Genetic control of resistance to cucumber mosaic virus in Cucurbita pepo

Segregation ratios in the F₂s of crosses between courgette cultivars and the pumpkin cv. Cinderella indicated that the resistance of the latter to cucumber mosaic virus (CMV), expressed as a failure to develop systemic symptoms, was controlled by two unlinked recessive genes. However, data from the backcross generations were not consistent with this. Biometrical analysis showed significant gene interactions, possibly between the genes for CMV resistance and the background genotype determining plant vigour and a gene dosage effect for resistance. The resistance has been successfully backcrossed into courgette breeding material.     

Metabolic alterations in cotyledons of Cucurbita pepo infected by cucumber mosaic virus

Changes in the capacities of enzymes in various metabolic pathwayshave been measured during infection of cotyledons of Cucurbitapepo L. with cucumber mosaic virus (CMV). Starch accumulationand low sucrose content, which are characteristic of the earlystages of infection, are reversed in the later stages of infection.The decline in starch correlated with a reduced capacity forstarch synthesis (ADP-glucose pyrophosphorylase) and a risein the capacity for starch degradation (total starch hydrolase,starch phosphorylase). ¹⁴CO₂ feeding experiments, conductedat saturating CO₂ concentration, show that the newly-assimilatedcarbon was lost at a lower rate from infected cotyledons andless was incorporated into structural carbohydrates, phosphorylatedintermediates plus organic acids, more into soluble sugars,amino acids and proteins. At a later stage of infection therewere dramatic increases in respiratory capacity and a substantialalteration of carbohydrate metabolism. The infection had a largestimulatory effect on the capacity for oxidative pentose-phosphatepathway (glucose-6-phosphate dehydrogenase, 6-phospho-gluconatedehydrogenase), glycolysis (ATP- and pyrophosphate-dependentphosphofructokinases), tri-carboxylic acid cycle (isocitratedehydrogenase, fumarate hydratase), anaplerotic reactions (NAD-dependentmalic enzyme, phosphoenolpyruvate car-boxylase) and oxidativeelectron transport (cytochrome c oxidase). While there wereno overall changes in photosynthetic rate (measured in saturatingCO₂), infection either reduced (Rubisco and glycerate kinase)or did not affect (chloroplastic fructose bis-phosphatase andhydroxypyruvate kinase) the capacities of the photosyntheticcarbon reduction pathway or the photosynthetic carbon oxidationpathway. Key words: Plant-virus interaction, sucrose, starch, enzymes, ¹⁴CO₂ incorporation, O₂ flux     

Complex spatial responses to cucumber mosaic virus infection in susceptible Cucurbita pepo cotyledons

Cucumber mosaic virus infection of its susceptible host Cucurbita pepo results in a program of biochemical changes after virus infection. Applying a spatial analysis to expanding infected lesions, we investigated the relationship between the changes in enzyme activity and gene expression. Patterns of altered expression were seen that could not be detected by RNA gel blot analysis. For all the host genes studied, there was a downregulation (shutoff) of expression within the lesion. In addition, two distinct types of upregulation were observed. The expression of heat shock protein 70 (HSP70) and NADP(+)-dependent malic enzyme (NADP-ME) showed induction in apparently uninfected cells ahead of the infection. This response was more localized than the upregulation exhibited by catalase expression, which occurred throughout the uninfected regions of the tissue. The experiments showed that virus infection induced immediate and subsequent changes in gene expression by the host and that the infection has the potential to give advance signaling of the imminent infection.     

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.     

Mutation of capsid protein phosphorylation sites abolishes cauliflower mosaic virus infectivity

The cauliflower mosaic virus (CaMV) capsid protein is derived by bidirectional processing of the precapsid protein (CP56). We expressed several derivatives of CP56 in Escherichia coli and used them as substrates for virus-associated kinase and casein kinase II purified from plant cells. Three serine residues located at the N terminus of the mature viral protein CP44 were identified as phosphorylation targets. A mutation of one of them in the viral context had little or no effect on viral infectivity, but a mutation of all three serines abolished infectivity. The mapping of phosphorylation sites in CP44, but not CP39 or CP37, and immunodetection of the Zn finger motif in CP44 and CP39, but not CP37, support the model that CP39 is produced from CP44 by N-terminal processing and CP37 is produced from CP39 by C-terminal processing. We discuss the possible role of phosphorylation in the processing and assembly of CaMV capsid protein.     

