Farnesyl/geranylgeranyl diphosphate synthases regulate the biosynthesis of alarm pheromone in a unique manner in the vetch aphid Megoura viciae.

Farnesyl/geranylgeranyl diphosphate synthases (FPPS/GGPPS) as the short-chain prenyltransferases catalyse the formation of the acyclic precursors (E)-FPP and (E)-GGPP for isoprenoid biosynthesis. Here, we first cloned the cDNAs encoding FPPS and GGPPS in the vetch aphid Megoura viciae (designated as MvFPPS and MvGGPPS). They had an open reading frame of 1185 and 930 bp in length, encoding 395 and 309 amino acids, with a theoretical isoelectric point of 6.52 and 6.21, respectively. Sequence alignment and phylogenetic analysis showed that MvFPPS and MvGGPPS shared the conserved aspartate-rich motifs characterized by all prenyltransferases identified to date and were clustered with their homologues in two large clades. RNA interference (RNAi) combined with gas chromatography/mass spectrometry (GC-MS) analysis showed that both MvFPPS and MvGGPPS were involved in the biosynthesis of alarm pheromone. Spatiotemporal expression profiling showed that the expression of MvFPPS and MvGGPPS was significantly higher in embryos than in other tissues. RNAi and GC-MS performed specifically in embryos corroborated the function of MvFPPS and MvGGPPS. In vitro, enzymatic activity assay and product analysis demonstrated that MvFPPS could catalysed the formation of (E)-FPP using DMAPP or (E)-GPP as the allylic cosubstrates in the presence of IPP, while MvGGPPS could only use (E)-GPP or (E)-FPP as cosubstrates. Functional interaction analysis using RNAi revealed that MvGGPPS exerts unidirectional functional compensation for MvFPPS. Moreover, it can regulate the biosynthesis of alarm pheromone by imposing a negative feedback regulation on MvFPPS. Our study helps to understand the molecular regulatory mechanism of terpenoid biosynthesis in the aphid.

Insect kinin mimics act as potential control agents for aphids: Structural modifications of Trp4.

Insect kinins are endogenous, biologically active peptides with various physiological functions. The use of insect kinins in plant protection is being evaluated by many groups. Some kinins have been chosen as lead compounds for pest control. We previously reported an insect kinin mimic IV-3 that had insecticidal activity. And by introducing a strong electron withdrawing group (-CF3 ) on the benzene ring (Phe2 ), we discovered a compound, L7 , with better activity than lead IV-3. In this work, taking L7 as the lead compound, we designed and synthesized 13 compounds to evaluate the influence of position 4 (Trp4 ) of insect kinin on insecticidal activity, by replacing the H atom on tryptophan with -CH3 and -Cl or substituting the indole ring of tryptophan with the benzene, naphthalene, pyridine, imidazole, cyclohexane, and alkyl carboxamides. The aphid bioassay results showed that the compounds M1 , M3 , and M5 were more active than the positive control, pymetrozine. Especially, replacing the side chain by an indole ring with 4-Cl substitution (M1 , LC50 = 0.0029 mmol/L) increased the aphicidal activity. The structure-activity relationships (SARs) indicated that the side chain benzene ring at this position may be important to the aphicidal activity. In addition, the toxicity prediction by Toxtree, and the toxicity experiments on Apis mellifera suggested that M1 was no toxicity risk on a non-target organism. It could be used as a selective and bee-friendly insecticide to control aphids.

Effects of sgRNAs, Promoters, and Explants on the Gene Editing Efficiency of the CRISPR/Cas9 System in Chinese Kale.

