Antennae-enriched expression of candidate odorant degrading enzyme genes in the turnip aphid, Lipaphis erysimi.

Aphids heavily rely on their olfactory system for foraging behavior. Odorant-degrading enzymes (ODEs) are essential in preserving the olfactory acuity of aphids by removing redundant odorants in the antennae. Certain enzymes within this group stand out as being enriched and/or biased expressed in the antennae, such as carboxylesterases (CXEs), cytochrome P450 (CYPs), glutathione S-transferases (GSTs), and UDP-glycosyltransferases (UGTs). Here, we performed a comparative transcriptome analysis of antennae and body tissue to isolate the antennal ODE genes of turnip aphid Lipaphis erysimi. A dataset of one CXE, seven CYPs, two GSTs, and five UGTs enriched in the antennae was identified and subjected to sequence analysis. Furthermore, qRT-PCR analyses showed that 13 ODE genes (LeCXE6, LeCYP4c1, LeCYP6a2, LeCYP6a13, LeCYP6a14.2, LeCYP6k1, LeCYP18a1, LeGST1, LeUGT1-7, LeUGT2B7, LeUGT2B13, LeUGT2C1.1, and LeUGT2C1.2) were specifically or significantly elevated in antennal tissues. Among these antennae-enriched ODEs, LeCYP4c1, LeCYP6a2, LeCYP6a13, LeCYP6a14.2, LeCYP18a1, LeUGT2B7, and LeUGT2B13 were found to exhibit significantly higher expression levels in alate aphids compared to apterous and nymph aphids, suggesting their putative role in detecting new host plant location. The results presented in this study highlight the identification and expression of ODE genes in L. erysimi, paving the path to investigate their functional role in odorant degradation during the olfactory processes.

Candidate odorant-binding protein and chemosensory protein genes in the turnip aphid Lipaphis erysimi.

The turnip aphid, Lipaphis erysimi Kaltenbach, inflicts heavy damage on cruciferous crops worldwide. In these insects, olfactory perception is crucial for mating, host location, and oviposition. Both odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) are responsible for the delivery of host odorants and pheromones during initial molecular interactions. In this study, antennal and body transcriptomes of L. erysimi were generated through the deep sequencing of RNA libraries. A dataset of 11 LeryOBP and four LeryCSP transcripts was identified among assembled unigenes and subjected to sequence analysis. Phylogenetic analysis found a one-to-one orthologous relationship between LeryOBP/LeryCSP and its corresponding homologs from other aphid species. Further quantitative real-time PCR analyses across developmental stages and tissues showed that five LeryOBP genes (i.e., LeryGOBP, LeryOBP6, LeryOBP7, LeryOBP9, and LeryOBP13) and LeryCSP10 were specifically or significantly elevated in the antennae compared with other tissues. Moreover, two transcripts (i.e., LeryGOBP and LeryOBP6) exhibited remarkably higher expression levels in alate aphids, implying their potentially functional role in the perception of new host plant locations. These results present the identification and expression of OBP/CSP genes in L. erysimi, providing valuable insights into their putative role in olfactory signal transduction.

Sex-specific transcription and DNA methylation landscapes of the Asian citrus psyllid, a vector of huanglongbing pathogens.

The relationship of DNA methylation and sex-biased gene expression is of high interest, it allows research into mechanisms of sexual dimorphism and the development of potential novel strategies for insect pest control. The Asian citrus psyllid, Diaphorina citri Kuwayama, is a major vector for the causative agents of Huanglongbing (HLB), which presents an unparalleled challenge to citrus production worldwide. Here, we identify the X chromosome of D. citri and investigate differences in the transcription and DNA methylation landscapes between adult virgin males and females. We find a large number of male-biased genes on the autosomes and a depletion of such on the X chromosome. We have also characterized the methylome of D. citri, finding low genome-wide levels, which is unusual for an hemipteran species, as well as evidence for both promoter and TE methylation. Overall, DNA methylation profiles are similar between the sexes but with a small number of differentially methylated genes found to be involved in sex differentiation. There also appears to be no direct relationship between differential DNA methylation and differential gene expression. Our findings lay the groundwork for the development of novel epigenetic-based pest control methods, and given the similarity of the D. citri methylome to some other insect species, these methods could be applicable across agricultural insect pests.

