Broad-spectrum Recognition Research Articles

A polydopamine coated magnetic spiral switchable composite material for photothermal sterilization and dual-color fluorescence detection of food-borne pathogens

A multifunctional magnetic composite material (MSp@PDA) featuring with helical structure, superparamagnetism, photothermal properties, and fluorescence quenching capability was synthesized through sequential magnetization and in-situ polydopamine coating on the Spirulina surface, then applied for multi-scenario application of pathogens capture, sterilization, and sensitive detection. The pathogens capture and sterilization was investigated by loading bacterial broad-spectrum recognition molecule (MPBA) on MSp@PDA, the capture efficiencies of 91.6 % and 94.2 % were obtained for Escherichia coli and Staphylococcus aureus in 15 minutes, respectively, with complete photothermal sterilization achieved within 4 minutes. The fluorescence real-time detection of E. coli and S. aureus was realized by connecting dual-color fluorescent carbon dots to MSp@PDA through specific aptamers (dCD-apt-MSp@PDA), the corresponding detection limits were 2 and 3 cfu·mL−1, respectively, with linear ranges of 101∼107 cfu·mL−1. The spiked recovery rates of target bacteria in actual samples were 97.4∼101.1 % and 98.2∼101.4 % for E. coli and S. aureus, respectively. The constructed capture, sterilization, and fluorescence detection methods demonstrated promising application value for on-site monitoring of food-borne pathogens in complex matrices.

A highly sensitive electrochemical aptasensor for detecting broad-spectrum estrogen molecules in clinical samples

The lack of sensitive and accurate monitoring methods for in vivo estrogen levels presents challenges for better prevention of estrogen-induced diseases. We have developed a label-free electrochemical aptasensor that demonstrates high sensitivity and selectivity for the broad-spectrum detection of estrogen molecules. This biosensor uses gold nanoparticles for electrochemical signal amplification and aptamers for broad-spectrum target recognition, enabling precise detection of trace amounts of estrogen in serum samples. The aptasensor demonstrates high sensitivity in estrogen detection, with a linear detection range of 0.01–1 nM and a minimum detection limit of 3 pM. It also exhibits excellent selectivity and interference resistance, with a detection error of less than 19 %, even in the presence of high concentrations of other biological substances. Additionally, molecular dynamics simulations were performed to provide insights into the molecular mechanism of aptamer broad-spectrum recognition and construction principles underlying the sensor. We anticipate that this aptasensor will serve as a robust, convenient, and cost-effective detection method, offering a valuable solution for the prevention of estrogen-induced diseases.

Broad-spectrum detection of benzimidazoles with lateral flow immunoassay: A computational chemistry-assisted hapten design strategy and explore of molecular recognition mechanism

Benzimidazoles (BMZs) are a class of veterinary drugs with a benzimidazole ring, the abuse of which poses a serious threat to ecological balance and human health. Consequently, the development of broad-spectrum antibodies and rapid assays are crucial for detecting BMZs in food samples. Herein, we scientifically designed three hapten structures, predicted the availability of the hapten with computational chemistry, and subsequently verified the broad-spectrum with immunological experiments. A broad-spectrum monoclonal antibody (6F10) was prepared based on the predicted hapten-2. Molecular recognition studies illustrated intricate interactions between mAb 6F10 binding to BMZs attributed to halogen bonds and π-π/π-alkyl interactions, revealing key amino acid sites and demonstrating the reliability of the hapten prediction strategies. Finally, a broad-spectrum, rapid, and sensitive lateral flow immunoassay based on aggregation-induced emission microspheres with high fluorescence intensity was established. The LOD values of the proposed method for eight kinds of BMZs were 0.027, 0.032, 0.058, 0.091, 0.087, 0.246, 0.369, and 0.311 ng mL−1, respectively. In this work, a hapten prediction strategy based on a computational chemistry method effectively guided the preparation of antibodies for broad-spectrum recognition of BMZs, and the molecular recognition studies verified the interaction of mAb 6F10 with BMZs, enabling broad-spectrum and sensitive detection of BMZs in milk.

Phenylboronic Acid-Modified Membrane-Like Magnetic Quantum Dots Enable the Ultrasensitive and Broad-Spectrum Detection of Viruses by Lateral Flow Immunoassay.

