Pelargonium zonate spot virus is transmitted vertically via seed and pollen in tomato.

In autumn 2007, a new disease with unknown etiology was observed in open-field tomato (Solanum lycopersicum) in the Lachish region of Israel. The symptoms included mild mosaic, leaf malformation, and severe stunting of the plants. The causal agent was readily transmitted mechanically from the sap of infected plants to indicator plants. Viral particles were purified from infected plants and cDNA was synthesized from RNA isolated from the particles. Cloning and sequencing of the cDNA showed 95% identity to RNA 3 of Pelargonium zonate spot virus (PZSV). Using reverse-transcription polymerase chain reaction, PZSV was detected in both seed and pollen grains of infected tomato plants. Attempts to disinfect seed by using hydrochloric acid and trisodium phosphate failed to eliminate this PZSV detection. Seed from infected tomato plants gave rise to infected seedlings with a seed-transmission rate of PZSV of 11 to 29%. Pollen grains collected from flowers of infected plants were used to hand pollinate healthy mother tomato plants. Although none of the pollinated mother plants became infected with PZSV, 29% of the seedlings produced from seed harvested from these plants were found to be infected. This is the first demonstration that PZSV is transmitted vertically via both pollen and seed in tomato plants.

Breakage of resistance to Cucumber mosaic virus by co-infection with Zucchini yellow mosaic virus: enhancement of CMV accumulation independent of symptom expression.

Resistance to the cucumovirus Cucumber mosaic virus (CMV) in cucumber cv. Delila was manifested as a very low level of accumulation of viral RNA and capsid protein, and an absence of CMV-induced symptoms. In addition, resistance was observed at the single cell level, with a reduction in accumulation of CMV RNAs, compared to accumulation in cells of the susceptible cucumber cv. Bet Alpha. Resistance to CMV in cv. Delila was broken by co-infection with the potyvirus Zucchini yellow mosaic virus (ZYMV). Resistance breakage in cv. Delila plants was manifested by an increase in the accumulation of (+) and (-) CMV RNA as well as CMV capsid protein, with no increase in the level of accumulation of ZYMV. Resistance breakage in the resistant cultivar by ZYMV also occurred at the single cell level. Thus, synergistic interactions known to occur between a potyvirus and a cucumovirus led to resistance breakage during a double infection. However, resistance breakage was not accompanied by an increase in disease symptoms beyond those induced by ZYMV itself. On co-inoculation with an asymptomatic variant of ZYMV-AG an enhancement of CMV infection occurred without disease manifestation. Consequently, intensification of viral RNA and capsid protein accumulation can occur without a corresponding increase in disease development, suggesting that different host genes regulate viral accumulation and disease development in the CMV-resistant cucumber plants.

Shoot production in squash (Cucurbita pepo) by in vitro organogenesis.

Seedling-derived cotyledon explants of squash ( Cucurbita pepo L.) of commercial cultivars True French, Ma'yan and Goldy were regenerated in vitro on Murashige and Skoog medium augmented with 1 mg/l benzyladenine. After 4 weeks in culture small shoots and buds regenerated only on the most proximal cotyledon edge. Culture on an elongation medium with a reduced cytokinin concentration (0.1 mg/l) with or without 1 mg/l gibberellic acid (GA(3)) facilitated the recovery of shoots. Fresh shoots could be recovered at each subculture of the regenerating mass. Peak productivity was during the third cycle of subculture, and shoot production ceased after the fifth subculture. Culture on elongation medium supplemented with GA(3) was 55% more effective with respect to overall shoot production than that on medium without GA(3), with 22 shoots recovered in total per explant from the former. Regeneration occurred under both light and dark conditions. All of the shoots tested were diploid. The shoots were rooted and transferred to the greenhouse where they grew and flowered normally.

Increase in Zucchini yellow mosaic virus Symptom Severity in Tolerant Zucchini Cultivars Is Related to a Point Mutation in P3 Protein and Is Associated with a Loss of Relative Fitness on Susceptible Plants.