Antennae-abundant expression of candidate cytochrome P450 genes associated with odorant degradation in the asian citrus psyllid, Diaphorina citri.

The Asian citrus psyllid, Diaphorina citri, is a notorious pest that is an efficient vector for Candidatus Liberibacter asiaticus (CLas), the causal agent of citrus huanglongbing (HLB). The olfactory system of insects is crucial for foraging and mating behavior. Antennae-abundant odorant degrading enzymes (ODEs), including cytochrome P450 (CYPs), are important in degrading redundant odorant molecules to recover the insect olfactory. In this study, to isolate the antennal CYP genes of D. citri, we generated four transcriptomes from female/male antennae and body through deep sequencing of RNA libraries. Seven DcCYP genes preferentially expressed in antennae were first identified by comparing the antennal and body transcriptomes. Phylogenetic analysis grouped four DcCYPs (DcCYP6a13, DcCYP6j1, DcCYP6k1, and DcCYP6a2) into the CYP3 class, whereas DcCYP4d2, DcCYP4c62, and DcCYP4d8 were clustered in the CYP4 clade. qRT-PCR analyses across developmental stages and tissues showed they were antennae-abundant in both genders and constantly expressed from the first instar nymph to the adult. The results presented here highlight the isolation and expression of CYP genes in D. citri antennae, providing valuable insights into their putative role in odorant degradation.

Identification of the extracellular metallo-endopeptidases ADAM and ADAMTS in the yellow fever mosquito Aedes aegypti.

The mosquito Aedes aegypti is a major vector for dengue, Zika, yellow fever, and chikungunya (CHIKV) viruses, which cause significant morbidity and mortality among human populations in the tropical regions of the world. Following ingestion of a viremic bloodmeal from a vertebrate host, an arbovirus needs to productively infect the midgut epithelium of the mosquito. De novo synthesized virions then exit the midgut by traversing the surrounding basal lamina (BL) in order to disseminate to secondary tissues and infect those. Once the salivary glands are infected, the virus is transmitted to a vertebrate host along with saliva released during probing of the mosquito. Midgut tissue distention due to bloodmeal ingestion leads to remodeling of the midgut structure and facilitates virus dissemination from the organ. Previously, we described the matrix-metalloproteinases (MMP) of Ae. aegypti as zinc ion dependent endopeptidases (Metzincins) and showed MMP activity during midgut BL rearrangement as a consequence of bloodmeal ingestion and subsequent digestion thereby affecting arbovirus dissemination from the midgut. Here we investigate the ADAM/ADAMTS of Ae. aegypti, which form another major group of multi-domain proteinases within the Metzincin superfamily and are active during extra-cellular matrix (ECM) remodeling. Seven different ADAM and five ADAMTS were identified in Ae. aegypti. The functional protein domain structures of the identified mosquito ADAM resembled those of human ADAM10, ADAM12, and ADAM17, while two of the five mosquito ADAMTS had human orthologs. Expression profiling of Ae. aegypti ADAM/ADAMTS in immature forms, whole body-females, midguts, and ovarian tissues showed transcriptional activity of the proteinases during metamorphosis, bloodmeal ingestion/digestion, and female reproduction. Custom-made antibodies to ADAM10a and ADAM12c showed that both were strongly expressed in midgut and ovarian tissues. Furthermore, transient silencing of ADAM12c significantly reduced the carcass infection rate with CHIKV at 24 h post-infection, while silencing of ADAM12a significantly increased viral titers in secondary tissues at the same time point. Our results indicate a functional specificity for several ADAM/ADAMTS in those selected mosquito tissues.

