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1.  Plant Nucleotide Binding Site–Leucine-Rich Repeat (NBS-LRR) Genes: Active Guardians in Host Defense Responses 
The most represented group of resistance genes are those of the nucleotide binding site–leucine-rich repeat (NBS-LRR) class. These genes are very numerous in the plant genome, and they often occur in clusters at specific loci following gene duplication and amplification events. To date, hundreds of resistance genes and relatively few quantitative trait loci for plant resistance to pathogens have been mapped in different species, with some also cloned. When these NBS-LRR genes have been physically or genetically mapped, many cases have shown co-localization between resistance loci and NBS-LRR genes. This has allowed the identification of candidate genes for resistance, and the development of molecular markers linked to R genes. This review is focused on recent genomics studies that have described the abundance, distribution and evolution of NBS-LRR genes in plant genomes. Furthermore, in terms of their expression, NBS-LRR genes are under fine regulation by cis- and trans-acting elements. Recent findings have provided insights into the roles of alternative splicing, the ubiquitin/proteasome system, and miRNAs and secondary siRNAs in the regulation of NBS-LRR gene expression at the post-transcriptional, post-translational and epigenetic levels. The possibility to use this knowledge for genetic improvement of plant resistance to pathogens is discussed.
doi:10.3390/ijms14047302
PMCID: PMC3645687  PMID: 23549266
NBS-LRR genes; gene evolution; plant breeding
2.  NB-LRR proteins: Pairs, pieces, perception, partners and pathways 
Current opinion in plant biology  2010;13(4):472-477.
SUMMARY OF RECENT ADVANCES
In plants, many of the innate immune receptors or disease resistance (R) proteins contain a NB-LRR (Nucleotide-binding site, Leucine-rich repeat) structure. The recent findings regarding NB-LRR signaling are summarized in this article. An emerging theme is that two NB-LRRs can function together to mediate disease resistance against pathogen isolates. Also, recent results delineate which NB-LRR protein fragments are sufficient to initiate defense signaling. Importantly, distinct fragments of different NB-LRRs are sufficient for function. Finally, we describe the new roles of accessory proteins and downstream host genes in NB-LRR signaling.
doi:10.1016/j.pbi.2010.04.007
PMCID: PMC2910844  PMID: 20483655
3.  Nucleotide diversity and linkage disequilibrium in 11 expressed resistance candidate genes in Lolium perenne 
BMC Plant Biology  2007;7:43.
Background
Association analysis is an alternative way for QTL mapping in ryegrass. So far, knowledge on nucleotide diversity and linkage disequilibrium in ryegrass is lacking, which is essential for the efficiency of association analyses.
Results
11 expressed disease resistance candidate (R) genes including 6 nucleotide binding site and leucine rich repeat (NBS-LRR) like genes and 5 non-NBS-LRR genes were analyzed for nucleotide diversity. For each of the genes about 1 kb genomic fragments were isolated from 20 heterozygous genotypes in ryegrass. The number of haplotypes per gene ranged from 9 to 27. On average, one single nucleotide polymorphism (SNP) was present per 33 bp between two randomly sampled sequences for the 11 genes. NBS-LRR like gene fragments showed a high degree of nucleotide diversity, with one SNP every 22 bp between two randomly sampled sequences. NBS-LRR like gene fragments showed very high non-synonymous mutation rates, leading to altered amino acid sequences. Particularly LRR regions showed very high diversity with on average one SNP every 10 bp between two sequences. In contrast, non-NBS LRR resistance candidate genes showed a lower degree of nucleotide diversity, with one SNP every 112 bp. 78% of haplotypes occurred at low frequency (<5%) within the collection of 20 genotypes. Low intragenic LD was detected for most R genes, and rapid LD decay within 500 bp was detected.
Conclusion
Substantial LD decay was found within a distance of 500 bp for most resistance candidate genes in this study. Hence, LD based association analysis is feasible and promising for QTL fine mapping of resistance traits in ryegrass.
doi:10.1186/1471-2229-7-43
PMCID: PMC1978496  PMID: 17683574
4.  Leucine-rich repeat (LRR) proteins 
Autophagy  2011;7(9):1082-1084.
The leucine-rich repeats (LRR)-containing domain is evolutionarily conserved in many proteins associated with innate immunity in plants, invertebrates and vertebrates. Serving as a first line of defense, the innate immune response is initiated through the sensing of pathogen-associated molecular patterns (PAMPs). In plants, NBS (nucleotide-binding site)-LRR proteins provide recognition of pathogen products of avirulence (AVR) genes. LRRs also promote interaction between LRR proteins as observed in receptor-coreceptor complexes. In mammals, toll-like receptors (TLRs) and NOD-like receptors (NLRs) through their LRR domain, sense molecular determinants from a structurally diverse set of bacterial, fungal, parasite and viral-derived components. In humans, at least 34 LRR proteins are implicated in diseases. Most LRR domains consist of 2–45 leucine-rich repeats, with each repeat about 20–30 residues long. Structurally, LRR domains adopt an arc or horseshoe shape, with the concave face consisting of parallel β-strands and the convex face representing a more variable region of secondary structures including helices. Apart from the TLRs and NLRs, most of the 375 human LRR proteins remain uncharacterized functionally. We incorporated computational and functional analyses to facilitate multifaceted insights into human LRR proteins and outline a few approaches here.
doi:10.4161/auto.7.9.16464
PMCID: PMC3901792  PMID: 21606681
systems biology; pathogen sensors; pathogen response; antibacterial; inflammation; autophagy; bioinformatics; computational biology
5.  Plant NB-LRR signaling: Upstreams and Downstreams 
Current opinion in plant biology  2011;14(4):365-371.
