A novel subviral agent associated with a geminivirus: the first report of a DNA satellite.

Numerous plant RNA viruses have associated with them satellite (sat) RNAs that have little or no nucleotide sequence similarity to either the viral or host genomes but are completely dependent on the helper virus for replication. We report here on the discovery of a 682-nt circular DNA satellite associated with tomato leaf curl geminivirus (TLCV) infection in northern Australia. This is the first demonstration that satellite molecules are not limited to RNA viral systems. The DNA satellite (TLCV sat-DNA) is strictly dependent for replication on the helper virus replication-associated protein and is encapsidated by TLCV coat protein. It has no significant open reading frames, and it shows no significant sequence similarity to the 2766-nt helper-virus genome except for two short motifs present in separate putative stem-loop structures: TAATATTAC, which is universally conserved in all geminiviruses, and AATCGGTGTC, which is identical to a putative replication-associated protein binding motif in TLCV. Replication of TLCV sat-DNA is also supported by other taxonomically distinct geminiviruses, including tomato yellow leaf curl virus, African cassava mosaic virus, and beet curly top virus. Therefore, this unique DNA satellite does not appear to strictly conform with the requirements that dictate the specificity of interaction of geminiviral replication-associated proteins with their cognate origins as predicted by the current model of geminivirus replication.

Plant virus DNA replication processes in Agrobacterium: insight into the origins of geminiviruses?

Agrobacterium tumefaciens, a bacterial plant pathogen, when transformed with plasmid constructs containing greater than unit length DNA of tomato leaf curl geminivirus accumulates viral replicative form DNAs indistinguishable from those produced in infected plants. The accumulation of the viral DNA species depends on the presence of two origins of replication in the DNA constructs and is drastically reduced by introducing mutations into the viral replication-associated protein (Rep or C1) ORF, indicating that an active viral replication process is occurring in the bacterial cell. The accumulation of these viral DNA species is not affected by mutations or deletions in the other viral open reading frames. The observation that geminivirus DNA replication functions are supported by the bacterial cellular machinery provides evidence for the theory that these circular single-stranded DNA viruses have evolved from prokaryotic episomal replicons.

ORF C4 of tomato leaf curl geminivirus is a determinant of symptom severity.

A tomato leaf curl virus (TLCV) mutant has been constructed in vitro that contains T-to-C replacements at nucleotides 2457 and 2463 within the C4 open reading frame (ORF). The mutations destroy the two possible initiator AUG codons for the C4 ORF without disrupting the coding capacity of the C1 ORF which entirely overlaps the C4 ORF. Agroinoculation of the C4 mutant TLCV into three alternative experimental hosts for the virus (Datura stramonium, Lycopersicon esculentum, and Nicotiana tabacum) gives rise to infections which show dramatically reduced symptoms when compared to a wild-type infection, while retaining wild-type levels of all viral DNA species. In most cases the mutations were stably inherited by progeny virus. However, a single tomato plant inoculated with the mutant developed phenotypically wild-type symptoms and was subsequently shown to contain progeny virus in which the mutation at position 2457 had reverted to wild-type sequence, indicating that this AUG may be the site of initiation of translation of the C4 product in the wild-type virus. The results suggest that the C4 ORF encodes a polypeptide which is not required by TLCV to replicate or to spread through the host plant, but is involved in symptom development.

Mapping of the polycistronic RNAs of tomato leaf curl geminivirus.

Four major transcripts from infected tomato were identified and mapped onto the monopartite genome of the geminivirus tomato leaf curl virus (TLCV). The C1, C2, and C3 ORFs are spanned by one transcript and a second internal RNA covered C2 and C3. Both these RNAs have their counterparts in the DNA A components of the bipartite subgroup of geminiviruses. The 5' ends of the virion-sense RNAs map either side of the first in-frame AUG of the V1 ORF. The 3' ends of the virion-sense RNAs are coterminal and overlap with the 3' ends of the complementary-sense RNAs. All of the RNAs have transcription regulatory sequences close to their mapped termini and the presence of overlapping transcripts suggests that temporal regulation of their synthesis may occur. The translation of these polycistronic RNAs is discussed in the light of the RNA mapping data.

Nucleotide sequence and genome organization of tomato leaf curl geminivirus.

