Physical interaction between the strawberry allergen Fra a 1 and an associated partner FaAP: Interaction of Fra a 1 proteins and FaAP

The strawberry fruit allergens Fra a 1.01E, Fra a 1.02 and Fra a 1.03 belong to the group of pathogenesis‐related 10 (PR‐10) proteins and are homologs of the major birch pollen Bet v 1 and apple allergen Mal d 1. Bet v 1 related proteins are the most extensively studied allergens but their physiological function in planta remains elusive. Since Mal d 1‐Associated Protein has been previously identified as interaction partner of Mal d 1 we studied the binding of the orthologous Fra a 1‐Associated Protein (FaAP) to Fra a 1.01E/1.02/1.03. As the C‐terminal sequence of FaAP showed strong auto‐activation activity in yeast 2‐hybrid analysis a novel time resolved DNA‐switching system was successfully applied. Fra a 1.01E, Fra a 1.02, and Fra a 1.03 bind to FaAP with K_D of 4.5 ± 1.1, 15 ± 3, and 11 ± 2 nM, respectively. Fra a 1.01E forms a dimer, whereas Fra a 1.02 and Fra a 1.03 bind as monomer. The results imply that PR‐10 proteins might be integrated into a protein‐interaction network and FaAP binding appears to be essential for the physiological function of the Fra a 1 proteins.     

Solution structure of PcFK1, a spider peptide active against Plasmodium falciparum

Psalmopeotoxin I (PcFK1) is a 33-amino-acid residue peptide isolated from the venom of the tarantula Psalmopoeus cambridgei. It has been recently shown to possess strong antiplasmodial activity against the intra-erythrocyte stage of Plasmodium falciparum in vitro. Although the molecular target for PcFK1 is not yet determined, this peptide does not lyse erythrocytes, is not cytotoxic to nucleated mammalian cells, and does not inhibit neuromuscular function. We investigated the structural properties of PcFK1 to help understand the unique mechanism of action of this peptide and to enhance its utility as a lead compound for rational development of new antimalarial drugs. In this paper, we have determined the three-dimensional solution structure by (1)H two-dimensional NMR means of recombinant PcFK1, which is shown to belong to the ICK structural superfamily with structural determinants common to several neurotoxins acting as ion channels effectors.     

Direct adventitious shoot formation on seedling radicles in seed cultures of strawberry

Mature seeds of strawberry (Fragaria x ananassa) were placed on Murashige and Skoog medium supplemented with 2.22 μM 6-benzyladenine. After four weeks of culture, and without an intervening callus phase, approximately 36% of the resulting seedling radicles had formed numerous adventitious buds near their tips. A few buds on each radicle developed into shoots, while others formed disorganized calli. Consequently, the seedlings exhibited shoot apices at both ends of the axis of polarity. Our overall results suggest that a considerable level of plasticity in organ determination occurs even in higher plants, and that exogenous growth regulators can cause a root primordium in the radicle to be converted to a shoot primordium.     

Pyr c 1, the major allergen from pear (Pyrus communis), is a new member of the Bet v 1 allergen family

Pear is known as an allergenic food involved in the ‘oral allergy syndrome’ which affects a high percentage of patients allergic to birch pollen. The aim of this study was to clone the major allergen of this fruit, to express it as bacterial recombinant protein and to study its allergenic properties in relation to homologous proteins and natural allergen extracts. The coding region of the cDNA was obtained by a PCR strategy, cloned, and the allergen was expressed as His-Tag fusion protein. The fusion peptide was removed by treatment with cyanogen bromide. Purified non-fusion protein was subjected to allergenicity testing by the enzyme allergosorbent test (EAST), Western blotting, competitive inhibition assays, and basophil histamine release. The deduced protein sequence shared a high degree of identity with other major allergens from fruits, nuts, vegetables, and pollen, and with a family of PR-10 pathogenesis related proteins. The recombinant (r) protein was recognised by specific IgE from sera of all pear-allergic patients (n=16) investigated in this study. Hence, the allergen was classified as a major allergen and named Pyr c 1. The IgE binding characteristics of rPyr c 1 appeared to be similar to the natural pear protein, as was demonstrated by EAST-inhibition and Western blot-inhibition experiments. Moreover, the biological activity of rPyr c 1 was equal to that of pear extract, as indicated by basophil histamine release in two patients allergic to pears. The related major allergens Bet v 1 from birch pollen and Mal d 1 from apple inhibited to a high degree the binding of IgE to Pyr c 1, whereas Api g 1 from celery, also belonging to this family, had little inhibitory effects, indicating epitope differences between Bet v 1-related food allergens. Unlimited amounts of pure rPyr c 1 are now available for studies on the structure and epitopes of pollen-related food allergens. Moreover, the allergen may serve as stable and standardised diagnostic material.     

