Molecular characterization of the largest mycoviral-like double-stranded RNAs associated with Amasya cherry disease, a disease of presumed fungal aetiology.

The sequence of the four large (L) double-stranded RNAs (dsRNAs) associated with Amasya cherry disease (ACD), which has a presumed fungal aetiology, is reported. ACD L dsRNAs 1 (5121 bp) and 2 (5047 bp) potentially encode proteins of 1628 and 1620 aa, respectively, that are 37% identical and of unknown function. ACD L dsRNAs 3 (4458 bp) and 4 (4303 bp) potentially encode proteins that are 68% identical and contain the eight motifs conserved in RNA-dependent RNA polymerases (RdRp) of dsRNA mycoviruses, having highest similarity with those of members of the family Totiviridae. Both terminal regions share extensive conservation in all four RNAs, suggesting a functional relationship between them. As ACD L dsRNAs 1 and 2 do not encode RdRps, both are probably replicated by those from either ACD L dsRNA 3 or 4. Partial characterization of the equivalent L dsRNAs 3 and 4 associated with cherry chlorotic rusty spot revealed essentially identical sequences.

Synthesis of genomic and subgenomic RNAs by a membrane-bound RNA-dependent RNA polymerase isolated from oat plants infected with cereal yellow dwarf virus.

A membrane-bound RNA-dependent RNA polymerase (RdRp) complex was isolated by differential sedimentation from oat plants infected with cereal yellow dwarf virus (CYDV). When incubated with 32P-labelled UTP, unlabelled ATP, CTP and GTP, and Mg2+ ions, the RdRp preparation catalysed the synthesis of double-stranded (ds) RNAs corresponding in size to the virus genomic RNA (5.7 kbp) and two putative subgenomic RNAs (2.8 and 0.7 kbp). Hybridisation using strand-specific hybridization targets showed that the 5.7-kbp dsRNA was labelled mainly in the plus strand, whereas the 2.8- and 0.7-kbp dsRNAs were labelled only in the minus strand. Genomic-length single-stranded, plus-strand RNA of 5.7 kb and single-stranded, plus-strand subgenomic RNAs of 2.8 and 0.7 kbp were detected in RNA isolated from oat plants infected with CYDV. Mapping experiments were consistent with the genomic and subgenomic RNAs having common 3' ends, but different 5' ends, whether produced in vitro or in vivo. The RdRp-encoding region of the CYDV genome was cloned and expressed in Escherichia coli, and the purified protein was used to raise antibodies in a rabbit. In immunoblots, the antibodies detected a protein of about 68 kDa in RdRp preparations from CYDV-infected oat plants, but not from equivalent preparations from healthy oats. As far as we are aware, this is the first report of an in vitro RNA synthesis system for a phloem-limited virus.

Analysis of the RNA of Potato yellow vein virus: evidence for a tripartite genome and conserved 3'-terminal structures among members of the genus Crinivirus.

Double-stranded RNA preparations produced from potato plants graft-inoculated with a Peruvian isolate of Potato yellow vein virus (PYVV; genus Crinivirus, family Closteroviridae) contain five RNA species denoted RNA 1, RNA 2, RNA 3, x and y of approximately 8, 5.3, 3.8, 2.0 and 1.8 kbp, respectively. The complete nucleotide sequences of PYVV RNAs 1, 2 and 3 and Northern hybridization analysis showed that PYVV RNA 1 contained the replication module and an additional open reading frame (p7), while two distinct species, RNAs 2 and 3, contain the Closteroviridae hallmark gene array. Pairwise comparisons and phylogeny of genome-encoded proteins showed that PYVV shares significant homology with other criniviruses but is most closely related to the Trialeurodes vaporariorum-vectored Cucumber yellows virus. Secondary structure prediction of the 3'-untranslated regions of all three PYVV RNAs revealed four conserved stem-loop structures and a 3'-terminal pseudoknot structure, also predicted for all fully characterized members of the genus Crinivirus and some members of the genera Closterovirus and Ampelovirus.

Molecular variation of Potato yellow vein virus isolates.

To evaluate the variation of Potato yellow vein virus from potato fields, 12 isolates were collected from Colombia and one was collected from Peru. Double-stranded RNA was extracted from the plants and used as a template for RT-PCR amplification of the coat protein ( CP) gene and, in separate reactions the C-terminal region of the heat shock protein 70 homologue ( Hsp70h) gene and the N-terminal region of the p60 open reading frame. The CP amplicons were subjected to single-strand conformation polymorphism (SSCP) analysis and, together with the other amplicon, nucleotide sequence analysis. These analyses suggested that there is low genetic diversity in the PYVV isolates examined and that the Peruvian isolate of PYVV may have originated in Colombia.

