MUTATIONAL ANALYSIS OF BEAN YELLOW DWARF VIRUS, A GEMINIVIRUS OF THE GENUS MASTREVIRUS THAT IS ADAPTED TO DICOTYLEDONOUS PLANTS

Bean yellow dwarf virus (BeYDV) is an atypical member of the geminivir us genus Mastrevirus that infects dicotyledonous plants. BeYDV DNA con tains six open reading frames (ORFs) with the capacity to encode prote ins in excess of 10 kDa, Two virion-sense ORFs (V1 and V2) and two com plementary-sense ORFs (C1 and C2) have homologues in all mastreviruses , while ORFs C3 and C4 are not conserved. To investigate their functio ns, each of the ORFs has been truncated by either frameshifting or the introduction of a stop codon. We demonstrate that an ORF V1 mutant re plicated efficiently in Nicotiana tabacum protoplasts but was unable t o systemically infect Phaseolus vulgaris and Datura stramonium, consis tent with a role for V1 protein in virus movement. However, the mutant was able to systemically infect Nicotiana benthamiana although the on set of symptoms was appreciably delayed in comparison with wild-type v irus. Disruption of ORF V2, encoding the coat protein, prevented syste mic infection of all three hosts but the mutant replicated in protopla sts. Both ORF C1 and ORF C2 were essential for replication in protopla sts. Modification of the complementary-sense splice donor and acceptor sequences also prevented replication. Removal of the intron prevented systemic infection, although the intronless mutant was able to produc e functional replication-associated protein (Rep) and replicated effic iently in protoplasts, ORFs C3 and C4 were not required for systemic i nfection. Our results indicate that four ORFs are spatially and functi onally conserved in mastreviruses that infect both monocotyledonous an d dicotyledonous plants.