Expressing an (E)-β-farnesene synthase in the chloroplast of tobacco affects the preference of green peach aphid and its parasitoid

(E)-β-Famesene(EβF) synthase catalyses the production of EβF,which for many aphids is the main or only component of the alarm pheromone causing the repellence of aphids and also functions as a kairomone for aphids' natural enemies.Many plants possess EβF synthase genes and can release EβF to repel aphids.In order to effectively recruit the plant-derived EβF synthase genes for aphid control,by using chloroplast transit peptide(CTP) of the small subunit of Rubisco(rbcS) from wheat(Triticum aestivum L.),we targeted AαβFS1,an EβF synthase gene from sweet wormwood(Artemisia annua L),to the chloroplast of tobacco to generate CTP + AαβFS1 transgenic lines.The CTP +AαβFS1 transgenic tobacco plants could emit EβF at a level up to 19.25 ng/day per g fresh tissues,4-12 fold higher than the AαβFS1 transgenic lines without chloroplast targeting.Furthermore,aphid/parasitoid behavioral bioassays demonstrated that the CTP + AαβFS1 transgenic tobacco showed enhanced repellence to green peach aphid(Myzus persicae) and attracted response of its parasitoid Diaeretiella rapae,thus affecting aphid infestation at two trophic levels.These data suggest that the chloroplast is an ideal subcellular compartment for metabolic engineering of plant-derived EβF synthase genes to generate a novel type of transgenic plant emitting an alarm pheromone for aphid control.     

N′-Isopropylnornicotine in Burley Tobacco (Nicotiana tabacum)

Allyl sulfides such as diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS), typical flavor components of Allium vegetables, have been shown to inhibit benzo[a]pyrene (B[a]P)-induced carcinogenesis in animal models. As a possible mechanism of this inhibition, the effect of these volatile substances on cytochrome P450 (CYP)1 (CYP1A1, 1A2 and 1B1)-mediated bioactivation of B[a]P was investigated using a human hepatoma cell model (HepG2). DADS and DATS inhibited the B[a]P-induced ethoxyresorufin O-deethylase (EROD) activity, a marker enzyme for CYP1, by 30-90% and 70-95% at 100-1,000 μM concentration, respectively. The cell viability, an indicator of the capacity to inhibit B[a]P bioactivation, was increased by treatments of 100-1,000 μM DADS and 10-100 μM DATS. Immunoblot results indicated that the B[a]P inducible CYP1A2 protein was suppressed by 100-1,000 μM of DADS and 10-100 μM of DATS, but CYP1A1 and 1B1 were not detectable in any microsomes. Analysis of B[a]P metabolites revealed that the level of 7,8-diol formed was significantly reduced in the DADS and DATS treated microsomes as compared to the control. The level of 9,10-diol and 4,5-diol formed was also lowered by the allyl sulfide treatments. These results suggest that the protective mechanism of allyl sulfides on B[a]P-induced carcinogenesis is possibly related with the modulation of CYP1-mediated bioactivation of B[a]P.     

Mapping Rm2 gene conferring resistance to the green peach aphid (Myzus persicae Sulzer) in the peach cultivar ��Rubira?��

The green peach aphid (GPA), Myzus persicae (Sulzer), is a widespread pest insect that significantly reduces yield in peach orchards [Prunus persica (L.) Batsch]. Chemical control of the GPA population in the orchards showed little efficiency because of the development of resistance to most classes of insecticides. Biological control partially gave convincing results. Breeding for resistant peach cultivars is therefore a serious option to take into account for the development of sustainable pest management. Among the few available resistance cultivars, the rootstock peach “Rubira?” shows a strong induced antixenosis-type GPA resistance. This was demonstrated segregating as a single dominant gene. In order to investigate the genetic basis of resistance and develop molecular tools useful in breeding programs, a F₂ population derived from “Rubira?” also segregating for leaf color was grown and scored for GPA resistance under contrasted environmental conditions. An SSR-based genetic linkage map composed of 120 SSR loci spanned over a distance of 497.8 cM was then established. The GPA resistance mapped to a single locus at the bottom end of linkage group 1. We propose to name Rm2 the dominant allele of the underlying gene. Additionally, a reciprocal translocation was identified near the Gr gene controlling leaf color. The red-leaf parent “Rubira?” was demonstrated responsible for the translocation. This study provides the basis for future molecular analysis for the use of Rm2 in peach breeding programs against GPA in peach orchards.     