Nucleotide sequence and genome organization of Cucurbit yellow stunting disorder virus RNA1.

The complete nucleotide sequence of Cucurbit yellow stunting disorder virus (CYSDV) RNA1, a member of the Crinivirus genus in the Closteroviridae, was determined. CYSDV RNA1 is 9126 nucleotides long and contains two overlapping open reading frames (ORFs) that encode the replication module, consisting of the putative papain-like cysteine proteinase, methyl transferase, helicase, and polymerase domains, a small 5 kDa hydrophobic protein and two further downstream ORFs potentially encoding proteins respectively 25 and 22 kDa in size. The genomic position and homology of the four domains comprising the replication module appear to be similar for all sequenced criniviruses but there is divergence in the downstream ORFs, in terms of number, size, position and sequence homology.

Development of a species-specific recA-based PCR test for Burkholderia fungorum.

The genus Burkholderia comprises over 28 species and species-specific, recA-based polymerase chain reaction (PCR) tests are available for several species, but not for some soil-inhabiting species including B. fungorum. Previous analysis of several novel rhizospheric, environmental isolates belonging to the B. cepacia complex suggested they may be closely related to B. fungorum. To discover any relationship between these isolates and B. fungorum we set out to clone and sequence a portion of the B. fungorum recA gene in order to design species-specific primer pairs for use in a recA-based PCR assay. Using a similar procedure we extended the recA-based PCR assay to identify B. sacchari and B. caledonica, two additional soil-inhabiting Burkholderia spp.

Diversity of Burkholderia isolates from woodland rhizosphere environments.

AIMS: Determination of genetic diversity among UK Burkholderia cepacia isolates from various environmental niches, principally woodland tree rhizospheres and onions. METHODS AND RESULTS: Genus determination was made using polymerase chain reaction (PCR) amplification and fatty acid methyl ester profiling. Genetic diversity was investigated by repetitive sequence genetic PCR fingerprinting. Several onion isolates were similar to clinical isolates but others were diverse. Some environmental isolates were possibly synonymous with B. cepacia and B. gladioli but most from woodland rhizospheres were distinct and clustered together. The 16S rRNA genes of representatives from these clusters were PCR amplified, sequenced and phylogenetically compared with all known Burkholderia and related species. This revealed that the rhizospheric isolates had closest affinity with Burkholderia spp. with known bioremediative and biocontrol capabilities and were unrelated to taxa comprising plant or human pathogenic strains. CONCLUSIONS: All of the analyses investigated revealed that environmental and onion isolates of B. cepacia complex bacteria are genetically diverse but that woodland rhizospheric isolates are related to each other and unrelated to plant or human pathogenic strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Woodland rhizospheric isolates of B. cepacia are potentially good candidates for use in bioremediation and biocontrol, as they appear distinct from plant or human pathogenic strains.

Incidence of the cblA major subunit pilin gene amongst Burkholderia species.

PCR-amplification has been used to screen 75 isolates of the Burkholderia cepacia complex for the cblA pilin gene. PCR-amplified products of the correct size (664 bp) were cloned and sequenced and the sequences compared. Apart from in the control, epidemic cystic fibrosis (CF)-associated B. cepacia lineage we also identified, for the first time, cblA genes in a unique, non-CF clinical isolate from France and a plant (onion) pathogenic isolate from Italy. The sequence of the cblA gene amplified from the clinical isolate was more diverged from the epidemic lineage than that amplified from the onion pathogenic isolate.

Mutation analysis of cis-elements in the 3'- and 5'-untranslated regions of satellite tobacco necrosis virus strain C RNA.

The putative, 3'-terminal stem-loop structure in satellite tobacco necrosis virus strain C (STNV-C) RNA constitutes an essential cis-acting structure for the promotion of negative-strand RNA synthesis and a single-stranded tail is also important. The putative, 5'-terminal stem-loop structure in STNV-C RNA is not essential for productive, plus-strand RNA accumulation but is required for optimal accumulation. Residues 2 and 3 are the minimal cis-acting sequences required for RNA synthesis. The RNA of chimeric mutants, which exchanged 3'- and 5'-untranslated regions between STNV-C and helper tobacco necrosis virus strain D RNAs, accumulated in protoplasts, implying similar replication mechanisms for both RNAs.

