Pathogenic variants in the NLRP3 LRR domain at position 861 are responsible for a boost-dependent atypical CAPS phenotype.

BACKGROUND: Cryopyrin-associated periodic syndrome (CAPS) is associated with NLRP3 pathogenic variants, mostly located in the NACHT (neuronal apoptosis inhibitor protein, MHC class 2 transcription activator, incompatibility locus protein from Podospora anserina, telomerase-associated protein) domain. Cold-induced urticarial rash is among the main clinical features. However, this study identified a series of 14 patients with pathogenic variants of the Y861 residue (p.Tyr861) of the LRR domain of NLRP3 and minimal prevalence of cold-induced urticarial rash. OBJECTIVES: This study aimed to address a possible genotype/phenotype correlation for patients with CAPS and to investigate at the cellular levels the impact of the Y861C substitution (p.Tyr861Cys) on NLRP3 activation. METHODS: Clinical features of 14 patients with CAPS and heterozygous substitution at position 861 in the LRR domain of NLRP3 were compared to clinical features of 48 patients with CAPS and pathogenic variants outside the LRR domain of NLRP3. IL-1ß secretion by PBMCs and purified monocytes from patients and healthy donors was evaluated following LPS and monosodium urate crystal stimulation. RESULTS: Patients with substitution at position 861 of NLRP3 demonstrated a higher prevalence of sensorineural hearing loss while being less prone to skin urticarial. In contrast to patients with classical CAPS, cells from patients with a pathogenic variant at position 861 required an activation signal to secrete IL-1ß but produced more IL-1ß during the early and late phase of secretion than cells from healthy donors. CONCLUSIONS: Pathogenic variants of Y861 of NLRP3 drive a boost-dependent oversecretion of IL-1ß associated with an atypical CAPS phenotype.

Tsw - A case study on structure-function puzzles in plant NLRs with unusually large LRR domains.

Plant disease immunity heavily depends on the recognition of plant pathogens and the subsequent activation of downstream immune pathways. Nod-like receptors are often crucial in this process. Tsw, a Nod-like resistance gene from Capsicum chinense conferring resistance against Tomato spotted wilt virus (TSWV), belongs to the small group of Nod-like receptors with unusually large LRR domains. While typical protein domain dimensions rarely exceed 500 amino acids due to stability constraints, the LRR of these unusual NLRs range from 1,000 to 3,400 amino acids and contain over 30 LRR repeats. The presence of such a multitude of repeats in one protein is also difficult to explain considering protein functionality. Interactions between the LRR and the other NLR domains (CC, TIR, NBS) take place within the first 10 LRR repeats, leaving the function of largest part of the LRR structure unexplained. Herein we discuss the structural modeling limits and various aspects of the structure-function relation conundrums of large LRRs focusing on Tsw, and raise questions regarding its recognition of its effector NSs and the possible inhibition on other domains as seen in other NLRs.

DeepLRR: An Online Webserver for Leucine-Rich-Repeat Containing Protein Characterization Based on Deep Learning.

Members of the leucine-rich repeat (LRR) superfamily play critical roles in multiple biological processes. As the LRR unit sequence is highly variable, accurately predicting the number and location of LRR units in proteins is a highly challenging task in the field of bioinformatics. Existing methods still need to be improved, especially when it comes to similarity-based methods. We introduce our DeepLRR method based on a convolutional neural network (CNN) model and LRR features to predict the number and location of LRR units in proteins. We compared DeepLRR with six existing methods using a dataset containing 572 LRR proteins and it outperformed all of them when it comes to overall F1 score. In addition, DeepLRR has integrated identifying plant disease-resistance proteins (NLR, LRR-RLK, LRR-RLP) and non-canonical domains. With DeepLRR, 223, 191 and 183 LRR-RLK genes in Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa ssp. Japonica) and tomato (Solanum lycopersicum) genomes were re-annotated, respectively. Chromosome mapping and gene cluster analysis revealed that 24.2% (54/223), 29.8% (57/191) and 16.9% (31/183) of LRR-RLK genes formed gene cluster structures in Arabidopsis, rice and tomato, respectively. Finally, we explored the evolutionary relationship and domain composition of LRR-RLK genes in each plant and distributions of known receptor and co-receptor pairs. This provides a new perspective for the identification of potential receptors and co-receptors.

Molecular phylogeny and dynamic evolution of disease resistance genes in the legume family.