Plant disease resistance proteins commonly belong to the nucleotide binding-leucine rich repeat (NB-LRR) protein family. These specialized immune proteins mediate recognition of diverse pathogen-derived effector proteins and initiate potent defense responses. NB-LRRs exhibit a multidomain architecture and each domain appears to have discrete functions depending on the stage of NB-LRR signaling. Novel proteins that were found to interact with the core HSP90 chaperone complex regulate accumulation and activation of NB-LRR immune receptors. Recent studies have also advanced our understanding of how accessory proteins contribute to NB-LRR activation. The dynamic nature of NB-LRR localization to different subcellular compartments before and after activation suggests that NB-LRRs may activate immune responses in multiple parts of the cell. In this review we highlight recent advances in understanding NB-LRR function.
doi:10.1016/j.pbi.2011.03.011
PMCID: PMC3155621  PMID: 21459033
6.  Resistance gene enrichment sequencing (RenSeq) enables reannotation of the NB-LRR gene family from sequenced plant genomes and rapid mapping of resistance loci in segregating populations 
The Plant Journal  2013;76(3):530-544.
Summary
RenSeq is a NB-LRR (nucleotide binding-site leucine-rich repeat) gene-targeted, Resistance gene enrichment and sequencing method that enables discovery and annotation of pathogen resistance gene family members in plant genome sequences. We successfully applied RenSeq to the sequenced potato Solanum tuberosum clone DM, and increased the number of identified NB-LRRs from 438 to 755. The majority of these identified R gene loci reside in poorly or previously unannotated regions of the genome. Sequence and positional details on the 12 chromosomes have been established for 704 NB-LRRs and can be accessed through a genome browser that we provide. We compared these NB-LRR genes and the corresponding oligonucleotide baits with the highest sequence similarity and demonstrated that ∼80% sequence identity is sufficient for enrichment. Analysis of the sequenced tomato S. lycopersicum ‘Heinz 1706’ extended the NB-LRR complement to 394 loci. We further describe a methodology that applies RenSeq to rapidly identify molecular markers that co-segregate with a pathogen resistance trait of interest. In two independent segregating populations involving the wild Solanum species S. berthaultii (Rpi-ber2) and S. ruiz-ceballosii (Rpi-rzc1), we were able to apply RenSeq successfully to identify markers that co-segregate with resistance towards the late blight pathogen Phytophthora infestans. These SNP identification workflows were designed as easy-to-adapt Galaxy pipelines.
doi:10.1111/tpj.12307
PMCID: PMC3935411  PMID: 23937694
NB-LRR; pathogen resistance; Solanaceae; target enrichment; next-generation sequencing; Solanum tuberosum Group Phureja clone DM1-3 516 R44; Solanum ruiz-ceballosii; Solanum berthaultii; Solanum lycopersicum; technical advance
7.  New insights in plant immunity signaling activation 
Current opinion in plant biology  2011;14(5):512-518.
Plant disease resistance can be triggered by specific recognition of microbial effectors by plant nucleotide binding-leucine rich repeat (NB-LRR) receptors. Over the last few years, many efforts have greatly improved the understanding of effector and NB-LRR function, but have left a lot of questions as to how effector perception activates NB-LRR induction of defense signaling. This review describes exciting new findings showing similarities and differences in function of diverse plant NB-LRR proteins in terms of pathogen recognition and where and how resistance proteins are activated. Localization studies have shown that some NB-LRRs can activate signaling from the cytosol while others act in the nucleus. Also, the structural determination of two NB-LRR signaling domains demonstrated that receptor oligomerization is fundamental for activation of resistance signaling.
doi:10.1016/j.pbi.2011.05.005
PMCID: PMC3191233  PMID: 21723182
8.  Interfamily Transfer of Dual NB-LRR Genes Confers Resistance to Multiple Pathogens 
PLoS ONE  2013;8(2):e55954.
