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Divergent Evolution of Plant NBS-LRR Resistance Gene Homologues in Dicot and Cereal Genomes
Abstract
The majority of plant disease resistance genes are members of very large multigene families. They encode structurally related proteins containing nucleotide binding site domains (NBS) and C-terminal leucine rich repeats (LRR). The N-terminal region of some resistance genes contain a short sequence called TIR with homology to the animal innate immunity factors, Toll and interleukin receptor-like genes. Only a few plant resistance genes have been functionally analyzed and the origin and evolution of plant resistance genes remain obscure. We have reconstructed gene phylogeny by exhaustive analysis of available genome and amplified NBS domain sequences. Our study shows that NBS domains faithfully predict whole gene structure and can be divided into two major groups. Group I NBS domains contain group-specific motifs that are always linked with the TIR sequence in the N terminus. Significantly, Group I NBS domains and their associated TIR domains are widely distributed in dicot species but were not detected in cereal databases. Furthermore, Group I specific NBS sequences were readily amplified from dicot genomic DNA but could not be amplified from cereal genomic DNA. In contrast, Group II NBS domains are always associated with putative coiled-coil domains in their N terminus and appear to be present throughout the angiosperms. These results suggest that the two main groups of resistance genes underwent divergent evolution in cereal and dicot genomes and imply that their cognate signaling pathways have diverged as well.
... Some R genes have a defense role against the diverse pathogens if number of effector targets is limited. The NBS-LRR proteins have been further subgrouped based on the presence or absence of an N-terminal Toll/Interleukin-1 Receptor (TIR) homology region 23, 95,110,116 . The proteins lacking a TIR have a coiledcoil (CC) motif in the N-terminal region 110 . ...
... The NBS-LRR proteins have been further subgrouped based on the presence or absence of an N-terminal Toll/Interleukin-1 Receptor (TIR) homology region 23, 95,110,116 . The proteins lacking a TIR have a coiledcoil (CC) motif in the N-terminal region 110 . Several R genes are clustered in plant genomes 55 . ...
... Studies related to comparisons and domain swaps among alleles at the L locus indicated that TIR domain may be involved in pathogen recognition. Data search of EST databases indicated absence of TIR-NBS-LRR-like proteins in monocots 95,110 . ...
The crop plants of the family Leguminosae are second to cereal crops of commercial importance on the basis of area harvested and total production worldwide. It is well known globally that many crops do not give good yield due to certain diseases existing in their plants. Nowadays, there is much emphasis on developing disease resistant varieties of crops and especially of commercial crops. Plants need to protect themselves against attack from viruses, microbes, invertebrates and even other plants. NBS-LRR (Nucleotide binding site-leucine rich repeats) genes belong to the largest plant disease resistance gene family and are responsible for plant resistance to pathogens. Studies of the NBS-LRR gene family in plants represent an intriguing challenge and can provide knowledge on the genomic and molecular mechanisms that form the basis of gene regulation and protein function. Their evolution at the gene and genomic level can be defined through ancient and numerous gene families. In the present study, beneficial concepts for generating basic and fundamental knowledge on the NBS-LRR plant disease resistance genes are discussed with emphasis on selected legume plants of commercial importance.
... The aminoterminal signalling domain is generally divided into two separate classes based on homology to either the signalling domain of Toll/Interleukin-1 Receptors (TIR) or the presence of a coiled-coil (CC) domain. These two distinct signalling components share common downstream signalling pathways, however both classes have also been observed to activate separate downstream components (Aarts et al. 1998;Falk et al. 1999;Meyers et al. 1999;Pan et al. 2000;Takken et al. 2006;Hofius et al. 2009). ...
... While both CC and TIR type NLRs (CNLs and TNLs, respectively) are widely distributed in dicots, canonical TNLs appear to be absent in monocots (Meyers et al. 1999;Pan et al. 2000;Meyers et al. 2002;Tarr et al. 2009). In addition, variations of the signalling domain-NBD-LRR (NLR) structure can be found in most plant species, with NBD-containing proteins lacking either the amino-terminal signalling domain or the carboxy-terminal LRR domain, or having juxtaposed non-canonical domains, extending their flexibility as signalling components or effector decoys for host proteins (Bonardi et al. 2012;Kroj et al. 2016). ...
... Clade 1 was highly enriched in TNLs (708/806), CNLs dominated clade 2 (307/510) and clade 4 (326/385), clade 5 was enriched for RNLs (62/87), and clade 3 contained mainly XNLs (211/245) (Supplemental table 7). The clear correlation between domain structure and the NBD-based phylogeny indicated that the NBD sequences contained sufficient information for inferring the evolutionary history of the plant NLR family, as previously suggested (Pan et al. 2000). ...
Nucleotide-binding site leucine-rich repeat resistance genes (NLRs) allow plants to detect microbial effectors. We hypothesized that NLR expression patterns would reflect organ-specific differences in effector challenge and tested this by carrying out a meta-analysis of expression data for 1,235 NLRs from 9 plant species. We found stable NLR root/shoot expression ratios within species, suggesting organ-specific hardwiring of NLR expression patterns in anticipation of distinct challenges. Most monocot and dicot plant species preferentially expressed NLRs in roots. In contrast, Brassicaceae species, including oilseed rape and the model plant Arabidopsis thaliana , were unique in showing NLR expression skewed towards the shoot across multiple phylogenetically distinct groups of NLRs. The Brassicaceae NLR expression shift coincides with loss of the endomycorrhization pathway, which enables intracellular root infection by symbionts. We propose that its loss offer two likely explanations for the unusual Brassicaceae NLR expression pattern: loss of NLR-guarded symbiotic components and elimination of constraints on general root defences associated with exempting symbionts from targeting. This hypothesis is consistent with the existence of Brassicaceae -specific receptors for conserved microbial molecules and suggests that Brassicaceae species are rich sources of unique antimicrobial root defences.
... Thus, a separate subclass is formed in the class nTNL genes which encompasses this RPW8 domain at their N terminal portion referred as RPW8-NBS-LRR class) (Dubey and Singh 2018). Presence of specific motifs of amino acids found in NBS domain along with position of intron and genomic distribution also demonstrates variation among the two groups (Pan et al. 2000). Genes belonging to Toll/interleukin-1 receptor NBS-LRR and Coil coil-NBS-LRR groups relies on Enhanced Diseased Susceptibilty1-or Nonexpressor of Pathogenesis-Related genes 1-signaling pathways (Wiermer et al. 2005). ...
... To study variation among the pathogen responsive CcNBS-LRRs phylogenetic analysis was carried out by constructing phylogenetic tree using protein sequences of all pathogenic responsive CcNBS-LRR genes (Pan et al. 2000). Very clear demarcation between TIR-type and non-TIR-type sequences was observed (Fig. 1). ...
We identified 41 pathogen responsive CcNBS-LRR genes which belonged to two major groups of NBS-LRR genes, i.e., TIR-NBS-LRR (6) and NON-TIR-NBS-LRR (35). Gene structures, motifs, cis-acting elements, and chromosomal distributions of these pathogen responsive CcNBS-LRRs were analyzed. Further, transcriptional regulation of 41 biotic stress responsive CcNBS-LRRs was recorded against Fusarium udum in a wilt resistant and a susceptible cultivar. 39 and 37 genes were differentially up-regulated in the resistant and susceptible cultivar, respectively. Two genes Cc143 and Cc150 were upregulated at all sampling periods in the resistant cultivar, whereas, in the susceptible cultivar four genes (Cc127, Cc129, Cc219, and Cc87) were constantly down-regulated and a single gene Cc255 was up-regulated at all sampling periods. The resistant cultivar also displayed rapid up-regulation (within 6 h) of the genes against F. udum. CcNBS 125, CcNBS 244, CcNBS 22, CcNBS 136, and CcNBS 143 were identified as major genes in the resistant cultivar as their relative gene expression increased by 33 folds (6 h), 31 folds (24 h), 27 folds (48 h), 25 folds (6 h), and 25(48 h) folds change, respectively, in comparison to the control. Similarly in the susceptible cultivar CcNBS 255, CcNBS 161, and CcNBS 143 were considered major gene as their relative gene expression enhanced 12 folds, 11 folds, and 11 folds, respectively, at 72 h post-inoculation. The study provided fundamental insight into the role of biotic stress responsive CcNBS-LRRs in defense responses against the wilt pathogen F. udum.
... The highly conserved NB domain regulates the protein ON/OFF state by binding and hydrolyzing ADP and GTP [19], and contains highly conserved motifs involved in intraand extra-molecular interactions [20,21]. These include the motifs hhGRExE, P-loop (Walker A/kinase 1), RNBS-A, kinase 2 (Walker B), kinase 3a, RNBS-B, RNBS-C, GLPL and RNBS-D and MHD [22,23]. The high level of conservation of this amino acid region makes the NB domain very useful for studying the genomic architecture of NB-LRR gene family [24]. ...
... To fight a multitude of phytopathogens, plants have diversified a wide defense arsenal from a successful supra-domain assembly originating 3,5 billion years ago [8,15]. About 35,000 NB-LRR genes were identified in 104 genomes using a protein domain search approach [8,23]. To minimize the risk of bias in R-gene identification, we used gene sets from the soft-masked versions of the genome assemblies [9,25]. ...
The nucleotide-binding and leucine-rich repeat (NB-LRR) genes, also known as resistance (R)-genes, play an important role in the activation of immune responses. In recent years, large-scale studies have been performed to highlight the diversification of plant NB-LRR repertories. It is well known that, to provide new functionalities, NB-LRR sequences are subject to duplication, domain fusions and acquisition and other kinds of mutations. Although some mechanisms that govern NB-LRR protein domain adaptations have been uncovered, to retrace the plant-lineage-specific evolution routes of R protein structure, a multi-genome comparative analysis was performed. This study allowed us to define groups of genes sharing homology relationships across different species. It is worth noting that the most populated groups contained well-characterized R proteins. The arsenal profile of such groups was investigated in five botanical families, including important crop species, to underline specific adaptation signatures. In addition, the dissection of 70 NB domains of well-characterized R-genes revealed the NB core motifs from which the three main R protein classes have been diversified. The structural remodeling of domain segments shaped the specific NB-LRR repertoires observed in each plant species. This analysis provided new evolutionary and functional insights on NB protein domain shuffling. Taken together, such findings improved our understanding of the molecular adaptive selection mechanisms occurring at plant R loci.
... AMPs are small peptides, mostly forming an alpha-helix or β sheet structure, and they confer protection against fungi and bacteria infections as well as certain viruses [6]. Plant genomes hold a wide range of genes for detecting pathogen attack and inducing adequate defense responses [7]. Several small basic peptides were identified from kernels of maize inbred line B73 and with antimicrobial properties [8]. ...
... Plant genomes hold a wide range of genes for detecting pathogen attacks and inducing adequate defense responses [7,30]. Z. mays L. inbred line B73 is regarded as a useful resource for the isolation of antimicrobial peptides. ...
Antimicrobial peptides (AMPs) are small molecules consisting of less than fifty residues of amino acids. Plant AMPs establish the first barrier of defense in the innate immune system in response to invading pathogens. The purpose of this study was to isolate new AMPs from the Zea mays L. inbred line B73 and investigate their antimicrobial activities and mechanisms against certain essential plant pathogenic bacteria. In silico, the Collection of Anti-Microbial Peptides (CAMPR3), a computational AMP prediction server, was used to screen a cDNA library for AMPs. A ZM-804 peptide, isolated from the Z. mays L. inbred line B73 cDNA library, was predicted as a new cationic AMP with high prediction values. ZM-804 was tested against eleven pathogens of Gram-negative and Gram-positive bacteria and exhibited high antimicrobial activities as determined by the minimal inhibitory concentrations (MICs) and the minimum bactericidal concentrations (MBCs). A con-focal laser scanning microscope observation showed that the ZM-804 AMP targets bacterial cell membranes. SEM and TEM images revealed the disruption and damage of the cell membrane morphology of Clavibacter michiganensis subsp. michiganensis and Pseudomonas syringae pv. tomato (Pst) DC3000 caused by ZM-804. In planta, ZM-804 demonstrated antimicrobial activity and prevented the infection of tomato plants by Pst DC3000. Moreover, four virulent phytopathogenic bacteria were prevented from inducing hypersensitive response (HR) in tobacco leaves in response to low ZM-804 concentrations. ZM-804 exhibits low hemolytic activity against mouse red blood cells (RBCs) and is relatively safe for mammalian cells. In conclusion, the ZM-804 peptide has a strong antibacterial activity and provides an alternative tool for plant disease control. Additionally, the ZM-804 peptide is considered a promising candidate for human and animal drug development.
... Plants utilize their potent arsenal of disease resistance genes or proteins (R-genes or R-proteins) as their defense mechanism. These vigilant protectors are responsible for detecting [4][5][6][7][8][9][10] and defeating invading pathogens with steadfast determination [11,12]. Oddly, the individual R-proteins can possess "Not Merely Conserved Domains" (NMCD) (See Methods 4) along with multifarious conserved motifs positioned within or beyond these domains, highlighting the intricate nature of their functional architecture, was introduced for the rst time in this experiment. ...
Background: In the quest to identify new resistance genes analogous to those found in other plant species, a novel primer designing strategy is introduced for the first time. Unlike traditional methods that rely on prior information about degeneracy positions, this new approach involves designing primers based on specific domain positions within the candidate resistance gene and eliminates the need for prior knowledge of degeneracy. By using this new approach, it becomes possible to uncover resistance genes and understand their functional interactions with pathogens. Additionally, this approach sheds light on the redundancy and diversity of resistance genes. Notably, this primer designing strategy exhibits remarkable sensitivity, allowing the detection of elusive low-abundance target sequences that were previously challenging to identify using degeneracy-based designs.
Results: The qPCR primers, designed using the novel approach of protein domain-specific regions, underwent standardization and validation in endpoint PCR. Subsequent melt curve analysis in qPCR revealed that out of the ten primers tested, six NB-ARC family protein domain-specific qPCR primers (NB-ARC_2, NB-ARC_3, NB-ARC_4, NB-ARC_8, NB-ARC_12, and NB-ARC_17) exhibited a single peak melt curve, indicating precise amplification of the conserved NB-ARC domain of the R-protein. This confirms their specificity and reliability for target detection, enabling the identification of new resistance gene analogues.
Conclusion: Our innovative protein domain-specific qPCR primer design approach allows for precise and accurate PCR amplification, overcoming the limitations of traditional degenerate primers. It enables targeted amplification of specific domain regions within resistance proteins, uncovering both conserved domains and novel resistance genes or gene analogs. The use of these primers also captures the redundancy of resistance genes, offering improved accuracy and reliability in target gene identification. This breakthrough represents a significant advancement in molecular biology research and opens new possibilities for identifying resistance gene analogs. To the best of our knowledge, this is the first report of identifying resistance gene analogs using “protein domain-specific” region based qPCR primer design approach.