The CRISPR/Cas9 system is extensively used for plant gene editing. This study developed an efficient CRISPR/Cas9 system for Chinese kale using multiple sgRNAs and two promoters to create various CRISPR/Cas9 vectors. These vectors targeted BoaZDS and BoaCRTISO in Chinese kale protoplasts and cotyledons. Transient transformation of Chinese kale protoplasts was assessed for editing efficiency at three BoaZDS sites. Notably, sgRNA: Z2 achieved the highest efficiency (90%). Efficiency reached 100% when two sgRNAs targeted BoaZDS with a deletion of a large fragment (576 bp) between them. However, simultaneous targeting of BoaZDS and BoaCRTISO yielded lower efficiency. Transformation of cotyledons led to Chinese kale mutants with albino phenotypes for boazds mutants and orange-mottled phenotypes for boacrtiso mutants. The mutation efficiency of 35S-CRISPR/Cas9 (92.59%) exceeded YAO-CRISPR/Cas9 (70.97%) in protoplasts, and YAO-CRISPR/Cas9 (96.49%) surpassed 35S-CRISPR/Cas9 (58%) in cotyledons. These findings introduce a strategy for enhancing CRISPR/Cas9 editing efficiency in Chinese kale.

Discovery of Novel Potential Aphid Repellents: Geranic Acid Esters Containing Substituted Aromatic Rings.

Aphids are one of the most damaging agricultural pests. For the sake of novel eco-friendly compounds with good activity for aphid control, a series of novel geranic acid esters containing substituted aromatic rings were designed by inverting ester groups of lead compounds. All compounds were characterized by HRMS, 1H-NMR, and 13C-NMR. In order to identify the effect of inversion ester groups on activity, a bioassay was conducted. The results showed that the repellent activity against Acyrthosiphon pisum (A. pisum) and the binding affinity with the odorant-binding protein 9 from A. pisum (ApisOBP9) of the compounds were increased after inversion of the ester groups. Particularly, 5f showed the best repellent activity (repellency proportion: 55.6%) and binding affinity (1/Ki: 0.49 µM). Meanwhile, the structure-activity relationships revealed that the introduction of meta-substitution of the benzene ring and halogen atoms, such as Cl and Br, facilitated the biological activity. The further molecular docking results demonstrated that hydrogen bonding interactions and hydrophobic interactions were vital for the binding affinity with ApisOBP9. Additionally, all compounds were predicted to be eco-friendly and their volatile physicochemical properties have been enhanced compared to the leads. The present results provide valuable clues for the further rational design of aphids' behavioral control agents.

Identification and Behavioral Assays of Alarm Pheromone in the Vetch Aphid Megoura viciae.

Aphids are destructive pests, and alarm pheromones play a key role in their chemical ecology. Here, we conducted a detailed analysis of terpenoids in the vetch aphid, Megoura viciae, and its host plant Pisum sativum using gas chromatography/mass spectrometry. Four major components, (-)-ß-pinene (49.74%), (E)-ß-farnesene (32.64%), (-)-α-pinene (9.42%) and ( +)-limonene (5.24%), along with trace amounts of ( +)-sabinene, camphene and α-terpineol) (3.14%) were found in the aphid. In contrast, few terpenoids were found in the host plant, consisting mainly of squalene (66.13%) and its analog 2,3-epoxysqualene (31.59%). Quantitative analysis of the four major terpenes in different developmental stages of the aphid showed that amounts of the monoterpenes increased with increasing stage, while the sesquiterpene amount peaked in the 3rd instar. (-)-ß-Pinene was the most abundant terpene at all developmental stages. Behavioral assays using a three-compartment olfactometer revealed that the repellency of single compounds varied in a concentration-dependent manner, but two mixtures [(-)-α-pinene: (-)-ß-pinene: (E)-ß-farnesene: ( +)-limonene = 1:44.4:6.5:2.2 or 1:18.4:1.3:0.8], were repellent at all concentrations tested. Our results suggest that (-)-α-pinene and (-)-ß-pinene are the major active components of the alarm pheromone of M. viciae, but that mixtures play a key role in the alarm response. Our study contributes to the understanding of the chemical ecology of aphids and may help design new control strategies against this aphid pest.

Novel (E)-ß-Farnesene Analogues Containing 2-Nitroiminohexahydro-1,3,5-triazine: Synthesis and Biological Activity Evaluation.