A major class of disease resistance (R) genes which encode nucleotide binding and leucine rich repeat (NB-LRR) proteins have been used in traditional breeding programs for crop protection. However, it has been difficult to functionally transfer NB-LRR-type R genes in taxonomically distinct families. Here we demonstrate that a pair of Arabidopsis (Brassicaceae) NB-LRR-type R genes, RPS4 and RRS1, properly function in two other Brassicaceae, Brassica rapa and Brassica napus, but also in two Solanaceae, Nicotiana benthamiana and tomato (Solanum lycopersicum). The solanaceous plants transformed with RPS4/RRS1 confer bacterial effector-specific immunity responses. Furthermore, RPS4 and RRS1, which confer resistance to a fungal pathogen Colletotrichum higginsianum in Brassicaceae, also protect against Colletotrichum orbiculare in cucumber (Cucurbitaceae). Importantly, RPS4/RRS1 transgenic plants show no autoimmune phenotypes, indicating that the NB-LRR proteins are tightly regulated. The successful transfer of two R genes at the family level implies that the downstream components of R genes are highly conserved. The functional interfamily transfer of R genes can be a powerful strategy for providing resistance to a broad range of pathogens.
doi:10.1371/journal.pone.0055954
PMCID: PMC3577827  PMID: 23437080
9.  Global expression analysis of nucleotide binding site-leucine rich repeat-encoding and related genes in Arabidopsis 
BMC Plant Biology  2007;7:56.
Background
Nucleotide binding site-leucine rich repeat (NBS-LRR)-encoding genes comprise the largest class of plant disease resistance genes. The 149 NBS-LRR-encoding genes and the 58 related genes that do not encode LRRs represent approximately 0.8% of all ORFs so far annotated in Arabidopsis ecotype Col-0. Despite their prevalence in the genome and functional importance, there was little information regarding expression of these genes.
Results
We analyzed the expression patterns of ~170 NBS-LRR-encoding and related genes in Arabidopsis Col-0 using multiple analytical approaches: expressed sequenced tag (EST) representation, massively parallel signature sequencing (MPSS), microarray analysis, rapid amplification of cDNA ends (RACE) PCR, and gene trap lines. Most of these genes were expressed at low levels with a variety of tissue specificities. Expression was detected by at least one approach for all but 10 of these genes. The expression of some but not the majority of NBS-LRR-encoding and related genes was affected by salicylic acid (SA) treatment; the response to SA varied among different accessions. An analysis of previously published microarray data indicated that ten NBS-LRR-encoding and related genes exhibited increased expression in wild-type Landsberg erecta (Ler) after flagellin treatment. Several of these ten genes also showed altered expression after SA treatment, consistent with the regulation of R gene expression during defense responses and overlap between the basal defense response and salicylic acid signaling pathways. Enhancer trap analysis indicated that neither jasmonic acid nor benzothiadiazole (BTH), a salicylic acid analog, induced detectable expression of the five NBS-LRR-encoding genes and one TIR-NBS-encoding gene tested; however, BTH did induce detectable expression of the other TIR-NBS-encoding gene analyzed. Evidence for alternative mRNA polyadenylation sites was observed for many of the tested genes. Evidence for alternative splicing was found for at least 12 genes, 11 of which encode TIR-NBS-LRR proteins. There was no obvious correlation between expression pattern, phylogenetic relationship or genomic location of the NBS-LRR-encoding and related genes studied.
Conclusion
Transcripts of many NBS-LRR-encoding and related genes were defined. Most were present at low levels and exhibited tissue-specific expression patterns. Expression data are consistent with most Arabidopsis NBS-LRR-encoding and related genes functioning in plant defense responses but do not preclude other biological roles.
doi:10.1186/1471-2229-7-56
PMCID: PMC2175511  PMID: 17956627
10.  Leucine-Rich repeat receptor kinases are sporadically distributed in eukaryotic genomes 
Background
Plant leucine-rich repeat receptor-like kinases (LRR-RLKs) are receptor kinases that contain LRRs in their extracellular domain. In the last 15 years, many research groups have demonstrated major roles played by LRR-RLKs in plants during almost all developmental processes throughout the life of the plant and in defense/resistance against a large range of pathogens. Recently, a breakthrough has been made in this field that challenges the dogma of the specificity of plant LRR-RLKs.
Results
We analyzed ~1000 complete genomes and show that LRR-RK genes have now been identified in 8 non-plant genomes. We performed an exhaustive phylogenetic analysis of all of these receptors, revealing that all of the LRR-containing receptor subfamilies form lineage-specific clades. Our results suggest that the association of LRRs with RKs appeared independently at least four times in eukaryotic evolutionary history. Moreover, the molecular evolutionary history of the LRR-RKs found in oomycetes is reminiscent of the pattern observed in plants: expansion with amplification/deletion and evolution of the domain organization leading to the functional diversification of members of the gene family. Finally, the expression data suggest that oomycete LRR-RKs may play a role in several stages of the oomycete life cycle.
Conclusions
In view of the key roles that LRR-RLKs play throughout the entire lifetime of plants and plant-environment interactions, the emergence and expansion of this type of receptor in several phyla along the evolution of eukaryotes, and particularly in oomycete genomes, questions their intrinsic functions in mimicry and/or in the coevolution of receptors between hosts and pathogens.
doi:10.1186/1471-2148-11-367
PMCID: PMC3268121  PMID: 22185365
11.  Characterization of a Novel Leucine-Rich Repeat Protein Antigen from Group B Streptococci That Elicits Protective Immunity  
Infection and Immunity  2005;73(3):1671-1683.