... The conserved motif of the NBS-ARC region such as P-loop, RNBS-A, Kinase2, Kinase3, RNBS-C and GLPLA were identified. The presence of Aspartic acid (D) at the end of the Kinase 2 motif differentiates TIR class from the non-TIR class of the R gene (Pan et al. 2000). Sequence variation in RNBS-A and RNBS-C motif was observed when compared with known TIR-NBS LRR gene such as L6, M and N. Inter-domain sequence variation is required for adaptability and maintains individuality among the R genes (Campbell 2003). ...
Fusarium wilt is a major threat to lentil production in India and worldwide. The presence of evolving virulent races has imposed the necessity of reliable management practices including breeding for resistance using unexplored germplasms. The magnitude of resistance by the plant is determined by rapid recognition of the pathogen and induction of defence genes. Resistance gene analogues have been key factors involved in the recognition and induction of defence response. In the present study, the expression of key RGA previously cloned was determined in three resistant accessions (L65, L83 and L90) and a susceptible accession (L27). The expression was assessed via qPCR at 24, 48 and 72 hpi against virulent race5 (CG-5). All the RGAs differentially transcribed in resistant and susceptible accession showed temporal variation. RGA Lc2, Lc8, Ln1 and Lo6 produced cDNA signals during early infection (24 hpi) predicting its involvement in recognition. LoRGA6 showed significant upregulation in L65 and L83 while downregulating in L27 and the full length of LoRGA6 loci was isolated by 5′ and 3′ RACE PCR. In-silico characterization revealed LoRGA6 loci code for 912 amino acids long polypeptide with a TIR motif at the N terminal and eight LRR motifs at the C terminal. The tertiary structure revealed a concave pocket-like structure at the LRR domain potentially involved in pathogen effectors interaction. The loci have ADP binding domain and ATPase activity. This has further paved the path for functional analysis of the loci by VIGS to understand the molecular mechanism of resistance.
... NLR proteins are multidomain proteins that possess a conserved architecture, including a C-terminal LRR domain, a central nucleotide-binding and oligomerization domain (NOD), and a variable N-terminal domain (Takken and Goverse, 2012). The N-terminal domains mainly include the Toll-interleukin 1 receptor (TIR)-like or coiledcoil (CC) types; thus, NLR proteins can be largely classified into TIR-NLR (TNL) and CC-NLR (CNL) based on differences in Nterminal structure (Pan et al., 2000;DeYoung and Innes, 2006). Many studies have established the CC and TIR domains as signaling modules (Lapin et al., 2019;Lapin et al., 2022;Zhang et al., 2022). ...
Plant nucleotide-binding and leucine-rich repeat (NLR) proteins are immune sensors that detect pathogen effectors and initiate a strong immune response. In many cases, single NLR proteins are sufficient for both effector recognition and signaling activation. These proteins possess a conserved architecture, including a C-terminal leucine-rich repeat (LRR) domain, a central nucleotide-binding (NB) domain, and a variable N-terminal domain. Nevertheless, many paired NLRs linked in a head-to-head configuration have now been identified. The ones carrying integrated domains (IDs) can recognize pathogen effector proteins by various modes; these are known as sensor NLR (sNLR) proteins. Structural and biochemical studies have provided insights into the molecular basis of heavy metal-associated IDs (HMA IDs) from paired NLRs in rice and revealed the co-evolution between pathogens and hosts by combining naturally occurring favorable interactions across diverse interfaces. Focusing on structural and molecular models, here we highlight advances in structure-guided engineering to expand and enhance the response profile of paired NLR-HMA IDs in rice to variants of the rice blast pathogen MAX-effectors (Magnaporthe oryzae AVRs and ToxB-like). These results demonstrate that the HMA IDs-based design of rice materials with broad and enhanced resistance profiles possesses great application potential but also face considerable challenges.
... The C terminal leucine-rich repeat (LRR) domain, which is usually composed of repeated units with hydrophobic amino acids, acts mostly as a ligand binding platform with autoinhibitory function (Martin et al., 2003). At the N terminus, NLR proteins carry either a coiled coil (CC) domain, or a domain that has homology to the Toll/Interleukin-1 Receptor (TIR) domain, whose role is to transduce an activation signal to downstream defense pathways (Pan et al., 2000). The recent solving of NLR protein structures greatly advanced our knowledge about their action and signaling (Ma et al., 2020;Martin et al., 2020). ...
The majority of plant disease resistance (R) genes encode nucleotide binding-leucine rich repeat (NLR) proteins. In melon, two closely linked NLR genes, Fom-1 and Prv, were mapped and identified as candidate genes that control resistance to Fusarium oxysporum f.sp. melonis races 0 and 2, and to papaya ringspot virus (PRSV), respectively. In this study, we validated the function of Prv and showed that it is essential for providing resistance against PRSV infection. We generated CRISPR/Cas9 mutants using Agrobacterium-mediated transformation of a PRSV-resistant melon genotype, and the T1 progeny proved susceptible to PRSV, showing strong disease symptoms and viral spread upon infection. Three alleles having 144, 154 and ~3 kb deletions, respectively, were obtained, all of which caused loss of resistance. Interestingly, one of the Prv mutant alleles, prvΔ154, encoding a truncated product, caused an extreme dwarf phenotype, accompanied by leaf lesions, high salicylic acid levels and defense gene expression. The autoimmune phenotype observed at 25 oC proved to be temperature-dependent, being suppressed at 32 oC. This is a first report on successful application of CRISPR/Cas9 to confirm R-gene function in melon. Such validation opens new opportunities for molecular breeding of disease resistance in this important vegetable crop.
... The amino-terminal of the typical R gene is linked to TIR (Toll/interleukin-1-like receptor) or CC (coiled coil) and is involved in defense signaling. They are differentiated based on the presence of aspartate (D) or tryptophan (W) at end of the kinase 2 motif in TIR and CC, respectively (Pan et al., 2000). We observed that forty-two isolated clones belonged to class TIR and the other three to non-TIR, which had previously not been found in Spanish lines (Yaish et al., 2004). ...
Fusarium wilt caused by Fusarium oxysporum f. sp. lentis (Fol) is the most devastating disease of lentil present worldwide. Identification of multi-race fusarium wilt resistance genes and their incorporation into existing cultivars will help to reduce yield losses. In the present study, 100 lentil germplasms belonging to seven lentil species were screened against seven prevalent races of Fol, and accessions IC201561 (Lens culinaris subsp. culinaris), EC714243 (L. c. subsp. odemensis), and EC718238 (L. nigricans) were identified as resistant. The typical R gene codes for the nucleotide-binding site and leucine-rich repeats (NBS-LRR) at the C terminal are linked to either the Toll/interleukin 1-like receptor (TIR) or coiled coil (CC) at the N terminal. In the present study, degenerate primers, designed from the NBS region amplifying the P-loop to the GLPLA motif, isolated forty-five resistance gene analogues (RGAs) from identified resistant accessions. The sequence alignment identified both classes of RGAs, TIR and non-TIR, based on the presence of aspartate (D) and tryptophan (W) at the end of the kinase motif, respectively. The phylogenetic analysis grouped the RGAs into six classes, from LRGA1 to LRGA6, which determined the diversity of the RGAs present in the host. Grouping of the RGAs identified from Lens nigricans, LnRGA 2, 9, 13 with I2 revealed the structural similarity with the fusarium resistance gene. The similarity index ranged from 27.85% to 86.98% among the RGAs and from 26.83% to 49.41% among the known R genes, I2, Gpa2, M, and L6. The active binding sites present along the conserved motifs grouped the RGAs into 13 groups. ADP/ATP, being the potential ligand, determines the ATP binding and ATP hydrolysis activity of the RGAs. The isolated RGAs can be used to develop markers linked to the functional R gene. Furthermore, expression analysis and full-length gene isolation pave the path to identifying the molecular mechanism involved in resistance.
... A total of 902 RGAs were identified from 15 flax chromosome-scale pseudomolecules (CDC Bethune v2) after the removal of redundancy. RGAugury categorized these RGAs into four major groups: NLR, RLK, RLP, and TM-CC This is different from most monocot species in which TNL genes have lost in the common ancestor of monocots during their genome evolution (Meyers et al. 1999;Pan et al. 2000;Akita and Valkonen 2002;Bai et al. 2002;Cannon et al. 2002;. A total of eight CNL and eleven TNL genes with complete structures were predicted in this assembly. ...
Genomic selection (GS) or genomic prediction (GP) is a type of marker-assisted selection that relies on genome-wide markers to predict genomic-estimated breeding values (GEBVs) of phenotypes. GS is quickly becoming a conventional approach in both plant and animal breeding to increase selection accuracy, reduce breeding cost and shorten breeding cycles. The concept of GS models was first developed using genome-wide random markers, with marker density being a key element in estimating the predictive ability in breeding populations. It is currently straightforward to generate high-density marker datasets thanks to the remarkable advances in genotyping technologies. Recent studies showed that high-density genome-wide random markers do not necessarily generate high genomic predictive ability in GS because the vast majority of markers are unrelated to the traits of interest, thus generating background noises and lowering the predictive ability. Alternatively, the use of quantitative trait loci (QTLs), identified through genome-wide association study (GWAS) methods, in GS models can significantly improve genomic predictive ability and reduce the genotyping cost of the test populations. Here, we present recent findings, discuss a few case studies, a QTL-based GS strategy and a genomic cross-predictions for flax breeding improvement.
... Currently, over 60% of R genes belong to a family that encodes nucleotide-binding site (NBS) and leucine-rich-repeat (LRR) domain receptors, well known as NBS-LRR or NLR genes (Kourelis and van der Hoorn, 2018). Based on the different N-terminal structural domains, NLR genes were classified into three subclasses, including TNL containing the TIR structural domain, CNL with the CC structural domain, and RNL characterized by the RPW8 structural domain (Whitham et al., 1994;Parker et al., 1997;Botella et al., 1998;Pan et al., 2000;Shao et al., 2014). The majority of CNL and TNL proteins function as detectors of pathogens' effectors (Kourelis and van der Hoorn, 2018), while RNL proteins typically act as "helper" NLRs and get involved in the downstream signaling of CNL and TNL proteins (Kourelis and van der Hoorn, 2018;Wang et al., 2020). ...
Introduction: Nucleotide-binding leucine-rich repeat (NLR) genes play a crucial role in green plants’ responding to various pathogens. Genome-scale evolutionary studies of NLR genes are important for discovering and applying functional NLR genes. However, little is known about the evolution of NLR genes in the Apiaceae family including agricultural and medical plants.
Methods: In this study, comparative genomic analysis was performed in four Apiaceae species to trace the dynamic evolutionary patterns of NLR genes during speciation in this family.
Results: The results revealed different number of NLR genes in these four Apiaceae species, namely, Angelica sinensis (95), Coriandrum sativum (183), Apium graveolens (153) and Daucus carota (149). Phylogenetic analysis demonstrated that NLR genes in these four species were derived from 183 ancestral NLR lineages and experienced different levels of gene-loss and gain events. The contraction pattern of the ancestral NLR lineages was discovered during the evolution of D. carota, whereas a different pattern of contraction after first expansion of NLR genes was observed for A. sinensis, C. sativum and A. graveolens.
Discussion: Taken together, rapid and dynamic gene content variation has shaped evolutionary history of NLR genes in Apiaceae species.
... A total of 902 RGAs were identified from 15 flax chromosome-scale pseudomolecules (CDC Bethune v2) after the removal of redundancy. RGAugury categorized these RGAs into four major groups: NLR, RLK, RLP, and TM-CC This is different from most monocot species in which TNL genes have lost in the common ancestor of monocots during their genome evolution (Meyers et al. 1999;Pan et al. 2000;Akita and Valkonen 2002;Bai et al. 2002;Cannon et al. 2002;. A total of eight CNL and eleven TNL genes with complete structures were predicted in this assembly. ...
Quantitative trait locus (QTL) mapping is a powerful statistical genetics approach to identify genomic regions and candidate genes associated with traits of interest in plants. Depending on the genetic populations and the theoretical considerations, either linkage map-based QTL mapping or linkage disequilibrium-based association mapping, commonly known as genome-wide association study (GWAS), is widely used QTL mapping strategies. Recently, several multi-locus statistical models have been developed and applied in crops, including flax, leading to the identification of large- and small-effect QTLs for complex traits. In the last decade, at least 21 QTL mapping studies were reported in flax. Using bi-parental populations or germplasm collections, more than 1000 unique QTLs or quantitative trait nucleotides (QTNs) have been reported for 37 traits, including seed yield and agronomic traits, fiber yield and quality, seed quality, and abiotic and biotic traits. Some candidate genes neighboring these QTLs/QTNs have also been identified. These results provide a large set of genomic resources and genomic tools for genomics-assisted breeding, such as marker-based selection and genomic selection to pyramid favorable alleles into cultivars.
... NBS-LRR proteins belonging to the non-TIR class mostly have the RPW8 domain, zinc finger, and coiled-coil (CC) N-terminal domain and are called CNL proteins (DeYoung and Innes 2006). This class is present in dicotyledonous and monocotyledonous plants (Pan et al. 2000). Red-rot-related NBS-LRR genes have a significant role in systemic acquired resistance by upregulating after C. falcatum inoculation (Ramesh Sundar et al. 2012). ...
Sugarcane is the major agro-industrial crop, which not only fulfills 80% of the world’s sugar needs but is also a valuable source of bioenergy. Crop yield and sugar recovery are continuously under threat owing to consistent infestation by diseases and insect pests. Plants respond to pathogen infection by the activation of constitutive or inducible defense systems, including expression of defense-related proteins, i.e. chitinase, glucanase, chitosanase, metallothionine, peroxidase, thaumatin, and endoproteinase. These pathogen-induced proteins are directly or indirectly involved in plant defense response. Other plant proteins involved in the plant defense system are NBS-LRR, glycoproteins, catalases, and WRKY proteins. Pathogenic diseases are recognized by NBS-LRR, and it induces the production of glycoproteins after infection, which disrupts the physiological activity of the pathogens and make them inactive. Likewise, catalases are involved in the detoxification of reactive oxygen species (ROS). WRKY transcription factors play a crucial role in plant defense systems by regulating PR genes. Molecular interventions provide a swift solution to combat these stresses. Various endogenous genes have been explored in sugarcane to play a pivotal role in biotic stress tolerance. Efforts have also been made to develop GMOs having the potential to survive fungal pathogen infections. Few have reached the commercialization scale, whereas others are at the infancy stage. This chapter highlights defense-related proteins in sugarcane and their potential role to mitigate pathogen infestation through advancements in molecular biology.
... Many crops including rice, B. distachyon, maize, sorghum, Arabidopsis, C. sativus, grapevine, popular, cabbage have a similar grouping of NBS genes (Li et al. 2010a;Guo et al. 2011;Wan et al. 2013;Goyal et al. 2020;Liu et al. 2021a, b). Furthermore, banana being a monocot, there are no TNL groups of NBS-LRR, which are usually present in dicots, and reports of TIR in monocots are scarce (Li et al. 2010b;Pan et al. 2000). ...
