Crystal structure of the potato leafroll virus coat protein and implications for viral assembly

Luteoviruses, poleroviruses, and enamoviruses are insect-transmitted, agricultural pathogens that infect a wide array of plants, including staple food crops. Previous cryo-electron microscopy studies of virus-like particles show that luteovirid viral capsids are built from a structural coat protein that organizes with T = 3 icosahedral symmetry. Here, we present the crystal structure of a truncated version of the coat protein monomer from potato leafroll virus at 1.80-Å resolution. In the crystal lattice, monomers pack into flat sheets that preserve the two-fold and three-fold axes of icosahedral symmetry and show minimal structural deviations when compared to the full-length subunits of the assembled virus-like particle. These observations have important implications in viral assembly and maturation and suggest that the CP N-terminus and its interactions with RNA play an important role in generating capsid curvature.     

Spread of potato leafroll virus is decreased from plants of potato clones in which virus accumulation is restricted

Tubers of eight potato clones infected with potato leafroll luteovirus (PLRV) were planted as ‘infectors’ in a field crop grown, at Invergowrie, of virus-free potato cv. Maris Piper in 1989. The mean PLRV contents of the infector clones, determined by enzyme-linked immunosorbent assay (ELISA) of leaf tissue, ranged from c. 65 to 2400 ng/g leaf. Myzus persicae colonised the crop shortly after shoot emergence in late May and established large populations on all plants, exceeding 2000/plant by 27 June. Aphid infestations were controlled on 30 June by insecticide sprays. Aphid-borne spread of PLRV from plants of the infector clones was assessed in August by ELISA of foliage samples from the neighbouring Maris Piper ‘receptors’. Up to 89% infection occurred in receptor plots containing infector clones with high concentrations of PLRV. Spread was least (as little as 6%) in plots containing infectors in which PLRV concentrations were low. Primary PLRV infection in guard areas of the crop away from infectors was 4%. Some receptor plants became infected where no leaf contact was established with the infectors, suggesting that some virus spread may have been initiated by aphids walking across the soil.     

The effects of temperature on the availability and acquisition of potato leafroll luteovirus by Myzus persicae

The concentration of potato leafroll luteovirus (PLRV) did not differ in potato plants with secondary infections grown at 15°C or 27°C. Detached leaves of plants grown at 15°C or 27°C were used as sources of PLRV for peach-potato aphids (Myzus persicae Sulz.) both at 15°C and 27°C. At comparable temperature during virus acquisition, aphids which fed on leaves of plants kept previously at 15°C contained more viral antigen detected by ELISA than aphids which fed on leaves of plants grown at 27°C. The aphids which acquired PLRV at 27°C contained evidently more viral antigen than those which acquired PLRV at 15°C. The greatest amount of PLRV was found in the aphids which acquired the virus at 27°C from the leaves of plants kept at 15°C. The ability of M. persicae to transmit PLRV to Physalis ftoridana Rydb. generally decreased with decrease in the amount of PLRV in vectors.     

Expression of the potato leafroll luteovirus coat protein gene in transgenic potato plants inhibits viral infection

Transgenic potato plants, cultivar Désirée, were produced that contained the coat protein gene of potato leafroll luteovirus (PLRV). The transformed potato plants expressed the PLRV coat protein (CP) RNA sequences but accumulation of coat protein in transgenic tissues could not be detected. Upon inoculation with PLRV, the PLRV CP RNA expressing potato plants showed a reduced rate of virus multiplication.     

Expression of Recombinant Potato leafroll virus Structural and Non‐structural Proteins for Antibody Production

Vector pMPM‐A4Ω and vectors pQE‐30 and pET‐45b(+) containing the 6x His‐tag sequence were used for expression of Potato leafroll virus (PLRV) structural and non‐structural proteins in Escherichia coli. Coat protein (CP) and RNA‐dependent RNA polymerase (RdRp)–fragments RdRp_43‐616 and RdRp_304‐537 were chosen for expression. A high level of CP and RdRp_304‐537 was obtained only in an expression system using pET‐45b(+) vector and E. coli Rosetta‐gami 2(DE3) cells. After purification, the His‐tagged PLRV proteins were used for immunization of rabbits.     

