Catalytically inactive long prokaryotic Argonaute systems employ distinct effectors to confer immunity via abortive infection.

Argonaute proteins (Agos) bind short nucleic acids as guides and are directed by them to recognize target complementary nucleic acids. Diverse prokaryotic Agos (pAgos) play potential functions in microbial defense. The functions and mechanisms of a group of full-length yet catalytically inactive pAgos, long-B pAgos, remain unclear. Here, we show that most long-B pAgos are functionally connected with distinct associated proteins, including nucleases, Sir2-domain-containing proteins and trans-membrane proteins, respectively. The long-B pAgo-nuclease system (BPAN) is activated by guide RNA-directed target DNA recognition and performs collateral DNA degradation in vitro. In vivo, the system mediates genomic DNA degradation after sensing invading plasmid, which kills the infected cells and results in the depletion of the invader from the cell population. Together, the BPAN system provides immunoprotection via abortive infection. Our data also suggest that the defense strategy is employed by other long-B pAgos equipped with distinct associated proteins.

Eastern honeybee Apis cerana sense cold flowering plants by increasing the static binding affinity of odorant-binding protein to cold floral volatiles from loquats.

Eastern honeybee Apis cerana has important ecological value for the cold flowering loquat flower pollination in early winter in East Asia. However, the low-temperature adaptive pollination mechanism has not yet been revealed. One odorant-binding protein, OBP2, had been found that could bind to some plant volatiles with strong affinity before. In this study, by using competitive fluorescence binding assay, we first measured the ligand-binding profiles of recombinant OBP2 protein with nine representative aroma chemical substances from loquat flowers. Thermodynamic results showed that three loquat volatiles, 4-Methoxybenzaldehyde, (E)-Ethyl cinnamate, and Methyl cinnamate, have the strongest binding affinity with OBP2 with the static process. And interestingly their binding affinity significantly increased at low temperature (285 K/12 °C) compared to high temperature (298 K/25 °C). In addition, site-directed mutagenesis results showed that Met55 and Lys51 may be the key amino acid sites in the electrostatic and hydrophobic interactions of OBP2 interacting with Methyl cinnamate, respectively. This study suggests that OBP2 is functionally similar and universal in binding to different flower volatiles at low temperatures. Our studies interpreted a novel olfactory mechanism of A. cerana sensing loquat floral volatiles in cold early winter, and enrich a theoretical molecular basis for the temperature-adaptive ecological mechanism of insects' pollination.

Unique dynamic mode between Artepillin C and human serum albumin implies the characteristics of Brazilian green propolis representative bioactive component.

As a representative bioactive component in Brazil green propolis, Artepillin C (ArtC; 3, 5-diprenyl-4-hydroxycinnamic acid) has been reported a wide variety of physiological activities including anti-tumor, anti-inflammatory, and antimicrobial activity etc. However, it seems incompatible that ArtC in vivo was characterized as low absorption efficiency and low bioavailability. In order to obtain the elucidation, we further investigated the physicochemical basis of ArtC interacting with human serum albumin (HSA) in vitro. We found a unique dynamic mode interaction between ArtC and HSA, which is completely different from other reported propolis bioactive components. Thermodynamic analysis showed that hydrophobic interactions and electrostatic forces are the main driving force. The competitive assay indicates that the binding site of ArtC with HSA is close to the Sudlow's site I. The findings of this study reveal the unique physicochemical transport mechanism of ArtC in the human body, which helps to further understand the uniqueness of the representative functional components of Brazilian green propolis in the human body.

Chemical structure of semiochemicals and key binding sites together determine the olfactory functional modes of odorant-binding protein 2 in Eastern honey bee, Apis cerana.

Insects can exhibit flexible olfaction that is sensitive to complex natural chemical environments. Odorant-binding proteins (OBPs) in insects' antennal chemosensilla can act as transporters of plant volatiles and pheromones across the sensillar lymph. Although the physiological functions of OBPs have been widely reported, it is still unclear how OBP binds to ligands with various structures in detail. Here, we further investigated the ligand-binding modes and characteristics of AcerOBP2 from the Eastern honey bee (Apis cerana). The results showed that, as a specific protein distributed below the base of chemosensilla on the antennal surface, AcerOBP2 was strongly bound with the candidate floral volatiles and bee pheromones. By docking analysis and site-directed mutagenesis, four different binding modes were found in the five AcerOBP2 mutants between six ligands. Two key amino acids, Ser123 and Lys51, play a key role in AcerOBP2 binding to odors, depending on the presence or absence of hydrogen bonds. In addition, the binding modes depend on their chemical structures and the binding poses of the diverse ligands. These results not only further prompted the functional basis of the relationship between the chemical structures of odorants and bee OBPs, but also revealed the complexity of the flexible behavioral modes of odor binding in insect olfactory systems.

Physicochemical Basis and Comparison of Two Type II Sex Pheromone Components Binding with Pheromone-Binding Protein 2 from Tea Geometrid, Ectropis obliqua.

