphatase (AP/S). The fusion proteins were tested for their ability to detect, or trap on enzymelinked immunosorbent assay (ELISA) plates, PLRV in extracts of infected Potato leaves. The tests done with the different scFv fusion proteins were compared with a standard triple-antibody sandwich (TAS)-ELISA that employs a rabbit polyclonal antibody preparation to coat microtiter plates and a monoclonal antibody, SCR3, to detect PLRV. Of 11 scFvCL fusion proteins, 7 detected PLRV as readily as SCR3 when used as detecting...

  • properties of a panel of single chain variable fragments against Potato Leafroll Virus obtained from two phage display libraries
    Journal of Virological Methods, 1999
    Co-Authors: K Harper, M. A. Mayo, R L Toth, L Torrance
    Abstract:

    Twelve single chain variable fragment (scFv) antibodies that bind to particles of Potato Leafroll Virus (PLRV) were obtained from two naive phage display libraries. Phages were selected against PLRV particles or dissociated PLRV particles immobilised onto tubes. Individual PLRV-binding scFv were identified by ELISA, after their expression either fused to the surface of phage particles, or as soluble scFv (scFv-c-myc), or as scFv-alkaline phosphatase fusion proteins (scFv-AP), obtained by subcloning into pSKAP/S. These procedures resulted in the isolation of scFv with different properties. For example, some of the scFv reacted strongly with Virus particles but not with dissociated capsid protein, which suggests that they had reacted with discontinuous epitopes. Others reacted with dissociated capsid proteins and SDS-denatured protein, which suggests that they had reacted with continuous epitopes. ScFv were also subcloned into pC(L) for expression as fusion proteins with human kappa constant region (scFv-C(L)). Expression of these constructs in Escherichia coli yielded 0.2-1 mg protein per litre of bacterial culture. The different scFv fusion proteins were evaluated in ELISA to detect PLRV in leaf extracts of Physalis floridana. Absorbance values obtained with the fusion proteins were greater than those obtained with the scFv-c-myc, and were similar to those obtained in assays done using monoclonal or polyclonal antibodies.

Robert R Martin - One of the best experts on this subject based on the ideXlab platform.