Nucleotide-binding Site Leucine-rich Repeat Genes Research Articles

The pan-NLRome analysis based on 23 genomes reveals the diversity of NLRs in Brassica napus.

Brassica napus, a globally significant oilseed crop, exhibits a wide distribution across diverse climatic zones. B. napus is being increasingly susceptible to distinct diseases, such as blackleg, clubroot and sclerotinia stem rot, leading to substantial reductions in yield. Nucleotide-binding site leucine-rich repeat genes (NLRs), the most pivotal family of resistance genes, can be effectively harnessed by identifying and uncovering their diversity to acquire premium disease-resistant gene resources. Here, we collected the genomes of 23 accessions and established the first comprehensive pan-NLRome in B. napus by leveraging multiple genomic resources. We observe significant variation in the number of NLR genes across different B. napus accessions, ranging from 189 to 474. Notably, TNL (TIR-NBS-LRR) genes constitute approximately half of the total count, indicating their predominant presence in B. napus. The number of NLRs in the C subgenome is significantly higher than that in the A subgenome, and chromosome C09 exhibits the highest density of NLR genes with featuring multiple NLR clusters. Domain analysis reveals that the integrated domains significantly enhance the diversity of NLRs, with B3 DNA binding, VQ, and zinc fingers being the most prevalent integrated domains. Pan-genomic analysis reveals that the core type of NLR genes, which is present in most accessions, constitutes approximately 58% of the total NLRs. Furthermore, we conduct a comparative analysis of the diversity of NLR genes across distinct ecotypes, leading to the identification of ecotype-specific NLRs and their integrated domains. In conclusion, our study effectively addresses the limitations of a single reference genome and provides valuable insights into the diversity of NLR genes in B. napus, thereby contributing to disease resistance breeding.

Identification of RppSLN from an Elite Landrace: A Major Locus Conferring Resistance to Southern Corn Rust in Maize (Zea mays L.).

Southern corn rust (SCR) is one of the most destructive foliar diseases in maize (Zea mays L.), resulting in significant yield losses. Therefore, the continuous identification of disease-resistant germplasm and the deployment of resistant hybrids is essential for durably controlling SCR. The objective of this research was to identify and characterize resistance loci against SCR in maize to expand disease management strategies. Here, we identified a maize landrace with high resistance to SCR 'Silunuo' (SLN) approaching complete immunity. We backcrossed it with a susceptible inbred line, N531, to generate a stable SCR-resistant introgression line N531_R. By crossing it with F35 (a susceptible inbred line), we created a large F2 segregating population and mapped a major SCR-resistant locus on chromosome 10, known as RppSLN. Based on the genome assembly and annotation, we found that RppSLN harbors two NBS-LRR (nucleotide binding site-leucine-rich repeat) genes, namely Zmays10G000430 and Zmays10G000440. These NBS-LRR genes were significantly induced during artificial inoculation with Puccinia polysora, suggesting that they might be candidate genes collectively contributing to the resistance level at this locus. In conclusion, this study identified a major SCR resistance locus directly isolated from a landrace, providing valuable support and information for expanding new disease-resistant germplasms and promoting the utilization of landraces.

Genome and transcriptome exploration reveals receptor-like kinases as potential resistance gene analogs against bacterial blight in pomegranate.

Pomegranate (Punica granatum L.) is a tropical fruit crop of pharma-nutritional importance. However, it faces farming challenges due to pests and diseases, particularly bacterial blight and wilt. Developing resistant cultivars is crucial for sustainable pomegranate cultivation, and understanding resistance's genetic basis is essential. We used an extensive resistance gene analogues (RGA) prediction tool to identify 958 RGAs, classified into Nucleotide Binding Site-leucine-rich repeat (NBS-LRR) proteins, receptor-like kinases (RLKs), receptor-like proteins (RLPs), Transmembrane coiled-coil (TM-CC), and nine non-canonical RGAs. RGAs were distributed across all eight chromosomes, with chromosome 02 containing the most RGAs (161), and chromosome 08 having the highest density (4.42 RGA/Mb). NBS-LRR genes were predominantly present on chromosomes 08 and 02, whereas RLKs and RLPs were primarily located on chromosomes 04 and 07. Gene ontology analysis revealed that 475 RGAs were associated with defence against various biotic stresses. Using RNAseq, we identified 120 differentially expressed RGAs, with RLKs (74) being prominent among the differentially expressed genes. The discovery of these RGAs is a significant step towards breeding pomegranates for pest and disease resistance. The differentially expressed RLKs hold promise for developing resistant cultivars against bacterial blight, thereby contributing to the sustainability of pomegranate cultivation.