Group B streptococci (GBS) usually behave as commensal organisms that asymptomatically colonize the gastrointestinal and urogenital tracts of adults. However, GBS are also pathogens and the leading bacterial cause of life-threatening invasive disease in neonates. While the events leading to transmission and disease in neonates remain unclear, GBS carriage and level of colonization in the mother have been shown to be significant risk factors associated with invasive infection. Surface antigens represent ideal vaccine targets for eliciting antibodies that can act as opsonins and/or inhibit colonization and invasion. Using a genetic screen for exported proteins in GBS, we identified a gene, designated lrrG, that encodes a novel LPXTG anchored surface antigen containing leucine-rich repeat (LRR) motifs found in bacterial invasins and other members of the LRR protein family. Southern blotting showed that lrrG was present in all GBS strains tested, representing the nine serotypes, and revealed the presence of an lrrG homologue in Streptococcus pyogenes. Recombinant LrrG protein was shown in vitro to adhere to epithelial cells in a dose-dependent manner, suggesting that it may function as an adhesion factor in GBS. More importantly, immunization with recombinant LrrG elicited a strong immunoglobulin G response in CBA/ca mice and protected against lethal challenge with virulent GBS. The data presented in this report suggest that this conserved protein is a highly promising candidate antigen for use in a GBS vaccine.
doi:10.1128/IAI.73.3.1671-1683.2005
PMCID: PMC1064916  PMID: 15731068
12.  Phylogenetic analyses of peanut resistance gene candidates and screening of different genotypes for polymorphic markers 
The nucleotide-binding-site-leucine-rich-repeat (NBS–LRR)-encoding gene family has attracted much research interest because approximately 75% of the plant disease resistance genes that have been cloned to date are from this gene family. Here, we describe a collection of peanut NBS–LRR resistance gene candidates (RGCs) isolated from peanut (Arachis) species by mining Gene Bank data base. NBS–LRR sequences assembled into TIR-NBS-LRR (75.4%) and non-TIR-NBS-LRR (24.6%) subfamilies. Total of 20 distinct clades were identified and showed a high level of sequence divergence within TIR-NBS and non-TIR-NBS subfamilies. Thirty-four primer pairs were designed from these RGC sequences and used for screening different genotypes belonging to wild and cultivated peanuts. Therefore, peanut RGC identified in this study will provide useful tools for developing DNA markers and cloning the genes for resistance to different pathogens in peanut.
doi:10.1016/j.sjbs.2009.12.007
PMCID: PMC3730907  PMID: 23961057
Resistance gene; NBS–LRR; Arachis hypogaea L.; Peanut diseases; Phylogentic and DNA markers
13.  Genome-wide identification, characterization and expression analysis of populus leucine-rich repeat receptor-like protein kinase genes 
BMC Genomics  2013;14:318.
Background
Leucine-rich repeat receptor-like kinases (LRR-RLKs) comprise the largest group within the receptor-like kinase (RLK) superfamily in plants. This gene family plays critical and diverse roles in plant growth, development and stress response. Although the LRR-RLK families in Arabidopsis and rice have been previously analyzed, no comprehensive studies have been performed on this gene family in tree species.
Results
In this work, 379 LRR-RLK genes were retrieved from the Populus trichocarpa genome and further grouped into 14 subfamilies based on their structural and sequence similarities. Approximately 82% (312 out of 379) of the PtLRR-RLK genes are located in segmental duplication blocks indicating the role of duplication process in the expansion of this gene family. The conservation and variation in motif composition and intron/exon arrangement among PtLRR-RLK subfamilies were analyzed to provide additional support for their phylogenetic relationship and more importantly to indicate the potential divergence in their functions. Expression profiling of PtLRR-RLKs showed that they were differentially expressed in different organs and tissues and some PtLRR-RLKs were specifically expressed in meristem tissues, which indicated their potential involvement in tissue development and differentiation. For most AtLRR-RLKs with defined functions, Populus homologues exhibiting similar expression patterns could be identified, which might indicate the functional conservation during evolution. Among 12 types of environmental cues analyzed by the genome-wide microarray data, PtLRR-RLKs showed specific responses to shoot organogenesis, wounding, low ammonium feeding, hypoxia and seasonal dormancy, but not to drought, re-watering after drought, flooding, AlCl3 treatment and bacteria or fungi treatments.
