Interrelationships between "Candidatus Phytoplasma asteris" and its leafhopper vectors (Homoptera: Cicadellidae).

The titer of chrysanthemum yellows phytoplasma (CYP, "Candidatus Phytoplasma asteris") in the three vector species Euscelis incisus Kirschbaum, Euscelidius variegatus Kirschbaum, and Macrosteles quadripunctulatus Kirschbaum (Homoptera: Cicadellidae) was measured after controlled acquisition from infected Chrysanthemum carinatum (Schousboe) (daisy) plants. Phytoplasma DNA was quantified in relation to insect DNA (genome units [GU] of phytoplasma DNA per ng of insect DNA) by using a quantitative real-time polymerase chain reaction (PCR) procedure. The increase in phytoplasma titer recorded in hoppers after they were transferred to plants that were nonhosts for CYP provides definitive evidence for phytoplasma multiplication in leafhoppers. CYP multiplication over time in M. quadripunctulatus was much faster than in E. incisus and E. variegatus. CYP titer was also highest in M. quadripunctulatus, and this was reflected in the latent period in the insect. The mean latent period of CYP in M. quadripunctulatus was 18 d versus 30 d in E. variegatus. M. quadripunctulatus was the most efficient vector, giving 100% transmission for single insects compared with 75-82% for E. incisus or E. variegatus, respectively. By sequential transmission, we analyzed the time course of transmission: E. variegatus were persistently infective for life or until shortly before death. Occasionally, leafhoppers failed to maintain continuity of infectivity even after completion of the latent period. PCR analysis of transmitter and nontransmitter E. variegatus adults showed that some nontransmitters were CYP positive, whereas others were CYP negative. These findings suggest that both midgut and salivary gland barriers play a role in transmission efficiency.

Aphid transmission of a potyvirus depends on suitability of the helper component and the N terminus of the coat protein.

The present study investigates the specificity of potyviruses for aphid species. Two potyviruses differing in their host range were used: Zucchini yellow mosaic virus (ZYMV) mainly infecting cucurbits and Turnip mosaic virus (TuMV) mainly infecting crucifers. Two sets of aphids species were used as vectors, one polyphagous (Myzus persicae and Aphis gossypii) and the other from crucifers (Brevicoryne brassicae and Lipaphis erysimi). Evidence is provided that the specificity between a vector and a potyvirus depends either on the affinity between the aphid species and the helper component (HC) protein used or on the affinity between the HC and the virions. The difference between the two potyviruses cannot be attributed to the DAG domain which is unaltered in both N termini of the CP. Therefore, a ZYMV full length clone served to exchange a fragment encoding for the N terminus of the ZYMV CP by that of TuMV. This partial exchange in the ZYMV CP, allowed the TuMV HC to transmit the chimeric virus but not the wild type ZYMV. The significance of the N terminus context of the CP in the specificity for the HC is discussed.

Comparison of newly isolated cuticular protein genes from six aphid species.

This paper reports on the first aphids' cuticular proteins. One gene (Mpcp1) was obtained by screening a cDNA library of Myzus persicae with antibodies to a lepidopteran cuticle protein. MpCP1 presents a putative signal peptide, a central extended R&R domain, flanked by N- and C-terminal repeats of alanine, tyrosine and proline. The mRNA of Mpcp1 could be detected in a larval and in adult stages. Primers based on Mpcp1 allowed isolating and comparing cuticle protein genes from five aphid species, but not from whitefly or thrips. Comparison revealed a high degree of similarity. Data from this paper suggest that this cuticle protein family is typical and predominant to aphids. The conformation of these cuticle proteins and the significance on particular properties of aphid cuticle is discussed.

The route of tomato spotted wilt virus inside the thrips body in relation to transmission efficiency.

