LRR Region Research Articles

Genetic Basis Identification of a NLR Gene, TaRGA5-like, That Confers Partial Powdery Mildew Resistance in Wheat SJ106.

Wheat powdery mildew is an important fungal disease that seriously jeopardizes wheat production, which poses a serious threat to food safety. SJ106 is a high-quality, disease-resistant spring wheat variety; this disease resistance is derived from Wheat-wheatgrass 33. In this study, the powdery mildew resistance genes in SJ106 were located at the end of chromosome 6DS, a new disease resistance locus tentatively named PmSJ106 locus. This interval was composed of a nucleotide-binding leucine-rich repeat (NLR) gene cluster containing 19 NLR genes. Five NLRs were tandem duplicated genes, and one of them (a coiled coil domain-nucleotide binding site-leucine-rich repeat (CC-NBS-LRR; CNL) type gene, TaRGA5-like) expressed 69-836-fold in SJ106 compared with the susceptible control. The genome DNA and cDNA sequences of TaRGA5-like were amplified from SJ106, which contain several nucleotide polymorphisms in LRR regions compared with susceptible individuals and Chinese Spring. Overexpression of TaRGA5-like significantly increased resistance to powdery mildew in susceptible receptor wheat Jinqiang5. However, Virus induced gene silence (VIGS) of TaRGA5-like resulted in only a small decrease of SJ106 in disease resistance, presumably compensated by other NLR duplicated genes. The results suggested that TaRGA5-like confers partial powdery mildew resistance in SJ106. As a member of the PmSJ106 locus, TaRGA5-like functioned together with other NLR duplicated genes to improve wheat resistance to powdery mildew. Wheat variety SJ106 would become a novel and potentially valuable germplasm for powdery mildew resistance.

Low temperature compensates for defective tapetum initiation to restore the fertility of the novel TGMS line ostms15.

In rice breeding, thermosensitive genic male sterility (TGMS) lines based on the tms5 locus have been extensively employed. Here, we reported a novel rice TGMS line ostms15 (Oryza sativa ssp. japonica ZH11) which show male sterility under high temperature and fertility under low temperature. Field evaluation from 2018 to 2021 revealed that its sterility under high temperature is more stable than that of tms5 (ZH11), even with occasional low temperature periods, indicating its considerable value for rice breeding. OsTMS15 encodes an LRR-RLK protein MULTIPLE SPOROCYTE1 (MSP1) which was reported to interact with its ligand to initiate tapetum development for pollen formation. In ostms15, a point mutation from GTA (Val) to GAA (Glu) in its TIR motif of the LRR region led to the TGMS phenotype. Cellular observation and gene expression analysis showed that the tapetum is still present in ostms15, while its function was substantially impaired under high temperature. However, its tapetum function was restored under low temperature. The interaction between mOsTMS15 and its ligand was reduced while this interaction was partially restored under low temperature. Slow development was reported to be a general mechanism of P/TGMS fertility restoration. We propose that the recovered protein interaction together with slow development under low temperature compensates for the defective tapetum initiation, which further restores ostms15 fertility. We used base editing to create a number of TGMS lines with different base substitutions based on the OsTMS15 locus. This work may also facilitate the mechanistic investigation and breeding of other crops.

The antibacterial activity and antibacterial mechanism analyses of an LRR-IG protein in the Chinese mitten crab, Eriocheir sinensis

Leucine-rich repeat and immunoglobulin domain containing protein (LRR-IG) family is an important class of immune molecules in invertebrates. Herein, a novel LRR-IG, named as EsLRR-IG5, was identified from Eriocheir sinensis. It contained typical structures of LRR-IG including an N-terminal LRR region and three IG domains. EsLRR-IG5 was ubiquitously expressed in all the tested tissues, and its transcriptional levels increased after being challenged with Staphylococcus aureus and Vibrio parahaemolyticus. Recombinant proteins of LRR and IG domains from the EsLRR-IG5 (named as rEsLRR5 and rEsIG5) were successfully obtained. rEsLRR5 and rEsIG5 could bind to both gram-positive bacteria and gram-negative bacteria as well as lipopolysaccharide (LPS) and peptidoglycan (PGN). Moreover, rEsLRR5 and rEsIG5 exhibited antibacterial activities against V. parahaemolyticus and V. alginolyticus and displayed bacterial agglutination activities against S. aureus, Corynebacterium glutamicum, Micrococcus lysodeikticus, V. parahaemolyticus and V. alginolyticus. The scanning electron microscopy (SEM) observation revealed that the membrane integrity of V. parahaemolyticus and V. alginolyticus was destroyed by rEsLRR5 and rEsIG5, which may lead to the leakage of cell contents and death. This study provided clues for further studies on the immune defense mechanism mediated by LRR-IG in crustaceans and provided candidate antibacterial agents for prevention and control of diseases in aquaculture.

