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Key message Novel disease resistance gene paralogues are generated by targeted chromosome cleavage of tandem duplicated NBS-LRR gene complexes and subsequent DNA repair in soybean. This study demonstrates accelerated diversification of innate immunity of plants using CRISPR. Abstract Nucleotide-binding-site-leucine-rich-repeat (NBS-LRR) gene families are key components of effector-triggered immunity. They are often arranged in tandem duplicated arrays in the genome, a configuration that is conducive to recombinations that will lead to new, chimeric genes. These rearrangements have been recognized as major sources of novel disease resistance phenotypes. Targeted chromosome cleavage by CRISPR/Cas9 can conceivably induce rearrangements and thus emergence of new resistance gene paralogues. Two NBS-LRR families of soy have been selected to demonstrate this concept: a four-copy family in the Rpp1 region (Rpp1L) and a large, complex locus, Rps1 with 22 copies. Copy-number variations suggesting large-scale, CRISPR/Cas9-mediated chromosome rearrangements in the Rpp1L and Rps1 complexes were detected in up to 58.8% of progenies of primary transformants using droplet-digital PCR. Sequencing confirmed development of novel, chimeric paralogs with intact open reading frames. These novel paralogs may confer new disease resistance specificities. This method to diversify innate immunity of plants by genome editing is readily applicable to other disease resistance genes or other repetitive loci.

NBS-LRR-type disease resistance gene-like cDNA, induced by salicylic acid (SA) was cloned from rye Secalecereale L. (2n = 14RR) var. Petkus, which has rust resistance genes such as Lr26, Sr31 andRr9. We designed primers based on the NBS region and performed PCR using Petkus genomic DNA as a template. Next, we TA-cloned a 532-bp DNA fragment containing five homologous amino acid sequences in the NBS region. The SA-treated rye showed strong expression of a transcript of approximately 3.5 knt in the Northern blots probed with the NBS fragment; however, no transcripts were observed with the untreated rye. We constructed a cDNA library of rye var. Petkus treated with SA, and then screened the cDNA library using the TA-cloned NBS fragments as a probe. The entire nucleotide sequence of a full length of rye NBS-LRR-containing class cDNA 3,446 bp was determined.

Plant resistance genes (R-genes) drive the immune responses of crops against specific pathotypes of disease-causing organisms. Over time, genetic diversity in R-genes and R-pseudogenes has arisen among different rice varieties. This bioinformatics study was carried out to (i) predict the full sets of candidate nucleotide-binding site leucine-rich repeat (NLR) R-genes present in six rice genomes, (ii) detect variation within candidate R-genes, (iii) identify potential selectable markers within and near to LRR genes among 75 diverse indica rice genomes. Four high quality indica genomes, plus the standard japonica and indica reference genomes, were analysed with widely available bioinformatic tools to identify candidate R-genes and R-pseudogenes. They were detected in clusters, consistent with previous studies. BLAST analysis of cloned protein sequences of 31 R-gene loci gave confidence in this approach for detection of cloned NLR R-genes. Approximately 10% of candidate R-genes were located within 1 kb of a microsatellite (SSR) marker. Sequence comparisons among indica rice genomes detected SNPs or InDels in 334 candidate rice R-genes. There were significantly more SNPs and InDels within the identified NLR R-gene candidates than in other types of gene. The genome-wide locations of candidate R-genes and their associated markers are presented here for the potential future development of improved disease-resistant varieties. Limitations of in silico approaches used for R-gene discovery are discussed.

Background Nucleotide-binding site-leucine-rich repeat (NBS-LRR) resistance genes are the largest class of plant resistance genes which play an important role in the plant defense response. These genes are better conserved than others and function as a recognition-based immune system in plants through their encoded proteins. Results Here, we report the effect of Magnaporthe oryzae, the rice blast pathogen inoculation in resistant BR2655 and susceptible HR12 rice cultivars. Transcriptomic profiling was carried out to analyze differential gene expression in these two cultivars. A total of eight NBS-LRR uncharacterized resistance proteins (RP1, RP2, RP3, RP4, RP5, RP6, RP7, and RP8) were selected in these two cultivars for in silico modeling. Modeller 9.22 and SWISS-MODEL servers were used for the homology modeling of eight RPs. ProFunc server was utilized for the prediction of secondary structure and function. The CDvist Web server and Interpro scan server detected the motif and domains in eight RPs. Ramachandran plot of eight RPs confirmed that the modeled structures occupied favorable positions. Conclusions From the present study, computational analysis of these eight RPs may afford insights into their role, function, and valuable resource for studying the intricate details of the plant defense mechanism. Furthermore, the identification of resistance proteins is useful for the development of molecular markers linked to resistance genes.

Background Bacterial fruit blotch (BFB), a disease caused by Acidovorax citrulli, results in significant economic losses in melon. The causal QTLs and genes for resistance to this disease have yet to be identified. Resistance (R)-genes play vital roles in resistance to plant diseases. Since the complete genome sequence of melon is available and genome-wide identification of R-genes has been performed for this important crop, comprehensive expression profiling may lead to the identification of putative candidate genes that function in the response to BFB. Results We identified melon accessions that are resistant and susceptible to BFB through repeated bioassays and characterized all 70 R-genes in melon, including their gene structures, chromosomal locations, domain organizations, motif distributions, and syntenic relationships. Several disease resistance-related domains were identified, including NBS, TIR, LRR, CC, RLK, and DUF domains, and the genes were categorized based on the domains of their encoded proteins. In addition, we profiled the expression patterns of the genes in melon accessions with contrasting levels of BFB resistance at 12 h, 1 d, 3 d, and 6 d after inoculation with A. citrulli. Six R-genes exhibited consistent expression patterns (MELO3C023441, MELO3C016529, MELO3C022157, MELO3C022146, MELO3C025518, and MELO3C004303), with higher expression levels in the resistant vs. susceptible accession. Conclusion We identified six putative candidate R-genes against BFB in melon. Upon functional validation, these genes could be targeted for manipulation via breeding and biotechnological approaches to improve BFB resistance in melon in the future.