Molecular characterization of the cucurbit yellow stunting disorder virus coat protein gene.

ABSTRACT Cucurbit yellow stunting disorder virus (CYSDV) is a partially characterized bipartite closterovirus transmitted by the tobacco whitefly (Bemisia tabaci). CYSDV has emerged as a serious pathogen in southeastern Spain and the Mediterranean Region, causing yellowing disease of cucumber and melon crops. Using a modified reverse-transcription polymerase chain reaction protocol with gel-extracted dsRNA templates, fragments of CYSDV RNA2 were amplified and cloned. Sequence analysis of the cloned fragments revealed open reading frames encoding the heat shock protein 70 homolog, two proteins of unknown function (p58 and p9), and the coat protein (CP) of the virus in a contiguous gene arrangement similar to that of lettuce infectious yellows virus (LIYV) RNA2. The complete CYSDV CP gene is 756 nt long and encodes a protein with a molecular mass of 28.5 kDa. A comparison of the amino acid sequence of the CYSDV CP gene with those of other closteroviruses revealed significant levels of similarity with sweet potato chlorotic stunt virus and LIYV (36 and 27%, respectively), both of which are members of the recently proposed Crinivirus genus of closteroviruses. The complete CYSDV CP gene was cloned into a bacterial expression vector, and the resulting fusion protein was purified and used to produce antiserum. Purified immunoglobulins specifically detected CYSDV in infected plant extracts, both in immunoblot and indirect enzyme-linked immunosorbent assays with a titer exceeding 2,000 times for both assays.

The nucleotide sequence of satellite tobacco necrosis virus strain C and helper-assisted replication of wild-type and mutant clones of the virus.

The complete nucleotide sequence of satellite tobacco necrosis virus strain C (STNV-C) was determined. The genome has a similar overall organization to two STNV isolates studied previously but differs significantly from them in the secondary structure of the translated and untranslated regions (UTRs). STNV-C RNA is naturally uncapped and contains 1221 nt: 101 nt in the 5' UTR, 606 nt in the capsid protein (CP) coding region and 514 nt in the 3' UTR. Using the known sequences of STNV-C and tobacco necrosis virus strain D (TNV-D) RNAs, full-length cDNA clones of both RNAs were constructed. Synthetic transcripts derived from STNV-C cDNA clones only replicated in plants and protoplasts when co-inoculated with TNV-D transcripts. A number of mutant clones in both the 3' and the 5' STNV-C RNA UTRs were constructed which disrupted putative cis-acting elements recognized by helper virus polymerase. Deletion analysis revealed an essential requirement of all 3' and 5' proximal sequences in the STNV-C UTRs for replication. However, an internal region in the 3' UTR could be deleted without loss of infectivity. Likewise, the entire STNV-C CP-encoding region could be deleted and replaced with a marker gene of a similar size without loss of transcript accumulation in plants.

Potato yellow mosaic geminivirus AC2 protein is a sequence non-specific DNA binding protein.

The AC2 protein of potato yellow mosaic geminivirus (PYMV) is by analogy with related geminiviruses thought to be a transcriptional activator protein. We have over-expressed the AC2 open reading frame in E. coli and purified the protein from bacterial extracts to near homogeneity. We have studied the interaction of the AC2 protein with DNA and from gel retardation assays shown that it binds both double-stranded (ds) and single-stranded (ss) DNA non-specifically. The binding to PYMV intergenic region ds DNA appeared to be independent of the presence of zinc ions and did not require the protein to be phosphorylated.

Beet pseudo-yellows virus is an authentic closterovirus.

We have previously used RT-PCR to amplify a 251 base pair fragment diagnostic for beet pseudo-yellows virus (BPYV) infection from several BPYV infected samples from Europe and the USA. This has now been shown by sequencing to represent a fragment of a heat shock protein (HSP)70 gene. Sequence analysis of the fragment revealed that it is closely similar to the C-terminal sequence of the HSP70 homologue gene of lettuce infectious yellows virus, a definitive closterovirus, and also has obvious sequence similarities to the HSP70 genes of the other previously sequenced closteroviruses citrus tristeza virus and beet yellows virus. This observation confirms that BPYV is an authentic closterovirus with a wide geographical distribution.

Pseudorecombination and complementation between potato yellow mosaic geminivirus and tomato golden mosaic geminivirus.