Transmission of potato leafroll virus in relation to the honeydew excretion of Myzus persicae

Honeydew excretion of single Myzus persicae nymphs on potato leafroll virus (PLVR)-infected Physalis floridana was studied during the acquisition access period (AAP) in relation to the efficiency of virus transmission.
With increasing length of the AAP, the percentage of nymphs that transmitted the virus increased. These nymphs produced significantly more honeydew droplets during the AAP on PLRV-infected P. floridana plants than nymphs which failed to transmit the virus. However, the number of honeydew droplets excreted during the AAP by transmitting nymphs did not affect the length of the latency period. Nymphs which infected the first test plant after a short latency period produced a similar amount of honeydew during the AAP to those with a longer latency period.
Honeydew excretion recorded on plants of varied age, showed that nymphs feeding on bottom leaves of infected plants produced more honeydew droplets than on comparable leaves of healthy plants. On infected plants, nymphs produced more honeydew droplets on bottom leaves with pronounced symptoms than on top leaves that hardly showed any symptom of PLRV infection.
The concentration of viral antigen measured by ELISA was lower in top leaves than in bottom leaves of infected plants. Nevertheless, nymphs feeding on top leaves transmitted the virus more efficiently than those which used bottom leaves as virus source. When bottom leaves were used as a virus source, the percentage of viruliferous nymphs decreased with plant age. These results indicate that the availability of virus for acquisition by aphids declines with increasing plant age and symptom severity.     

Azadirachta indica metabolites interfere with the host-endosymbiont relationship and inhibit the transmission of potato leafroll virus by Myzus persicae

The effects of neem (Azadirachta indica A. Juss) seed kernel extracts (NSKE) and azadirachtin on the ability of Myzus persicae (Sulz.) to transmit potato leafroll luteovirus (PLRV) was studied. Moreover, it was investigated whether treatments with these compounds would exert an effect on larval growth and mortality, and on the aphid intracellular symbionts. Endosymbiotic bacteria play an essential role in the performance of aphids, and in luteovirus transmission by aphids. NSKE and azadirachtin were offered to one-day-old M. persicae nymphs via a membrane feeding system. The neem metabolites displayed a 100% mortality at doses higher than 2560 ppm. At intermediate doses, ranging between 320 and 2560 ppm, larval growth and mortality were affected in a dose-dependent manner. The transmission of PLRV by M. persicae was inhibited by 55–90%. The endosymbiont population of the aphid was clearly affected by a treatment with neem metabolites as the release of their most abundant protein, Buchnera GroEL, into the haemocoel of the aphid was inhibited. Moreover, morphological aberrations on the bacterial endosymbionts were observed in aphids which fed on 2560 ppm of azadirachtin. At doses lower than 160 ppm of NSKE or azadirachtin, the endosymbiont population of M. persicae, and mortality, growth and feeding behaviour were similar to that of the untreated groups of aphids. However, PLRV transmission was still inhibited by 40–70%. The possible targets of the neem metabolites in the aphid are discussed.     

Effects of sub-lethal imidacloprid levels on potato leafroll virus transmission by Myzus persicae

The effects of sub-lethal imidacloprid concentrations on acquisition and inoculation of potato leafroll virus (PLRV) by Myzus persicae (Sulzer) (Hemiptera: Aphididae) were investigated. In experiments using two aphid clones to acquire PLRV from infected potatoes, virus transmission declined significantly with increasing concentrations of imidacloprid. The same was true in experiments using imidacloprid-treated Physalis floridana Rydb. as acquisition sources. When viruliferous M. persicae were placed on uninfected, imidacloprid-treated P. floridana, there were significant declines in PLRV transmission. Sub-lethal concentrations of imidacloprid clearly inhibited both acquisition and inoculation of PLRV by M. persicae, either through poisoning, temporary intoxication, and/or antifeedant effects.     

Resistance to potato leafroll luteovirus can be greatly improved by combining two independent types of heritable resistance

Twelve potato clones were exposed to infection by aphids with potato leafroll luteovirus (PLRV) in three field trials in order to assess their resistance to infection. Up to 92% of the plants of some clones became infected, although other clones were relatively resistant to infection and one clone remained virus-free in all three trials. The resistance of the same 12 clones to PLRV multiplication was assessed in glasshouse-grown plant: lants were graft-inoculated and their daughter tubers were used to grow plants with secondary infection. High concentrations of PLRV were found in some clones (c. 1700 ng/g leaf) while in others much less virus accumulated (as little as 60 ng/g leaf). However, clones in which little virus accumulated were not necessarily those which were most resistant to infection in the field, and there was no association between the two types of resistance. Nevertheless, both types of resistance were found in some clones. The clone G8107(1), which remained virus-free in all the field exposure trials, was also the most resistant to PLRV multiplication. The combination of these two types of resistance in cultivars should help to eliminate the spread of PLRV in crops.     