Integrated transcriptome sequencing and RNA interference reveals molecular changes in Diaphorina citri after exposure to validamycin.

Validamycin has been widely used as a specific competitive inhibitor of trehalase. In our previous research, validamycin significantly inhibited trehalase activity and chitin synthesis in Diaphorina citri, resulting in abnormal phenotypes. However, the mechanism of validamycin's action on D. citri remains unclear. Here, using a comparative transcriptome analysis, 464 differentially expressed genes (DEGs) in D. citri were identified after validamycin treatment. A Gene Ontology enrichment analysis revealed that these DEGs were mainly involved in "small molecule process", "structural molecule activity" and "transition metal ion binding". DEGs involved in chitin metabolism, cuticle synthesis and insecticide detoxification were validated by reverse transcription quantitative polymerase chain reaction. The RNA interference of D. citri chitinase-like protein ENO3 and D. citri cuticle protein 7 genes significantly affected D. citri molting. Moreover, the recombinant chitinase-like protein ENO3 exhibited a chitin-binding property, and an antimicrobial activity against Bacillus subtilis. This study provides a first insight into the molecular changes in D. citri after exposure to validamycin and identifies two effective RNA interference targets for D. citri control.

Transcriptome Analyses of Diaphorina citri Midgut Responses to Candidatus Liberibacter Asiaticus Infection.

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae), is an important transmission vector of the citrus greening disease Candidatus Liberibacter asiaticus (CLas). The D. citri midgut exhibits an important tissue barrier against CLas infection. However, the molecular mechanism of the midgut response to CLas infection has not been comprehensively elucidated. In this study, we identified 778 differentially expressed genes (DEGs) in the midgut upon CLas infection, by comparative transcriptome analyses, including 499 upregulated DEGs and 279 downregulated DEGs. Functional annotation analysis showed that these DEGs were associated with ubiquitination, the immune response, the ribosome, endocytosis, the cytoskeleton and insecticide resistance. KEGG enrichment analysis revealed that most of the DEGs were primarily involved in endocytosis and the ribosome. A total of fourteen DEG functions were further validated by reverse transcription quantitative PCR (RT-qPCR). This study will contribute to our understanding of the molecular interaction between CLas and D. citri.

Zika Virus Dissemination from the Midgut of Aedes aegypti is Facilitated by Bloodmeal-Mediated Structural Modification of the Midgut Basal Lamina.

The arboviral disease cycle requires that key tissues in the arthropod vector become persistently infected with the virus. The midgut is the first organ in the mosquito that needs to be productively infected with an orally acquired virus. Following midgut infection, the virus then disseminates to secondary tissues including the salivary glands. Once these are productively infected, the mosquito is able to transmit the virus to a vertebrate host. Recently, we described the midgut dissemination pattern for chikungunya virus in Aedes aegypti. Here we assess the dissemination pattern in the same mosquito species for Zika virus (ZIKV), a human pathogenic virus belonging to the Flaviviridae. ZIKV infection of secondary tissues, indicative of dissemination from the midgut, was not observed before 72 h post infectious bloodmeal (pibm). Virion accumulation at the midgut basal lamina (BL) was only sporadic, although at 96-120 h pibm, virions were frequently observed between strands of the BL indicative of their dissemination. Our data suggest that ZIKV dissemination from the mosquito midgut occurs after digestion of the bloodmeal. Using gold-nanoparticles of 5 nm and 50 nm size, we show that meal ingestion leads to severe midgut tissue distention, causing the mesh width of the BL to remain enlarged after complete digestion of the meal. This could explain how ZIKV can exit the midgut via the BL after bloodmeal digestion. Ingestion of a subsequent, non-infectious bloodmeal five days after acquisition of an initial, dengue 4 virus containing bloodmeal resulted in an increased number of virions present in the midgut epithelium adjacent to the BL. Thus, subsequent bloodmeal ingestion by an infected mosquito may primarily stimulate de novo synthesis of virions leading to increased viral titers in the vector.

