B. D. Harrison

Revision of taxonomic criteria for species demarcation in the family Geminiviridae, and an updated list of begomovirus species.

Members of the family Geminiviridae characteristically have circular single-stranded DNAgenomes packaged within twinned (so-called geminate) particles. Geminiviruses are currentlydivided into four genera on the basis of their genome organizations and biological properties[2,20].Thosethathaveamonopartitegenomeandaretransmittedbyleafhoppervectors,primarilyto monocotyledonous plants, are included in the genus Mastrevirus, of which Maize streak virus isthe type species. Viruses that have monopartite genomes distinct from those of the mastrevirusesand that are transmitted by leafhopper vectors to dicotyledonous plants are included in thegenus Curtovirus, with Beet curly top virus as the type species. The genus Topocuvirus, recentlyrecognized by the International Committee on Taxonomy of Viruses (ICTV) [18], has only onemember (also the type species), Tomato pseudo-curly top virus, which has a monopartite genomeandistransmittedbyatreehoppervectortodicotyledonousplants.ThegenusBegomoviruscontainsviruses that are transmitted by the whitefly Bemisia tabaci (Gennadius) to dicotyledonous plants,with Bean golden yellow mosaic virus (originally Bean golden mosaic virus – Puerto Rico)asthetype species. Many begomoviruses have bipartite genomes (DNA A and DNA B components),although numerous begomoviruses with a monopartite genome occur in the Old World, and thereare some for which a single component is not infectious yet no DNA B component has been found.Geminiviruses cause significant yield losses to many crop plants throughout the world [5, 7].Because of their economic importance and the relative ease with which their DNA genomescan be cloned, many geminiviruses have been isolated and characterized. Guidelines for naming

Virus variation in relation to resistance-breaking in plants

In plant viruses, genomic variation caused by mutation is enhanced by recombination, pseudo-recombination and acquisition of extra genomic components. Genomic RNA typically has a high mutation rate, and individual virus isolates consist of swarms of mutants, with the consensus sequence changing in response to selection pressure. Recombination is found among field isolates of some RNA viruses, especially tobraviruses. Among DNA viruses, field isolates of geminiviruses show unexpected nucleotide diversity and frequency of recombination, some of it interspecific. Recombination among whitefly-transmitted geminiviruses (begomoviruses) occurring in the same geographical region contributes to their evolutionary divergence, as a group. Several (perhaps potentially all) viral genes can encode an avirulence factor that elicits resistance controlled by a cognate dominant host gene: such factors include viral coat protein, RNA polymerase, movement protein and proteinase. Some resistance-breaking virus variants have merely a single nonsynonymous nucleotide replacement in their avirulence gene but, with more durable resistances, virulence necessitates two or even multiple replacements. The probability of a resistance-breaking variant appearing and spreading also depends on its biological fitness in the absence of the host resistance gene, and on the type of resistance and number of resistance genes to be overcome. Viruses can have particular difficulty in overcoming strong resistance controlled either by multiple recessive genes or by coat-protein transgenes. Resistance acting at the RNA level is exemplified by post-transcriptional gene silencing induced by transgenic viral sequences. This resistance may be broken by variants with >10% nucleotide non-identity distributed along the sequence. Our ability to identify, and potentially to create, durable virus resistance is increasing steadily.

Multiple infection, recombination and genome relationships among begomovirus isolates found in cotton and other plants in Pakistan

Begomoviruses occur in many plant species in Pakistan and are associated with an epidemic of cotton leaf curl disease that has developed since 1985. PCR analysis with primer pairs specific for each of four already sequenced types of DNA-A of cotton leaf curl virus (CLCuV-PK types a, 26, 72b and 804a), or for okra yellow vein mosaic virus (OYVMV), indicated that many individual naturally infected plants of cotton and other malvaceous species contained two or three begomovirus sequences. Similarly, sequence differences among overlapping fragments of begomovirus DNA-A, amplified from individual naturally infected plants, indicated much multiple infection in malvaceous and non-malvaceous species. Some cotton plants contained DNA-A sequences typical of begomoviruses from non-malvaceous species, and some non-malvaceous plants contained sequences typical of CLCuV-PK. Some DNA-A sequences were chimaeric; they each included elements typical of different types of CLCuV-PK, or of different malvaceous and/or non-malvaceous begomoviruses. Often an apparent recombination site occurred at the origin of replication. No complete CLCuV-PK DNA-A sequence was found in malvaceous or non-malvaceous species collected in Pakistan outside the area of the cotton leaf curl epidemic but chimaeric sequences, including a part that was typical of CLCuV-PK DNA-A, did occur there. We suggest that recombination among such pre-existing sequences was crucial for the emergence of CLCuV-PK. Recombination, following multiple infection, could also explain the network of relationships among many of the begomoviruses found in the Indian subcontinent, and their evolutionary divergence, as a group, from begomoviruses causing similar diseases in other geographical regions.

