Rapid screening for dominant negative mutations in the beet necrotic yellow vein virus triple gene block proteins P13 and P15 using a viral replicon

Point mutations were introduced into the genes encoding the triple gene boc k 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 RN A 3-based replicon. For P13, three types of mutation inhibited the movement function: non-synomynous mutations in the N- and C-terminal hydrophobic do mains, 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 t he "boundary" mutant P13-A12 strongly inhibited movement of wild-type virus when expressed from the co-inoculated replicon. Similar experiments with P 15 detected four movement-defective mutants which strongly inhibited cell-t o-cell movement of wild-type BNYVV when the mutants were expressed from a c o-inoculated replicon. Beta vulgaris transformed with two of these P15 muta nts were highly resistant to fungus-mediated infection with BNYVV.