Salivary gland transcriptomics of the cotton aphid Aphis gossypii and comparative analysis with other sap-sucking insects.

Aphids are sap-sucking insects responsible for crop losses and a severe threat to crop production. Proteins in the aphid saliva are integral in establishing an interaction between aphids and plants and are responsible for host plant adaptation. The cotton aphid, Aphis gossypii (Hemiptera: Aphididae) is a major pest of Gossypium hirsutum. Despite extensive studies of the salivary proteins of various aphid species, the components of A. gossypii salivary glands are unknown. In this study, we identified 123,008 transcripts from the salivary gland of A. gossypii. Among those, 2933 proteins have signal peptides with no transmembrane domain known to be secreted from the cell upon feeding. The transcriptome includes proteins with more comprehensive functions such as digestion, detoxification, regulating host defenses, regulation of salivary glands, and a large set of uncharacterized proteins. Comparative analysis of salivary proteins of different aphids and other insects with A. gossypii revealed that 183 and 88 orthologous clusters were common in the Aphididae and non-Aphididae groups, respectively. The structure prediction for highly expressed salivary proteins indicated that most possess an intrinsically disordered region. These results provide valuable reference data for exploring novel functions of salivary proteins in A. gossypii with their host interactions. The identified proteins may help develop a sustainable way to manage aphid pests.

Salinity stress alters plant-mediated interactions between above- and below-ground herbivores.

Below-ground herbivory impacts plant development and often induces systemic responses in plants that affect the performance and feeding behavior of above-ground herbivores. Meanwhile, pest-damaged root tissue can enhance a plant's susceptibility to abiotic stress such as salinity. Yet, the extent to which herbivore-induced plant defenses are modulated by such abiotic stress has rarely been studied. In this study, we examine whether root feeding by larvae of the turnip moth, Agrotis segetum (Lepidoptera: Noctuidae) affects the performance of the above-ground, sap-feeding aphid Aphis gossypii (Hemiptera: Aphididae) on cotton, and assess whether those interactions are modulated by salinity stress. In the absence of salinity stress, A. segetum root feeding does not affect A. gossypii development. On the other hand, under intense salinity stress (i.e., 600 mM NaCl), A. segetum root feeding decreases aphid development time by 16.1 % and enhances fecundity by 72.0 %. Transcriptome, metabolome and bioassay trials showed that root feeding and salinity stress jointly trigger the biosynthesis of amino acids in cotton leaves. Specifically, increased titers of valine in leaf tissue relate to an enhanced performance of A. gossypii. Taken together, salinity stress alters the interaction between above- and below-ground feeders by changing amino acid accumulation. Our findings advance our understanding of how plants cope with concurrent biotic and abiotic stressors, and may help tailor plant protection strategies to varying production contexts.

Combining transcriptome and metabolome analysis to understand the response of sorghum to Melanaphis sacchari.

BACKGROUND: The sorghum aphid Melanaphis sacchari (Zehntner) (Homoptera: Aphididae) is an important insect in the late growth phase of sorghum (Sorghum bicolor L.). However, the mechanisms of sorghum response to aphid infestation are unclear. RESULTS: In this paper, the mechanisms of aphid resistance in different types of sorghum varieties were revealed by studying the epidermal cell structure and performing a transcriptome and metabolome association analysis of aphid-resistant and aphid-susceptible varieties. The epidermal cell results showed that the resistance of sorghum to aphids was positively correlated with epidermal cell regularity and negatively correlated with the intercellular space and leaf thickness. Transcriptome and metabolomic analyses showed that differentially expressed genes in the resistant variety HN16 and susceptible variety BTX623 were mainly enriched in the flavonoid biosynthesis pathway and differentially expressed metabolites were mainly related to isoflavonoid biosynthesis and flavonoid biosynthesis. The q-PCR results of key genes were consistent with the transcriptome expression results. Meanwhile, the metabolome test results showed that after aphidinfestation, naringenin and genistein were significantly upregulated in the aphid-resistant variety HN16 and aphid-susceptible variety BTX623 while luteolin was only significantly upregulated in BTX623. These results show that naringenin, genistein, and luteolin play important roles in plant resistance to aphid infestation. The results of exogenous spraying tests showed that a 1‰ concentration of naringenin and genistein is optimal for improving sorghum resistance to aphid feeding. CONCLUSIONS: In summary, the physical properties of the sorghum leaf structure related to aphid resistance were studied to provide a reference for the breeding of aphid-resistant varieties. The flavonoid biosynthesis pathway plays an important role in the response of sorghum aphids and represents an important basis for the biological control of these pests. The results of the spraying experiment provide insights for developing anti-aphid substances in the future.

The Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway contributes to the defense against bacterial infection in the pea aphid.

The Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling is activated by infections of bacteria, fungi, viruses and parasites and mediated cellular and humoral immune responses. In the pea aphid Acyrthosiphon pisum little is known about the function of JAK/STAT signaling in its immune system. In this study, we first showed that expression of genes in the JAK/STAT signaling, including the receptors Domeless1/2, Janus kinase (JAK) and transcriptional factor Stat92E, is up-regulated upon bacteria Escherichia coli and Staphylococcus aureus and fungus Beauveria bassiana infections. After knockdown of expression of these genes by means of dsRNA injection, the aphids harbored more bacteria and suffered more death after infected with E. coli and S. aureus, but showed no significant change after B. bassiana infection. Our study suggests the JAK/STAT signaling contributes to the defense against bacterial infection in the pea aphid.

S431F mutation on AChE1 and overexpression of P450 genes confer high pirimicarb resistance in Sitobion miscanthi.

Sitobion miscanthi is a destructive wheat pest responsible for significant wheat yield losses. Pirimicarb, one of the most important representatives of N, N-dimethylcarbamate insecticides, is widely used to control wheat aphids. In present work, heterozygous S431F mutation of acetylcholinesterase 1 (AChE1) was identified and verified in three pirimicarb-resistant S. miscanthi populations (two field populations (HA and HS, >955.8-fold) and one lab-selected population (PirR, 486.1-fold)), which has not been reported in S. miscanthi yet. The molecular docking results revealed that AChE1 containing the S431F mutation of S. miscanthi (SmAChE1S431F) showed higher free binding energy to three insecticides (pirimicarb, omethoate, and methomyl) than wild-type AChE1 of S. miscanthi (SmAChE1). Enzyme kinetic and inhibition experiments showed that the recombinant SmAChE1S431F was more insensitive to pirimicarb and omethoate than the recombinant SmAChE1. Furthermore, two overexpression P450 genes (CYP6K1 and CYP6A14) associated with pirimicarb resistance of S. miscanthi were verified by RNAi. These results suggested both target alteration and enhanced metabolism contributed to high pirimicarb resistance of S. miscanthi in the field and laboratory. These findings lay a foundation for further elucidating the mechanism of pirimicarb resistance in S. miscanthi, and have important implications for the resistance management of S. miscanthi control.

microRNA-3037 targeting CYP6CY2 confers imidacloprid resistance to Sitobion miscanthi (Takahashi).

The wheat aphid Sitobion miscanthi is a dominant and destructive pest in agricultural production. Insecticides are the main substances used for effective control of wheat aphids. However, their extensive application has caused severe resistance of wheat aphids to some insecticides; therefore, exploring resistance mechanisms is essential for wheat aphid management. In the present study, CYP6CY2, a new P450 gene, was isolated and overexpressed in the imidacloprid-resistant strain (SM-R) compared to the imidacloprid-susceptible strain (SM-S). The increased sensitivity of S. miscanthi to imidacloprid after knockdown of CYP6CY2 indicates that it could be associated with imidacloprid resistance. Subsequently, the posttranscriptional regulation of CYP6CY2 in the 3' UTR by miR-3037 was confirmed, and CYP6CY2 participated in imidacloprid resistance. This finding is critical for determining the role of P450 in relation to the resistance of S. miscanthi to imidacloprid. It is of great significance to understand this regulatory mechanism of P450 expression in the resistance of S. miscanthi to neonicotinoids.