Although lateral flow immunochromatographic assay (LFIA) is an effective point-of-care testing technology, it still cannot achieve broad-spectrum and ultrasensitive detection of viruses. Herein, we propose a multiplex LFIA platform using a two-dimensional graphene oxide (GO)-based magnetic fluorescent nanofilm (GF@DQD) as a multifunctional probe and 4-aminophenylboronic acid (APBA) as a broad-spectrum recognition molecule for viral glycoprotein detection. GF@DQD-APBA with enhanced magnetic/fluorescence properties and universal capture ability for multiple viruses was easily prepared through the electrostatic adsorption of one layer of density-controlled Fe3O4 nanoparticles (NPs) and thousands of small CdSe/ZnS-MPA quantum dots (QDs) on a monolayer GO sheet followed by chemical coupling with APBA on the QD surface. The GF@DQD-APBA probe enabled the universal capture and specific determination of different target viruses on the test strip through an arbitrary combination with the antibody-modified LFIA strip, thus greatly improving detection efficiency and reducing the cost and difficulty of multiplex LFIA for viruses. The proposed technique can simultaneously and sensitively diagnose three newly emerged viruses within 20 min with detection limits down to the pg/mL level. The excellent practicability of GF@DQD-APBA-LFIA was also demonstrated in the detection of 34 clinical specimens positive for SARS-CoV-2, revealing its potential for epidemic control and on-site viral detection.

Identification and characterization of three monoclonal antibodies targeting the SFTSV glycoprotein and displaying a broad spectrum recognition of SFTSV-related viruses

Identification and characterization of three monoclonal antibodies targeting the SFTSV glycoprotein and displaying a broad spectrum recognition of SFTSV-related viruses

Screening and Identification of Anti-Idiotypic Nanobody Capable of Broad-Spectrum Recognition of the Toxin Binding Region of Lepidopteran Cadherins and Mimicking Domain II of Cry2Aa Toxin.

The widespread use of Bacillus thuringiensis toxins as insecticides has brought about resistance problems. Anti-idiotypic nanobody approaches provide new strategies for resistance management and toxin evolution. In this study, the monoclonal antibody generated against the receptor binding region Domain II of Cry2Aa toxin was used as a target to screen materials with insecticidal activity. After four rounds of screening, anti-idiotypic nanobody 1C12 was obtained from the natural alpaca nanobody phage display library. To better analyze the activity of 1C12, soluble 1C12 was expressed by the Escherichia coli BL21 (DE3). The results showed that 1C12 not only binds the midgut brush border membrane vesicles (BBMV) of two lepidopteran species and cadherin CR9-CR11 of three lepidopteran species but also inhibits Cry2Aa toxins from binding to CR9-CR11. The insect bioassay showed that soluble 1C12 caused 25.65% and 23.61% larvae mortality of Helicoverpa armigera and Plutella xylostella, respectively. Although 1C12 has low insecticidal activity, soluble 1C12 possesses the ability to screen a broad-spectrum recognition of the toxin binding region of lepidopteran cadherins and can be used for the identification of the toxin binding region of other lepidopteran cadherins and the subsequent evolution of Cry2Aa toxin. The present study demonstrates a new strategy to screen for the production of novel insecticides.

Development of a Magnetically-Assisted SERS Biosensor for Rapid Bacterial Detection.

Ultrasensitive bacterial detection methods are crucial to ensuring accurate diagnosis and effective clinical monitoring, given the significant threat bacterial infections pose to human health. The aim of this study is to develop a biosensor with capabilities for broad-spectrum bacterial detection, rapid processing, and cost-effectiveness. A magnetically-assisted SERS biosensor was designed, employing wheat germ agglutinin (WGA) for broad-spectrum recognition and antibodies for specific capture. Gold nanostars (AuNSs) were sequentially modified with the Raman reporter molecules and WGA, creating a versatile SERS tag with high affinity for a diverse range of bacteria. Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) antibody-modified Fe3O4 magnetic gold nanoparticles (MGNPs) served as the capture probes. Target bacteria were captured by MGNPs and combined with SERS tags, forming a "sandwich" composite structure for bacterial detection. AuNSs, with a core size of 65 nm, exhibited excellent storage stability (RSD=5.6%) and demonstrated superior SERS enhancement compared to colloidal gold nanoparticles. Efficient binding of S. aureus and P. aeruginosa to MGNPs resulted in capture efficiencies of 89.13% and 85.31%, respectively. Under optimized conditions, the developed assay achieved a limit of detection (LOD) of 7 CFU/mL for S. aureus and 5 CFU/mL for P. aeruginosa. The bacterial concentration (10-106 CFU/mL) showed a strong linear correlation with the SERS intensity at 1331 cm-1. Additionally, high recoveries (84.8% - 118.0%) and low RSD (6.21% - 11.42%) were observed in spiked human urine samples. This study introduces a simple and innovative magnetically-assisted SERS biosensor for the sensitive and quantitative detection of S. aureus or P. aeruginosa, utilizing WGA and antibodies. The developed biosensor enhances the capabilities of the "sandwich" type SERS biosensor, offering a novel and effective platform for accurate and timely clinical diagnosis of bacterial infections.