Short-term studies of 15NO3 - and 15NH4 + uptake by micropropagated strawberry shoots cultured with or without CO2 enrichment

The uptake of ¹⁵NO₃ ^- and ¹⁵NH₄ ⁺ has been examined in 5-,10- and 28-day-old micropropagated strawberry (Fragaria x ananassa Duch. cv. Kent) shoots rooted in one-half strength Murashige and Skoog (MS) liquid medium on cellulose plugs (Sorbarods). The results indicated that the plantlets absorbed both NO₃ ^- and NH₄ ⁺ during the culture with a greater uptake of NH₄ ⁺ at 5 days of culture. Furthermore, a pronounced reduction in NO₃ ^- and NH₄ ⁺ uptake at 10 and 28 days of culture was observed within 6 h of the short-term uptake study. This reduction could be explained by the low CO₂ concentration in test tubes during the photoperiod, since no reduction in nitrogen uptake occurred in the CO₂ enriched condition. The results are interpreted as an indication of the important role for photosynthetic CO₂ fixation in the process of nitrogen uptake by the plantlets during the rooting stage.Contribution No. CRH 82, Centre de Recherche en Horticulture, F.S.A.A., Université Laval, Québec.     

Solution structure of HI1506, a novel two-domain protein from Haemophilus influenzae

HI1506 is a 128-residue hypothetical protein of unknown function from Haemophilus influenzae. It was originally annotated as a shorter 85-residue protein, but a more detailed sequence analysis conducted in our laboratory revealed that the full-length protein has an additional 43 residues on the C terminus, corresponding with a region initially ascribed to HI1507. As part of a larger effort to understand the functions of hypothetical proteins from Gram-negative bacteria, and H. influenzae in particular, we report here the three-dimensional solution NMR structure for the corrected full-length HI1506 protein. The structure consists of two well-defined domains, an alpha/beta 50-residue N-domain and a 3-alpha 32-residue C-domain, separated by an unstructured 30-residue linker. Both domains have positively charged surface patches and weak structural homology with folds that are associated with RNA binding, suggesting a possible functional role in binding distal nucleic acid sites.     

Solution structure of Escherichia coli Par10: The prototypic member of the Parvulin family of peptidyl-prolyl cis/trans isomerases

E. coli Par10 is a peptidyl-prolyl cis/trans isomerase (PPIase) from Escherichia coli catalyzing the isomerization of Xaa-Pro bonds in oligopeptides with a broad substrate specificity. The structure of E. coli Par10 has been determined by multidimensional solution-state NMR spectroscopy based on 1207 conformational constraints (1067 NOE-derived distances, 42 vicinal coupling-constant restraints, 30 hydrogen-bond restraints, and 68 phi/psi restraints derived from the Chemical Shift Index). Simulated-annealing calculations with the program ARIA and subsequent refinement with XPLOR yielded a set of 18 convergent structures with an average backbone RMSD from mean atomic coordinates of 0.50 A within the well-defined secondary structure elements. E. coli Par10 is the smallest known PPIase so far, with a high catalytic efficiency comparable to that of FKBPs and cyclophilins. The secondary structure of E. coli Par10 consists of four helical regions and a four-stranded antiparallel beta-sheet. The N terminus forms a beta-strand, followed by a large stretch comprising three alpha-helices. A loop region containing a short beta-strand separates these helices from a fourth alpha-helix. The C terminus consists of two more beta-strands completing the four-stranded anti-parallel beta-sheet with strand order 2143. Interestingly, the third beta-strand includes a Gly-Pro cis peptide bond. The curved beta-strand forms a hydrophobic binding pocket together with alpha-helix 4, which also contains a number of highly conserved residues. The three-dimensional structure of Par10 closely resembles that of the human proteins hPin1 and hPar14 and the plant protein Pin1At, belonging to the same family of highly homologous proteins.     