Validation of CAPS marker WR003 for the leaf rust resistance gene Lr1 and the molecular evolution of Lr1 in wheat

The wheat leaf rust resistance gene Lr1 encodes a typical coiled-coil nucleotide-binding site leucine-rich repeat (CC-NBS-LRR) of resistance protein containing 1 344 amino acids. WR003, a cleaved amplified polymorphic sequence (CAPS) marker is derived from the LRR regions of Lr1. In this study, a worldwide collection of 120 Aegilops tauschii accessions and 282 hexaploid wheat varieties was screened for Lr1 alleles using WR003, and the specificity of WR003 for Lr1 was confirmed by pathogenicity tests and genotype analysis. The sequence alignment and phylogenetic tree analysis of 38 Lr1 haplotypes provided a further view of the molecular evolution of Lr1. The results showed that there were very few polymorphisms between the Lr1 alleles from Ae. tauschii and hexaploid wheat with the same resistance phenotype. The polymorphisms of the Lr1 haplotypes were mainly between the different resistance lines, rather than between the different ploidy levels. These results indicate that Lr1 originated from Ae. tauschii and differentiated into resistant and susceptible genotypes before its introgression into hexaploid wheat. Therefore, it is likely that wheat Lr1 has at least two major variants for disease resistance and susceptibility, and except for certain point mutations, few gene conversions and genetic re-combinations occurred during the hexaploid wheat domestication.

In silico modelling and interactive profiling of BPH resistance NBS-LRR proteins with salivary specific proteins of rice planthoppers

Brown planthopper resistance NBS-LRR specific R genes (Bph9, Bph14, Bph18, Bph26) have been reported in rice. However, the physical interaction between the expressed BPH R proteins with planthopper salivary proteins remains unclear. In this study, we summarize the interaction of major BPH R proteins with various salivary proteins of rice planthoppers (BPH, WBPH & SBPH) using in-silico molecular docking studies. We also reveal the phylogentic relationship across the major R proteins of rice to provide an overview on the functional mimicry of R proteins in rice resistance. BPH specific R genes were clustered with other R genes of rice on chromosome 12 (Bph9, Bph18, Bph26) and 3 (Bph14) providing clues on the functional evolution of plant R genes against different insect pests and pathogens. Motif analysis showed the predominant motifs as CC, NBS and LRR regions. Bph9, Bph18 and Bph26 proteins exhibited high degree of sequence similarity in their CC and NBS region and are considered as functional alleles of BPH resistance at chromosome 12. Interaction between BPH R proteins and salivary components occurred at both NBS and LRR region. The salivary protein dipeptidyl peptidase IV from SBPH followed by carboxylesterase (BPH and WBPH) exhibited higher docking score and hydrogen bond with BPH R proteins. The interacting residues of the NBS-LRR region varied with the salivary proteins indicating the specificity in recognizing individual insect associated molecules. Our results provide first confirmation about the interaction between NBS LRR R proteins and salivary components of rice planthoppers using in silico approaches. The study provides new avenues to investigate the mechanism of interaction of R proteins and insect salivary components in the context of adaptation and evolutionary biology.

Development of candidate gene-based markers and map-based cloning of a dominant rust resistance gene in cultivated groundnut (Arachis hypogaea L.)

A dominant rust resistance gene, VG 9514-Rgene was isolated through map-based cloning. Sequence analysis revealed non-synonymous mutations in the TIR, NBS and LRR region of the R-protein. Candidate gene-based markers from these SNPs revealed complete co-segregation of the isolated VG 9514-Rgene with rust resistance in a RIL population and confirmed their map position in between FRS 72 and SSR_GO340445 markers in arahy03 chromosome. Blastp search of VG 9514-Rprotein detected Arahy.T6DCA5 with >80.0% identity that localized at 142,544,745.0.142,549,184 in arahy03 chromosome. Ka/Ks calculation revealed that VG 9514-Rgene had undergone positive selection compared to four homologous genes in the groundnut genome. Homology based structure modelling of this R-protein revealed a typical consensus three-dimensional folding of TIR-NBS-LRR protein. Non-synonymous mutations in susceptible version of R-protein were mapped and found E268Q mutation in hhGRExE motif, Y309F in RNBS-A motif and I579T in MHD motif of NB-ARC domain are probable candidates for loss of function.

