Fate of African Cassava Mosaic Virus Coat Protein Deletion Mutants after Agroinoculation

Abstract

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.

The infectivity of dimeric potato yellow mosaic geminivirus clones in different hosts

“…2, lane 1 and results not shown). These results are similar to those found with two isolates of T G M V (Elmer et al, 1988;Frischmuth et al, 1993), ACMV (Klinkenberg et al, 1989), AbMV (Evans & Jeske, 1993) and Indian cassava mosaic virus (Frischmuth et al, 1993) and illustrates that as with these other bipartite geminiviruses, the PYMV D N A A component encodes all of the viral proteins necessary for viral D N A replication. Following agroinoculation of tomato and potato leaf discs with a mixture of pP2A and pP2B extracts of both were found to contain virus-specific, replicative supercoiled and genomic ssDNA of both PYMV DNA (Fig.…”

“…The ability of cloned dimeric PYMV DNAs to replicate in agroinoculated leaf discs derived from the same plants used in the whole plant agroinoculation experiments above was assayed as described previously (Elmer et al, 1988;Klinkenberg et al, 1989) in accordance with established procedures (Horsch & Klee, 1986). Axenically grown potato plants (Adams et al, 1985) were used as a source of sterile leaf tissue in preference to greenhouse-grown plants.…”

“…tuberosum L. cv. Desiree) either by mechanical inoculation (Roberts et al, 1988) or stem agroinoculation (Klinkenberg et al, 1989). In some experiments the potato plants were decapitated prior to agroinoculation (Klinkenberg et al, 1989) and sugar beet plants were inoculated by crown needle-puncture (Briddon et al, 1989).…”

Mutagenesis of the AC3 open reading frame of African cassava mosaic virus DNA A reduces DNA B replication and ameliorates disease symptoms

“…The deleted region in the progeny DNA A was replaced by cloning vector DNA sequestered from pUC DNA surrounding the DNA A fragment in the inoculated construct. Size reversion of ACMV DNA A has been shown previously for systemic infections resulting with coat protein deletion mutants (Etessami et al, 1989;Klinkenberg et al, 1989). Klinkenberg et al (1989) showed that the size reversion observed for ACMV DNA A with the coat protein deletion mutants was produced following systemic spread, whereas in dividing callus ceils the deleted DNA A was replicated.…”

Replication promiscuity of DNA-β satellites associated with monopartite begomoviruses; deletion mutagenesis of the Ageratum yellow vein virus DNA-β satellite localizes sequences involved in replication

“…Binary vector clones were introduced into Agrobacterium tumefaciens strain GV3850 (Zambryski et al, 1983) and used to agroinoculate Nicotiana benthamiana and A. conyzoides plants as described by Saunders et al (2004). N. benthamiana leaf disks were prepared and maintained as described by Klinkenberg et al (1989) and DNA was extracted from plant tissues and analysed by Southern blot hybridization as described by Saunders et al (2004). Equal amounts of DNA were loaded in each lane.…”