Genetic diversity in vector populations influences the transmission efficiency of an important plant virus.

The transmission efficiency of aphid-vectored plant viruses can differ between aphid populations. Intra-species diversity (genetic variation, endosymbionts) is a key determinant of aphid phenotype; however, the extent to which intra-species diversity contributes towards variation in virus transmission efficiency is unclear. Here, we use multiple populations of two key aphid species that vector barley yellow dwarf virus (BYDV) strain PAV (BYDV-PAV), the grain aphid (Sitobion avenae) and the bird cherry-oat aphid (Rhopalosiphum padi), and examine how diversity in vector populations influences virus transmission efficiency. We use Illumina sequencing to characterize genetic and endosymbiont variation in multiple Si. avenae and Rh. padi populations and conduct BYDV-PAV transmission experiments to identify links between intra-species diversity in the vector and virus transmission efficiency. We observe limited variation in the transmission efficiency of Si. avenae, with transmission efficiency consistently low for this species. However, for Rh. padi, we observe a range of transmission efficiencies and show that BYDV transmission efficiency is influenced by genetic diversity within the vector, identifying 542 single nucleotide polymorphisms that potentially contribute towards variable transmission efficiency in Rh. padi. Our results represent an important advancement in our understanding of the relationship between genetic diversity, vector-virus interactions, and virus transmission efficiency.

PacBio Hi-Fi genome assembly of Sipha maydis, a model for the study of multipartite mutualism in insects.

Dependence on multiple nutritional endosymbionts has evolved repeatedly in insects feeding on unbalanced diets. However, reference genomes for species hosting multi-symbiotic nutritional systems are lacking, even though they are essential for deciphering the processes governing cooperative life between insects and anatomically integrated symbionts. The cereal aphid Sipha maydis is a promising model for addressing these issues, as it has evolved a nutritional dependence on two bacterial endosymbionts that complement each other. In this study, we used PacBio High fidelity (HiFi) long-read sequencing to generate a highly contiguous genome assembly of S. maydis with a length of 410 Mb, 3,570 contigs with a contig N50 length of 187 kb, and BUSCO completeness of 95.5%. We identified 117 Mb of repetitive sequences, accounting for 29% of the genome assembly, and predicted 24,453 protein-coding genes, of which 2,541 were predicted enzymes included in an integrated metabolic network with the two aphid-associated endosymbionts. These resources provide valuable genetic and metabolic information for understanding the evolution and functioning of multi-symbiotic systems in insects.

Design, Synthesis, and Bioactivity of Trifluoroethylthio-Substituted Phenylpyrazole Derivatives.

As the first marketed phenylpyrazole insecticide, fipronil exhibited remarkable broad-spectrum insecticidal activity. However, it poses a significant threat to aquatic organisms and bees due to its high toxicity. Herein, 35 phenylpyrazole derivatives containing a trifluoroethylthio group on the 4 position of the pyrazole ring were designed and synthesized. The predicted physicochemical properties of all of the compounds were within a reasonable range. The biological assay results revealed that compound 7 showed 69.7% lethality against Aedes albopictus (A. albopictus) at the concentration of 0.125 mg/L. Compounds 7, 7g, 8d, and 10j showed superior insecticidal activity for the control of Plutella xylostella (P. xylostella). Notably, compound 7 showed similar insecticidal activity against Aphis craccivora (A. craccivora) compared with fipronil. Potential surface calculation and molecular docking suggested that different lipophilicity and binding models to the Musca domestica (M. domestica) gamma-aminobutyric acid receptors may be responsible for the decreased activity of the tested derivatives. Toxicity tests indicated that compound 8d (LC50 = 14.28 mg/L) induced obviously 14-fold lower toxicity than fipronil (LC50 = 1.05 mg/L) on embryonic-juvenile zebrafish development.

Computational Modeling Oriented Substructure Splicing Application in the Identification of Thiazolidine Derivatives as Potential Low Honeybee Toxic Neonicotinoids.