ABSTRACT Zucchini yellow mosaic virus (ZYMV, Potyvirus) is a very damaging cucurbit virus worldwide. Interspecific crosses with resistant Cucurbita moschata have led to the release of "resistant" zucchini squash (C. pepo) F(1) hybrids. However, although the resistance is almost complete in C. moschata, the commercial C. pepo hybrids are only tolerant. ZYMV evolution toward increased aggressiveness on tolerant hybrids was observed in the field and was obtained experimentally. Sequence comparisons and recombination experiments revealed that a point mutation in the P3 protein of ZYMV was enough to induce tolerance breaking. Competition experiments were performed between quasi-isogenic wild-type, and aggressive variants of ZYMV distinguished by monoclonal antibodies. The aggressive mutants were more fit than wild-type strains in mixed infections of tolerant zucchini, but they presented a drastic fitness loss in mixed infections of susceptible zucchini or melon. Thus, the ability to induce severe symptoms in tolerant zucchini is related to a genetic load in susceptible zucchini, but also on other susceptible hosts. This represents the first quantitative study of the fitness cost associated with tolerance breaking for a plant virus. Thus, although easily broken, the tolerance might prove durable in some conditions if the aggressive variants are counterselected in susceptible crops.

A new approach for weed control in a cucurbit field employing an attenuated potyvirus-vector for herbicide resistance.

Expression of bar, a phosphinothricin acetyltransferase, in plant tissues, leads to resistance of these plants to glufosinate ammonium based herbicides. We have created a bar expressing, attenuated zucchini yellow mosaic potyvirus-vector, AGII-Bar, to enable herbicide use in cucurbit fields. The parental vector, ZYMV-AGII, has been rendered environmentally safe by both disease-symptom attenuation and aphid-assisted virus transmission abolishment. The recombinant AGII-Bar virus-encoding cDNA, when inoculated on diverse cucurbits was highly infectious, accumulated to similar levels as AGII, and elicited attenuated AGII-like symptoms. Potted cucurbits inoculated with AGII-Bar became herbicide resistant about a week post-inoculation. Herbicide resistance was sustained in squash over a period of at least 26 days and for at least 60 days in cucumber grown in a net-house under commercial conditions. To test the applicability of AGII-Bar use in a weed-infested field, a controlled experiment including more than 450 plants inoculated with this construct, was performed. Different dosages of glufosinate ammonium were sprayed, 2 weeks after planting, on the foliage of melons, cucumbers, squash, and watermelons. AGII-Bar provided protection to all inoculated plants, of every variety tested, at each dosage applied, including the highest doses that totally eradicated weeds. This study demonstrates that AGII-Bar can be utilized to facilitate weed control in cucurbits and exemplifies the practical potential of attenuated virus-vector use in agriculture.

A nonviral peptide can replace the entire N terminus of zucchini yellow mosaic potyvirus coat protein and permits viral systemic infection.

Systematic deletion and peptide tagging of the amino-terminal domain (NT, ~43 amino acids) of an attenuated zucchini yellow mosaic potyvirus (ZYMV-AGII) coat protein (CP) were used to elucidate its role in viral systemic infection. Deletion mutants truncated by 8, 13, and 33 amino acid residues from the CP-NT 5' end were systemically infectious and produced symptoms similar to those of the AGII virus. Tagging these deletion mutants with either human c-Myc (Myc) or hexahistidine peptides maintained viral infectivity. Similarly, addition of these peptides to the intact AGII CP-NT did not affect viral life cycle. To determine which parts, if any, of the CP-NT are essential for viral systemic infection, a series of Myc-tagged mutants with 8 to 43 amino acids removed from the CP-NT were constructed. All Myc-tagged CP-NT deletion mutants, including those from which virtually all the viral CP-NT had been eliminated, were able to encapsidate and cause systemic infection. Furthermore, chimeric viruses with deletions of up to 33 amino acids from CP-NT produced symptoms indistinguishable from those caused by the parental AGII virus. In contrast to CP-NT Myc fusion, addition of the foot-and-mouth disease virus (FMDV) immunogenic epitope to AGII CP-NT did not permit systemic infection. However, fusion of the Myc peptide to the N terminus of the FMDV peptide restored the capability of the virus to spread systemically. We have demonstrated that all CP-NT fused peptides were exposed on the virion surface, masking natural CP immunogenic determinants. Our findings demonstrate that CP-NT is not essential for ZYMV spread and that it can be replaced by an appropriate foreign peptide while maintaining systemic infectivity.