Fine Mapping and Functional Analysis of Major QTL, CRq for Clubroot Resistance in Chinese Cabbage (Brassica rapa ssp. pekinensis)

Clubroot disease caused by Plasmodiophora brassicae is one of the major threats to Brassica crops. New clubroot resistant varieties of Chinese cabbage (B. rapa ssp. pekinensis) have been developed through breeding, but the underlying genetic mechanism of clubroot resistance is still unclear. In this study, two Chinese cabbage DH lines, clubroot-resistant Y635-10 and susceptible Y177-47 were crossed to develop F2 population for fine mapping and cloning resistance gene CRq. After sequence analysis, the expression vector was constructed by gateway technology and transferred into Arabidopsis thaliana for functional characterization. Bulked segregant analysis sequencing (BSA-seq) confirmed that CRq is located in the 80 kb genomic region on chromosome A03 between markers GC30-FW/RV and BGA. In silico tools confirmed that the gene length was 3959 bp with 3675 bp coding sequences (CDs), and it has three exons and two introns. In addition, we found 72bp insertion in the third exon of CRq in the susceptible line. We developed and verified functional marker Br-insert1, by which genotyping results showed that 72bp insertion might lead to the destruction of the LRR region of Y177-47, resulting in a loss of resistance relative to clubroot. The results of genetic transformation showed that the roots for wild-type Arabidopsis thaliana were significantly enlarged compared with T2 generation transgenic Arabidopsis after treatment by P. brassicae spores, and transgenic Arabidopsis had certain resistance. Therefore, CRq is a candidate gene of clubroot disease resistance in Chinese cabbage, which could be used as a reference for elucidating disease resistance mechanisms and the marker-assisted breeding of clubroot resistant varieties.

Comprehensive Description of Monoallelic GP1BA Variants Associated with Thrombocytopenia

GP1BA encodes the glycoprotein (GP) 1bα subunit of the GP1b/IX/V complex, the platelet receptor for von Willebrand factor (VWF) and a mediator of outside-in signalling that contributes to platelet activation. Biallelic variants in GPIBA that prevent surface expression of GPIB/IX/V cause the recessive disorder Bernard Soulier Syndrome (BSS; OMIM #23100) in which there is often severe bleeding caused by thrombocytopenia and platelet dysfunction. Thrombocytopenia has also been associated with monoallelic missense variants in the GP1bα R loop (residues 227-242) which enhance VWF binding (platelet-type von Willebrand disease (pVWD); OMIM #177820). Other monoallelic GPIBA missense variants such as the Bolzano variant (p.Ala172Val; OMIM #153670) have been associated with dominant thrombocytopenia without altered VWF levels. In order to better describe the relationship between monoallelic GPIBA variants and thrombocytopenia we evaluated data from the NIHR BioResource for Rare Diseases, (NBR-RD) and the Inherited Platelet Disorders programmes at the Universities of Birmingham (UK), Murcia (Spain) and Toronto (Canada) in which patients with unexplained platelet disorders underwent diagnostic next generation sequencing. Using the BeviMed method for genetic association we compared the genotypes at rare nonsynoymous variants of 105 unrelated thrombocytopenia cases (platelet count (PLT) <130x109/L or the human phenotype ontology term 'HP 001873 thrombocytopenia') in the NBR-BR with those of 10,152 unrelated participants without thrombocytopenia. There was a strong statistical association (posterior probability 0.97) between dominantly inherited GPIBA variants and thrombocytopenia. Considering the five variants within the NBR-BR that drove this association alongside genotype data from the other case collections, we identified a total of 21 different monoallelic GPIBA coding variants that were rare (gnomAD allele frequency <1/1000) and had a CADD pathogenicity score >15. These were present in 29 index cases (18 females, median age 37 years) with unexplained thrombocytopenia and included the variant predicting the p.Asn150Ser substitution which was identified in seven independent index cases. The variants comprised 18 missense, two inframe insertions (one with an additional missense change) and one insertion resulting in a frame shift. Seven had been previously associated with thrombocytopenia. Sixteen of the variants predicted amino acid substitutions or single residue insertions within the GP1bα leucine rich repeat region (LRR; residues 48-200) or in adjacent regions of the LRR N- and C-terminal caps. Cases with variants in the LRR region typically displayed mild mucocutaneous bleeding, abnormal bleeding after minor trauma or surgery and heavy menstrual bleeding. The platelet count was typically 60-100 x109/L and mean platelet volume greater than 11 fL. There were two missense variants in the GP1bα R loop that have been previously associated with pVWD and two further previously unreported missense variants in the cytoplasmic domain. The final variant was a monoallelic single nucleotide insertion predicted to cause a frame shift and absent expression of GP1bα and has been previously reported as a biallelic variant associated with BSS. These data indicate that in a multicentre cross-sectional cohort of cases with otherwise unexplained thrombocytopenia, monoallelic missense GPIBA variants affecting the LRR or adjacent regions were more prevalent than R loop variants associated with pVWD or variants causing absent GP1bα expression associated with biallelic BSS. The LRR region contains the GP1bα-VWF binding site which is predicted to be disrupted by the missense variants observed in this study. The LRR region is also the location of the GP1bα Bolzano variant and six other monoallelic variants previously associated with thrombocytopenia in previous single case reports. These observations extend the repertoire of GPIBA variants associated with thrombocytopenia and although requiring experimental confirmation of pathogenicity, suggest a common molecular pathogenesis for dominant GPIBA-associated thrombocytopenia in which reduced circulating platelet numbers arises from defective GP1bα-VWF interactions. Figure Disclosures Turro: Tachyon Ventures: Other: Partner & Scientific Director.