The route of tomato spotted wilt virus (TSWV) in the body of its vectors, Frankliniella occidentalis and Thrips tabaci (Thysanoptera: Thripidae) was studied during their development. First instar larvae were allowed, immediately upon hatching, to acquire virus from mechanically infected Datura stramonium plants for 24 h. The rate of transmission by adults was determined in inoculation access feeding test on Emilia sonchifolia leaf disks. Thrips tissues were analysed for infection at 24 h intervals after the acquisition-access feeding period, and assayed by the whole-mount immuno-fluorescent staining technique. The virus was initially detected in the proximal midgut region in larvae of both species, and then in the second and third midgut regions, foregut, and salivary glands. Occasionally the first infections of the salivary glands were already detected in one-day-old second stage larvae. The intensity of the infection in the various organs of the thrips of each species was positively related to the transmission efficiency. In both thrips populations good agreement was found between the percentage of second instar larvae and adults with at least one infected salivary gland lobe and the percentage of transmitting adults. These results support the contention that the virus must reach the salivary glands before thrips pupation in order to be transmitted by old second instar larvae and adults.

Distribution and Transmission of Iris yellow spot virus.

Iris yellow spot virus (IYSV), a new tospovirus associated with a disease in onion (Allium cepa) that is known to growers in Israel as "straw bleaching," was identified and further characterized by host range, serology, electron microscopy, and molecular analysis of the nucleocapsid gene. The transmissibility of IYSV by Thrips tabaci and Frankliniella occidentalis was studied. IYSV was efficiently transmitted by T. tabaci from infected to healthy onion seedlings and leaf pieces. Two biotypes of F. occidentalis, collected from two different locations in Israel, failed to transmit the virus. Surveys to relate the incidence of thrips populations to that of IYSV were conducted in onion fields. They revealed that the onion thrips T. tabaci was the predominant thrips species, and that its incidence was strongly related to that of IYSV. Forty-five percent of the thrips population collected from IYSV-infected onion and garlic fields in Israel transmitted the virus. IYSV was not transmitted to onion seedlings from infected mother plants through the seed, and was not located in bulbs of infected plants.

Lisianthus Leaf Necrosis: A New Disease of Lisianthus Caused by Iris yellow spot virus.

Unusual viral symptoms were seen on lisianthus (Eustoma russellianum) grown in the Besor area in Israel. Symptoms included necrotic spots and rings on leaves and systemic necrosis. Preliminary analyses suggested that the disease was caused by a tospovirus. Virus particles typical of a tospovirus were observed with electron microscopy in samples taken only from symptomatic leaves. Double-antibody sandwich enzyme-linked immunosorbent assay tests of leaf sap, extracted from lisianthus and mechanically inoculated indicator plants, gave a strong positive reaction to Iris yellow spot virus (IYSV). Polyclonal antibodies prepared against IYSV enabled specific detection of the virus in crude sap from infected plants. Western blot analysis showed that IYSV was serologically distinct from Tomato spotted wilt virus (TSWV). Primers specific to the nucleocapsid gene of IYSV were used in a reverse transcription-polymerase chain reaction assay (RT-PCR) to verify the presence of IYSV. RT-PCR gave an expected PCR product of approximately 850 bp. The sequence of the cloned nucleocapsid gene confirmed the identity of IYSV, thus confirming IYSV infection of lisianthus. This is the first report of IYSV infection in dicotyledons.

First Report of Impatiens Necrotic Spot Tospovirus (INSV) in Israel.