Engineering zucchini yellow mosaic potyvirus as a non-pathogenic vector for expression of heterologous proteins in cucurbits.

Plant virus vectors provide an attractive biotechnological tool for the transient expression of foreign genes in whole plants. As yet there has been no use of recombinant viruses for the improvement of commercial crops. This is mainly because the viruses used to create vectors usually cause significant yield loss and can be transmitted in the field. A novel attenuated zucchini yellow mosaic potyvirus (AG) was used for the development of an environmentally safe non-pathogenic virus vector. The suitability of AG as an expression vector in plants was tested by analysis of two infectious viral constructs, each containing a distinct gene insertion site. Introduction of a foreign viral coat protein gene into AG genome between the P1 and HC-Pro genes, resulted in no expression in planta. In contrast, the same gene was stably expressed when inserted between NIb and CP genes, suggesting that this site is more suitable for a gene vector. Virus-mediated expression of reporter genes was observed in squash and cucumber leaves, stems, roots and edible fruit. Furthermore, AG stably expressed human interferon-alpha 2, an important human anti-viral drug, without affecting plant development and yield. Interferon biological activity was measured in cucumber and squash fruit. Together, these data corroborate a biotechnological utility of AG as a non-pathogenic vector for the expression of a foreign gene, as a benefit trait, in cucurbits and their edible fruit.

Biological and molecular characterization of a new cucurbit-infecting tobamovirus.

ABSTRACT An uncharacterized virus was isolated from greenhouse-grown cucumber plants. Biological and serological data described in the present study indicated that the virus belonged in the genus Tobamovirus. The host range of the virus included several plant species within the family Cucurbitaceae. The virus designated Cucumber fruit mottle mosaic virus (CFMMV) causes severe mottling or mosaic on cucumber fruits, and its fast spread within greenhouses could lead to significant economic losses in cucumber crops. The genome of CFMMV has been completely sequenced and its genome organization was typical of a Tobamovirus. However, its sequence was distinct from other described viruses within the group of cucurbit-infecting Tobamoviruses. Comparisons of sequences and phylogenetic analysis suggested that the cucurbit-infecting Tobamoviruses be separated into two subgroups: subgroup I comprising the strains and isolates referred to in the literature as Cucumber green mottle mosaic virus (CGMMV) (CV3, CV4, CGMMV-W, CGMMV-SH, and CGMMV-Is) and subgroup II comprising CFMMV, Kyuri green mottle mosaic virus (KGMMV), and the Yodo strain of CGMMV, which is closely related to KGMMV and may be considered a strain of it.

Identification of a novel plant virus promoter using a potyvirus infectious clone.

A putative promoter from the strawberry vein banding caulimovirus (SVBV) genome was identified by its ability to drive infection with full-length cDNA of the zucchini yellow mosaic RNA potyvirus (ZYMV). A high rate of infection was obtained with the cDNA under control of the SVBV promoter using particle bombardment technology. The SVBV promoter shows 60% homology to the cauliflower mosaic virus 35S promoter in the domain spanning the conserved motifs of CCACT (at -83) and the TATA box (at -31), to the transcription start. The 3'-end one-third of the putative promoter (328 bp) was sufficient to invoke full infectivity with the ZYMV clone, and drove transient reporter gene expression in Solanaceae and Cucurbitaceae transformed with a binary plant transformation vector. Stable expression of a reporter gene (GUS) under control of the truncated SVBV promoter was shown in transformed tobacco shoots in roots, leaves and stems.

Characterisation of genetically modified cucumber mosaic virus expressing histidine-tagged 1a and 2a proteins.

