Grapevine fanleaf virus (GFLV)‐specific antibodies confer GFLV and Arabis mosaic virus (ArMV) resistance in Nicotiana benthamiana

Nölke, Greta; Cobanov, P.; Uhde‐Holzem, Kerstin; Reustle, Götz M.; Fischer, Rainer; Schillberg, Stefan

doi:10.1111/j.1364-3703.2008.00510.x

Cited by 36 publications

(25 citation statements)

References 45 publications

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“…Finally, ELISA readings at OD 405 nm were measured using the Synergy HT reader (BioTek Instruments, Bad Friedrichshall, Germany). The mice with the strongest immune responses were selected for further immunization with 50 lg of protein but no adjuvant and the production of hybridomas as described (Nölke et al 2009). …”

Section: Production Of Monoclonal Antibodiesmentioning

confidence: 99%

Biochemical properties of the matrix metalloproteinase NtMMP1 from Nicotiana tabacum cv. BY-2 suspension cells

Mandal

Fischer

Schillberg

et al. 2010

Planta

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A zinc-dependent matrix metalloproteinase (NtMMP1) found in the plasma membrane of Nicotiana tabacum cv. Bright Yellow 2 (BY-2) suspension cells is thought to be responsible for the degradation of recombinant proteins secreted into the culture supernatant. We have characterized the proteolytic activity of NtMMP1 by expressing a recombinant derivative lacking the C-terminal transmembrane domain in yeast. After purifying the protein by affinity chromatography, its autocatalytic activity was analyzed using monoclonal antibodies raised against its N-terminal and C-terminal portions. Both the unprocessed and processed forms of NtMMP1 displayed caseinolytic activity and N-terminal sequencing identified an autocatalytic cleavage site within the sequence motif HFSFFP, which is similar to the corresponding sequences of the human matrix metalloproteinases stromelysin-1 (MMP-3) and stromelysin-2 (MMP-10). Unlike all other matrix metalloproteinases investigated so far, NtMMP1 contains a disulfide bond within its propeptide thus rendering the proenzyme catalytically active. Kinetic analysis of NtMMP1 with a synthetic substrate revealed a K(m) of 10.55 +/- 0.9 microM, a k(cat) of 0.6 +/- 0.01 s(-1) and maximum activity at pH 7.5. We found that NtMMP1 degrades Desmodus rotundus salivary plasminogen activator alpha 1 (DSPAalpha1), a biopharmaceutical protein, that has proven difficult to produce in tobacco BY-2 cells. This provides a likely explanation for the frequent instability of secreted recombinant biopharmaceuticals produced in plant suspension cell cultures. Our data suggest new avenues that can be explored to improve the production of pharmaceutical proteins in plants and plant cells.

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Section: Production Of Monoclonal Antibodiesmentioning

confidence: 99%

Biochemical properties of the matrix metalloproteinase NtMMP1 from Nicotiana tabacum cv. BY-2 suspension cells

Mandal

Fischer

Schillberg

et al. 2010

Planta

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“…The magnetic immunodetection of plant viruses was demonstrated using GFLV as a model. GFLV-specific murine mAbFL 6 was produced using a monoclonal hybridoma cell line (39), and its integrity was verified by SDS-PAGE, followed by staining with Coomassie brilliant blue, and by Western blot analysis. This revealed typical 150-kDa bands under native conditions and separate 55-kDa and 20-kDa bands representing the heavy and light chains, respectively, under denaturing conditions (data not shown).…”

Section: Resultsmentioning

confidence: 99%

Simple and Portable Magnetic Immunoassay for Rapid Detection and Sensitive Quantification of Plant Viruses

Rettcher

Jungk

Kühn

et al. 2015

Appl Environ Microbiol

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c Plant pathogens cause major economic losses in the agricultural industry because late detection delays the implementation of measures that can prevent their dissemination. Sensitive and robust procedures for the rapid detection of plant pathogens are therefore required to reduce yield losses and the use of expensive, environmentally damaging chemicals. Here we describe a simple and portable system for the rapid detection of viral pathogens in infected plants based on immunofiltration, subsequent magnetic detection, and the quantification of magnetically labeled virus particles. Grapevine fanleaf virus (GFLV) was chosen as a model pathogen. Monoclonal antibodies recognizing the GFLV capsid protein were immobilized onto immunofiltration columns, and the same antibodies were linked to magnetic nanoparticles. GFLV was quantified by immunofiltration with magnetic labeling in a double-antibody sandwich configuration. A magnetic frequency mixing technique, in which a two-frequency magnetic excitation field was used to induce a sum frequency signal in the resonant detection coil, corresponding to the virus concentration within the immunofiltration column, was used for high-sensitivity quantification. We were able to measure GFLV concentrations in the range of 6 ng/ml to 20 g/ml in less than 30 min. The magnetic immunoassay could also be adapted to detect other plant viruses, including Potato virus X and Tobacco mosaic virus, with detection limits of 2 to 60 ng/ml.