Plasmid engineering of aphid alarm pheromone in tobacco seedlings affects the preference of aphids.

Plants producing sufficient amount of aphid alarm pheromone by expressing (E)-ß-Farnesene (EßF) synthase gene may contribute to plant protection by reducing aphid populations. However, terpene biosynthesis varies among plant species and developmental stages. In the present study, volatile headspace analysis of tobacco seedlings with MaßFS1 (an EßF synthase from the Asian peppermint Mentha asiatica) failed to generate EßF. We further targeted MaßFS1 to the tobacco plastid, using a chloroplast targeting sequence, either with or without the AtFPS1 gene for the biosynthesis of the precursor farnesyl diphosphate. When both MaßFS1 and AtFPS1 genes were targeted to the chloroplast, low levels of EßF were detected in stably transformed tobacco seedlings; resulting in specific repellence of the green peach aphid, Myzus persicae. These data indicate that redirecting the EßF biosynthetic pathway from its natural cytosolic location to the chloroplast is a valid strategy. This redirecting strategy may be very useful for other crop plants that do not naturally produce EßF or other repellent volatiles.

[Metabolic engineering of (E)-ß-farnesene synthase genes for aphid-resistant genetically modified plants].

Aphids are major agricultural pests that cause significant yield losses of crops each year. (E)-ß-farnesene (EßF), as the main component of the aphid alarm pheromones, can interrupt aphid feeding and cause other conspecies in the vicinity to become agitated or disperse from their host plant. Furthermore, EßF can function as a kairomone in attracting aphid predators. EßF synthase genes, which encode enzymes that convert farnesyl diphosphate (FPP) to the acyclic sesquiterpene EßF, have been isolated and characterized from peppermint (Mentha × piperita and Mentha asiatica), Yuzu (Citrus junos), Douglas fir (Pseudotsuga menziesii), sweet wormwood (Artemisia annua) and chamomile (Matricaria recutita), respectively. Transgenic plant overexpressing EßF synthase genes has been one of the most efficient strategies for aphid management. In this review, the current statuses of transgenic plants engineered for aphid resistance were summarized. The plant-derived EßF synthase genes with their potential roles in aphid management via genetic-modified (GM) approaches were reviewed. The existing problem in GM plants with EßF synthase gene, such as low EßF emission was usually detected in the transgenic plant, was discussed and the development direction in this area was proposed.

RNA interference of two glutathione S-transferase genes, Diaphorina citri DcGSTe2 and DcGSTd1, increases the susceptibility of Asian citrus psyllid (Hemiptera: Liviidae) to the pesticides fenpropathrin and thiamethoxam.

BACKGROUND: The Asian citrus psyllid, Diaphorina citri Kuwayama, is an important agricultural pest of citrus globally. Foliar application of chemical insecticides is the most widely used option for reducing D. citri populations. Knockdown of glutathione S-transferase (GST) in several insect species leads to increased susceptibility to insecticides; however, information about the detoxifying role of GST genes in D. citri is unavailable. RESULTS: Via a sequence homology search, we isolated and characterized three DcGST genes (DcGSTd1, DcGSTe1 and DcGSTe2) from D. citri. Phylogenetic analysis grouped DcGSTd1 into the delta class of GST genes, whereas DcGSTe1 and DcGSTe2 were clustered in the epsilon clade. Gene expression analysis revealed that chlorpyrifos treatment increased the mRNA levels of DcGSTe1 and fenpropathrin enhanced the expression level of DcGSTd1, while DcGSTe2 was significantly up-regulated after exposure to thiamethoxam at a dose of 30% lethal concentration (LC30). RNA interference (RNAi) of DcGSTe2 and DcGSTd1 followed by an insecticide bioassay increased the mortalities of thiamethoxam-treated psyllids by 23.0% and fenpropathrin-treated psyllids by 15.0%. In contrast, knockdown of DcGSTe1 did not significantly increase the susceptibility of D. citri to any of these three insecticides. Further, feeding with double-stranded RNA (dsDcGSTe2-d1) interfusion co-silenced DcGSTe2 and DcGSTd1 expression in D. citri, and led to an increase of susceptibility to both fenpropathrin and thiamethoxam. CONCLUSION: The findings suggest that DcGSTe2 and DcGSTd1 play unique roles in detoxification of the pesticides thiamethoxam and fenpropathrin. In addition, co-silencing by creating a well-designed dsRNA interfusion against multiple genes was a good RNAi strategy in D. citri. © 2017 Society of Chemical Industry.

