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.     

Genes for resistance to zucchini yellow mosaic in tropical pumpkin

Four cultigens of Cucurbita moschata resistant to zucchini yellow mosaic virus were crossed with the susceptible 'Waltham Butternut' and with each other in order to clarify the mode of inheritance of resistance and relationships among the genes involved. Five loci were segregating, with genes for resistance Zym-0 and Zym-4 carried by 'Nigerian Local' and one of them also carried by 'Nicklow's Delight,' Zym-1 carried by 'Menina,' and zym-6 carried by 'Soler.' A recessive gene carried by 'Waltham Butternut,' zym-5, is complementary with the dominant Zym-4 of 'Nigerian Local,' that is, the resistance conferred by Zym-4 is only expressed in zym-5/zym-5 individuals. Gene zym-6 appears to be linked to either Zym-0 or Zym-4, and it is also possible that Zym-1 is linked to one of them as well.     

Inheritance of resistance to zucchini yellow mosaic virus and watermelon mosaic virus in watermelon

High resistance to zucchini yellow mosaic virus-China strain (ZYMV-CH) and moderate resistance to watermelon mosaic virus (WMV) were found in a selection of PI 595203 (Citrullus lanatus var. lanatus), an Egusi type originally collected in Nigeria. Mixed inoculations showed primarily that these two viruses have no cross-protection. This fact may explain the high frequency of mixed infection often observed in commercial fields. When plants were inoculated with a mixture of the two viruses, the frequency of plants resistant to ZYMV was lower than expected, indicating that WMV infection may reduce the ability of a plant to resist ZYMV. We studied inheritance of resistance to ZYMV-CH and WMV, using crosses between a single-plant selection of PI 595203 and the ZYMV-susceptible watermelon inbreds 9811 and 98R. According to virus ratings of the susceptible parents, the resistant parent, and the F1, F2, and BC1 generations, resistance to ZYMV-CH was conferred by a single recessive gene, for which the symbol zym-CH is suggested. The high tolerance to WMV was controlled by at least two recessive genes.     

Multiple alleles for zucchini yellow mosaic virus resistance at the zym locus in cucumber

 Sources of resistance to several potyviruses have been identified and characterized within the cucumber (Cucumis sativus L.) germplasm. Resistance to zucchini yellow mosaic virus (ZYMV) is present in inbred lines derived from the Dutch hybrid Dina (Dina-1) and from the Chinese cultivar ‘Taichung Mou Gua’ (TMG-1). Tests of allelism indicated that the genes for resistance to ZYMV in TMG-1 and Dina-1 are at the same locus; however, the two genotypes exhibited different phenotypes in response to cotyledon inoculation with ZYMV. Dina-1 exhibited a distinct veinal chlorosis and accumulation of virus limited to the first and/or second true leaves, while TMG-1 remained symptom-free and did not accumulate virus. The distinct veinal chlorosis phenotype in Dina-1 was dominant to the symptom-free phenotype in TMG-1 and was shown not to be due to a separate gene. These results indicate that a series of alleles differing in effectiveness and dominance relationships occurs at the zym locus such that Zym>zym ^Dina>zym ^TMG-1. In addition to ZYMV resistance, TMG-1 is also resistant to watermelon mosaic virus (WMV), the watermelon strain of papaya ringspot virus (PRSV-W) and the Moroccan watermelon mosaic virus (MWMV); the WMV and MWMV resistances are at the same locus, or tightly linked to the zym locus. Dina-1 also was found to be resistant to PRSV-W and MWMV. The gene for MWMV resistance in Dina-1 appeared to be at the same locus or tightly linked (<1% recombination) to the gene for ZYMV resistance. In contrast to the response to ZYMV inoculation, Dina-1 does not exhibit distinct veinal chlorosis when inoculated with PRSV-W or MWMV. Collectively, these observations suggest that the gene(s) conferring resistance to ZYMV, WMV, and MWMV may be part of a gene cluster for potyvirus resistance in cucumber. Received: 12 November 1996 / Accepted: 25 April 1997     

Analysis of the autoproteolytic activity of the recombinant helper component proteinase from zucchini yellow mosaic virus

The multifunctional helper component proteinase (HC-Pro) of potyviruses contains an autoproteolytic function that, together with the protein 1 (P1) and NIa proteinase, processes the polyprotein into mature proteins. In this study, we analysed the autoproteolytic active domain of zucchini yellow mosaic virus (ZYMV) HC-Pro. Several Escherichia coli-expressed MBP:HC-Pro:GFP mutants containing deletions or point mutations at either the N- or C-terminus of the HC-Pro protein were examined. Our results showed that amino acids essential for the proteolytic activity of ZYMV HC-Pro are distinct from those of the tobacco etch virus HC-Pro, although the amino acid sequences in the proteolytic active domain are conserved among potyviruses.     