Genome-Wide Identification of Nucleotide-Binding Site–Leucine-Rich Repeat Gene Family in Cymbidium ensifolium and Expression Profiles in Response to Fusarium Wilt Infection

Genome-Wide Identification of Nucleotide-Binding Site–Leucine-Rich Repeat Gene Family in Cymbidium ensifolium and Expression Profiles in Response to Fusarium Wilt Infection

Cytological and molecular characterization of wheat lines carrying leaf rust and stem rust resistance genes Lr24 and Sr24

Previous studies showed that Australian wheat cultivars Janz and Sunco carry leaf rust and stem rust resistance genes Lr24 and Sr24 derived from Thinopyrum ponticum chromosome arm 3AgL. However, the size of the alien segments carrying Lr24 and Sr24 in the lines were not determined. In this study, we used non-denaturing fluorescence in situ hybridization (ND-FISH), genomic in situ hybridization (GISH), and PCR-based landmark unique gene (PLUG) markers to visualize the alien segments in Janz and Sunco, and further compared them with the segments in US cultivars Agent and Amigo. The fraction length (FL) of the alien translocation in Agent was 0.70–1.00, whereas those in Janz, Sunco, and Amigo were smaller, at FL 0.85–1.00. It was deduced that the alien gene RAg encoding for red grain color and rust resistance genes Lr24 and Sr24 on chromosome arm 3AgL were in bins of FL 0.70–0.85 and 0.85–1.00, respectively. We retrieved and extracted nucleotide-binding site-leucine-rich repeat (NBS-LRR) receptor genes corresponding to the region of Lr24 and Sr24 on chromosomes 3E, and 3J, 3Js and 3St from the reference genome sequences of Th. elongatum and Th. intermedium, respectively. A set of molecular markers developed for Lr24 and Sr24 from those extracted NBS-LRR genes will provide valuable information for fine mapping and cloning of these genes.

Genome-wide characterization of the NBLRR gene family provides evolutionary and functional insights into blast resistance in pearl millet (Cenchrusamericanus (L.) Morrone).

The investigation is the first report on genome-wide identification and characterization of NBLRR genes in pearl millet. We have shown the role of gene loss and purifying selection in the divergence of NBLRRs in Poaceae lineage and candidate CaNBLRR genes for resistance to Magnaporthe grisea infection. Plants have evolved multiple integral mechanisms to counteract the pathogens' infection, among which plant immunity through NBLRR (nucleotide-binding site, leucine-rich repeat) genes is at the forefront. The genome-wide mining in pearl millet (Cenchrus americanus (L.) Morrone) revealed 146 CaNBLRRs. The variation in the branch length of NBLRRs showed the dynamic nature of NBLRRs in response to evolving pathogen races. The orthology of NBLRRs showed a predominance of many-to-one orthologs, indicating the divergence of NBLRRs in the pearl millet lineage mainly through gene loss events followed by gene gain through single-copy duplications. Further, the purifying selection (Ka/Ks < 1) shaped the expansion of NBLRRs within the lineage of pear millet and other members of Poaceae. Presence of cis-acting elements, viz. TCA element, G-box, MYB, SARE, ABRE and conserved motifs annotated with P-loop, kinase 2, RNBS-A, RNBS-D, GLPL, MHD, Rx-CC and LRR suggests their putative role in disease resistance and stress regulation. The qRT-PCR analysis in pearl millet lines showing contrasting responses to Magnaporthe grisea infection identified CaNBLRR20, CaNBLRR33, CaNBLRR46 CaNBLRR51, CaNBLRR78 and CaNBLRR146 as putative candidates. Molecular docking showed the involvement of three and two amino acid residues of LRR domains forming hydrogen bonds (histidine, arginine and threonine) and salt bridges (arginine and lysine) with effectors. Whereas 14 and 20 amino acid residues of CaNBLRR78 and CaNBLRR20 showed hydrophobic interactions with 11 and 9 amino acid residues of effectors, Mg.00g064570.m01 and Mg.00g006570.m01, respectively. The present investigation gives a comprehensive overview of CaNBLRRs and paves the foundation for their utility in pearl millet resistance breeding through understanding of host-pathogen interactions.

The Soybean Rpp3 Gene Encodes a TIR-NBS-LRR Protein that Confers Resistance to Phakopsora pachyrhizi.