Unexpected effects of chitinases on the peach-potato aphid (Myzus persicae Sulzer) when delivered via transgenic potato plants (Solanum tuberosum Linné) and in vitro

With the aim of producing insect-resistant potato plants, internode explants of Solanum tuberosum L. cv. Désirée were transformed with an Agrobacterium strain C58pMP90 containing an insect (Phaedon cochleariae: Coleoptera, Chrysomelidae) chitinase gene and the neomycin phosphotransferase (nptII) gene as selectable marker, both under the control of the viral CaMV 35S promoter. Three transformed potato lines (CH3, CH5 and CH25) exhibiting the highest chitinolytic activities were selected for feeding experiments with the peach-potato aphid, Myzus persicae (Sulzer), under controlled photoperiod and temperature conditions. Aphids fed on transgenic potato plants showed a reduced pre-reproductive period and an enhanced daily fecundity. Transgenic potato lines did not affect nymphal mortality, but improved several biological parameters related to aphid populations growth. Artificial diets were used to provide active (1, 10, 100 and 500 g ml^–1) and inactive (500 g ml^–1) bacterial (Serratia marcescens) chitinase to M. persicae. These compounds increased nymph survival at all active chitinase doses when compared to the control diet, while inactive chitinase did not. Although the pre-reproductive period was slightly shortened and the daily fecundity slightly higher, active and inactive chitinase provided as food led a reduction from 1 to 1.5 day populations doubling time. Therefore chitinase activity was responsible for the probiotic effects on aphids. Our results question the relevance of a chitinase-based strategy in the context of potato culture protection.     

Discrimination of alarm pheromone (E)-β-farnesene by aphid odorant-binding proteins

OBPs have been recently demonstrated to be required for odour perception in insects and directly involved in odour discrimination. In aphids they might represent new interesting targets for the control of their population in agriculture. Based on sequence information available in the EST database, we have cloned four genes encoding odorant-binding proteins (OBP) in Acyrthosiphon pisum and homologous genes in other aphid species. Unlike OBPs from other orders of insects, that are greatly divergent, in aphids these proteins have been found to be highly conserved, with differences between species limited to only few amino acid substitutions. On the contrary, similarities between OBP sequences of the same species are poor with 31% or less of identical amino acids. Three selected OBPs (OBP1, OBP3 and OBP8) have been expressed in bacteria and purified. Ligand-binding experiments have shown similar behaviour of the three proteins towards several organic compounds, but also some significant selectivities. In particular, (E)-β-farnesene, the alarm pheromone and its related compound farnesol exhibited good affinity to OBP3, but did not bind the other two proteins. We suggest that OBP3 could mediate response of aphids to the alarm pheromone.     

Occurrence of Rachiplusia nu (Guenée) (Lepidoptera: Noctuidae) on tobacco (Nicotiana tabacum L.) in Rio Grande do Sul, Brazil

The occurrence of Rachiplusia nu (Guenée) is registered for the first time in tobacco [Nicotiana tabacum L. (Solanaceae)]. The specimens were collected in a Virginia tobacco field, in Venancio Aires, State of Rio Grande do Sul. Caterpillars occurred in large infestations, causing important damage to tobacco leaves, and demanding chemical control to minimize losses. This occurrence indicates that this species can feed on Virginia tobacco, which corresponds to 80% of the tobacco area of Rio Grande do Sul, and so presents high risk to become an important pest in tobacco fields.     

Enlarged euchromatic chromosomes (“megachromosomes”) in hybrids between Nicotiana tabacum and N. plumbaginifolia

The gigantic chromosomes (megachromosomes) described previously as occurring spontaneously in hybrid combinations between N. tabacum and species of the Tomentosae section of Nicotiana were due to an enlargement of heterochromatic segments introduced from the latter into a N. tabacum background. Only chromosomes with large heterochromatic segments became megachromosomes and the enlarged parts themselves showed at interphase and prophase the intense staining characteristic of heterochromatin. Euchromatic arms of the same chromosomes did not undergo enlargement.In contrast, megachromosomes described here for N. tabacum x N. plumbaginifolia hybrids originate from chromosomes which have no heterochromatic blocks. These megachromosomes are not recognizable at interphase and when distinguished at prophase are found to be stained lightly like the rest of the euchromatin.The mode of origin of megachromosomes is still unknown. Spontaneous chromosome breakage is frequent in all hybrids in which megachromosomes are found and is probably associated in some way with their formation, but an origin of megachromosomes by breakage and end-to-end fusion of broken strands is unlikely. This leaves as a possibility an origin by repeated replication from the same template.Other examples of very large chromosomes with characteristics of megachromosomes found in the literature are briefly discussed. They all arose in atypical situations of interspecific hydribization, exposure to mutagens or in tumors and cell cultures.Paper number 4748 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, North Carolina     

The Sesquiterpenes(E)-?-Farnesene and (E)-α-Bergamotene Quench Ozone but Fail to Protect the Wild Tobacco Nicotiana attenuata from Ozone,UVB, and Drought Stresses