In order to discover novel eco-friendly compounds with good activity for aphid control, (E)-ß-farnesene (EßF), the main component of the aphid alarm pheromone, was chosen as the lead compound. By introducing a 2-nitroimino-hexahydro-1,3,5-triazine moiety (abbreviated NHT) to replace the unstable conjugated double bond system of EßF, a series of novel EßF analogues containing the NHT moiety were synthesized via the reaction of substituted NHT rings with (E)-1-chloro-3,7-dimethylocta-2,6-diene. All the compounds were characterized by ¹H-NMR, (13)C-NMR, IR, and high resolution mass spectroscopy (HRMS). The bioassay results showed that all the analogues displayed different repellent and aphicidal activities against green peach aphid (Myzus persicae). Particularly, the analogue 4r exhibited obvious repellent activity (repellent proportion: 78.43%) and similar aphicidal activity against M. persicae (mortality: 82.05%) as the commercial compound pymetrozine (80.07%). A preliminary structure-activity relationship (SAR) study was also performed, which offered valuable clues for the design of further new EßF analogues.

Understanding the onion aphid Neotoxoptera formosana's (Hemiptera: Aphididae) interaction with Allium (Asparagales: Amaryllidaceae) crops: a comprehensive study of feeding patterns and life history traits.

The onion aphid, Neotoxoptera formosana, poses a significant threat to Allium crops worldwide, causing considerable economic losses and quality degradation. To develop effective pest management strategies, it is crucial to understand the feeding behavior and life history of this pest on different Allium crops. In this study, the electrical penetration graph (EPG) technique was used to monitor the thorn-feeding behavior of the onion aphid on 4 Allium crops: leek, chive, garlic, and shallot. The EPG data revealed distinct feeding patterns, with garlic and shallots being more preferred hosts than chives. Additionally, the aphids primarily fed on the phloem in garlic and shallots. Analysis of life history trait showed that chives provided the most favorable conditions for aphid development and reproduction, while leek exhibited relatively unfavorable conditions. Examination of leaf histology also revealed differences among the crops, which may influence aphid feeding behavior. This study provides valuable insights into the interaction between the onion aphid and different Allium crops, aiding in the development of comprehensive pest control strategies to minimize crop damage and economic losses. The use of advanced techniques like EPG contributes to a more detailed understanding of aphid behavior and shows promise for improving pest management in other plant-pest interactions.

Synergism of (E)-ß-farnesene and Its Analogue to Insecticides against the Green Peach Aphid Myzus persicae.

With high aphid-repellent activity but low stability, (E)-ß-farnesene (EßF), the major component of the aphid alarm pheromone, can be used as a synergist to insecticides. Some EßF analogues possess both good aphid-repellent activity and stability, but the synergistic effect and related mechanism are still unclear. Therefore, this study investigated the synergistic effect and underlying mechanism of the EßF and its analogue against the aphid Myzus persicae. The results indicated that EßF and the analogue showed significantly synergistic effects to different insecticides, with synergism ratios from 1.524 to 3.446. Mechanistic studies revealed that EßF and the analogue exhibited effective repellent activity, significantly upregulated target OBP genes by 161 to 731%, increased aphid mobility, and thereby enhanced contact with insecticides. This research suggests that the EßF analogue represents a novel synergist for insecticides, with the potential for further application in aphid control owing to its enhanced bioactivity and the possibility of reducing insecticide doses.

Rational design, synthesis and binding mechanisms of novel benzyl geranate derivatives as potential eco-friendly aphid repellents.

BACKGROUND: The push-pull strategy is considered as a promising eco-friendly method for pest management. Plant volatile organic compounds (PVOCs) act as semiochemicals constitute the key factor in implementing this strategy. Benzyl alcohol and geraniol, as functional PVOCs, were reported to regulate insect behavior, showing the potential application in pest control. Using geraniol as lead, a geraniol derivative 5i with fine repellent activity was discovered in our previous work. In order to explore novel, eco-friendly aphid control agents, a series of benzyl geranate derivatives was designed and synthesized using 5i as the lead and benzyl alcohol as the active fragment. RESULTS: Benzyl alcohol was firstly evaluated to have repellent activity to Acyrthosiphon pisum. Based on this repellent fragment, a series of novel benzyl geranate derivatives was rationally designed and synthesized using a scaffold-hopping strategy. Among them, compound T9, with a binding affinity (Kd = 0.43 µm) and a substantial repellency of 64.7% against A. pisum, is the most promising compound. Molecule docking showed that hydrophobic and hydrogen-bonding interactions substantially influenced the binding affinity of compounds with ApisOBP9. Additionally, T9 exhibited low-toxicity to honeybees and ladybugs. CONCLUSION: Using a simple scaffold-hopping strategy combined with active fragment benzyl alcohol, a new derivative T9, with high aphid-repellency and low-toxicity to nontarget organisms, can be considered as a novel potential eco-friendly aphid control agent for sustainable agriculture. © 2023 Society of Chemical Industry.

