Leucine-rich Repeat Motifs Research Articles

M6A-related genes and their role in Parkinson’s disease: Insights from machine learning and consensus clustering

Parkinson disease (PD) is a chronic neurological disorder primarily characterized by a deficiency of dopamine in the brain. In recent years, numerous studies have highlighted the substantial influence of RNA N6-methyladenosine (m6A) regulators on various biological processes. Nevertheless, the specific contribution of m6A-related genes to the development and progression of PD remains uncertain. In this study, we performed a differential analysis of the GSE8397 dataset in the Gene Expression Omnibus database and selected important m6A-related genes. Candidate m6A-related genes were then screened using a random forest model to predict the risk of PD. A nomogram model was built based on the candidate m6A-related genes. By employing a consensus clustering method, PD was divided into different m6A clusters based on the selected significant m6A-related genes. Finally, we performed immune cell infiltration analysis to explore the immune infiltration between different clusters. We performed a differential analysis of the GSE8397 dataset in the Gene Expression Omnibus database and selected 11 important m6A-related genes. Four candidate m6A-related genes (YTH Domain Containing 2, heterogeneous nuclear ribonucleoprotein C, leucine-rich pentatricopeptide repeat motif containing protein and insulin-like growth factor binding protein-3) were then screened using a random forest model to predict the risk of PD. A nomogram model was built based on the 4 candidate m6A-related genes. The decision curve analysis indicated that patients can benefit from the nomogram model. By employing a consensus clustering method, PD was divided into 2 m6A clusters (cluster A and cluster B) based on the selected significant m6A-related genes. The immune cell infiltration analysis revealed that cluster A and cluster B exhibit distinct immune phenotypes. In conclusion, m6A-related genes play a significant role in the development of PD and our study on m6A clustering may potentially guide personalized treatment strategies for PD in the future.

Dual transcriptional characterization of spinach and Peronospora effusa during resistant and susceptible race-cultivar interactions

BackgroundSpinach downy mildew, caused by the obligate oomycete pathogen, Peronospora effusa remains a major concern for spinach production. Disease control is predominantly based on development of resistant spinach cultivars. However, new races and novel isolates of the pathogen continue to emerge and overcome cultivar resistance. Currently there are 20 known races of P. effusa. Here we characterized the transcriptomes of spinach, Spinacia oleracea, and P. effusa during disease progression using the spinach cultivar Viroflay, the near isogenic lines NIL1 and NIL3, and P. effusa races, R13 and R19, at 24 h post inoculation and 6 days post inoculation. A total of 54 samples were collected and subjected to sequencing and transcriptomic analysis.ResultsDifferentially expressed gene (DEG) analysis in resistant spinach interactions of R13-NIL1 and R19-NIL3 revealed spinach DEGs from protein kinase-like and P-loop containing families, which have roles in plant defense. The homologous plant defense genes included but were not limited to, receptor-like protein kinases (Spiol0281C06495, Spiol06Chr21559 and Spiol06Chr24027), a BAK1 homolog (Spiol0223C05961), genes with leucine rich repeat motifs (Spiol04Chr08771, Spiol04Chr01972, Spiol05Chr26812, Spiol04Chr11049, Spiol0084S08137, Spiol03Chr20299) and ABC-transporters (Spiol02Chr28975, Spiol06Chr22112, Spiol06Chr03998 and Spiol04Chr09723). Additionally, analysis of the expression of eight homologous to previously reported downy mildew resistance genes revealed that some are differentially expressed during resistant reactions but not during susceptible reactions. Examination of P. effusa gene expression during infection of susceptible cultivars identified expressed genes present in R19 or R13 including predicted RxLR and Crinkler effector genes that may be responsible for race-specific virulence on NIL1 or NIL3 spinach hosts, respectively.ConclusionsThese findings deliver foundational insight to gene expression in both spinach and P. effusa during susceptible and resistant interactions and provide a library of candidate genes for further exploration and functional analysis. Such resources will be beneficial to spinach breeding efforts for disease resistance in addition to better understanding the virulence mechanisms of this obligate pathogen.