Banana is an important food crop that is susceptible to a wide range of pests and diseases that can reduce yield and quality. The primary objective of banana breeding programs is to increase disease resistance, which requires the identification of resistance (R) genes. Despite the fact that resistant sources have been identified in bananas, the genes, particularly the nucleotide-binding site (NBS) family, which play an important role in protecting plants against pathogens, have received little attention. As a result, this study included a thorough examination of the NBS disease resistance gene family’s classification, phylogenetic analysis, genome organization, evolution, cis-elements, differential expression, regulation by microRNAs, and protein–protein interaction. A total of 116 and 43 putative NBS genes from M. acuminata and M. balbisiana, respectively, were identified and characterized, and were classified into seven sub-families. Structural analysis of NBS genes revealed the presence of signal peptides, their sub-cellular localization, molecular weight and pI. Eight commonly conserved motifs were found, and NBS genes were unevenly distributed across multiple chromosomes, with the majority of NBS genes being located in chr3 and chr1 of the A and B genomes, respectively. Tandem duplication occurrences have helped bananas’ NBS genes spread throughout evolution. Transcriptome analysis of NBS genes revealed significant differences in expression between resistant and susceptible cultivars of fusarium wilt, eumusae leaf spot, root lesion nematode, and drought, implying that they can be used as candidate resistant genes. Ninety miRNAs were discovered to have targets in 104 NBS genes from the A genome, providing important insights into NBS gene expression regulation. Overall, this study offers a valuable genomic resource and understanding of the function and evolution of NBS genes in relation to rapidly evolving pathogens, as well as providing breeders with selection targets for fast-tracking breeding of banana varieties with more durable resistance to pathogens.
... Members of the non-TNL class usually contain a predicted coiled-coil (CC) domain and belong to the CC-NBS-LRR (CNL) class [2,3]. It is known that TNLs can provide resistance to diseases in solanaceous and brassicaceous species, while CNLs are more common in dicotyledon and cereal crops [4,5]. Studies have revealed insights into defense responses to viruses in maize [6], tomato [7], potato [8], tobacco [9], etc. ...
Blackcurrant reversion virus (BRV) is the most destructive mite-transmitted pathogen in blackcurrants. The understanding of the resistance to BRV is limited, hindering and delaying the selection process. To identify the resistance (R) gene for BRV resistance, a gene expression analysis based on de novo blackcurrant cv. Aldoniai comparative transcriptome analysis (mock- and BRV-inoculated samples at 2 and 4 days post-inoculation (dpi)) was performed. In this study, 111 annotated clusters associated with pathogenesis according to conservative R gene domains were identified. In virus-infected samples, only Cluster-12591.33361 showed significant expression at 4 dpi. The expression profiles of this cluster were significantly associated with the presence of BRV particles in plant tissues, making it a putative R gene in the dominant resistance strategy in the BRV–Ribes nigrum interaction. The newly identified gene R.nigrum_R belongs to the CC-NBS-LRR class and has 63.9% identity with RPM1 in Populus spp. This study provides new insights on dominant putative R genes related to resistance to BRV in R. nigrum, which could aid targeted research and genetic improvement in breeding programs of blackcurrants.
... Nucleotide-binding leucine-rich repeats (NBS LRR/NLR) are the most significant type of R gene (Van Ooijen et al. 2007). Phylogenetic analysis of the NBS gene was reassembled by an extensive study of resistance gene analogs (RGAs) available in various genomes and NBS domains (Pan et al. 2000). Depending on the presence or absence of multiple domains at the N-terminal (amino-terminal) region, NL proteins are classified into two subclasses; the Toll/ interleukin receptor-nucleotide-binding site-leucine-rich repeats (TIR-NBS-LRR/TNL) and coiled-coil nucleotide-binding site-leucine-rich repeats (CNL). ...
Barley is regarded as the globe’s fourth major cereal crop. A variety of airborne, seedborne, and soilborne infective agents attack barley, causing a variety of barley diseases and substantial losses in agricultural output. Brown and yellow rusts, smut, net blotches, spot blotches, barley yellow dwarf, and molya disease are among the most serious diseases. In general, employing integrated disease management approaches is the best way to handle barley diseases. Growing resistant or tolerant varieties with the fewest foliar fungicides is the most effective approach for barley disease treatments. However, managing soilborne pathogens in barley plants is problematic due to a deficiency in distinguishing symptoms for diagnosis and the absence of fungicides or nematicides that are effective for these pathogens. Recently, nanotechnology has driven the advancement of creative concepts and agricultural productivity with a broad scope for managing plant infections and pests. The antimicrobial properties of metallic and metal oxide nanoparticulates such as silver, selenium, titanium dioxide, zinc oxide, and iron oxide have been extensively researched. In this chapter, we go over barley disease and the role of nanomaterials in reducing the incidence of disease and diagnosis, as well as barley seed germination, physiology, and nutritional quality of barley grain.KeywordsLeaf rust diseaseNet Blotch diseasePowdery mildewBarley yellow dwarfBarley smutSpot blotchFungicidesNanoparticulate
... , kinase-2 (LLVL DDVW/D) , Pan [8] , NBS-LRR non-TIR-NBS-LRR NBS , [9] , NBS , NBS , NBS [10] , RAS (rat sarcoma) ATPase G NBS [2] , NBS ATP GTP , G , , , ...
The large group of plant disease resistance (R) genes that share similar structures possesss a predicted nucleotide-binding site (NBS) domain. NBS domains of this class of R genes show highly conserved amino acid motifs, which makes it possible to isolate resistance gene analogs (RGAs) by PCR with degenerate primers. According to the conserved motifs in the NBS regions of the three typical NBS-LRR type resistance genes (RPS2, N, and L6), five degenerate and one non-degenerate primers were designed to correspond to the P-loop motif in the sense direction, while nine degenerate plus one non-degenerate primers were made corresponding to the HD motif in the anti-sense direction. Then, the homologous PCR was used to amplify NBS sequences from genomic DNA and cDNA using sugarcane variety NCo376 with smut resistance. In all, eleven RGAs were obtained, five from DNA (EF059973, EF059974, EF059975, EF059976, and EF059977) and six from cDNA (EF155648, EF155649, EF155650, EF155651, EF155652, and EF155653). Sequence analysis showed that RGAs comprised the conserved domains P-loop, Kinase-2a, Kinase-3a and HD, which was conserved in NBS-LRR type disease resistance gene. Cluster analysis showed that eleven RGAs and RPS2 and XA1 were clustered into one group, and N and L6 were divided into another group. Further, amino acid sequences showed that their last amino acid in alignment was residue W in LLVLDDV(W/D) motif, which is typical to non-TIR-NBS-LRR type gene. It was suggested that only non-TIR-NBS-LRR but not TIR-NBS-LRR type resistance genes existed in sugarcane genome. One RGA termed PIC (EF059974) was selected randomly for function validation through Real-time PCR. The result showed that expression of PIC gene could to some extent be influenced by U.scitaminea, SA and H2O2, and had the characteristics of constitutive expression and tissue-specific. The RGA cloned in this experiment may provide the shortcut for cloning of sugarcane disease resistance gene.
... NLRs are classified into three types, based on the domain that occupies the N-terminal region:, the Toll/interleukin-1 receptor (TIR)-NBS-LRRs (referred to as TNLs), the coiled-coil (CC)-NBS-LRRs (referred to as CNLs) and the resistance to powdery mildew8-like domain (RPW8)-NBS-LRRs (referred to as RNLs) (Shao et al. 2016(Shao et al. , 2019Van Ghelder et al. 2019;Xue et al. 2020). So far, no TNL proteins have been detected in monocotyledonous plants (Glowacki et al. 2011;Marone et al. 2013;Van Ghelder et al. 2019) because they have been lost from the monocot lineage after dicotyledon/monocotyledon separation (Pan et al. 2000). The NBS domain is the core portion of the ~ 300 amino acid NB-ARC domain and comprises rigorously ordered conserved motifs (Meyers et al. 1999(Meyers et al. , 2003Tameling et al. 2002). ...
Key message
The date palm NBS-encoding family of resistance genes has a wide set of structural features and distinct domains. Recent duplication and relaxed selection have both played major roles in biotic stress adaptation. Several genes that we discussed will aid in genetic improvement.
Abstract
Date palm, Phoenix dactylifera L., is one of the crucial socio-economic plants in the irrigable deserts of South West Asia and North Africa and its sweet edible fruits are traded globally. Crop yield is still vulnerable to a variety of biotic stressors. The largest class of disease resistance (R) genes in plants involves genes encoding nucleotide-binding site (NBS) domains. In the current study, we performed a genome-wide investigation of the structural diversity, phylogenetic relationships and functional attributes of the NBS-encoding R gene family in P. dactylifera, using comparison with banana, Musa acuminata. In date palm and banana, 123 and 85 regular NBS-encoding protein sequences, respectively, were identified. All proteins in both species lacked the TIR domain, while proteins with the LRR domain (NBS-LRR subfamily) represented one-third of the NBS protein complement. In addition to the NBS, LRR, and TIR, other atypical domains were discovered and their probable roles in the molecular mechanisms of host-pathogen recognition were widely discussed, providing insight into the functional flexibility of the NBS gene family in date palm. Motif discovery in the full NBS complement from date palm and banana revealed matches to the eight primary motifs of the NB-ARC domain, with slight variation in motif frequencies among both species. Thirty eight (38) date palm NBS (PdNBS) gene paralogous pairs were identified, accounting for almost two-thirds (61.7%) of the total PdNBS genes, demonstrating the relevance of duplication in the expansion of this gene family in date palm. The majority of these duplication pairings (73.68%) had a Ka/Ks greater than 0.3, implying that selection was relaxed, allowing for some functional divergence following duplication. Seven pairs of orthologs were identified between PdNBSs and known disease resistance genes from several plant species, shedding light on the function of date palm R genes. Finally, we found that 23 PdNBSs were supported by expression evidence from expressed sequence tags (ESTs). The findings of this study generate a detailed overview of date palm NBS genes and offer potential functional candidates for disease resistance.
... TIRs are grouped into the canonical TIR and TIR2 subclasses. Though TIR2-NB proteins are found in monocot plants, TNLs with canonical TIR domains have been detected in dicot but not in monocot plants (7,8). Adenosine diphosphate/adenosine triphosphate exchange at the NB domain (9,10) and oligomerization of NLRs (11)(12)(13)(14)(15)(16) trigger ETI signaling. ...
Throughout their evolution, plant nucleotide-binding leucine-rich-repeat receptors (NLRs) have acquired widely divergent unconventional integrated domains that enhance their ability to detect pathogen effectors. However, the functional dynamics that drive the evolution of NLRs with integrated domains (NLR-IDs) remain poorly understood. Here, we reconstructed the evolutionary history of an NLR locus prone to unconventional domain integration and experimentally tested hypotheses about the evolution of NLR-IDs. We show that the rice ( Oryza sativa ) NLR Pias recognizes the effector AVR-Pias of the blast fungal pathogen Magnaporthe oryzae . Pias consists of a functionally specialized NLR pair, the helper Pias-1 and the sensor Pias-2, that is allelic to the previously characterized Pia pair of NLRs: the helper RGA4 and the sensor RGA5. Remarkably, Pias-2 carries a C-terminal DUF761 domain at a similar position to the heavy metal–associated (HMA) domain of RGA5. Phylogenomic analysis showed that Pias-2/RGA5 sensor NLRs have undergone recurrent genomic recombination within the genus Oryza , resulting in up to six sequence-divergent domain integrations. Allelic NLRs with divergent functions have been maintained transspecies in different Oryza lineages to detect sequence-divergent pathogen effectors. By contrast, Pias-1 has retained its NLR helper activity throughout evolution and is capable of functioning together with the divergent sensor-NLR RGA5 to respond to AVR-Pia. These results suggest that opposite selective forces have driven the evolution of paired NLRs: highly dynamic domain integration events maintained by balancing selection for sensor NLRs, in sharp contrast to purifying selection and functional conservation of immune signaling for helper NLRs.
... On another hand, the 18 research articles pertain to the subtopics 'PRR' (12) and 'NLR' (6). The six articles focused on NLRs reported on their phylogenetic diversity (Meyers et al., 2003;Pan et al., 2000), on the mechanism of effector indirect recognition (Axtell and Staskawicz, 2003), on the key role of helper NLRs (Castel et al., 2019), or on the structural arrangements and activation mechanisms (Wang et al., 2019a(Wang et al., , 2019b. ...
Molecular plant immunity is a dynamic research field that broadly addresses how plants interact with their associated organisms and defend themselves against pests and pathogens. Here, we aimed at providing readers with a snapshot of influential molecular plant immunity research by identifying and analyzing 170 highly influential publications in molecular plant immunity (hereafter called HIPPYs) published in this field between 2000 and 2019. Our analysis draws a broad analytical knowledge of influential scientific advances in the field as well as of the research community that made them. We notably show that HIPPYs are shared by a small, structured, and connected research community. The HIPPYs address coherent research questions using a handful of key model objects (i.e., organisms or molecules) and report findings and concepts that contribute to our integrated understanding of the molecular interactions between plants and their associated organisms. Our ‘HIP in’ (‘highly influential publication in’ ...) method is easily transposable to other large research areas and may help early career researchers to gain a broader knowledge of their field of interest.
[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .
... Inactive NLRs are in an ADP-bound state, as they likely have either a binding preference for ADP over ATP or an intrinsic property for ATP hydrolysis (Tameling et al. 2006). Plant NLRs possess the conserved Walker-B motif in NBD, which is crucial for ATP hydrolysis (Pan et al. 2000;Meyers et al. 2003). Similar to APAF-1 (Reubold et al. 2009), RPP1 was shown to exhibit greater ATPase activity in its catalytically inactive form than effector-activated form , supporting its intrinsic ATPase activity. ...
Animals and plants have NLRs (nucleotide-binding leucine-rich repeat receptors) that recognize the presence of pathogens and initiate innate immune responses. In plants, there are three types of NLRs distinguished by their N-terminal domain: the CC (coiled-coil) domain NLRs, the TIR (Toll/interleukin-1 receptor) domain NLRs and the RPW8 (resistance to powdery mildew 8)-like coiled-coil domain NLRs. CC-NLRs (CNLs) and TIR-NLRs (TNLs) generally act as sensors of effectors secreted by pathogens, while RPW8-NLRs (RNLs) signal downstream of many sensor NLRs and are called helper NLRs. Recent studies have revealed three dimensional structures of a CNL (ZAR1) including its inactive, intermediate and active oligomeric state, as well as TNLs (RPP1 and ROQ1) in their active oligomeric states. Furthermore, accumulating evidence suggests that members of the family of lipase-like EDS1 (enhanced disease susceptibility 1) proteins, which are uniquely found in seed plants, play a key role in providing a link between sensor NLRs and helper NLRs during innate immune responses. Here, we summarize the implications of the plant NLR structures that provide insights into distinct mechanisms of action by the different sensor NLRs and discuss plant NLR-mediated innate immune signalling pathways involving the EDS1 family proteins and RNLs.
