Nicotiana benthamiana was transformed with a single copy of a tandem repeat of subgenomic DNA B isolated from plants infected with a Kenyan isolate of the bipartite geminivirus African cassava mosaic virus. Symptoms in transformed plants were less severe than in nontransformed controls when challenged with virus or cloned DNA of Kenyan or Nigerian isolates. Symptom amelioration was associated with the mobilization and amplification of the subgenomic DNA, producing a comparable reduction in the amount of DNA specific to each genomic component. The disproportionate reduction in the levels of full-length components (DNA A, 20%; DNA B, 70%) indicates that the episomally replicating subgenomic DNA has been amplified at the expense of full-length DNA B to three times the level of the latter. Serial infection of transformants resulted in a further decrease in symptom severity and viral DNA levels. No differences were observed in the severity of symptoms or levels of viral DNA when transformants and controls were challenged with the related geminiviruses beet curly top virus and tomato golden mosaic virus, demonstrating the specific nature of the interaction. Analysis of infected tissue showed that tomato golden mosaic virus was unable to amplify the subgenomic DNA. However, since the production of subgenomic DNA is possibly a common feature of the bipartite geminiviruses, this approach might contribute to the production of plants showing increased tolerance to a number of economically important viral diseases.

SUMMARYWe have investigated the behaviour of coat protein deletion mutants of the geminivirus African cassava mosaic virus (ACMV) when introduced into Nicotiana benthamiana by agroinoculation. In dividing callus tissue, replicating mutant DNA A, in the absence of DNA B, remained subgenomic in size. However, systemic infection of plants was associated with the rapid reversion of the deletion mutants to native component size, as happened when the mutants were introduced into the host by more conventional mechanical inoculation procedures. The results contrast with those reported for tomato golden mosaic virus (TGMV) for which similar mutants, agroinoculated into N. benthamiana, remained subgenomic. The results indicate that the inoculation route is not responsible for the different behaviour of ACMV and TGMV mutants and illustrate a significant difference between the interaction of these otherwise closely related geminiviruses with a common host that might be exploited for the investigation of the determinants of DNA replication and spread.

Insertion and deletion mutagenesis of both extended open reading frames (ORFs) of cassava latent virus DNA 2 destroys infectivity. Infectivity is restored by coinoculating constructs that contain single mutations within different ORFs. Although frequent intermolecular recombination produces dominant parental-type virus, mutants can be retained within the virus population indicating that they are competent for replication and suggesting that rescue can occur by complementation of trans acting gene products. By cloning specific fragments into DNA 1 coat protein deletion vectors we have delimited the DNA 2 coding regions and provide substantive evidence that both are essential for virus infection. Although a DNA 2 component is unique to whitefly-transmitted geminiviruses, the results demonstrate that neither coding region is involved solely in insect transmission. The requirement for a bipartite genome for whitefly-transmitted geminiviruses is discussed.