Lepidopteran geometrid moth can produce complex Type II sex pheromone components to attract males and trigger mating behavior. Although several sex pheromone components have been identified, it remains unclear whether their physicochemical roles in sex pheromone sensing are the same. Therefore, we utilized tea geometrid ( Ectropis obliqua) as an example model to investigate and compare the physicochemical basis of two key Type II sex pheromone components, cis-6,7-epoxy-(3Z,9Z)-3,9-octadecadiene ( Z3 Z9-6,7-epo-18:Hy) and ( Z, Z, Z)-3,6,9-octadecatriene (Z3Z6Z9-18:Hy), interacting with pheromone-binding protein 2 ( EoblPBP2) from E. obliqua. Multispectral, thermodynamic, docking, and site-directed mutagenesis indicated that the major sex pheromone component Z3Z9-6,7-epo-18:Hy is more susceptible to pH-tuned than the minor component Z3Z6Z9-18:Hy, whereas Z3Z6Z9-18:Hy seems to be more susceptible to temperature and amino acid mutations than Z3Z9-6,7-epo-18:Hy. Our study suggests that different components of Type II sex pheromone play different binding characters under specific conditions in the physicochemical behavior. This deeply supplements the theoretical knowledge of Type II pheromones involved in the recognition and discrimination in the Lepidopteran sex pheromones family.

Schaftoside Interacts With NlCDK1 Protein: A Mechanism of Rice Resistance to Brown Planthopper, Nilaparvata lugens.

Brown planthopper (BPH) Nilaparvata lugens Stål is a serious insect pest of rice in Asian countries. Active compounds have close relationship with rice resistance against BPH. In this study, HPLC, MS/MS, and NMR techniques were used to identify active compounds in total flavonoids of rice. As a result, a BPH resistance-associated compound, Peak 1 in HPLC chromatogram of rice flavonoids, was isolated and identified as schaftoside. Feeding experiment with artificial diet indicated that schaftoside played its role in a dose dependent manner, under the concentration of 0.10 and 0.15 mg mL-1, schaftoside showed a significant inhibitory effect on BPH survival (p < 0.05), in comparison with the control. The fluorescent spectra showed that schaftoside has a strong ability to bind with NlCDK1, a CDK1 kinase of BPH. The apparent association constant KA for NlCDK1 binding with schaftoside is 6.436 × 103 L/mol. Docking model suggested that binding of schaftoside might affect the activation of NlCDK1 as a protein kinase, mainly through interacting with amino acid residues Glu12, Thr14 and Val17 in the ATP binding element GXGXXGXV (Gly11 to Val18). Western blot using anti-phospho-CDK1 (pThr14) antibody confirmed that schaftoside treatment suppressed the phosphorylation on Thr-14 site of NlCDK1, thus inhibited its activation as a kinase. Therefore, this study revealed the schaftoside-NlCDK1 interaction mode, and unraveled a novel mechanism of rice resistance against BPH.

Various Bee Pheromones Binding Affinity, Exclusive Chemosensillar Localization, and Key Amino Acid Sites Reveal the Distinctive Characteristics of Odorant-Binding Protein 11 in the Eastern Honey Bee, Apis cerana.

Odorant-binding proteins (OBPs) are the critical elements responsible for binding and transporting odors and pheromones in the sensitive olfactory system in insects. Honey bees are representative social insects that have complex odorants and pheromone communication systems relative to solitary insects. Here, we first cloned and characterized OBP11 (AcerOBP11), from the worker bees antennae of Eastern honey bee, Apis cerana. Based on sequence and phylogenetic analysis, most sequences homologous to AcerOBP11 belong to the typical OBPs family. The transcriptional expression profiles showed that AcerOBP11 was expressed throughout the developmental stages and highly specifically expressed in adult antennae. Using immunofluorescence localization, AcerOBP11 in worker bee's antennae was only localized in the sensilla basiconica (SB) near the fringe of each segment. Fluorescence ligand-binding assay showed that AcerOBP11 protein had strong binding affinity with the tested various bee pheromones components, including the main queen mandibular pheromones (QMPs), methyl p-hydroxybenzoate (HOB), and (E)-9-oxo-2-decanoic acid (9-ODA), alarm pheromone (n-hexanol), and worker pheromone components. AcerOBP11 also had strong binding affinity to plant volatiles, such as 4-Allylveratrole. Based on the docking and site-directed mutagenesis, two key amino acid residues (Ile97 and Ile140) were involved in the binding of AcerOBP11 to various bee pheromones. Taken together, we identified that AcerOBP11 was localized in a single type of antennal chemosensilla and had complex ligand-binding properties, which confer the dual-role with the primary characteristics of sensing various bee pheromones and secondary characteristics of sensing general odorants. This study not only prompts the theoretical basis of OBPs-mediated bee pheromones recognition of honey bee, but also extends the understanding of differences in pheromone communication between social and solitary insects.

Combinatorial multispectral, thermodynamics, docking and site-directed mutagenesis reveal the cognitive characteristics of honey bee chemosensory protein to plant semiochemical.