Novel homozygous mutation in the FBXL4 gene is associated with mitochondria DNA depletion syndrome-13

BackgroundMitochondrial DNA depletion syndrome-13 (MTDPS13) is caused by mutations in FBXL4 (F-box and leucine-rich repeat protein 4), a nuclear gene encoding an F-box protein that plays a role in maintaining mtDNA integrity and stability. MethodsWe identified a novel homozygous FBXL4 gene mutation, c.993dupA (p.L332Tfs*3), in a 1-year-old girl of Han Chinese descent. We performed three-dimensional protein structural analysis and targeted mtDNA next-generation sequencing. We analysed FBXL4 expression and mitochondrial DNA level, and reviewed mutations reported in FBXL4-related literature. ResultsThis mutation resulted in premature termination of translation and loss of 288 amino acids from C-terminus. A three-dimensional structural analysis revealed that conserved LRR domains were lost in mutant FBXL4 protein, which likely affected its ability to form protein-protein interactions. There were no differences in FBXL4 mRNA expression levels between the patient and her parents. There were no mtDNA mutations in either the patient or her parents. However, ND1/GAPDH ratio in lymphocytes and urine, which represents mtDNA/nuclear DNA ratio, showed that the number of mitochondrial genomes was significantly lower in the patient than in her parents or wild-type subjects. ConclusionHomozygous FBXL4 gene mutation, c.993dupA, can cause mitochondrial dysfunction, and LRR region is especially important for FBXL4 protein function.

LRR domain of NLRX1 protein delivery by dNP2 inhibits T cell functions and alleviates autoimmune encephalomyelitis.

Multiple sclerosis (MS) is a demyelinating inflammatory disease of the central nervous system (CNS), which is a chronic progressive disease and is caused by uncontrolled activation of myelin antigen specific T cells. It has high unmet medical needs due to the difficulty of efficient drug delivery into the CNS to control tissue inflammation. In this study, we demonstrate that a fusion protein of NOD-like receptor family member X1 (NLRX1) and blood brain barrier (BBB)-permeable peptide, dNP2 ameliorates experimental autoimmune encephalomyelitis (EAE).Methods: We purified recombinant LRR or NBD regions of NLRX1 protein conjugated with dNP2. To examine intracellular delivery efficiency of the recombinant protein, we incubated the proteins with Jurkat T cells or murine splenic T cells and their delivery efficiency was analyzed by flow cytometry. To investigate the therapeutic efficacy in an EAE model, we injected the recombinant protein into mice with 3 different treatment schemes e.g., prevention, semi-therapeutic, and therapeutic. To analyze their functional roles in T cells, we treated MACS-sorted naïve CD4 T cells with the proteins during their activation and differentiation into Th1, Th17, and Treg cells.Results: dNP2-LRR protein treatment showed significantly higher delivery efficiency than TAT-LRR or LRR alone in Jurkat T cells and mouse splenic T cells. In all three treatment schemes of EAE experiments, dNP2-LRR administration showed ameliorated tissue inflammation and disease severity with reduced number of infiltrating T cells producing inflammatory cytokines such as IFNγ. In addition, dNP2-LRR inhibited T cell activation, cytokine production, and Th1 differentiation.Conclusion: These results suggest that dNP2-LRR is a novel agent, which regulates effector T cell functions and could be a promising molecule for the treatment of CNS autoimmune diseases such as multiple sclerosis.