BACKGROUND: Legumes are the second-most important crop family in agriculture for its economic and nutritional values. Disease resistance (R-) genes play an important role in responding to pathogen infections in plants. To further increase the yield of legume crops, we need a comprehensive understanding of the evolution of R-genes in the legume family. RESULTS: In this study, we developed a robust pipeline and identified a total of 4,217 R-genes in the genomes of seven sequenced legume species. A dramatic diversity of R-genes with structural variances indicated a rapid birth-and-death rate during the R-gene evolution in legumes. The number of R-genes transiently expanded and then quickly contracted after whole-genome duplications, which meant that R-genes were sensitive to subsequent diploidization. R proteins with the Coiled-coil (CC) domain are more conserved than others in legumes. Meanwhile, other types of legume R proteins with only one or two typical domains were subjected to higher rates of loss during evolution. Although R-genes evolved quickly in legumes, they tended to undergo purifying selection instead of positive selection during evolution. In addition, domestication events in some legume species preferentially selected for the genes directly involved in the plant-pathogen interaction pathway while suppressing those R-genes with low occurrence rates. CONCLUSIONS: Our results provide insights into the dynamic evolution of R-genes in the legume family, which will be valuable for facilitating genetic improvements in the disease resistance of legume cultivars.

TNL genes in peach: insights into the post-LRR domain.

BACKGROUND: Plants develop sustainable defence responses to pathogen attacks through resistance (R) genes contributing to effector-triggered immunity (ETI). TIR-NB-LRR genes (TNL genes) constitute a major family of ETI R genes in dicots. The putative functions or roles of the TIR, NB and LRR domains of the proteins they encode (TNLs) are well documented, but TNLs also have a poorly characterised C-terminal region, the function of which is unknown in most cases. We characterised this prevalent stress-response protein family in a perennial plant, using the genome of peach (Prunus persica), the model Prunus species. The first TNL gene from this genus to be cloned, the Ma gene, confers complete-spectrum resistance to root-knot nematodes (RKNs) and encodes a protein with a huge C-terminal region with five duplicated post-LRR (PL) domains. This gene was the cornerstone of this study. RESULTS: We investigated the role of this C-terminal region, by first describing the frequency, distribution and structural characteristics of i) TNL genes and ii) their PL domains in the peach genome, using the v1.0 Sanger sequence together with the v2.0 sequence, which has better genome annotation due to the incorporation of transcriptomic data. We detected 195 predicted TNL genes from the eight peach chromosomes: 85 % of these genes mapped to chromosomes 1, 2, 7 and 8. We reconstructed the putative structure of the predicted exons of all the TNL genes identified, and it was possible to retrieve the PL domains among two thirds of the TNL genes. We used our predicted TNL gene sequences to develop an annotation file for use with the Gbrowse tool in the v2.0 genome. The use of these annotation data made it possible to detect transcribed PL sequences in two Prunus species. We then used consensus sequences defined on the basis of 124 PL domains to design specific motifs, and we found that the use of these motifs significantly increased the numbers of PL domains and correlative TNL genes detected in diverse dicot genomes. Based on PL signatures, we showed that TNL genes with multiple PL domains were rare in peach and the other plants screened. The five-PL domain pattern is probably unique to Ma and its orthologues within Prunus and closely related genera from the Rosaceae and was probably inherited from the common ancestor of these plants in the subfamily Spiraeoideae. CONCLUSIONS: The first physical TNL gene map for Prunus species can be used for the further investigation of R genes in this genus. The PL signature motifs are a complementary tool for the detection of TNL R genes in dicots. The low degree of similarity between PL domains and the neighbouring LRR exons and the specificity of PL signature motifs suggest that PL and LRR domains have different origins, with PL domains being specific to TNL genes, and possibly essential to the functioning of these genes in some cases. Investigations of the role of the oversized Ma PL region, in ligand binding or intramolecular interactions for example, may help to enrich our understanding of NB-LRR-mediated plant immunity to RKNs.

Comparative analysis of resistance gene analogues encoding NBS-LRR domains in cotton.