... Angiosperm NLR genes can be divided into three subclasses, namely, TIR-NBS-LRR (TNL), CC-NBS-LRR (CNL), and RPW8-NBS-LRR (RNL), based on the identity of the N-terminal domain, which can be one of the three types, namely, Toll/Interleukin-1 receptor-like (TIR), coiled-coil (CC), and resistance to powdery mildew 8 (RPW8; Parker et al., 1997;Pan et al., 2000;Shao et al., 2014). Most TNL and CNL proteins function as pathogen sensors, either directly recognizing pathogenic effector proteins or indirectly monitoring the status shift of host proteins targeted by effectors (Kourelis and van der Hoorn, 2018). ...
Magnoliids are the third-largest group of angiosperms and occupy a critical position in angiosperm evolution. In the past years, due to the lack of sequenced genomes, the disease resistance gene (R gene) profile of magnoliids remains poorly understood. By the genome-wide identification of 1,832 NLR genes from seven magnoliid genomes, we built a framework for the evolution of magnoliid R genes. TNL genes were completely absent from five magnoliids, presumably due to immune pathway deficiencies. A total of 74 ancestral R genes (70 CNLs, 3 TNLs, and 1 RNL) were recovered in a common ancestor of magnoliids, from which all current NLR gene repertoires were derived. Tandem duplication served as the major drive for NLR genes expansion in seven magnoliid genomes, as most surveyed angiosperms. Due to recent rapid expansions, most magnoliids exhibited “a first expansion followed by a slight contraction and a further stronger expansion” evolutionary pattern, while both Litsea cubeba and Persea americana showed a two-times-repeated pattern of “expansion followed by contraction.” The transcriptome analysis of seven different tissues of Saururus chinensis revealed a low expression of most NLR genes, with some R genes displaying a relatively higher expression in roots and fruits. Overall, our study sheds light on the evolution of NLR genes in magnoliids, compensates for insufficiency in major angiosperm lineages, and provides an important reference for a better understanding of angiosperm NLR genes.
... One type contains a coiled-coil (CC) motif for N terminals capable of participating in protein-protein interactions (Maekawa et al., 2011). The second form of NBS-LRR lacks the CC, whereas the Nterminus region has a TIR domain that shares homological features with a protein like Drosophila Toll Interleukin-1 mammals (TIR) (Pan et al., 2000). CNL and TNL comprise two families, usually found at the N-terminus of the R-protein, and differentiate themselves in a domain structure (Marone et al., 2013). ...
Objective
Using disease-resistant genes is the most effective strategy for protecting crops and ensuring agricultural production, or and protection against infections of different pathogens. Under biotic and abiotic stresses, NB-ARC proteins play a critical role in regulating several critical plant metabolic processes and pathways.
Methods
NB-ARC identification and characterization in soybean are still in their infancy, even though R genes have been characterized by various major crop plants. NB-ARC encoding (R) genes in the soybean genome were identified and characterized in silico.
Results
The 103 NB-ARC genes were computationally identified in the soybean genome, randomly distributed on all soybean chromosomes except 5, 10, and 17. Phylogenetic analysis classified the NB-ARC proteins into nine primary groups. However, synteny analysis results of NB-ARC genes of soybean found the best orthologous hit in the A. thaliana representing sequence conservation up to 80%. Soybean NB-ARC genes displayed a plurality of introns between one to seven among the family members. Although their genomic regions have different sizes, a relatively conserved genetic structure was observed within phylogenetic tree groups. Twenty different domains were kept in a group-specific manner, together with the presence of the NB-ARC signatory. Moreover, the transcriptome based-data expression analysis suggested that NB-ARC genes in between non-pathogens and pathogens after the inoculation of Fusarium oxysporum (biotic stress) in the soybean transcriptome, supporting the conjecture of NB-ARC genes have disease resistance functions in the soybean genome and revealing the potential involvement of these genes in the conserved pathways of the biotic-stress-response.
Conclusion
This genome-wide in silico/ computational analysis will be used for accelerating NB-ARC members used for functional characterization, especially under biotic and abiotic stresses.
... On another hand, the 18 research articles pertain to the subtopics 'PRR' (12) and 'NLR' (6). The six articles focused on NLRs reported on their phylogenetic diversity (Meyers et al., 2003;Pan et al., 2000), on the mechanism of effector indirect recognition (Axtell and Staskawicz, 2003), on the key role of helper NLRs (Castel et al., 2019), or on the structural arrangements and activation mechanisms (Wang et al., 2019a(Wang et al., , 2019b. The 12 papers focused on PRRs reported the identification of the chitin receptors CEBip (Kaku et al., 2006) and CERK1 (Miya et al., 2007), the discovery of the bacterial elongation factor Tu (Ef-tu) as an elicitor of plant immunity (Kunze et al., 2004) and then the identification of EF-tu receptor EFR (Zipfel et al., 2006), the identification of the FLS2 locus responsible for Flagellin detection and its importance in plant immunity (Gomez-Gomez and Boller, 2000;, the identification of the FLS2 co-receptor BAK1 (Chinchilla et al., 2007), and the structural arrangement of the FLS2-BAK1-flg22 complex (Sun et al., 2013). ...
... The plantspecific NLR gene family originated and diverged in the common ancestor of green plants . Three NLR gene subclasses, TIR-NBS-LRR (TNL), CC-NBS-LRR (CNL), and RPW8-NBS-LRR (RNL), have been characterized based on the N-terminal domains of the encoded NLR proteins: the Toll/ Interleukin-1 receptor/resistance (TIR) domain, the coiled-coil (CC) domain, and the resistance to powdery mildew 8 (RPW8) domain, respectively (Whitham et al., 1994;Parker et al., 1997;Botella et al., 1998;Pan et al., 2000;Shao et al., 2014). Most CNL and TNL proteins function as pathogen detectors, either interacting directly with pathogen effectors or monitoring the state alteration of host proteins targeted by the effectors (Kourelis and van der Hoorn, 2018). ...
Nucleotide-binding leucine-rich repeat (NLR) genes comprise the largest family of plant disease resistance genes. Angiosperm NLR genes are phylogenetically divided into the TNL, CNL, and RNL subclasses. NLR copy numbers and subclass composition vary tremendously across angiosperm genomes. However, the evolutionary associations between genomic NLR content and ecological adaptation, or between NLR content and signal transduction components, are poorly characterized due to limited genome availability. Here, we established an angiosperm NLR atlas (ANNA, https://biobigdata.nju.edu.cn/ANNA/), which includes NLR genes from over 300 angiosperm genomes. Using ANNA, we revealed that NLR copy numbers differ up to 66-fold among closely related species due to rapid gene loss and gain. Interestingly, NLR contraction was associated with adaptations to aquatic, parasitic, and carnivorous lifestyles. The convergent NLR reduction in aquatic plants resembles the lack of NLR expansion during the long-term evolution of green algae before the colonization of land. A co-evolutionary pattern between NLR subclasses and plant immune-pathway components was also identified, suggesting that immune pathway deficiencies may drive TNL loss. Finally, we recovered a conserved TNL lineage that may function independently of the EDS1-SAG101-NRG1 module. Our findings provide new insights into the evolution of NLR genes in the context of ecological adaptation and genome content variation.
... Dominant R genes can be classified into two groups, the major class encoding NB and LRR domain (NLR) proteins and all others having a different architecture (de Ronde et al., 2014). Furthermore, the NLR R genes can be divided according to their functional domain at the N-terminal part of the protein, either encoding a CC or a Toll and interleukin-1 receptor (TIR) domain (Moffett, 2009;Pan et al., 2000). Both domains are supposed to be involved in recognition of the Avr determinant (Rairdan et al., 2008). ...
Sugar beet cultivation is dependent on an effective control of beet necrotic yellow vein virus (BNYVV, family Benyviridae), which causes tremendous economic losses in sugar production. As the virus is transmitted by a soilborne protist, the use of resistant cultivars is currently the only way to control the disease. The Rz2 gene product belongs to a family of proteins conferring resistance towards diverse pathogens in plants. These proteins contain coiled-coil and leucine-rich repeat domains. After artificial inoculation of homozygous Rz2 resistant sugar beet lines, BNYVV and beet soilborne mosaic virus (BSBMV, family Benyviridae) were not detected. Analysis of the expression of Rz2 in naturally infected plants indicated constitutive expression in the root system. In a transient assay, coexpression of Rz2 and the individual BNYVV-encoded proteins revealed that only the combination of Rz2 and triple gene block protein 1 (TGB1) resulted in a hypersensitive reaction (HR)-like response. Furthermore, HR was also triggered by the TGB1 homologues from BSBMV as well as from the more distantly related beet soilborne virus (family Virgaviridae). This is the first report of an R gene providing resistance across different plant virus families.
... NLRs are intracellular immune receptors, which instigate defensive signaling pathways following perception of a specific effector protein, either through direct interaction with the effector or through monitoring the state of an intermediate protein (15)(16)(17). The canonical NLR structure comprises a C-terminal leucine-rich repeat domain, a central nucleotide-binding NB-ARC domain, and an N-terminal coiled-coil (CC or CC R (18)) or Toll/interleukin-1 receptor (TIR) domain (19)(20)(21). Recent studies have identified noncanonical domains in multiple NLR proteins from different plant species (22)(23)(24). ...
Microbial plant pathogens secrete effector proteins which manipulate the host to promote infection. Effectors can be recognised by plant intracellular nucleotide-binding leucine-rich repeat (NLR) receptors, initiating an immune response. The AVR-Pik effector from the rice blast fungus Magnaporthe oryzae is recognised by a pair of rice NLR receptors, Pik-1 and Pik-2. Pik-1 contains a non-canonical integrated heavy metal-associated (HMA) domain, which directly binds AVR-Pik to activate plant defences. The host targets of AVR-Pik are also HMA domain-containing proteins, namely heavy metal-associated isoprenylated plant proteins (HIPPs) and heavy metal-associated plant proteins (HPPs). Here, we demonstrate that one of these targets interacts with a wider set of AVR-Pik variants compared to the Pik-1 HMA domains. We define the biochemical and structural basis of the interaction between AVR-Pik and OsHIPP19, and compare the interaction to that formed with the HMA domain of Pik-1. Using analytical gel filtration and surface plasmon resonance, we show that multiple AVR-Pik variants, including the stealthy variants AVR-PikC and AVR-PikF which do not interact with any characterised Pik-1 alleles, bind to OsHIPP19 with nanomolar affinity. The crystal structure of OsHIPP19 in complex with AVR-PikF reveals differences at the interface that underpin high-affinity binding of OsHIPP19-HMA to a wider set of AVR-Pik variants than achieved by the integrated HMA domain of Pik-1. Our results provide a foundation for engineering the HMA domain of Pik-1 to extend binding to currently unrecognised AVR-Pik variants and expand disease resistance in rice to divergent pathogen strains.
... Single nucleotide polymorphisms (SNPs) significantly associated with root-knot nematode Meloidogyne javanica (MJ) resistance through genome-wide association study (GWAS) and In addition to also identifying a locus for resistance on chr 13 in the current study, we also identified with GWAS using SNPs markers, loci for resistance to MJ on chr 7 and chr 20, and significantly narrowed down the regions to identify candidate resistance genes in each locus. According to Pan, Wendel, and Fluhr (2000), most of the resistance genes are characterized by the presence of Cterminal LRRs and a central nucleotide binding site (NBS) domain. The presence of proteins containing LRRs at the chr 7 region supports the potential of this SNP to be added to the MJ resistance soybean-breeding program. ...
The root‐knot nematode (RKN) species Meloidogyne javanica (MJ) causes substantial root damage and yield loss in susceptible soybean [Glycine max (L.) Merr.] cultivars. In this study, a genome wide association study (GWAS) was undertaken to identify genomic regions controlling RKN resistance in a soybean association‐mapping panel, using single nucleotide polymorphism (SNP) markers and haplotype information. This study was carried out with a sample (n = 124) from the Coodetec soybean gene bank, including some commercial Brazilian varieties. For the phenotypic evaluations MJ inoculum was obtained from roots of cultivar CD 206, a highly susceptible soybean used to maintain MJ for studies at Coodetec. The cultivars CD 208 and BRS CONQUISTA were used as resistant checks while CD 206, R7, and Nidera NA 5909 RG were used as susceptible checks and all 124 lines were genotyped with 6,000 SNP markers. MJ resistance determinants in the soybean panel were linked to SNPs with significant effects, with nine SNPs distributed along chromosomes 7, 13, and 20. The observed average MJ resistance in the group of soybean entries with haplotype GCT, ACC, ACT, and GCC in the markers Gm20_44446670_G_A, Gm13_29739984_C_A and Gm07_8166605_T_C, respectively, represents moderately resistant genotypes. Gm13_29739984_C_A and Gm07_8166605_T_C discriminated resistant and susceptible cultivars better than the marker Gm20_44446670_G_A. These results are particularly promising for soybean‐breeding programs, by not only identifying new resistance‐associated haplotypes, but also suggesting new genetic mechanisms in RKN‐resistance sources.
... In the wild emmer wheat a marker-based analysis shows that NLRs exhibit higher differentiation (FST = 0.58) than other markers (FST= 0.38) (Sela et al., 2009). Within a single genus and especially among crops, the number of NLR can differ dramatically between species Pan, Wendel & Fluhr, 2000;Guo et al., 2011) suggesting that the NLR family experiences a rapid birth-anddeath process ). ...
Nucleotide binding site, Leucine-rich repeat Receptors (NLRs), are canonical resistance (R) genes in plants, fungi and animals, functioning as central (helper) and peripheral (sensor) genes in a signalling network. We investigate NLR evolution during the colonisation of novel habitats in a model tomato species, Solanum chilense.
We used R-gene enrichment sequencing (RENSeq) to obtain polymorphism data at NLRs of 140 plants sampled across 14 populations covering the whole species range. We inferred the past demographic history of habitat colonisation by resequencing whole genomes from three S. chilense plants from three key populations, and performing Approximate Bayesian Computation using data from the 14 populations.
Using these parameters we simulated the genetic differentiation statistics distribution expected under neutral NLR evolution, and identified small subsets of outlier NLRs exhibiting signatures of selection across populations.
NLRs under selection between habitats are more often helper genes, while those showing signatures of adaptation in single populations are more often sensor-NLRs. Thus, centrality in the NLR network does not constrain NLR evolvability, and new mutations in central genes in the network are key for R gene adaptation during colonisation of different habitats.
... Association analysis was performed using morphological data of 150 oil palm germplasm and genotypic data of 54 SSR markers and Q matrix obtained from structure by using software TASSEL (Bradbury et al. 2007). The markertrait association analysis was conducted using TASSEL 3.0 software along with the general linear model (GLM), mixed linear model (MLM) procedures (Pan et al. 2000). The significant threshold for the association was set at different levels P \ 0.01 and P \ 0.001. ...
