Putative Pattern Recognition Receptors Research Articles

Use of Open Surface Plasmon Resonance (OpenSPR) to Characterize the Binding Affinity of Protein-Protein Interactions.

Surface Plasmon Resonance(SPR) is a label-free optical technique to assess protein-protein interaction kinetics and affinities in a real-time setting. Traditionally, Biacore SPR employs a continuous film of gold to detect any change in the angle of re-emitted light when the refractive index of a ligand conjugated to the flat gold surface is altered by its interaction with a local analyte. In contrast, the Nicoya Lifesciences' OpenSPR technology uses gold nanoparticles to detect small changes in the absorbance peak wavelength of a conjugated ligand after its engagement by an analyte. Specifically, when broadband white light is shone onto the gold nanoparticles, it produces a strong resonance absorbance peak corresponding to the refractive index of a ligand conjugated to the surface of gold nanoparticles. Upon its interaction with an analyte, however, the absorbance wavelength peak of the conjugated ligand will be changed and timely recorded as sensorgrams of dynamic ligand-analyte interactions. Thus, the improvement in the detection method (from traditional detection of changes in the angle of re-emitted light to the contemporary detection of changes in the wavelength of the absorbance peak) features OpenSPR as a cost-effective and user-friendly technique for in-depth characterization of protein-protein interactions. Here, we describe the detailed method that we used to characterize procathepsin L (pCTS-L) interactions with two putative pattern recognition receptors (TLR4 and RAGE) using the 1st generation of Nicoya Lifesciences' OpenSPR instrument with a 1-channel detection. Key features • Nicoya OpenSPR is a benchtop small-size equipment that provides in-depth label-free binding kinetics and affinity measurement for protein-protein interactions in real-time fashion. • This technology is relatively intuitive and user-friendly for scientists at any skill level. • OpenSPR sensors employ nanotechnology to reduce the cost of manufacturing complex optical hardware and Sensor Chips, and similarly reduce the consumption of precious analyte samples. • The manufacturer provides online training for OpenSPR (Catalog: TRAIN-REMOTE) and TraceDrawer (Catalog: TRAIN-TD) to customer scientists.

Comparative transcriptome of compatible and incompatible interaction of Erysiphe pisi and garden pea reveals putative defense and pathogenicity factors.

Comparative transcriptome analysis of Erysiphe pisi-infected pea (Pisum sativum) genotypes JI-2480 (resistant) and Arkel (susceptible) at 72 hours post-inoculation (hpi) was carried to detect molecular components involved in compatible and incompatible interactions. Differential gene expression was observed in Arkel and JI-2480 genotype at 72 hpi with E. pisi isolate (Ep01) using EdgeR software. Out of 32 217 transcripts, 2755 transcripts showed significantly altered gene expression in case of plants while 530 were related to E. pisi (P <0.05). The higher transcript number of differentially expressed genes demonstrated peak activity of pathogenicity genes in plants at 72 hpi. Glycolysis was observed to be the major pathway for energy source during fungal growth. Differential gene expression of plant transcripts revealed significant expression of putative receptor and regulatory sequences involved in defense in the resistant, JI-2480 compared to susceptible, Arkel genotype. Expression of genes involved in defense and hormonal signaling, genes related to hypersensitive response, reactive oxygen species and phenylpropanoid pathway in JI-2480 indicated their crucial role in disease resistance against E. pisi. Down-regulation of transcription factors like-WRKY-28 and up-regulation of several putative pattern recognition receptors in JI-2480 compared to Arkel also suggested activation of host-mediated defense responses against E. pisi in pea.