Distribution of Determinants for Symptom Production, Host Range and Nematode Transmissibility between the two RNA Components of Raspberry Ringspot Virus

Summary Raspberry ringspot virus has two RNA species, of mol. wt. about 2.4 × 106 (RNA-1) and 1.4 × 106 (RNA-2). In experiments with four naturally occurring strains, virus hybrids were made by mixing RNA-1 and RNA-2 preparations from different strains. Parent strains were regenerated by crossing appropriate hybrids. In the crosses, both serological specificity and transmissibility by the nematode Longidorus elongatus were determined by RNA-2, suggesting that the protein surface of the virus particles is involved in the transmission process. Ability of isolates to cause systemic yellowing in Petunia hybrida, previously found to be also controlled by RNA-2, was shown to be associated with distinctive ultrastructural changes in the chloroplasts. Severity of systemic symptoms in Chenopodium quinoa and other herbaceous hosts, ability to infect Lloyd George raspberry and ability to invade the non-inoculated leaves of Phaseolus vulgaris, were all determined by RNA-1. Both RNA species played a part in determining lesion type in inoculated leaves of Chenopodium amaranticolor and C. quinoa, and in some crosses the two kinds of hybrid were respectively less virulent and more virulent than either parent. The determinant for systemic symptoms in Petunia hybrida that is carried by RNA-2 was not expressed when in association with RNA-1 from the strain able to infect Lloyd George raspberry. Some genes of raspberry ringspot virus are probably pleiotropic.

Evidence that Potato Leafroll Virus RNA is Positive-stranded, is Linked to a Small Protein and Does Not Contain Polyadenylate

Summary RNA extracted from particles of potato leafroll virus (PLRV) infected tobacco mesophyll protoplasts. Treating the RNA with proteinase K did not abolish its infectivity. In messenger-dependent rabbit reticulocyte lysate, PLRV RNA induced the synthesis of specific polypeptides: a major product of mol. wt. 71000 but no product the size of coat protein. PLRV RNA is therefore positive-stranded. A genome-linked protein (apparent mol. wt. 7000) was detected in preparations of PLRV RNA but no polyadenylate sequence was found. These features may prove to be characteristic of luteoviruses.

Serological Relationships and Genome Homologies among Geminiviruses

Summary In immunosorbent electron microscopy (ISEM) tests, strong relationships were detected between five whitefly-transmitted geminiviruses: African cassava mosaic (ACMV), bean golden mosaic, euphorbia mosaic, squash leaf curl and tomato golden mosaic. Among five leafhopper-transmitted geminiviruses, beet curly top and tobacco yellow dwarf viruses were distantly related but no relationship was detected between either chloris striate mosaic, maize streak or wheat dwarf viruses and any of the other four. No relationship was detected between any whitefly-transmitted and any leafhopper-transmitted virus. A similar pattern of relationships was found by spot hybridization experiments in which extracts from infected leaves were tested with probes for ACMV DNA-1 or DNA-2. Imperfect nucleotide sequence homologies were found between ACMV DNA-1, which contains the particle protein gene, and the DNA of five other whitefly-transmitted viruses: bean golden mosaic, tomato golden mosaic, tobacco leaf curl, tomato leaf curl and tomato yellow leaf curl, the last three of which are not sap-transmissible. Thus, relationships were established between sap-transmissible and sap non-transmissible geminiviruses. No homologies were detected with a full-length probe for ACMV DNA-2. Extracts from plants infected with three leafhopper- transmitted viruses (beet curly top, maize streak and wheat dwarf) did not react with probes for ACMV DNA-1 or DNA-2. Because each of the leafhopper-transmitted geminiviruses has a different vector species whereas the whitefly-transmitted geminiviruses all have the same vector, Bemisia tabaci, the genome homologies and antigenic relationships detected among members of the group could be explained if their coat proteins have a key role in transmission by vectors.