Oil palm (Elaeis guineensis Jacq.) is a perennial vegetable and a high oil-yielding crop (4–6 t/ha). There is a large scope for increasing the oil yield by selecting elite planting material for breeding programme in germplasm evaluation, characterization and utilization. In the present study, a diverse range of 150 oil palm genotypes were characterized using 12 quantitative variables with 54 genomic microsatellite markers. A wide variation was observed in the morphological traits among indigenous populations. Highly significant and positive correlations were observed between vegetative dry matter (VDM) and total dry matter (TDM) (0.862), and height and height increment (0.838). The first two principal component analyses explained 67.7% of total variation among morphological traits. The genotypes IC0610001-59 (Pune-2) and IC0610001-60 (Pune-2) were found highly promising based on less height increment, more TDM with high yield. For the mapping study, general linear model (GLM) approach, quantitative-trait loci (QTL) for annual height increment, number of bunches, bunch yield and bunch index were linked to simple-sequence repeat (SSR) loci mEgCIR3649 with phenotypic variance of 15.08, 10.43, 11.74, 15.39. TDM and VDM were linked to mEgCIR0192 (27.34 and 24.19%), mEgCIR3684 (16.84 and 18.30%), SPSC00163 (18.8 and 15.39%) and mEgCIR0555 (16.47 and 18.81%), with at a significant threshold (P) level of ≤0.001 and by mixed linear model (MLM) approach. TDM was linked to mEgCIR0555 with phenotypic variance of 20.72%, bunch yield and bunch index were linked to mEgCIR2813 at phenotypic variance of 17.11% and 12.88%, respectively, at a significant threshold (P) level of ≤0.01.
... For example, the crystal structure of the CC domain for the barley mildew A (Mla10) resistance protein shows a rodshaped homodimer [17]. The CC domain consists of seven residue repeats known as heptads [24]. The heptad has seven positions that are known as a-b-c-d-e-f-g. ...
Resistance proteins are the most effective weapons of plants against pathogen invasion since they can recognize the corresponding pathogen effectors or associated proteins to activate plant immune response. Up to date, greater than seventy resistance proteins have been identified from different plant species. Most resistance proteins contain conserved domains such as the nucleotide-binding sites, the leucine-rich repeat, the coiled-coil domain and others. These domains play significant roles in resistance proteins interaction with effector proteins from pathogens and inactivating signals involved in innate immunity. This review highlights illuminating the structure and function of the isolated plant resistance proteins in different plant-pathogen interaction systems.
... However, all NBS-encoding genes of rice are of the non-TIR class. The TIR type has not been detected in any grass species (Pan et al. 2000). The genome-wide studies have demonstrated that the TNL subfamily is abundant in dicots while absent in cereals (monocots) (Zhou et al. 2004). ...
Oilseed Brassica crops play an important role in the vegetable oil economy of India and the world. There is an urgently felt need to increase their yield levels across varying growing conditions to meet the ever-growing demand for edible oil. The production level fluctuates widely over time and space mainly due to the challenges posed by various biotic and abiotic stresses. To combat these stresses that are very complex in nature, we need to have multi-pronged strategy by combining agronomical and breeding approaches. A major shift in yield level of any crop plant with increased tolerance to biotic and abiotic stresses is possible only with extensive genetic manipulation through breeding, and the breeding efforts in turn will require the continued collection, conservation, evaluation, and deployment of diverse crop genetic resources in a targeted way. Various activities enshrined in the management of rapeseed-mustard genetic resources are systematically explained in this chapter with an aim firstly to sensitize the readers about the importance of PGR and then to explain the principles and methodologies underlying the various activities involved in PGR management with special emphasis on rapeseed-mustard group of crops. Status of rapeseed-mustard germplasm in India and the world in terms of their number, characterization, evaluation, utilization so far, and future thrust area required are also discussed in this chapter.
... 8,9 It was well known that the NBS-LRR protein encodes 3 main domains-N-terminal, NBS, and LRR domains. 5,9,10 2 ...
NBS-LRR (nucleotide-binding site and leucine-rich repeat) is one of the largest resistance gene families in plants. The completion of the genome sequencing of wild tomato Solanum pimpinellifolium provided an opportunity to conduct a comprehensive analysis of the NBS-LRR gene superfamily at the genome-wide level. In this study, gene identification, chromosome mapping, and phylogenetic analysis of the NBS-LRR gene family were analyzed using the bioinformatics methods. The results revealed 245 NBS-LRRs in total, similar to that in the cultivated tomato. These genes are unevenly distributed on 12 chromosomes, and ~59.6% of them form gene clusters, most of which are tandem duplications. Phylogenetic analysis divided the NBS-LRRs into 2 subfamilies (CNL-coiled-coil NBS-LRR and TNL-TIR NBS-LRR), and the expansion of the CNL subfamily was more extensive than the TNL subfamily. Novel conserved structures were identified through conserved motif analysis between the CNL and TNL subfamilies. Compared with the NBS-LRR sequences from the model plant Arabidopsis thaliana, wide genetic variation occurred after the divergence of S. pimpinellifolium and A thaliana. Species-specific expansion was also found in the CNL subfamily in S. pimpinellifolium. The results of this study provide the basis for the deeper analysis of NBS-LRR resistance genes and contribute to mapping and isolation of candidate resistance genes in S. pimpinellifolium.
Along with the emergence of green plants on this planet one billion years ago, the nucleotide binding site leucine-rich repeat (NLR) gene family originated and diverged into at least three subclasses. Two of them, with either characterized N-terminal toll/interleukin-1 receptor (TIR) or coiled-coil (CC) domain, serve as major types of immune receptor of effector-triggered immunity (ETI) in plants, whereas the one having a N-terminal Resistance to powdery mildew8 (RPW8) domain, functions as signal transfer component to them. In this review, we briefly summarized the history of identification of diverse NLR subclasses across Viridiplantae lineages during the establishment of NLR category, and highlighted recent advances on the evolution of NLR genes and several key downstream signal components under the background of ecological adaption.
Flax (Linum usitatissimum L.) is an important crop as the sources of fiber and seed oil. Annually, various pathogens cause significant loss to flax production. Thus, finding the underlying genetic bases for plant resistance to pathogens is essential for plant geneticists and breeders. Several types of resistance gene analogs (RGAs) such as NBS-LRR, RLK, RLP, and TM-CC play roles as pathogen invasion sensors or in the signal transduction pathways of hypersensitive response. Genome-wide RGA prediction facilitates resistance gene identification, gene cloning, and biological function verification. Because of significant structural features and conserved domains and motifs exist in various RGAs, computational approaches are effective for predicting and screening genome-wide RGAs. In this context, some bioinformatics pipelines for RGA prediction have been developed. This chapter reviews the recent progress of bioinformatics pipelines and their applications in flax resistance gene studies.
Plant immunity largely relies on intracellular NLR immune receptors. Some plant NLRs carry integrated domains (IDs) that mimic authentic pathogens effector targets. We report here the identification of a genetically linked NLR‐ID/NLR pair: BnRPR1 and BnRPR2 in Brassica napus. The NLR‐ID carries two ID fusions and the mode of action of the pair conforms to the proposed “integrated sensor/decoy” model. The two NLRs interact and the heterocomplex localizes in the plant‐cell nucleus and nucleolus. However, the BnRPRs pair does not operate through a negative regulation as it was previously reported for other NLR‐IDs. Cell death is induced only upon co‐expression of the two proteins and it is dependent on the helper genes EDS1 and NRG1. The nuclear localization of both proteins seems to be essential for cell death activation, while the IDs of BnRPR1 are dispensable for this purpose. In summary, we describe a new pair of NLR‐IDs with interesting features in relation to its regulation and the cell death activation.
In plant-pathogen interactions, signal activation and transduction confer resistance in plants against various pathogens. Communication between host and pathogen is the prime step for a pathogen to cause infection. The molecular basis of pathogen response in plants depends on the pathogen types. Hypersensitive reactions usually result from Avr-R interactions that restrict pathogens’ development through cell death. These avr genes can be recognized directly and indirectly by the resistance (R) gene. The NBS-LRR family is an important resistance gene (R gene) family in plants, which is divided into subclasses. Resistant gene analogues (RGAs) are candidates for R genes that have a significant role in defense response against disease-causing pathogens and are classified into two classes. The first class is based on the immediate recognition of a pathogen called resistance genes (R genes), while the second class is based on the defense response generated by recognition events. Hence, this chapter attempts to delineate a comprehensive overview of resistance genes, their classes, identification, and characterization in plants.KeywordsDisease resistanceR genesResistance genes analogsPlant-pathogen interaction
Fusarium wilt caused by Fusarium oxysporum f. sp. lentis (Fol) is the most devastating disease of lentil present worldwide and in India. Identification of multi-race fusarium wilt resistance genes and incorporation into existing cultivar will help to reduce yield loss. In the present study, a hundred lentil germplasm were screened against seven prevalent races of Fol and accession IC201561 , EC714243 and EC718238 were identified resistant. The typical R gene codes for the nucleotide-binding site and leucine-rich repeats (NBS-LRR) at the C terminal linked to either Toll/interleukin 1- like receptor (TIR) or coiled-coil (CC) at the N terminal. In the present study degenerate primers designed from the NBS region amplifying P-loop to GLPLA motif isolated forty-five resistance gene analogues (RGA) from identified resistant accessions. The sequence alignment identified both classes of RGA, TIR and non-TIR based on the presence of Aspartate (D) and Tryptophan (W) at the end of kinase motif respectively. The phylogenetic analysis grouped RGA into six classes, LRGA1 to LRGA6 determining the diversity of RGA present in the host. Grouping of RGA identified from Lens nigricans, LnRGA 2, 9, 13 with I2 reveals a probable role in Fusarium resistance. The similarity index of 27.85% to 86.98% was found among RGA and 26.83% to 49.41% between known R genes, I2, Gpa2, M and L6. Active binding sites present along the conserved motifs have grouped the RGA into 13 groups. ADP/ATP being the potential ligand determines ATP binding and ATP hydrolysis activity of RGA. The isolated RGA can be used in developing marker linked to the functional R gene. Further, expression analysis and full-length gene isolation further pave path to identifying the molecular mechanism involved in resistance.
Banana is one of the major food crops and its production is subject to many pests and diseases. Banana breeding exploits wild relatives and progenitor species for the introgression of resistant genes (R) into cultivated varieties to overcome these hurdles. With advances in sequencing technologies, whole-genome sequences are available for many Musa spp. and many of them are potential donors of disease resistance genes. Considering their potential role, R genes from these species were explored to develop an user-friendly open-access database that will be useful for studying and implementing disease resistance in bananas. MusaRgene database is complemented with complete details of 3598 R genes identified from eight Musa spp. and rice, Arabidopsis, sorghum along with its classification and separate modules on its expression under various stresses in resistant and susceptible cultivars and corresponding SSRs are also provided. This database can be regarded as the primary resource of information on R genes from bananas and their relatives. R genes from other allele mining studies are also incorporated which will enable the identification of its homolog in related Musa spp. MusaRgene database will aid in the identification of genes and markers associated, cloning of full-length R genes, and genetic transformation or gene editing of the R genes in susceptible cultivars. Multiple R genes can also be identified for pyramiding the genes to increase the level of resistance and durability. Overall, this database will facilitate the understanding of defense mechanisms in bananas against biotic or abiotic stresses leading to the development of promising disease-resistant varieties.
NBS domain-containing sequences from the Agave tequilana transcriptome shotgun assembly were identified, characterized, and classified based on their physicochemical properties and motif structure, which resulted in a differential response to Lasiodiplodia sp. infection. Agave tequilana is an important crop in Mexico that is susceptible to many pathogens and adverse conditions. In plants, NBS-LRR genes are involved in the physiological response to pathogenic infection. Forty-six partial NBS-LRR sequences were identified in the transcriptome shotgun assembly and were classified into five subclasses (CNL, CN, NL, N, and L) belonging to the non-TIR class in the NBS-LRR family. The identified sequences encode functional NBS-LRR proteins based on physicochemical properties, gene structure and motif analysis, functional annotation, and gene ontology. Phylogenetic analysis showed that these genes were clustered into seven groups (Groups I-VII). These groups were under diversifying selection pressure (Ka/Ks rates < 1) except for Group V (Ka/Ks rate = 1.23) which formed more recently (9 Mya). Specific primers designed for Groups I, II and V showed that the expression response to pathogenic Lasiodiplodia strains varied among the different NBS-LRR gene groups. The highest NBS-LRR gene transcript induction was obtained at 48 h, and the expression peaks were preceded by an increase in the concentration of endogenous salicylic acid, which has been associated with the activation of some NBS-LRR genes, suggesting that each group may have a specific defence response function.
Toll-like receptors (TLRs) are an important class of molecules involved in non-specific immunity, and they are also the bridge connecting between non-specific immunity and specific immunity. As a vital member of TLR family TLR9 can be activated by bacterial DNA and induce the production of inflammatory cytokines. In this study, a full length of TLR9 homologue of 3677 bp in Nibea albiflora (named as NaTLR9, GenBank accession no: MN125017.1) was characterized, and its ORF was 3180 bp encoding 1059 amino acid residues with a calculated molecular weight of 121.334 kDa (pI = 6.29). Several leucine-rich repeated sequences (LRR domain) and conservative TIR domain were found in NaTLR9, which was mainly expressed in dendritic cells and macrophages. The phylogenetic and synteny analysis further revealed high sequence identity of NaTLR9 with its counterparts of other teleost, confirming their correct nomenclature and conservative during evolution as an important pattern recognition receptor. The NaTLR9-TIR-pEGFP-N1 fusion protein showed green fluorescence and mainly distributed in the cytoplasm. After co-transfection of NaTLR9-TIR-pEGFP-N1 and NaMyD88-pDsRED-Monomer-N1, green fluorescence obviously overlapped with red and changed into yellowish-green, which suggested that there might be the interaction between homologous NaTLR9-TIR and MyD88. Based on this result the pCDNA3.1-NaTLR9-TIR-flag and pcMV-NaMyD88-TIR-Myc plasmids were co-transfected into 293T cells for the immunoprecipitation test. According to Western blot, TLR9 and MyD88 protein could interact with each other. Furthermore, NaTLR9 was ubiquitously expressed in all the investigated tissues, most abundantly in head kidney, followed by stomach, spleen, liver and gill, but lower in muscle. The vitro immune stimulation experiments revealed that Pseudomonas plecoglossicida and polyinosinic-polycytidylic acid [Poly (I:C)] induced higher levels of NaTLR9 mRNA expression with the peaks of 9.52 times at 2 h and 39.91 times at 24 h compared with the control group respectively. The functional domains (LRRs and TIR, named NaTLR9-TIR and NaTLR9-LRR respectively) of NaTLR9 were expressed and purified, the recombinant proteins both could bind three kinds of typical aquatic pathogenic bacteria (Vibrio. parahaemolyticus, Vibrio alginolyticus, and Vibrio harveyi), which showed that NaTLR9 could couple to bacteria by its function domains. The aforementioned results indicated that NaTLR9 played a significant role in the defense against pathogenic bacteria infection in innate immune response of sciaenidae fish, which may provide some further understandings of the regulatory mechanisms in the teleostean innate immune system.