Effect of abscisic acid on chilling injury of zucchini squash

The endogenous levels of abscisic acid (ABA) in zucchini squash were increased by temperature conditioning at 10°C for 2 days. This temperature conditioning treatment reduced the severity of chilling injury in the squash during subsequent storage at 2.5°C. The ABA levels remained higher in treated squash than in untreated samples throughout the storage. Direct treatments of squash with ABA at 0.5 and 1.0 mM before storage at 2.5°C increased ABA levels in the tissue and were also effective in reducing chilling injury.     

Effect of abscisic acid on chilling injury of zucchini squash

The endogenous levels of abscisic acid (ABA) in zucchini squash were increased by temperature conditioning at 10°C for 2 days. This temperature conditioning treatment reduced the severity of chilling injury in the squash during subsequent storage at 2.5°C. The ABA levels remained higher in treated squash than in untreated samples throughout the storage. Direct treatments of squash with ABA at 0.5 and 1.0 mM before storage at 2.5°C increased ABA levels in the tissue and were also effective in reducing chilling injury.     

Intraspecific variation in zucchini yellow mosaic virus transmission by Myzus persicae and the impact of aphid host plant

Three isofemale lines of Myzus persicae (Sulzer), two lines collected from and reared on a brassicaceous host, and one line collected from and reared on a malvaceous host, were evaluated for their efficiency of transmitting Zucchini yellow mosaic virus (family Potyviridae, genus Potyvirus, ZYMV). In the first experiment, the transmission efficiencies of two clones from Brassicaceae (B1 and B2) were 52.0 and 60.8%, respectively, and these transmissions were not significantly different. In a second experiment, the transmission efficiencies of the clone on Malvaceae (M1) and clone B2 were significantly different at 35.6 and 55.7%, respectively. Further experiments evaluated host-related mechanisms that may have contributed to the differential transmissions observed between clones M1 and B2. Studies on short-term feeding showed that aphids continuously reared on okra, Abelmoschus esculentus (L.) Moench (malvaceous host), and those that were reared on okra and allowed a 24-h preacquisition feeding period on mustard, Brassica juncea (L.) Czern (brassicaceous host), had significantly lower transmission than aphids continuously maintained on mustard. Aphids reared on mustard and allowed a 24-h preacquisition feeding period on okra had intermediate transmission efficiency. In long-term host association studies, we found that aphids reared on mustard had significantly higher transmission efficiency than those reared on okra, and aphids reared first on okra and then switched to mustard had a transmission efficiency that was intermediate and not significantly different from the other two treatments. Our study reveals the existence of intraspecific variation in the transmission of ZYMV by M. persicae, and it suggests that to accurately assess the transmission capability of ZYMV by this species, multiple clones should be examined. Furthermore, the host plant on which the aphid is reared as well as the host plant on which it feeds just before virus acquisition contribute to ZYMV transmission efficiency of M. persicae.     

Characterisation of a variant of zucchini yellow fleck virus (ZYFV), a potyvirus causing a wilt disease of melons in Israel*

A flexuous filamentous virus 750 nm in length was isolated from field-grown melons (Cucumis melo L.) and squirting cucumber (Ecballium elaterium L.). Infection of melon seedlings of most commercial varieties resulted in a lethal wilting, but late infections gave dieback. The virus infected systemically all economically important cucurbit crops. Its coat protein consists of a single polypeptide, 34 500 D in size, encapsidating a single stranded RNA genome of 10.1 kb. Antiserum against the Italian zucchini yellow fleck virus (ZYFV) reacted specifically with the Israeli isolate in both ISEM and Western blot. The virus was aphid transmissible in a non-persistent manner. Based on host range, host reaction and serological data, it is suggested that the virus described is an Israeli variant of ZYFV.     

Inheritance of resistance to watermelon mosaic virus in the cucumber line TMG-1: tissue-specific expression and relationship to zucchini yellow mosaic virus resistance

The inbred cucumber (Cucumis sativus L.) line TMG-1 is resistant to three potyviruses:zucchini yellow mosaic virus (ZYMV), watermelon mosaic virus (WMV), and the watermelon strain of papaya ringspot virus (PRSV-W). The genetics of resistance to WMV and the relationship of WMV resistance to ZYMV resistance were examined. TMG-1 was crossed with WI-2757, a susceptible inbred line. F₁, F₂ and backcross progeny populations were screened for resistance to WMV and/or ZYMV. Two independently assorting factors conferred resistance to WMV. One resistance was conferred by a single recessive gene from TMG-1 (wmv-2). The second resistance was conferred by an epistatic interaction between a second recessive gene from TMG-1 (wmv-3) and either a dominant gene from WI-2757 (Wmv-4) or a third recessive gene from TMG-1 (wmv-4) located 20–30 cM from wmv-3. The two resistances exhibited tissue-specific expression. Resistance conferred by wmv-2 was expressed in the cotyledons and throughout the plant. Resistance conferred by wmv-3 + Wmv-4 (or wmv-4) was expressed only in true leaves. The gene conferring resistance to ZYMV appeared to be the same as, or tightly linked to one of the WMV resistance genes, wmv-3.     