First report of Squash leaf curl china virus causing mosaic symptoms on summer squash (Cucurbita pepo) grown in Varanasi district of India

Severe incidence of a mosaic disease was observed on summer squash (Cucurbita pepo), commonly called pepo, grown in Varanasi during June–September of Khariff season 2007. Symptoms observed were mosaic, puckering on the leaves, wartiness on fruits, general stunting of plants and low yield. PCR amplification with degenerate primers designed to target the conserved sequences of coat protein gene of whitefly transmitted geminiviruses showed ～800 bp fragment in all symptomatic samples tested, indicating the association of a geminivirus with the disease. Nucleotide sequence analysis of the amplified fragment showed 99% identity with pumpkin isolate of squash leaf curl china virus (SLCCNV) from Lucknow. It showed 85–96.7% homology with other isolates of SLCCNV from India and abroad. Phylogenetic analysis revealed the isolate on pepo from Varanasi clustered with SLCCNV isolates on pumpkin from Lucknow and Coimbatore.     

Inheritance of parthenocarpy in summer squash (Cucurbita pepo L.)

The inheritance of the tendency to set parthenocarpic fruit in the summer squash (Cucurbita pepo L.) line Whitaker was studied. Two parental lines, Whitaker (parthenocarpic) and Caserta (non-parthenocarpic), and the F1 and F2 generations and backcrosses to both parents were tested. The parthenocarpic tendency of individual plants was scored on a scale from 1 (non-parthenocarpic fruit) to 5 (parthenocarpic fruit). The Whitaker line produced parthenocarpic fruit and had a mean score of 4.2, whereas Caserta did not set parthenocarpic fruit and had a score of 1.55. The heritability estimates indicated that genetic gains from selection were feasible. The additive-dominant model showed a good fit, with epistasis being negligible or nonexistent. The hypothesis of monogenic inheritance with incomplete dominance was not rejected within the degree of dominance range from 0.2 to 0.5. These results indicate that parthenocarpy is controlled by a single locus, with incomplete dominance in the direction of parthenocarpic expression.     

Evidence for channel mediated transport of boric acid in squash (Cucurbita pepo)

Boron (B) is taken up by plant roots as undissociated boric acid which is a non-electrolyte of similar size to urea and other non-electrolytes. In animal systems, non-electrolytes are transported across biological membranes through aquaporins or through non-aquaporin channels. In artificial lipids boric acid is known to diffuse directly through the lipid bilayer at a rate that is determined by lipid composition. A possible role for channel proteins in in-vitro B uptake is suggested by recent work in which B uptake into isolated membrane vesicles was inhibited by channel blockers and by demonstration that the expression of the plant channel protein PIP1 in Xenopus oocytes increases boric acid uptake by 30%. This study examines whether B transport is a channel-mediated process in intact plants. In the presence of the channel inhibitors HgCl₂, phloretin, and DIDS, B uptake by squash plants was reduced by 40–90% by HgCl₂ (as HgCl₂ varied from 50 M to 1 mM), 44% by phloretin (250 M) and 58% by DIDS (250 M). The effect of Hg ions on B uptake was reversed by 2-mercaptoethanol. The addition of other non-electrolytes in size ranges similar to boric acid inhibited B uptake to various degrees. Addition of urea resulted in 54% decrease in B uptake, while, acetamide, formamide, thiourea and glycerol inhibited uptake by 50, 35, 53 and 44%, respectively. The effect of HgCl₂ on B uptake was greater at high B concentrations than at low B concentrations. These data and information from in-vivo studies suggest two possible mechanisms of B uptake: passive diffusion through lipid bilayers and channel-mediated transport.     

Oligogenic inheritance for resistance to Zucchini yellow mosaic virus in Cucurbita pepo

‘True French’ is an open‐pollinated cultivar of the Zucchini (Courgette) Group of Cucurbita pepo and is susceptible to Zucchini yellow mosaic virus (ZYMV). Using C. moschata‘Menina’ as the source of ZYMV resistance and following six generations of backcrossing, a true‐breeding line nearly isogenic to ‘True French’, designated 381e, was recovered that carried ZYMV resistance, albeit not at as high a level as in ‘Menina’. ‘True French’ and accession 381e were crossed, and their reciprocal F₁, F², and backcross progenies were grown in a chamber and inoculated with a highly virulent, non‐aphid‐transmissible strain of ZYMV. Nearly all F¹ plants and all plants of the backcross to 381e were classified as resistant. Segregation to resistant and susceptible individuals occurred in the backcross to the susceptible parent, in accordance with a 3:5 three‐gene ratio of resistant: susceptible. The F₂ segregated in accordance with a ratio of 45 resistant : 19 susceptible, which would be obtained if there was one major gene for resistance, Zym‐1 (Zym), and two other genes, herein designated Zym‐2 and Zym‐3, both of which for complementary to Zym‐1. The presence of Zym‐1 and either Zym‐2 or Zym‐3 is necessary for resistance to be expressed in young plants, but the presence of all three might be necessary for resistance to continue to be expressed during subsequent development of the plants. Evidently, Zym‐2 and Zym‐3 are ubiquitous in C. moschata but their susceptible alleles are much more common in C. pepo. As the level of resistance of 381e to ZYMV is not as high as that of C. moschata‘Menina’, additional, as yet unidentified, genes must be involved in conferring high resistance to this virus.     