Soybean rust is an economically significant disease caused by the fungus Phakopsora pachyrhizi that negatively impacts soybean (Glycine max [L.] Merr.) production throughout the world. Susceptible plants infected by P. pachyrhizi develop tan-colored lesions on the leaf surface that give rise to funnel-shaped uredinia as the disease progresses. While most soybean germplasm is susceptible, seven genetic loci (Rpp1 to Rpp7) that provide race-specific resistance to P. pachyrhizi (Rpp) have been identified. Rpp3 was first discovered and characterized in the soybean accession PI 462312 (Ankur), and it was also determined to be one of two Rpp genes present in PI 506764 (Hyuuga). Genetic crosses with PI 506764 were later used to fine-map the Rpp3 locus to a 371-kb region on chromosome 6. The corresponding region in the susceptible Williams 82 (Wm82) reference genome contains several homologous nucleotide binding site-leucine rich repeat (NBS-LRR) genes. To identify Rpp3, we designed oligonucleotide primers to amplify Rpp3 candidate (Rpp3C) NBS-LRR genes at this locus from PI 462312, PI 506764, and Wm82 using polymerase chain reaction (PCR). Five Rpp3C genes were identified in both Rpp3-resistant soybean lines, and co-silencing these genes compromised resistance to P. pachyrhizi. Gene expression analysis and sequence comparisons of the Rpp3C genes in PI 462312 and PI 506764 suggest that a single candidate gene, Rpp3C3, is responsible for Rpp3-mediated resistance. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2024.

Genome-wide identification and characterization of NBLRR genes in finger millet (Eleusine coracana L.) and their expression in response to Magnaporthe grisea infection

BackgroundThe nucleotide binding site leucine rich repeat (NBLRR) genes significantly regulate defences against phytopathogens in plants. The genome-wide identification and analysis of NBLRR genes have been performed in several species. However, the detailed evolution, structure, expression of NBLRRs and functional response to Magnaporthe grisea are unknown in finger millet (Eleusine coracana (L.) Gaertn.).ResultsThe genome-wide scanning of the finger millet genome resulted in 116 NBLRR (EcNBLRRs1-116) encompassing 64 CC-NB-LRR, 47 NB-LRR and 5 CCR-NB-LRR types. The evolutionary studies among the NBLRRs of five Gramineae species, viz., purple false brome (Brachypodium distachyon (L.) P.Beauv.), finger millet (E. coracana), rice (Oryza sativa L.), sorghum (Sorghum bicolor L. (Moench)) and foxtail millet (Setaria italica (L.) P.Beauv.) showed the evolution of NBLRRs in the ancestral lineage of the target species and subsequent divergence through gene-loss events. The purifying selection (Ka/Ks < 1) shaped the expansions of NBLRRs paralogs in finger millet and orthologs among the target Gramineae species. The promoter sequence analysis showed various stress- and phytohormone-responsive cis-acting elements besides growth and development, indicating their potential role in disease defence and regulatory mechanisms. The expression analysis of 22 EcNBLRRs in the genotypes showing contrasting responses to Magnaporthe grisea infection revealed four and five EcNBLRRs in early and late infection stages, respectively. The six of these nine candidate EcNBLRRs proteins, viz., EcNBLRR21, EcNBLRR26, EcNBLRR30, EcNBLRR45, EcNBLRR55 and EcNBLRR76 showed CC, NB and LRR domains, whereas the EcNBLRR23, EcNBLRR32 and EcNBLRR83 showed NB and LRR somains.ConclusionThe identification and expression analysis of EcNBLRRs showed the role of EcNBLRR genes in assigning blast resistance in finger millet. These results pave the foundation for in-depth and targeted functional analysis of EcNBLRRs through genome editing and transgenic approaches.

Identification, characterization, and validation of NBS-encoding genes in grass pea.