The genome of tomato leaf curl virus (TLCV) from Australia was cloned and its complete nucleotide sequence determined. It is a single circular ssDNA of 2766 nucleotides containing the consensus nonanucleotide sequence present in all geminiviruses. It has six open reading frames with an organization resembling that of certain other dicotyledonous plant-infecting monopartite geminiviruses, i.e. tomato yellow leaf curl and beet curly top viruses. The regulatory sequences present indicate a bidirectional mode of transcription. A dimeric TLCV DNA clone was constructed in a binary vector and used to agroinoculate three different host species. Typical virus infections were produced, confirming that the single DNA component is sufficient for infectivity.

Common identity of grapevine viroids from USA and Australia revealed by PCR analysis.

Pairs of viroid-specific oligonucleotide primers were selected and used in separate reverse transcription reactions coupled with the polymerase chain reaction to obtain DNA products of predetermined sizes characteristic of each viroid. The reaction conditions allowed efficient incorporation of small amounts of 32P-dATP which enabled rapid detection of the products in polyacrylamide gels. Using this method as well as probe hybridization, the presence of grapevine yellow speckle viroids 1 and 2 (previously known as GV1B) in grapevine samples from California was demonstrated, and it was established that the Australian grapevine viroid occurs in California. These comparisons provide the basis for uniform nomenclature of grapevine viroids found in different geographical regions.

Detection of virus-associated dsRNA from leafroll infected grapevines.

A simple procedure is described for reproducible detection of double stranded (ds) RNAs in leafroll infected grapevines. The procedure involves the extraction of tissues by a medium which preferentially yields dsRNA. The RNA is purified by CF11 cellulose chromatography and gel electrophoresis. The dsRNAs varied in size in different vines. In the cases tested they did not cross hybridize and occurred at higher concentrations in stem cortex tissues than in leaves. They were not detectable in healthy vines, could be passaged with the disease to healthy plants by graft inoculation and removed by virus elimination procedures. These observations indicated that the dsRNAs are of viral origin and that a number of viruses are associated with the grapevine leafroll disease.

Two related viroids cause grapevine yellow speckle disease independently.

We have confirmed that two closely related circular RNA molecules previously named grapevine yellow speckel viroid (GYSV) and grapevine viroid 1B (GV1B) are indeed viroids. Electron microscopy after spreading under non-denaturing conditions revealed that GYSV has a rod-like structure typical of viroids. Purified GYSV and GV1B replicated independently in inoculated grapevine seedlings and some of the infected plants developed yellow speckle symptoms indicating that both viroids can cause grapevine yellow speckle disease. Plus-sense RNA transcripts derived from a dimeric GYSV cDNA clone induced yellow speckle symptoms in a grapevine seedling confirming the role of GYSV in the yellow speckle disease. Two oligonucleotide probes were synthesized for the detection of the two related viroids. The probes which could detect each viroid individually were used to assess correlations between the occurrence of these viroids and the incidence of the disease.

Isolation of three viroids and a circular RNA from grapevines.

Analysis of nucleic acids from grapevine tissues by two-dimensional gel electrophoresis demonstrated the presence of two bands of circular RNA. The smaller RNA contained about 300 nucleotide residues and was identified as hop stunt viroid by nucleotide sequencing. The larger RNA band was a mixture of species and contained similar amounts of two components, referred to as RNA 1a and RNA 1b, and in addition a trace amount of citrus exocortis viroid (CEV) which became detectable only after inoculation of the mixture to tomato. The identity of CEV was determined by probe hybridization and nucleotide sequencing. Both RNAs 1a and 1b are distinct from CEV and have estimated sizes larger than those of CEV and other viroids reported so far. RNA 1a preparations were infectious in cucumber and in tomato and the recovered viroid had unique properties. We have provisionally named this viroid Australian grapevine viroid. Evidence for the autonomous replication of RNA 1b was not obtained.

Nucleic acid extraction and virus detection in grapevine.

Three extraction media for the isolation of nucleic acids from grapevines, a tissue high in polyphenols and other materials that interfere with nucleic acid extraction, were compared. When phenol was present in the initial extraction media only a small yield of soluble RNA and no high molecular weight rRNA was obtained. In the absence of phenol in conventional salt and detergent-based extraction media, rRNAs were extracted, but a major proportion of the RNAs were broken down. Using Na-perchlorate, a chaotropic salt, in a rapid procedure, it was possible to extract both high and low molecular weight RNA efficiently. This procedure enabled the detection of viral RNA, which could not be detected following phenol extraction, at the picogram levels, by dot-blot hybridization.