R. H. A. Coutts

Effects of Mutagenesis in vitro on the Ability of Cloned Tomato Golden Mosaic Virus DNA to Infect Nicotiana benthamiana Plants

Summary Insertion mutations introduced in vitro into cloned DNA of tomato golden mosaic virus that considerably shortened the length of the open reading frames (ORFs) AL1, AL2/AL3, BL1 or BR1, abolished the ability of the DNA to infect Nicotiana benthamiana seedlings. Mutants in which ORF AR1 was similarly shortened by an insertion or a 28 bp deletion were infectious, showing that the formation of coat protein or virions is not required for replication and systemic spread of virus DNA, although the appearance of symptoms was delayed in infections with the deletion mutant. Mutants with larger deletions (178 bp to 603 bp) in ORF AR1 were not infectious. Infections could be initiated with mixtures of AL1 and AL2/AL3 mutants, or BL1 and BR1 mutants, primarily as a result of complementation, although a low proportion of wild-type DNA A molecules regenerated by recombination or reversion was detected in the progeny of infection with the DNA A mutants.

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 others DNA B component. The ability of PYMV and TGMV to mediate the systemic movement of each others 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.

The nucleotide sequence of the infectious cloned DNA components of potato yellow mosaic virus

The complete nucleotide sequence of a Venezuelan isolate of potato yellow mosaic virus (PYMV) has been determined, showing it to be typical of subgroup I geminiviruses in that it is whitefly-transmitted, has a circular, bipartite ssDNA genome and possesses bidirectionally orientated open reading frames (ORFs). The two genomic components have little sequence similarity apart from a common region of 268 nucleotides (nt) which is almost identical. Analysis of ORFs revealed six potential coding regions encoding proteins of Mr greater than 10K, four in PYMV A (2593 nt) and two in PYMV B (2547 nt), which are preceded by regulatory transcription elements and have polyadenylation signals present at the ends. Amino acid sequence alignments of PYMV DNA ORF-encoded proteins with those encoded by other previously sequenced geminivirus ORFs show that PYMV is closely related to those geminiviruses isolated from the New World, especially in the putative coat protein gene regions.

Agroinfection of Nicotiana spp. with cloned DNA of tomato golden mosaic virus

Summary Head-to-tail dimers of cloned DNA A and DNA B of tomato golden mosaic virus (TGMV) were integrated between the T-DNA border sequences of a broad host range binary vector and transferred into cells of Nicotiana benthamiana seedlings using an Agrobacterium tumefaciens-mediated delivery system. Most of the inoculated plants developed golden-yellow mosaic and leaf curling symptoms typical of TGMV infection. Extracts of infected leaves were shown to contain both double-stranded and single-stranded TGMV DNA forms of genome length and the virus capsid polypeptide. Infection was also achieved by inoculating plants with mixtures of Agrobacterium strains containing dimers of DNA A or DNA B, with a strain containing a partial dimer of DNA A and a dimer of DNA B and with a strain containing a dimer of DNA B and a partial dimer of DNA A with a 603 bp deletion in the coat protein gene. In the latter case, mosaic symptoms were mild and leaves did not curl. Transgenic N. tabacum cv. Samsun plants containing head-to-tail dimers of DNA A (A2 plants) or DNA B (B2 plants) were produced by transformation with Ti plasmid vectors. A2 plants and B2 plants were agroinfected with dimeric DNA B and dimeric DNA A, respectively. In both cases, symptoms typical of TGMV infection were induced and viral single-stranded DNA of both components was detected in the systemically infected tissue.

Priming of Complementary DNA Synthesis in Vitro by Small DNA Molecules Tightly Bound to Virion DNA of Wheat Dwarf Virus

Summary DNA isolated from purified preparations of wheat dwarf virus (WDV) has been shown to contain tightly bound small DNA molecules which can act as primers for the synthesis of full-length complementary DNA in vitro. The small DNA molecules are bound in the terminating intergenic region of the WDV genome between the end of an open reading frame encoding a putative protein of M r 17292 and a conserved ‘A-T’ box containing putative transcriptional polyadenylation signals. Evidence that the small DNA molecules contain ribonucleotides at their 5′ termini is presented and their possible role in the priming of virus DNA synthesis in vivo is discussed.

Agroinfection of Triticum aestivum with Cloned DNA of Wheat Dwarf Virus

Summary A head-to-tail dimer of cloned DNA from a Swedish isolate of wheat dwarf virus (WDV) was integrated between the T-DNA border sequences of a broad host range binary vector and transferred into cells of wheat seedlings using an Agrobacterium-mediated delivery system. Two-thirds of the inoculated plants developed a systemic infection. Extracts of infected leaves were shown to contain the virus double-stranded (supercoiled, open circular and linear) and single-stranded DNA forms of unit genome length and the virus capsid polypeptide. The results demonstrate the infectivity of a previously sequenced clone of WDV DNA.

Expression of Potato Virus X Resistance Gene Rx in Potato Leaf Protoplasts

Summary Protoplasts derived from shoot cultures of potato cv. Cara, which carries immunity gene Rx, supported only limited virus multiplication after inoculation with particles or RNA of isolate DX, a group 3 strain of potato virus X, as compared to similarly inoculated protoplasts of cultivars King Edward and Pentland Ivory which lack Rx. The Cara protoplasts were, however, able to support extensive replication of the resistance-breaking strain HB. This strain-specific resistance did not appear to be mediated by a failure or inhibition of the uncoating mechanism or of virus assembly.

Tomato aspermy virus has an evolutionary relationship with other tripartite RNA plant viruses

The entire RNA 3 (2214 nucleotides) of a chrysanthemum isolate of tomato aspermy virus (C-TAV) has been cloned and its sequence determined. C-TAV possesses two open reading frames which encode a 3a protein (277 amino acids) and a coat protein (229 amino acids). Computer-assisted comparisons were made between C-TAV RNA 3 and its predicted protein sequences and those of two other tripartite RNA viruses, cucumber mosaic virus (CMV) and brome mosaic virus (BMV). Results from this study suggest that a close evolutionary relationship exists between C-TAV, Q-CMV and BMV. Divergence of nucleotide and amino acid sequences between these viruses is not reflected at the level of the predicted secondary structure of the encoded proteins, where conservation is strong.

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.

Molecular variation of Potato yellow vein virus isolates

Summary.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.