Sustainable Natural Resources for Aphid Management: ß-Ionone and Its Derivatives as Promising Ecofriendly Botanical-Based Products.

ß-Ionone, sustainably derived from Petunia hybrida as a natural bioresource, was identified as a lead compound for integrated aphid management. A series of ß-ionone derivatives containing ester groups were designed and synthesized for the purpose of discovering renewable botanical-based products. The odorant-binding protein (OBP) binding test indicated that ß-ionone and its derivatives displayed binding affinities with Acyrthosiphon pisum OBP9 (ApisOBP9) and Harmonia axyridis OBP15 (HaxyOBP15). Bioactivity assays revealed that most ß-ionone derivatives exhibited a higher repellent activity than that of ß-ionone. ß-Ionone and derivatives 4g and 4l displayed attractiveness to H. axyridis. Specifically, 4g was a highly promising derivative, possessing good repellent activity against A. pisum and attractiveness to H. axyridis. Molecular dynamics simulations revealed that integrating the hydrophobic ester group into the ß-ionone framework strengthened the van der Waals interactions of 4g with ApisOBP9/HaxyOBP15, improving the binding affinity with OBPs and producing higher push-pull activity than ß-ionone; 4g also had low toxicity toward nontarget organisms. Thus, 4g is a potential ecofriendly, botanical-based option for aphid management.

Functional characterization of alkaline phosphatases involved alarm pheromone in the vetch aphid Megoura viciae.

The alkaline phosphatases (ALPs) are highly promiscuous enzymes and have been extensively investigated in mammals for their medical significance, but their functional promiscuity is relatively poorly understood in insects. Here, we first identified four ALP genes (designated as MvALP1-4) in the vetch aphid Megoura viciae that contained one alkaline phosphatase site, three metal-binding sites, and varied other functional sites. Phylogenetic analysis, molecular docking and the spatiotemporal expression profiling of MvALP1-4 were very different, indicating a promiscuous functionality. We also found that MvALP4 involved the biosynthesis of aphid alarm pheromones (EßF) in vitro and in vivo. Finally, transcriptome analysis in the stimulated and unstimulated aphids supported the involvement of MvALPs in the biosynthesis of aphid alarm pheromones. Our study identified a multifunctional ALP involved terpene synthase enzyme activity in the aphid, which contributes to the understanding of the functional plasticity of ALPs in insects.

Functional analysis of odorant-binding proteins for the parasitic host location to implicate convergent evolution between the grain aphid and its parasitoid Aphidius gifuensis.

(E)-ß-farnesene (EBF) is a typical and ecologically important infochemical in tri-trophic level interactions among plant-aphid-natural enemies. However, the molecular mechanisms by which parasitoids recognize and utilize EBF are unclear. In this study, we functionally characterized 8 AgifOBPs in Aphidifus gifuensis, one dominant endo-parasitoid of wheat aphid as well as peach aphid in China. Among which, AgifOBP6 was the only OBP upregulated by various doses of EBF, and it showed a strong binding affinity to EBF in vitro. The lack of homology between AgifOBP6 and EBF-binding proteins from aphids or from other aphid natural enemies supported that this was a convergent evolution among insects from different orders driven by EBF. Molecular docking of AgifOBP6 with EBF revealed key interacting residues and hydrophobic forces as the main forces. AgifOBP6 is widely expressed among various antennal sensilla. Furthermore, two bioassays indicated that trace EBF may promote the biological control efficiency of A. gifuensis, especially on winged aphids. In summary, this study reveals an OBP (AgifOBP6) that may play a leading role in aphid alarm pheromone detection by parasitoids and offers a new perspective on aphid biological control by using EBF. These results will improve our understanding of tri-trophic level interactions among plant-aphid-natural enemies.

