369
October 1999
Family: Closteroviridae
Genus: Crinivirus
Species: Lettuce infectious yellows virus
Acronym: LIYV


Lettuce infectious yellows virus

Bryce W. Falk
Department of Plant Pathology, University of California, 1 Shields Ave., Davis, CA 95616, USA

Tongyan Tian
Department of Plant Pathology, University of California, 1 Shields Ave., Davis, CA 95616, USA

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

First described by Duffus et al. (1986).

LIYV virions are morphologically polar long flexuous filaments, about 12 × 800-900 nm. They are probably of two types, one for each of the two genomic ssRNA species, RNA-1 (8,118 nt) and RNA-2 (7,193 nt). LIYV infections are phloem-limited within the plant host, and the virus is not mechanically transmissible. It is transmitted from plant to plant by the sweet potato whitefly, Bemisia tabaci, in a semi-persistent manner, the insects remaining viruliferous for only 3 days or less. LIYV has a wide host range among dicotyledonous plants and has caused significant economic losses in important food crops, including cucurbits and lettuce. The geographical incidence is limited primarily to southwestern USA, where incidence in recent years has declined dramatically.

Main Diseases

LIYV was originally discovered in 1981 when commercial lettuce plantings in southwestern USA exhibited sudden and severe yellowing and stunting. Coincident with the discovery of LIYV was the appearance of the sweet potato whitefly, Bemisia tabaci, which built up high populations during the summer. Many autumn-planted vegetable crops, including lettuce, sugarbeets, crucifers and cucurbits, were subsequently affected by LIYV. In the early 1980's, 100% of susceptible crop plants were affected and LIYV caused economic agricultural losses of $20 million in a single growing season (Duffus et al., 1986; Wisler et al., 1998). In recent years the incidence of LIYV has declined dramatically.

All commercial cultivars of lettuce, and of various cucurbits, including squash, cantaloupes, watermelons and other melons, are susceptible to LIYV. There is no good genetic resistance developed for these crop hosts.

Geographical Distribution

The known geographical incidence of LIYV is somewhat limited. So far, field infection has been reported only from California and Arizona, apparently limited to locations where the whitefly, B. tabaci, is established. LIYV has also been reported from one location (Pennsylvania) where B. tabaci was present in greenhouse-grown lettuce (Brown & Stanghellini, 1988).

Host Range and Symptomatology

The host ranges of LIYV, and of its principal vector, B. tabaci, are wide and overlap in many plant species. Plants in several families including the Solanaceae, Cucurbitaceae, Chenopodiaceae, Brassicaceae and Compositae are good hosts for both LIYV and B. tabaci (Duffus et al., 1986). The virus is not transmissible by inoculation of sap but has been transmitted by whitefly inoculation to plants in at least 45 species and 15 families (Duffus et al., 1986). Most LIYV-infected plants, including lettuce, cucurbits and Chenopodium spp., show typical interveinal yellowing of older leaves as the primary symptom (Fig.1 & Fig.2). Interveinal areas eventually become completely yellow while the veins remain green (Fig.1). Symptom-bearing leaves remain turgid. Symptoms induced by LIYV infection are similar to those seen on plants infected by beet western yellows virus (BWYV; family Luteoviridae, genus Polerovirus). Symptoms appear as early as 14 days post-inoculation, depending on environmental conditions. Symptoms develop sooner and more intensely on plants grown under long days and high light intensities.

Diagnostic species

Lactuca sativa (lettuce, cv. Summer Bibb) plants are very good for diagnosis. Young plants (2-3 cm) are very susceptible to LIYV and are also favoured by the B. tabaci vector. Symptom development is very consistent: the plants show initial interveinal chlorotic spots and distinct yellowing symptoms on older leaves 14-20 days post-inoculation. By 25-30 days post-inoculation, the interveinal areas of older leaves become completely yellow-white while the veins remain green (Fig.1). These symptoms can sometimes be confused with those caused by nutritional disorders, or by other whitefly-transmitted viruses also within the family Closteroviridae, or by BWYV.

Chenopodium murale. Yellowing of older leaves 20-24 days post-inoculation (Fig.2).

Nicotiana benthamiana. Yellowing on older leaves, but often only 4-6 weeks post-inoculation.