Zucchini yellow mosaic virus (ZYMV; potyviridae) represents a major pathogen of Cucurbitaceae crops. ZYMV resistance in melon PI 414723 is conditioned by a dominant allele at the Zym locus. This resistant accession restricts viral spread and does not develop mosaic symptoms, but necrosis sometimes develops in response to inoculation. In previous studies, Zym has been mapped to linkage group II of the melon genetic map. In the present study, positional cloning of the locus was undertaken, starting from the CM-AG36 SSR marker at approximately 2 cm distance. We utilized five mapping populations that share the same resistant parent, PI 414723, and analyzed a total of 1630 offspring, to construct a high-resolution genetic map of the Zym locus. Two melon BAC libraries were used for chromosome walking and for developing new markers closer to the resistance gene by BAC-end sequencing. A BAC contig was constructed, and we identified a single BAC clone, from the ZYMV susceptible genotype MR-1, that physically encompasses the resistance gene. A second clone was isolated from another susceptible genotype, WMR 29, and the two clones were fully sequenced and annotated. Additional markers derived from the sequenced region delimited the region to 17.6 kb of a sequence that harbors a NAC-like transcription factor and, depending on the genotype, either two or three R-gene homologs with a CC-NBS-LRR structure. Mapping was confirmed by saturating the map with SNP markers using a single mapping population. The same region was amplified and sequenced also in the ZYMV resistant genotype PI 414723. Because numerous polymorphic sites were noted between genotypes, we could not associate resistance with a specific DNA polymorphism; however, this study enables molecular identification of Zym and paves the way to functional studies of this important locus.

The root-knot nematode (RKN) Meloidogyne incognita severely reduces yields of pepper (Capsicum annuum) worldwide. A single dominant locus, Me7, conferring RKN resistance was previously mapped on the long arm of pepper chromosome P9. In the present study, the Me7 locus was fine mapped using an F2 population of 714 plants derived from a cross between the RKN-susceptible parent C. annuum ECW30R and the RKN-resistant parent C. annuum CM334. CM334 exhibits suppressed RKN juvenile movement, suppressed feeding site enlargement and significant reduction in gall formation compared with ECW30R. RKN resistance screening in the F2 population identified 558 resistant and 156 susceptible plants, which fit a 3:1 ratio confirming that this RKN resistance was controlled by a single dominant gene. Using the C. annuum CM334 reference genome and BAC library sequencing, fine mapping of Me7 markers was performed. The Me7 locus was delimited between two markers G21U3 and G43U3 covering a physical interval of approximately 394.7 kb on the CM334 chromosome P9. Nine markers co-segregated with the Me7 gene. A cluster of 25 putative nucleotide-binding site and leucine-rich repeat (NBS-LRR)-type disease resistance genes were predicted in the delimited Me7 region. We propose that RKN resistance in CM334 is mediated by one or more of these NBS-LRR class R genes. The Me7-linked markers identified here will facilitate marker-assisted selection (MAS) for RKN resistance in pepper breeding programs, as well as functional analysis of Me7 candidate genes in C. annuum.

High expression of plant nucleotide binding site leucine-rich repeat (NBS-LRR) defense genes is often lethal to plant cells, a phenotype perhaps associated with fitness costs. Plants implement several mechanisms to control the transcript level of NBS-LRR defense genes. As negative transcriptional regulators, diverse miRNAs target NBS-LRRs in eudicots and gymnosperms. To understand the evolutionary benefits of this miRNA-NBS-LRR regulatory system, we investigated the NBS-LRRs of 70 land plants, coupling this analysis with extensive small RNA data. A tight association between the diversity of NBS-LRRs and miRNAs was found. The miRNAs typically target highly duplicated NBS-LRRs. In comparison, families of heterogeneous NBS-LRRs were rarely targeted by miRNAs in Poaceae and Brassicaceae genomes. We observed that duplicated NBS-LRRs from different gene families periodically gave birth to new miRNAs. Most of these newly emerged miRNAs target the same conserved, encoded protein motif of NBS-LRRs, consistent with a model of convergent evolution for these miRNAs. By assessing the interactions between miRNAs and NBS-LRRs, we found nucleotide diversity in the wobble position of the codons in the target site drives the diversification of miRNAs. Taken together, we propose a co-evolutionary model of plant NBS-LRRs and miRNAs hypothesizing how plants balance the benefits and costs of NBS-LRR defense genes.

SUMMARY The broad host range necrotrophic fungus Sclerotinia sclerotiorum is a devastating pathogen of many oil and vegetable crops. Plant genes conferring complete resistance against S. sclerotiorum have not been reported. Instead, plant populations challenged by S. sclerotiorum exhibit a continuum of partial resistance designated as quantitative disease resistance (QDR). Because of their complex interplay and their small phenotypic effect, the functional characterization of QDR genes remains limited. How broad host range necrotrophic fungi manipulate plant programmed cell death is for instance largely unknown. Here, we designed a time‐resolved automated disease phenotyping pipeline enabling high‐throughput disease lesion measurement with high resolution, low footprint at low cost. We could accurately recover contrasted disease responses in several pathosystems using this system. We used our phenotyping pipeline to assess the kinetics of disease symptoms caused by seven S. sclerotiorum isolates on six A. thaliana natural accessions with unprecedented resolution. Large effect polymorphisms common to the most resistant A. thaliana accessions identified highly divergent alleles of the nucleotide‐binding site leucine‐rich repeat gene LAZ5 in the resistant accessions Rubezhnoe and Lip‐0. We show that impaired LAZ5 expression in laz5.1 mutant lines and in A. thaliana Rub natural accession correlate with enhanced QDR to S. sclerotiorum. These findings illustrate the value of time‐resolved image‐based phenotyping for unravelling the genetic bases of complex traits such as QDR. Our results suggest that S. sclerotiorum manipulates plant sphingolipid pathways guarded by LAZ5 to trigger programmed cell death and cause disease., Significance Statement We developed an image‐based method to measure plant quantitative disease resistance over time with high resolution and throughput. We applied it to reveal Arabidopsis LAZ5 as a susceptibility gene to the necrotrophic fungal pathogen Sclerotinia sclerotiorum using only six plant genotypes. LAZ5 belongs to a family of typical resistance genes, suggesting that S. sclerotiorum exploits classical resistance pathways to cause disease.