Solution structure of Vibrio cholerae protein VC0424: a variation of the ferredoxin-like fold

The structure of Vibrio cholerae protein VC0424 was determined by NMR spectroscopy. VC0424 belongs to a conserved family of bacterial proteins of unknown function (COG 3076). The structure has an alpha-beta sandwich architecture consisting of two layers: a four-stranded antiparallel beta-sheet and three side-by-side alpha-helices. The secondary structure elements have the order alphabetaalphabetabetaalphabeta along the sequence. This fold is the same as the ferredoxin-like fold, except with an additional long N-terminal helix, making it a variation on this common motif. A cluster of conserved surface residues on the beta-sheet side of the protein forms a pocket that may be important for the biological function of this conserved family of proteins.     

Molecular investigations of pathogenesis-related Bet v 1 homologues in Passiflora (Passifloraceae)

The major birch pollen allergen, Bet v 1, responsible for allergic reactions in many areas of the world, is homologous to a large number of pathogenesis-related proteins (PRs), identified as PR10. As part of a long-range investigation of these types of proteins and of evolution in Passiflora,DNA sequences from eight Bet v 1 homologue isoforms were obtained from five species of this genus in Brazil, and their sequences compared among themselves and with 30 others from 8 different species, classified in different taxonomic units. The objective was a first characterization of these PRs in wild passionflowers, and their use for evolutionary and applied investigations. High interspecific, but low intraspecific variability was observed, as expected from multigenic families subjected to concerted evolution. The relationships obtained both within Passiflora and between it and seven other genera probably best reflect functional similarities than evolutionary history.     

Localization of strawberry (Fragaria x ananassa) and Methylobacterium extorquens genes of strawberry flavor biosynthesis in strawberry tissue by in situ hybridization

Strawberry flavor is one of the most popular fruit flavors worldwide, with numerous applications in the food industry. In addition, the biosynthetic origin of the most important strawberry flavor components, such as 2,5-dimethyl-4-hydroxy-2H-furan-3-one (DMHF), is a challenging research area. DMHF's precursor, 2-hydroxy-propanal (or lactaldehyde), is biosynthesized by the endophytic bacterium Methylobacterium extorquens (M. extorquens). In particular, the alcohol dehydrogenase (ADH) enzymes of M. extorquens are involved in the biogenesis of DMHF precursors since they have the capacity to oxidize the strawberry-derived 1,2-propanediol to lactaldehyde. In this study, the expression of the endophytic ADH and the plant DMHF biosynthesis genes was examined in the tissues of raw and ripe strawberry receptacles by in situ hybridization. The presence of endophytic bacteria was studied in the same tissues by probes targeting bacterial 16S ribosomal ribonucleic acid. Hybridization signals of probes specific for endophytic ADH and plant DMHF biosynthesis genes, as well as bacteria-specific probes, were detected in the same locations. The probes were localized near the plasma membranes or intercellular spaces of cortical and vascular tissues of the receptacle, and intracellularly in the tissues of achenes. By localizing the expression of the endophytic methanol ADH and plant DMHF biosynthesis genes to the same tissues, we have reinforced our original hypothesis that an intimate symbiotic relationship between strawberry and endophytic cells exists and leads to the biosynthesis of DMHF.     

A novel member of the split betaalphabeta fold: Solution structure of the hypothetical protein YML108W from Saccharomyces cerevisiae

As part of the Northeast Structural Genomics Consortium pilot project focused on small eukaryotic proteins and protein domains, we have determined the NMR structure of the protein encoded by ORF YML108W from Saccharomyces cerevisiae. YML108W belongs to one of the numerous structural proteomics targets whose biological function is unknown. Moreover, this protein does not have sequence similarity to any other protein. The NMR structure of YML108W consists of a four-stranded beta-sheet with strand order 2143 and two alpha-helices, with an overall topology of betabetaalphabetabetaalpha. Strand beta1 runs parallel to beta4, and beta2:beta1 and beta4:beta3 pairs are arranged in an antiparallel fashion. Although this fold belongs to the split betaalphabeta family, it appears to be unique among this family; it is a novel arrangement of secondary structure, thereby expanding the universe of protein folds.     