Nicotiana clevelandii. Characteristic and distinct yellowing on older leaves starting about 3 weeks post-inoculation.

Symptoms on the above plants are helpful for diagnosis but are not sufficient on their own; they must be supported by evidence of transmission by B. tabaci, and/or by the results of molecular diagnostic tests.

Propagation species

Propagation of LIYV requires the use of plants that are good hosts for both LIYV and its vector, B. tabaci. Both L. sativa and C. murale are good propagation hosts, although LIYV-infected lettuce plants cannot be easily maintained for longer than about 2.5 months, whereas infected C. murale plants can be kept for 4-5 months. C. murale and N. clevelandii (but not lettuce) are useful for producing quantities of plant material sufficient for virion and/or ds-RNA purification. Additional plants suitable for propagating and maintaining LIYV for longer periods of time include sugarbeets (Beta vulgaris) and Malva parviflora. B. tabaci readily acquires LIYV from these species.

Assay species

Lactuca sativa cv. Summer Bibb. A good host for studies with the whitefly vector.

Strains

All LIYV isolates characterized so far were collected from southern California, and no distinct strains have been reported. A number of whitefly-transmitted viruses that cause similar symptoms on several plant species (i.e. cucurbit yellow stunting disorder virus (CYSDV), beet pseudo-yellows virus (BPYV) and lettuce chlorosis virus (LCV)) are now recognized to be distinct viruses (e.g. Wisler et al., 1998). They can be differentiated from LIYV by vector transmission specificity, plant host range, or by using molecular diagnostic methods such as the reverse transcription polymerase chain reaction (RT-PCR), and RNA hybridization analyses (Tian et al., 1996).

Transmission by Vectors

The principal vector is the sweet potato whitefly, Bemisia tabaci (Fig.3; Duffus et al., 1986). The transmission relationship can be classified as semi-persistent (non-circulative). In tests with adult B. tabaci (Duffus et al., 1986), the minimum reported acquisition access threshold was 10 min, but efficiency of transmission increased with longer acquisition access periods. The minimum inoculation threshold reported for B. tabaci reared on LIYV-infected plants was 1 h, but longer inoculation access periods resulted in increased transmission efficiency. No latent period appears to be required between acquisition and inoculation, and the maximum reported period of retention of LIYV by B. tabaci was 3 days. There is no evidence for passage of LIYV through the moult, or transovarially from viruliferous females to progeny. The silverleaf whitefly, B. argentifolii, also has been shown to transmit LIYV, but it is about 100 times less efficient than B. tabaci and, though widespread, does not appear to be an important natural vector (Cohen et al., 1992).

Transmission through Seed

None reported.

Transmission by Dodder

None reported.

Serology

LIYV virions are moderately immunogenic. Polyclonal antibodies have been produced in rabbits by using purified virions as immunogens (Duffus et al., 1986). These antibodies have proved useful in ELISA for detecting LIYV in several plant species. Antisera have also been produced to the LIYV capsid protein (CP), the diverged copy of the capsid protein (CPm), the HSP70 homologue and the p59 protein, by using bacterially expressed proteins as immunogens. These antisera have proved useful for detecting LIYV antigens in analyses such as immunoblotting and immunogold labeling coupled with transmission electron microscopy (Klaassen et al., 1994; Medina et al., 1998; Tian et al., 1999). No other plant viruses serologically related to LIYV have been identified.

Relationships

LIYV is the type member of the genus Crinivirus in the family Closteroviridae. The only other genus in this family, Closterovirus, contains such viruses as citrus tristeza virus and beet yellows virus. Viruses in the two genera have many molecular and biological properties in common, but those in the genus Closterovirus have monopartite ssRNA genomes whereas those in the genus Crinivirus have bipartite ssRNA genomes.

At the present time the genus Crinivirus includes LIYV and six other definitive members as well as several tentative members (Martelli et al., 2000). All viruses in this genus are whitefly-transmitted. LIYV is the only member for which the complete nucleotide sequences of the genomic RNAs have been determined (Klaassen et al., 1995). However, sequence data obtained so far for other members of this genus suggest that they have genomic organizations similar to that of LIYV.