Comparative transcriptome analysis reveals defense responses against soft rot in Chinese cabbage

Pectobacterium carotovorum ssp. carotovorum (Pcc) is a necrotrophic bacterial species that causes soft rot disease in Chinese cabbage. In this study, plants harboring the resistant mutant sr gene, which confers resistance against Pcc, were screened from an 800 M2 population mutated by ethyl methane sulfonate (EMS) and scored in vitro and in vivo for lesion size. The transcript profiles showed ~512 differentially expressed genes (DEGs) between sr and WT plants occurring between 6 and 12 h postinoculation (hpi), which corresponded to the important defense regulation period (resistance) to Pcc in Chinese cabbage. The downstream defense genes (CPK, CML, RBOH MPK3, and MPK4) of pathogen pattern-triggered immunity (PTI) were strongly activated during infection at 12 hpi in resistant mutant sr; PTI appears to be central to plant defense against Pcc via recognition by three putative pattern recognition receptors (PRRs; BrLYM1-BrCERK1, BrBKK1/SERK4-PEPR1, BrWAKs). Pcc triggered the upregulation of the jasmonic acid (JA) and ethylene (ET) biosynthesis genes in mutant sr, but auxins and other hormones may have affected some negative signals. Endogenous hormones (auxins, JAs, and SA), as well as exogenous auxins (MEJA and BTH), were also verified as functioning in the immune system. Concurrently, the expression of glucosinolate and lignin biosynthesis genes was increased at 12 hpi in resistant mutant sr, and the accumulation of glucosinolate and lignin also indicated that these genes have a functional defensive role against Pcc. Our study provides valuable information and elucidates the resistance mechanism of Chinese cabbage against Pcc infection.

Two Lysin-Motif Receptor Kinases, Gh-LYK1 and Gh-LYK2, Contribute to Resistance against Verticillium wilt in Upland Cotton.

Lysin-motif (LysM) receptor kinases (LYKs) play essential roles in recognition of chitin and activation of defense responses against pathogenic fungi in the model plants Arabidopsis and rice. The function of LYKs in non-model plants, however, remains elusive. In the present work, we found that the transcription of two LYK-encoding genes from cotton, Gh-LYK1 and Gh-LYK2, was induced after Verticillium dahliae infection. Virus-induced gene silencing (VIGS) of Gh-LYK1 and Gh-LYK2 in cotton plants compromises resistance to V. dahliae. As putative pattern recognition receptors (PRRs), both Gh-LYK1 and Gh-LYK2 are membrane-localized, and all three LysM domains of Gh-LYK1 and Gh-LYK2 are required for their chitin-binding ability. However, since Gh-LYK2, but not Gh-LYK1, is a pseudo-kinase and, on the other hand, the ectodomain (ED) of Gh-LYK2 can induce reactive oxygen species (ROS) burst in planta, Gh-LYK2 and Gh-LYK1 may contribute differently to cotton defense. Taken together, our results establish that both Gh-LYK1 and Gh-LYK12 are required for defense against V. dahliae in cotton, possibly through different mechanisms.

The immunotranscriptome of the Caribbean reef-building coral Pseudodiploria strigosa.

The viability of coral reefs worldwide has been seriously compromised in the last few decades due in part to the emergence of coral diseases of infectious nature. Despite important efforts to understand the etiology and the contribution of environmental factors associated to coral diseases, the mechanisms of immune response in corals are just beginning to be studied systematically. In this study, we analyzed the set of conserved immune response genes of the Caribbean reef-building coral Pseudodiploria strigosa by Illumina-based transcriptome sequencing and annotation of healthy colonies challenged with whole live Gram-positive and Gram-negative bacteria. Searching the annotated transcriptome with immune-related terms yielded a total of 2782 transcripts predicted to encode conserved immune-related proteins that were classified into three modules: (a) the immune recognition module, containing a wide diversity of putative pattern recognition receptors including leucine-rich repeat-containing proteins, immunoglobulin superfamily receptors, representatives of various lectin families, and scavenger receptors; (b) the intracellular signaling module, containing components from the Toll-like receptor, transforming growth factor, MAPK, and apoptosis signaling pathways; and (3) the effector module, including the C3 and factor B complement components, a variety of proteases and protease inhibitors, and the melanization-inducing phenoloxidase. P. strigosa displays a highly variable and diverse immune recognition repertoire that has likely contributed to its resilience to coral diseases.