Siah2- and LRSAM1-mediated K63-linked ubiquitination of snakehead vesiculovirus nucleoprotein facilitates viral replication.

Ubiquitination of viral protein plays an important role in viral replication. However, the ubiquitination of the nucleoprotein (N) of negative-strand RNA viruses has rarely been investigated. This study aimed at investigating the ubiquitination of the N protein of a fish rhabdovirus SHVV (snakehead vesiculovirus), identifying the related host E3 ubiquitin ligases, and determining the role of SHVV N ubiquitination and host E3 ubiquitin ligases in viral replication. We found that SHVV N was ubiquitinated mainly via K63-linked ubiquitination, which was mediated by host E3 ubiquitin ligases Siah2 (Siah E3 ubiquitin protein ligase 2) and LRSAM1 (leucine-rich repeat and sterile alpha motif containing 1). The data suggested that Siah2 and LRSAM1 were hijacked by SHVV to ubiquitinate the N protein for viral replication, which exhibited novel anti-SHVV targets for drug design.

Alternative splicing of a potato disease resistance gene maintains homeostasis between growth and immunity.

Plants possess a robust and sophisticated innate immune system against pathogens and must balance growth with rapid pathogen detection and defense. The intracellular receptors with nucleotide-binding leucine-rich repeat (NLR) motifs recognize pathogen-derived effector proteins and thereby trigger the immune response. The expression of genes encoding NLR receptors is precisely controlled in multifaceted ways. The alternative splicing (AS) of introns in response to infection is recurrently observed but poorly understood. Here we report that the potato (Solanum tuberosum) NLR gene RB undergoes AS of its intron, resulting in 2 transcriptional isoforms, which coordinately regulate plant immunity and growth homeostasis. During normal growth, RB predominantly exists as an intron-retained isoform RB_IR, encoding a truncated protein containing only the N-terminus of the NLR. Upon late blight infection, the pathogen induces intron splicing of RB, increasing the abundance of RB_CDS, which encodes a full-length and active R protein. By deploying the RB splicing isoforms fused with a luciferase reporter system, we identified IPI-O1 (also known as Avrblb1), the RB cognate effector, as a facilitator of RB AS. IPI-O1 directly interacts with potato splicing factor StCWC15, resulting in altered localization of StCWC15 from the nucleoplasm to the nucleolus and nuclear speckles. Mutations in IPI-O1 that eliminate StCWC15 binding also disrupt StCWC15 re-localization and RB intron splicing. Thus, our study reveals that StCWC15 serves as a surveillance facilitator that senses the pathogen-secreted effector and regulates the trade-off between RB-mediated plant immunity and growth, expanding our understanding of molecular plant-microbe interactions.

Specificities and redundancies in the NEL family of bacterial E3 ubiquitin ligases of Salmonella enterica serovar Typhimurium.

Salmonella enterica serovar Typhimurium expresses two type III secretion systems, T3SS1 and T3SS2, which are encoded in Salmonella pathogenicity island 1 (SPI1) and SPI2, respectively. These are essential virulent factors that secrete more than 40 effectors that are translocated into host animal cells. This study focuses on three of these effectors, SlrP, SspH1, and SspH2, which are members of the NEL family of E3 ubiquitin ligases. We compared their expression, regulation, and translocation patterns, their role in cell invasion and intracellular proliferation, their ability to interact and ubiquitinate specific host partners, and their effect on cytokine secretion. We found that transcription of the three genes encoding these effectors depends on the virulence regulator PhoP. Although the three effectors have the potential to be secreted through T3SS1 and T3SS2, the secretion of SspH1 and SspH2 is largely restricted to T3SS2 due to their expression pattern. We detected a role for these effectors in proliferation inside fibroblasts that is masked by redundancy. The generation of chimeric proteins allowed us to demonstrate that the N-terminal part of these proteins, containing the leucine-rich repeat motifs, confers specificity towards ubiquitination targets. Furthermore, the polyubiquitination patterns generated were different for each effector, with Lys48 linkages being predominant for SspH1 and SspH2. Finally, our experiments support an anti-inflammatory role for SspH1 and SspH2.