Movement of soil-applied imidacloprid and thiamethoxam into nectar and pollen of squash (Cucurbita pepo)

There has been recent interest in the threat to bees posed by the use of systemic insecticides. One concern is that systemic insecticides may translocate from the soil into pollen and nectar of plants, where they would be ingested by pollinators. This paper reports on the movement of two such systemic neonicotinoid insecticides, imidacloprid and thiamethoxam, into the pollen and nectar of flowers of squash (Cucurbita pepo cultivars "Multipik," "Sunray" and "Bush Delicata") when applied to soil by two methods: (1) sprayed into soil before seeding, or (2) applied through drip irrigation in a single treatment after transplant. All insecticide treatments were within labeled rates for these compounds. Pollen and nectar samples were analyzed using a standard extraction method widely used for pesticides (QuEChERS) and liquid chromatography mass spectrometric analysis. The concentrations found in nectar, 10 ± 3 ppb (mean ± s.d) for imidacloprid and 11 ± 6 ppb for thiamethoxam, are higher than concentrations of neonicotinoid insecticides in nectar of canola and sunflower grown from treated seed, and similar to those found in a recent study of neonicotinoids applied to pumpkins at transplant and through drip irrigation. The concentrations in pollen, 14 ± 8 ppb for imidacloprid and 12 ± 9 ppb for thiamethoxam, are higher than those found for seed treatments in most studies, but at the low end of the range found in the pumpkin study. Our concentrations fall into the range being investigated for sublethal effects on honey bees and bumble bees.     

Rubella virus capsid protein modulates viral genome replication and virus infectivity

The structural proteins (SP) of the Togaviridae can be deleted in defective interfering RNAs. The dispensability of viral SP has allowed construction of noninfectious viral expression vectors and replicons from viruses of the Alphavirus and Rubivirus genera. Nevertheless, in this study, we found that the SP of rubella virus (RUB) could enhance expression of reporter genes from RUB replicons in trans. SP enhancement required capsid protein (CP) expression and was not due to RNA-RNA recombination. Accumulation of minus- and plus-strand RNAs from replicons was observed in the presence of SP, suggesting that SP specifically affects RNA synthesis. By using replicons containing an antibiotic resistance gene, we found 2- to 50-fold increases in the number of cells surviving selection in the presence of SP. The increases depended significantly on the amount of transfected RNA. Small amounts of RNA or templates that replicated inefficiently showed more enhancement. The infectivity of infectious RNA was increased by at least 10-fold in cells expressing CP. Moreover, virus infectivity was greatly enhanced in such cells. In other cells that expressed higher levels of CP, RNA replication of replicons was inhibited. Thus, depending on conditions, CP can markedly enhance or inhibit RUB RNA replication.     

Chemical reactivity of brome mosaic virus capsid protein

Viral particles are biological machines that have evolved to package, protect, and deliver the viral genome into the host via regulated conformational changes of virions. We have developed a procedure to modify lysine residues with S-methylthioacetimidate across the pH range from 5.5 to 8.5. Lysine residues that are not completely modified are involved in tertiary or quaternary structural interactions, and their extent of modification can be quantified as a function of pH. This procedure was applied to the pH-dependent structural transitions of brome mosaic virus (BMV). As the reaction pH increases from 5.5 to 8.5, the average number of modified lysine residues in the BMV capsid protein increases from 6 to 12, correlating well with the known pH-dependent swelling behavior of BMV virions. The extent of reaction of each of the capsid protein's lysine residues has been quantified at eight pH values using coupled liquid chromatography-tandem mass spectrometry. Each lysine can be assigned to one of three structural classes identified by inspection of the BMV virion crystal structure. Several lysine residues display reactivity that indicates their involvement in dynamic interactions that are not obvious in the crystal structure. The influence of several capsid protein mutants on the pH-dependent structural transition of BMV has also been investigated. Mutant H75Q exhibits an altered swelling transition accompanying solution pH increases. The H75Q capsids show increased reactivity at lysine residues 64 and 130, residues distal from the dimer interface occupied by H75, across the entire pH range.     

Genetics of resistance to red pumpkin beetle (A ulacophora foveicollis) in summer squash (Cucurbita pepo L.)