Grass pea is a promising crop with the potential to provide food and fodder, but its genomics has not been adequately explored. Identifying genes for desirable traits, such as drought tolerance and disease resistance, is critical for improving the plant. Grass pea currently lacks known R-genes, including the nucleotide-binding site-leucine-rich repeat (NBS-LRR) gene family, which plays a key role in protecting the plant from biotic and abiotic stresses. In our study, we used the recently published grass pea genome and available transcriptomic data to identify 274 NBS-LRR genes. The evolutionary relationships between the classified genes on the reported plants and LsNBS revealed that 124 genes have TNL domains, while 150 genes have CNL domains. All genes contained exons, ranging from 1 to 7. Ten conserved motifs with lengths ranging from 16 to 30 amino acids were identified. We found TIR-domain-containing genes in 132 LsNBSs, with 63 TIR-1 and 69 TIR-2, and RX-CCLike in 84 LsNBSs. We also identified several popular motifs, including P-loop, Uup, kinase-GTPase, ABC, ChvD, CDC6, Rnase_H, Smc, CDC48, and SpoVK. According to the gene enrichment analysis, the identified genes undergo several biological processes such as plant defense, innate immunity, hydrolase activity, and DNA binding. In the upstream regions, 103 transcription factors were identified that govern the transcription of nearby genes affecting the plant excretion of salicylic acid, methyl jasmonate, ethylene, and abscisic acid. According to RNA-Seq expression analysis, 85% of the encoded genes have high expression levels. Nine LsNBS genes were selected for qPCR under salt stress conditions. The majority of the genes showed upregulation at 50 and 200μM NaCl. However, LsNBS-D18, LsNBS-D204, and LsNBS-D180 showed reduced or drastic downregulation compared to their respective expression levels, providing further insights into the potential functions of LsNBSs under salt stress conditions. They provide valuable insights into the potential functions of LsNBSs under salt stress conditions. Our findings also shed light on the evolution and classification of NBS-LRR genes in legumes, highlighting the potential of grass pea. Further research could focus on the functional analysis of these genes, and their potential use in breeding programs to improve the salinity, drought, and disease resistance of this important crop.

An NBS-LRR protein in the Rpp1 locus negates the dominance of Rpp1-mediated resistance against Phakopsora pachyrhizi in soybean.

The soybean Rpp1 locus confers resistance to Phakopsora pachyrhizi, causal agent of rust, and resistance is usually dominant over susceptibility. However, dominance of Rpp1-mediated resistance is lost when a resistant genotype (Rpp1 or Rpp1b) is crossed with susceptible line TMG06_0011, and the mechanism of this dominant susceptibility (DS) is unknown. Sequencing the Rpp1 region reveals that the TMG06_0011 Rpp1 locus has a single nucleotide-binding site leucine-rich repeat (NBS-LRR) gene (DS-R), whereas resistant PI 594760B (Rpp1b) is similar to PI 200492 (Rpp1) and has three NBS-LRR resistance gene candidates. Evidence that DS-R is the cause of DS was reflected in virus-induced gene silencing of DS-R in Rpp1b/DS-R or Rpp1/DS-R heterozygous plants with resistance partially restored. In heterozygous Rpp1b/DS-R plants, expression of Rpp1b candidate genes was not significantly altered, indicating no effect of DS-R on transcription. Physical interaction of the DS-R protein with candidate Rpp1b resistance proteins was supported by yeast two-hybrid studies and in silico modeling. Thus, we conclude that suppression of resistance most likely does not occur at the transcript level, but instead probably at the protein level, possibly with Rpp1 function inhibited by binding to the DS-R protein. The DS-R gene was found in other soybean lines, with an estimated allele frequency of 6% in a diverse population, and also found in wild soybean (Glycine soja). The identification of a dominant susceptible NBS-LRR gene provides insight into the behavior of NBS-LRR proteins and serves as a reminder to breeders that the dominance of an R gene can be influenced by a susceptibility allele.

Pan-genome and transcriptome analyses provide insights into genomic variation and differential gene expression profiles related to disease resistance and fatty acid biosynthesis in eastern black walnut (Juglans nigra).

Walnut (Juglans) species are used as nut crops worldwide. Eastern black walnut (EBW, Juglans nigra), a diploid, horticultural important woody species is native to much of eastern North America. Although it is highly valued for its wood and nut, there are few resources for understanding EBW genetics. Here, we present a high-quality genome assembly of J. nigra based on Illumina, Pacbio, and Hi-C technologies. The genome size was 540.8Mb, with a scaffold N50 size of 35.1Mb, and 99.0% of the assembly was anchored to 16 chromosomes. Using this genome as a reference, the resequencing of 74 accessions revealed the effective population size of J. nigra declined during the glacial maximum. A single whole-genome duplication event was identified in the J. nigra genome. Large syntenic blocks among J. nigra, Juglans regia, and Juglans microcarpa predominated, but inversions of more than 600kb were identified. By comparing the EBW genome with those of J. regia and J. microcarpa, we detected InDel sizes of 34.9Mb in J. regia and 18.3Mb in J. microcarpa, respectively. Transcriptomic analysis of differentially expressed genes identified five presumed NBS-LRR (NUCLEOTIDE BINDING SITE-LEUCINE-RICH REPEAT) genes were upregulated during the development of walnut husks and shells compared to developing embryos. We also identified candidate genes with essential roles in seed oil synthesis, including FAD (FATTY ACID DESATURASE) and OLE (OLEOSIN). Our work advances the understanding of fatty acid bioaccumulation and disease resistance in nut crops, and also provides an essential resource for conducting genomics-enabled breeding in walnut.