Expression characteristics and inhibitory activity of a leucine-rich repeat (LRR)-only protein in the Chinese mitten crab, Eriocheir sinensis

The leucine-rich repeat (LRR) domain is a crucial structure in a variety of immune related proteins and displays multiple immune functions. In this study, the open reading frame (ORF) of an LRR-only protein was cloned from the Chinese mitten crab, Eriocheir sinensis (EsLRRop1). The protein sequence of EsLRRop1 contained seven LRR motifs, three LRR-TYP motifs and an LRRCT motif. Tissue distribution exhibited that EsLRRop1 mainly expressed in nervous tissues including thoracic ganglion, eyestalk and brain while showed relatively lower transcriptional level in hemocyte. Based on the above expression characteristics, the responses of EsLRRop1 to the challenge of Vibrio parahaemolyticus and Staphylococcus aureus were tested. The result showed that the transcript of EsLRRop1 in thoracic ganglion and eyestalk up-regulated after being challenged with S. aureus, while it decreased post injection with V. parahaemolyticus. The transcript of EsLRRop1 in hemocytes up-regulated sharply at 3 h and decreased at 12 h and 24 h after being challenged with V. parahaemolyticus, while it decreased at 12 h and 24 h post injection with S. aureus. The recombinant protein of EsLRRop1 (His-EsLRRop1) displayed binding activities to V. alginolyticus, V. harveyi, V. parahaemolyticus, S. aureus, Corynebacterium glutamicum and Micrococcus lysodeikticus as well as lipopolysaccharide (LPS) and peptidoglycan (PGN). Moreover, the His-EsLRRop1 exhibited inhibitory activity against V. parahaemolyticus and V. harveyi with minimum inhibitory concentration (MIC) of 3.57–7.14 μM and 7.14–14.28 μM, respectively. These results provide theoretical basis for the application of EsLRRop1 in inhibiting bacteria in aquaculture practice.

Three-Dimensional Modeling of CpG DNA Binding with Matrix Lumican Shows Leucine-Rich Repeat Motif Involvement as in TLR9-CpG DNA Interactions.

Lumican is an extracellular matrix proteoglycan known to regulate toll-like receptor (TLR) signaling in innate immune cells. In experimental settings, lumican suppresses TLR9 signaling by binding to and sequestering its synthetic ligand, CpG-DNA, in non-signal permissive endosomes. However, the molecular details of lumican interactions with CpG-DNA are obscure. Here, the 3-D structure of the 22 base-long CpG-DNA (CpG ODN_2395) bound to lumican or TLR9 were modeled using homology modeling and docking methods. Some of the TLR9-CpG ODN_2395 features predicted by our model are consistent with the previously reported TLR9-CpG DNA crystal structure, substantiating our current analysis. Our modeling indicated a smaller buried surface area for lumican-CpG ODN_2395 (1803 Å2) compared to that of TLR9-CpG ODN_2395 (2094 Å2), implying a potentially lower binding strength for lumican and CpG-DNA than TLR9 and CpG-DNA. The docking analysis identified 32 amino acids in lumican LRR1-11 interacting with CpG ODN_2395, primarily through hydrogen bonding, salt-bridges, and hydrophobic interactions. Our study provides molecular insights into lumican and CpG-DNA interactions that may lead to molecular targets for modulating TLR9-mediated inflammation and autoimmunity.

LvCD14L Acts as a Novel Pattern Recognition Receptor and a Regulator of the Toll Signaling Pathway in Shrimp.