With the aim of identifying novel neonicotinoid insecticides with low bee toxicity, a series of compounds bearing thiazolidine moiety, which has been shown to be low bee toxic, were rationally designed through substructure splicing strategy and evaluated insecticidal activities. The optimal compounds A24 and A29 exhibited LC50 values of 30.01 and 17.08 mg/L against Aphis craccivora, respectively. Electrophysiological studies performed on Xenopus oocytes indicated that compound A29 acted on insect nAChR, with EC50 value of 50.11 µM. Docking binding mode analysis demonstrated that A29 bound to Lymnaea stagnalis acetylcholine binding protein through H-bonds with the residues of D_Arg55, D_Leu102, and D_Val114. Quantum mechanics calculation showed that A29 had a higher highest occupied molecular orbit (HOMO) energy and lower vertical ionization potential (IP) value compared to the high bee toxic imidacloprid, showing potentially low bee toxicity. Bee toxicity predictive model also indicated that A29 was nontoxic to honeybees. Our present work identified an innovative insecticidal scaffold and might facilitate the further exploration of low bee toxic neonicotinoid insecticides.

Rational Design of Triazinone Derivatives with Low Bee Toxicity Based on the Binding Mechanism of Neonicotinoids to Apis mellifera.

Bees, one of the most vital pollinators in the ecosystem and agriculture, are currently threatened by neonicotinoids. To explore the molecular mechanisms of neonicotinoid toxicity to bees, the different binding modes of imidacloprid, thiacloprid, and flupyradifurone with nicotinic acetylcholine receptor (nAChR) α1ß1 and cytochrome P450 9Q3 (CYP9Q3) were studied using homology modeling and molecular dynamics simulations. These mechanisms provided a basis for the design of compounds with a potential low bee toxicity. Consequently, we designed and synthesized a series of triazinone derivatives and assessed their bioassays. Among them, compound 5a not only displayed substantially insecticidal activities against Aphis glycines (LC50 = 4.40 mg/L) and Myzus persicae (LC50 = 6.44 mg/L) but also had low toxicity to Apis mellifera. Two-electrode voltage clamp recordings further confirmed that compound 5a interacted with the M. persicae nAChR α1 subunit but not with the A. mellifera nAChR α1 subunit. This work provides a paradigm for applying molecular toxic mechanisms to the design of compounds with low bee toxicity, thereby aiding the future rational design of eco-friendly nicotinic insecticides.

Intracellular defensive symbiont is culturable and capable of transovarial, vertical transmission.

Insects frequently form heritable associations with beneficial bacteria that are vertically transmitted from parent to offspring. Long-term vertical transmission has repeatedly resulted in genome reduction and gene loss, rendering many such bacteria incapable of establishment in axenic culture. Among aphids, heritable endosymbionts often provide context-specific benefits to their hosts. Although these associations have large impacts on host phenotypes, experimental approaches are often limited by an inability to cultivate these microbes. Here, we report the axenic culture of Candidatus Fukatsuia symbiotica strain WIR, a heritable bacterial endosymbiont of the pea aphid, Acyrthosiphon pisum. Whole-genome sequencing revealed similar genomic features and high sequence similarity to previously described strains, suggesting that the cultivation techniques used here may be applicable to Ca. F. symbiotica strains from distantly related aphids. Microinjection of cultured Ca. F. symbiotica into uninfected aphids revealed that it can reinfect developing embryos and that infections are maintained in subsequent generations via transovarial maternal transmission. Artificially infected aphids exhibit phenotypic and life history traits similar to those observed for native infections. Our results show that Ca. F. symbiotica may be a useful tool for experimentally probing the molecular mechanisms underlying host-symbiont interactions in a heritable symbiosis. IMPORTANCE: Diverse eukaryotic organisms form stable, symbiotic relationships with bacteria that provide benefits to their hosts. While these associations are often biologically important, they can be difficult to probe experimentally because intimately host-associated bacteria are difficult to access within host tissues, and most cannot be cultured. This is especially true for the intracellular, maternally inherited bacteria associated with many insects, including aphids. Here, we demonstrate that a pea aphid-associated strain of the heritable endosymbiont, Candidatus Fukatsuia symbiotica, can be grown outside of its host using standard microbiology techniques and can readily re-establish infection that is maintained across host generations. These artificial infections recapitulate the effects of native infections, making this host-symbiont pair a useful experimental system.