Group selective aptamers: Broad-spectrum recognition of target groups in Cronobacter species and implementation of electrochemical biosensors as receptors

Aptamers are a versatile class of receptors with a high affinity and selectivity for specific targets. Although their ability to recognize individual targets has been extensively studied, some scenarios require the development of receptors capable of identifying all target groups. This study investigated the use of aptamers to achieve the broad-spectrum recognition of groups instead of individual targets. Aptamers were screened for selectively distinct groups of Cronobacter species associated with foodborne diseases. Seven Cronobacter spp. were divided into Group A (C. sakazakii, C. malonaticus, C. turicensis, and C. muytjensii) and Group B (C. dublinensis, C. condimenti, and C. universalis). Aptamers with exclusive selectivity for each group were identified, allowing binding to the species within their designated group while excluding those from the other group. The screened aptamers demonstrated reliable affinity and specificity with dissociation constants ranging from 1.3 to 399.7 nM for Group A and 4.0–24.5 nM for Group B. These aptamers have also been successfully employed as receptors in an electrochemical biosensor platform, enabling the selective detection of each group based on the corresponding aptamer (limit of detection was 7.8 and 3.2 CFU for Group A and Group B, respectively). The electrochemical sensor effectively detected the extent of infection in each group in powdered infant formula samples. This study highlights the successful screening and application of group-selective aptamers as sensing receptors, emphasizing their potential for diverse applications in different fields such as food safety, environmental monitoring, and clinical diagnostics, where the selective biosensing of target groups is crucial.

Tumor Microenvironment-Inspired Glutathione-Responsive Three-Dimensional Fibrous Network for Efficient Trapping and Gentle Release of Circulating Tumor Cells

Detection of circulating tumor cells (CTCs) is important for early cancer diagnosis, prediction of postoperative recurrence, and individualized treatment. However, it is still challenging to achieve efficient capture and gentle release of CTCs from the complex peripheral blood due to their rarity and fragility. Herein, inspired by the three-dimensional (3D) network structure and high glutathione (GSH) level of the tumor microenvironment (TME), a 3D stereo (3D-G@FTP) fibrous network is developed by combining the liquid-assisted electrospinning method, gas foaming technique, and metal-polyphenol coordination interactions to achieve efficient trapping and gentle release of CTCs. Compared with the traditional 2D@FTP fibrous scaffold, the 3D-G@FTP fibrous network could achieve higher capture efficiency (90.4% vs 78.5%) toward cancer cells in a shorter time (30 min vs 90 min). This platform showed superior capture performance toward heterogeneous cancer cells (HepG2, HCT116, HeLa, and A549) in an epithelial cell adhesion molecule (EpCAM)-independent manner. In addition, the captured cells with high cell viability (>90.0%) could be gently released under biologically friendly GSH stimulus. More importantly, the 3D-G@FTP fibrous network could sensitively detect 4-19 CTCs from six kinds of cancer patients' blood samples. We expect this TME-inspired 3D stereo fibrous network integrating efficient trapping, broad-spectrum recognition, and gentle release will promote the development of biomimetic devices for rare cell analysis.