Among the terpenes, isoprene (C₅) and monoterpene hydrocarbons (C₁₀) have been shown to ameliorate abiotic stress in a number of plant species via two proposed mechanisms: membrane stabilization and direct antioxidant effects. Sesquiterpene hydrocarbons (C₁₅) not only share the structural properties thought to lend protective qualities to isoprene and monoterpene hydrocarbons, but also react rapidly with ozone, suggesting that sesquiterpenes may similarly enhance tolerance of abiotic stresses. To test whether sesquiterpenes protect plants against ozone, UVB light, or drought, we used transgenic lines of the wild tobacco Nicotiana attenuata. The transgenic plants expressed a maize terpene synthase gene (ZmTPS10) which produced a blend of (E)-ß-farnesene and (E)-α-bergamotene, or a point mutant of the same gene (ZmTPS10M) which produced (E)-ß-farnesene alone,. (E)-ß-farnesene exerted a local, external, and transient ozone-quenching effect in ozone-fumigated chambers, but we found no evidence that enhanced sesquiterpene production by the plant inhibited oxidative damage, or maintained photosynthetic function or plant fitness under acute or chronic stress. Although the sesquiterpenes (E)-ß-farnesene and (E)-α-bergamotene might confer benefits under intermittent heat stress, which was not tested, any roles in relieving abiotic stress may be secondary to their previously demonstrated functions in biotic interactions.     

Dynamic changes in photosynthesis and chlorophyll fluorescence in Nicotiana tabacum infested by Bemisia tabaci (Middle East–Asia Minor 1) nymphs

Bemisia tabaci Middle East–Asia Minor 1 (MEAM1) is a worldwide pest. To determine whether MEAM1 nymphs produce the same symptoms in different host plants, we measured the plant growth and chlorophyll content of tobacco and cotton plants that were infested by MEAM1 nymphs. Furthermore, to investigate the spatial and temporal changes in photosynthesis caused by MEAM1 nymphs, the net photosynthetic rate (Pn) and chlorophyll a fluorescence of local and systemic tobacco leaves were assayed at 8, 11, 14, and 20 days after MEAM1 adult removal, which represent the stages of 1st, 2nd, 3rd, and 4th instar nymphs, respectively. The results showed that MEAM1 nymph infestation reduced the plant height and internode length of tobacco at 14 and 20 days, as well as the dry weight of infested and systemic tobacco leaves. However, MEAM1 nymph infestation did not affect the plant height or internode length of cotton. Also, the dry weight and chlorophyll and carotenoid content of infested and systemic leaves of cotton plants were not influenced by MEAM1 nymph infestation. However, the contents of chlorophyll a and b and carotenoids in infested tobacco leaves decreased over time; the chlorophyll a content of systemic tobacco leaves decreased at 11, 14, and 20 days. The chlorophyll and carotenoid contents in infested and systemic leaves of cotton plants were not influenced by MEAM1 nymph infestation. In addition, the Pn of infested tobacco leaves decreased at 14 and 20 days, while the Pn in systemic tobacco leaves decreased after 11 days. The greatest decrease in performance index on absorption basis (PI _ABS ) of infested and systemic tobacco leaves occurred on day 14. The fluorescence intensity at 2 ms (J peak) and 300 μs (K peak) increased on day 14, which indicates that 3rd instar nymphs caused serious damage to the primary photochemical reactions and donor side of PSII. These results suggest that MEAM1 nymph infestation had different effects on tobacco and cotton plants. The infestation caused spatial and temporal changes in photosynthesis in tobacco plants. The lower chlorophyll a content may have been related to the lower net photosynthetic rate of systemic and infested tobacco leaves. The decreased stability of the oxygen-evolving complex and the reaction center of PSII and the decrease in electron transport were the main reasons for the decrease in the level of photosynthesis in tobacco leaves caused by MEAM1 nymphs during various stages of infestation.     

Cell-free protein synthesis of membrane (1,3)-β-d-glucan (curdlan) synthase: Co-translational insertion in liposomes and reconstitution in nanodiscs