Nucleotide-binding site-leucine-rich repeat (NBS-LRR) domain proteins are immune sensors and play critical roles in plant disease resistance. In this study, we cloned and characterized a novel NBS-LRR gene ZmNBS25 in maize. We found that ZmNBS25 could response to pathogen inoculation and salicylic acid (SA) treatment in maize, and transient overexpression of ZmNBS25 induced a hypersensitive response in tobacco. High-performance liquid chromatography (HPLC) analysis showed that, compared to control plants, ZmNBS25 overexpression (ZmNBS25-OE) in Arabidopsis and rice resulted in higher SA levels. By triggering the expression of certain defense-responsive genes, ZmNBS25-OE enhanced the resistance of Arabidopsis and rice to Pseudomonas syringae pv. tomato DC3000 and sheath blight disease, respectively. Moreover, we found little change of grain size and 1000-grain weight between ZmNBS25-OE rice lines and controls. Together, our results suggest that ZmNBS25 can function as a disease resistance gene across different species, being a valuable candidate for engineering resistance in breeding programs.

Wilt caused by Verticillium dahliae significantly reduces cotton yields, as host resistance in commercially cultivated Gossypium species is lacking. Understanding the molecular basis of disease resistance in non-commercial Gossypium species could galvanize the development of Verticillium wilt resistance in cultivated species. Nucleotide-binding site leucine-rich repeat (NBS-LRR) proteins play a central role in plant defence against pathogens. In this study, we focused on the relationship between a locus enriched with eight NBS-LRR genes and Verticillium wilt resistance in G. barbadense. Independent virus-induced gene silencing of each of the eight NBS-LRR genes in G. barbadense cultivar Hai 7124 revealed that silencing of GbaNA1 alone compromised the resistance of G. barbadense to V. dahliae isolate Vd991. In cultivar Hai 7124, GbaNA1 could be induced by V. dahliae isolate Vd991 and by ethylene, jasmonic acid and salicylic acid. Nuclear protein localization of GbaNA1 was demonstrated by transient expression. Sequencing of the GbaNA1 orthologue in nine G.hirsutum accessions revealed that all carried a non-functional allele, caused by a premature peptide truncation. In addition, all 10 G. barbadense and nine G. hirsutum accessions tested carried a full-length (∼1140 amino acids) homologue of the V. dahliae race 1 resistance gene Gbve1, although some sequence polymorphisms were observed. Verticillium dahliae Vd991 is a non-race 1 isolate that lacks the Ave1 gene. Thus, the resistance imparted by GbaNA1 appears to be mediated by a mechanism distinct from recognition of the fungal effector Ave1.

Verticillium wilt caused by Verticillium dahliae results in severe losses in cotton, and is economically the most destructive disease of this crop. Improving genetic resistance is the cleanest and least expensive option to manage Verticillium wilt. Previously, we identified the island cotton NBS-LRR-encoding gene GbaNA1 that confers resistance to the highly virulent V. dahliae isolate Vd991. In this study, we expressed cotton GbaNA1 in the heterologous system of Arabidopsis thaliana and investigated the defense response mediated by GbaNA1 following inoculations with V. dahliae. Heterologous expression of GbaNA1 conferred Verticillium wilt resistance in A. thaliana. Moreover, overexpression of GbaNA1 enabled recovery of the resistance phenotype of A. thaliana mutants that had lost the function of GbaNA1 ortholog gene. Investigations of the defense response in A. thaliana showed that the reactive oxygen species (ROS) production and the expression of genes associated with the ethylene signaling pathway were enhanced significantly following overexpression of GbaNA1. Intriguingly, overexpression of the GbaNA1 ortholog from Gossypium hirsutum (GhNA1) in A. thaliana did not induce the defense response of ROS production due to the premature termination of GhNA1, which lacks the encoded NB-ARC and LRR motifs. GbaNA1 therefore confers Verticillium wilt resistance in A. thaliana by the activation of ROS production and ethylene signaling. These results demonstrate the functional conservation of the NBS-LRR-encoding GbaNA1 in a heterologous system, and the mechanism of this resistance, both of which may prove valuable in incorporating GbaNA1-mediated resistance into other plant species.

The vast majority of plant disease resistance (R) genes encode nucleotide binding site-leucine-rich repeat (NBS-LRR) proteins, which specifically determine the plant immune response and have been demonstrated to be targets of several microRNA (miRNA) families. The fungus Fusarium oxysporum f. sp. lycopersici (FOL) causes vascular wilt disease in tomato worldwide. Here, we explored a possible role for FGR3 in tomato defense against FOL. FRG3 is a predicted NBS-LRR like gene that is targeted by slmiR482e-3p, a member of slmiR482 miRNA family. Northern blot data demonstrated that all seven members of the slmiR482 family were regulated in diverse ways after infection by FOL. The ability of FRG3 to be regulated by slmiR482e-3p was confirmed at the transcript level by co-expression studies in Nicotiana benthamiana. A virus-induced gene silencing (VIGS) approach revealed that FRG3 confers resistance to the Motelle tomato cultivar. Taken together, our study has identified a novel R gene, FRG3, which is targeted by slmiR482e-3p at the transcript level, and is necessary for resistance to tomato wilt disease in planta.