Conclusions
This study provides the first comprehensive genomic analysis of the Populus LRR-RLK gene family. Segmental duplication contributes significantly to the expansion of this gene family. Populus and Arabidopsis LRR-RLK homologues not only share similar genetic structures but also exhibit comparable expression patterns which point to the possible functional conservation of these LRR-RLKs in two model systems. Transcriptome profiling provides the first insight into the functional divergence among PtLRR-RLK gene subfamilies and suggests that they might take important roles in growth and adaptation of tree species.
doi:10.1186/1471-2164-14-318
PMCID: PMC3682895  PMID: 23663326
Populus trichocarpa; Leucine-rich repeat receptor-like kinase (LRR-RLK); Phylogenetic analysis; Motif elicitation; Expression profiling
14.  RNA-Seq Analysis of a Soybean Near-Isogenic Line Carrying Bacterial Leaf Pustule-Resistant and -Susceptible Alleles 
Bacterial leaf pustule (BLP) disease is caused by Xanthomonas axonopodis pv. glycines (Xag). To investigate the plant basal defence mechanisms induced in response to Xag, differential gene expression in near-isogenic lines (NILs) of BLP-susceptible and BLP-resistant soybean was analysed by RNA-Seq. Of a total of 46 367 genes that were mapped to soybean genome reference sequences, 1978 and 783 genes were found to be up- and down-regulated, respectively, in the BLP-resistant NIL relative to the BLP-susceptible NIL at 0, 6, and 12h after inoculation (hai). Clustering analysis revealed that these genes could be grouped into 10 clusters with different expression patterns. Functional annotation based on gene ontology (GO) categories was carried out. Among the putative soybean defence response genes identified (GO:0006952), 134 exhibited significant differences in expression between the BLP-resistant and -susceptible NILs. In particular, pathogen-associated molecular pattern (PAMP) and damage-associated molecular pattern (DAMP) receptors and the genes induced by these receptors were highly expressed at 0 hai in the BLP-resistant NIL. Additionally, pathogenesis-related (PR)-1 and -14 were highly expressed at 0 hai, and PR-3, -6, and -12 were highly expressed at 12 hai. There were also significant differences in the expression of the core JA-signalling components MYC2 and JASMONATE ZIM-motif. These results indicate that powerful basal defence mechanisms involved in the recognition of PAMPs or DAMPs and a high level of accumulation of defence-related gene products may contribute to BLP resistance in soybean.
doi:10.1093/dnares/dsr033
PMCID: PMC3223079  PMID: 21987089
bacterial leaf pustules; disease resistance; RNA-Seq analysis; soybean
15.  Probing the roles of LRR RLK genes in Arabidopsis thaliana roots using a custom T-DNA insertion set 
Plant Molecular Biology  2011;76(1-2):69-83.
Leucine-rich repeat receptor-like protein kinases (LRR RLKs) represent the largest group of Arabidopsis RLKs with approximately 235 members. A minority of these LRR RLKs have been assigned to diverse roles in development, pathogen resistance and hormone perception. Using a reverse genetics approach, a collection of homozygous T-DNA insertion lines for 69 root expressed LRR RLK genes was screened for root developmental defects and altered response after exposure to environmental, hormonal/chemical and abiotic stress. The obtained data demonstrate that LRR RLKs play a role in a wide variety of signal transduction pathways related to hormone and abiotic stress responses. The described collection of T-DNA insertion mutants provides a valuable tool for future research into the function of LRR RLK genes.
Electronic supplementary material
The online version of this article (doi:10.1007/s11103-011-9769-x) contains supplementary material, which is available to authorized users.
doi:10.1007/s11103-011-9769-x
PMCID: PMC3097349  PMID: 21431781
LRR RLK; Arabidopsis; Root; Development; Hormone; stress
16.  A genome-wide genetic map of NB-LRR disease resistance loci in potato 
Like all plants, potato has evolved a surveillance system consisting of a large array of genes encoding for immune receptors that confer resistance to pathogens and pests. The majority of these so-called resistance or R proteins belong to the super-family that harbour a nucleotide binding and a leucine-rich-repeat domain (NB-LRR). Here, sequence information of the conserved NB domain was used to investigate the genome-wide genetic distribution of the NB-LRR resistance gene loci in potato. We analysed the sequences of 288 unique BAC clones selected using filter hybridisation screening of a BAC library of the diploid potato clone RH89-039-16 (S. tuberosum ssp. tuberosum) and a physical map of this BAC library. This resulted in the identification of 738 partial and full-length NB-LRR sequences. Based on homology of these sequences with known resistance genes, 280 and 448 sequences were classified as TIR-NB-LRR (TNL) and CC-NB-LRR (CNL) sequences, respectively. Genetic mapping revealed the presence of 15 TNL and 32 CNL loci. Thirty-six are novel, while three TNL loci and eight CNL loci are syntenic with previously identified functional resistance genes. The genetic map was complemented with 68 universal CAPS markers and 82 disease resistance trait loci described in literature, providing an excellent template for genetic studies and applied research in potato.
Electronic supplementary material
The online version of this article (doi:10.1007/s00122-011-1602-z) contains supplementary material, which is available to authorized users.
doi:10.1007/s00122-011-1602-z
PMCID: PMC3135832  PMID: 21590328
17.  A nematode demographics assay in transgenic roots reveals no significant impacts of the Rhg1 locus LRR-Kinase on soybean cyst nematode resistance 
BMC Plant Biology  2010;10:104.