Small but strong: herbivory by sap-feeding insect reduces plant progeny growth but enhances direct and indirect anti-herbivore defenses.

The transmission of resistance traits to herbivores across subsequent generations is an important strategy employed by plants to enhance their fitness in environments with high herbivore pressure. However, our understanding of the impact of maternal herbivory on direct and indirect induced chemical defenses of progeny, as well as the associated costs, is currently limited to herbivory by leaf-chewing insects. In this study, we investigated the transgenerational effects of a sap-feeding insect, the green peach aphid Myzus persicae, on direct and indirect chemical defenses of bell pepper plants (Capsicum annuum), and whether the effects entail costs to plant growth. Aphid herbivory on parental plants led to a reduced number of seeds per fruit, which exhibited lower germination rates and produced smaller seedlings compared to those from non-infested parental plants. In contrast, the progeny of aphid-infested plants were less preferred as hosts by aphids and less suitable than the progeny of non-infested plants. This enhanced resistance in the progeny of aphid-infested plants coincided with elevated levels of both constitutive and herbivore-induced total phenolic compounds, compared to the progeny of non-infested plants. Furthermore, the progeny of aphid-infested plants emitted herbivore-induced plant volatiles (HIPVs) that were more attractive to the aphid parasitoid Aphidius platensis than those emitted by the progeny of non-infested plants. Our results indicate that herbivory by sap-feeding insect induces transgenerational resistance on progeny bell pepper plants, albeit at the expense of vegetative growth.

Bioaccumulation and transferreing for impacts on Cd and Pb by aphid consumption of the broad bean, Vicia faba L, in soil heavy metal pollution.

Heavy metal pollution threatens human and ecological health. Heavy metals can exist in the soil for a long time and migrate to organisms along the food chain. However, only a few studies have investigated the effects of a single stress on broad beans. Here, we aimed to characterize Cd and Pb bioaccumulation, at varying concentrations, in the broad bean, Vicia faba L. We also determined how the bioaccumulated metals are impacted by aphids that consume the plant. No significant difference was noted in the germination rates of broad beans at the early stage of planting (after 8 days), but eventually, the germination rates of broad beans at all time points first decreased and then increased, and the highest inhibition efficiency was observed in the T3 group (12.5 mg/L Cd2+ + 50 mg/L Pb2+). Fourteen days after planting, there was no significant difference in seedling height between the T5 (50 mg/L Cd2+ + 200 mg/L Pb2+) and control groups; however, that in the other groups decreased significantly and there was no dependence between stress concentration and inhibition efficiency. In addition, both Cd and Pb in the soil could be transferred to broad beans, and the concentration of Pb in the roots of broad beans was greater than that of Cd, whereas the opposite was observed in the stems and leaves. Notably, under mixed stress, aphids could significantly reduce the content of Cd in broad beans; similarly, the Pb content in the roots and stems of broad beans decreased significantly after being infested with aphids but increased significantly in the leaves. Further, the aphid infestation decreased the Pb content in the soil and the soil Cd content in the highest concentration group (T5 group) (50 mg/L Cd2+ + 200 mg/L Pb2+). These results highlight the necessity of focusing on the effect of insects on heavy metal remediation in plants and provide a new perspective for reducing plant Cd toxicity.

Hesperidin as a Species-Specific Modifier of Aphid Behavior.