Biological active cDNA clones of cucumber mosaic virus (CMV) RNAs 1 and 2 were modified by addition of sequences that encode hexahistidine (His-tag) at the amino- (N-) or carboxy- (C-) terminus of the 1a and 2a proteins. These proteins are essential components of the viral RNA-dependent RNA polymerase (RdRp). In all but one case, addition of the His-tag did not significantly affect the yields of the corresponding viruses and the His-tag-encoding sequences were maintained after mechanical passages. No differences were observed among the in vitro activities of the modified vs. wild-type viral RdRps. Subcellular fractionation showed that 2a protein was found both membrane-associated and in the 30,000 x g soluble fraction. Both termini of the native His-tag 2a protein could bind to a resin containing nickel-nitrilotriacetic acid (Ni(2+)-NTA). Detergent-treated RdRp containing C-terminal His-tagged 1a and 2a proteins was chromatographed on Ni(2+)-NTA resin. The activity of the eluted RdRp was template- dependent, in contrast to pre-chromatography fractions. However, only a small proportion of the viral RdRp as well as numerous host proteins bound to and eluted from the resin under non-denaturing conditions.

A Point Mutation in the FRNK Motif of the Potyvirus Helper Component-Protease Gene Alters Symptom Expression in Cucurbits and Elicits Protection Against the Severe Homologous Virus.

Sequence comparison had previously shown three amino acid changes in conserved motifs in the 455-amino acid sequence of the helper component-protease (HC-Pro) between a severe field strain of Zucchini yellow mosaic virus (ZYMV-NAT) and a mild field strain of ZYMV (ZYMV-WK). In this study, exchange of fragments and site-directed mutagenesis within the HC-Pro gene in an infectious clone of ZYMV enabled the effects of the mutations on symptom expression to be mapped. The substitution of Ile for Arg at position 180 in the conserved motif Phe-Arg-Asn-Lys (FRNK) of potyviruses was found to affect symptom expression. Infection of cucurbits with the engineered ZYMV (ZYMV-AG) that contained this mutation caused a dramatic symptom change from severe to mild in squash and to a symptom-free appearance in cucumber, melon, and watermelon. The Ile to Arg mutation was found to be stable, and no revertant virus was found after several passages through plants after long incubation periods. The AG strain was detected 4 days postinoculation and accumulated in cucurbits to a level and with kinetics similar to that of the wild-type ZYMV-AT strain. Cucurbit plants infected with the AG strain were protected against infection by the severe strain.

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.

Transgenic resistance to cucumber mosaic virus in tomato: blocking of long-distance movement of the virus in lines harboring a defective viral replicase gene.

ABSTRACT Tomato breeding lines were transformed with a defective replicase gene from RNA 2 of cucumber mosaic virus (CMV). A total of 63 transformants from five tomato genotypes were evaluated for resistance to CMV strains. The responses of R1 transgenic offspring fit into three categories: fully susceptible lines (44%), fully resistant lines (8%), and an intermediate-type mixture of susceptible and resistant seedlings in variable proportions (48%). Further characterization of the response of two highly resistant lines was performed by mechanical inoculation, aphid transmission, or grafting experiments. No virus was detected in noninoculated leaves from these lines, although a low level of virus accumulated initially in the inoculated leaf. The homozygous R2 plants and further generations that were evaluated (up to R5) showed resistance to the Fny-CMV strain, two Israeli isolates tentatively classified as subgroup IA, and K-CMV (a representative of subgroup IB). These lines were partially resistant to LS-CMV (a representative of subgroup II) when a high-virus-titer inoculum was used. Expression of the viral transgene was verified in these lines; however, the expected translation product was not detectable. In grafting experiments, we demonstrated that CMV virions were blocked in their ability to move from infected rootstocks of nontransformed tomato or tobacco into the transgenic scions. Interestingly, virions could not move through a transgenic intersection into the upper scion. These results provide an additional indication that replicase-mediated resistance affects long-distance movement.

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.

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.

A cDNA clone from a defective RNA of citrus tristeza virus is infective in the presence of the helper virus.