Background
Soybean cyst nematode (Heterodera glycines, SCN) is the most economically damaging pathogen of soybean (Glycine max) in the U.S. The Rhg1 locus is repeatedly observed as the quantitative trait locus with the greatest impact on SCN resistance. The Glyma18g02680.1 gene at the Rhg1 locus that encodes an apparent leucine-rich repeat transmembrane receptor-kinase (LRR-kinase) has been proposed to be the SCN resistance gene, but its function has not been confirmed. Generation of fertile transgenic soybean lines is difficult but methods have been published that test SCN resistance in transgenic roots generated with Agrobacterium rhizogenes.
Results
We report use of artificial microRNA (amiRNA) for gene silencing in soybean, refinements to transgenic root SCN resistance assays, and functional tests of the Rhg1 locus LRR-kinase gene. A nematode demographics assay monitored infecting nematode populations for their progress through developmental stages two weeks after inoculation, as a metric for SCN resistance. Significant differences were observed between resistant and susceptible control genotypes. Introduction of the Rhg1 locus LRR-kinase gene (genomic promoter/coding region/terminator; Peking/PI 437654-derived SCN-resistant source), into rhg1- SCN-susceptible plant lines carrying the resistant-source Rhg4+ locus, provided no significant increases in SCN resistance. Use of amiRNA to reduce expression of the LRR-kinase gene from the Rhg1 locus of Fayette (PI 88788 source of Rhg1) also did not detectably alter resistance to SCN. However, silencing of the LRR-kinase gene did have impacts on root development.
Conclusion
The nematode demographics assay can expedite testing of transgenic roots for SCN resistance. amiRNAs and the pSM103 vector that drives interchangeable amiRNA constructs through a soybean polyubiqutin promoter (Gmubi), with an intron-GFP marker for detection of transgenic roots, may have widespread use in legume biology. Studies in which expression of the Rhg1 locus LRR-kinase gene from different resistance sources was either reduced or complemented did not reveal significant impacts on SCN resistance.
doi:10.1186/1471-2229-10-104
PMCID: PMC3095272  PMID: 20529370
18.  Genome Wide Analysis of Nucleotide-Binding Site Disease Resistance Genes in Brachypodium distachyon 
Nucleotide-binding site (NBS) disease resistance genes play an important role in defending plants from a variety of pathogens and insect pests. Many R-genes have been identified in various plant species. However, little is known about the NBS-encoding genes in Brachypodium distachyon. In this study, using computational analysis of the B. distachyon genome, we identified 126 regular NBS-encoding genes and characterized them on the bases of structural diversity, conserved protein motifs, chromosomal locations, gene duplications, promoter region, and phylogenetic relationships. EST hits and full-length cDNA sequences (from Brachypodium database) of 126 R-like candidates supported their existence. Based on the occurrence of conserved protein motifs such as coiled-coil (CC), NBS, leucine-rich repeat (LRR), these regular NBS-LRR genes were classified into four subgroups: CC-NBS-LRR, NBS-LRR, CC-NBS, and X-NBS. Further expression analysis of the regular NBS-encoding genes in Brachypodium database revealed that these genes are expressed in a wide range of libraries, including those constructed from various developmental stages, tissue types, and drought challenged or nonchallenged tissue.
doi:10.1155/2012/418208
PMCID: PMC3368180  PMID: 22693425
19.  Multiple Functions of the Leucine-Rich Repeat Protein LrrA of Treponema denticola  
Infection and Immunity  2004;72(8):4619-4627.
The gene lrrA, encoding a leucine-rich repeat protein, LrrA, that contains eight consensus tandem repeats of 23 amino acid residues, has been identified in Treponema denticola ATCC 35405. A leucine-rich repeat is a generally useful protein-binding motif, and proteins containing this repeat are typically involved in protein-protein interactions. Southern blot analysis demonstrated that T. denticola ATCC 35405 expresses the lrrA gene, but the gene was not identified in T. denticola ATCC 33520. In order to analyze the functions of LrrA in T. denticola, an lrrA-inactivated mutant of strain ATCC 35405 and an lrrA gene expression transformant of strain ATCC 33520 were constructed. Characterization of the mutant and transformant demonstrated that LrrA is associated with the extracytoplasmic fraction of T. denticola and expresses multifunctional properties. It was demonstrated that the attachment of strain ATCC 35405 to HEp-2 cell cultures and coaggregation with Tannerella forsythensis were attenuated by the lrrA mutation. In addition, an in vitro binding assay demonstrated specific binding of LrrA to a portion of the Tannerella forsythensis leucine-rich repeat protein, BspA, which is mediated by the N-terminal region of LrrA. It was also observed that the lrrA mutation caused a reduction of swarming in T. denticola ATCC 35405 and consequently attenuated tissue penetration. These results suggest that the leucine-rich repeat protein LrrA plays a role in the attachment and penetration of human epithelial cells and coaggregation with Tannerella forsythensis. These properties may play important roles in the virulence of T. denticola.
doi:10.1128/IAI.72.8.4619-4627.2004
PMCID: PMC470683  PMID: 15271922
20.  Direct Bacterial Killing In Vitro by Recombinant Nod2 Is Compromised by Crohn's Disease-Associated Mutations 
PLoS ONE  2010;5(6):e10915.