In January 1999, Anemone coronaria L. imported from Europe and grown in open fields near Jerusalem in Israel developed foliar ringspots and foliar necrosis. Within a few weeks of the first appearance of these symptoms, further anemone plants in the surrounding area were affected and seriously damaged. Impatiens necrotic spot tospovirus (INSV) was detected in affected plants by enzyme-linked immunosorbent assay (ELISA; anti-INSV monoclonal antibodies were provided by H. T. Hsu, USDA, Beltsville, MD, and a polyclonal antibody to INSV was purchased from Loewe, Otterfing, Germany). Crude sap extracted from symptomatic tissue was mechanically transmitted to Emilia spp., Petunia hybrida, Nicotian glutinosa, N. benthamiana, and N. rustica plants that developed symptoms characteristic of INSV infection (1). ELISAs of leaf sap extracted from anemone plants and mechanically inoculated indicator plants gave a strong positive reaction to INSV. Leaf-dip preparations prepared from leaf samples of anemone plants were analyzed by transmission electron microscopy (TEM). Virus particles typical of a tospovirus were observed in samples taken only from symptomatic plants. TEM studies with ultrathin sections of infected anemone and Emilia spp. leaves revealed the presence of tospovirus-like particles. This first report of INSV interception in Israel brings the count of the Tospovirus members in Israel to three, including tomato spotted wilt virus (TSWV), which was found in the past to infect anemone and other ornamental crops, and the Iris yellow spot tospovirus, infecting onion (2). INSV is known to occur in Europe and in the U.S., mostly in flowers grown in greenhouses. The virus is transmitted by the Western flower thrips (WFT; Frankliniella occidentalis Pergande). Although all infected plants were destroyed, precautions to prevent further introduction of the virus must be made. INSV might spread by the WFT, which is abundant in Israel year round, and might also infect other greenhouse or field crops. References: (1) M. Daughtrey et al. Plant Dis. 81:1220, 1997. (2) A. Gera et al. Plant Dis. 82:127, 1998.

Use of modified plum pox virus coat protein genes developed to limit heteroencapsidation-associated risks in transgenic plants.

Aphid transmission of a non-aphid-transmissible strain of zucchini yellow mosaic virus (ZYMV-NAT) occurs in transgenic plants expressing the plum pox potyvirus (PPV) coat protein (CP) gene. Heteroencapsidation has been shown to be responsible for this modification in the epidemiological characteristics of the infecting virus. In order to prevent this biological risk, several modified PPV CP constructs were produced that were designed to interfere with heteroencapsidation itself or to block aphid transmission of heteroencapsidated virions. These constructs were first expressed in Escherichia coli in order to check for the accumulation of pseudo-particles by electron microscopy. Virus-like particles (VLPs) were found with the full-length CP and with a PPV CP lacking the DAG amino acid triplet involved in aphid transmission. However, no VLPs were observed with CP lacking R220, Q221 or D264, amino acids known to be essential for the assembly of other potyvirus CPs. Transgenic Nicotiana benthamiana lines expressing the different PPV CP constructs were infected with ZYMV-NAT. Aphid transmission assays performed with these plants demonstrated that the strategies developed here provide an effective means of minimizing the biological risks associated with heteroencapsidation.

Recombination of engineered defective RNA species produces infective potyvirus in planta.

Recombination occurred between viral genomes when squash plants were cobombarded with mixtures of engineered disabled constructs of a zucchini yellow mosaic potyvirus. Single and double recombinants were detected in the progeny. Genes involved in the recombination process and the mechanisms of recombination were studied in potyviruses for the first time.

Mutations in the HC-Pro gene of zucchini yellow mosaic potyvirus: effects on aphid transmission and binding to purified virions.