Aphid-repellent, ladybug-attraction activities, and binding mechanism of methyl salicylate derivatives containing geraniol moiety.

BACKGROUND: Aphids have been mainly controlled by traditional chemical insecticides, resulting in unamiable risk to the environment over the last decades. Push-pull strategy is regarded as a promising eco-friendly approach for aphid management through repelling aphid away and attracting their natural enemy. Methyl salicylate (MeSA), one of typical HIPVs (herbivore-induced plant volatiles), can repel aphids and attract ladybugs. Our previous studies discovered a new lead compound 3e, a salicylate-substituted carboxyl (E)-ß-farnesene derivative that had effective aphid-repellent activity. However, whether 3e has attractive activity to ladybug like MeSA is unknown. Meanwhile, to discover a new derivative for both deterring aphid and recruiting ladybug is meaningful for green control of aphids. RESULTS: Through the structural optimization of 3e, 14 new derivatives were designed and synthesized. Among them, compounds 4e and 4i had good aphid (Acyrthosiphon pisum) repellent activity, and compounds 3e, 4e and 4i had significant ladybug (Harmonia axyridis) attractive activity to males. Particularly, 4i exhibited manifest attractive effect on the females as well. Binding mechanism showed that 4i not only bound effectively with the aphid (Acyrthosiphon pisum) target ApisOBP9 thanks to its multiple hydrophobic interactions and hydrogen-bond, but also had strong binding affinity with ladybug target HaxyOBP15 due to the suitable steric space. Additionally, 4i displayed low toxicity to bee Apis mellifera. CONCLUSION: Compound 3e does exhibit attractive activity to male ladybug as MeSA. However, the new derivative 4i, with both pleasant aphid-repellent and ladybug-attraction activities, can be considered as a novel potential push-pull candidate for aphid control in sustainable agriculture. © 2022 Society of Chemical Industry.

Kairomonal Effect of Aphid Alarm Pheromones and Analogs on the Parasitoid Diaeretiella rapae.

Aphid alarm pheromones, as important semiochemicals, not only mediate behavioral response of aphids, but can also act as kairomones to attract their natural enemies. The sesquiterpene (E)-ß-farnesene (EßF), the major alarm pheromone component of most aphid species, has been shown to have a kairomonal effect on the predators of aphids, but other alarm pheromone components, especially the monoterpenes and analogs, are rarely investigated. Here, two EßF analogs were successfully synthesized via the nucleophilic substitution reaction, and we then examined the kairomonal effects of four alarm pheromone components and two EßF analogs on the aphid parasitoid, Diaeretiella rapae. In olfactory bioassays, D. rapae females generally showed no significant behavioral response to these alarm pheromone components and analogs under low concentrations (0.1 µg/µL). Nevertheless, their olfactory response to these compounds gradually enhanced with increasing concentrations. Among the four pheromone components, EßF showed the highest attractive activity, but the parasitoid preferred blends over single compounds. Moreover, the response time decreased as the concentration increased. We confirmed the kairomonal effect of monoterpene alarm pheromone components and their blends, in addition to EßF, on the natural enemies of aphids. This is the first report that the blend of alarm pheromone components and their analogs has a stronger kairomonal effect than do the single components on the natural enemies of aphids. This study contributes to our understanding of the mechanisms involved in the regulation of parasitoid behaviors by kairomones and provides a promising opportunity for designing kairomones for the aphid parasitoid to mediate aphid populations in the field.

Spatial Expression Analysis of Odorant Binding Proteins in Both Sexes of the Aphid Parasitoid Aphidius gifuensis and Their Ligand Binding Properties.