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“…Moreover, to construct a functional scFv, the influence of the V H and V L orientation on the binding activity and structure should also be considered. Some reports have shown that transgenic plants expressing an scFv that specifically detects plant virus proteins exhibit viral resistance and tolerance (59,60). Therefore, future studies should be performed to examine transgenic plants expressing the broad-spectrum C4 scFv that may achieve wideranging antibody-meditated resistance to potyviruses.…”

Section: Discussionmentioning

confidence: 99%

A Strategy for Generating a Broad-Spectrum Monoclonal Antibody and Soluble Single-Chain Variable Fragments against Plant Potyviruses

Liu

Lin

et al. 2015

Appl Environ Microbiol

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Potyviruses are major pathogens that often cause mixed infection in calla lilies. To reduce the time and cost of virus indexing, a detection method for the simultaneous targeting of multiple potyviruses was developed by generating a broad-spectrum monoclonal antibody (MAb) for detecting the greatest possible number of potyviruses. The conserved 121-amino-acid core regions of the capsid proteins of Dasheen mosaic potyvirus (DsMV), Konjak mosaic potyvirus (KoMV), and Zantedeschia mild mosaic potyvirus (ZaMMV) were sequentially concatenated and expressed as a recombinant protein for immunization. After hybridoma cell fusion and selection, one stable cell line that secreted a group-specific antibody, named C4 MAb, was selected. In the reaction spectrum test, the C4 MAb detected at least 14 potyviruses by indirect enzyme-linked immunosorbent assay (I-ELISA) and Western blot analysis. Furthermore, the variable regions of the heavy (V H ) and light (V L ) chains of the C4 MAb were separately cloned and constructed as single-chain variable fragments (scFvs) for expression in Escherichia coli. Moreover, the pectate lyase E (PelE) signal peptide of Erwinia chrysanthemi S3-1 was added to promote the secretion of C4 scFvs into the medium. According to Western blot analysis and I-ELISA, the soluble C4 scFv (V L -V H ) fragment showed a binding specificity similar to that of the C4 MAb. Our results demonstrate that a recombinant protein derived from fusion of the conserved regions of viral proteins has the potential to produce a broad-spectrum MAb against a large group of viruses and that the PelE signal peptide can improve the secretion of scFvs in E. coli. The genus Potyvirus, one of the largest groups of plant viruses, includes 158 species, many of which are economically important, such as Potato virus Y (PVY) and Plum pox virus (1, 2). Most potyviruses infect various host plants and are spread worldwide by aphid vectors and mechanical inoculation (3). Potyviruses have a single-stranded, positive-sense RNA genome of approximately 10 kb in length that encodes a polyprotein translated from a large open reading frame (ORF) (3) and a trans-frame movement-related protein (P3N-PIPO) encoded from the 5=-terminal portion of P3 and the PIPO cistrons (4, 5). During proteolytic processing, the large polyprotein can be separated into 10 mature proteins that are involved in virus replication, movement, and virion assembly (3). Potyviral capsid proteins (CPs) not only form the viral particle but also participate in cell-to-cell and systemic movement (6, 7). The variable N-and C-terminal regions of the CPs exposed on the virion surface are required for long-distance transport, are involved in aphid transmission (DAG motif), and may also assist in cell-to-cell spreading (7-10), whereas the conserved core regions of CPs are necessary for virion assembly and the cell-to-cell movement of potyviruses (6, 7).In Taiwan, the viral disease caused by potyviruses is one of the limiting factors for production of calla lilies. At least five potyvirus...

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Grapevine fanleaf virus (GFLV)‐specific antibodies confer GFLV and Arabis mosaic virus (ArMV) resistance in Nicotiana benthamiana

Cited by 36 publications

References 45 publications

Biochemical properties of the matrix metalloproteinase NtMMP1 from Nicotiana tabacum cv. BY-2 suspension cells

Biochemical properties of the matrix metalloproteinase NtMMP1 from Nicotiana tabacum cv. BY-2 suspension cells

Simple and Portable Magnetic Immunoassay for Rapid Detection and Sensitive Quantification of Plant Viruses

A Strategy for Generating a Broad-Spectrum Monoclonal Antibody and Soluble Single-Chain Variable Fragments against Plant Potyviruses

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