395
March 2003
Family: Geminiviridae
Genus: Topocuvirus
Species: Tomato pseudo-curly top virus
Acronym: TPCTV


Tomato pseudo-curly top virus

Rob Briddon
Department of Disease and Stress Biology, John Innes Centre, Colney Lane, Norwich, NR4 7UH, UK

Contents

Introduction
Main Diseases
Geographical Distribution
Host Range and Symptomatology
Strains
Transmission by Vectors
Transmission through Seed
Transmission by Grafting
Transmission by Dodder
Serology
Nucleic Acid Hybridization
Relationships
Stability in Sap
Purification
Properties of Particles
Particle Structure
Particle Composition
Properties of Infective Nucleic Acid
Molecular Structure
Genome Properties
Satellites
Relations with Cells and Tissues
Ecology and Control
Notes
References
Acknowledgements
Figures

Introduction

A disease of tomato in Florida with symptoms resembling those of the curtovirus Beet curly top virus (BCTV) was first decribed in 1950 (Stoner & Hogan, 1950) although it was known in the western area of the state as early as 1944 (Giddings et al., 1951). Only upon the identification of the treehopper vector of the disease was the term "pseudo-curly top" used to distinguish this disease from that caused by BCTV.

Brief description

A virus with twinned (geminate) particles approx. 18 x 30nm containing a single species of circular single-stranded DNA. The major hosts are in the Solanaceae and the virus is the only member of the Geminiviridae known to be transmitted by a treehopper.

Main Diseases

The only significant commercial losses due to TPCTV occur in tomatoes. "Pseudo-curly top" disease of tomatoes, caused by TPCTV, is a minor problem as the main tomato growing season in Florida is during cooler months when vector populations are low (Mead, 1986; Simons, 1962).

Geographical Distribution

TPCTV occurs exclusively across southern Florida and appears limited by the geographical extent of its vector, Micrutalis malleifera (Mead, 1986).

Host Range and Symptomatology

TPCTV is reported to be readily graft transmissible from tomato to tomato but not mechanically transmissible (Stoner & Hogan,1950; Giddings et al., 1951).

The host range of TPCTV is restricted to dicotyledonous plant species. The natural host range includes the weeds nightshade (Solanum nigrum L.), ragweed (Ambrosia spp.) Datura stramonium and common chickweed (Stellaria media (L. Cyr.). Affected crop plants include tomato (Lycopersicon esculentum Mill.) and beans (Phaseolus vulgaris L.) Experimental hosts include Nicotiana benthamiana, nightshade, lettuce (Lactuca sativa L.) and eggplant (Solanum melongena L.). Although early investigations reported sugarbeet (Beta vulgaris L.) to be susceptible to TPCTV, this was not confirmed using infectious clones of the virus (Briddon et al., 1996).

The symptoms of TPCTV infections in many hosts resemble those of the curtovirus Beet curly top virus and include initial vein clearing, followed by swelling of the veins, upward or downward rolling of the leaf margins and chlorosis (Fig.1; Fig.2; Fig.3). In the majority of susceptible species, leaves become leathery and brittle and both flowering and fruit set are greatly reduced (Briddon et al., 1996; McDaniel and Tsai, 1990; Simons, 1962).

The most useful propagation and insect transmission assay host is nightshade, as this is a prefered host of the insect vector.

Transmission by Vectors

TPCTV is transmitted exclusively by the the treehopper Micrutalis malleifera Fowler (Homoptera: Membracidae) (Fig.4). The virus has a persistent relationship with this insect and is believed to be circulative/non-propagative (typical of the relationship of all other geminiviruses with their insect vectors) although this has not been shown for TPCTV. The minimum acquisition access period and minimum transmission access period are reported to be approximately 30 minutes. Once they have acquired virus, insects remain virulliferous for life, although virus titre decreases with time when the insect is maintained on healthy plants that are not hosts of the virus (suggestive of a non-propagative interaction). The latent period between acquisition and ability to transmit the virus was estimated to be 24 to 48 hours (Simons, 1962).

Transmission through Seed

Geminiviruses are believed not to be transmitted through seed.

Serology

The production of a polyclonal antiserum against partially purified TPCTV virions has been reported (McDaniel & Tsai, 1990) but this was of only moderate quality, requiring extensive cross-absorption with healthy plant tissue before use in indirect ELISA tests.