Stability in Sap

Not reported.

Purification

Virions can be purified from LIYV-infected plants by using procedures that protect the long, filamentous particles, but also facilitate their release from phloem tissues. Two protocols have been reported (Duffus et al., 1986; Klaassen et al., 1994), but the latter is the most useful. Use plants (N. clevelandii, N. benthamiana and/or C. murale) that are about 4 weeks post-inoculation and show good symptoms characteristic of LIYV infection. Grind 25 g fresh or frozen leaf material in liquid nitrogen, and add the resulting powder to 100 ml 0.1 M Tris-HCl, pH 7.4, plus 0.5% sodium sulphite and 0.5% 2-mercaptoethanol. Add Triton X-100 to 2% (v/v) and stir the mixture at moderate to slow speed for 1-2 h at 4 °C. Centrifuge the solution at 10,000 g for 10 min and transfer the supernatant fluid to polycarbonate ultracentrifuge bottles (45 Ti rotor), underlaying the solution in each tube with 6 ml 20% sucrose (prepared in extraction buffer minus the 2- mercaptoethanol). Centrifuge for 1 h at 4 °C and 93,000 g. Resuspend the pellets thoroughly in 0.1 M Tris-HCl, pH 7.4. Add Triton X-100 to 2% (v/v) and stir at 4 °C for several hours. Centrifuge at 10,000 g, retain the supernatant fluid and recover the virions by ultracentrifugation, resuspending the final pellet in 0.01 M Tris plus 0.001 M EDTA, pH 7.4 (TE). This sample should contain concentrated, partially purified LIYV virions. If necessary (i.e. for antiserum production), the virions can be further purified by centrifugation in caesium sulphate/ sucrose step gradients (see Klaassen et al., 1994; Gumpf et al., 1981).

Properties of Particles

LIYV virions are stable in the presence of non-ionic detergents such as Triton X-100, but not in the presence of organic solvents. The virions can be readily disrupted to release genomic RNA by treatment with 1% sodium dodecyl sulphate and phenol. Purified LIYV virions can be stored in TE buffer at 4 °C for up to 4 weeks and remain infectious. The sedimentation coefficient and buoyant density have not been determined.

Particle Structure

LIYV virions are long flexuous filaments, about 12 × 800-900 nm. They are morphologically polar, the minor capsid protein (CPm; see below), which makes up about 10% of the protein in the virion, being localized to only one end (Fig.4). Each virion contains a single molecule of ssRNA, and there are probably two types of virion in which the two genomic RNA species are separately encapsidated. Two virion populations of different lengths have been reported for viruses in the genus Crinivirus (Martelli et al., 2000).

Particle Composition

Nucleic acid: The genome is composed of two species of positive-sense ssRNA: RNA-1 (8,118 nucleotides) and RNA-2 (7,193 nucleotides) (accession numbers U15440 and U15441 respectively). The 5' terminus of each genomic RNA is probably capped but the 3' termini contain no obvious structural components. There are two regions of homology between the two genomic RNAs: (i) the first 5 nucleotides at the 5' terminus of each genomic segment are the same (5'-GGUAA); (ii) each RNA has an identical 23-nucleotide sequence which occurs at about 97 (RNA-1) or 136 (RNA-2) nucleotides from the 5' terminus (Klaassen et al., 1995). The virion RNAs are infective when inoculated to Nicotiana spp. protoplasts (Klaassen et al., 1996).

Protein: The virions are composed of two structural proteins: the major capsid protein (CP; Mr 27,800) and the diverged duplicate capsid protein or minor capsid protein (CPm; Mr 52,300). The CP constitutes approximately 90% of the total virion protein, the remaining 10% consisting of the CPm, which is localized to only one end of the virion (Fig.4). Current evidence suggests that CPm is a whitefly transmission determinant (Tian et al., 1999). Purified virions also have associated with them at least two other LIYV-encoded proteins, the HSP70 homologue and the p59 (Tian et al., 1999). The significance of the association of these proteins with LIYV virions is unknown.

Genome Properties

The LIYV bipartite genome is large for plant viruses, having a total size of 15,311 nucleotides. The complete nucleotide sequences of the two LIYV genomic RNAs have been determined and sequence analyses have allowed identification of ORFs as well as predictions for functions of some of the encoded proteins.

