Rapid screening for dominant negative mutations in the beet necrotic yellow vein virus triple gene block proteins P13 and P15 using a viral replicon |
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Authors: | Lauber Emmanuelle Janssens Laurence Weyens G. Jonard G. Richards K.E. Lefèbvre M. Guilley H. |
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Affiliation: | (1) Institut de Biologie Moléculaire des Plantes du CNRS et de l'Université Louis Pasteur, 12 rue du Général Zimmer, 67084 Strasbourg Cedex, France;(2) ADVANTA Biotechnology Department, SES EUROPE N.V./S.A, Industriepark, Soldatenplein Z2 no 15, B-3300 Tienen, Belgium |
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Abstract: | Point mutations were introduced into the genes encoding the triple gene bock movement proteins P13 and P15 of beet necrotic yellow vein virus (BNYVV). Mutations which disabled viral cell-to-cell movement in Chenopodium quinoa were then tested for their ability to act as dominant negative inhibiters of movement of wild-type BNYVV when expressed from a co-inoculated BNYVV RNA 3-based replicon. For P13, three types of mutation inhibited the movement function: non-synomynous mutations in the N- and C-terminal hydrophobic domains, a mutation at the boundary between the N-terminal hydrophobic domain and the central hydrophilic domain (mutant P13-A12), and mutations in the conserved sequence motif in the central hydrophilic domain. However, only the boundary mutant P13-A12 strongly inhibited movement of wild-type virus when expressed from the co-inoculated replicon. Similar experiments with P15 detected four movement-defective mutants which strongly inhibited cell-to-cell movement of wild-type BNYVV when the mutants were expressed from a co-inoculated replicon. Beta vulgaris transformed with two of these P15 mutants were highly resistant to fungus-mediated infection with BNYVV. |
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Keywords: | beet necrotic yellow vein virus BNYVV cell-to-cell movement dominant negative mutation sugar beet triple gene block transgenic |
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