Aegilops tauschii is the donor of the D subgenome of hexaploid wheat and an important genetic resource. The reference-quality genome sequence Aet v4.0 for Ae. tauschii acc. AL8/78 was therefore an important milestone for wheat biology and breeding. Further advances in sequencing acc. AL8/78 and release of the Aet v5.0 sequence assembly are reported here. Two new optical maps were constructed and used in the revision of pseudomolecules. Gaps were closed with Pacific Biosciences long-read contigs, decreasing the gap number by 38,899. Transposable elements and protein-coding genes were reannotated. The number of annotated high-confidence genes was reduced from 39,635 in Aet v4.0 to 32,885 in Aet v5.0. A total of 2,245 biologically important genes, including those affecting plant phenology, grain quality, and tolerance of abiotic stresses in wheat was manually annotated and disease-resistance genes were annotated by a dedicated pipeline. Disease-resistance genes encoding nucleotide-binding site domains, receptor-like protein kinases, and receptor-like proteins were preferentially located in distal chromosome regions, whereas those encoding transmembrane coiled-coil proteins were dispersed more evenly along the chromosomes. Discovery, annotation, and expression analyses of microRNA (miRNA) precursors, mature miRNAs, and phasiRNAs are reported, including miRNA target genes. Other small RNAs, such as hc-siRNAs and tRFs, were characterized. These advances enhance the utility of the Ae. tauschii genome sequence for wheat genetics, biotechnology, and breeding.
Nucleotide-binding site-leucine-rich repeat receptor (NLR) genes comprise the largest family of plant disease resistance genes. NLR genes are phylogenetically divided into the TNL, CNL, and RNL subclasses. NLR copy numbers and subclass composition vary tremendously across angiosperm genomes. However, the evolutionary associations between genomic NLR content and plant lifestyle, or between NLR content and signal transduction components, are poorly characterized due to limited genome availability. Here, we established an angiosperm NLR atlas (ANNA, http://compbio.nju.edu.cn/app/ANNA/), which includes NLR genes from over 300 angiosperm genomes. Using ANNA, we revealed that NLR copy numbers differ up to 66-fold among closely related species due to rapid gene loss and gain. Interestingly, NLR contraction was associated with adaptations to aquatic, parasitic, and carnivorous lifestyles. The convergent NLR reduction in aquatic plants resembles the long-term evolutionary silence of NLR genes in green algae before the colonization of land. A co-evolutionary pattern between NLR subclasses and plant immune-pathway components was also identified, suggesting that immune pathway deficiencies may drive TNL loss. Finally, we recovered a conserved TNL lineage that may function independently of the RNL pathway. Our findings provide new insights into the evolution of NLR genes in the context of plant lifestyles and genome content variation.
The advent of rapid and inexpensive DNA sequencing has led to an explosion of data that must be transformed into knowledge about genome organization and function. Gene prediction is customarily the starting point for genome analysis. This paper presents a bioinformatics study of the oil palm genome, including a comparative genomics analysis, database and tools development, and mining of biological data for genes of interest. We annotated 26,087 oil palm genes integrated from two gene-prediction pipelines, Fgenesh++ and Seqping. As case studies, we conducted comprehensive investigations on intronless, resistance and fatty acid biosynthesis genes, and demonstrated that the current gene prediction set is of high quality. 3,672 intronless genes were identified in the oil palm genome, an important resource for evolutionary study. Further scrutiny of the oil palm genes revealed 210 candidate resistance genes involved in pathogen defense. Fatty acids have diverse applications ranging from food to industrial feedstock, and we identified 42 key genes involved in fatty-acid biosynthesis in oil palm mesocarp and kernel. These results provide an important resource for studies on plant genomes and a theoretical foundation for marker-assisted breeding of oil palm and related crops.
As the largest family among the plant resistance (R) proteins, the nucleotide-binding site-leucine-rich repeat (NBS-LRR) proteins play significant roles in the defense of pathogens. The completion and improvement of banana reference sequence make a systematic insight into the banana NBS-LRR protein family possible. In this study, a total of 98 NBS-LRR proteins were identified from the banana genome and clustered into eight classes in the phylogenetic tree. NBS-LRR genes were unevenly distributed on all 11 chromosomes of banana. Typical NB-ARC (nucleotide binding-APAF-1, disease resistance proteins, CED-4) and LRR motifs were found in all NBS-LRR proteins and a small number of introns existed in most NBS-LRR genes. Transcriptional profiles of NBS-LRRs were investigated in Fusarium oxysporum f. sp. cubense (Foc) susceptible and resistant banana cultivars BX and HDJ challenged with Foc race 1 (Foc 1) and tropical race 4 (Foc TR4). Among 31 representative NBS-LRRs detected in this study, 12 members showed stronger transcriptional stimulation in HDJ than in BX, while in contrast, eight members exhibited higher expression in BX than in HDJ. This study advances our understanding of roles of banana NBS-LRRs in the defense of Fusarium wilt and will contribute to the genetic improvement of banana resistance.
Genetics of plant disease, as explained by Flor, is based on the hypothesis that resistance genes confer resistance against pathogens that express matching avirulence genes. Avirulence genes of the pathogen encode a protein product that is conditionally recognized directly or indirectly only by those plants that contain the complementary resistance gene. At the gene level, plant resistance to pathogens can be divided into qualitative and quantitative disease resistance, conditioned by major gene(s) and multiple genes with minor effects, respectively. The genomic approaches to resistance gene initially started with genomic sequencing of Arabidopsis and rice. Genes containing a nucleotide-binding site (NBS) constitute around 80% of the plant resistance gene families. Monocot plants possess non-TIR type while both TIR and non-TIR types of NBS genes are the characteristics of resistance gene in dicots. A set of NBS-encoding genes at the genome level has been identified in more than 30 angiosperms. The number of identified NBS-encoding genes ranges from 200 in Arabidopsis thaliana to 581 in Oryza sativa. In Brassica, the whole-genome gene expression and methylation studies have been proved to be very useful in the direction of uncovering the structure, function, and evolutionary origin of resistance genes. A number of NBS-encoding genes have been identified and sequenced in Brassica species for various important diseases, which will provide a base for candidate gene approach for cloning the quantitative trait loci for disease resistance. A genome-wide comparison of putatively functional NBS-encoding genes in B. napus, B. rapa, and B. oleracea identified 464, 202, and 146 putatively functional NBS-encoding genes, respectively, with genes unevenly distributed in several clusters. As our knowledge about the molecular mechanisms of the plant immunity signaling pathway and the role of resistance genes will amplify, we will gain more effective and durable strategies to overcome pathogen attack.
Hairiness, which is a phenotypic trait common among land plants, primarily affects the stem, leaf, and floral organs. Plant hairiness is associated with complex functions. For example, glume hairiness in wheat is related to the resistance to biotic and abiotic stresses, and may also influence human health. In the present study, two pairs of near-isogenic lines (NILs) for glume hairiness, which were derived from a cross between a Tibetan semi-wild wheat accession (Triticum aestivum ssp. tibetanum Q1028) and a common wheat cultivar (T. aestivum ‘Zhengmai 9023’), underwent a glume transcriptome analysis. We detected 27,935 novel genes, of which 18,027 were annotated. Additionally, 488 and 600 differentially expressed genes (DEGs) were detected in NIL1 and NIL2, respectively, with 37 DEGs detected in both NIL pairs. Moreover, 987 and 1,584 single nucleotide polymorphisms (SNPs) were detected in NIL1 and NIL2, respectively, with 39 SNPs detected in both NIL pairs, of which most were located in the Hairy glume (Hg) gene region on chromosome arm 1AS. The annotation of the DEGs with gene ontology terms revealed that genes associated with hairiness in Arabidopsis and rice were similarly enriched. The possible functions of these genes related to glume hairiness were examined. The study results provide useful information for identifying candidate genes and the fine-mapping of Hg in the wheat genome.
The tomato Cf-2 and Cf-5 genes confer resistance to Cladosporium fulvum and map to a complex locus on chromosome 6. The Cf-5 gene has been isolated and is predicted to encode a largely extracytoplasmic protein containing 32 leucine-rich repeats (LRRs), resembling the previously isolated Cf-2 gene, which has 38 LRRs. Three haplotypes of this locus from Lycopersicon esculentum, L. pimpinellifolium, and L. esculentum var cerasiforme were compared, and five additional homologs of Cf-5 were sequenced. All share extensive sequence identity, particularly within the C-terminal portions of the predicted proteins. In striking contrast to the Cf-9 gene family, six of seven homologs in the Cf-2/Cf-5 gene family vary in LRR copy number, ranging from 25 to 38 LRRs. Cf-5 and one adjacent homolog differ by only two LRRs. Recombination events that vary the LRR copy number in this region could provide a mechanism for the generation of new specificities for recognition of different ligands. A recombination breakpoint between the Cf-2 and Cf-5 loci was fully characterized and shown to be intragenic.
Disease resistance in plants is a desirable economic trait. A number of disease resistance genes from various plant species have been cloned so far. The gene products of some of these can be distinguished by the presence of an N-terminal nucleotide binding site and a C-terminal stretch of leucine-rich repeats. Although these gene products are structurally related, the DNA sequences are poorly conserved. Only parts of the nucleotide binding site share enough DNA identity to design primers for polymerase chain reaction amplification of related DNA sequences. Such primers were used to amplify different resistance-gene-like (RGL) DNA fragments from Arabidopsis thaliana accessions Landsberg erecta and Columbia. Almost all cloned DNA fragments were genetically closely linked with known disease resistance loci. Most RGL fragments were found in a clustered or dispersed multi-copy sequence organization, supporting the supposed correlation of RGL sequences and disease resistance loci.
The rice Xa21 gene, which confers resistance to Xanthomonas oryzae pv. oryzae race 6, was isolated by positional cloning. Fifty transgenic rice plants carrying the cloned Xa21 gene display high levels of resistance to the pathogen. The sequence of the predicted protein, which carries both a leucine-rich
repeat motif and a serine-threonine kinase-like domain, suggests a role in cell surface recognition of a pathogen ligand and
subsequent activation of an intracellular defense response. Characterization of Xa21 should facilitate understanding of plant disease resistance and lead to engineered resistance in rice.
Plants can recognize pathogens through the action of disease resistance (R) genes, which confer resistance to pathogens expressing
unique corresponding avirulence (avr) genes. The molecular basis of this gene-for-gene specificity is unknown. The Arabidopsis
thaliana RPM1 gene enables dual specificity to pathogens expressing either of two unrelated Pseudomonas syringae avr genes.
Despite this function, RPM1 encodes a protein sharing molecular features with recently described single-specificity R genes.
Surprisingly, RPM1 is lacking from naturally occurring, disease-susceptible Arabidopsis accessions.
The Pto gene in tomato confers resistance to races of Pseudomonas syringae pv. tomato that carry the avirulence gene avrPto. A yeast artificial chromosome clone that spans the Pto region was identified and used to probe a leaf complementary DNA (cDNA) library. A cDNA clone was isolated that represents a gene family, at least six members of which genetically cosegregate with Pto. When susceptible tomato plants were transformed with a cDNA from this family, they were resistant to the pathogen. Analysis of the amino acid sequence revealed similarity to serine-threonine protein kinases, suggesting a role for Pto in a signal transduction pathway.
The products of plant disease resistance genes are postulated to recognize invading pathogens and rapidly trigger host defense responses. Here we describe isolation of the resistance gene N of tobacco that mediates resistance to the viral pathogen tobacco mosaic virus (TMV). The N gene was isolated by transposon tagging using the maize Activator transposon. A genomic DNA fragment containing the N gene conferred TMV resistance to TMV susceptible tobacco. Sequence analysis of the N gene shows that it encodes a protein of 131.4 kDa with an amino-terminal domain similar to that of the cytoplasmic domain of the Drosophila Toll protein and the interleukin-1 receptor (IL-1R) in mammals, a nucleotide-binding site (NBS), and 14 [corrected] imperfect leucine-rich repeats (LRR). The sequence similarity of N, Toll, and IL-1R suggests that N mediates rapid gene induction and TMV resistance through a Toll-IL-1-like pathway.
The tomato Cf-9 gene confers resistance to infection by races of the fungus Cladosporium fulvum that carry the avirulence
gene Avr9. The Cf-9 gene was isolated by transposon tagging with the maize transposable element Dissociation. The DNA sequence
of Cf-9 encodes a putative membrane-anchored extracytoplasmic glycoprotein. The predicted protein shows homology to the receptor
domain of several receptor-like protein kinases in Arabidopsis, to antifungal polygalacturonase-inhibiting proteins in plants,
and to other members of the leucine-rich repeat family of proteins. This structure is consistent with that of a receptor that
could bind Avr9 peptide and activate plant defense.
In tomato, resistance to Pseudomonas syringae pv. tomato (Pst) strains expressing the avirulence gene avrPto requires the presence of at least two host genes, designated Pto and Prf. Here we report that Prf encodes a protein with leucine-zipper, nucleotide-binding, and leucine-rich repeat motifs, as are found in a number of resistance gene products from other plants. prf mutant alleles (4) were found to carry alterations within the Prf coding sequence. A genomic fragment containing Prf complemented a prf mutant tomato line both for resistance to Pst strains expressing avrPto and for sensitivity to the insecticide Fenthion. Prf resides in the middle of the Pto gene cluster, 24 kb from the Pto gene and 500 bp from the Fen gene.
The tobacco N and Arabidopsis RPS2 genes, among several recently cloned disease-resistance genes, share highly conserved structure, a nucleotide-binding site (NBS). Using degenerate oligonucleotide primers for the NBS region of N and RPS2, we have amplified and cloned the NBS sequences from soybean. Each of these PCR-derived NBS clones detected low-or moderate-copy soybean DNA sequences and belongs to 1 of 11 different classes. Sequence analysis showed that all PCR clones encode three motifs (P-loop, kinase-2, and kinase-3a) of NBS nearly identical to those in N and RPS2. The intervening region between P-loop and kinase-3a of the 11 classes has high (26% average) amino acid sequence similarity to the N gene although not as high (19% average) to RPS2. These 11 classes represent a superfamily of NBS-containing soybean genes that are homologous to N and RPS2. Each class or subfamily was assessed for its positional association with known soybean disease-resistance genes through near-isogenic line assays, followed by linkage analysis in F2 populations using restriction fragment length polymorphisms. Five of the 11 subfamilies have thus far been mapped to the vicinity of known soybean genes for resistance to potyviruses (Rsv1 and Rpv), Phytophthora root rot (Rps1, Rps2, and Rps3), and powdery mildew (rmd). The conserved N- or RPS2-homologous NBS sequences and their positional associations with mapped soybean-resistance genes suggest that a number of the soybean disease-resistance genes may belong to this superfamily. The candidate subfamilies of NBS-containing genes identified by genetic mapping should greatly facilitate the molecular cloning of disease-resistance genes.