In China, Aphidius gifuensis is one of the most common endoparasitoids of the green peach aphid Myzus persicae and grain aphid Sitobion miscanthi in the field. Insect odorant-binding proteins (OBPs) play vital roles in odor perception during feeding, host searching, mating and oviposition. In addition, some OBPs are involved in other physiological processes such as gustation and reproduction. In the present study, a comparative antennal transcriptomic analysis was applied between male and female A. gifuensis. The spatial expression patterns among antennae, heads, thoraxes, abdomens and legs of OBPs in both sexes were further profiled. Fifteen AgifOBPs were predicted, and 14 of them were identified by gene cloning, including 12 classic OBPs and 2 min-C OBPs. As expected, all OBPs were mainly expressed at high levels in antennae, heads or legs which are sensory organs and tissues. Finally, ligand binding properties of 2 OBPs (AgifOBP7 and AgifOBP9) were further evaluated. Female leg specifically expressed AgifOBP9 displays a broad and high binding property to aphid alarm pheromones, plant green volatiles and aphid sex pheromones (Ki < 10 µΜ). However, female leg specifically expressed AgifOBP7 displays poor affinity for all tested ligands except CAU-II-11 ((E)-3,7-dimethylocta-2,6-dien-1-yl-2-hydroxy-3-methoxybenzoate), a reported (E)-ß-farnesene (EBF) analog with an exceptionally high binding affinity (Ki = 1.07 ± 0.08 µΜ). In summary, we reported the spatial expression pattern of the OBP repertoire in A. gifuensis, and further studied the binding properties of OBP7 and OBP9, which are mainly expressed in female legs, laying the foundation for the dissection of the contribution of OBPs to chemosensation in A. gifuensis.

A chromosome-level genome assembly of Neotoxoptera formosana (Takahashi, 1921) (Hemiptera: Aphididae).

Neotoxoptera formosana (Takahashi), the onion aphid, is an oligophagous pest that mainly feeds on plants from the Allium genus. It sucks nutrients from the plants and indirectly acts as a vector for plant viruses. This aphid causes severe economic losses to Allium tuberosum agriculture in China. To better understand the host plant specificity of N. formosana on Allium plants and provide essential information for the control of this pest, we generated the entire genome using Pacific Biosciences long-read sequencing and Hi-C data. Six chromosomes were assembled to give a final size of 372.470 Mb, with an N50 scaffold of 66.911 Mb. The final draft genome assembly, from 192 Gb of raw data, was approximately 371.791 Mb in size, with an N50 contig of 24.99 Kb and an N50 scaffold of 2.637 Mb. The average GC content was 30.96%. We identified 73 Mb (31.22%) of repetitive sequences, 14,175 protein-coding genes, and 719 noncoding RNAs. The phylogenetic analysis showed that N. formosana and Pentalonia nigronervosa are sister groups. We found significantly expanded gene families that were involved in the THAP domain, the DDE superfamily endonuclease, zinc finger, immunity (ankyrin repeats), digestive enzyme (serine carboxypeptidase) and chemosensory receptor. This genome assembly could provide a solid foundation for future studies on the host specificity of N. formosana and pesticide-resistant aphid management.

Using single cell type proteomics to identify Al-induced proteomes in outer layer cells and interior tissues in the apical meristem/cell division regions of tomato root-tips.