Characterization and diagnostic marker development for Yr28-rga1 conferring stripe rust resistance in wheat

Stripe rust is one of the most devastating wheat diseases and developing resistant cultivars is the most economical way to manage this disease. More than 80 stripe rust resistance genes (Yr genes) have formally been mapped. However, a lack of diagnostic markers has partially restricted the deployment of some resistance genes in molecular breeding. Among them, Yr28 showed high-temperature adult plant resistance (HT-APR) to stripe rust in the field, but it is rarely employed in wheat breeding. Here, Yr28-rga1 was found and isolated through the co-segregated marker mwg634 of Yr28. The gene sequence of Yr28-rga1-4A and Yr28-rga1-4B were almost the same between PI648511 and other wheat germplasm, including Chinese Spring (CS), Avocet, Yanzhan1, and Chuanyu12, but Yr28-rga1-4D showed only 88% identity between PI648511 and the others. Further, the high similarity in conserved domains to other wheat R proteins indicated that Yr28-RGA1 was a typical NBS-LRR protein. But the low identity in amino sequences also suggested that it was a novel gene. The specific 3-D protein structure in LRR regions also indicated that Yr28-rga1-4D from PI648511 might have different functional responses to disease. Furthermore, two gene-specific markers of cib-Yr28M1 and cib-Yr28M2 were developed for Yr28-rga1. Only 24 accessions showed identity to PI648511 on Yr28-rga1 locus in a panel of 672 wheat germplasms. The two diagnostic markers also revealed the positive correlation of Yr28-rga1 and stripe rust resistance (R2 > 0.8; p < 0.01) in field testing. These results indicated that the newly characterized Yr28-rga1 was a novel NBS-LRR gene conferring stripe rust resistance. The two diagnostic markers would be useful for marker assisted selection in wheat breeding against stripe rust.

Variation in the LRR region of Pi54 protein alters its interaction with the AvrPi54 protein revealed by in silico analysis.

Rice blast, caused by the ascomycete fungus Magnaporthe oryzae is a destructive disease of rice and responsible for causing extensive damage to the crop. Pi54, a dominant blast resistance gene cloned from rice line Tetep, imparts a broad spectrum resistance against various M. oryzae isolates. Many of its alleles have been explored from wild Oryza species and landraces whose sequences are available in the public domain. Its cognate effector gene AvrPi54 has also been cloned from M. oryzae. Complying with the Flor's gene-for-gene system, Pi54 protein interacts with AvrPi54 protein following fungal invasion leading to the resistance responses in rice cell that prevents the disease development. In the present study Pi54 alleles from 72 rice lines were used to understand the interaction of Pi54 (R) proteins with AvrPi54 (Avr) protein. The physiochemical properties of these proteins varied due to the nucleotide level polymorphism. The ab initio tertiary structures of these R- and Avr- proteins were generated and subjected to the in silico interaction. In this interaction, the residues in the LRR region of R- proteins were shown to interact with the Avr protein. These R proteins were found to have variable strengths of binding due to the differential spatial arrangements of their amino acid residues. Additionally, molecular dynamic simulations were performed for the protein pairs that showed stronger interaction than Pi54tetep (original Pi54 from Tetep) protein. We found these proteins were forming h-bond during simulation which indicated an effective binding. The root mean square deviation values and potential energy values were stable during simulation which validated the docking results. From the interaction studies and the molecular dynamics simulations, we concluded that the AvrPi54 protein interacts directly with the resistant Pi54 proteins through the LRR region of Pi54 proteins. Some of the Pi54 proteins from the landraces namely Casebatta, Tadukan, Varun dhan, Govind, Acharmita, HPR-2083, Budda, Jatto, MTU-4870, Dobeja-1, CN-1789, Indira sona, Kulanji pille and Motebangarkaddi cultivars show stronger binding with the AvrPi54 protein, thus these alleles can be effectively used for the rice blast resistance breeding program in future.

