Satellite DNA beta overrides the pathogenicity phenotype of the C4 gene of tomato leaf curl virus but does not compensate for loss of function of the coat protein and V2 genes.

We have investigated the ability of satellite DNA beta to complement mutations in the CP, V2 and C4 genes of the monopartite begomovirus, tomato leaf curl virus, which are potentially involved in movement. A mutation in the coat protein was not complemented by DNA beta. Mutations of the C4 and V2 genes attenuated and abolished symptoms, respectively. In the presence of the C4 mutant, but not the V2 mutant, DNA beta induced typical symptoms, confirming that the satellite encodes a dominant symptom determinant. In contrast to the C4 mutant, DNA beta did not enhance the viral DNA levels of the V2 mutant, suggesting that V2 is required for this phenomenon. The significance of these findings is discussed based on our present understanding of the functions of the viral genes and DNA beta.

Characterization and transient replication of tomato leaf curl virus defective DNAs.

Distinct subgenomic DNA species known as defective (df) DNA molecules were found in plants infected with tomato leaf curl virus (TLCV). Four df DNAs derived from TLCV Type and Darwin 1 strains were found to contain large deletions that disrupt all of the viral genes required for viral replication, encapsidation and spread. However, the viral origin of replication (ori), including the replication-associated protein (Rep) binding domains, was present in all four df DNAs. Co-agroinfection of leaf strips with tandem repeat constructs of the viral and df DNAs resulted in their replication in the presence of the respective TLCV strain. However, the df DNAs failed to move in whole plants when co-inoculated with TLCV. The df DNAs were shown to be associated with TLCV coat protein, which may indicate encapsidation. Mutational analysis showed the minimum sequence requirements for df DNA replication by TLCV to be the intergenic region containing the Rep-binding domains.

Variants of Coconut cadang-cadang viroid isolated from an African oil palm (Elaies guineensis Jacq.) in Malaysia.

Variants of Coconut cadang-cadang viroid have been identified in a plantation oil palm growing in Malaysia. Three size classes are described, comprising 297, 293, and 270 nt. Compared with the 296-nt form of coconut cadang-cadang viroid (CCCVd), all variants substituted C31 --> U in the pathogenicity domain and A175 --> C in the right-hand terminus. Other mutations and deletions accounted for the different sizes. These are the first sequences reported for variants of Coconut cadang-cadang viroid in a species other than coconut palm, and this is the first evidence that variants closely related to CCCVd occur outside the Philippines.

Improved detection of grapevine leafroll-associated virus 1 by magnetic capture hybridisation RT-PCR on a conserved region of viral RNA.

We report the development of a sensitive diagnostic method for the detection of the grapevine leafroll-associated virus 1 (GLRaV-1). We have considered the current shortcoming in detection of GLRaV-1 to be linked to two factors, sequence variation in the viral RNA and low template concentration. Sequence information available allowed the selection of optimal target sequences for detection by RT-PCR, having high copy number and low levels of sequence variation. This was combined with the use of magnetic capture hybridisation to allow the removal of RT-PCR inhibitors and the addition of 100-fold excess template RNA to a single RT-PCR. The reproducibility of the technique was confirmed using field samples.

Molecular Characterization of Tomato and Chili Leaf Curl Begomoviruses from Pakistan.