Relationships

Serologically, TPCTV was found to be closely related to the leafhopper-transmitted Beet curly top virus (McDaniel &Tsai, 1990), although comparison of the coat protein sequences of the two viruses shows only low levels of sequence similarity (Briddon et al., 1996; Fig 5).

Based upon the nucleotide sequence of its genome, TPCTV appears to be a recombinant virus with the complementary-sense genes having the same arrangement as and showing high levels of sequence similarity to the equivalent genes of the curto- and begomoviruses (which also infect only dicotyledonous plant species). The sequences of the virion-sense genes (the V2 and coat protein genes) of TPCTV are distinct, although some low level sequence similarity is evident between coat protein sequences of geminiviruses in different genera (Fig.5), possibly due to a shared particle structure. The relationships among the coat proteins of geminiviruses reflect the fact that the coat protein determines vector specificity (Briddon et al., 1990) and TPCTV is the only know geminivirus with a treehopper vector.

Purification

A method for the partial purifiation of TPCTV has been described by McDaniel and Tsai (1990). Approx.100g of leaves and stems of TPCTV-infected nightshade were harvested and homogenised in a blender at 4°C with 3 to 4 volumes of extraction buffer (100mM Tris-HCl [pH 7.2], 500mM glucose, 0.05% 2-mercaptoethanol). The homogenate was filtered through four layers of muslin and extracted with 1/10 volume chloroform and centrifuged at 15,000g for 15 minutes. The aqueous phase was filtered through a polyester fiber filter and centrifuged at 100,000g for 3 hours. Pellets were resuspended in 5 to 6 ml of resuspension buffer (100mM Tris-HCl [pH7.2], 0.05% Triton X-100) overnight. The resulting suspension was clarified by centrifugation at 12,000g for 10 minutes and the supernatant layered onto two 10-40% linear sucrose density gradients in 100mM Tris-HCl [pH7.2]. Gradients were centrifuged at 100,000g for 3 hours at 10°C. The single opalescent band was removed with a large-bore needle and syringe. Finally the virus was pelleted, following dilution with 5 volumes of Tris-HCl [pH 7.2], by centrifugation at 100,000g for 3 hours. Pellets were resuspended in a small volume of 100mM Tris-HCl [pH 7.2].

Particle Structure

Virions are geminate (twinned), approximately 30nm by 18nm, consist of two quasi-icosahedra, and have no envelope. Like other geminiviruses, the particle is assumed to consist of 22 capsomeres each containing five units of the coat protein.

Particle Composition

Nucleic acid: Each geminate particle contains a single, covalently-closed, circular ssDNA copy of the 2861 nucleotide viral genome.

Protein: Geminiviruses encode only a single structural protein (the coat protein). The virion comprises 110 copies of this 26.9 kDa protein.

Genome Properties

The complete nucleotide sequence of a single clone of TPCTV DNA has been determined (EMBL acc. no. X84735; Briddon et al., 1996). The encapsidated genome of TPCTV is a single-stranded, circular DNA molecule 2861 nucleotides in length. Typical of all geminiviruses, the genome of TPCTV has within a non-coding region a predicted hairpin structure containing the loop sequence TAATATTAC (the nonanucleotide sequence), which is the origin of virion-strand DNA replication and is nicked by the replication associated protein (Rep) during initiation of replication. The genome encodes two open reading frames (ORFs) in the positive (virion)-sense and four in the negative (complementary)-sense (Fig.6). The functions of the products encoded by ORFs V2, C2, C3 and C4 have not been determined for TPCTV. The predicted ORF C2, C3 and C4 proteins of TPCTV have good sequence similarity to those encoded by the positionally equivalent genes of Beet curly top virus (BCTV) which have been shown to have a function in pathogenicity, in enhancing replication and in initiation of cell division, respectively (Hormuzdi & Bisaro, 1995; Latham et al., 1997). The predicted ORF V2 product has no significant sequence similarity to other geminivirus proteins. The positionally analogous protein of BCTV is involved in virus movement (Hormuzdi & Bisaro, 1993). TPCTV ORF V1 encodes the coat protein (CP), the only structural protein, which is involved in particle formation, cell-to-cell movement and interaction with the insect vector. ORF C1 encodes the replication-associated protein (Rep), the only virus-encoded protein required for viral DNA replication. Rep is a rolling-circle replication initiator protein. Viral DNA replication is achieved using host encoded replication machinery. Although replication of TPCTV has not been studied, it is likely to be very similar to that of other geminiviruses. Initially the viral DNA is used as a template for synthesis of the second (complementary) strand to generate a double-stranded replicative form (RF). The RF then serves as a template for viral strand synthesis which is inititated by Rep-mediated cleavage at the nonanucletide sequence (Heyraud et al., 1993; Laufs et al., 1995; Stanley, 1995). Rep then resolves the concatameric virion-strand DNA into circular monomeric forms by cleavage and ligation.