Hesperidin is a highly bioactive natural flavonoid whose role in ecological interactions is poorly known. In particular, the effects of hesperidin on herbivores are rarely reported. Flavonoids have been considered as prospective biopesticides; therefore, the aim of the present study was to examine the influence of hesperidin on the host plant selection behavior of three aphid (Hemiptera: Aphididae) species: Acyrthosiphon pisum Harrris, Rhopalosiphum padi (L.), and Myzus persicae (Sulz.). The aphid host plants were treated with 0.1% and 0.5% ethanolic solutions of hesperidin. Aphid probing behavior in the no-choice experiment was monitored using electropenetrography and aphid settling on plants in the choice experiment was recorded. The results demonstrated that hesperidin can be applied as a pre-ingestive, ingestive, and post-ingestive deterrent against A. pisum, as an ingestive deterrent against R. padi, and as a post-ingestive deterrent against M. persicae using the relatively low 0.1% concentration. While in A. pisum the deterrent effects of hesperidin were manifested as early as during aphid probing in peripheral plant tissues, in M. persicae, the avoidance of plants was probably the consequence of consuming the hesperidin-containing phloem sap.

Design, Synthesis, and Insecticidal Activity of Pyridino[1,2-a]pyrimidines Containing Indole Moeites at the 1-Position.

Research on mesoionic structures in pesticide design has gained significant attention in recent years. However, the 1-position of pyridino[1,2-a]pyrimidine is usually designed with 2-chlorothiazole, 2-chloropyridine, or cyano moieties commonly found in neonicotinoid insecticides. In order to enrich the available pharmacophore library, here, we disclose a series of new pyridino[1,2-a]pyrimidine mesoionics bearing indole-containing substituents at the 1-position. Most of these target compounds are confirmed to have good insecticidal activity against aphids through bioevaluation. In addition, a three-dimensional structure-activity relationship model is established to allow access to optimal compound F45 with an LC50 value of 2.97 mg/L. This value is comparable to the property achieved by the positive control triflumezopyrim (LC50 = 2.94 mg/L). Proteomics and molecular docking analysis suggest that compound F45 has the potential to modulate the functioning of the aphid nervous system through its interaction with neuronal nicotinic acetylcholine receptors. This study expands the existing pharmacophore library for the future development of new mesoionic insecticides based on 1-position modifications of the pyridino[1,2-a]pyrimidine scaffold.

Ecological divergence despite common mating sites: Genotypes and symbiotypes shed light on cryptic diversity in the black bean aphid species complex.

Different host plants represent ecologically dissimilar environments for phytophagous insects. The resulting divergent selection can promote the evolution of specialized host races, provided that gene flow is reduced between populations feeding on different plants. In black bean aphids belonging to the Aphis fabae complex, several morphologically cryptic taxa have been described based on their distinct host plant preferences. However, host choice and mate choice are largely decoupled in these insects: they are host-alternating and migrate between specific summer host plants and shared winter hosts, with mating occurring on the shared hosts. This provides a yearly opportunity for gene flow among aphids using different summer hosts, and raises the question if and to what extent the ecologically defined taxa are reproductively isolated. Here, we analyzed a geographically and temporally structured dataset of microsatellite genotypes from A. fabae that were mostly collected from their main winter host Euonymus europaeus, and additionally from another winter host and fourteen summer hosts. The data reveals multiple, strongly differentiated genetic clusters, which differ in their association with different summer and winter hosts. The clusters also differ in the frequency of infection with two heritable, facultative endosymbionts, separately hinting at reproductive isolation and divergent ecological selection. Furthermore, we found evidence for occasional hybridization among genetic clusters, with putative hybrids collected more frequently in spring than in autumn. This suggests that similar to host races in other phytophagous insects, both prezygotic and postzygotic barriers including selection against hybrids maintain genetic differentiation among A. fabae taxa, despite a common mating habitat.

Characterization of a new greenbug resistance gene Gb9 in a synthetic hexaploid wheat.