Background
A homeostatic relationship with the intestinal microflora is increasingly appreciated as essential for human health and wellbeing. Mutations in the leucine-rich repeat (LRR) domain of Nod2, a bacterial recognition protein, are associated with development of the inflammatory bowel disorder, Crohn's disease. We investigated the molecular mechanisms underlying disruption of intestinal symbiosis in patients carrying Nod2 mutations.
Methodology/Principal Findings
In this study, using purified recombinant LRR domains, we demonstrate that Nod2 is a direct antimicrobial agent and this activity is generally deficient in proteins carrying Crohn's-associated mutations. Wild-type, but not Crohn's-associated, Nod2 LRR domains directly interacted with bacteria in vitro, altered their metabolism and disrupted the integrity of the plasma membrane. Antibiotic activity was also expressed by the LRR domains of Nod1 and other pattern recognition receptors suggesting that the LRR domain is a conserved anti-microbial motif supporting innate cellular immunity.
Conclusions/Significance
The lack of anti-bacterial activity demonstrated with Crohn's-associated Nod2 mutations in vitro, supports the hypothesis that a deficiency in direct bacterial killing contributes to the association of Nod2 polymorphisms with the disease.
doi:10.1371/journal.pone.0010915
PMCID: PMC2879363  PMID: 20531959
21.  LRRC4, a Putative Tumor Suppressor Gene, Requires a Functional Leucine-rich Repeat Cassette Domain to Inhibit Proliferation of Glioma Cells In Vitro by Modulating the Extracellular Signal-regulated Kinase/Protein Kinase B/Nuclear Factor-κB Pathway 
Molecular Biology of the Cell  2006;17(8):3534-3542.
We have previously reported that the LRRC4 gene, which contains a conserved leucine-rich repeat (LRR) cassette and an immunoglobulin (Ig) IgC2 domain, is associated with glioma suppression both in vitro and in vivo. The present study provides evidence that the conspicuous absence of LRRC4 in high-grade gliomas directly contributes to the increasing tumor grade. The loss of LRRC4 in U251 cells is caused by the loss of homozygosity at chromosome 7q32-ter. It was also found that LRRC4 requires a functional LRR cassette domain to suppress U251 cell proliferation. In the LRR cassette domain, the third LRR motif of the core LRR is found to be indispensable for the function of LRRC4. The inhibitory effect of LRRC4 is accompanied by a decrease in the expression of pERK, pAkt, pNF-κBp65, signal transducer and activator of transcription protein-3 (STAT3), and mutant p53, and an increase in the expression of c-Jun NH2-terminal kinase (JNK)2 and p-c-Jun, suggesting that LRRC4 plays a major role in suppressing U251 cell proliferation by regulating the extracellular signal-regulated kinase (ERK)/Akt/NF-κBp65, STAT3, and JNK2/c-Jun pathways. In conclusion, LRRC4 may act as a novel candidate of tumor suppressor gene. Therefore, the loss of LRRC4 function may be an important event in the progression of gliomas.
doi:10.1091/mbc.E05-11-1082
PMCID: PMC1525233  PMID: 16723503
22.  Plant NBS-LRR proteins: adaptable guards 
Genome Biology  2006;7(4):212.
Plant nucleotide-binding site leucine-rich repeat (NBS-LRR) proteins are a large family involved in disease resistance; they may monitor the status of proteins targeted by pathogens.
The majority of disease resistance genes in plants encode nucleotide-binding site leucine-rich repeat (NBS-LRR) proteins. This large family is encoded by hundreds of diverse genes per genome and can be subdivided into the functionally distinct TIR-domain-containing (TNL) and CC-domain-containing (CNL) subfamilies. Their precise role in recognition is unknown; however, they are thought to monitor the status of plant proteins that are targeted by pathogen effectors.
doi:10.1186/gb-2006-7-4-212
PMCID: PMC1557992  PMID: 16677430
23.  Breaking restricted taxonomic functionality by dual resistance genes 
Plant Signaling & Behavior  2013;8(6):e24244.