The effect on yield in courgette and marrow of the mild strain of zucchini yellow mosaic virus used for cross-protection

The effects on yield in courgette and marrow (Cucurbita pepo) crops resulting from inoculation with the mild strain of zucchini yellow mosaic virus (ZYMV:WK), have been determined in polythene-house trials and in three years of outdoor, commercial field trials. In polythene-house trials ZYMV:WK inoculated plants were up to 10 days later in flowering than uninoculated plants and their cumulative yields were between 5% and 26% less than uninoculated plants depending on the cultivar. In most field trials cumulative yields from inoculated plants were between 4% and 38% less than uninoculated plants depending on the site and cultivar, but in one trial the yield was 7% higher from inoculated plants. In all experiments, courgette and marrow fruits harvested from ZYMV:WK inoculated plants were symptomless and indistinguishable from fruit harvested from uninoculated plants. The mild leaf symptoms induced by ZYMV:WK infection did not intensify to severe leaf symptoms and where there were natural outbreaks of severe ZYMV infection, fruits from inoculated plants remained symptomless whilst those from uninoculated plants were severely affected and unmarketable.     

Nitrogen response surface models of zucchini squash,head lettuce and potato

Plants grown at 5 nitrate (N) levels ranging from 2–36 mmol L^–1 for lettuce and 2–43 mmol L^–1 for zucchini squash and potato were harvested over the growth period to maturity at a minimum of 2 week intervals. Gamma × cubic response surface models fitted actual dry matter growth data quite precisely (R²>0.98) from which growth and dry matter partitioning could be derived.Total dry matter growth was very responsive to N and maximum growth was predicted to occur at an N level of 14.2 mmol L^–1 for lettuce, 18.1 mmol L^–1 for zucchini squash and 11.6 mmol L^–1 for potato. Growth declined at higher N levels for all species. For zucchini squash, both high and low N levels which reduced growth increased partitioning of dry matter to fruit. For potato, partitioning of dry matter to tubers tended to increase at high levels of N, particularly at maturity. For lettuce, the N level producing the highest dry matter yield partitioned the highest ratio of dry matter to head.The highest fresh yields of zucchini squash fruit, lettuce head and potato tubers were recorded at N levels of 14, 5 and 11 and 7 mmol L^–1 respectively. The effect of N on quality followed a similar trend.     

Resistances to turnip mosaic potyvirus in Arabidopsis thaliana.

The responses of a collection of Arabidopsis thaliana ecotypes to mechanical inoculation with turnip mosaic potyvirus were assessed. The virus induced characteristic severe symptoms of infection in systemically infected plants. Resistance was found in four ecotypes: Bay-0, Di-0, Er-0, and Or-0. Enzyme-linked immunosorbent assay results of the resistant ecotypes suggested that ecotypes Di-0, Er-0, and Or-0 actually consist of mixed genotypes with resistances acting at different levels in the virus life cycle. Another form of resistance was found in ecotype Bay-0, for which several lines of evidence indicated an interference with viral cell-to-cell movement in the inoculated leaves.     

Correlation of reduced chilling injury with Increased spermine and spermidine levels in zucchini squash

By means of a biparametric cytofluorimetric analysis it is possible to distribute meristematic plant cells in a variety of cell cycle sub-compartments, unidentifiable by DNA measurements alone. In this work, an asynchronous proliferating cell population of pea root meristems was divided into different sub-compartments of the cell cycle phases, i.e. G1A, G1B, S. G2A and G2B on the basis of their DNA-nuclear protein content. By means of the same biparametric analysis, differentiated mesophyll cells and quiescent cells of embryo roots, indicated as G0 and G2Q, were distinguished from cycling cells by their low nuclear protein content. These results conform to those of some analyses performed on animal cells in culture and show that it is possible to get a major insight into cell cycle kinetics and its control in a natural system such as root meristem.     

Fourier transform infrared spectra of zucchini squash stored at chilling or non-chilling temperatures

Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that might be suitable.Fourier transform infrared spectroscopy (FTIR) was used to investigate spectral changes in the epidermis of zucchini squash resulting from low temperature storage-chilling (5°C) or non-chilling (15°C). Increased levels of fluid (water) were found in the tissue after 2 days of 5°C storage. This increase was reversed to harvest levels when chilled squash were warmed to room temperature for 1 day. After 3 days at 5°C and 1 day at room temperature, a further increase in fluid levels was found in the epidermis. Squash chilled for 3 days were apparently beyond recovery as indicated by spectral changes, although visual symptoms of chilling injury were not apparent until another 3 days of exposure to 5°C. Spectra of epidermis tissues from squash stored at 15°C indicated a pattern of increased non-reversible fluid accumulation when storage was prolonged. These results suggest that FTIR spectroscopy may be a rapid way to detect changes in chilled tissues before the eventual appearance of visible symptoms.