Polymorphisms and Evolution of Solanum bulbocastanum Genes for Broad-Spectrum Resistance to Phytophthora infestans

For two decades, Solanum bulbocastanum Dun. has been challenging potato geneticists with its durable resistance to Phytophthora infestans Mont. de Bary, the causal agent of late blight, the most devastating potato disease. In this study, highly specific SCAR (sequence characterized amplified region) markers for three S. bulbocastanum genes of broad-spectrum resistance to P. infestans,RB/Rpi-blb1,Rpi-blb2 and Rpi-blb3, were employed to screen a vast collection of wild tuber-bearing species from several series of Solanum L. section Petota Dumort. and also potato hybrids and varieties. In S. bulbocastanum and S. stoloniferum Schlechtd. et Bche, the sequences of the markers Rpi-blb1-820 and Rpi-sto1-890, which recognize the LRR and CC regions respectively differed inconsiderably in the orthologous genes RB/Rpi-blb1 and Rpi-sto1. These two markers were tightly linked in diverse wild Solanum genotypes. These markers as well as RB-226, another marker recognizing the LRR region of the Rpi-blb1 gene, were obviously related to high resistance to P. infestans. One more marker recognizing the CC region of the gene, RB-629, was found in much wider range of Solanum genotypes; it was not related to resistance to P. infestans and is therefore considered non-specific. In addition to Rpi-blb1 = Rpi-sto1, the Rpi-blb2 and Rpi-blb3 genes were registered in both S. bulbocastanum and S. stoloniferum accessions. In most cases, three bulbocastanum genes were not linked together. The markers specific for Rpi-blb1 = Rpi-sto1 genes were found in the Solanum series Bulbocastana (Rydb.) Hawkes (genome designation BB) and Longipedicellata Buk. (genome designation AABB) and were absent from five Pinnatisecta Rydb. species also comprising genome B. The marker for Rpi-blb2 was found in the series Bulbocastana and Longipedicellata and also in three accessions of Pinnatisecta, while the Rpi-blb3 gene, the orthologue of widely spread R2 gene for resistance to P. infestans, was present in all three Solanum series. In contrast with Rpi-blb1 = Rpi-sto1, two other functional RB homologues, Rpi-bt1 in S. bulbocastanum and RBver in S. verrucosum Schlechtd. (genome AA), apparently diverged earlier in the evolution of tuber-bearing Solanum species. Our evidence on functional Rpi-blb1 = Rpi-sto1 genes in three Petota series substantiates the assumption that the B genome of tetraploid Longipedicellata evolved from diploid Bulbocastana rather than from diploid Pinnatisecta progenitors. Similar to wild Solanum species, the markers Rpi-blb1-820 and Rpi-sto1-890 were tightly linked in advanced potato hybrids and established varieties bred from S. stoloniferum as the conventional source of bulbocastanum genes for late blight resistance, but did not always match the markers for the Rpi-blb2 and Rpi-blb3 genes and the marker for the Rysto gene as an independent indicator of stoloniferum germplasm.

Genetic signatures of plant resistance genes with known function within and between species

Plant disease resistance (R) genes have undergone significant evolutionary divergence to cope with rapid changes in pathogens. These highly variable evolutionary patterns may have contributed to diversity in R gene protein families or structures. Here, the evolutionary patterns of 76 identified R genes and their homologs were investigated within and between plant species. Results demonstrated that nucleotide binding sites and leucine-rich-repeat genes located in loci with complex evolutionary histories tended to evolve rapidly, have high variation in copy numbers, exhibit high levels of nucleotide variation and frequent gene conversion events, and also exhibit high non-synonymous to synonymous substitution ratios in LRR regions. However, non-NBS-LRR R genes are relatively well conserved with constrained variation and are more likely to participate in the basic defense system of hosts. In addition, both conserved and highly divergent evolutionary patterns were observed for the same R genes and were consistent with inter- and intra-specific distributions of some R genes. These results thus indicate either continuous or altered evolutionary patterns between and within species. The present investigation is the first attempt to investigate evolutionary patterns among all clearly functional R genes. The results reported here thus provide a foundation for future plant disease studies.

Cryo-EM structures of the human volume-regulated anion channel LRRC8.