Disease resistance genes (R genes), as part of the plant defense system, have coevolved with corresponding pathogen molecules. The main objectives of this project were to identify non-Toll interleukin receptor, nucleotide-binding site, leucine-rich repeat (nTNL) genes and elucidate their evolutionary divergence across six plant genomes. Using reference sequences from Arabidopsis, we investigated nTNL orthologs in the genomes of common bean, Medicago, soybean, poplar, and rice. We used Hidden Markov Models for sequence identification, performed model-based phylogenetic analyses, visualized chromosomal positioning, inferred gene clustering, and assessed gene expression profiles. We analyzed 908 nTNL R genes in the genomes of the six plant species, and classified them into 12 subgroups based on the presence of coiled-coil (CC), nucleotide binding site (NBS), leucine rich repeat (LRR), resistance to Powdery mildew 8 (RPW8), and BED type zinc finger domains. Traditionally classified CC-NBS-LRR (CNL) genes were nested into four clades (CNL A-D) often with abundant, well-supported homogeneous subclades of Type-II R genes. CNL-D members were absent in rice, indicating a unique R gene retention pattern in the rice genome. Genomes from Arabidopsis, common bean, poplar and soybean had one chromosome without any CNL R genes. Medicago and Arabidopsis had the highest and lowest number of gene clusters, respectively. Gene expression analyses suggested unique patterns of expression for each of the CNL clades. Differential gene expression patterns of the nTNL genes were often found to correlate with number of introns and GC content, suggesting structural and functional divergence.

Nucleotide-binding site and leucine-rich repeat (NBS-LRR) genes represent the largest class of disease resistance genes in plants. The genome sequence of common bean (Phaseolus vulgaris L.) provides valuable data for determining the genomic organization of NBS-LRR genes. However, data on the NBS-LRR genes in common bean are limited. A total of 178 NBS-LRR-type genes and 145 partial genes (with or without a NBS) located on 11 common bean chromosomes were identified from the common bean genome database. Nucleotide-binding site and leucine-rich repeat (NBS-LRR) genes represent the largest and most important disease resistance genes in plants. The genome sequence of the common bean (Phaseolus vulgaris L.) provides valuable data for determining the genomic organization of NBS-LRR genes. However, data on the NBS-LRR genes in the common bean are limited. In total, 178 NBS-LRR-type genes and 145 partial genes (with or without a NBS) located on 11 common bean chromosomes were identified from genome sequences database. Furthermore, 30 NBS-LRR genes were classified into Toll/interleukin-1 receptor (TIR)-NBS-LRR (TNL) types, and 148 NBS-LRR genes were classified into coiled-coil (CC)-NBS-LRR (CNL) types. Moreover, the phylogenetic tree supported the division of these PvNBS genes into two obvious groups, TNL types and CNL types. We also built expression profiles of NBS genes in response to anthracnose and common bacterial blight using qRT-PCR. Finally, we detected nine disease resistance loci for anthracnose (ANT) and seven for common bacterial blight (CBB) using the developed NBS-SSR markers. Among these loci, NSSR24, NSSR73, and NSSR265 may be located at new regions for ANT resistance, while NSSR65 and NSSR260 may be located at new regions for CBB resistance. Furthermore, we validated NSSR24, NSSR65, NSSR73, NSSR260, and NSSR265 using a new natural population. Our results provide useful information regarding the function of the NBS-LRR proteins and will accelerate the functional genomics and evolutionary studies of NBS-LRR genes in food legumes. NBS-SSR markers represent a wide-reaching resource for molecular breeding in the common bean and other food legumes. Collectively, our results should be of broad interest to bean scientists and breeders.

Bacterial fruit blotch (BFB) causes losses in melon marketable yield. However, until now, there has been no information about the genetic loci responsible for resistance to the disease or their pattern of inheritance. We determined the inheritance pattern of BFB resistance from a segregating population of 491 F2 individuals raised by crossing BFB-resistant (PI 353814) and susceptible (PI 614596) parental accessions. All F1 plants were resistant to Acidovorax citrulli strain KACC18782, and F2 plants segregated with a 3:1 ratio for resistant and susceptible phenotypes, respectively, in a seedling bioassay experiment, indicating that BFB resistance is controlled by a monogenic dominant gene. In an investigation of 57 putative disease-resistance related genes across the melon genome, only the MELO3C022157 gene (encoding TIR-NBS-LRR domain), showing polymorphism between resistant and susceptible parents, revealed as a good candidate for further investigation. Cloning, sequencing and quantitative RT-PCR expression of the polymorphic gene MELO3C022157 located on chromosome 9 revealed multiple insertion/deletions (InDels) and single nucleotide polymorphisms (SNPs), of which the SNP A2035T in the second exon of the gene caused loss of the LRR domain and truncated protein in the susceptible accession. The InDel marker MB157-2, based on the large (504 bp) insertion in the first intron of the susceptible accession, was able to distinguish resistant and susceptible accessions among 491 F2 and 22 landraces/inbred accessions with 98.17% and 100% detection accuracy, respectively. This novel PCR-based, co-dominant InDel marker represents a practical tool for marker-assisted breeding aimed at developing BFB-resistant melon accessions.