A peptide Epitope − Synthetic hydrogel polymer conjugate that mimics insecticidal protein Receptors. Application in environmental and biological analysis

Monitoring transgenic crops (GMOs) is essential to track cross-breeding and ensure regulatory compliance but multiple protein variants now engineered into important crops challenges conventional antibody detection. Analysis of Bacillus thuringiensis (Bt) Cry protein − insect receptor complexes identified a peptide epitope, NITIHITDTNNK, with high sequence similarity to cadherin-like receptors across a family of Bt Cry proteins for broad-spectrum recognition. The peptide lacks the necessary physical, mechanical and chemical properties for practical applications. Conjugation of the peptide to a synthetic polymer creates a hybrid material that imparts robustness, adhesion, processability and polyvalence to the epitope. An epitope − hydrogel polymer conjugate library is created and screened for Bt Cry protein binding. A candidate with high adsorption capacity (∼995 mg g−1), fast adsorption kinetics (within 2 min), broad-spectrum specificity, and pH-responsive catch-and-release is identified as a selective sequestrant for extraction and isolation of Bt Cry variants from water and soil samples. An enrichment factor of 9.0 ∼ 12.5 and a linear range of 0.09 ∼ 0.45 µg g−1 was obtained for soil samples. The biotinylated epitope − polymer conjugate, a recognition element entirely different from an antibody, is integrated into an enzyme-linked immunosorbent assay (ELISA) for identification of cotton and rice crops transgenic with a variety of Bt Cry variants. The established ELISA assay exhibits wide linear range (0 ∼ 50 ng mL−1) and high accuracy (95.4 ∼ 108 %) for GMO cotton leaves. However, to distinguish GMO rice from non-GMO ones, background interference still needs to be overcome. We are now working for a more effective diagnostic for GMO rice.

Targeted preparation and recognition mechanism of broad-spectrum antibody specific to Aconitum alkaloids based on molecular modeling and its application in immunoassay

Ester-type Aconitum alkaloids (AAs), the main medicinal ingredients of Aconitum L. herbs, could cause brain and heart damage in humans and animals and have raised concerns worldwide. In the present study, we aimed to produce a high-performance and broad-spectrum antibody and establish an immunoassay method of ester-type AAs, 3-succinyl aconitine (ACO-HS) was selected as an optimal hapten from five designed haptens comparing the similarity of stereo structure, electronic distribution, and physicochemical properties using the computer-aided molecular modeling technology. The monoclonal antibody (mAb) 1A9 exhibited broad-spectrum recognition specificity of 15 ester-type AAs was obtained and had a high sensitivity with the binding affinity (half-maximum inhibition concentration, IC50) of 0.73–130.36 μg L−1. Through molecular docking, it was found that mAb 1A9 and ester-type AAs showed a semi-enveloped structure through hydrogen bonds and hydrophobicity interaction. The amino acid residues that responsible for recognition were ARG107, GLU55, PRO113, VAL36, and SER64, and the critical structures to be recognized of AAs were acetyl group, benzoyl group, and N-linked carbon chains. The developed indirect competitive enzyme-linked immunosorbent assay (icELISA) based on mAb 1A9 allowed a sensitive determination of 15 ester-type AAs with the limit of detection (LOD) of 0.21–43.72 μg L−1, and it was suitable for the analysis of ester-type AAs in various Aconitum L. samples. These results provided an effective strategy for the preparation of targeted broad-spectrum antibodies of small molecules and proposed an icELISA method available for rapid, sensitive, and high-throughput detection of toxic ester-type AAs in Aconitum L. herbs.

CgHMGB1 functions as a broad-spectrum recognition molecule to induce the expressions of CgIL17-5 and Cgdefh2 via MAPK or NF-κB signaling pathway in Crassostrea gigas

High-mobility group box 1 (HMGB1), a highly conserved nucleoprotein, functions in immune recognition, inflammation and antibacterial immunization in vertebrates. In the present study, the mediation mechanism of CgHMGB1 in activating MAPK and NF-κB/Rel signaling pathways to induce the expressions of immune effectors was investigated. CgHMGB1 mRNA was detected in all tested developmental stages from fertilized egg to D-larvae, with the higher expressions in 4 cells and 8 cells stages. CgHMGB1 proteins were mainly distributed in haemocyte granulocytes. The expressions of CgHMGB1 mRNA in haemocytes increased significantly after Vibrio splendidus stimulation, and CgHMGB1 protein translocated into the haemocyte cytoplasm and release into cell-free haemolymph. The phosphorylation of CgERK and CgP38 were induced, the nuclear translocation of CgRel were promoted, and the mRNA expressions of CgIL17-5 and Cgdefh2 increased significantly after rCgHMGB1 treatment. Obvious branchial swelling and cilium shedding were observed after rCgHMGB1 treatment. rCgHMGB1 exhibited binding activity to different polysaccharides, bacteria, and fungi. rCgHMGB1 also displayed obvious antibacterial activity to V. splendidus and E. coli. These results indicated that CgHMGB1 functioned as an immune recognition molecule to recognize various PAMPs and bacteria to induce the mRNA expressions of CgIL17-5 and Cgdefh2 via the activation of MAPK and NF-κB signaling pathways in oysters.