RNA-1 encodes for only three ORFs (Fig.5; Klaassen et al., 1995). The first, ORF 1A, begins at nucleotide 98 and can encode a protein of Mr 217,254. This protein contains a predicted papain-like protease as well as characteristic methyl transferase (MTR) and helicase (HEL) motifs. ORF 1B overlaps ORF 1A, and its encoded protein contains characteristic RNA-dependent RNA polymerase (RdRp) motifs. ORFs 1A and 1B are probably translated together via a +1 ribosomal frameshift, which occurs in the overlapping region. RNA-1 ORF 2 overlaps slightly ORF 1B and encodes a protein of Mr ca. 32,000. Computer sequence comparisons showed no similar proteins in existing databases and the function of this protein is unknown.

RNA-2 contains seven ORFs (Fig.5; Klaassen et al., 1995). After a 326-nucleotide untranslated region, RNA-2 ORF 1 encodes a small, hydrophobic protein (Mr 4,600) of unknown function. RNA-2 ORF 2 encodes for the heat-shock protein 70 homologue (Mr 62,300). ORF 3 encodes for a protein of Mr 59,200 of unknown function. ORF 4 encodes for a small Mr 9,000 protein, and ORF 5 encodes for the Mr 27,800 CP. ORF 6 encodes for the Mr 52,300 CPm and ORF 7 encodes for a Mr 26,000 protein of unknown function. The RNA-2 ORFs 1, 2, 3, 5 and 6 compose the five ORF hallmark gene array which is characteristic of all viruses found in the family Closteroviridae (Dolja et al., 1994; Martelli et al., 2000).

The above characteristics suggest that RNA-1 encodes replication-associated proteins whereas RNA-2 encodes for proteins involved in other functions (i.e. movement in planta, encapsidation, vector transmission, etc.). Indeed, LIYV RNA-1 is competent alone for replication in protoplasts whereas RNA-2 is dependent on co-infection with RNA-1 (Klaassen et al., 1996).

With the exception of RNA-1 ORF 1B (described above), other internal ORFs appear to be expressed during infection via subgenomic RNAs. Double-stranded RNAs also can be readily isolated from LIYV-infected plants, and northern hybridization analysis using specific cloned cDNA probes corresponding to each LIYV ORF has allowed mapping of the ds- and subgenomic RNAs. However, these analyses also showed that LIYV-infected plants contain abundant defective RNAs (D-RNAs). So far D-RNAs have been identified only for LIYV RNA-2.

Relations with Cells and Tissues

LIYV infections are phloem-limited within plant hosts, and phloem cells contain virion aggregates, typical closterovirus vesiculated inclusion bodies and plasmalemma deposits (Hoeffert et al., 1988; Pinto et al., 1988). All these types of inclusion body are formed when mesophyll protoplasts are inoculated with LIYV (Medina et al., 1998). However, when protoplasts are inoculated with LIYV RNA-1 alone, only the characteristic vesiculated inclusion bodies are formed. The virion aggregates and plasmalemma deposits are found only in protoplasts infected by both RNA-1 and RNA-2 (Medina et al., 1998). Plasmalemma deposits are not common for other plant viruses in the family Closteroviridae and their functions in LIYV are unknown. Within plants, plasmalemma deposits are often found associated with pit fields. In plants and protoplasts, plasmalemma deposits are often associated with masses of virions oriented perpendicular to the plasmalemma; this has led to speculation (Pinto et al., 1988) that these structures may be involved in trafficking LIYV virions within plant cells.

Ecology and Control

Although epidemic and of considerable importance in southwestern USA in the early 1980's, LIYV is no longer economically important in the USA. During the early 1980's LIYV was associated with high populations of B. tabaci. B. tabaci populations increased to high densities on summer crops such as cotton (a non-host for LIYV), and on indigenous weeds. As cotton was harvested in late summer, B. tabaci moved from cotton to newly emerging crop hosts (including autumn-planted melons and lettuce) and weed hosts. Whitefly populations continued to increase on melons, which also were susceptible to LIYV. This led to tremendous populations of B. tabaci, and LIYV infections were extremely high in susceptible crops such as lettuce. In theory, control could be achieved by elimination of late summer cotton or possibly autumn melons so as to prevent the large population increases of B. tabaci and subsequent epidemic spread of LIYV. This was not done but in the early 1990's a shift occurred in the whitefly population. B. tabaci was largely displaced by B. argentifolii, the latter being a relatively poor vector of LIYV. LIYV is no longer a significant economic problem in southwestern USA.