Double-stranded RNA delivery through soaking mediates silencing of the muscle protein 20 and increases mortality to the Asian citrus psyllid, Diaphorina citri.

BACKGROUND: Asian citrus psyllid, Diaphorina citri Kuwayama, is the most important economic pest of citrus because it transmits Candidatus Liberibacter asiaticus (CLas), the causal agent of huanglongbing (HLB). Silencing genes by RNA interference (RNAi) is a promising approach for controlling D. citri. RNAi-based insect management strategies depend on the selection of suitable target genes. RESULTS: The muscle protein 20 gene DcMP20 was characterized from D. citri in an effort to impair proper muscle development through RNAi. Phylogenetic analysis showed that DcMP20 was more closely related to MP20 from Drosophila compared with its counterpart from other insect species. Developmental expression analysis revealed that transcription of DcMP20 was development dependent and reached a maximum level in the last instar (fourth-fifth) of the nymphal stage. The extent of RNAi in D. citri was dose dependent, with dsRNA-DcMP20 at 75 ng µL-1 being sufficient to knock down endogenous DcMP20 expression, which resulted in significant mortality and reduced body weight that positively correlated with the silencing of DcMP20. No effect was found when dsRNA-GFP or water was used, indicating the specific effect of dsRNA-DcMP20. CONCLUSION: Our results suggest that dsRNA can be delivered to D. citri through soaking, and DcMP20 is an effective RNAi target to be used in the management of D. citri. © 2017 Society of Chemical Industry.

RNA Interference of the Ecdysone Receptor Genes EcR and USP in Grain Aphid (Sitobion avenae F.) Affects Its Survival and Fecundity upon Feeding on Wheat Plants.

RNA interference (RNAi) has been widely used in functional genomics of insects and received intensive attention in the development of RNAi-based plants for insect control. Ecdysone receptor (EcR) and ultraspiracle protein (USP) play important roles in molting, metamorphosis, and reproduction of insects. EcR and USP orthologs and their function in grain aphid (Sitobion avenae F.) have not been documented yet. Here, RT-PCR, qRT-PCR, dsRNA feeding assay and aphid bioassay were employed to isolate EcR and USP orthologs in grain aphid, investigate their expression patterns, and evaluate the effect of RNAi on aphid survival and fecundity, and its persistence. The results indicated that SaEcR and SaUSP exhibited similar expression profiles at different developmental stages. Oral administration of dsRNAs of SaEcR and dsSaUSP significantly decreased the survival of aphids due to the down-regulation of these two genes, respectively. The silencing effect was persistent and transgenerational, as demonstrated by the reduced survival and fecundity due to knock-down of SaEcR and SaUSP in both the surviving aphids and their offspring, even after switching to aphid-susceptible wheat plants. Taken together, our results demonstrate that SaEcR and SaUSP are essential genes in aphid growth and development, and could be used as RNAi targets for wheat aphid control.

Generation of Marker- and/or Backbone-Free Transgenic Wheat Plants via Agrobacterium-Mediated Transformation.