ELISA-Based Method for Variant-Independent Detection of Total Microcystins and Nodularins via a Multi-immunogen Approach.

Required routine monitoring of microcystins (MCs) and nodularins (NODs) in water samples, as posed by U.S. EPA Unregulated Contaminant Monitoring Rule 4, demands cost-effective, reliable, and sensitive detection methods. To target as many MC and NOD variants as possible, we developed an indirect competitive enzyme-linked immunosorbent assay (ELISA) with group-specific monoclonal antibodies for variant-independent detection of total MCs and NODs. In this ELISA method, the mice monoclonal antibodies presenting both high affinities and broad-spectrum recognition capabilities against MCs and NODs were self-produced by designing MC hapten-based multi-immunogens to minimize specificity for the particular variant. Their high affinities and variant-independent binding capabilities against MCs and NODs were validated by both wet lab and in silico methods. The developed ELISA method achieved a limit of detection of below 0.3 μg/L for 13 MC/NOD variants, well with the reported best cross-reactivities of 60-127% relative to MC-LR. As a case study, this ELISA method was used to map the variations of intracellular and extracellular total MCs/NODs in the Luoma Lake drinking water source, China, in July, 2020. Its capability to measure total MCs/NODs with high sensitivity and high throughput in a simple and affordable way would truly be a disruptive technology capable of changing our understanding of bloom/toxin dynamics and having obvious implications for monitoring efforts.

Two fibrinogen-related proteins (FREPs) in the razor clam (Sinonovacula constricta) with a broad recognition spectrum and bacteria agglutination activity

Fibrinogen-related proteins (FREPs) that contain only the fibrinogen-related domain are likely involved in pathogen recognition. In this study, we identified two FREPs from the razor clam (Sinonovacula constricta), called ScFREP-1 and ScFREP-2, and investigated their roles in the immune response. Both ScFREP-1 and ScFREP-2 contained a fibrinogen-related domain at the C-terminal. ScFREP-1 and ScFREP-2 mRNAs were detected in all adult clam tissues tested, with the highest expression levels in the gill and mantle, respectively. Their expression levels were significantly upregulated after microbe infection. Recombinant ScFREPs could bind Gram-positive and Gram-negative bacteria as well as some pathogen-associated molecular patterns (PAMPs), and they could agglutinate those bacteria. These results showed that ScFREPs functioned as potential pattern recognition receptors to mediate immune response by recognizing PAMPs and agglutinating invasive microbes.

A broadly neutralizing monoclonal antibody induces broad protection against heterogeneous PRRSV strains in piglets

Neutralizing antibodies (NAbs) have attracted attention as tools for achieving PRRSV control and prevention, but viral antigenic variation undermines the abilities of NAbs elicited by attenuated PRRSV vaccines to confer full protection against heterogeneous PRRSV field isolates. As demonstrated in this study, the monoclonal antibody (mAb) mAb-PN9cx3 exhibited broad-spectrum recognition and neutralizing activities against PRRSV-1 and PRRSV-2 strains in vitro. Furthermore, in vivo experiments revealed that the administration of two 10-mg doses of mAb-PN9cx3 before and after the inoculation of piglets with heterologous PRRSV isolates (HP-PRRSV-JXA1 or PRRSV NADC30-like strain HNhx) resulted in significant reduction of the PRRSV-induced pulmonary pathological changes and virus loads in porcine alveolar macrophages (PAMs) compared with the results obtained with mAb-treated isotype controls. Moreover, minimal hilar lymph node PRRSV antigen levels were observed in mAb-PN9cx3-treated piglets. A transcriptome profile analysis of PAMs extracted from lung tissues of piglets belonging to different groups (except for antibody-isotype controls) indicated that mAb-PN9cx3 treatment reversed the PRRSV infection-induced alterations in expression profiles. A gene ontology (GO) enrichment analysis of these genes traced their functions to pathways that included the immune response, inflammatory response, and response to steroid hormone, and their functions in oogenesis and positive regulation of angiogenesis have been implicated in PRRSV pathogenesis. Overall, NADC30-like HNhx infection affected more gene pathways than HP-PRRSV infection. In conclusion, our research describes a novel immunologic approach involving the use of mAbs that confer cross-protection against serious illness resulting from infection with heterogeneous PRRSV-2 isolates, which is a feat that has not yet been achieved through vaccination. Ultimately, mAb-PN9cx3 will be a powerful addition to our current arsenal for achieving PRRSV prevention and eradication.