Ecology and Control

Disease caused by TPCTV is only a minor problem. Removal of alternative weed hosts of the virus around susceptible crops is recommended for control of the disease (Simons, 1959).

Notes

Prior to the identification of the vector,TPCTV was widely confused with the curtovirus Beet curly top virus (BCTV) due to the similarity in symptoms they induce. The diseases caused by both viruses were described as "curly top". Subsequent to the identification of the vector of TPCTV, the associated disease was described as "pseudo-curly top" and the viral agent believed to be responsible was named pseudo-curly top virus.

TPCTV is of little economic significance and occurs in only a small geographic area. The symptoms of this virus, particularly in solanaceous hosts and possibly sugarbeet, are indistinguishable from those induced by BCTV. In cases where identification is necessary this is best achieved using the vector or by sequence analysis. TPCTV is of great interest to those studying the evolution of virus-vector interactions and the inheritance of vector competence during speciation of phytophagous insects (Nault, 1997).

References

  1. Briddon, Bedford, Tsai & Markham, Virology 219: 387, 1996.
  2. Briddon, Pinner, Stanley & Markham,Virology 177: 85, 1990.
  3. Giddings, Bennett & Harrison, Phytopathology 41: 415, 1951.
  4. Heyraud, Matzeit, Schaeffer, Schell & Gronenborn, Biochimie 75: 605, 1993.
  5. Hormuzdi & Bisaro,Virology 193: 900, 1993.
  6. Hormuzdi & Bisaro,Virology 206: 1044, 1995.
  7. Laufs, Traut, Heyraud, Matzeit, Rogers, Schell & Gronenborn, Proceedings of the National Academy of Sciences, USA 92: 3879, 1995.
  8. Latham, Saunders, Pinner & Stanley, Plant Journal 11: 1273, 1997.
  9. Mead, Florida Department of Agriculture and Consumer Services - Division of Plant Industry, Entomology Circular no. 283, 1986.
  10. McDaniel & Tsai, Plant Disease 74: 17, 1990.
  11. Nault, Annals of the Entomological Society of America 90: 521, 1997.
  12. Simons, Proceedings of the Florida State Horticultural Society 71: 31, 1959.
  13. Simons, Journal of Economic Entomology 55: 358, 1962.
  14. Stanley, Virology 206: 707, 1995.
  15. Stoner & Hogan, Florida Agricultural Experimental Station Annual Report: 206, 1950.


Figure 1

Symptoms of a TPCTV infection of Nicotiana benthamiana, showing the typical upward curling of the leaf margins and swelling of the veins on the undersides of the leaves.

Figure 2

Symptoms of a TPCTV infection of tomato, showing the upward curling of the leaf margins.

Figure 3

Symptoms of a TPCTV infection of nightshade, showing the upward curl of the edges of leaves with swelling and distortion of the veins on the undersides of leaves.

Figure 4

Micrutalis malleifera Fowler, the treehopper vector of TPCTV.

Figure 5

Alignment of the predicted amino acid sequence of the coat protein of Tomato pseudo- curly top (TPCTV) with those of Beet curly top virus (BCTV; genus Curtovirus; leafhopper-transmitted), African cassava mosaic virus (ACMV; genus Begomovirus, whitefly-transmitted), Maize streak virus (MSV; genus Mastrevirus; monocot-infecting, leafhopper-transmitted) and Tobacco yellow dwarf virus (TYDV; genus Mastrevirus; dicot-infecting, leafhopper-transmitted). The sequence of the coat protein of TPCTV and amino acids of the coat proteins of the other viruses which show identity with those of TPCTV are shown in red.

Figure 6

Predicted coding regions in TPCTV DNA. All open reading frames start with an ATG triplet. Their position and orientation are indicated by arrows within the circle. Red and blue triangles indicate the position and orientation of potential polyadenylation signals [(A/G)ATAA] and promoter regions [TATA(T/A)AA], respectively. The predicted hairpin structure with the nonanucleotide loop sequence is at position O.