Transmission of zucchini yellow mosaic virus (ZYMV) by aphids was examined by introducing mutations within the highly conserved proline-threonine-lysine (PTK) motif of the helper component proteinase (HC-Pro) using a cDNA full-length clone. Replacement of proline by alanine (ATK) in the PTK motif abolished transmission almost completely both from plants and from membranes. Substitution of the basic lysine by glutamic acid (PTE) did not reduce the rate of transmission compared with the wild-type. Replacement of threonine by valine (PVK) or serine (PSK) resulted in a rate of transmission that was lower than that of the wild-type. The rate was lower for PSK than for PVK. Western blot comparison did not permit attribution of HC-Pro functionality in transmission to its level in the host. The HC-Pro of strains that effected transmission (with the wild-type PTK motif, and with the mutated PTE and PVK motifs) could also bind in vitro to virions of ZYMV. HC-Pro with a PSK motif, which was less effective in assisting transmission, could bind only weakly to virions, while HC-Pro of the almost non-transmissible strains (with PAK and ATK motifs) did not bind at all. Interestingly, positive binding was recorded for transmission-defective ZYMV-Ct, which has a PTK motif but has glutamic acid instead of lysine in the lysine-leucine-serine-cysteine (KLSC) motif. These findings support the 'bridge hypothesis', and confirm the binding of the HC-Pro to the virion. The possible role of the PTK and KLSC motifs in binding to the virus and to the mouthparts of the aphid is discussed.

Affinity purification of HC-Pro of potyviruses with Ni2+-NTA resin.

The HC-Pro of zucchini yellow mosiac virus (ZYMV) was found to bind to Ni2+-NTA resin with or without His-tagging. The binding stringency was similar to that observed in proteins with a zinc finger motif like the HC-Pro. Using this characteristic we developed an efficient and rapid method (2-3 h) for purification of the HC-Pro of several potyviruses. A dominant protein of about 150 kDa was extracted and identified as the HC-Pro of ZYMV by means of immunoblotting. About 150 microg of HC-Pro were partially purified from the soluble fraction of 1 g of leaves. High titers of HC-Pro protein were obtained from plants infected with four potyviruses [ZYMV, watermelon mosaic virus II (WMVII), papaya ringspot virus (PRSV) and turnip mosaic virus (TuMV)]. The HC-Pros of potato virus Y (PVY) and tobacco vein mottling virus (TVMV) did not bind to the Ni2+-NTA resin. The ZYMV-HC-Pro purified by the Ni2+-NTA resin could bind in vitro to ZYMV virions blotted onto a membrane. All the HC-Pros which had been successfully purified by the Ni2+-NTA resin were bound in vitro to membrane-blotted ZYMV coat protein. However, only the HC-Pros of ZYMV and WMVII were able to mediate aphid transmission of purified ZYMV virions. The purification procedure described herein is efficient and convenient, and enables HC-Pro for a number of potyviruses to be obtained in larger amounts and at higher purity than possible by means of most existing methods, based on ultracentrifugation.

Iris Yellow Spot Tospovirus Detected in Onion (Allium cepa) in Israel.

During March 1997, 20 to 30% of field-grown onion (Allium cepa), observed in Bet Shean Valley, Israel, had unusual viral symptoms of straw-colored ringspots on leaves and flower stalks. Leaf samples were analyzed by transmission electron microscopy (EM) of leaf dip preparations. Typical tospovirus-like particles were observed only with samples taken from symptomatic plants. Crude sap from symptomatic tissue was mechanically transmitted to Nicotiana benthamiana, Chenopodium quinoa, and Gomphrena globosa. On inoculated plants of N. benthamiana, chlorotic spots developed on inoculated leaves, followed by systemic necrosis, 4 and 7 days post inoculation (DPI), respectively. On inoculated plants of C. quinoa and G. globosa, necrotic local lesions developed by 4 to 5 DPI. EM studies with ultrathin sections of infected onion and N. benthamiana leaves revealed the presence of tospovirus-like particles. Virus was purified from mechanically infected N. benthamiana and identified as Iris yellow spot tospovirus (IYSV) by Western blots (immunoblots) and enzyme-linked immunosorbent assay (ELISA) (anti-IYSV antiserum was provided by D. Peters, Wageningen, the Netherlands). A high incidence of the disease observed in the surrounding fields and in other onion-growing areas in Israel was associated with large populations of the onion thrips (Thrips tabaci). Although characteristic symptoms have been noted on a frequent basis, effects on yield have yet to be determined. IYSV is known to occur in the Netherlands, where it has been occasionally detected in Iris (1) and leek (A. Porrum) (J. Verhoeven, personal communication). The detection of IYSV in Israel and the wide distribution of thrips in the natural vegetation may be an important constraint on onion and other bulb-crop production in Israel. Reference: (1) A. F. L. M. Derks and M. E. C. Lemmers. Acta Hortic. 432:132, 1996.