Recombinant Bacteriophage Cell-Binding Domain Proteins for Broad-Spectrum Recognition of Methicillin-Resistant Staphylococcus aureus Strains.

Methicillin-resistant Staphylococcus aureus (MRSA) has been well-recognized as one of the most common multiresistant bacteria threatening human health. Broad-spectrum recognition of multiple MRSA strains can meet the urgent demands for efficient diagnosis and subsequent decision of relevant treatment of MRSA-induced infections. Here, recombinant cell-binding domain (CBD) and green fluorescent protein-fused CBD of MRSA bacteriophage were expressed in soluble form. Distinct from the strain-specific MRSA bacteriophage, both recombinant CBD proteins displayed broad-spectrum recognition capability toward all five staphylococcal cassette chromosome mec types of MRSA. Furthermore, they did not display any lytic activity toward the host bacteria, which facilitated the capture of whole MRSA cells with ideal flexibility for downstream manipulation and tracing. For demonstration of their application potential, a flow cytometry method employing the recombinant CBD proteins as the recognition agents was established to detect MRSA within a dynamic range of 1.5 × 102 to 1.5 × 106 cfu mL-1. The method can exclude potential interference from methicillin-sensitive Staphylococcus aureus strains and other bacterial species. The recombinant CBD proteins were also successfully employed in antibiotic susceptibility testing of MRSA with a microplate-based method. The obtained results were consistent with those by the standard broth microdilution method. The satisfying results demonstrated their great application potential in clinical diagnosis and treatment of MRSA-induced infections.

A novel invertebrate toll-like receptor with broad recognition spectrum from thick shell mussel Mytilus coruscus

Toll-like receptors (TLRs) are a category of most well recognized pattern recognition molecules that act on a vital role in both innate and adaptive immunity. In the present study, a novel toll-like receptor (McTLRw) was identified and characterized in thick shell mussel Mytilus coruscus. McTLRw possesses one intracellular Toll/interleukin-1 (IL-1) receptor (TIR) domain, one transmembrane region (TM), one leucine rich repeat N-terminal domain (LRR_NT) and a few of leucine-rich repeats (LRRs), which all are common in typical TLRs. McTLRw transcripts were constitutively expressed in all examined tissues with higher expression levels in immune related tissues, and were significantly induced in haemocytes with the challenges of live Vibrio alginolyticus, lipopolysaccharide (LPS), peptidoglycans (PGN) and β-glucan (GLU), but not induced by polyinosinic-polycytidylic acid (poly I:C). rMcTLRw exhibited affinity to LPS, PGN and GLU while no affinity to poly I:C. Further, the downstream of TLR signaling pathway myeloid differentiation factor 88a (MyD88a), interleukin-1 receptor-associated kinase-4 (IRAK4) and tumor necrosis factor receptor-associated factor 6 (TRAF6) were significantly repressed in McTLRw silenced mussels while challenged with LPS. These results collectively indicated that McTLRw is one member of TLR family and involved in immune response to against invaders by taking participate in TLR mediated signaling pathway.

A novel globular C1q domain containing protein (C1qDC-7) from Crassostrea gigas acts as pattern recognition receptor with broad recognition spectrum