Horizontal transfer of antibiotic resistance genes to animals and vertical transfer of herbicide resistance genes to the weedy relatives are perceived as major biosafety concerns in genetically modified (GM) crops. In this study, five novel vectors which used gusA and bar as a reporter gene and a selection marker gene, respectively, were constructed based on the pCLEAN dual binary vector system. Among these vectors, 1G7B and 5G7B carried two T-DNAs located on two respective plasmids with 5G7B possessing an additional virGwt gene. 5LBTG154 and 5TGTB154 carried two T-DNAs in the target plasmid with either one or double right borders, and 5BTG154 carried the selectable marker gene on the backbone outside of the T-DNA left border in the target plasmid. In addition, 5BTG154, 5LBTG154, and 5TGTB154 used pAL154 as a helper plasmid which contains Komari fragment to facilitate transformation. These five dual binary vector combinations were transformed into Agrobacterium strain AGL1 and used to transform durum wheat cv Stewart 63. Evaluation of the co-transformation efficiencies, the frequencies of marker-free transgenic plants, and integration of backbone sequences in the obtained transgenic lines indicated that two vectors (5G7B and 5TGTB154) were more efficient in generating marker-free transgenic wheat plants with no or minimal integration of backbone sequences in the wheat genome. The vector series developed in this study for generation of marker- and/or backbone-free transgenic wheat plants via Agrobacterium-mediated transformation will be useful to facilitate the creation of "clean" GM wheat containing only the foreign genes of agronomic importance.

RNAi-mediated plant protection against aphids.

Aphids (Aphididae) are major agricultural pests that cause significant yield losses of crop plants each year by inflicting damage both through the direct effects of feeding and by vectoring harmful plant viruses. Expression of double-stranded RNA (dsRNA) directed against suitable insect target genes in transgenic plants has been shown to give protection against pests through plant-mediated RNA interference (RNAi). Thus, as a potential alternative and effective strategy for insect pest management in agricultural practice, plant-mediated RNAi for aphid control has received close attention in recent years. In this review, the mechanism of RNAi in insects and the so far explored effective RNAi target genes in aphids, their potential applications in the development of transgenic plants for aphid control and the major challenges in this regard are reviewed, and the future prospects of using plant-mediated RNAi for aphid control are discussed. This review is intended to be a helpful insight into the generation of aphid-resistant plants through plant-mediated RNAi strategy. © 2016 Society of Chemical Industry.

Molecular characterization of two isoforms of a farnesyl pyrophosphate synthase gene in wheat and their roles in sesquiterpene synthesis and inducible defence against aphid infestation.

Aphids are important pests of wheat (Triticum aestivum) that affect crop production globally. Herbivore-induced emission of sesquiterpenes can repel pests, and farnesyl pyrophosphate synthase (FPS) is a key enzyme involved in sesquiterpene biosynthesis. However, fps orthologues in wheat and their functional roles in sesquiterpene synthesis and defence against aphid infestation are unknown. Here, two fps isoforms, Tafps1 and Tafps2, were identified in wheat. Quantitative real-time polymerase chain reaction (qRT-PCR) and in vitro catalytic activity analyses were conducted to investigate expression patterns and activity. Heterologous expression of these isoforms in Arabidopsis thaliana, virus-induced gene silencing (VIGS) in wheat and aphid behavioural assays were performed to understand the functional roles of these two isoforms. We demonstrated that Tafps1 and Tafps2 played different roles in induced responses to aphid infestation and in sesquiterpene synthesis. Heterologous expression in A. thaliana resulted in repulsion of the peach aphid (Myzus persicae). Wheat plants with these two isoforms transiently silenced were significantly attractive to grain aphid (Sitobion avenae). Our results provide new insights into induced defence against aphid herbivory in wheat, in particular, the different roles of the two Tafps isoforms in both sesquiterpene biosynthesis and defence against aphid infestation.

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

(E)-ß-Farnesene (EßF) synthase catalyses the production of EßF, 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 EßF synthase genes and can release EßF to repel aphids. In order to effectively recruit the plant-derived EßF 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 AaßFS1, an EßF synthase gene from sweet wormwood (Artemisia annua L.), to the chloroplast of tobacco to generate CTP + AaßFS1 transgenic lines. The CTP + AaßFS1 transgenic tobacco plants could emit EßF at a level up to 19.25 ng/day per g fresh tissues, 4-12 fold higher than the AaßFS1 transgenic lines without chloroplast targeting. Furthermore, aphid/parasitoid behavioral bioassays demonstrated that the CTP + AaßFS1 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 EßF synthase genes to generate a novel type of transgenic plant emitting an alarm pheromone for aphid control.