Genome wide identification and evolutionary analysis of vat like NBS-LRR genes potentially associated with resistance to aphids in cotton.

Nucleotide Binding Site - Leucine Rich Repeat (NBS-LRR) genes play a significant role in plant defense against biotic stresses and are an integral part of signal transduction pathways. Vat gene has been well reported for their role in resistance to Aphis gossypii and viruses transmitted by them. Despite their importance, Vat like NBS-LRR resistance genes have not yet been identified and studied in cotton species. This study report hundreds of orthologous Vat like NBS-LRR genes from the genomes of 18 cotton species through homology searches and the distribution of those identified genes were tend to be clustered on different chromosome. Especially, in a majority of the cases, Vat like genes were located on chromosome number 13 and they all shared two conserved NBS-LRR domains, one disease resistant domain and several repeats of LRR on the investigated cotton Vat like proteins. Gene ontology study on Vat like NBS-LRR genes revealed the molecular functions viz., ADP and protein binding. Phylogenetic analysis also revealed that Vat like sequences of two diploid species, viz., G. arboreum and G. anomalum, were closely related to the sequences of the tetraploids than all other diploids. The Vat like genes of G. aridum and G. schwendimanii were distantly related among diploids and tetraploids species. Various hormones and defense related cis-acting regulatory elements were identified from the 2kb upstream sequences of the Vat like genes implying their defensive response towards the biotic stresses. Interestingly, G. arboreum and G. trilobum were found to have more regulatory elements than larger genomes of tetraploid cotton species. Thus, the present study provides the evidence for the evolution of Vat like genes in defense mechanisms against aphids infestation in cotton genomes and allows further characterization of candidate genes for developing aphid and aphid transmitted viruses resistant crops through cotton breeding.

Genome-Wide Identification, Characterization, and Comparative Analysis of NLR Resistance Genes in Coffea spp.

The largest family of disease resistance genes in plants are nucleotide-binding site leucine-rich repeat genes (NLRs). The products of these genes are responsible for recognizing avirulence proteins (Avr) of phytopathogens and triggering specific defense responses. Identifying NLRs in plant genomes with standard gene annotation software is challenging due to their multidomain nature, sequence diversity, and clustered genomic distribution. We present the results of a genome-wide scan and comparative analysis of NLR loci in three coffee species (Coffea canephora, Coffea eugenioides and their interspecific hybrid Coffea arabica). A total of 1311 non-redundant NLR loci were identified in C. arabica, 927 in C. canephora, and 1079 in C. eugenioides, of which 809, 562, and 695 are complete loci, respectively. The NLR-Annotator tool used in this study showed extremely high sensitivities and specificities (over 99%) and increased the detection of putative NLRs in the reference coffee genomes. The NLRs loci in coffee are distributed among all chromosomes and are organized mostly in clusters. The C. arabica genome presented a smaller number of NLR loci when compared to the sum of the parental genomes (C. canephora, and C. eugenioides). There are orthologous NLRs (orthogroups) shared between coffee, tomato, potato, and reference NLRs and those that are shared only among coffee species, which provides clues about the functionality and evolutionary history of these orthogroups. Phylogenetic analysis demonstrated orthologous NLRs shared between C. arabica and the parental genomes and those that were possibly lost. The NLR family members in coffee are subdivided into two main groups: TIR-NLR (TNL) and non-TNL. The non-TNLs seem to represent a repertoire of resistance genes that are important in coffee. These results will support functional studies and contribute to a more precise use of these genes for breeding disease-resistant coffee cultivars.