Leucine-rich repeat (LRR) is a structural motif has important recognition function in immune receptors, such as Tolls and NOD-like receptors (NLRs). The immune-related LRR proteins can be divided into two categories, LRR-containing proteins and LRR-only proteins. The latter contain LRR motifs while they are without other functional domains. However, the functional mechanisms of the LRR-only proteins were still unclear in invertebrates. Here, we identified a gene encoding a secretory LRR-only protein, which possessed similarity with vertebrate CD14 and was designated as LvCD14L, from the Pacific whiteleg shrimp Litopenaeus vannamei. Its transcripts in shrimp hemocytes were apparently responsive to the infection of Vibrio parahaemolyticus. Knockdown of LvCD14L with dsRNA resulted in significant increase of the viable bacteria in the hepatopancreas of shrimp upon V. parahaemolyticus infection. Further functional studies revealed that LvCD14L could bind to microorganisms' PAMPs, showed interaction with LvToll1 and LvToll2, and regulated the expression of LvDorsal and LvALF2 in hemocytes. These results suggest that LvCD14L functions as a pattern recognition receptor and activates the NF-κB pathway through interaction with LvTolls. The present study reveals a shrimp LvCD14L-Tolls-NF-κB signaling pathway like the CD14/TLR4/NF-κB signaling pathway in mammalians, which enriches the functional mechanism of secretory LRR-only immune receptors during pathogens infection in invertebrates.

Role of Nod factor receptors and its allies involved in nitrogen fixation.

Lysin motif (LysM)-receptor-like kinase (RLK) and leucine-rich repeat(LRR)-RLK mediated signaling play important roles in the development and regulation of root nodule symbiosis in legumes. The availability of water and nutrients in the soil is a major limiting factor affecting crop productivity. Plants of the Leguminosae family form a symbiotic association with nitrogen-fixing Gram-negative soil bacteria, rhizobia for nitrogen fixation. This symbiotic relationship between legumes and rhizobia depends on the signal exchange between them. Plant receptor-like kinases (RLKs) containing lysin motif (LysM) and/or leucine-rich repeat (LRR) play an important role in the perception of chemical signals from rhizobia for initiation and establishment of root nodule symbiosis (RNS) that results in nitrogen fixation. This review highlights the diverse aspects of LysM-RLK and LRR receptors including their specificity, functions, interacting partners, regulation, and associated signaling in RNS. The activation of LysM-RLKs and LRR-RLKs is important for ensuring the successful interaction between legume roots and rhizobia. The intracellular regions of the receptors enable additional layers of signaling that help in the transduction of signals intracellularly. Additionally, symbiosis receptor-like kinase (SYMRK) containing the LRR motif acts as a co-receptor with Nod factors receptors (LysM-RLK). Cleavage of the malectin-like domain from the SYMRK ectodomain is a mechanism for controlling SYMRK stability. Overall, this review has discussed different aspects of legume receptors that are critical to the perception of signals from rhizobia and their subsequent role in creating the mutualistic relationship necessary for nitrogen fixation. Additionally, it has been discussed how crucial it is to extrapolate the knowledge gained from model legumes to crop legumes such as chickpea and common bean to better understand the mechanism underlying nodule formation in crop legumes. Future directions have also been proposed in this regard.

Identification of LRRC8/VRAC channel structural elements required for regulation by cell volume increase and intracellular ionic strength.