X-ray structure of Na-ASP-2, a pathogenesis-related-1 protein from the nematode parasite, Necator americanus, and a vaccine antigen for human hookworm infection

Human hookworm infection is a major cause of anemia and malnutrition of adults and children in the developing world. As part of on-going efforts to control hookworm infection, The Human Hookworm Vaccine Initiative has identified candidate vaccine antigens from the infective L3 larval stages of the parasite, including a family of pathogenesis-related (PR) proteins known as the Ancylostoma-secreted proteins (ASPs). A novel crystal structure of Na-ASP-2, a PR-1 protein secreted by infective larvae of the human hookworm Necator americanus, has been solved to resolution limits of 1.68 A and to an R-factor of 17% using the recombinant protein expressed in and secreted by Pichia pastoris. The overall fold of Na-ASP-2 is a three-layer alphabetaalpha sandwich flanked by an N-terminal loop and a short, cysteine-rich C terminus. Our structure reveals a large central cavity that is flanked by His129 and Glu106, two residues that are well conserved in all parasitic nematode L3 ASPs. Na-ASP-2 has structural and charge similarities to chemokines, which suggests that Na-ASP-2 may be an extra-cellular ligand of an unknown receptor. Na-ASP-2 is a useful homology model for NIF, a natural antagonistic ligand of CR3 receptor. From these modeling studies, possible binding modes were predicted. In addition, this first structure of a PR-1 protein from parasitic helminths may shed light on the molecular basis of host-parasite interactions.     

Solution structure of the rhodanese homology domain At4g01050(175-295) from Arabidopsis thaliana

The three-dimensional structure of the rhodanese homology domain At4g01050(175-195) from Arabidopsis thaliana has been determined by solution nuclear magnetic resonance methods based on 3043 upper distance limits derived from NOE intensities measured in three-dimensional NOESY spectra. The structure shows a backbone root mean square deviation to the mean coordinates of 0.43 A for the structured residues 7-125. The fold consists of a central parallel beta-sheet with five strands in the order 1-5-4-2-3 and arranged in the conventional counterclockwise twist, and helices packing against each side of the beta-sheet. Comparison with the sequences of other proteins with a rhodanese homology domain in Arabidopsis thaliana indicated residues that could play an important role in the scaffold of the rhodanese homology domain. Finally, a three-dimensional structure comparison of the present noncatalytic rhodanese homology domain with the noncatalytic rhodanese domains of sulfurtransferases from other organisms discloses differences in the length and conformation of loops that could throw light on the role of the noncatalytic rhodanese domain in sulfurtransferases.     

Anthocyanin from strawberry (Fragaria ananassa) with the novel aglycone, 5-carboxypyranopelargonidin

An anthocyanin, 1, with the novel 4-substituted aglycone, 5-carboxypyranopelargonidin, was isolated in small amounts from the acidified, methanolic extract of strawberries, Fragaria ananassa Duch., by preparative HPLC after purification by partition against ethyl acetate, Amberlite XAD-7 and Sephadex LH-20 column chromatography. It was identified mainly by 2D NMR spectroscopy and electrospray LC-MS as the 3-O-beta-glucopyranoside of 5-carboxy-2-(4-hydroxyphenyl)-3,8-dihydroxy-pyrano[4,3,2-de]-1-benzopyrylium, an anthocyanidin which is homologous to 5-carboxypyranomalvidin (vitisidin A) reported in red wines and 5-carboxypyranocyanidin recently isolated from red onions. By comparison of UV-Vis absorption spectra, 1 showed in contrast to 2, pelargonidin 3-O-beta-glucopyranoside, a local absorption peak around 360 nm, a hypsochromic shift (8 nm) of the visible absorption maximum, and lack of a distinct UV absorption peak around 280 nm. The similarities between the absorption spectra of 1 in various acidic and neutral buffer solutions implied restricted formation of the instable colourless equilibrium forms, which are typical for most anthocyanins in weakly acidic solutions. The molar absorptivity (epsilon) of 1 varied little with pH contrary to similar values of for instance the major anthocyanin in strawberry, 2. However, 2 revealed higher epsilon-values than 1 at all pH values except 5.1. At pH 5.1, the epsilon-value of 1 (6250) was nearly four times the corresponding value of 2 (1720), which showed the potential of 5-carboxypyranopelargonidin derivatives as colorants in solutions with pH around 5. The colours of 1 and 2 in buffered solutions with pH 1.1 and pH 6.9 have been described by the CIELAB coordinates h(ab) (hue angle), C* (chroma), and L* (lightness).     