Resistance gene homologs (RGHs) were isolated from the switchgrass variety Alamo by a combination of polymerase chain reaction and expressed sequence tag (EST) database mining. Fifty-eight RGHs were isolated by polymerase chain reaction and 295 RGHs were identified in 424,545 switchgrass ESTs. Four nucleotide binding site−leucine-rich repeat RGHs were selected to investigate RGH haplotypic diversity in seven switchgrass varieties chosen for their representation of a broad range of the switchgrass germplasm. Lowland and upland ecotypes were found to be less similar, even from nearby populations, than were more distant populations with similar growth environments. Most (83.5%) of the variability in these four RGHs was found to be attributable to the within-population component. The difference in nucleotide diversity between and within populations was observed to be small, whereas this diversity is maintained to similar degrees at both population and ecotype levels. The results also revealed that the analyzed RGHs were under positive selection in the studied switchgrass accessions. Intragenic recombination was detected in switchgrass RGHs, thereby demonstrating an active genetic process that has the potential to generate new resistance genes with new specificities that might act against newly-arising pathogen races.

Soybean dwarf virus (SbDV) causes serious dwarfing, yellowing and sterility in soybean (Glycine max). The soybean cv. Adams is tolerant to SbDV infection in the field and exhibits antibiosis to foxglove aphid (Aulacorthum solani), which transmits SbDV. This antibiosis (termed “aphid resistance”) is required for tolerance to SbDV in the field in segregated progenies of Adams. A major quantitative trait locus, Raso1, is reported for foxglove aphid resistance. Our objectives were to fine map Raso1 and to reveal whether Raso1 alone is sufficient to confer both aphid resistance and SbDV tolerance. We introduced Raso1 into cv. Toyomusume by backcrossing and investigated the degree of aphid antibiosis to foxglove aphid and the degree of tolerance to SbDV in the field. All Raso1-introduced backcross lines showed aphid resistance. Interestingly, only one Raso1-introduced backcross line (TM-1386) showed tolerance to SbDV in the field. The results demonstrated Raso1 alone is sufficient to confer aphid resistance but insufficient for SbDV tolerance. Tolerance to SbDV was indicated to require additional gene(s) to Raso1. Additionally, Raso1 was mapped to a 63-kb interval on chromosome 3 of the Williams 82 sequence assembly (Glyma1). This interval includes a nucleotide-binding site–leucine-rich repeat encoding gene and two other genes in the Williams 82 soybean genome sequence.

Gummy stem blight (GSB) causes enormous losses to melon (Cucumis melo L.) production worldwide. We aimed to develop useful molecular markers linked to GSB resistance. In this study, 168 F2 plants were obtained from the F1 population of a cross between the GSB-susceptible &lsquo, Cornell ZPPM 339&rsquo, and the GSB-resistant &lsquo, PI482399&rsquo, lines. A 3:1 ratio of susceptible and resistant genotypes was observed in the F2 population, indicating control by a single recessive gene. Nucleotide-binding site leucine-rich repeat (NBS-LRR) genes confer resistance against insects and diseases in cucurbits including melon. We cloned and sequenced the TIR-NBS-LRR-type resistance gene MELO3C022157, located on melon chromosome 9, from resistant and susceptible lines. Sequence analysis revealed deletions in the first intron, a 2-bp frameshift deletion from the second exon and a 7-bp insertion in the 4th exon of the resistant line. We developed two insertion/deletion (InDel) markers, GSB9-kh-1 and GSB9-kh-2, which were found in the first intron of MELO3C022157 linked to GSB resistance. We validated these markers with the F2 population and inbred lines. These InDels may be used to facilitate marker-assisted selection of GSB resistance in melon. However, functional analysis of overexpressing and/or knock-down mutants is needed to confirm the frameshift mutation.

Eucalyptus grandis is a commercially important hardwood species and is known to be susceptible to a number of pests and pathogens. Determining mechanisms of defense is therefore a research priority. The published genome for E. grandis has aided the identification of one important class of resistance (R) genes that incorporate nucleotide binding sites and leucine-rich repeat domains (NBS-LRR). Using an iterative search process we identified NBS-LRR gene models within the E. grandis genome. We characterized the gene models and identified their genomic arrangement. The gene expression patterns were examined in E. grandis clones, challenged with a fungal pathogen (Chrysoporthe austroafricana) and insect pest (Leptocybe invasa). One thousand two hundred and fifteen putative NBS-LRR coding sequences were located which aligned into two large classes, Toll or interleukin-1 receptor (TIR) and coiled-coil (CC) based on NB-ARC domains. NBS-LRR gene-rich regions were identified with 76% organized in clusters of three or more genes. A further 272 putative incomplete resistance genes were also identified. We determined that E. grandis has a higher ratio of TIR to CC classed genes compared to other woody plant species as well as a smaller percentage of single NBS-LRR genes. Transcriptome profiles indicated expression hotspots, within physical clusters, including expression of many incomplete genes. The clustering of putative NBS-LRR genes correlates with differential expression responses in resistant and susceptible plants indicating functional relevance for the physical arrangement of this gene family. This analysis of the repertoire and expression of E. grandis putative NBS-LRR genes provides an important resource for the identification of novel and functional R-genes; a key objective for strategies to enhance resilience.

Plant disease resistance genes (R-genes) play a critical role in the defense response to pathogens. Barley is one of the most important cereal crops, having a genome recently made available, for which the diversity and evolution of R-genes are not well understood. The main objectives of this research were to conduct a genome-wide identification of barley Coiled-coil, Nucleotide-binding site, Leucine-rich repeat (CNL) genes and elucidate their evolutionary history. We employed a Hidden Markov Model using 52 Arabidopsis thaliana CNL reference sequences and analyzed for phylogenetic relationships, structural variation, and gene clustering. We identified 175 barley CNL genes nested into three clades, showing (a) evidence of an expansion of the CNL-C clade, primarily due to tandem duplications; (b) very few members of clade CNL-A and CNL-B; and (c) a complete absence of clade CNL-D. Our results also showed that several of the previously identified mildew locus A (MLA) genes may be allelic variants of two barley CNL genes, MLOC_66581 and MLOC_10425, which respond to powdery mildew. Approximately 23% of the barley CNL genes formed 15 gene clusters located in the extra-pericentromeric regions on six of the seven chromosomes; more than half of the clustered genes were located on chromosomes 1H and 7H. Higher average numbers of exons and multiple splice variants in barley relative to those in Arabidopsis and rice may have contributed to a diversification of the CNL-C members. These results will help us understand the evolution of R-genes with potential implications for developing durable resistance in barley cultivars.