Maintenance of cell volume against osmotic change is crucial for proper cell functions. Leucine-rich repeat-containing 8 proteins are anion-selective channels that extrude anions to decrease the cell volume on cellular swelling. Here, we present the structure of human leucine-rich repeat-containing 8A, determined by single-particle cryo-electron microscopy. The structure shows a hexameric assembly, and the transmembrane region features a topology similar to gap junction channels. The LRR region, with 15 leucine-rich repeats, forms a long, twisted arc. The channel pore is located along the central axis and constricted on the extracellular side, where highly conserved polar and charged residues at the tip of the extracellular helix contribute to permeability to anions and other osmolytes. Two structural populations were identified, corresponding to compact and relaxed conformations. Comparing the two conformations suggests that the LRR region is flexible and mobile, with rigid-body motions, which might be implicated in structural transitions on pore opening.

Signatures of balancing selection in toll-like receptor (TLRs) genes \u2013 novel insights from a free-living rodent

Selective pressure from pathogens is considered a key selective force driving the evolution of components of the immune system. Since single components of the immune system may interact with many pathogens, and single pathogens may be recognized by multiple components of the immune system, gaining a better understanding of the mechanisms of parasite-driven selection requires the study of multiple genes and pathogens. Toll-like receptors (TLRs) are a large gene family that code for antigen-presenting components of the innate immune response. In the present paper we characterize polymorphism and signatures of selection in seven TLRs in free-living bank voles Myodes glareolus. We report the first evidence of balancing selection in several TLR genes, supported by positive values of Fu and Li’s D* in TLR2 and TLR5, and positive values of Tajima’s D in LRR regions within TLR1 and TLR2. We further found significant associations between amino-acid alleles of TLR1 and TLR5 and susceptibility to infection with the blood pathogen Bartonella. Interestingly, selection patterns in TLRs presenting virus-derived motifs (TLR7 and TLR9) differed considerably from those interacting with bacterial PAMPs. In contrast to the highly variable TLRs presenting bacterial motifs, TLR7 and TLR9 had low polymorphism and displayed signatures of directional selection. These findings suggest different functional responses across the TLR gene family and highlight the complexity of parasite-driven selection.

Identification of Mi-1 homologs in various Solanum species

In Ecuador, several solanaceous crops, including naranjilla (Solanum quitoense), are attacked by the nematode Meloidogyne sp. Resistant cultivars are not available and there is a need to identify potential sources of resistance that can be incorporated into breeding programs. The Mi-1 gene from S. peruvianum is known to confer resistance to Meloidogyne in tomato (S. lycopersicum).&#x0D; In this study, 42 plant accessions of wild and cultivated Solanum species were screened to identify the presence of this gene, its diversification, expression, and evidence of gene recombination. The Mi-1 gene (exon 3) was identified in 34 accessions. Sixteen alleles were identified, that encode 15 different amino acid sequences, all of which encode NBS-LRR proteins and share 72-96 % homology with the tomato Mi-1 protein. The Mi-1 locus was highly polymorphic; most polymorphisms tend to accumulate in the NBS rather than in the LRR region. Genetic recombination was detected among the tomato and potato relate d sequences, but it was absent in the naranjilla group. Homologs identified expressed in both non-infected roots and leaves of some accessions, suggesting the constitutive expression of the gene.

Class B CpG-ODN2006 is highly associated with IgM and antimicrobial peptide gene expression through TLR9 pathway in yellowtail Seriola lalandi