Sequences of cloned resistance genes from a wide range of plant taxa reveal significant similarities in sequence homology and structural motifs. This is observed among genes conferring resistance to viral, bacterial, and fungal pathogens. In this study, oligonucleotide primers designed for conserved sequences from coding regions of disease resistance genes N (tobacco), RPS2 (Arabidopsis) and L6 (flax) were used to amplify related sequences from soybean [Glycine max (L.) Merr.]. Sequencing of amplification products indicated that at least nine classes of resistance gene analogs (RGAs) were detected. Genetic mapping of members of these classes located them to eight different linkage groups. Several RGA loci mapped near known resistance genes. A bacterial artificial chromosome library of soybean DNA was screened using primers and probes specific for eight RGA classes and clones were identified containing sequences unique to seven classes. Individual bacterial artificial chromosomes contained 2-10 members of single RGA classes. Clustering and sequence similarity of members of RGA classes suggests a common process in their evolution. Our data indicate that it may be possible to use sequence homologies from conserved motifs of cloned resistance genes to identify candidate resistance loci from widely diverse plant taxa.
Analysis of viral and bacterial pathogenesis has revealed common themes in the ways in which plants and animals respond to pathogenic agents. Pathogenic bacteria use macromolecule delivery systems (types III and IV) to deliver microbial avirulence proteins and transfer DNA-protein complexes directly into plant cells. The molecular events that constitute critical steps of plant-pathogen interactions seem to involve ligand-receptor mechanisms for pathogen recognition and the induction of signal transduction pathways in the plant that lead to defense responses. Unraveling the molecular basis of disease resistance pathways has laid a foundation for the rational design of crop protection strategies.
We introduce a general probabilistic model of the gene structure of human genomic sequences which incorporates descriptions of the basic transcriptional, translational and splicing signals, as well as length distributions and compositional features of exons, introns and intergenic regions. Distinct sets of model parameters are derived to account for the many substantial differences in gene density and structure observed in distinct C + G compositional regions of the human genome. In addition, new models of the donor and acceptor splice signals are described which capture potentially important dependencies between signal positions. The model is applied to the problem of gene identification in a computer program, GENSCAN, which identifies complete exon/intron structures of genes in genomic DNA. Novel features of the program include the capacity to predict multiple genes in a sequence, to deal with partial as well as complete genes, and to predict consistent sets of genes occurring on either or both DNA strands. GENSCAN is shown to have substantially higher accuracy than existing methods when tested on standardized sets of human and vertebrate genes, with 75 to 80% of exons identified exactly. The program is also capable of indicating fairly accurately the reliability of each predicted exon. Consistently high levels of accuracy are observed for sequences of differing C + G content and for distinct groups of vertebrates.
The BLAST programs are widely used tools for searching protein and DNA databases for sequence similarities. For protein comparisons,
a variety of definitional, algorithmic and statistical refinements described here permits the execution time of the BLAST
programs to be decreased substantially while enhancing their sensitivity to weak similarities. A new criterion for triggering
the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program
that runs at approximately three times the speed of the original. In addition, a method is introduced for automatically combining
statistically significant alignments produced by BLAST into a position-specific score matrix, and searching the database using
this matrix. The resulting Position-Specific Iterated BLAST (PSIBLAST) program runs at approximately the same speed per iteration
as gapped BLAST, but in many cases is much more sensitive to weak but biologically relevant sequence similarities. PSI-BLAST
is used to uncover several new and interesting members of the BRCT superfamily.
The discovery of sequence homology between the cytoplasmic domains of Drosophila Toll and human interleukin 1 receptors has sown the conviction that both molecules trigger related signaling pathways tied to the nuclear translocation of Rel-type transcription factors. This conserved signaling scheme governs an evolutionarily ancient immune response in both insects and vertebrates. We report the molecular cloning of a class of putative human receptors with a protein architecture that is similar to Drosophila Toll in both intra- and extracellular segments. Five human Toll-like receptors--named TLRs 1-5--are probably the direct homologs of the fly molecule and, as such, could constitute an important and unrecognized component of innate immunity in humans. Intriguingly, the evolutionary retention of TLRs in vertebrates may indicate another role--akin to Toll in the dorsoventralization of the Drosophila embryo--as regulators of early morphogenetic patterning. Multiple tissue mRNA blots indicate markedly different patterns of expression for the human TLRs. By using fluorescence in situ hybridization and sequence-tagged site database analyses, we also show that the cognate Tlr genes reside on chromosomes 4 (TLRs 1, 2, and 3), 9 (TLR4), and 1 (TLR5). Structure prediction of the aligned Toll-homology domains from varied insect and human TLRs, vertebrate interleukin 1 receptors and MyD88 factors, and plant disease-resistance proteins recognizes a parallel beta/alpha fold with an acidic active site; a similar structure notably recurs in a class of response regulators broadly involved in transducing sensory information in bacteria.
The Xa1 gene in rice confers resistance to Japanese race 1 of Xanthomonas oryzae pv. oryzae, the causal pathogen of bacterial blight (BB). We isolated the Xa1 gene by a map-based cloning strategy. The deduced amino acid sequence of the Xa1 gene product contains nucleotide binding sites (NBS) and a new type of leucine-rich repeats (LRR); thus, Xa1 is a member of the NBS-LRR class of plant disease-resistance genes, but quite different from Xa21, another BB-resistance gene isolated from rice. Interestingly, Xa1 gene expression was induced on inoculation with a bacterial pathogen and wound, unlike other isolated resistance genes in plants, which show constitutive expression. The induced expression may be involved in enhancement of resistance against the pathogen.
The Arabidopsis genes EDS1 and NDR1 were shown previously by mutational analysis to encode essential components of race-specific disease resistance. Here, we examined the relative requirements for EDS1 and NDR1 by a broad spectrum of Resistance (R) genes present in three Arabidopsis accessions (Columbia, Landsberg-erecta, and Wassilewskija). We show that there is a strong requirement for EDS1 by a subset of R loci (RPP2, RPP4, RPP5, RPP21, and RPS4), conferring resistance to the biotrophic oomycete Peronospora parasitica, and to Pseudomonas bacteria expressing the avirulence gene avrRps4. The requirement for NDR1 by these EDS1-dependent R loci is either weak or not measurable. Conversely, three NDR1-dependent R loci, RPS2, RPM1, and RPS5, operate independently of EDS1. Another RPP locus, RPP8, exhibits no strong exclusive requirement for EDS1 or NDR1 in isolate-specific resistance to P. parasitica, although resistance is compromised weakly by eds1. Similarly, resistance conditioned by two EDS1-dependent RPP genes, RPP4 and RPP5, is impaired partially by ndr1, implicating a degree of pathway cross-talk. Our results provide compelling evidence for the preferential utilization of either signaling component by particular R genes and thus define at least two disease resistance pathways. The data also suggest that strong dependence on EDS1 or NDR1 is governed by R protein structural type rather than pathogen class.
The Mi locus of tomato confers resistance to root knot nematodes. Tomato DNA spanning the locus was isolated as bacterial artificial chromosome clones, and 52 kb of contiguous DNA was sequenced. Three open reading frames were identified with similarity to cloned plant disease resistance genes. Two of them, Mi-1.1 and Mi-1.2, appear to be intact genes; the third is a pseudogene. A 4-kb mRNA hybridizing with these genes is present in tomato roots. Complementation studies using cloned copies of Mi-1.1 and Mi-1.2 indicated that Mi-1.2, but not Mi-1.1, is sufficient to confer resistance to a susceptible tomato line with the progeny of transformants segregating for resistance. The cloned gene most similar to Mi-1.2 is Prf, a tomato gene required for resistance to Pseudomonas syringae. Prf and Mi-1.2 share several structural motifs, including a nucleotide binding site and a leucine-rich repeat region, that are characteristic of a family of plant proteins, including several that are required for resistance against viruses, bacteria, fungi, and now, nematodes.
Arabidopsis COP1 acts as a repressor of photomorphogenesis in darkness, and light stimuli abrogate the repressive ability and nuclear abundance of COP1. COP1 has three known structural modules: an N-terminal RING-finger, followed by a predicted coiled-coil and C-terminal WD-40 repeats. A systematic study was undertaken to dissect the functional roles of these three COP1 domains in light control of Arabidopsis seedling development. Our data suggest that COP1 acts primarily as a homodimer, and probably dimerizes through the coiled-coil domain. The RING-finger and the coiled-coil domains can function independently as light-responsive modules mediating the light-controlled nucleocytoplasmic partitioning of COP1. The C-terminal WD-40 domain functions as an autonomous repressor module since the overexpression of COP1 mutant proteins with intact WD-40 repeats are able to suppress photomorphogenic development. This WD-40 domain-mediated repression can be at least in part accounted for by COP1's direct interaction with and negative regulation of HY5, a bZIP transcription factor that positively regulates photomorphogenesis. However, COP1 self-association is a prerequisite for the observed interaction of the COP1 WD-40 repeats with HY5. This work thus provides a structural basis of COP1 as a molecular switch.
In "gene-for-gene" interactions between plants and their pathogens, incompatibility (no disease) requires a dominant or semidominant resistance (R) gene in the plant, and a corresponding avirulence (Avr) gene in the pathogen. Many plant/pathogen interactions are of this type. R genes are presumed to (a) enable plants to detect Avr-gene-specified pathogen molecules, (b) initiate signal transduction to activate defenses, and (c) have the capacity to evolve new R gene specificities rapidly. Isolation of R genes has revealed four main classes of R gene sequences whose products appear to activate a similar range of defense mechanisms. Discovery of the structure of R genes and R gene loci provides insight into R gene function and evolution, and should lead to novel strategies for disease control.
The hypersensitive response, or HR, is a form of cell death often associated with plant resistance to pathogen infection. Reactive oxygen intermediates and ion fluxes are proximal responses probably required for the HR. Apoptosis as defined in animal systems is, thus far, not a strict paradigm for the HR. The diversity observed in plant cell death morphologies suggests that there may be multiple pathways through which the HR can be triggered. Signals from pathogens appear to interfere with these pathways. HR may play in plants the same role as certain programmed cell deaths in animals with respect to restricting pathogen growth. In addition, the HR could regulate the defense responses of the plant in both local and distant tissues.
Pathogen resistance ( R ) genes of the NBS-LRR class (for nucleotide binding site and leucine-rich repeat) are found in many plant species and confer resistance to a diverse spectrum of pathogens. Little is known about the mechanisms that drive NBS-LRR gene evolution in the host‐pathogen arms race. We cloned the RPP8 gene (for resistance to Peronospora parasitica ) and compared the structure of alleles at this locus in resistant Landsberg erecta (L er -0) and susceptible Columbia (Col-0) accessions. RPP8-L er encodes an NBS-LRR protein with a putative N-terminal leucine zipper and is more closely related to previously cloned R genes that confer resistance to bacterial pathogens than it is to other known RPP genes. The RPP8 haplotype in L er -0 contains the functional RPP8-L er gene and a nonfunctional homolog, RPH8A. In contrast, the rpp8 locus in Col-0 contains a single chimeric gene, which was likely derived from unequal crossing over between RPP8-L er and RPH8A ancestors within a L er -like haplotype. Sequence divergence among RPP8 family members has been accelerated by positive selection on the putative ligand binding region in the LRRs. These observations indicate that NBS-LRR molecular evolution is driven by the same mechanisms that promote rapid sequence diversification among other genes involved in non-self-recognition.
Recognition of pathogens by plants is mediated by several distinct families of functionally variable but structurally related disease resistance (R) genes. The largest family is defined by the presence of a putative nucleotide binding domain and 12 to 21 leucine-rich repeats (LRRs). The function of these LRRs has not been defined, but they are speculated to bind pathogen-derived ligands. We have isolated a mutation in the Arabidopsis RPS5 gene that indicates that the LRR region may interact with other plant proteins. The rps5-1 mutation causes a glutamate-to-lysine substitution in the third LRR and partially compromises the function of several R genes that confer bacterial and downy mildew resistance. The third LRR is relatively well conserved, and we speculate that it may interact with a signal transduction component shared by multiple R gene pathways.
In many interactions between plants and their pathogens, resistance to infection is specified by plant resistance (R) genes and corresponding pathogen avirulence (Avr) genes. In tomato, the Cf-4 and Cf-9 resistance genes map to the same location but confer resistance to Cladosporium fulvum through recognition of different avirulence determinants (AVR4 and AVR9) by a molecular mechanism that has yet to be determined. Here, we describe the cloning and characterization of Cf-4, which also encodes a membrane-anchored extracellular glycoprotein. Cf-4 contains 25 leucine-rich repeats, which is two fewer than Cf-9. The proteins have >91% identical amino acids. DNA sequence comparison suggests that Cf-4 and cf-9 are derived from a common progenitor sequence. Amino acid differences distinguishing Cf-4 and Cf-9 are confined to their N termini, delimiting a region that determines the recognitional specificity of ligand binding. The majority of these differences are in residues interstitial to those of the leucine-rich repeat consensus motif. Many of these residues are predicted to form a solvent-exposed surface that can interact with the cognate ligand. Both Cf-4 and Cf-9 are located within a 36-kb region comprising five tandemly duplicated homologous genes. These results provide further insight into the molecular basis of pathogen perception by plants and the organization of complex R gene loci.
Plant disease resistance genes operate at the earliest steps of pathogen perception. The Arabidopsis RPP5 gene specifying resistance to the downy mildew pathogen Peronospora parasitica was positionally cloned. It encodes a protein that possesses a putative nucleotide binding site and leucine-rich repeats, and its product exhibits striking structural similarity to the plant resistance gene products N and L6. Like N and L6, the RPP5 N-terminal domain resembles the cytoplasmic domains of the Drosophila Toll and mammalian interleukin-1 transmembrane receptors. In contrast to N and L6, which produce predicted truncated products by alternative splicing, RPP5 appears to express only a single transcript corresponding to the full-length protein. However, a truncated form structurally similar to those of N and L6 is encoded by one or more other members of the RPP5 gene family that are tightly clustered on chromosome 4. The organization of repeated units within the leucine-rich repeats encoded by the wild-type RPP5 gene and an RPP5 mutant allele provides molecular evidence for the heightened capacity of this domain to evolve novel configurations and potentially new disease resistance specificities.
The major diversification of flowering plants (angiosperms) in the Early
Cretaceous, between about 130 and 90 million years ago, initiated
fundamental changes in terrestrial ecosystems and set in motion
processes that generated most of the extant plant diversity. New
palaeobotanical discoveries, combined with recent phylogenetic analyses
of morphological and molecular data, have clarified the initial phases
of this radiation and changed our perspective on early angiosperm
evolution, though important issues remain unresolved.