The nucleotide-binding site (NBS)–leucine-rich repeat (LRR) gene family is crucially important for offering resistance to pathogens. To explore evolutionary conservation and variability of NBS-LRR genes across grass species, we identified 88, 107, 24, and 44 full-length NBS-LRR genes in sorghum, rice, maize, and Brachypodium , respectively. A comprehensive analysis was performed on classification, genome organization, evolution, expression, and regulation of these NBS-LRR genes using sorghum as a representative of grass species. In general, the full-length NBS-LRR genes are highly clustered and duplicated in sorghum genome mainly due to local duplications. NBS-LRR genes have basal expression levels and are highly potentially targeted by miRNA. The number of NBS-LRR genes in the four grass species is positively correlated with the gene clustering rate. The results provided a valuable genomic resource and insights for functional and evolutionary studies of NBS-LRR genes in grass species.

Sequence conservation among resistance genes (R genes) was exploited to identify 47 R gene analogues (RGAs) from sugar beet (Beta vulgaris L.). Using degenerate primers, 11 RGAs were amplified from genomic DNA and 7 from leaf or beet cDNA. Twenty-nine were selected from an EST sequencing program. Twenty-one RGAs contained structures similar to the nucleotide binding site (NBS) – leucine rich repeat (LRR) domain, a motif commonly found in several R genes. Among the remaining RGAs, 19 revealed similarity to the serine (threonine) protein kinase domain of R genes, 4 showed features related to the LRR region of the rice disease resistance gene Xa21, 1 RGA resembled the sugar beet nematode resistance gene Hs1pro-1, and 2 had homologies to other gene products associated with disease resistance. For 20 EST-derived RGAs, transcript levels were compared in leaf and root tissue revealing organ-specific transcription in 7 cases. Thirty-three RGAs were spread over all nine sugar beet chromosomes, except for a cluster of nine closely linked RGAs on chromosome 7. The analysis of linkage between RGAs and loci for rhizomania and Cercospora resistance identified alleles associated with resistance in both cases.Key words: RGA, Beta vulgaris, NBS–LRR, genetic linkage map, molecular marker.

Resistance genes (R-genes) are essential for long-lived organisms such as forest trees, which are exposed to diverse herbivores and pathogens. In short-lived model species, R-genes have been shown to be involved in species isolation. Here, we studied more than 400 trees from two natural hybrid zones of the European Populus species Populus alba and Populus tremula for microsatellite markers located in three R-gene clusters, including one cluster situated in the incipient sex chromosome region. The results indicate that all three R-gene clusters present extensive linkage disequilibrium (LD). Outlier tests suggest balancing selection as a potential driver of R-gene diversity, but divergent selection was also detected. Nucleotide-binding site leucine-rich repeat (NBS-LRR) type R-gene clusters exhibit low species differentiation and appear to be affected by shared selection pressures between species, recurrent interspecific gene flow, or both. Our results are consistent with a role for R-gene clusters in the evolution of chromosome XIX, recently put forward as an incipient sex chromosome in Populus. © 2015, Springer-Verlag Berlin Heidelberg.

Disease resistance genes (R-genes) encode proteins involved in detecting pathogen attack and activating downstream defense molecules. Recent availability of soybean genome sequences makes it possible to examine the diversity of gene families including disease-resistant genes. The objectives of this study were to identify coiled-coil NBS-LRR (= CNL) R-genes in soybean, infer their evolutionary relationships, and assess structural as well as functional divergence of the R-genes. Profile hidden Markov models were used for sequence identification and model-based maximum likelihood was used for phylogenetic analysis, and variation in chromosomal positioning, gene clustering, and functional divergence were assessed. We identified 188 soybean CNL genes nested into four clades consistent to their orthologs in Arabidopsis. Gene clustering analysis revealed the presence of 41 gene clusters located on 13 different chromosomes. Analyses of the Ks-values and chromosomal positioning suggest duplication events occurring at varying timescales, and an extrapericentromeric positioning may have facilitated their rapid evolution. Each of the four CNL clades exhibited distinct patterns of gene expression. Phylogenetic analysis further supported the extrapericentromeric positioning effect on the divergence and retention of the CNL genes. The results are important for understanding the diversity and divergence of CNL genes in soybean, which would have implication in soybean crop improvement in future.

The resistance gene Pi-ta has been effectively used to control rice blast disease, but some populations of cultivated and wild rice have evolved resistance. Insights into the evolutionary processes that led to this resistance during crop domestication may be inferred from the population history of domesticated and wild rice strains. In this study, we applied a recently developed statistical method, time-dependent Poisson random field model, to examine the evolution of the Pi-ta gene in cultivated and weedy rice. Our study suggests that the Pi-ta gene may have more recently introgressed into cultivated rice, indica and japonica, and U.S. weedy rice from the wild species, O. rufipogon. In addition, the Pi-ta gene is under positive selection in japonica, tropical japonica, U.S. cultivars and U.S. weedy rice. We also found that sequences of two domains of the Pi-ta gene, the nucleotide binding site and leucine-rich repeat domain, are highly conserved among all rice accessions examined. Our results provide a valuable analytical tool for understanding the evolution of disease resistance genes in crop plants.