The purpose of this study was to characterize the TLR9 gene from yellowtail (Seriola lalandi) and evaluate its functional activity using the class B Cytosine-phosphate-guanine-oligodeoxynucleotide2006 (CpG-ODN2006) in an in vivo experiment after one-week immunostimulation. The gene expressions of TLR9, Immunoglobulin M (IgM), antimicrobial peptides and cytokines were evaluated by real time PCR, and humoral immune parameters were analyzed in serum. The TLR9 nucleotide sequence from yellowtail was obtained using the whole-genome shotgun sequencing method and bioinformatics tools. The yellowtail full-length cDNA sequence of SlTLR9 was 3789 bp in length, including a 66-bp 5′-untranslated region (UTR), a 3′-UTR of 528 bp, and an open reading frame (ORF) of 3192 bp translatable to 1064 amino acid showing a high degree of similarity with the counterparts of other fish species and sharing common structural architecture of the TLR family, including LRR domains, one C-terminal LRR region, and a TIR domain. Gene expression studies revealed the constitutive expression of TLR9 mRNA in all analyzed tissues; the highest levels were observed in intestine, liver and spleen where they play an important role in the fish immune system. The expression levels of TLR9 after B class CpG-ODN2006 (the main TLR9-agonist) was significantly up-regulated in all analyzed tissues, with the high expression observed in spleen followed by intestine and skin. The CpG-B has been shown as a potent B cell mitogen, and interestingly, IgM mRNA transcript was up-regulated in spleen and intestine, which was highly correlated with TLR9 after CpG-ODN2006 stimulation. The antimicrobial peptides, piscidin and NK-lysine, were up-regulated in spleen and gill after CpG-ODN2006 injection with a high correlation (r ≥ 0.82) with TLR9 gene expression. Cytokine genes were up-regulated in spleen, intestine and skin after CpG-ODN was compared with the control group. No significant correlation was observed between TLR9 and IL-1β, TNF-α and Mx gene expressions. The results showed that CpG-ODN2006 intraperitoneal injection enhanced lysozyme, peroxidase and superoxide dismutase activities in serum and demonstrated that CpG-ODN2006 can induce a specific immune response via TLR9 in which IgM and antimicrobial peptides must have an important role in the defense mechanisms against infections in yellowtail.

Identification of a three-base deletion in the Pi2 locus, and development of functional marker for marker-assisted resistance selection

Rice blast causes huge losses of rice and is the limiting factor in rice production. Host resistance improvement using marker-assisted selection (MAS) is considered to be the most economic and effective way to overcome rice blast. In this study, an allele mining strategy was used to find variation of the Pi2 blast-resistance gene from different rice varieties. In some varieties, there was an out-of-frame deletion of 3 base-pairs (bp) at the 1543–1545 site of the Pi2 gene (the “deletion” allele). Deletion of these 3 bp causes the loss of cysteine–arginine and their replacement with glycine in the protein product. As a consequence the predicted protein structure is changed, especially in the LRR region which is important to plant blast resistance. Plants with the deletion allele showed poor resistance compared to those with the non-deletion variant. Haplotype analysis showed that the deletion of these 3 bp was common only in indica rice, and the rice varieties with 3 bp deletion perform poor rice blast resistance, which indicates that the deletion allele variant of Pi2 occurred recently and was artificially selected. We developed a functional molecular marker Pi2-1, spanning base pairs 1543–1545 of the Pi2 gene, which clearly distinguished between rice varieties with good or inferior blast resistance. Finally, Pi2-1 was used for rice blast MAS in the Huazhan/R900 F2 segregation population, and excellent plants with good rice blast resistance were selected.

Identification, molecular evolution of toll-like receptors in a Tibetan schizothoracine fish (Gymnocypris eckloni) and their expression profiles in response to acute hypoxia

Hypoxia plays an important role in regulating a variety of physiological responses as well as in pathological situations, but to date the roles of Toll-like receptors (TLRs) in fish in response to hypoxia are still poorly understood. Here, we sequenced the transcriptome of G. eckloni and identified the members of TLR family by scanning transcriptome, and then investigated the expression profiles of a complete set of TLRs in G. eckloni in response to acute hypoxia (4 h at DO = 0.3 ± 0.1 mg/L). The de novo-assembled transcriptome consisted of a total of 162,235 transcripts, further clustered into 110,231 unigenes. Based on the transcriptome, a total of 18 TLRs were identified in G. eckloni, and of them three TLRs (TLR5, TLR8 and TLR22) possessed two distinct paralogous genes. The duplicated genes of TLR22 were discovered for the first time in cyprinid fish, but did not origin from a recent duplication event. Of them TLR22b may be specific for schizothoracine fish, at least for G. eckloni. Phylogenetic analysis supported the classification of TLRs into six families as in other vertebrates but was partly different from the previous study. The sliding window analysis showed strong signals of positive selection in TLR2, TLR 4, TLR 5a, TLR 7, TLR 19, TLR 20, TLR 21, TLR 22a and TLR 22b, but most codons under positive selection were located in the putative LRR regions. The mRNA expression of most TLRs in head kidney, spleen and gill decreased significantly or remained unchanged under acute hypoxia, whereas acute hypoxia increased expressions of TLR2 and TLR3 in head kidney, of TLR8a, TLR12 and TLR19 in spleen, and of TLR1 in gill, suggesting tissues-specific expressions of TLRs play important roles in mediating innate immune responses for host defense against tissue damages or physiological changes induced by hypoxia.