In plants, resistance to pathogens is frequently determined by dominant resistance genes, whose products are proposed to recognize pathogen-encoded avirulence gene (Avr) products. The tomato resistance locus Cf-2 was isolated by positional cloning and found to contain two almost identical genes, each conferring resistance to isolates of tomato leaf mould (C. fulvum) expressing the corresponding Avr2 gene. The two Cf-2 genes encode protein products that differ from each other by only three amino acids and contain 38 leucine-rich repeat (LRR) motifs. Of the LRRs, 20 show extremely conserved alternating repeats. The C-terminus of Cf-2 carries regions of pronounced homology to the protein encoded by the unlinked Cf-9 gene. We suggest that this conserved region interacts with other proteins involved in activating plant defense mechanisms.
Many ATP- and GTP-binding proteins have a phosphate-binding loop (P-loop), the primary structure of which typically consists of a glycine-rich sequence followed by a conserved lysine and a serine or threonine. The three-dimensional structures of several ATP- and GTP-binding proteins containing P-loops have now been solved. In this review current knowledge of P-loops is discussed with the additional aim of illustrating the fascinating relationship between protein sequence, structure and function.
The Toll gene of Drosophila, a maternal effect gene that plays a central role in the establishment of the embryonic dorsal-ventral pattern, has been cloned using P element tagging. A 5.3 kb poly(A)+ ovarian transcript from the cloned region was purified by hybrid selection with the cloned DNA. This purified transcript complements the Toll mutant phenotype when injected into Toll- embryos, which proves that it is the Toll transcript. The sequence of cDNAs suggests that the Toll protein is an integral membrane protein with a cytoplasmic domain and a large extracytoplasmic domain. The putative extracytoplasmic domain contains at least 15 repeats of a 24 amino acid, leucine-rich sequence found in both human and yeast membrane proteins.
Introduction to Computational Biology: Maps, Sequencesand Genomes. Chapman Hall, 1995.[WF74] R.A. Wagner and M.J. Fischer. The String to String Correction Problem. Journal of the ACM, 21(1):168--173, 1974.[WM92] S. Wu and U. Manber. Fast Text Searching Allowing Errors. Communicationsof the ACM, 10(35):83--91, 1992.73Bibliography[KOS+00] S. Kurtz, E. Ohlebusch, J. Stoye, C. Schleiermacher, and R. Giegerich.Computation and Visualization of Degenerate Repeats in CompleteGenomes. In ...
The L6 rust resistance gene from flax was cloned after tagging with the maize transposable element Activator. The gene is predicted to encode two products of 1294 and 705 amino acids that result from alternatively spliced transcripts. The longer product is similar to the products of two other plant disease resistance genes, the tobacco mosaic virus resistance gene N of tobacco and the bacterial resistance gene RPS2 of Arabidopsis. The similarity involves the presence of a nucleotide (ATP/GTP) binding site and several other amino acid motifs of unknown function in the N-terminal half of the polypeptides and a leucine-rich region in the C-terminal half. The truncated product of L6, which lacks most of the leucine-rich C-terminal region, is similar to the truncated product that is predicted from an alternative transcript of the N gene. The L6, N, and RPS2 genes, which control resistance to three widely different pathogen types, are the foundation of a class of plant disease resistance genes that can be referred to as nucleotide binding site/leucine-rich repeat resistance genes.
In plants, resistance to a pathogen is frequently correlated with a genetically defined interaction between a plant resistance gene and a corresponding pathogen avirulence gene. A simple model explains these gene-for-gene interactions: avirulence gene products generate signals (ligands), and resistance genes encode cognate receptors. The A. thaliana RPS2 gene confers resistance to the bacterial pathogen P. syringae carrying the avirulence gene avrRpt2. A map-based positional cloning strategy was used to identify RPS2. The identification of RPS2 was verified using a newly developed transient assay for RPS2 function and by genetic complementation in transgenic plants. RPS2 encodes a novel 105 kDa protein containing a leucine zipper, a nucleotide-binding site, and 14 imperfect leucine-rich repeats.
Plant disease resistance genes function is highly specific pathogen recognition pathways. PRS2 is a resistance gene of Arabidopsis
thaliana that confers resistance against Pseudomonas syringae bacteria that express avirulence gene avrRpt2. RPS2 was isolated
by the use of a positional cloning strategy. The derived amino acid sequence of RPS2 contains leucine-rich repeat, membrane-spanning,
leucine zipper, and P loop domains. The function of the RPS2 gene product in defense signal transduction is postulated to
involve nucleotide triphosphate binding and protein-protein interactions and may also involve the reception of an elicitor
produced by the avirulent pathogen.
From an analysis of current data on 16 protein structures with defined nucleotide-binding sites consensus motifs were determined for the peptide segments that form such nucleotide-binding sites. This was done by using the actual residues shown to contact ligands in the different protein structures, plus an additional 50 sequences for various kinases. Three peptide segments are commonly required to form the binding site for ATP or GTP. Binding motif Kinase-1a is found in almost all sequences examined, and functions in binding the phosphates of the ligand. Variant versions, comparable to Kinase-1a, are found in a subset of proteins and appear to be related to unique functions of those enzymes. Motif Kinase-2 contains the conserved aspartate that coordinates the metal ion on Mg-ATP. Motif Kinase-3 occurs in at least four versions, and functions in binding the purine base or the pentose. Two protein structures show ATP-binding at a separate regulatory site, formed by the motifs Regulatory-1 and Regulatory-2. Structures for adenylate kinase and guanylate kinase show three different sequence motifs that form the binding site for a nucleoside monophosphate (NMP). NMP-1 and NMP-2 bind to the pentose and phosphate of the bound ligand. NMP-1 is found in almost all the kinases that phosphorylate AMP, CMP, GMP, dTMP, or UMP. NMP-3a is found in kinases for AMP, GMP, and UMP, while NMP-3b binds only GMP. For the binding of NTPs, three distinct types of nucleotide-binding fold structures have been described. Each structure is associated with a particular function (e.g. transfer of the gamma-phosphate, or of the adenylate to an acceptor) and also with a particular spatial arrangement of the three Kinase segments evident in the linear sequence for the protein.
Plant genes for pathogen resistance can be used to engineer disease resistant crops. Oligonucleotides were designed from sequence motifs conserved between resistance genes of tobacco and Arabidopsis thaliana and used as PCR primers in potato DNA. Amplification products were obtained that were homologous to known resistance genes and linked without recombination with the nematode resistance locus Gro1 and the Phytophthora infestans resistance locus R7 of potato. Map positions of PCR-derived potato gene fragments were also correlated with resistance loci of the related tomato and tobacco genomes. Our results indicate that plant resistance genes that are effective against nematodes, fungi, viruses and bacteria may be isolated based on common sequence motifs and PCR methodology.
SEA VIEW and PHYLO_WIN are two graphic tools for X Windows-Unix computers dedicated to sequence alignment and molecular phylogenetics. SEA VIEW is a sequence alignment editor allowing manual or automatic alignment through an interface with CLUSTALW program. Alignment of large sequences with extensive length differences is made easier by a dot-plot-based routine. The PHYLO_WIN program allows phylogenetic tree building according to most usual methods (neighbor joining with numerous distance estimates, maximum parsimony, maximum likelihood), and a bootstrap analysis with any of them. Reconstructed trees can be drawn, edited, printed, stored, evaluated according to numerous criteria. Taxonomic species groups and sets of conserved regions can be defined by mouse and stored into sequence files, thus avoiding multiple data files. Both tools are entirely mouse driven. On-line help makes them easy to use. They are freely available by anonymous ftp at biom3.univ-lyon1.fr/pub/mol_phylogeny or http: //acnuc.univ-lyon1.fr/, or by e-mail to [email protected]
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Characterization of plant resistance genes is an important step in understanding plant defense mechanisms. Fusarium oxysporum f sp lycopersici is the causal agent of a vascular wilt disease in tomato. Genes conferring resistance to plant vascular diseases have yet to be described molecularly. Members of a new multigene family, complex I2C, were isolated by map-based cloning from the I2 F. o. lycopersici race 2 resistance locus. The genes show structural similarity to the group of recently isolated resistance genes that contain a nucleotide binding motif and leucine-rich repeats. Importantly, the presence of I2C antisense transgenes abrogated race 2 but not race 1 resistance in otherwise normal plants. Expression of the complete sense I2C-1 transgene conferred significant but partial resistance to F. o. lycopersici race 2. All members of the I2C gene family have been mapped genetically and are dispersed on three different chromosomes. Some of the I2C members cosegregate with other tomato resistance loci. Comparison within the leucine-rich repeat region of I2C gene family members shows that they differ from each other mainly by insertions or deletions.
The past several years have seen significant advances in our ability to recognize coiled coils from protein sequences and model their structures. New methods include a detection program based on pairwise residue correlations, a program that distinguishes two-stranded from three-stranded coiled coils and a routine for modelling the coordinates of the core residues in coiled coils. Several widely noted predictions, among them those for heterotrimeric G proteins and for cartilage oligomeric matrix protein, have been confirmed by crystal structures, and several new predictions have been made, including a model for the still hypothetical right-handed coiled coil.
The Cre3 gene confers a high level of resistance to the root endoparasitic nematode Heterodera avenae in wheat. A DNA marker cosegregating with H. avenae resistance was used as an entry point for map-based cloning of a disease resistance gene family at the Cre3 locus. Two related gene sequences have been analysed at the Cre3 locus. One, identified as a cDNA clone, encodes a polypeptide with a nucleotide binding site (NBS) and a leucine-rich region; this member of the disease resistance gene family is expressed in roots. A second Cre3 gene sequence, cloned as genomic DNA, appears to be a pseudogene, with a frame shift caused by a deletion event. These two genes, related to members of the cytoplasmic NBS-leucine rich repeat class of plant disease resistance genes were physically mapped to the distal 0.06 fragment of the long arm of wheat chromosome 2D and cosegregated with nematode resistance.
We report here the purification of the third protein factor, Apaf-3, that participates in caspase-3 activation in vitro. Apaf-3 was identified as a member of the caspase family, caspase-9. Caspase-9 and Apaf-1 bind to each other via their respective NH2-terminal CED-3 homologous domains in the presence of cytochrome c and dATP, an event that leads to caspase-9 activation. Activated caspase-9 in turn cleaves and activates caspase-3. Depletion of caspase-9 from S-100 extracts diminished caspase-3 activation. Mutation of the active site of caspase-9 attenuated the activation of caspase-3 and cellular apoptotic response in vivo, indicating that caspase-9 is the most upstream member of the apoptotic protease cascade that is triggered by cytochrome c and dATP.
We used conserved domains in the major class (nucleotide binding site plus leucine-rich repeat) of dicot resistance (R) genes to isolate related gene fragments via PCR from the monocot species rice and barley. Peptide sequence comparison of dicot R genes and monocot R-like genes revealed shared motifs but provided no evidence for a monocot-specific signature. Mapping of these genes in rice and barley showed linkage to genetically characterized R genes and revealed the existence of mixed clusters, each harboring at least two highly dissimilar R-like genes. Diversity was detected intraspecifically with wide variation in copy number between varieties of a particular species. Interspecific analyses of R-like genes frequently revealed nonsyntenic map locations between the cereal species rice, barley, and foxtail millet although tight collinear gene order is a hallmark of monocot genomes. Our data suggest a dramatic rearrangement of R gene loci between related species and implies a different mechanism for nucleotide binding site plus leucine-rich repeat gene evolution compared with the rest of the monocot genome.
Genetic mapping of wheat, maize, and rice and other grass species with common DNA probes has revealed remarkable conservation of gene content and gene order over the 60 million years of radiation of Poaceae. The linear organization of genes in some nine different genomes differing in basic chromosome number from 5 to 12 and nuclear DNA amount from 400 to 6,000 Mb, can be described in terms of only 25 "rice linkage blocks." The extent to which this intergenomic colinearity is confounded at the micro level by gene duplication and micro-rearrangements is still an open question. Nevertheless, it is clear that the elucidation of the organization of the economically important grasses with larger genomes, such as maize (2n = 10, 4,500 Mb DNA), will, to a greater or lesser extent, be predicted from sequence analysis of smaller genomes such as rice, with only 400 Mb, which in turn may be greatly aided by knowledge of the entire sequence of Arabidopsis, which may be available as soon as the turn of the century. Comparative genetics will provide the key to unlock the genomic secrets of crop plants with bigger genomes than Homo sapiens.
The nucleotide binding site (NBS) is a characteristic domain of many plant resistance gene products. An increasing number of NBS-encoding sequences are being identified through gene cloning, PCR amplification with degenerate primers, and genome sequencing projects. The NBS domain was analyzed from 14 known plant resistance genes and more than 400 homologs, representing 26 genera of monocotyledonous, dicotyle-donous and one coniferous species. Two distinct groups of diverse sequences were identified, indicating divergence during evolution and an ancient origin for these sequences. One group was comprised of sequences encoding an N-terminal domain with Toll/Interleukin-1 receptor homology (TIR), including the known resistance genes, N, M, L6, RPP1 and RPP5. Surprisingly, this group was entirely absent from monocot species in searches of both random genomic sequences and large collections of ESTs. A second group contained monocot and dicot sequences, including the known resistance genes, RPS2, RPM1, I2, Mi, Dm3, Pi-B, Xa1, RPP8, RPS5 and Prf. Amino acid signatures in the conserved motifs comprising the NBS domain clearly distinguished these two groups. The Arabidopsis genome is estimated to contain approximately 200 genes that encode related NBS motifs; TIR sequences were more abundant and outnumber non-TIR sequences threefold. The Arabidopsis NBS sequences currently in the databases are located in approximately 21 genomic clusters and 14 isolated loci. NBS-encoding sequences may be more prevalent in rice. The wide distribution of these sequences in the plant kingdom and their prevalence in the Arabidopsis and rice genomes indicate that they are ancient, diverse and common in plants. Sequence inferences suggest that these genes encode a novel class of nucleotide-binding proteins.
Over the past five years, the structures of more than 20 proteins containing coiled-coil domains have been solved to high resolution. This has provided many new insights into the structure of coiled coils, their discontinuities, their relationship with other helical bundles and the problems connected with their prediction from protein sequences.
Different requirements for EDS1 and NDR1 by disease resistance genes define at least two R gene-mediated signaling pathways in Arabidopsis Gapped BLAST and PSI-BLAST: A new gen-eration of protein database search programs
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A re-ceptor kinase-like protein encoded by the rice disease resistance gene Characterization of the tomato Cf-4 gene for resistance to Cladosporium fulvum identifies se-quences that determine recognitional specificity in Cf-4 and Cf-9
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The product of the tobacco mosaic virus resistance gene N: Similarity to Toll and the interleukin-1 receptor Toll-like receptor-2 mediates lipopolysaccharide-induced cellular signalling
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