Summary Resistance to red pumpkin beetle in summer squash was found to be controlled by polygenes. Diallel and Triple test cross analysis revealed the preponderance of non-additive and additive gene effects for resistance respectively. Absence of epistasis for resistance was indicated by both tests.     

Pollination value of male bees: the specialist bee Peponapis pruinosa (Apidae) at summer squash (Cucurbita pepo)

Male bees can be abundant at flowers, particularly floral hosts of those bee species whose females are taxonomic pollen specialists (oligolecty). Contributions of male bees to host pollination are rarely studied directly despite their prevalence in a number of pollination guilds, including those of some crop plants. In this study, males of the oligolectic bee, Peponapis pruinosa Say, were shown to be effective pollinators of summer squash, Cucurbita pepo L. Seven sequential visits from male P. pruinosa maximized squash fruit set and growth. This number of male visits accumulated during the first hour of their foraging and mate searching at flowers soon after sunrise. Pollination efficacy of male P. pruinosa and their abundances at squash flowers were sufficient to account for most summer squash production at our study sites, and by extrapolation, to two-thirds of all 87 North American farms and market gardens growing squashes that were surveyed for pollinators by collaborators in the Squash Pollinators of the Americas Survey. We posit that the substantial pollination value of male Peponapis bees is a consequence of their species' oligolecty, their mate seeking strategy, and some extreme traits of Cucurbita flowers (massive rewards, flower size, phenology).     

Trypsin inhibitors in summer squash (Cucurbita pepo) seeds. Isolation, purification and partial characterization of three inhibitors

Three trypsin inhibitors were isolated from summer squash (Cucurbita pepo) seeds and purified to homogeneity by fractionation with ammonium sulphate and methanol, ion-exchange chromatography and gel filtration. All three inhibitors have lysine at their active site. Two of them (II, IV) show the same isoelectric point (at pH 5.6), amino acid composition and molecular mass (3259). The third inhibitor (III) of molecular mass of 3654 and isoelectric point at 4.9 has additionally one histidine residue and two glutamic acid residues more per molecule.     

The capsid protein of satellite Panicum mosaic virus contributes to systemic invasion and interacts with its helper virus

Satellite panicum mosaic virus (SPMV) depends on its helper Panicum mosaic virus (PMV) for replication and spread in host plants. The SPMV RNA encodes a 17-kDa capsid protein (CP) that is essential for formation of its 16-nm virions. The results of this study indicate that in addition to the expression of the full-length SPMV CP from the 5'-proximal AUG start codon, SPMV RNA also expresses a 9.4-kDa C-terminal protein from the third in-frame start codon. Differences in solubility between the full-length protein and its C-terminal product were observed. Subcellular fractionation of infected plant tissues showed that SPMV CP accumulates in the cytosol, cell wall-, and membrane-enriched fractions. However, the 9.4-kDa protein exclusively cofractionated with cell wall- and membrane-enriched fractions. Earlier studies revealed that the 5'-untranslated region (5'-UTR) from nucleotides 63 to 104 was associated with systemic infection in a host-specific manner in millet plants. This study shows that nucleotide deletions and insertions in the 5'-UTR plus simultaneous truncation of the N-terminal part of the CP impaired SPMV spread in foxtail millet, but not in proso millet plants. In contrast, the expression of the full-length version of SPMV CP efficiently compensated the negative effect of the 5'-UTR deletions in foxtail millet. Finally, immunoprecipitation assays revealed the presence of a specific interaction between the capsid proteins of SPMV and its helper virus (PMV). Our findings show that the SPMV CP has several biological functions, including facilitating efficient satellite virus infection and movement in millet plants.     

The serine proteinase inhibitor from summer squash (Cucurbita pepo): some structural features, stability and proteolytic degradation

The serine proteinase inhibitor from summer squash seeds (CPTI-II) with Mr of about 3250 contains three disulphide bridges and is unusually resistant to denaturing agents (e.g. 10% trichloroacetic acid at about 100 degrees C), thermolysin and proteinase V8 from Staphylococcus aureus. The inhibitor is digested by pepsin; the digestion of the virgin form proceeds more rapidly than when the peptide bond of the reactive site is broken. The inhibitor is not specifically reduced by sodium borohydride at pH 8.8, and almost full reactivation of the inhibitor reduced by dithiothreitol takes place at pH 8.15 in the presence of EDTA and the reduced + oxidized glutathione system. The inhibitor was crystallized from methanol. CD spectra point to the occurrence of beta-turns in the secondary structure of the inhibitor.     