Responses of strawberry fruit to 1-Methylcyclopropene (1-MCP) and ethylene

1-Methylcyclopropene (1-MCP), a competitive inhibitorof ethylene action, binds to the ethylene receptor toregulate tissue responses to ethylene. In this work,we investigated the effects of 1-MCP and exogenousethylene on ripening, respiration rate, ionicconductivity and peroxidase activity in strawberryfruit. Strawberry fruit can ripen without exogenousethylene treatment, but exogenous ethylene inducessecondary ripening processes. Results indicated thatstimulation of respiration by ethylene wasdose-dependent. Fruit colour development and softeningwere slightly accelerated by ethylene, but changes insoluble solid content were not. 1-MCP may/may notaffect the respiratory rise induced by exogenousethylene dependent on fruit maturity. Cycloheximide(CHI) reduced the ethylene-induced respiratoryincrease. Combinations of 1-MCP and CHI reducedrespiration more than CHI alone. 1-MCP and CHI did notinfluence the primary respiratory change in nonethylene-treated fruit. This indicates that ethyleneinduced respiratory increase may involve an ethylenereceptor in early harvested fruit, but not in laterharvested fruit. Exogenous ethylene stimulatedrespiration by regulating new respiratory enzyme(s)synthesis in strawberry fruit. Ethylene induced anionic leakage increase, and this was positivelycorrelated to fruit water loss and peroxidaseactivity. These results suggest that non-climactericfruit, such as strawberry, may have different ethylenereceptor(s) and/or ethylene receptor(s) may havedifferent regulatory functions. It may be thesecondary effect of ethylene to stimulate respirationin strawberry. Non-climacteric fruit ripening may berelated to the development of active oxygen species(AOS) induced by postharvest stress.     

Biological control by Clonostachys rosea as a key component in the integrated management of strawberry gray mold

Gray mold, caused by Botrytis cinerea, is an important strawberry disease. As gray mold control is difficult, there is a need to evaluate integrated methods to successfully manage the disease. The efficiency of integrating Clonostachys rosea sprays, fungicide sprays, and crop debris removal to manage gray mold was evaluated in field experiments conducted in 2006 and 2007. Leaf colonization by C. rosea (LAC), average number of B. cinerea conidiophores (ANC), gray mold incidence in both flowers (Iflower) and fruits (Ifruit), and yield were evaluated weekly. In both years, LAC was higher in the treatments with no fungicide. When compared to the check, ANC, Iflower and Ifruit were most reduced in treatments that included C. rosea sprays. Maximal reductions were achieved with the combination of C. rosea sprays, fungicide sprays and debris removal (96.62%, 86.54% and 65.33% reductions of ANC, Iflower and Ifruit, respectively). Otherwise, maximal yield (103.14% increase as compared to the check) was achieved with the combination of the three treatments. With just C. rosea sprays, ANC, Iflower and Ifruit were reduced by 92.01%, 68.48% and 65.33%, respectively, whereas yield was increased by 75.15%. Considering the individual effects, application of C. rosea was the most efficient treatment. Chemical control was effective only in plots without debris removal. Elimination of crop debris was the least effective method in reducing gray mold incidence in both flowers and fruits. The integrated control approach enhanced the efficacy of the individual methods of gray mold control and provided high strawberry yield. An important component of this integrated approach it the biological control with C. rosea.     