VRACs mediate volume regulatory Cl- and organic solute efflux and are encoded by Lrrc8a-e genes. Native LRRC8/VRACs are heteromers with unknown stoichiometry comprising the essential LRRC8A subunit and another paralog. We utilize a chimeric protein approach to define LRRC8/VRAC structure-function relationships. Functional chimeric channels with physiologically relevant properties require the presence of the first intracellular loop (IL1) of LRRC8A and the first extracellular loop of another paralog. Replacement of the C-terminus of LRRC8C or LRRC8E with the C-terminus of LRRC8A (8A(C-term)) gives rise to robust plasma membrane anion channel activity. 8C-8A(C-term) and 8E-8A(C-term) chimeric channels exhibit greatly reduced sensitivity to reduced intracellular ionic strength (IS) and cannot be activated by hypotonicity-induced cell volume increases of up to 4-fold. However, both channels could be activated by rapid, stepwise volume increases of 2-2.5-fold induced by positive pressure applied to the patch pipette. Addition of the LRRC8A IL1 to both chimeras restored normal sensitivity to hypotonicity-induced volume increases and IS. A cryo-EM structure of a LRRC8 chimeric channel comprising LRRC8C with 25 amino acids unique to the LRRC8A IL1, 8C-8A(IL125), suggested a possible functional interaction between 8A(IL125) and leucine-rich repeat motif 5, LRR5. Deletion of 9 cytoplasm-facing LRR5 amino acids or substitution with alanine greatly reduced volume and IS sensitivity of 8C-8A(IL125) channels. Point mutations in the 8A(IL125) region identified two unique lysine residues required for channel regulation. We conclude that the LRRC8 C-terminus is 1) required for channel volume and IS sensing, 2) that channel regulation requires a functional interaction between the unique 8A(IL125) region and LRR5 and 3) that LRR5 and the 8A(IL125) region are key components of the LRRC8/VRAC volume and IS sensing machinery.

An LRR domain-containing membrane protein gene in rotifer Brachionus plicatilis: Sequence feature, expression pattern, and ligands binding activity

Leucine-rich repeat (LRR) domains mediate multiple innate immune responses via protein-ligand and protein-protein interactions, but their exact roles in invertebrates are poorly understood. Herein, an LRR domain-containing transmembrane protein (BpLRRm) was identified in the rotifer Brachionus plicatilis. The 1069 bp BpLRRm nucleotide sequence contains a 942 bp open reading frame (ORF) encoding a 313 amino acid polypeptide with four LRR motifs harbouring the LXXLXXLXLXXNXLXXL motif, and a transmembrane domain. Treatment with 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) decreased BpLRRm mRNA levels at 3 h, but they increased thereafter and peaked at 12 h. Lipopolysaccharide (LPS) treatment first increased BpLRRm mRNA levels at 3 h, but levels returned to normal at 12 h, then increased and peaked at 24 h. Recombinant BpLRRm protein bound pathogen-related molecular patterns (PAMPs), including LPS, peptidoglycan (PGN), glucan (GLU) and polyinosinic-polycytidylic acid (poly IC), in a dose-dependent manner. Thus, BpLRRm might function as a pattern recognition receptor (PRR) in the innate immunity of B. plicatilis, and mediate responses to environmental pollution.

Leptospiral Leucine-Rich Repeat Protein-Based Lateral Flow for Assessment of Canine Leptospiral Immunoglobulin G.

The recombinant, modified leucine-rich repeat protein rhKU_Sej_LRR_2271 has been suggested as a candidate for leptospiral vaccine development since it was predicted to be a transmembrane protein containing leucine-rich repeat motifs and immunogenic epitopes. The immunogenic epitopes showed binding affinities with lower IC50 values than peptides of known antigenic proteins, e.g., LipL32. Moreover, this protein was immunoreactive with hyperimmune sera against several serovars. In this study, we aimed to develop a lateral flow strip test using the rhKU_Sej_LRR_2271 protein for the detection of anti-leptospiral IgG in dogs. The lateral flow assay was performed with 184 dog plasma samples and evaluated with a culture method, 16S ribosomal RNA gene (rss) analysis real-time PCR, and LipL32 ELISA. The culture method failed to detect leptospires in the dog blood samples. Six of nine symptomatic dogs gave positive results with the real-time PCR assay. The lateral flow assay and LipL32 ELISA gave positive results with 59 and 50 dogs, respectively. The sensitivity, specificity, and accuracy of the rhKU_Sej_LRR_2271 lateral flow strip test were 70.00, 82.09, and 78.80%, respectively, when compared with LipL32 ELISA. There was a significant association between the LipL32 ELISA and the rhKU_Sej_LRR_2271 lateral flow assay. The rhKU_Sej_LRR_2271 lateral flow strip test has therefore demonstrated a good potential to detect anti-leptospiral IgG in dogs.