Aluminum (Al) toxicity primarily targets the root tips, inhibiting root growth and function and leading to crop yield losses on acidic soils. Previously we reported using laser capture microdissection (LCM) proteomics to identify Al-induced proteins in the outer layer cells in the transitional zone of tomato root-tips. This study aims to further characterize Al-induced proteomic dynamics from the outer to interior tissues, thus providing a panoramic view reflecting Al resistance in the root tip as a whole in tomatoes. Three types of cells were isolated via LCM from the basal 350-400 µm (below cell elongation regions) of root tips using tomato (Solanum lycopersicum) 'Micro-Tom' plants. Type I and Type II were from Al-treated plants. Type I included cells of the outer three layers, i.e., the epidermis and cortex initials and the quiescent center (QC) in root apical meristem (RAM), and Type II possessed the interior tissues of the same region. Type III contained cells from the non-Al-treated root tips collected in the same region as Type I. Two tandem mass tag (TMT) proteomics analyses with three biological replicates for each sample type were conducted. The TMTexp1 (comparing Type I and Type II) identified 6575 quantifiable proteins and 178 different abundance proteins (DAPs). The TMTexp2 (comparing Type I and Type III) identified 7197 quantifiable proteins and 162 DAPs. Among all quantified proteins (7685) from the two TMT experiments, 6088 (79%) proteins, including 313 DAPs (92% of the 340 total), were identified in all tissues. A model reflecting the tissue-specific Al-resistance mechanism was proposed, in which the level of the citrate transporter MATE protein, involved in Al exclusion, accumulated to the highest level in the outer-layer cells but decreased toward the interior of root-tips (which concurs with the tissue-specific importance in Al resistance). Proteins for biosynthesis of ethylene and jasmonic acid, proteolytic enzymes, stress-responsive proteins, and cell wall modeling were affected by Al treatment, some in a cell type-specific manner. The KEGG metabolite pathways enriched with these DAPs changed depending on the cell types. This study demonstrated the advantage of using the tissue/cell-specific analysis for identifying proteins and their dynamic changes directly associated with Al resistance in the root-tip region. The proteomics datasets have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (https://www.ebi.ac.uk/pride/) with the dataset identifier as PXD021994 under project title: Proteomics studies of outer and inner cellular layers of tomato root-tips for Al stress, Project DOI: 10.6019/ PXD021994; and PXD018234 under Project title: Al-induced root proteomics changes in stress-acclimated tomato plant, Project DOI: https://doi.org/10.6019/PXD018234. SIGNIFICANCE: This paper presents the method of using laser capture microdissection (LCM) to collect homogenous cell-type specific tissue samples from the outer layers and inner central regions of tomato root-tips. The tandem mass tag-proteomics analysis showed that the outer-layer cells expressed proteomes that were different from the inner tissues of Al-treated root-tips; proteins related to resistance/tolerance to Al toxicity were highly accumulated in the outer-layer cells. Furthermore, the Al-treated outer-layer cells expressed proteomes which were different from the non-Al treated counterpart cells. This study has provided the first dataset of proteins differentiating from the outer to inner layers of cells in Al-treated root-tips. It provided convincing experimental evidences demonstrating the single-cell type proteomics as a powerful analytical approach to identify Al tolerance mechanisms in plants. The analytical procedure of LCM-tandem mass tag-quantitative proteomics analysis has a broad application for proteomics analysis of spatially separated cells in complex tissues.

Novel Temporal Expression Patterns of EBF-Binding Proteins in Wing Morphs of The Grain Aphid Sitobion miscanthi.

High chemosensitivity of insects to volatile organic compounds (VOC) stimuli is mediated by odorant binding proteins (OBPs). In aphids, three OBPs (OBP3, OBP7 and OBP9) are E-ß-farnesene (EBF)-binding proteins. Winged aphids are generally more sensitive than wingless aphids to VOCs, thus, wing presence is a phenotypic correlate of olfaction sensitivity. Here, we investigate the detailed temporal expression of these EBF-binding proteins and two other OBPs (OBP6 and OBP10), in the grain aphid Sitobion miscanthi 0 h, 2 h, 1 day, 3 days, 10 days, and 20 days after adult emergence. Both winged and wingless aphids were examined to further uncover phenotypic specification. Then, the expression patterns before and after EBF induction were analyzed. Throughout adulthood, only OBP7 had significantly higher antennal expression in winged aphids; however, there was no significant difference in the antennal expression of OBP3 between wing morphs at most time points. Except it was lower in newly emerged winged aphids but increased rapidly to the same level in wingless aphids at 1 day. OBP9 did not differ in expression between the morphs and was the only OBP that did not exhibit an expression trough at the beginning of the adult stage (0 h). The expression of OBP9 remained relatively stable and high throughout the adult stage in both phenotypes, showing the highest level among the three EBF-binding proteins. After EBF induction, its expression was further up-regulated in both morphs. Therefore, this protein may be an important molecule for EBF recognition in aphids. OBP7 strongly responded to EBF but only in winged aphids, suggesting that this protein is important in the more sensitive EBF recognition process of winged aphids. In addition, the antennal expression level of OBP3 did not respond to EBF induction. These findings revealed a temporal expression pattern of OBPs in aphids and showed that figuring out the pattern is critical for correctly selecting morphs and sampling times, which will support the discovery of reliable findings and allow solid conclusions to be drawn. Our findings also inspire on the interaction mode of the three EBF-binding proteins in relation to EBF perception in aphids.