NB-LRR-type disease resistance (R) genes have been used in traditional breeding programs for crop protection. However, functional transfer of NB-LRR-type R genes to plants in taxonomically distinct families to establish pathogen resistance has not been successful. Here we demonstrate that a pair of Arabidopsis (Brassicaceae) NB-LRR-type R genes, RPS4 and RRS1, properly function in two other Brassicaceae, Brassica rapa and B. napus, but also in two Solanaceae, Nicotiana benthamiana and tomato (Solanum lycopersicum). The solanaceous plants transformed with RPS4/RRS1 confer bacterial effector-specific immunity responses. Furthermore, RPS4 and RRS1, which confer resistance to a fungal pathogen Colletotrichum higginsianum in Brassicaceae, also protect against Colletotrichum orbiculare in cucumber (Cucurbitaceae). Thus the successful transfer of two R genes at the family level overcomes restricted taxonomic functionality. This implies that the downstream components of R genes must be highly conserved and interfamily utilization of R genes can be a powerful strategy to combat pathogens.
doi:10.4161/psb.24244
PMCID: PMC3907395  PMID: 23518587
Colletotrichum higginsianum; Pseudomonas syringae; R gene; RPS4; RRS1; Ralstonia solanacearum; restricted taxonomic functionality
24.  Identification and Phylogenetic Analysis of a CC-NBS-LRR Encoding Gene Assigned on Chromosome 7B of Wheat 
Hexaploid wheat displays limited genetic variation. As a direct A and B genome donor of hexaploid wheat, tetraploid wheat represents an important gene pool for cultivated bread wheat. Many disease resistant genes express conserved domains of the nucleotide-binding site and leucine-rich repeats (NBS-LRR). In this study, we isolated a CC-NBS-LRR gene locating on chromosome 7B from durum wheat variety Italy 363, and designated it TdRGA-7Ba. Its open reading frame was 4014 bp, encoding a 1337 amino acid protein with a complete NBS domain and 18 LRR repeats, sharing 44.7% identity with the PM3B protein. TdRGA-7Ba expression was continuously seen at low levels and was highest in leaves. TdRGA-7Ba has another allele TdRGA-7Bb with a 4 bp deletion at position +1892 in other cultivars of tetraploid wheat. In Ae. speltoides, as a B genome progenitor, both TdRGA-7Ba and TdRGA-7Bb were detected. In all six species of hexaploid wheats (AABBDD), only TdRGA-7Bb existed. Phylogenic analysis showed that all TdRGA-7Bb type genes were grouped in one sub-branch. We speculate that TdRGA-7Bb was derived from a TdRGA-7Ba mutation, and it happened in Ae. speltoides. Both types of TdRGA-7B participated in tetraploid wheat formation. However, only the TdRGA-7Bb was retained in hexaploid wheat.
doi:10.3390/ijms140815330
PMCID: PMC3759862  PMID: 23887654
wheat; NBS-LRR gene; gene mutation; molecular evolution
25.  A 1,681-locus consensus genetic map of cultivated cucumber including 67 NB-LRR resistance gene homolog and ten gene loci 
BMC Plant Biology  2013;13:53.
Background
Cucumber is an important vegetable crop that is susceptible to many pathogens, but no disease resistance (R) genes have been cloned. The availability of whole genome sequences provides an excellent opportunity for systematic identification and characterization of the nucleotide binding and leucine-rich repeat (NB-LRR) type R gene homolog (RGH) sequences in the genome. Cucumber has a very narrow genetic base making it difficult to construct high-density genetic maps. Development of a consensus map by synthesizing information from multiple segregating populations is a method of choice to increase marker density. As such, the objectives of the present study were to identify and characterize NB-LRR type RGHs, and to develop a high-density, integrated cucumber genetic-physical map anchored with RGH loci.
Results
From the Gy14 draft genome, 70 NB-containing RGHs were identified and characterized. Most RGHs were in clusters with uneven distribution across seven chromosomes. In silico analysis indicated that all 70 RGHs had EST support for gene expression. Phylogenetic analysis classified 58 RGHs into two clades: CNL and TNL. Comparative analysis revealed high-degree sequence homology and synteny in chromosomal locations of these RGH members between the cucumber and melon genomes.
Fifty-four molecular markers were developed to delimit 67 of the 70 RGHs, which were integrated into a genetic map through linkage analysis. A 1,681-locus cucumber consensus map including 10 gene loci and spanning 730.0 cM in seven linkage groups was developed by integrating three component maps with a bin-mapping strategy. Physically, 308 scaffolds with 193.2 Mbp total DNA sequences were anchored onto this consensus map that covered 52.6% of the 367 Mbp cucumber genome.
Conclusions
Cucumber contains relatively few NB-LRR RGHs that are clustered and unevenly distributed in the genome. All RGHs seem to be transcribed and shared significant sequence homology and synteny with the melon genome suggesting conservation of these RGHs in the Cucumis lineage. The 1,681-locus consensus genetic-physical map developed and the RGHs identified and characterized herein are valuable genomics resources that may have many applications such as quantitative trait loci identification, map-based gene cloning, association mapping, marker-assisted selection, as well as assembly of a more complete cucumber genome.
doi:10.1186/1471-2229-13-53
PMCID: PMC3626583  PMID: 23531125
Cucumber; Cucumis sativus; NB-LRR; Resistance gene homolog; Genetic mapping; Comparative mapping; Map integration

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