Leaf curl or yellowing symptoms, typical of those caused by begomovirus infection, are commonly observed in chili (Capsicum annuum) and tomato (Lycopersicon esculentum) plantings in Pakistan. One chili sample with leaf curl symptoms was collected in 1998 in Multan (Punjab Province), and two tomato samples with leaf curl and yellowing symptoms were collected from Islamabad and Dargai (North West Frontier Province) in 2000 and 2001, respectively. Virus DNA was first amplified by polymerase chain reaction using the degenerate primer pair PAL1v1978/PAR1c715 (3). The expected 1.4-kb PCR products were obtained from the three samples. Based on the sequences of the 1.4-kb DNA products, specific primers were designed to complete each of the DNA-A sequences. Two primer pairs, DNABLC1/DNABLV2 and DNABLC2/DNABLV2, were used for the detection of DNA-B (2). The genome of the tomato leaf curl isolate from Islamabad contained a DNA-A of 2,739 nucleotides (GenBank Accession No. AF448059), a DNA-B of 2,728 nucleotides (GenBank Accession No. AY150304), and had 94% nucleotide identity in the common region. The genome of the tomato leaf curl isolate from Dargai contained a DNA-A of 2,740 nucleotides (GenBank Accession No. AF448058), a DNA-B of 2,686 nucleotides (GenBank Accession No. AY150305), and had 96% nucleotide identity in the common region. Each of the tomato isolates contained eight predicted open-reading frames (ORFs) (AV1, AV2, AV3, AC1, AC2, AC3, AC4, and AC5) in the DNA-A and two predicted ORFs (BV1 and BC1) in the DNA-B. The DNA-A nucleotide sequence identity of the Islamabad isolate and Dargai tomato isolate is 96% and that of DNA-B is 88%. Sequence comparisons with begomovirus sequences available in the GenBank sequence database showed that these two tomato virus isolates had the highest sequence identity with Tomato leaf curl New Delhi virus-Severe (GenBank Accession No. U15015) from northern India (more than 95% for DNA-A and less than 90% for DNA-B). The DNA-A of the virus associated with chili leaf curl from Pakistan (GenBank Accession No. AF336806) consists of 2,754 nucleotides, containing six predicted ORFs (AV1, AV2, AC1, AC2, AC3, and AC4). The chili virus was unrelated to the two tomato begomovirus isolates from Pakistan, with which it shares less than 75% nucleotide identity. Sequence comparisons show highest sequence identity (87%) with Tomato leaf curl Bangladesh virus (GenBank Accession No. AF188481). DNA-beta of 1.3 kb was detected in the chili begomovirus isolate using Beta01/Beta02 primers (1). There was no evidence for the presence of a DNA-B in the chili begomovirus isolate when tested by the two DNA-B specific primer pairs. Based on DNA sequence comparisons, the chili leaf curl virus from Pakistan, to our knowledge, constitutes a distinct, new monopartite begomovirus. References: (1) R. W. Briddon et al. Mol. Biotechnol. 20:315, 2002. (2) S. K. Green et al. Plant Dis. 85:1286, 2001. (3) M. R. Rojas et al. Plant Dis. 77:340, 1993.

Molecular Characterization of a New Begomovirus Associated with Tomato Yellow Leaf Curl and Eggplant Yellow Mosaic Diseases in Thailand.

Leaf curl and yellowing symptoms on tomato, and yellow mosaic symptoms on eggplant, are frequently observed in Kanchanaburi Province, Thailand. DNA was extracted from leaves of 13 symptomatic tomato and six symptomatic eggplant samples by the method of Gilbertson et al (1). Viral DNA was amplified by polymerase chain reaction (PCR) using the begomovirus-specific degenerate primer pair PAL1v1978/PAR1c715 (3), which amplified a 1.4-kb fragment of DNA-A. All samples, except one eggplant sample, yielded the expected product. The 1.4-kb PCR products of one tomato and one eggplant sample were cloned and sequenced. Both begomoviruses from tomato and eggplant contained a DNA-B component, amplified using two degenerate primer pairs, DNABLC1/DNABLV2 and DNABLC2/DNABLV2 (2). Based on sequences of the DNA products amplified by the degenerate primer pairs, specific primers were designed to complete the DNA-A and DNA-B sequences. Computer-assisted sequence comparisons were performed with geminivirus sequences available in the laboratory at the Asian Vegetable Research and Development Center, Taiwan and in the GenBank sequence database. Both tomato (GenBank Accession Nos. AF511529 and AF511528) and eggplant (GenBank Accession Nos. AF511530 and AF511527) virus isolates contain the conserved geminivirus sequence-TAATATTAC on the DNA-A and B. Based on the high sequence identities of 99% DNA-A and 98% DNA-B, these two virus isolates are considered to be the same species. Although the genome organization of these two isolates was the same as Tomato yellow leaf curl virus Thailand (TYLCTHV; GenBank Accession Nos. X63015 and X63016), including six open reading frames (ORFs) on the DNA-A (AV1, AV2, AC1, AC2, AC3, and AC4) and two ORFs on the DNA-B (BV1 and BC1), sequence comparisons showed highest DNA-A sequence identity (73%) with Ageratum yellow vein virus from Singapore (GenBank Accession No. X74516) and highest DNA-B identity (77%) with the TYLCTHV (X63016). To our knowledge, these tomato- and eggplant-infecting viruses from Thailand constitute a distinctly novel bipartite Begomovirus species. References: (1) R. L. Gilbertson et al. J. Gen. Virol. 72:2843, 1991. (2) S. K. Green et al. Plant Dis. 85:1286, 2001. (3) M. R. Rojas et al. Plant Dis. 77:340, 1993.