Engineering plants for aphid resistance: current status and future perspectives.

KEY MESSAGE: The current status of development of transgenic plants for improved aphid resistance, and the pros and cons of different strategies are reviewed and future perspectives are proposed. Aphids are major agricultural pests that cause significant yield losses of crop plants each year. Excessive dependence on insecticides for aphid control is undesirable because of the development of insecticide resistance, the potential negative effects on non-target organisms and environmental pollution. Transgenic plants engineered for resistance to aphids via a non-toxic mode of action could be an efficient alternative strategy. In this review, the distribution of major aphid species and their damages on crop plants, the so far isolated aphid-resistance genes and their applications in developments of transgenic plants for improved aphid resistance, and the pros and cons of these strategies are reviewed and future perspectives are proposed. Although the transgenic plants developed through expressing aphid-resistant genes, manipulating plant secondary metabolism and plant-mediated RNAi strategy have been demonstrated to confer improved aphid resistance to some degree. So far, no aphid-resistant transgenic crop plants have ever been commercialized. This commentary is intended to be a helpful insight into the generation and future commercialization of aphid-resistant transgenic crops in a global context.

Metabolic engineering of plant-derived (E)-ß-farnesene synthase genes for a novel type of aphid-resistant genetically modified crop plants.

Aphids are major agricultural pests that cause significant yield losses of crop plants each year. Excessive dependence on insecticides for long-term aphid control is undesirable because of the development of insecticide resistance, the potential negative effects on non-target organisms and environmental pollution. Transgenic crops engineered for resistance to aphids via a non-toxic mode of action could be an efficient alternative strategy. (E)-ß-Farnesene (EßF) synthases catalyze the formation of EßF, which for many pest aphids is the main component of the alarm pheromone involved in the chemical communication within these species. EßF can also be synthesized by certain plants but is then normally contaminated with inhibitory compounds. Engineering of crop plants capable of synthesizing and emitting EßF could cause repulsion of aphids and also the attraction of natural enemies that use EßF as a foraging cue, thus minimizing aphid infestation. In this review, the effects of aphids on host plants, plants' defenses against aphid herbivory and the recruitment of natural enemies for aphid control in an agricultural setting are briefly introduced. Furthermore, the plant-derived EßF synthase genes cloned to date along with their potential roles in generating novel aphid resistance via genetically modified approaches are discussed.

(E)-ß-farnesene synthase genes affect aphid (Myzus persicae) infestation in tobacco (Nicotiana tabacum).

Aphids are major agricultural pests which cause significant yield losses of the crop plants each year. (E)-ß-farnesene (EßF) is the alarm pheromone involved in the chemical communication between aphids and particularly in the avoidance of predation. In the present study, two EßF synthase genes were isolated from sweet wormwood and designated as AaßFS1 and AaßFS2, respectively. Overexpression of AaßFS1 or AaßFS2 in tobacco plants resulted in the emission of EßF ranging from 1.55 to 4.65 ng/day/g fresh tissues. Tritrophic interactions involving the peach aphids (Myzus persicae), predatory lacewings (Chrysopa septempunctata) demonstrated that the transgenic tobacco expressing AaßFS1 and AaßFS2 could repel peach aphids, but not as strongly as expected. However, AaßFS1 and AaßFS2 lines exhibited strong and statistically significant attraction to lacewings. Further experiments combining aphids and lacewing larvae in an octagon arrangement showed transgenic tobacco plants could repel aphids and attract lacewing larvae, thus minimizing aphid infestation. Therefore, we demonstrated a potentially valuable strategy of using EßF synthase genes from sweet wormwood for aphid control in tobacco or other economic important crops in an environmentally benign way.