The nucleotide-binding-site-leucine-rich-repeat (NBS–LRR)-encoding gene family has attracted much research interest because approximately 75% of the plant disease resistance genes that have been cloned to date are from this gene family. Here, we describe a collection of peanut NBS–LRR resistance gene candidates (RGCs) isolated from peanut (Arachis) species by mining Gene Bank data base. NBS–LRR sequences assembled into TIR-NBS-LRR (75.4%) and non-TIR-NBS-LRR (24.6%) subfamilies. Total of 20 distinct clades were identified and showed a high level of sequence divergence within TIR-NBS and non-TIR-NBS subfamilies. Thirty-four primer pairs were designed from these RGC sequences and used for screening different genotypes belonging to wild and cultivated peanuts. Therefore, peanut RGC identified in this study will provide useful tools for developing DNA markers and cloning the genes for resistance to different pathogens in peanut.

Background Plant nucleotide-binding site (NBS)-leucine-rich repeat (LRR) proteins encoded by resistance genes play an important role in the responses of plants to various pathogens, including viruses, bacteria, fungi, and nematodes. In this study, a comprehensive analysis of NBS-encoding genes within the whole cucumber genome was performed, and the phylogenetic relationships of NBS-encoding resistance gene homologues (RGHs) belonging to six species in five genera of Cucurbitaceae crops were compared. Results Cucumber has relatively few NBS-encoding genes. Nevertheless, cucumber maintains genes belonging to both Toll/interleukine-1 receptor (TIR) and CC (coiled-coil) families. Eight commonly conserved motifs have been established in these two families which support the grouping into TIR and CC families. Moreover, three additional conserved motifs, namely, CNBS-1, CNBS-2 and TNBS-1, have been identified in sequences from CC and TIR families. Analyses of exon/intron configurations revealed that some intron loss or gain events occurred during the structural evolution between the two families. Phylogenetic analyses revealed that gene duplication, sequence divergence, and gene loss were proposed as the major modes of evolution of NBS-encoding genes in Cucurbitaceae species. Compared with NBS-encoding sequences from the Arabidopsis thaliana genome, the remaining seven TIR familes of NBS proteins and RGHs from Cucurbitaceae species have been shown to be phylogenetically distinct from the TIR family of NBS-encoding genes in Arabidopsis, except for two subfamilies (TIR4 and TIR9). On the other hand, in the CC-NBS family, they grouped closely with the CC family of NBS-encoding genes in Arabidopsis. Thus, the NBS-encoding genes in Cucurbitaceae crops are shown to be ancient, and NBS-encoding gene expansions (especially the TIR family) may have occurred before the divergence of Cucurbitaceae and Arabidopsis. Conclusion The results of this paper will provide a genomic framework for the further isolation of candidate disease resistance NBS-encoding genes in cucumber, and contribute to the understanding of the evolutionary mode of NBS-encoding genes in Cucurbitaceae crops.

Background R genes are a key component of genetic interactions between plants and biotrophic bacteria and are known to regulate resistance against bacterial invasion. The most common R proteins contain a nucleotide-binding site and a leucine-rich repeat (NBS-LRR) domain. Some NBS-LRR genes in the soybean genome have also been reported to function in disease resistance. In this study, the number of NBS-LRR genes was found to correlate with the number of disease resistance quantitative trait loci (QTL) that flank these genes in each chromosome. NBS-LRR genes co-localized with disease resistance QTL. The study also addressed the functional redundancy of disease resistance on recently duplicated regions that harbor NBS-LRR genes and NBS-LRR gene expression in the bacterial leaf pustule (BLP)-induced soybean transcriptome. Results A total of 319 genes were determined to be putative NBS-LRR genes in the soybean genome. The number of NBS-LRR genes on each chromosome was highly correlated with the number of disease resistance QTL in the 2-Mb flanking regions of NBS-LRR genes. In addition, the recently duplicated regions contained duplicated NBS-LRR genes and duplicated disease resistance QTL, and possessed either an uneven or even number of NBS-LRR genes on each side. The significant difference in NBS-LRR gene expression between a resistant near-isogenic line (NIL) and a susceptible NIL after inoculation of Xanthomonas axonopodis pv. glycines supports the conjecture that NBS-LRR genes have disease resistance functions in the soybean genome. Conclusions The number of NBS-LRR genes and disease resistance QTL in the 2-Mb flanking regions of each chromosome was significantly correlated, and several recently duplicated regions that contain NBS-LRR genes harbored disease resistance QTL for both sides. In addition, NBS-LRR gene expression was significantly different between the BLP-resistant NIL and the BLP-susceptible NIL in response to bacterial infection. From these observations, NBS-LRR genes are suggested to contribute to disease resistance in soybean. Moreover, we propose models for how NBS-LRR genes were duplicated, and apply Ks values for each NBS-LRR gene cluster.

Resistance genes are among the most important gene classes for plant breeding purposes being responsible for activation of plant defense mechanisms. Among them, the nucleotide binding site-leucine rich repeat (NBS-LRR) class R-genes are the most abundant and actively found in all types of plants. Insilico characterization of EST database resulted in the detection of 28 NBS types R-gene sequences in Curcuma longa. All the 28 sequences represented the NB-ARC domain, 21 of which were found to have highly conserved motif characteristics and categorized as regular NBS genes. The Open Reading Frames varied from 361 (CL.CON.3566) to 112 (CL.CON.1267) with an average of 279 amino acids. Most alignment occurred with monocots (67.8%) with emphasis on Oryza sativa and Zingiber sequences. All best alignments with dicots occurred with Arabidopsis thaliana, Populus trichocarpa and Medicago sativa. These detected NBS type Rgenes from Curcuma longa can be used as a valuable resource for molecular marker development, molecular mapping of R-genes, and identification of resistance gene analogs and functional and evolutionary characterization of NBS-LRR-encoding resistance genes in asexually reproducing plants.