The first isolated collagen gene of the root-knot nematode Meloidogyne javanica is developmentally regulated.

The nematode's surface comprises a multilayered cuticle, which consists mainly of collagen proteins. We identified, cloned and characterized the first cuticular collagen gene, Mjcol-3, of the plant-parasitic nematode Meloidogyne javanica. The gene putatively encodes a 32.4-kDa collagen protein, including a propeptide which possesses a subtilisin-like protease-cleavage site. Six introns were identified in the gene sequence, with three slightly different acceptor-splicing sites. The basic structure of the predicted MJCOL-3 protein sequence is highly similar to that of the Caenorhabditis elegans DPY-7, with 65.9% identity between the two amino acid sequences. Relative to DPY-7, the putative MJCOL-3 protein has a shorter carboxy-terminus. This non-conserved feature may indicate different contributions of DPY-7 and MJCOL-3 collagens to the structure of the cuticle. Mjcol-3 is developmentally regulated: transcripts were found mainly in preparasitic developing eggs, less in parasitic third- and fourth-stage juveniles and young females shortly after the fourth molt, and much less in females before egg-laying.

Characterization of epitopes on zucchini yellow mosaic potyvirus coat protein permits studies on the interactions between strains.

Monoclonal antibodies (MAbs) raised against the coat protein of zucchini yellow mosaic potyvirus (ZYMV) were characterized by epitope mapping using synthetic oligopeptides. Two mutant viruses with a mutation in the amino acid sequence important for epitope recognition in vitro were obtained by site-directed mutagenesis of a full-length cDNA of ZYMV. Two MAbs, CC11 and DD2, could distinguish specifically between these mutants in mixed infections, or after sequential inoculations of muskmelons. Sequential inoculations of the mutants and analysis with MAbs CC11 and DD2 revealed that cross-protection was established between these quasi-isogenic strains within 48 h.

Particle bombardment drastically increases the infectivity of cloned DNA of zucchini yellow mosaic potyvirus.

An infectious full-length cDNA clone of the RNA genome of the potyvirus zucchini yellow mosaic virus (ZYMV) was constructed under the control of the cauliflower mosaic virus 35S promoter. All squash, cucumber, melon and watermelon plants inoculated with the cloned cDNA of ZYMV by particle bombardment become infected. Bombardment technology is 10(6)-fold more effective than mechanical inoculation. Due to the great increase in efficiency, ineffective constructs now became infective (i.e. cDNA under the control of the 35S promoter without the NOS terminator; with an addition of 127 nucleotides at the 5' end of the viral cDNA; uncapped transcripts), and the infectivity of capped-transcripts was maximized. Inoculation by particle bombardment produced visual symptoms rapidly (3-4 days), allowing the detection of viral coat protein and virions after 2 and 3 days in systemically infected leaves and inoculated cotyledons respectively.

Mutations in the helper component protease gene of zucchini yellow mosaic virus affect its ability to mediate aphid transmissibility.

