Identification of a novel circular single-stranded DNA associated with cotton leaf curl disease in Pakistan

Recent reports have suggested that cotton leaf curl virus (CLCuV), a gemini virus of the genus Begomovirus, may be responsible for cotton leaf curl dis ease in Pakistan. However, the causal agent of the disease remains unclear as CLCuV genomic components resembling begomovirus DNA A are unable to indu ce typical disease symptoms when reintroduced into plants. All attempts to isolate a genomic component equivalent to begomovirus DNA B have been unsuc cessful. Here, we describe the isolation and characterisation of a novel ci rcular single-stranded (ss) DNA associated with naturally infected cotton p lants. In addition to a component resembling DNA A, purified geminate parti cles contain a smaller unrelated ssDNA that we refer to as DNA 1. DNA 1 was cloned from double-stranded replicative form of the viral DNA isolated fro m infected cotton plants. Blot hybridisation using probes specific for eith er CLCuV DNA or DNA 1 was used to demonstrate that both DNAs co-infect natu rally infected cotton plants from different geographical locations. DNA 1 w as detected in viruliferous Bemisia tabaci and in tobacco plants infected u nder laboratory conditions using B. tabaci, indicating that it is transmitt ed by whiteflies. Sequence analysis showed that DNA 1 is approximately half the size of CLCuV DNA but shares no homology, indicating that it is not a defective geminivirus component. DNA 1 has some homology to a genomic compo nent of members of Nanoviridae, a family of DNA viruses that are normally t ransmitted by aphids or planthoppers. DNA 1 encodes a homologue of the nano virus replication-associated protein (Rep) and has the capacity to autonomo usly replicate in tobacco. The data suggest that a nanovirus-like DNA has b ecome whitefly-transmissible as a result of its association with a geminivi rus and that cotton leaf curl disease may result from a mutually dependent relationship that has developed between members of two distinct DNA virus f amilies that share a similar replication strategy. (C) 1999 Academic Press.