Greenbug [Schizaphis graminum (Rondani)] is a serious insect pest that not only damages cereal crops, but also transmits several destructive viruses. The emergence of new greenbug biotypes in the field makes it urgent to identify novel greenbug resistance genes in wheat. CWI 76364 (PI 703397), a synthetic hexaploid wheat (SHW) line, exhibits greenbug resistance. Evaluation of an F2:3 population from cross OK 14319 × CWI 76364 indicated that a dominant gene, designated Gb9, conditions greenbug resistance in CWI 76364. Selective genotyping of a subset of F2 plants with contrasting phenotypes by genotyping-by-sequencing identified 25 SNPs closely linked to Gb9 on chromosome arm 7DL. Ten of these SNPs were converted to Kompetitive allele-specific polymerase chain reaction (KASP) markers for genotyping the entire F2 population. Genetic analysis delimited Gb9 to a 0.6-Mb interval flanked by KASP markers located at 599,835,668 bp (Stars-KASP872) and 600,471,081 bp (Stars-KASP881) on 7DL. Gb9 was 0.5 cM distal to Stars-KASP872 and 0.5 cM proximal to Stars-KASP881. Allelism tests indicated that Gb9 is a new greenbug resistance gene which confers resistance to greenbug biotypes C, E, H, I, and TX1. TX1 is one of the most widely virulent biotypes and has overcome most known wheat greenbug resistance genes. The introgression of Gb9 into locally adapted wheat cultivars is of economic importance, and the KASP markers developed in this study can be used to tag Gb9 in cultivar development.

Salivary Protein Cyclin-Dependent Kinase-like from Grain Aphid Sitobion avenae Suppresses Wheat Defense Response and Enhances Aphid Adaptation.

Aphids are insect pests that suck phloem sap and introduce salivary proteins into plant tissues through saliva secretion. The effector of salivary proteins plays a key role in the modulation of host plant defense responses and enhancing aphid host adaptation. Based on previous transcriptome sequencing results, a candidate effector cyclin-dependent kinase-like (CDK) was identified from the grain aphid Sitobion avenae. In this study, the function of SaCDK in wheat defense response and the adaptation of S. avenae was investigated. Our results showed that the transient overexpression of SaCDK in tobacco Nicotiana benthamiana suppressed cell death triggered by mouse pro-apoptotic protein-BAX or Phytophthora infestans PAMP-INF1. SaCDK, delivered into wheat cells through a Pseudomonas fluorescens-mediated bacterial type III secretion system, suppressed callose deposition in wheat seedlings, and the overexpression of SaCDK in wheat significantly decreased the expression levels of salicylic acid and jasmonic acid signaling pathway-related genes phenylalanine ammonia lyase (PAL), pathogenesis-related 1 protein (PR1), lipoxygenase (LOX) and Ω-3 fatty acid desaturase (FAD). In addition, aphid bioassay results showed that the survival and fecundity of S. avenae were significantly increased while feeding on the wheat plants carrying SaCDK. Taken together, our findings demonstrate that the salivary protein SaCDK is involved in inhibiting host defense response and improving its host adaptation, which lays the foundation to uncover the mechanism of the interaction of cereal aphids and host plants.

Computer-driven Evolution of Myrosinase from the Cabbage Aphid for Efficient Production of (R)-Sulforaphane.

Myrosinase (Myr) catalyzes the hydrolysis of glucosinolates, yielding biologically active metabolites. In this study, glucoraphanin (GRA) extracted from broccoli seeds was effectively hydrolyzed using a Myr-obtained cabbage aphid (Brevicoryne brassicae) (BbMyr) to produce (R)-sulforaphane (SFN). The gene encoding BbMyr was successfully heterologously expressed in Escherichia coli, resulting in the production of 1.6 g/L (R)-SFN, with a remarkable yield of 20.8 mg/gbroccoli seeds, achieved using recombination E. coli whole-cell catalysis under optimal conditions (pH 4.5, 45 °C). Subsequently, BbMyr underwent combinatorial simulation-driven mutagenesis, yielding a mutant, DE9 (N321D/Y426S), showing a remarkable 2.91-fold increase in the catalytic efficiency (kcat/KM) compared with the original enzyme. Molecular dynamics simulations demonstrated that the N321D mutation in loopA of mutant DE9 enhanced loopA stability by inducing favorable alterations in hydrogen bonds, while the Y426S mutation in loopB decreased spatial resistance. This research lays a foundation for the environmentally sustainable enzymatic (R)-SFN synthesis.