The globular C1q domain containing (C1qDC) proteins are a family of versatile pattern recognition receptors (PRRs) to bind various ligands by their globular C1q (gC1q) domain. In the present study, a novel globular C1qDC (CgC1qDC-7) was characterized from Pacific oyster Crassostrea gigas. The open reading frame of CgC1qDC-7 was of 555 bp, encoding a polypeptide of 185 amino acids. Phylogenetic analysis indicated that CgC1qDC-7 shared high homology with C1qDCs from Crassostrea virginica, Mytilus galloprovincialis, and Mizuhopecten yessoensis. The mRNA transcripts of CgC1qDC-7 were widely expressed in all the tested tissues including mantle, gonad, gills, adductor muscle, hemocytes, hepatopancreas and labial palps, with the highest expression level in hemocytes and gills. The recombinant protein of CgC1qDC-7 (rCgC1qDC-7) exhibited binding activity towards Gram-negative bacteria (Vibrio splendidus, V. anguillarum, Escherichia coli, V. alginolyticus, and Aeromonas hydrophila), Gram-positive bacteria (Micrococcus luteus and Staphylococcus aureus) and fungi (Pichia pastoris and Yarrowia lipolytica), and displayed strongest binding affinity towards Gram-negative bacteria V. splendidus and V. anguillarum. It also exhibited affinity to vital pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS), peptidoglycan (PGN), mannan (MAN) and Poly (I:C) with high affinity towards LPS and PGN, and low affinity to MAN and Poly (I:C). These results collectively indicated that CgC1qDC-7 was a novel PRR in C. gigas with high binding affinity towards LPS and PGN as well as Gram-negative bacteria.

Glycans as Modulators of Plant Defense Against Filamentous Pathogens.

Plants and microbes utilize glycoconjugates as structural entities, energy reserves for cellular processes, and components of cellular recognition or binding events. The structural heterogeneity of carbohydrates in such systems is a result of the ability of the carbohydrate biosynthetic enzymes to reorient sugar monomers in a variety of forms, generating highly complex, linear, branched, or hierarchical structures. During the interaction between plants and their microbial pathogens, the microbial cell surface glycans, cell wall derived glycans, and glycoproteins stimulate the signaling cascades of plant immune responses, through a series of specific or broad spectrum recognition events. The microbial glycan-induced plant immune responses and the downstream modifications observed in host-plant glycan structures that combat the microbial attack have garnered immense interest among scientists in recent times. This has been enabled by technological advancements in the field of glycobiology, making it possible to study the ongoing co-evolution of the microbial and the corresponding host glycan structures, in greater detail. The new glycan analogs emerging in this evolutionary arms race brings about a fresh perspective to our understanding of plant–pathogen interactions. This review discusses the role of diverse classes of glycans and their derivatives including simple sugars, oligosaccharides, glycoproteins, and glycolipids in relation to the activation of classical Pattern-Triggered Immunity (PTI) and Effector-Triggered Immunity (ETI) defense responses in plants. While primarily encompassing the biological roles of glycans in modulating plant defense responses, this review categorizes glycans based on their structure, thereby enabling parallels to be drawn to other areas of glycobiology. Further, we examine how these molecules are currently being used to develop new bio-active molecules, potent as priming agents to stimulate plant defense response and as templates for designing environmentally friendly foliar sprays for plant protection.

Leptosphaeria maculans AvrLm9: a new player in the game of hide and seek with AvrLm4-7.

Blackleg disease of Brassica napus caused by Leptosphaeria maculans (Lm) is largely controlled by the deployment of race-specific resistance (R) genes. However, selection pressure exerted by R genes causes Lm to adapt and give rise to new virulent strains through mutation and deletion of effector genes. Therefore, a knowledge of effector gene function is necessary for the effective management of the disease. Here, we report the cloning of Lm effector AvrLm9 which is recognized by the resistance gene Rlm9 in B.napus cultivar Goéland. AvrLm9 was mapped to scaffold 7 of the Lm genome, co-segregating with the previously reported AvrLm5 (previously known as AvrLmJ1). Comparison of AvrLm5 alleles amongst the 37 re-sequenced Lm isolates and transgenic complementation identified a single point mutation correlating with the AvrLm9 phenotype. Therefore, we renamed this gene as AvrLm5-9 to reflect the dual specificity of this locus. Avrlm5-9 transgenic isolates were avirulent when inoculated on the B.napus cultivar Goéland. The expression of AvrLm5-9 during infection was monitored by RNA sequencing. The recognition of AvrLm5-9 by Rlm9 is masked in the presence of AvrLm4-7, another Lm effector. AvrLm5-9 and AvrLm4-7 do not interact, and AvrLm5-9 is expressed in the presence of AvrLm4-7. AvrLm5-9 is the second Lm effector for which host recognition is masked by AvrLm4-7. An understanding of this complex interaction will provide new opportunities for the engineering of broad-spectrum recognition.