Molecular characterization of an LRR-only protein gene in Pacific white shrimp Litopenaeus vannamei: Sequence feature, expression pattern, and protein activity

Leucine-rich repeat (LRR)-only proteins have been proved to be involved in the innate immune responses as they could mediate protein-protein or protein-ligand interactions. In the present study, a novel LRR-only protein (LvLRRop-1) was identified and characterized from Pacific white shrimp Litopenaeus vannamei. The complete cDNA sequence of LvLRRop-1 contains an open reading frame (ORF) of 1488 bp, which encoded a polypeptide of 495 amino acids with a predicted molecular mass of 55.67 kDa and a calculated theoretical isoelectric point of 6.435. There are five LRR motifs, six LRR_TYP motifs in the protein sequence of LvLRRop-1 with consensus signature sequences of LxxLxxLxLxxNxL. The LvLRRop-1 mRNA transcripts could be detected in all the tested tissues, including eyestalk, gill, gonad, heart, hemocytes, hepatopancreas, intestine, muscle, nerve and stomach, especially highest in hemocytes and hepatopancreas. The mRNA transcripts of LvLRRop-1 increased within the first 6 h in hemocytes and hepatopancreas after Vibrio parahaemolyticus or white spot syndrome virus (WSSV) challenges. The recombinant LvLRRop-1 could bind four typical pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide (LPS), peptidoglycan (PGN), glucan (GLU) and polycytidine-polycytidylic acid (poly IC), in a dose-dependent manner, and inhibit the growth of bacteria Micrococcus luteus. These data indicated that LvLRRop-1 could play a pivotal role in the innate immune response of shrimps as a kind of pattern recognition receptor (PRR).

Genetic Insight into Disease Resistance Gene Clusters by Using Sequencing-Based Fine Mapping in Sunflower (Helianthus annuus L.).

Rust and downy mildew (DM) are two important sunflower diseases that lead to significant yield losses globally. The use of resistant hybrids to control rust and DM in sunflower has a long history. The rust resistance genes, R13a and R16, were previously mapped to a 3.4 Mb region at the lower end of sunflower chromosome 13, while the DM resistance gene, Pl33, was previously mapped to a 4.2 Mb region located at the upper end of chromosome 4. High-resolution fine mapping was conducted using whole genome sequencing of HA-R6 (R13a) and TX16R (R16 and Pl33) and large segregated populations. R13a and R16 were fine mapped to a 0.48 cM region in chromosome 13 corresponding to a 790 kb physical interval on the XRQr1.0 genome assembly. Four disease defense-related genes with nucleotide-binding leucine-rich repeat (NLR) motifs were found in this region from XRQr1.0 gene annotation as candidate genes for R13a and R16. Pl33 was fine mapped to a 0.04 cM region in chromosome 4 corresponding to a 63 kb physical interval. One NLR gene, HanXRQChr04g0095641, was predicted as the candidate gene for Pl33. The diagnostic SNP markers developed for each gene in the current study will facilitate marker-assisted selections of resistance genes in sunflower breeding programs.

The RECEPTOR-LIKE PROTEIN53 immune complex associates with LLG1 to positively regulate plant immunity.

Pattern recognition receptors (PRRs) sense ligands in pattern-triggered immunity (PTI). Plant PRRs include numerous receptor-like proteins (RLPs), but many RLPs remain functionally uncharacterized. Here, we examine an Arabidopsis thaliana RLP, RLP53, which positively regulates immune signaling. Our forward genetic screen for suppressors of enhanced disease resistance1 (edr1) identified a point mutation in RLP53 that fully suppresses disease resistance and mildew-induced cell death in edr1 mutants. The rlp53 mutants showed enhanced susceptibility to virulent pathogens, including fungi, oomycetes, and bacteria, indicating that RLP53 is important for plant immunity. The ectodomain of RLP53 contains leucine-rich repeat (LRR) motifs. RLP53 constitutively associates with the LRR receptor-like kinase SUPPRESSOR OF BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE (BAK1)-INTERACTING RECEPTOR KINASE1 (SOBIR1) and interacts with the co-receptor BAK1 in a pathogen-induced manner. The double mutation sobir1-12 bak1-5 suppresses edr1-mediated disease resistance, suggesting that EDR1 negatively regulates PTI modulated by the RLP53-SOBIR1-BAK1 complex. Moreover, the glycosylphosphatidylinositol (GPI)-anchored protein LORELEI-LIKE GPI-ANCHORED PROTEIN1 (LLG1) interacts with RLP53 and mediates RLP53 accumulation in the plasma membrane. We thus uncovered the role of a novel RLP and its associated immune complex in plant defense responses and revealed a potential new mechanism underlying regulation of RLP immune function by a GPI-anchored protein.

