Single-stranded DNA from abutilon mosaic virus is present in the plastids of infected Abutilon sellovianum

Abstract

The leaf mosaic of Abutilon selovianum is caused by abutilon mosaic virus (AbMV), a geminivirus that has a circular single-stranded DNA genome. DNA was isolated from intact plastids of AbMV-infected and noninfected plants. Plastids from infected plants were shown to contain the single-stranded AbMV DNA by Southern-blot hybridization experiments that used a probe made from highly purified AbMV DNA. The possibility of adsorption of AbMV virions or viral DNA onto the plastid envelope was ruled out by several in vitro experiments with DNase I and protease. Furthermore, the lamellar system of plastids from AbMV-infected plants was degenerated and substituted by amorphous electron-dense material. The transport of AbMV DNA across the plastid envelope has implications for the development of a chloroplast transformation vector.Geminiviruses are plant viruses that contain single-stranded DNA (ssDNA) encapsidated in twin particles (1,2). The genomes of some geminiviruses have been cloned and sequenced (for review, see refs. 3 and 4). Geminiviruses have been localized in the vascular-bundle area of infected plants in general (5) and, in one case (chloris striate mosaic virus), also in the remaining mesophyll (6). Virus-like twin particles have mainly been found in the nuclei (5) but, to our knowledge, never in plastids.The whitefly-transmitted abutilon mosaic virus (AbMV) is classified as a geminivirus (7). The disease is transmitted by the whitefly Bemisia tabaci, which is not indigenous to Europe; therefore, cuttings were used for propagation of infected plants. The corresponding virus-like twin particles had been found (8,9) in the nuclei of the phloem-associating cells of various host plants. Structures called "chains of pearls" that contain the AbMV DNA and transmit the disease can be isolated from AbMV-infected plants (10).After infecting Malva parviflora, comparable filaments could be detected in the chloroplasts of mesophyll cells, because they form paracrystalline inclusions (8, 9).The appearance of virus-associated structures in plastids could explain why cytological symptoms of the disease are predominant in the mesophyll chloroplasts, whereas they are generally absent from the plastids of the vascular-bundle area (11). Therefore, the question arises whether AbMV DNA is present in the plastids. Our analysis has shown that it is. The tissue and organelle specificity of the virus are of central importance ifgeminiviruses are to be used as genetic vectors. MATERIALS AND METHODSIsolation and Purification of Plastids. Plant material from uninfected Abutilon sellovianum Regel and AbMV-infected Abutilon sellovianum var. marmorata Regel was homogenized in an isotonic medium of 0.4 M sorbitol/44 mM Mes, pH 6.1/10 mM NaCl/8 mM EDTA/1 mM MnCl2/1 mM MgC2/0.5 mM KH2PO4/2 mM sodium ascorbate/2 mM cysteine/3 mM dithioerythritol/1% bovine serum albumin/ 0.01% polyvinylpyrrolidone 10 in a Waring blender at 0-2°C and filtered through 100-gm and 20-,um nylon gauze. The plastids were pelleted by centrifugation in a swinging-...

A phylogenetic and evolutionary justification for three genera of Geminiviridae

“…Clear evidence for rolling circle replication in geminiviruses has been provided by Stenger et al [42]. There is also compelling evidence that AbMV closely associates with chloroplasts if it does not actually replicate in them [9,10]. These lines of evidence would suggest a geminiviral origin as a phage or bacterial plasmid: it is therefore interesting that Petty et al [31] have shown that a geminivirus CP promoter functions efficiently in E. coli.…”

The induction of stromule formation by a plant DNA-virus in epidermal leaf tissues suggests a novel intra- and intercellular macromolecular trafficking route

“…The role of chloroplasts in the life cycle of plant DNA-viruses needs to be examined. In studies of cellular alterations induced by various geminiviruses in systemically infected plants, dramatic morphological changes in the ultrastructure of chloroplasts were identified, such as vesiculated entities, reduced starch and chlorophyll content, accumulation of fibrillar inclusions, virus-like particles, and vDNA within plastids (Esau, 1933; Jeske and Werz, 1978, 1980a,b; Schuchalter-Eicke and Jeske, 1983; Jeske and Schuchalter-Eicke, 1984; Jeske, 1986; Gröning et al, 1987, 1990; Rushing et al, 1987; Channarayappa et al, 1992). For AbMV it was shown that the severity of chloroplast structure remodeling was dependent on light intensity, and diurnal and seasonal conditions.…”

“…Nevertheless, the detection of vDNA, fibrillar inclusions, or virus-like particles within chloroplasts, suggests other functions of this interplay. Until now, only AbMV vDNA was detected in purified plastids from infected plants (Gröning et al, 1987, 1990). An artificial co-purification was excluded by thermolysin and DNase I treatment.…”

Movement of DNA across the chloroplast envelope: Implications for the transfer of promiscuous DNA

“…There is convincing evidence that several plant virus nucleic acids, and T-DNA from Agrobacterium tumefaciens can become localized in chloroplasts (DeBlock et al 1985;Gröning et al 1987;Schoelz and Zaitlin 1989;Gröning et al 1990;Venkateswarlu and Nazar 1991). However, it is not known whether a vector mediated mechanism might be involved in the transport of these nucleic acids into chloroplasts.…”