In the chemoreceptive system of insects, there are always some soluble binding proteins, such as some antennal-specific chemosensory proteins (CSPs), which are abundantly distributed in the chemosensory sensillar lymph. The antennal-specific CSPs usually have strong capability to bind diverse semiochemicals, while the detailed interaction between CSPs and the semiochemicals remain unclear. Here, by means of the combinatorial multispectral, thermodynamics, docking and site-directed mutagenesis, we detailedly interpreted a binding interaction between a plant semiochemical ß-ionone and antennal-specific CSP1 from the worker honey bee. Thermodynamic parameters (ΔH < 0, ΔS > 0) indicate that the interaction is mainly driven by hydrophobic forces and electrostatic interactions. Docking prediction results showed that there are two key amino acids, Phe44 and Gln63, may be involved in the interacting process of CSP1 to ß-ionone. In order to confirm the two key amino acids, site-directed mutagenesis were performed and the binding constant (KA) for two CSP1 mutant proteins was reduced by 60.82% and 46.80% compared to wild-type CSP1. The thermodynamic analysis of mutant proteins furtherly verified that Phe44 maintained an electrostatic interaction and Gln63 contributes hydrophobic and electrostatic forces. Our investigation initially elucidates the physicochemical mechanism of the interaction between antennal-special CSPs in insects including bees to plant semiochemicals, as well as the development of twice thermodynamic analysis (wild type and mutant proteins) combined with multispectral and site-directed mutagenesis methods.

Physicochemical Evidence on Sublethal Neonicotinoid Imidacloprid Interacting with an Odorant-Binding Protein from the Tea Geometrid Moth, Ectropis obliqua.

Nowadays the excessive usage of neonicotinoid insecticides always results in residues in Chinese tea fields. It is not clear whether the insecticide residue at the sublethal level influences the physiological processes of tea pests. Here, we provide evidence of interaction between the neonicotinoid imidacloprid and a general odorant-binding protein, EoblGOBP2, from the tea geometrid moth, Ectropis obliqua. The interacting process was demonstrated through multiple fluorescence spectra, UV absorption spectra, circular dichroism (CD) spectra, molecular docking, etc. The binding mode was determined to be static (from 300 to 310 K) and dynamic quenching (from 290 to 300 K). The binding distance was calculated to be 6.9 nm on the basis of FRET theory. According to the thermodynamic analysis, the process was mainly driven by enthalpy (ΔH < 0), and hydrogen bond and van der Waals interactions were the main driving forces in the static and dynamic binding cases, respectively. Moreover, synchronous fluorescence spectra and CD spectra analysis showed stretching of the EoblGOBP2 peptide chains with a decreasing α-helix when imidacloprid was added. Molecular docking was applied and predicted that two hydrogen bonds were formed between imidacloprid and Arg110 in the mature peptide of EoblGOBP2. Moreover, when the absolute amounts of EoblGOBP2 in the moth antennae were measured and calculated by using real-time PCR, it was estimated that imidacloprid at sublethal level (about 0.233 and 0.175 ng/male and female moth antennae, respectively) inhibited the binding of a tea volatile, E-2-hexenal, to EoblGOBP2 at about half. This study indicates that neonicotinoid insecticide at sublethal level may still affect the olfactory cognition of the tea geometrid moth to volatile compounds from tea leaves.

Sublethal doses of neonicotinoid imidacloprid can interact with honey bee chemosensory protein 1 (CSP1) and inhibit its function.

As a frequently used neonicotinoid insecticide, imidacloprid can impair the chemoreceptive behavior of honey bees even at sublethal doses, while the physiochemical mechanism has not been further revealed. Here, multiple fluorescence spectra, thermodynamic method, and molecular docking were used to study the interaction and the functional inhibition of imidacloprid to the recombinant CSP1 protein in Asian honey bee, Apis cerana. The results showed that the fluorescence intensity (λem = 332 nm) of CSP1 could be significantly quenched by imidacloprid in a dynamic mode. During the quenching process, ΔH > 0, ΔS > 0, indicating that the acting forces of imidacloprid with CSP1 are mainly hydrophobic interactions. Synchronous fluorescence showed that the fluorescence of CSP1 was mainly derived from tryptophan, and the hydrophobicity of tryptophan decreased with the increase of imidacloprid concentration. Molecular docking predicted the optimal pose and the amino acid composition of the binding process. Circular dichroism (CD) spectra showed that imidacloprid reduced the α-helix of CSP1 and caused the extension of the CSP1 peptide chain. In addition, the binding of CSP1 to floral scent ß-ionone was inhibited by nearly 50% of the apparent association constant (KA) in the presence of 0.28-2.53 ng/bee of imidacloprid, and the inhibition rate of nearly 95% at 3.75 ng/bee of imidacloprid at sublethal dose level. This study initially revealed the molecular physiochemical mechanism that sublethal doses of neonicotinoid still interact and inhibit the physiological function of the honey bees' chemoreceptive system.