Hypervariable genes in Grapevine leafroll associated virus 1.

Analysis of nucleotide sequences of 10 open reading frames from the Grapevine leafroll associated virus 1 (GLRaV-1), a tentative member of the genus Closterovirus, revealed the presence of an unusually high degree of sequence variation in ORFs 3, 6 and 7 encoding a homologue of heat shock protein 70 and two diverged copies of the coat protein (CPd1 and CPd2), respectively. Overall, 75 clones corresponding to ORFs 3, 6 and 7 were sequenced and 1916 nucleotide changes were recorded relative to the published sequence. Surprisingly, none of the changes resulted in a frame shift or stop codon and there was a trend for the conservation of amino acids or change to amino acids having similar physiochemical properties. The CPd2 gene was particularly variable with a mutation seen in 60% of the nucleotide positions in one or more of the 1.1-kb cDNA clones sequenced. These observations suggest that GLRaV-1 may exist in the form of a heterogeneous population, possibly resulting from the lack of selective pressure and from mixing of virus strains due to viticulture practices of vegetative propagation and grafting over the centuries.

Nucleotide sequence and organization of ten open reading frames in the genome of grapevine leafroll-associated virus 1 and identification of three subgenomic RNAs.

The genome of Grapevine leafroll-associated virus 1 (GLRaV-1) was cloned and the sequence of 12394 nts determined. It contains 10 major open reading frames (ORFs) and a 3'-non-coding region lacking a poly(A) tract. The first ORF (ORF 1a) encodes a putative RNA helicase at the C-terminal portion of an apparently larger protein. The downstream ORF, 1b, overlaps ORF 1a and lacks an initiation codon. This ORF encodes an RNA-dependent RNA polymerase of M(r) 59276. ORF 2 encodes a small hydrophobic protein of M(r) 6736, and ORF 3 encodes a homologue of the HSP70 family of heat shock proteins and has an M(r) of 59500. ORF 4 encodes a protein with an M(r) of 54648 that shows similarity to the corresponding proteins of other closteroviruses. ORF 5 encodes the viral coat protein (CP) with an M(r) of 35416. The identity of this ORF as the CP gene was confirmed by expression in Escherichia coli and testing with the viral antibody. ORFs 6 and 7 code for two CP-related products with M(r) of 55805 and 50164, respectively. ORFs 8 and 9 encode proteins of M(r) 21558 and 23771 with unknown functions. Using DNA probes to different regions of the GLRaV-1 sequence, three major 3'-coterminal subgenomic RNA species were identified and mapped on the GLRaV-1 genome. Phylogenetic analyses of the individual genes of GLRaV-1 demonstrated a closer relationship between GLRaV-1 and GLRaV-3 than with other closteroviruses.

Synthesis of infectious viroids and other circular RNAs.

Viroids are small autonomously replicating RNAs that share structural features with other subviral circular single-stranded RNAs of plants. Viroids and other circular single-stranded RNAs can be synthesised in vitro by a PCR-based procedure using a simple set of reactions. Two end-to-end primers are selected from a desired region of the viroid, one for the synthesis of the first strand cDNA and another for the production of the second strand DNA. The second primer contains an 18 nucleotide T7 promoter at its 5' end, and is selected such that the G nucleotide at the transcription start site represents a G in the viroid. Linked reverse transcription-PCR results in linear double-stranded DNA consisting of the viroid sequence and the T7 promoter. Run-off transcription of the PCR product allows the synthesis of exact-length linear viroid RNA which can be circularised by T4 RNA ligase following an enzymic modification of the 5' triphosphate to a monophosphate. This procedure results in authentic viroid molecules and obviates the need for construction and cloning of DNA in the form of tandem repeats for infectivity tests. It also allows PCR-based manipulation of circular RNAs, thus greatly simplifying structure-function analyses of viroid molecules.