A membrane-embedded curdlan synthase (CrdS) from Agrobacterium is believed to catalyse a repetitive addition of glucosyl residues from UDP-glucose to produce the (1,3)-β-d-glucan (curdlan) polymer. We report wheat germ cell-free protein synthesis (WG-CFPS) of full-length CrdS containing a 6xHis affinity tag and either Factor Xa or Tobacco Etch Virus proteolytic sites, using a variety of hydrophobic membrane-mimicking environments. Full-length CrdS was synthesised with no variations in primary structure, following analysis of tryptic fragments by MALDI-TOF/TOF Mass Spectrometry. Preparative scale WG-CFPS in dialysis mode with Brij-58 yielded CrdS in mg/ml quantities. Analysis of structural and functional properties of CrdS during protein synthesis showed that CrdS was co-translationally inserted in DMPC liposomes during WG-CFPS, and these liposomes could be purified in a single step by density gradient floatation. Incorporated CrdS exhibited a random orientation topology. Following affinity purification of CrdS, the protein was reconstituted in nanodiscs with Escherichia coli lipids or POPC and a membrane scaffold protein MSP1E3D1. CrdS nanodiscs were characterised by small-angle X-ray scattering using synchrotron radiation and the data obtained were consistent with insertion of CrdS into bilayers. We found CrdS synthesised in the presence of the Ac-AAAAAAD surfactant peptide or co-translationally inserted in liposomes made from E. coli lipids to be catalytically competent. Conversely, CrdS synthesised with only Brij-58 was inactive. Our findings pave the way for future structural studies of this industrially important catalytic membrane protein.     

(E,E)-α-Farnesene as a host-induced plant volatile that attracts Apanteles taragamae (Hymenoptera: Braconidae) to host-infested cucumber plants

In tritrophic interactions between cucumber plants, the cucumber moth Diaphania indica Saunders (Lepidoptera: Crambidae) and a larval parasitoid Apanteles taragamae Viereck (Hymenoptera: Braconidae), female A. taragamae may use herbivore-induced plant volatiles (HIPVs) to locate their host. However, the specific compound or blend of chemicals attracting A. taragamae remains unknown. In this study, differences in volatiles released from uninfested, mechanically damaged and host-infested cucumber plants were examined by the headspace volatile collection method. Responses of the larval parasitoid A. taragamae to the volatile extracts were examined in a four-arm olfactometer. We also investigated the attraction of female A. taragamae to a single compound identified as an HIPV from host-infested cucumber plants. Parasitoids discriminated between the volatiles from uninfested, host-infested and mechanically damaged plants. Chemical analysis of headspace volatiles from host-infested cucumber plants showed that (E,E)-α-farnesene was released as a major component (73.1%). When (E,E)-α-farnesene was tested alone in the range of 1.7–170?ng, female parasitoids responded to 17?ng only. Therefore, tritrophic interactions between A. taragamae and D. indica appear to be partly mediated by (E,E)-α-farnesene.     

Comparison of short- and long-feed transmission of the cauliflower mosaic virus Cabb-S strain and SΔII hybrid by two species of aphid: Myzus persicae (Sulzer) and Brevicoryne brassicae (L.)

The cauliflower mosaic virus (CaMV) hybrid SΔII, partially deleted in ORFII, loses its transmissibility by the aphid Myzus persicae on 5-min acquisition feed. We have also shown that it is not transmitted after 8-h acquisition feed. The same occurs with Brevicoryne brassicae. Therefore, the aphid transmission factor (ATF) is involved in both means of transmission and in both aphid species. M. persicae can acquire CaMV Cabb-S strain in less than 20 s. M. persicae is a more efficient vector during a short feed than during a long feed, contrary to B. brassicae which transmits better during a long feed.     

The location of (1→3)-β-glucans in the walls of pollen tubes of Nicotiana alata using a (1→3)-β-glucan-specific monoclonal antibody

The location of the (13)--glucan, callose, in the walls of pollen tubes in the style of Nicotiana alata Link et Otto was studied using specific monoclonal antibodies. The antibodies were raised against a laminarinhaemocyanin conjugate. One antibody selected for further characterization was specific for (13)--glucans and showed no binding activity against either a cellopentaose-bovine serum albumin (BSA) conjugate or a (13, 14)--glucan-BSA conjugate. Binding was inhibited by (13)--oligoglucosides (DP, 3–6) with maximum competition being shown by laminaripentaose and laminarihexaose, indicating that the epitope included at least five (13)--linked glucopyranose residues. The monoclonal antibody was determined to have an affinity constant for laminarihexaose of 2.7. 10⁴M^–1. When used with a second-stage gold-labelled, rabbit anti-mouse antibody, the monoclonal antibody probe specifically located the (13)--glucan in the inner wall layer of thin sections of the N. alata pollen tubes.Abbreviations BSA bovine serum albumin - PBS phosphate-buffered saline - ELISA enzyme linked immunosorbent assay - DP degree of polymerization - PVC polyvinyl chloride P.J.M. is an Australian Postdoctoral Research Fellow. We wish to thank Joan Hoogenraad for her technical assistance with the tissue culture, and Althea Wright for her assistance in the preparation of this paper.     

2′-(E)-O-p-coumaroylgalactaric acid and 2′-(E)-O-feruloylgalactaric acid in citrus

2′-(E)-O-p-Coumaroyl- and 2′-(E)-O-feruloylgalactaric acids, hitherto unknown in nature, have been isolated and identified from orange peel.