In vivo expression of a full-length cDNA copy of potato leafroll virus (PLRV) in protoplasts and transgenic plants

A full-length cDNA copy (PLRV_fl) of potato leafroll virus (PLRV) was constructed and examined in vivo for its biological activities by transient expression experiments with plasmid DNA or in vitro transcribed RNA. In addition, PLRV_fl cDNA was stably introduced into the genome of potato plants by Agrobacterium-mediated leaf disc transformation. Both transient and stable expression of PLRV_fl resulted in the synthesis of genomic and subgenomic PLRV RNAs. Transgenic plants accumulated the 17-kDa movement protein and displayed the typical symptoms of PLRV infection. This is the first example of the constitutive expression of a phloem-limited virus in planta. Received: 20 August 1996 / Accepted: 24 September 1996     

The role of volunteer potatoes in the spread of potato virus YN in ware crops of cv. Record

Surveys were made for the presence of potato virus Y (PVY) in the planted seed and harvested tubers in ware potato crops of cv. Record grown at three sites in England in 1994 (survey 1) and seven sites in 1995 (survey 2). PVY was not found in samples of planted seed, but high levels of infection were found in many, but not all, harvested crops. However, plants of volunteer potatoes (VP) (i.e. plants arising from tubers or true seed derived from previous crops and surviving in the soil) were frequently found to be infected. Infection in tubers harvested from crops in the first survey ranged from 2–52%. In 1995, VP were collected from two of the three English sites where potato crops had been grown the previous season and also from a site in Scotland where PVY infection in an experimental crop of cv. Record had been monitored in 1994. The percentages of infected VP ranged from 2–54%. PVY^N was the predominant strain found in sampled VP, with only two plants (out of 300 infected) containing PVY^O. In the second survey, VP were assessed within the 1995 ware crops and were found at four sites, at which they comprised between 4–8% of emerged potato plants. Between 31–93% of VP were infected. Again, PVY^N was the predominant strain with one plant containing PVY^O and another PVY^C (out of 189 infected). A sample of harvested tubers from each site was also tested for PVY. At those sites which had many infected VP, the harvested crop contained a large percentage of infected tubers, ranging from 60–97%. Two sites which had not previously been used for cropping potatoes had no VP and a very low incidence of PVY infection in the harvested tubers (1% and 2%). However, although no VP were found at one site, 31% of harvested tubers were infected, suggesting that alternative inoculum sources may be important.     

Dynamics of Myzus persicae arrestment by volatiles from Potato leafroll virus‐infected potato plants during disease progression

Green peach aphid, Myzus persicae (Sulzer) (Hemiptera: Aphididae), an important pest of potato (Solanum tuberosum L.) (Solanaceae), preferentially settles on Potato leafroll virus (PLRV)‐infected potato plants as compared with non‐infected ones, primarily in response to volatile organic compounds (VOCs) released by the plants. In this study, we examined the dynamics of these effects, measuring arrestment of apterous M. persicae in response to VOC from upper, middle, and lower leaflets of PLRV‐infected potato plants at the same stage in disease progression (4 weeks after inoculation), but inoculated at 1, 3, or 5 weeks after transplanting (WAT). Sham‐inoculated plants were used as controls and VOC were collected and quantified. Aphid arrestment was greater on PLRV‐infected plants inoculated at 1 and 3 WAT as compared with sham‐inoculated plants, but this preference was reversed in plants inoculated at 5 WAT. Relative arrestment of M. persicae by infected plants and VOC release was greater for lower and middle leaflets than for upper leaflets at 1 and 3 WAT compared to sham‐inoculated plants. The reverse was observed in plants inoculated at 5 WAT. Findings indicate that aphid preference is influenced by VOC release from PLRV‐ or sham‐inoculated potato plants and that VOC emissions and aphid preference depend upon the age at inoculation and leaf position within the potato plants. The implications of these dynamics in vector behavior for spread of PLRV in the field in natural and managed systems are discussed.     

The symptomless leaf infection with grapevine leafroll associated virus 3 in grown in vitro plants as a simple model system for investigation of viral effects on photosynthesis

The photosynthetic changes evaluated by oxygen evolution, chlorophyll fluorescence, photoacoustics, and delayed fluorescence (DF) were studied in leaves of grown in vitro for 8 weeks grapevine plants (Vitis vinifera) infected by grapevine leafroll-associated virus 3 (GLRaV-3). The infected leaves were characterized during the viral infection without visible disease symptoms. The symptomless infection led to a decrease in plant biomass. The non-photochemical fluorescence quenching, qN, declined, whereas the photochemical quenching, qP, and the Chl a/b ratio were not significantly affected. Photoacoustic and oxygen evolution measurements showed that the energy storage and oxygen evolution rate decreased in the infected leaves. Enhanced alternative electron sinks during the symptomless viral infection were also estimated. The changes in fluorescence and DF temperature curves demonstrated an enhanced stability of the thylakoid membranes in the infected leaves. This effect was clearly expressed at high actinic light intensities. The viral infected in vitro grown grapevine plants were used in the present study as a simplified model system that allow to avoid the involvement of different environmental factors that could interfere with the GLRaV infection and the virus-grapevine interactions. Thus, the 'pure' impact of the viral infection on photosynthesis could be investigated.