Reduced insecticide susceptibility of the wheat aphid Sitobion miscanthi after infection by the secondary bacterial symbiont Hamiltonella defensa.

BACKGROUND: Bacterial symbionts in insects, especially aphids, have a major influence on host adaptation. We previously showed that infection with the secondary symbiont Hamiltonella defensa increases the fitness of the wheat aphid Sitobion miscanthi, yielding increases in fitness parameters such as adult weight and offspring number. However, whether H. defensa affects the sensitivity of host aphids to insecticides remains unknown. RESULTS: We tested the effects of H. defensa on host aphid susceptibility to the insecticides chlorpyrifos methyl, imidacloprid, cyantraniliprole and acetamiprid. Our results showed that compared with Hamiltonella-free aphid clones, Hamiltonella-infected aphid clones exhibited lower sensitivity to most of the tested insecticides at low concentrations. Quantitative polymerase chain reaction showed that the density of H. defensa in the infected clones was slightly decreased at 24 h but then sharply increased until the late stage after treatment with the different insecticides. H. defensa in the host aphids was detected by fluorescence in situ hybridization and was localized to the aphid hindgut. Levels of the detoxification enzymes acetylcholinesterase, glutathione transferase and carboxylesterase were significantly higher in Hamiltonella-infected clones than in Hamiltonella-free clones. CONCLUSIONS: The findings indicated that infection with H. defensa reduced aphid susceptibility to the investigated insecticides at low concentrations, potentially by increasing detoxification enzyme activity in the host. Therefore, symbiont-mediated insecticide resistance should be taken into account when performing resistance-monitoring studies. Studies of symbiont-mediated insecticide resistance may enhance our understanding of the emergence of insecticide resistance in agricultural systems. © 2020 Society of Chemical Industry.

Bioactivities of synthetic salicylate-substituted carboxyl (E)-ß-Farnesene derivatives as ecofriendly agrochemicals and their binding mechanism with potential targets in aphid olfactory system.

BACKGROUND: The aphid alarm pheromone, (E)-ß-farnesene (EßF), is a natural product secreted from the aphid cornicle as a signal to warn companions of danger. Odorant binding proteins (OBPs) are the vital targets in insect signal transduction pathways. To improve bioactivity of EßF as more economic and stable aphid control agents, EßF derivatives containing an active substructure, salicylic acid moiety, were designed, synthesized, and evaluated for their bioactivities in a structure-function study under laboratory conditions. RESULTS: EßF derivatives, (E)-3,7-dimethylocta-2,6-dien-1-yl-2-hydroxy-3-methylbenzoate and (E)-3,7-dimethylocta-2,6-dien-1-yl-2-hydroxy-3-methoxybenzoate showed outstanding aphid-repellent activity at a dose of 5 µg against Acyrthosiphon pisum (repellency proportions of 67.3% and 71.2%, respectively) and Myzus persicae (repellency proportions of 80.0% and 74.4%, respectively) in laboratory. EßF and most of its derivatives bound strongly to ApisOBP9 with a higher affinity than those of the reported potential targets AphisOBP3 and ApisOBP7. The binding affinities to these three ApisOBPs were generally consistent with the in vivo aphid-repellent activity. A molecular docking study suggested that the hydrophobic effect was crucial for the interactions between the derivatives and the OBPs. CONCLUSION: New EßF derivatives containing salicylic acid moiety and their repellent activity, binding mechanism with three potential OBPs are presented. A new OBP, ApisOBP9, was characterized as a potential EßF and EßF derivatives binding protein for the first time. The hydrophobic nature of these analogues is responsible for their activity. Two analogues 3b and 3e with outstanding aphid-repellent activity could be new leads for aphid control agents.