International audience; As the largest class of disease resistance R genes, the genes encoding nucleotide binding site and leucine-rich repeat proteins (“NBS-LRR genes”) play a critical role in defending plants from a multitude of pathogens and pests. The diversity of NBS-LRR genes was examined in the Populus trichocarpa draft genome sequence. The NBS class of genes in this perennial tree is large and diverse, comprised of ∼400 genes, at least twice the complement of Arabidopsis. The NBS family can be divided into multiple subfamilies with distinct domain organizations. It includes 119 Coiled-Coil-NBS-LRR genes, 64 TIR-NBS-LRR genes, 34 BED-finger-NBS-LRR, and both truncated and unusual NBS- and NBS-LRR-containing genes. The transcripts of only 34 NBS-LRR genes were detected in rust-infected and non-infected leaves using a whole-genome oligoarray. None showed an altered expression two days post inoculation.

Degenerate oligonucleotide primers, designed based on conserved regions of Nucleotide Binding Site (NBS) domains from previously cloned plant resistance genes, were used to isolate Resistance Gene Analogues (RGAs) from wild and cultivated strawberries. Seven distinct families of RGAs of the NBS-LRR type were identified from two related wild species, Fragaria vesca and F. chiloensis, and six different Fragaria x ananassa cultivars. With one exception (GAV-3), the deduced amino acid sequences of strawberry RGAs showed strong similarity to TIR (Toll Interleukin I Receptor)-type R genes from Arabidopsis, tobacco and flax, suggesting the existence of common ancestors. GAV-3 seemed to be more closely related to the non-TIR type. Further studies showed that the recombination level and the ratio of non-synonymous to synonymous substitutions within families were low. These data suggest that NBS-encoding sequences of RGAs in strawberry are subject to a gradual accumulation of mutations leading to purifying selection, rather than to a diversifying process. The present paper is the first report on RGAs in strawberry. Fil: Martinez Zamora, Martin Gustavo. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Castagnaro, Atilio Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Diaz Ricci, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; Argentina

Background Biofuels extracted from the seeds of Camelina sativa have recently been used successfully as environmentally friendly jet-fuel to reduce greenhouse gas emissions. Camelina sativa is genetically very close to Arabidopsis thaliana, and both are members of the Brassicaceae. Although public databases are currently available for some members of the Brassicaceae, such as A. thaliana, A. lyrata, Brassica napus, B. juncea and B. rapa, there are no public Expressed Sequence Tags (EST) or genomic data for Camelina sativa. In this study, a high-throughput, large-scale RNA sequencing (RNA-seq) of the Camelina sativa transcriptome was carried out to generate a database that will be useful for further functional analyses. Results Approximately 27 million clean “reads” filtered from raw reads by removal of adaptors, ambiguous reads and low-quality reads (2.42 gigabase pairs) were generated by Illumina paired-end RNA-seq technology. All of these clean reads were assembled de novo into 83,493 unigenes and 103,196 transcripts using SOAPdenovo and Trinity, respectively. The average length of the transcripts generated by Trinity was 697 bp (N50 = 976), which was longer than the average length of unigenes (319 bp, N50 = 346 bp). Nonetheless, the assembly generated by SOAPdenovo produced similar number of non-redundant hits (22,435) with that of Trinity (22,433) in BLASTN searches of the Arabidopsis thaliana CDS sequence database (TAIR). Four public databases, the Kyoto Encyclopedia of Genes and Genomes (KEGG), Swiss-prot, NCBI non-redundant protein (NR), and the Cluster of Orthologous Groups (COG), were used for unigene annotation; 67,791 of 83,493 unigenes (81.2%) were finally annotated with gene descriptions or conserved protein domains that were mapped to 25,329 non-redundant protein sequences. We mapped 27,042 of 83,493 unigenes (32.4%) to 119 KEGG metabolic pathways. Conclusions This is the first report of a transcriptome database for Camelina sativa, an environmentally important member of the Brassicaceae. We showed that C. savita is closely related to Arabidopsis spp. and more distantly related to Brassica spp. Although the majority of annotated genes had high sequence identity to those of A. thaliana, a substantial proportion of disease-resistance genes (NBS-encoding LRR genes) were instead more closely similar to the genes of other Brassicaceae; these genes included BrCN, BrCNL, BrNL, BrTN, BrTNL in B. rapa. As plant genomes are under long-term selection pressure from environmental stressors, conservation of these disease-resistance genes in C. sativa and B. rapa genomes implies that they are exposed to the threats from closely-related pathogens in their natural habitats.

Our understanding of inducible plant defense responses was greatly increased by the characterization of disease resistance genes from a variety of plant–pathogen interaction models. In the paper we aimed to characterize conserved motifs in the NBS domains, to determine their evolutionary fate. Using combined iterative data-mining approaches we effectively expanded the dataset, but the number of NBS-LRR sequences obtained was much lower than projected. We interpret this as evidence supporting low basal transcription levels as expected for R genes situated at the very start of signal amplification cascades. Motif analysis showed all key motifs of the NBS-LRR CNL domain present, with no evidence for TNL type sequences as previously observed for monocotyledonous taxa. Significant overlap between Triticeae CNL members and CNL R genes from other taxa was found. Three recently diverged clades of paralogous NBS-core sequences for barley was tested for gene-conversion events, but none was found, contradicting results from Arabidopsis where even ectopic gene-conversion events were detected. Non-synonymous to synonymous substitution rate ratio tests for comparing evolution of recent paralogous and homeologous duplications showed that the NBS-core domain of three barley paralogue clades were under strong purifying selection in contrast to the wheat go35 CNL gene, where four different non-synonymous substitutions were found. Only synonymous differences were seen between sequences obtained from two diploid wheat ancestors ( A. tauschii and A. speltoides ). We consider this as evidence supporting a divergence-before-duplication model of R gene evolution.