A possible role for FRM-1, a C. elegans FERM family protein, in embryonic development

FRM-1 is a member of the FERM protein superfamily containing a FERM domain, which is a highly conserved protein-protein interaction module found in most eukaryotes. Although FRM-1 is thought to be involved in linking intracellular proteins to membrane proteins, the specific role for FRM-1 remains to be elucidated. In an effort to explore the biological function of FRM-1, we examined the phenotype of frm-1(tm4168) mutant worms. We observed that frm-1(tm4168) worms have a delayed hatching phenotype. Twelve hours after being laid, when virtually all wild-type eggs had hatched, only 64% of frm-1(tm4168) eggs had hatched. About 3% of frm-1(tm4168) eggs failed to hatch, even 3 days after they had been laid. We also found that frm-1(tm4168) mutants displayed a temperature-sensitive sterility phenotype. About 13% of frm-1(tm4168) worms were unable to produce eggs or produced nonviable eggs at 25°C. In contrast, less than 1% of wild-type animals were sterile at this temperature. At 20°C, neither the mutant nor wild type appeared to be sterile. Western blot analysis indicates that FRM-1 is expressed throughout the developmental stages with the strongest expression at the egg stage. Immunostaining experiments revealed that FRM-1 is mainly localized to the plasma membrane of most if not all cells at an early embryonic stage and to the plasma membrane of P cells during the late embryonic stages. GFP fusion experiments showed that FRM-1 can be expressed in the pharynx and intestine at the larval and adult stages. Our data suggest that FRM-1 may participate in diverse biological processes, including embryonic development.     

Solution structure of APETx2, a specific peptide inhibitor of ASIC3 proton-gated channels

Acid-sensing ion channels (ASIC) are proton-gated sodium channels that have been implicated in pain transduction associated with acidosis in inflamed or ischemic tissues. APETx2, a peptide toxin effector of ASIC3, has been purified from an extract of the sea anemone Anthopleura elegantissima. APETx2 is a 42-amino-acid peptide cross-linked by three disulfide bridges. Its three-dimensional structure, as determined by conventional two-dimensional 1H-NMR, consists of a compact disulfide-bonded core composed of a four-stranded beta-sheet. It belongs to the disulfide-rich all-beta structural family encompassing peptide toxins commonly found in animal venoms. The structural characteristics of APETx2 are compared with that of PcTx1, another effector of ASIC channels but specific to the ASIC1a subtype and to APETx1, a toxin structurally related to APETx2, which targets the HERG potassium channel. Structural comparisons, coupled with the analysis of the electrostatic characteristics of these various ion channel effectors, led us to suggest a putative channel interaction surface for APETx2, encompassing its N terminus together with the type I-beta turn connecting beta-strands III and IV. This basic surface (R31 and R17) is also rich in aromatic residues (Y16, F15, Y32, and F33). An additional region made of the type II'-beta turn connecting beta-strands I and II could also play a role in the specificity observed for these different ion effectors.     

Reduction of ribulose-1,5-bisphosphate carboxylase/oxygenase efficiency by the presence of sucrose during the tissue culture of strawberry plantlets

Summary In order to identify the physiological and biochemical events leading to the negative effects of the presence of sucrose in culture medium on the photosynthetic capacity of plantlets cultivated in vitro, time course in photosynthesis, metabolite pool sizes, and ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) activity were investigated in strawberry (Fragaria x ananassa Duch. cv. Kent) plantlets following their transfer to medium with or without sucrose. When the plantlets grown in medium without sucrose were transferred to a similar medium with 30 g liter⁻¹ sucrose, their net photosynthesis decreased and their level of phosphorylated compounds increased with time. In addition, initial catalytic turnover, total catalytic turnover, and the activation state of ribulose-1,5-bisphosphate carboxylase decreased in these plantlets. Conversely, when the plantlets grown in medium with 30 g liter⁻¹ sucrose were transferred to a similar medium without sucrose, their net photosynthesis slowly increased with time and their level of phosphorylated compounds slowly decreased. A slow increase with time of initial catalytic turnover, total catalytic turnover, and the activation state of ribulose-1,5-bisphosphate carboxylase was also observed in these plantlets. The results of the present paper suggest that the reduced photosynthetic capacity of strawberry plantlets cultivated in vitro in the presence of sucrose is the consequence of a reduction in the efficiency of ribulose-1,5-bisphosphate carboxylase/oxygenase due to its deactivation and the possible presence of putative inhibitors of carboxylation sites.