Two LRR-Only Proteins Involved in Antibacterial Defense and Prophenoloxidase System of Swimming Crab Portunus trituberculatus

The leucine-rich repeat (LRR) motif is evolutionarily conserved in many pattern recognition receptors. Compared to the reported LRR proteins with multiple functional domains, the role of LRR-only proteins merely containing LRR motifs remain largely unexplored. In this study, two LRR-only proteins, PtLRR1 and PtLRR2, were identified from the swimming crab Portunus trituberculatus. Five LRR motifs with a consensus sequence LxxLxxLxLxxNxL were found in their encoded peptides. Both PtLRR1 and PtLRR2 were dominantly expressed in the hepatopancreas and showed a time-dependent response post bacteria and virus stimulation. The recombinant PtLRR1 could bind to various PAMPs, including LPS, PGN, and GLU. PtLRR1 and PtLRR2 displayed different regulatory activities in inducing the expression of inflammation and proPO system-related genes. Knockdown of PtLRR2 led to the decreased expression of the tested cytokines and adapter, while PtLRR1 knockdown enhanced the expression of serine proteases, serine protease homologues, and proPO genes. In addition, knockdown of PtLRR1 or PtLRR2 reduced the clearance activity of Vibrio but upregulated the expression levels of AMPs and key genes of Toll, IMD, and JNK pathways. These results suggest that PtLRR1 and PtLRR2 could act as potential immune receptors and regulate antibacterial immunity in crab.

TuRLK1, a leucine-rich repeat receptor-like kinase, is indispensable for stripe rust resistance of YrU1 and confers broad resistance to multiple pathogens.

BackgroundYrU1 is a nucleotide-binding site (NBS) and leucine-rich repeat (LRR) protein (NLR), with additional ankyrin-repeat and WRKY domains and confers effective resistance to stripe rust fungus Puccinia striiformis f. sp. Tritici (Pst). YrU1 was positionally cloned in the progenitor species of the A genome of bread wheat, Tricicum urartu, recently. However, the molecular mechanism and components involved in YrU1-mediated resistance are not clear.ResultsIn this study, we found that the transcript level of TuRLK1, which encodes a novel leucine-rich repeat receptor-like kinase, was up-regulated after inoculation with Pst in the presence of YrU1, through RNA-seq analysis in T. urartu accession PI428309. TuRLK1 contained only a small number of LRR motifs, and was localized in the plasma-membrane. Transient expression of TuRLK1 induced hypersensitive cell death response in N. benthamiana leaves. Silencing of TuRLK1, using barley stripe mosaic virus (BSMV)-induced gene silencing (VIGS) system in PI428309 that contains YrU1, compromised the resistance against stripe rust caused by Pst CY33, indicating that TuRLK1 was required for YrU1-activated plant immunity. Furthermore, overexpression of TuRLK1 could enhance powdery mildew resistance in bread wheat and Arabidopsis thaliana after inoculating with the corresponding pathogens.ConclusionsOur study indicates that TuRLK1 is required for immune response mediated by the unique NLR protein YrU1, and likely plays an important role in disease resistance to other pathogens.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12870-022-03679-6.

Diversity and similarity of wheat powdery mildew resistance among three allelic functional genes at the Pm60 locus.