Notes

Differentiation of LIYV from other viruses in the genus Crinivirus can be difficult if symptoms on affected plants are the primary diagnostic approach. The best way of identifying LIYV is to use nucleic acid-based diagnostic methods such as RNA hybridization or RT-PCR. Specific probes and nucleotide sequences for designing RT-PCR primers are available for LIYV and many of the viruses in the genus Crinivirus (e.g. Tian et al., 1996). These are making it much easier to work with LIYV and related viruses.

References

  1. Brown & Stanghellini, Plant Disease 72: 453, 1988.
  2. Célix, López-Sesé, Almarza, Gómez-Guillamón & Rodríguez-Cerezo, Phytopathology 86: 1370, 1996.
  3. Cohen, Duffus & Liu, Phytopathology 82: 86, 1992.
  4. Dolja, Karasev & Koonin, Annual Review of Phytopathogy 32: 261, 1994.
  5. Duffus, Larsen & Liu, Phytopathology 76: 97, 1986.
  6. Gumpf, Bar-Joseph & Dodds, Phytopathology 71: 878, 1981.
  7. Hoeffert, Pinto & Fail, Journal of Ultrastructure and Molecular Structure Research 98:243, 1988.
  8. Klaassen, Boeshore, Dolja & Falk, Journal of General Virology 75: 1525, 1994.
  9. Klaassen, Boeshore, Koonin, Tian & Falk, Virology 208: 99, 1995.
  10. Klaassen, Mayhew, Fisher & Falk, Virology 222: 169, 1996.
  11. Martelli et al., Family Closteroviridae, in Virus Taxonomy. Seventh Report of the International Committee on Taxonomy of Viruses, eds M.H.V. van Regenmortel et al., San Diego: Academic Press, 2000, IN PRESS.
  12. Medina, Tian, Wierzchos & Falk, Journal of General Virology 78: 2325, 1998.
  13. Pinto, Hoeffert & Fail, Journal of Ultrastructure and Molecular Structure Research 100: 245, 1988.
  14. Tian, Klaassen, Soong, Wisler, Duffus & Falk, Phytopathology 86: 1167, 1996.
  15. Tian, Rubio, Yeh, Crawford & Falk, Journal of General Virology 80: 1111, 1999.
  16. Wisler, Duffus, Liu & Li, Plant Disease 82: 270, 1998.


Figure 1

Leaves from lettuce (Lactuca sativa) plants showing typical symptoms of LIYV infection. Note interveinal yellowing and green veins.

Figure 2

Leaves from Chenopodium murale plants showing typical symptoms of LIYV infection. Note interveinal yellowing and green veins.

Figure 3

The LIYV whitefly vector, Bemisia tabaci. Note winged adults and nymphs on leaf surface.

Figure 4

Immunogold labelling of LIYV virions, treated with (A), antiserum to the CP; (B) and (C), antiserum to the CPm; (D) pre-immune antiserum. Labelling was done with 10-nm gold particles conjugated with goat anti-rabbit antibodies to (A), CP; (B) and (C), CPm. Arrow in (A) indicates a virion terminal region not coated with CP antiserum. Arrows in (B) and (C) indicate virion termini coated with CPm antiserum. Bars represent 224 nm. Reprinted with the permission of the Society for General Microbiology from Tian et al. (1999).

Figure 5

Schematic representation of the LIYV genome. Rectangles represent ORFs. P-PRO, papain-like protease; MTR, methyltransferase; HEL, RNA helicase; RDRP, RNA-dependent RNA polymerase; HSP70, homologue of HSP70 proteins; CP, capsid protein; and CPm, diverged duplicate of the capsid protein (minor capsid protein). Proteins of unknown function are indicated by their respective molecular weight × 10-3 (i.e. P32 = a protein of unknown function of Mr 32,000).