A naturally occurring defective RNA of 2379 nt (D2.3) from the VT strain of citrus tristeza closterovirus (CTV) was cloned and sequenced. The D2.3 RNA is a fusion of two regions of 1521 and 858 nt from the 5' and 3' ends of the CTV genome, respectively. A cDNA clone of D2.3 RNA was tagged by the insertion of a 0.47 kb chimeric DNA fragment and the recombinant cDNA was inserted downstream of the cauliflower mosaic virus 35S promoter. The resulting construct was bombarded into CTV-infected tissue, which was then grafted onto virus-free plants. The presence of recombinant RNA in systemically infected leaves was demonstrated by RT-PCR. Sequencing the RT-PCR products synthesized from double-stranded RNA confirmed the presence of the chimeric segment used for tagging. This is the first report of an infectious cDNA molecule derived from CTV D-RNA.

Characterization of cucumber mosaic virus. III. Localization of sequences in the movement protein controlling systemic infection in cucurbits.

Cucumber mosaic virus (CMV), generated from biologically active cDNA clones of Fny-CMV RNA 1 plus 2 and Sny-CMV RNA 3, derived from the Fny- and Sny-strains of CMV, was able to infect tobacco but not squash plants systemically. In squash, viral RNA, movement protein, and coat protein all accumulated in the inoculated cotyledons. The lack of systemic infection was associated with a reduced rate of cell-to-cell movement within the cotyledons. The restricted movement mapped to two sequence changes in the codons of amino acids 51 and 240 of the Sny-CMV 3a gene. These same sequence changes previously were shown to be associated with high levels of 3a protein accumulation and chronic vs acute, cyclic infection typical of Sny-CMV vs Fny-CMV [Gal-on et al. (1996). Virology 226, 354-361]. Fny-CMV, mutated in the codons of 3a gene amino acids 51 and 240, was still able to infect several solanaceous hosts (tobacco, tomato, and pepper) systemically, but did not elicit a typical CMV systemic infection on any of several cucurbit hosts (cucumber, melon, or squash). The significance of the location of amino acid positions 51 and 240 in the 3a movement protein is discussed.

Simple hand-held devices for the efficient infection of plants with viral-encoding constructs by particle bombardment.

An efficient method for infection of plants with a cloned potyvirus by particle bombardment has been described (Gal-On et al., 1995). A simplified method is described now whereby a vaccuum chamber and helium propulsive gas are not required to achieve a high efficiency of infection. The new device-the 'HandGun'--is hand-held, and easily constructed from readily available materials. With this technique it is possible to bombard soft plants and seedings that do not survive particle bombardment by other devices. bombardment of C. pepo plants with a full length clone of zucchini yellow mosaic potyvirus results in approximately 100% infection at 100 pg cDNA per plant using air or helium to propel the microprojectiles. The HandGun is 10(5)-fold more efficient than mechanical inoculation. Tungsten and gold were found to be the most efficient materials tested for use as microprojectiles. Crude extracts of plasmids from E. coli were found to be effective, as well as column-purified cDNA. A functional, simple version of the HandGun--'the Blowpipe'--was also constructed, which does not require an electrically controlled valve. Plants can be inoculated with plant viruses from sap with the HandGun.

Characterization of cucumber mosaic virus. II. Identification of movement protein sequences that influence its accumulation and systemic infection in tobacco.

Tobacco plants infected by the Sny strain of cucumber mosaic virus (CMV) showed chronic symptoms rather than cycles of acute disease followed by no symptoms, as seen for many strains such as Fny-CMV. The Sny-CMV 3a movement protein (MP) accumulated at 10 to 50 times the level of the Fny-CMV MP in the first few systemically infected leaves showing disease symptoms. The increased production of movement protein was not associated with an increase in synthesis of viral RNA and/or capsid protein. The correlation of high levels of accumulated MP and chronic infection in tobacco mapped to the 3a gene of Sny-CMV. Sequence analysis of the 3a gene of Sny-CMV showed that there were three nucleotide changes that altered amino acid sequences within the MP. Analysis of the kinetics of infection and level of MP accumulation for various CMV RNA 3 chimeric constructs and site-directed mutants of RNA 3 showed that chronic infection and high MP accumulation were associated with nucleotide sequence changes at two positions within codons 51 and 240 of the MP gene. These changes in the MP of Fny-CMV resulted in an altered subcellular distribution of the 3a protein, as also was observed for the Sny-CMV 3a protein. In both cases, considerably more 3a protein was associated with a fraction containing membranes. The potential relationship between membrane association and chronic infection is discussed.