Cultivated wheat is continually exposed to various pathogens. Blumeria graminis f. sp. tritici (Bgt) causes powdery mildew disease and significant yield loss. Pm60 was cloned from Triticum urartu and confers race-specific powdery mildew resistance in wheat. Pm60a and Pm60b are allelic variants of Pm60 and have two leucine-rich repeat motifs deletions and insertions, respectively, which were detected in other T. urartu accessions. Through map-based cloning, virus-induced gene silencing, and stable transformation assays, we demonstrated that Pm60a and Pm60b conferred Bgt E09 resistance resembling that provided by Pm60. However, the homozygous Pm60a (but not Pm60 or Pm60b) transformants driven by the native promoters lacked race-specific resistance when they were inoculated with Bgt E18. As all three T. urartu accessions contained the three foregoing alleles, they had high resistance to Bgt E18. Pyramiding Pm60a with either of the allelic genes in F1 plants did not cause mutual allele suppression or interference with Bgt E18 resistance. Deletion (but not insertion) of the two leucine-rich repeat motifs in Pm60a substantially narrowed the resistance spectrum. In T. urartu accession PI428210, we identified another locus adjacent to Pm60a and resistant to Bgt E18. Characterization of the alleles at the Pm60 locus revealed their diversity and similarity and may facilitate wheat breeding for resistance to powdery mildew disease caused by B. graminis f. sp. tritici.

Numerous variants of leucine rich repeats in proteins from nucleo-cytoplasmic large DNA viruses

Leucine rich repeats (LRRs) occurring in tandem are 20–29 amino acids long. Eleven LRR types have been recognized. Sequence features of LRRs from viruses were investigated using over 600 LRR proteins from 89 species. Directly before, metagenome data of nucleo-cytoplasmic large dsDNA viruses (NCLDVs) have been published; the 2,074 NCLDVs encode 199,021 proteins. From the NCLDVs 547 LRR proteins were identified and 502 were used for analysis. Various variants of known LRR types were identified in viral LRRs. A comprehensive analysis of TpLRR and FNIP that belong to an LRR type was first performed. The repeating unit lengths (RULs) in five types are 19 residues which is the shortest among all LRRs. The RULs of eight LRR types including FNIP are one to five residues shorter than those of the known, corresponding LRR types. The conserved hydrophobic residues such as Leu, Val or Ile in the consensus sequences are frequently substituted by cysteine at one or two positions. Four unique LRR motifs that are different from those identified previously are observed. The present study enhances the previous result. An evolutionary scenario of short or unique LRR was discussed.

Two non-mammalian toll-like receptors (TLR21 and TLR22) from golden pompano (Trachinotus ovatus): Molecular cloning, gene characterization and expression analysis

Toll-like receptors (TLRs), important pattern recognition receptors (PRRs), involve in recognizing pathogen-associated molecular patterns (PAMPs) and play crucial roles in the host innate immunity. In the present study, two non-mammalian TLRs, troTLR21 and troTLR22, were identified and characterized from golden pompano (Trachinotus ovatus), a marine teleost with great economic values. The two TLRs were typical TLRs, containing several motifs conserved in TLRs family including leucine-rich repeat (LRR) motifs, a transmembrane domain, and a toll/interleukin-1 receptor (TIR) domain. The two TLRs shared high sequence identity with their counterparts in other teleosts. Phylogenetic tree analysis showed that troTLR21 and troTLR22 were well clustered with other fish TLR21 and TLR22, respectively. Quantitative real-time PCR (qPCR) revealed that the two TLRs were ubiquitously expressed in all investigated tissues, with higher transcription levels in the immune-related tissues (spleen and head kidney (HK)). Further, the expressions of troTLR21 and troTLR22 were significantly up-regulated in HK and spleen following Vibrio alginolyticus, lipopolysaccharide (LPS), and polyinosinic: polycytidylic acid (polyI:C) stimulation. Our results suggested that the two TLRs involve in the immune response against pathogen invasion in golden pompano, providing solid basis for further study the functions of fish-specific TLRs.