Polyadenylate in the RNA of Five Nepoviruses

Summary RNA extracted from particles of five nepoviruses [raspberry ringspot (RRV), strawberry latent ringspot, tobacco ringspot (TRSV), tomato black ring (TBRV) and tomato ringspot viruses] was bound to oligo(dT)-cellulose in buffers of high ionic strength (HS) whereas RNA from particles of tobacco mosaic virus or tobacco rattle virus was not. This suggests that nepovirus RNA molecules contain polyadenylate [poly(A)]. At least 97% of the infective RNA molecules of TRSV and TBRV were bound in HS buffer and eluted in buffer of low ionic strength. The two species of genome RNA of RRV and TBRV were bound equally to oligo(dT)-cellulose, but the satellite RNA (RNA-3) of TBRV was bound less avidly and to a smaller extent than the genome RNA. When 3H-borohydride-labelled TRSV RNA was digested with ribonucleases A + T1 about 29% of the radioactivity bound to oligo(dT)-cellulose, presumably as polyadenylate. Adenosine trialcohol was the only nucleoside trialcohol detected in alkali digests of borohydride-labelled RNA. Thus polyadenylate is probably located at the 3′-termini of the RNA molecules.

Replication of RNA-1 of tomato black ring virus independently of RNA-2.

Summary In hybridization experiments, using complementary DNA (cDNA) copies of the two genome parts of tomato black ring virus (TBRV RNA-1 and RNA-2), no sequence homology between the two RNA species was detected. When tobacco mesophyll protoplasts were inoculated with purified middle component particles, which contain only RNA-2, no replication of TBRV RNA could be detected. However, when they were inoculated with purified bottom component particles, which contain only RNA-1, extracts made 24 or 48 h later contained RNA that had the same mobility as RNA-1 in polyacrylamide-agarose gels, and that hybridized to cDNA copies of RNA-1. Thus RNA-1 can replicate in protoplasts that do not contain RNA-2. Moreover, this RNA-1 was capable, when mixed with nucleoprotein particles containing RNA-2, of inducing the formation of local lesions in Chenopodium amaranticolor leaves, and therefore was intact and attached to the genome-linked protein. The genome-linked protein of nepoviruses is probably virus-coded, and its production in protoplasts inoculated with bottom component particles therefore suggests that RNA-1 contains the gene for this protein.

Evidence for two functional RNA species and a 'satellite' RNA in tomato black ring virus.

Our recent studies have shown that raspberry ringspot virus (R/1:2.4/43 + 1.4/30 (or 2 × 1.4/46):S/S:S/Ne, nepovirus group) has two functional RNA species, of mol. wt. 2.4 × 106 (RNA-1) and 1.4 × 106 (RNA-2) (Harrison, Murant & Mayo, 1972a, b; Murant et al. 1972). There is also evidence that tomato top necrosis (Bancroft, 1968), tobacco ringspot (Harrison et al. 1972a) and cherry leaf roll (Jones & Mayo, 1972) viruses have divided genomes and we suggested (Harrison et al. 1972a) that this property is characteristic of nepoviruses. In this paper we present results with another nepovirus, tomato black ring virus (TBRV), in which we have found three RNA components. Two of these seem essential for infection and presumably comprise the genome of the virus. The third seems to be a ‘satellite’ RNA, analogous to that found in some cultures of tobacco ringspot virus (Schneider, 1969, 1971).

Tobacco Rattle Virus in Tobacco Mesophyll Protoplasts: Infection and Virus Multiplication

Summary Poly-l-ornithine greatly increased infection of protoplasts by tobacco rattle virus (TRV), and had the largest effect when incubated with virus particles for at least 10 min before inoculation. Using a final concentration of 1 µg/ml TRV particles and 1 to 1.5 µg/ml poly-l-ornithine in 0.025 m-phosphate buffer, pH 6.0, to inoculate mesophyll protoplasts of tobacco cv. Xanthi by the ‘indirect’ method, up to 98% of the intact protoplasts became infected. When the protoplasts were stored overnight at 5 °C before inoculation, 95% became infected. In protoplasts kept at 22 °C after inoculation, about half the yield of infective particles was produced during the first day and almost all the remainder during the second. The final yield was about 2 × 105 long virus particles and 6 × 105 short particles per infected protoplast. Fluorescent antibody staining showed that TRV coat protein antigen accumulated throughout the cytoplasm. In electron micrographs, the long TRV particles were associated with mitochondria whereas the short particles were generally dispersed in the cytoplasm. Infective RNA was produced after inoculation with long particles but TRV coat protein antigen, and long and short TRV particles, were made only in protoplasts inoculated with both kinds of particle; infection was not detected in protoplasts inoculated with short particles alone.