Physiological and metabolic changes of Cucurbita pepo leaves in response to zucchini yellow mosaic virus (ZYMV) infection and salicylic acid treatments.

The changes of some physiological and biochemical parameters in pumpkin (Cucurbita pepo cv Eskandarani) leaves associated with zucchini yellow mosaic virus (ZYMV) infection and the effect of exogenous application of salicylic acid (SA) were studied in this paper. In comparison to the untreated leaves, ZYMV infected leaves showed many symptoms, including severe mosaic, size reduction, stunting and deformation. Results from analysis of physiological parameters indicated that viral infection and SA treatments affected metabolism. Viral infection decreased pigment, protein and carbohydrate levels. But with all SA treatments, the protein and carbohydrate contents are noticeably increased. Moreover, the other biochemical parameters showed variable alterations. The peroxidase (POX, EC 1.11.1.7) activity and proline contents were induced by both viral infection and SA treatments. In addition, protein patterns represent some newly synthesized polypeptides which reflect formation of pathogenesis related proteins in all treatments. SA treatment increases the plant resistance against ZYMV. This can be noticed through reduction of percentage of the infected plants, decrease in disease severity and virus concentration of the plants treated with SA then inoculated with virus. All results show significant changes in metabolism affected by either viral infection or SA treatments and also indicate that exogenous SA plays an important role in induction of defense mechanism against ZYMV infection.     

Crop/weed microgametophyte competition in Cucurbita pepo (Cucurbitaceae)

Prior studies of crop/weed gene flow in Cucurbita pepo have demonstrated that pollinators can deposit mixed pollen loads. The fate of microgametophytes emerging from these pollen mixtures is unknown. In an effort to define the relationship between pollen mixture composition and progeny composition, experimental plants representing a zucchini cultivar and a cross-compatible free-living type were genotyped with an isozyme marker and grown under greenhouse conditions. Weighed mixtures of weed/crop pollen were applied to stigmas of both types in differing ratios. Subsequent fertilizations were tracked by an electrophoretic screen of over 1,600 progeny from 39 fruits. Equal mixtures of weed/crop pollen do not produce a corresponding suite of progeny. Pollen of the pistillate parent is favored, and this advantage increases as the proportion of pollen from the pistillate parent increases. However, increasing the proportion of zucchini pollen does not produce a corresponding increase in fertilization success when the free-living type is used as pistillate parent. These results indicate that microgametophyte competition in both domesticated and free-living Cucurbita gynoecia could be a significant component of gene flow dynamics. The nature of this competition can only be defined by experiments that control numerous variables, both paternal and maternal, that might be involved. The tendency for crop/weed hybrids to occur at different portions of the ovary suggests that interactions involving two factors—microgametophyte growth rate and gynoecium structure—might play a significant role.     

The interaction of sodium dodecyl sulfate with cucumber green mottle mosaic virus protein and tobacco mosaic virus protein

The binding of sodium dodecyl sulfate to coat protein subunits of cucumber green mottle mosaic virus and tobacco mosaic virus was studied by equilibrium dialysis. The amount of dodecyl sulfate bound to the cucumber virus protein in 0.1 m phosphate buffer (pH 7.2) was found to be 1.55 g/g, which was the same value as that obtained with the tobacco virus protein. The presence of 8 m urea markedly decreased the degree of binding of dodecyl sulfate to the proteins. The amount of binding to the cucumber virus protein was reduced to 0.56 g/g, and that to the tobacco virus protein decreased to 0.8 g/g. The net charges of both proteins were negative at neutral pH and the amount of negative charge of the cucumber virus protein, obtained from the potentiometric titration curves, was larger than that of the tobacco virus protein, either in the native state or in the denatured state. In dodecyl sulfate/polyacrylamide gel electrophoresis the cucumber virus protein migrated faster than the tobacco virus protein. On the other hand, in the presence of 8 m urea, the electrophoretic migration rate of the cucumber virus protein was equal to that of the tobacco virus protein. Sedimentation equilibrium experiments in 6 m guanidinium chloride gave molecular weights of 17,700 and 17,200 for the tobacco mosaic virus and the cucumber virus proteins, respectively. These results suggest that the effective negative charge density of the cucumber virus protein-dodecyl sulfate complex is higher than that of the tobacco virus proteindodecyl sulfate complex in 0.1% dodecyl sulfate solution. The conformation of both proteins was investigated by circular dichroism measurements. Both proteins have a slightly higher degree of α-helix content in dodecyl sulfate solution than in the native state. The addition of 8 m urea to both proteins while in this solution induced a change in conformation to one having a much smaller degree of ordered structure, although the change in the cucumber virus protein was more intense than that in the tobacco virus protein.