Pseudorecombinants made by exchanging the cloned, infectious genome components (DNAs A and B) of potato yellow mosaic geminivirus (PYMV) and the common strain (cs) of tomato golden mosaic geminivirus (csTGMV) are not infectious in their common host Nicotiana benthamiana. In an N. benthamiana leaf disc assay neither PYMV DNA A nor TGMV DNA A transreplicated each other's DNA B component. The ability of PYMV and TGMV to mediate the systemic movement of each other's DNA A was investigated following coinoculation of N. benthamiana with both genome components of one virus (the helper virus) and DNA A of the other virus (the dependent virus). Movement of the dependent virus DNA A in both cases illustrates interchangeability between the DNA B-encoded movement proteins of New World geminiviruses which infect solanaceous hosts. We have studied this genetic interchangeability further in separate co-agroinoculation experiments with N. benthamiana plants using TGMV DNA A to complement mutations in PYMV open reading frame (ORF) AC2, which encodes a protein that trans-activates the expression of virion sense promoters, and in PYMV ORF AC3, which specifies a protein that enhances viral DNA replication. TGMV DNA A complemented a PYMV AC2 mutant and restored its infectivity and it also complemented a PYMV AC3 mutant and restored the reduced DNA phenotype.

Mutational analysis of potato yellow mosaic geminivirus.

Mutations have been inserted into the virion and complementary sense ORFs encoding proteins with M(r)s in excess of 9 kDa of both DNA A and DNA B of potato yellow mosaic geminivirus (PYMV). Wild-type and mutant monomeric clones were tested for their ability to replicate, produce PYMV-specific DNA, spread and cause symptoms in Nicotiana benthamiana plants following biolistic inoculation. Dimeric clones of the DNA A mutants were also investigated by agroinoculation of leaf discs. In contrast to N. benthamiana plants agroinoculated with PYMV DNA A, in which the wild-type DNA A component was capable of limited independent replication and spread, both excised DNA A and B components were required for DNA replication and symptom development in plants inoculated by the biolistic method. Mixtures of both genomic components were also infectious for potato plants following biolistic inoculation. Mutations in ORFs AL1, AL2, BR1 and BL1 resulted in clones incapable of infecting N. benthamiana plants. However, the AL2 mutation, but not the AL1 mutation, allowed viral DNA replication in leaf discs. Mutations to both the AR1 and AL3 ORFs produced clones which were infectious in plants but showed a considerable delay in the production of attenuated symptoms as compared to wild-type infections. Mutating the AL3 ORF dramatically reduced viral DNA replication in both whole plants and leaf discs. Mutations to the AL4 ORF produced clones which were as infectious for both N. benthamiana and potato plants as the wild-type clones. Our results are compared with those from mutagenesis studies on related bipartite geminiviruses.

The tobacco necrosis virus p7a protein is a nucleic acid-binding protein.

The two centrally located open reading frames (ORFs) of necroviruses may, by analogy with the similarly located and related ORFs of carmoviruses, be expected to have a function in virus movement in plants. In the case of tobacco necrosis virus (TNV) strain D these proteins both have a molecular mass of approximately 7 kDa and are thus known as p7a and p7b. We overexpressed p7a in Escherichia coli, separated it from bacterial proteins and renatured it on gels, and showed that p7a was able to bind single-stranded RNA and single-stranded DNA, but was unable to bind double-stranded DNA. These protein-nucleic acid complexes were stable at moderately high salt concentrations. Protein p7b could not be expressed in a number of bacterial systems. We speculate that in TNV, unlike some other viruses which encode a single movement protein with separate functional domains for RNA binding and plasmodesmatal targeting, p7a and p7b may respectively provide these functions on separate proteins.

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

Head-to-tail dimeric clones of both DNA A and DNA B of potato yellow mosaic geminivirus (PYMV) were constructed. These constructs were infectious when inoculated onto Nicotiana benthamiana plants either as DNA or by agroinoculation and were also infectious for tomato plants by agroinoculation. The dimers were not infectious for potato plants following inoculation by either method. Symptom induction required both DNA A and DNA B but agroinoculation with DNA A alone resulted in virus spread in 30% of the inoculated N. benthamiana plants. Leaf disc explants of N. benthamiana, tomato and potato could all be infected by agroinoculation indicating that the method of delivery of the DNA to intact potato plants was unsuitable for successful inoculation rather than an inherent inability of the virus to replicate/spread in potato per se. Neither whole plants nor leaf discs of sugar beet supported the replication of PYMV DNA.