The nucleotide sequence of the helper component protease (HC-Pro) genes of three zucchini yellow mosaic virus (ZYMV) strains has been compared with that of a helper-deficient strain of ZYMV-HC. The comparisons revealed three unique deduced amino acid differences. Two of these mutations were located in regions which are conserved in other potyviruses. The role of these mutations in aphid transmissibility was examined by exchanging DNA fragments of part of the deficient HC-Pro gene with the respective section within the gene of the infectious full-length clone of the aphid-transmissible ZYMV. The first exchange included two of the three mutations, the first coding for a change from Asp to Gly (in a non-conserved region) and the second coding for a change from Arg to Ile [within the Phe-Arg-Asp-Lys (FRNK) conserved box]. This exchange resulted in a reduced transmission (20.6% for the mutated virus compared with 57.4% in the normal ZYMV when acquired from plants and 37.2% compared with 83.1%, respectively, when acquired from membranes). The second exchange incorporated a single mutation [conferring a change from Thr to Ala within the Pro-Thr-Lys (PTK) conserved box]. This single mutation resulted in almost total loss of HC activity in aphid transmission both from plants and from membranes. The Lys residue in the conserved Lys-Ile-Thr-Cys (KITC) box, which is related to loss of HC activity in potato virus Y, tobacco vein mottling virus and in the Michigan strain of ZYMV, is unchanged in the helper-deficient ZYMV. It is therefore proposed that more than one site in HC-Pro may be functionally related to aphid transmissibility. The possible reasons for the role of these mutations in helper activity in aphid transmission of ZYMV are discussed.

A zucchini yellow mosaic virus coat protein gene mutation restores aphid transmissibility but has no effect on multiplication.

An aphid-transmissible (AT) and two non-aphid-transmissible (NAT) isolates of zucchini yellow mosaic virus (ZYMV) were studied. The predicted amino acid sequences of the coat protein (CP) of the three virus isolates were analysed and compared. The NAT isolates differed from the AT isolate in having a Thr instead of an Ala residue at position 10 in the conserved Asp-Ala-Gly triplet in the N-terminal region of CP. Aphid transmissibility was restored in a progeny virus derived from an infectious clone of the ZYMV-NAT isolate in which Thr was changed back to Ala by site-directed mutagenesis. However this mutation did not have any effect on the multiplication rate in squash, which was significantly higher than that of the AT isolate. The involvement of this mutation in aphid transmission and virus multiplication is discussed.

Infectious in vitro RNA transcripts derived from cloned cDNA of the cucurbit potyvirus, zucchini yellow mosaic virus.

A full-length cDNA clone of the RNA genome of the cucurbit potyvirus zucchini yellow mosaic virus (ZYMV) was constructed downstream from a bacteriophage T7 RNA polymerase promoter. A single extra guanosine residue not present in ZYMV RNA was added to the 5' and 3' ends. Capped (m7GpppG) ZYMV RNA transcripts were infectious in 10 of 91 Cucurbita pepo test plants; uncapped RNA transcripts were not infectious. The appearance of symptoms in plants inoculated with the infectious transcript was delayed for more than a week compared to plants inoculated with native viral RNA. The progeny virions recovered from infected plants had the same biological properties (aphid non-transmissibility and typical symptoms) as the parental virus. The progeny virions also reacted positively with ZYMV antiserum and ZYMV-specific probes by dot blot hybridization. The authenticity of the progeny virus was verified by identifying a specific molecular marker (C substituted for T in the 3' non-coding region) using nucleotide sequence analysis.

A point mutation in the coat protein abolishes aphid transmissibility of a potyvirus.

A nonaphid transmissible (NAT) variant of tobacco vein mottling virus (TVMV) was used to test the hypothesis that the viral coat protein (CP) plays a role in determining aphid transmissibility. Comparison of the nucleotide sequences in the coat protein cistron of an aphid transmissible isolate (TVMV-AT) with that of TVMV-NAT revealed a single nucleotide difference (G----A) at position 8445; this alters a single amino acid residue (G----E) at position 2747. A cDNA fragment representing the CP region of TVMV-NAT was substituted into the CP region of a full-length cDNA clone of TVMV-AT, and transcribed RNA was inoculated to tobacco plants. Aphids were unable to transmit the resultant hybrid virus which had the TVMV-NAT coat protein, although the concentration and infectivity of the hybrid virus in the source plants were similar to those of TVMV-AT. This is the first direct demonstration that a CP mutation affects aphid transmissibility of a potyvirus.