INTERACTION BETWEEN A GEMINIVIRUS REPLICATION PROTEIN AND ORIGIN DNA IS ESSENTIAL FOR VIRAL REPLICATION

The geminivirus, tomato golden mosaic virus (TGMV), encodes one protei n, AL1, that is absolutely required for viral DNA replication. AL1 int eracts with the TGMV DNA genome by binding specifically to the viral o rigin of replication. We have investigated the nature and significance of AL1/origin interactions in vitro and in vivo by using competitive DNA binding and transient replication assays. Competition assays estab lished that a 13-base pair (bp) element (5'-GGTAGTAAGGTAG) containing two 5-bp direct repeat motifs separated by a 3-bp central core constit utes a high affinity AL1 binding site. DNAs containing intact 3' repea t sequences plus core (TAAGGTAG and ccTAGTAAGGTAG) were stronger compe titors for AL1 binding than DNAs containing intact 5' repeat sequences plus core (GGTAGTAA and GGTAGTA-AccTAG), thereby demonstrating that A L1 interacts differently with the repeat motifs. Replication in tobacc o protoplasts established that the AL1 binding site is an essential ci s-acting element for viral replication. No replication was detected fo r DNAs containing mutations in either of the repeat motifs of the AL1 recognition sequence when AL1 was provided in trans from a plant gene expression vector. In contrast, a DNA with a mutation in the 5' repeat motif (ccTAGTAAGGTAG) replicated when both AL1 and AL3, a TGMV protei n involved in viral DNA accumulation, were provided in trans. No repli cation was detected for a DNA containing a mutation in the 3' repeat m otif (GGTAGTAAccTAG) in the presence of AL1 and AL3. The in vitro and in vivo results suggest that binding of AL1 to the 3' repeat element i s an essential step in DNA replication, while binding to the 5' repeat element may serve to enhance viral replication.