Efficient cloning of cDNA from grapevine leafroll-associated virus 4 and demonstration of probe specificity by the viral antibody.

Using a random-PCR method, a cDNA clone (LR4) was constructed from the replicative form dsRNA of grapevine leafroll-associated virus 4 (GLRaV-4). Northern blot analysis showed hybridization of LR4 to dsRNA in an extract of a Thompson Seedless grapevine clone from which GLRaV-4 was isolated originally by Hu et al. (1990). The cDNA clone was sequenced and shown to be specific to GLRaV-4 by reverse-transcription-PCR using GLRaV-4 particles enriched by the virus antibody coupled to magnetic beads. Reverse-transcription-PCR was used successfully to screen different varieties of grapevines for the virus. Western blot analysis of GLRaV-4 extracts from different varieties of infected grapevines revealed two distinct species of capsid protein with estimated Mr of either 35500 or 38000 depending on the variety used. Both proteins reacted with polyclonal as well as monoclonal antibodies.

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.

The termini of a new citrus viroid contain duplications of the central conserved regions from two viroid groups.

A citrus viroid associated with dwarfing, CVdIIIA, has been sequenced and its 294 nucleotide residues can be arranged to form the typical rod-like secondary structure of other viroids with 71% of nucleotides base-paired. CVdIIIA has greatest sequence similarity with apple scar skin viroid (ASSVd; 69%) and has the central sequence which is conserved in the ASSVd group. CVdIIIA is the smallest member of the ASSVd group but contains the terminal conserved region shared by all viroids over 300 nucleotides. The two ends of CVdIIIA are highly unusual in that each end appears to be derived from the conserved central core region of a different viroid group.

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.

Mutagenesis of the virion-sense open reading frames of tomato leaf curl geminivirus.

A series of frame shift, deletion, and inversion mutants in the virion-sense open reading frames (ORFs) of the monopartite geminivirus tomato leaf curl virus have been constructed and their ability to replicate, produce single-stranded DNA, spread, and cause symptoms in tomato plants has been investigated. Disruptions in the V1 ORF lead to symptomless, systemic infections with a reduced titer of all viral DNA forms while interruptions in the V2 (coat protein) ORF disrupted spread of the virus. Mutagenesis of the virion-sense ORFs did not affect the replication of viral double-stranded DNA, although both V1 and V2 products appear to play a role in the accumulation of viral single-stranded DNA.

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.

Analysis of sequence variation in grapevine yellow speckle viroid 1 reveals two distinct alternative structures for the pathogenic domain.

The nucleotide sequences of 24 full-length cDNA clones prepared from a field isolate of grapevine yellow speckle viroid 1 (GYSV 1) have been determined and compared with that of the previously published GYSV 1 sequence. A large number of sequence variations were observed, the majority of which occurred in the pathogenicity domain (P domain) of the viroid. The GYSV 1 variants could be divided into two types each containing a distinct secondary structure within the P domain. Monomeric exact-length RNA transcripts produced from individual clones belonging to both types were infectious in viroid-free grapevines and produced homogenous populations of progeny viroid in vivo.

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.

In vitro synthesis of an infectious viroid: analysis of the infectivity of monomeric linear CEV.

Infectious monomers of citrus exocortis viroid (CEV) were synthesized in vitro precisely to predetermined sequences in microgram quantities without resorting to cloning procedures. Amplification of CEV double-stranded cDNAs fused with a T7 RNA polymerase promoter was followed by transcription of the DNA resulting in the production of an infectious linear CEV monomer. This is the first demonstration of an infectious unit length viroid synthesized in vitro. Transcripts containing 3'-OH terminal groups were infectious, demonstrating that a 2',3'-cyclic phosphate terminus is not a prerequisite for viroid infectivity as previously suggested. Conversion of the 5'-triphosphate terminus to either 5'-monophosphate or 5'-OH had little effect on infectivity. The linear RNA could be circularized using T4 RNA ligase to produce an authentic CEV molecule. This procedure, which results in the production of biologically active RNA, would allow routine application of oligonucleotide-directed mutagenesis to the study of viroids and other circular RNAs. It would also enable the in vitro synthesis and mutagenesis of infectious viral RNAs containing a 5'-G residue.