Sensing of Gram-positive bacteria in Drosophila: GNBP1 is needed to process and present peptidoglycan to PGRP-SA

Genetic evidence indicates that Drosophila defense against Gram-positive bacteria is mediated by two putative pattern recognition receptors acting upstream of Toll, namely Gram-negative binding protein 1 (GNBP1) and peptidoglycan recognition protein SA (PGRP-SA). Until now however, the molecular recognition proceedings for sensing of Gram-positive pathogens were not known. In the present, we report the physical interaction between GNBP1 and PGRP-SA using recombinant proteins. GNBP1 was able to hydrolyze Gram-positive peptidoglycan (PG), while PGRP-SA bound highly purified PG fragments (muropeptides). Interaction between these proteins was enhanced in the presence of PG or muropeptides. PGRP-SA binding depended on the polymerization status of the muropeptides, pointing to constraints in the number of PGRP-SA molecules bound for signaling initiation. We propose a model whereby GNBP1 presents a processed form of PG for sensing by PGRP-SA and that a tripartite interaction between these proteins and PG is essential for downstream signaling.

Innate immunity in the malaria vector Anopheles gambiae: comparative and functional genomics.

The resurgence of malaria is at least partly attributed to the absence of an effective vaccine, parasite resistance to antimalarial drugs and resistance to insecticides of the anopheline mosquito vectors. Novel strategies are needed to combat the disease on three fronts: protection (vaccines), prophylaxis/treatment (antimalarial drugs) and transmission blocking. The latter entails either killing the mosquitoes (insecticides), preventing mosquito biting (bednets and repellents), blocking parasite development in the vector (transmission blocking vaccines), genetic manipulation or chemical incapacitation of the vector. During the past decade, mosquito research has been energized by several breakthroughs, including the successful transformation of anopheline vectors, analysis of gene function by RNAi, genome-wide expression profiling using DNA microarrays and, most importantly, sequencing of the Anopheles gambiae genome. These breakthroughs helped unravel some of the mechanisms underlying the dynamic interactions between the parasite and the vector and shed light on the mosquito innate immune system as a set of potential targets to block parasite development. In this context, putative pattern recognition receptors of the mosquito that act as positive and negative regulators of parasite development have been identified recently. Characterizing these molecules and others of similar function, and identifying their ligands on the parasite surface, will provide clues on the nature of the interactions that define an efficient parasite-vector system and open up unprecedented opportunities to control the vectorial capacity of anopheline mosquitoes.

In Vivo RNA Interference Analysis Reveals an Unexpected Role for GNBP1 in the Defense against Gram-positive Bacterial Infection in Drosophila Adults

The Drosophila immune system discriminates between different classes of infectious microbes and responds with pathogen-specific defense reactions via the selective activation of the Toll and the immune deficiency (Imd) signaling pathways. The Toll pathway mediates most defenses against Gram-positive bacteria and fungi, whereas the Imd pathway is required to resist Gram-negative bacterial infection. Microbial recognition is achieved through peptidoglycan recognition proteins (PGRPs); Gram-positive bacteria activate the Toll pathway through a circulating PGRP (PGRP-SA), and Gram-negative bacteria activate the Imd pathway via PGRP-LC, a putative transmembrane receptor, and PGRP-LE. Gram-negative binding proteins (GNBPs) were originally identified in Bombyx mori for their capacity to bind various microbial compounds. Three GNBPs and two related proteins are encoded in the Drosophila genome, but their function is not known. Using inducible expression of GNBP1 double-stranded RNA, we now demonstrate that GNBP1 is required for Toll activation in response to Gram-positive bacterial infection; GNBP1 double-stranded RNA expression renders flies susceptible to Gram-positive bacterial infection and reduces the induction of the antifungal peptide encoding gene Drosomycin after infection by Gram-positive bacteria but not after fungal infection. This phenotype induced by GNBP1 inactivation is identical to a loss-of-function mutation in PGRP-SA, and our genetic studies suggest that GNBP1 acts upstream of the Toll ligand Spätzle. Altogether, our results demonstrate that the detection of Gram-positive bacteria in Drosophila requires two putative pattern recognition receptors, PGRP-SA and GNBP1.