BACKGROUND: Plant production is severely affected by biotic and abiotic stresses R-genes exhibit resistance against a range of diseases and pathogens in plants. The nucleotide binding site and leucine rich repeat (NBS-LRR) class of R-genes is the most comprehensively studied in terms of sequence evolution and genome distribution. The differential response for resistance against biotic and abiotic stress has been observed in cultivated and wild relatives of the genus Gossypium. RESULTS: Efforts have been made to address the recent evolution of NBS-LRR sequences within Gossypium hirsutum and resistance gene analogue (RGA) sequences derived from G. arboreum and G. raimondii. The % identity and phylogenetic analysis of NBS-LRR-encoded RGAs from tetraploid New World cotton and its diploid ancestors G. raimondii and G. arboreum suggest that the evolution of NBS-LRR-encoding sequences in G. hirsutum occurred by gradual accumulation of mutants that led to positive selection and a slow rate of divergence within distinct R-gene families. CONCLUSION: The allotetraploid genome of cotton, after separating from its diploid parents, experienced polyploidisation, natural and artificial selection, hybrid necrosis, duplication and recombination which became the reason to shed off and evolve new genes for its survival. These driving forces influenced the development of genomic architecture that make it susceptible to diseases and pathogens as compared to donor parents.

Heme interplay between IlsA and IsdC: Two structurally different surface proteins from Bacillus cereus.

BACKGROUND: Iron is an essential element for bacterial growth and virulence. Because of its limited bioavailability in the host, bacteria have adapted several strategies to acquire iron during infection. In the human opportunistic bacteria Bacillus cereus, a surface protein IlsA is shown to be involved in iron acquisition from both ferritin and hemoproteins. IlsA has a modular structure consisting of a NEAT (Near Iron transporter) domain at the N-terminus, several LRR (Leucine Rich Repeat) motifs and a SLH (Surface Layer Homology) domain likely involved in anchoring the protein to the cell surface. METHODS: Isothermal titration calorimetry, UV-Vis spectrophotometry, affinity chromatography and rapid kinetics stopped-flow measurements were employed to probe the binding and transfer of hemin between two different B. cereus surface proteins (IlsA and IsdC). RESULTS: IlsA binds hemin via the NEAT domain and is able to extract heme from hemoglobin whereas the LRR domain alone is not involved in these processes. A rapid hemin transfer from hemin-containing IlsA (holo-IlsA) to hemin-free IsdC (apo-IsdC) is demonstrated. CONCLUSIONS: For the first time, it is shown that two different B. cereus surface proteins (IlsA and IsdC) can interact and transfer heme suggesting their involvement in B. cereus heme acquisition. GENERAL SIGNIFICANCE: An important role for the complete Isd system in heme-associated bacterial growth is demonstrated and new insights into the interplay between an Isd NEAT surface protein and an IlsA-NEAT-LRR protein, both of which appear to be involved in heme-iron acquisition in B. cereus are revealed.

High-resolution crystal structure of the leucine-rich repeat domain of the human tumour suppressor PP32A (ANP32A).

Acidic leucine-rich nuclear phosphoprotein 32A (PP32A) is a tumour suppressor whose expression is altered in many cancers. It is an apoptotic enhancer that stimulates apoptosome-mediated caspase activation and also forms part of a complex involved in caspase-independent apoptosis (the SET complex). Crystals of a fragment of human PP32A corresponding to the leucine-rich repeat domain, a widespread motif suitable for protein-protein interactions, have been obtained. The structure has been refined to 1.56 Šresolution. This domain was previously solved at 2.4 and 2.69 Šresolution (PDB entries 2je0 and 2je1, respectively). The new high-resolution structure shows some differences from previous models: there is a small displacement in the turn connecting the first α-helix (α1) to the first ß-strand (ß1), which slightly changes the position of α1 in the structure. The shift in the turn is observed in the context of a new crystal packing unrelated to those of previous structures.

Molecular characterization and comparative sequence analysis of defense-related gene, Oryza rufipogon receptor-like protein kinase 1.

Many of the plant leucine rich repeat receptor-like kinases (LRR-RLKs) have been found to regulate signaling during plant defense processes. In this study, we selected and sequenced an LRR-RLK gene, designated as Oryza rufipogon receptor-like protein kinase 1 (OrufRPK1), located within yield QTL yld1.1 from the wild rice Oryza rufipogon (accession IRGC105491). A 2055 bp coding region and two exons were identified. Southern blotting determined OrufRPK1 to be a single copy gene. Sequence comparison with cultivated rice orthologs (OsI219RPK1, OsI9311RPK1 and OsJNipponRPK1, respectively derived from O. sativa ssp. indica cv. MR219, O. sativa ssp. indica cv. 9311 and O. sativa ssp. japonica cv. Nipponbare) revealed the presence of 12 single nucleotide polymorphisms (SNPs) with five non-synonymous substitutions, and 23 insertion/deletion sites. The biological role of the OrufRPK1 as a defense related LRR-RLK is proposed on the basis of cDNA sequence characterization, domain subfamily classification, structural prediction of extra cellular domains, cluster analysis and comparative gene expression.