Genome-wide characterization of NBS-LRR family genes and expression analysis under powdery mildew stress in Lagenaria siceraria

As the largest group of plant resistance genes, the nucleotide-binding site–leucine-rich repeat (NBS-LRR) genes play a critical role in plant disease defense system. The bottle gourd is a distinctive horticultural crop of the Cucurbitaceae family and powdery mildew is one of the major diseases jeopardizing crop production and quality. In this study, we have identified 84 NBS-LRR genes and divided into seven subfamilies. Phylogenetic tree and motif analysis grouped these genes into two major clusters with toll/interleukin-1 like receptor (TIR) domain or a coiled-coil domain. Subsequently, duplication events, motif sequence, orthologous gene cluster, protein-protein interaction, cis-elements were analyzed. 12 pairs of tandem duplicated genes and two pairs of segmental duplicated genes were detected in NBS-LRR family. 14 singleton genes and 62 functional cis-elements were found. The structure of 27 proteins were constructed by homology modeling and divided into five different types. 34 differentially expressed genes of NBS-LRR genes were found in PM-resistant and PM-susceptible line. Expression of NBS-LRR genes were validated by qRT-PCR under PM stress and gene Lsi04g015960 with powdery mildew8 (RPW8) domain was significantly up-regulated in PM-resistant variety. Our study provides fundamental insights of NBS-LRR family genes in bottle gourd and find a potential candidate gene Lsi04g015960 for PM tolerance breeding.

In-silico genome-wide identification and characterisation of NBS-LRR gene family in banana Musa acuminata

Various common diseases caused by bacteria and viruses are increasing threats to the production of bananas worldwide. Besides the practical application of cultivation techniques, intensive exploitation of resistant genes, most of which belong to the NBS-LRR(Nucleotide-binding site - Leucine-rich repeat) gene family, in fortifying banana’s disease resistance, has been considered as a sustainable approach. The recently published genome assembly of Musa acuminata, a species of the Musa genus native to Southern Asia, has facilitated the genome-wide identification and characterisation of NBS-LRR genes. In the present study, an in-silicoapproach was employed to identify and characterise 97 NBS-LRR genes and their evolution in M. acuminatagenome. Except Ma_NBS_083 belonging to the RNLsub-family, other identified genes are members of the sub-family CNL and most of them contain only one exon. Sixty-six (66) NBS-LRR genes (68%) were mapped on 11 banana chromosomes, among them 38 genes (39%) located in clusters. Phylogenetic analysis in combination with conserved motif identification classified the banana NBS-LRR genes into seven groups. This study provides novel insight into the NBS-LRR gene family in bananas as a potential resource for further research in functional evaluation and utilisation of resistant genes.

NLR diversity and candidate fusiform rust resistance genes in loblolly pine

Resistance to fusiform rust disease in loblolly pine (Pinus taeda) is a classic gene-for-gene system. Early resistance gene mapping in the P. taeda family 10-5 identified RAPD markers for a major fusiform rust resistance gene, Fr1. More recently, single nucleotide polymorphism (SNP) markers associated with resistance were mapped to a full-length gene model in the loblolly pine genome encoding for a nucleotide-binding site leucine-rich repeat (NLR) protein. NLR genes are one of the most abundant gene families in plant genomes and are involved in effector-triggered immunity. Inter- and intraspecies studies of NLR gene diversity and expression have resulted in improved disease resistance. To characterize NLR gene diversity and discover potential resistance genes, we assembled de novo transcriptomes from 92 loblolly genotypes from across the natural range of the species. In these transcriptomes, we identified novel NLR transcripts that are not present in the loblolly pine reference genome and found significant geographic diversity of NLR genes providing evidence of gene family evolution. We designed capture probes for these NLRs to identify and map SNPs that stably cosegregate with resistance to the SC20-21 isolate of Cronartium quercuum f.sp. fusiforme (Cqf) in half-sib progeny of the 10-5 family. We identified 10 SNPs and 2 quantitative trait loci associated with resistance to SC20-21 Cqf. The geographic diversity of NLR genes provides evidence of NLR gene family evolution in loblolly pine. The SNPs associated with rust resistance provide a resource to enhance breeding and deployment of resistant pine seedlings.

NLR1 is a strong candidate for the Rm3 dominant green peach aphid (Myzus persicae) resistance trait in peach.

The green peach aphid (GPA), Myzus persicae, is a polyphagous, sap-sucking aphid and a vector of many plant viruses. In peach, Prunus persica, three individual dominant GPA resistance loci have been genetically defined (Rm1-3), but knowledge of the underlying genes is limited. In this study, we focused on the Rm3 locus. Bulk segregant analysis (BSA) mapping in segregating progeny populations delimited Rm3 to an interval spanning 160kb containing 21 genes on chromosome 1. RNA-seq data provided no evidence of candidate genes, but chromosomal structural variations were predicted around a nucleotide-binding site-leucine-rich repeat (NLR) gene (ppa000596m) within the Rm3 fine-mapping interval. Following bacterial artificial chromosome (BAC) library construction for a GPA-resistant peach cultivar and the sequencing of three target BAC clones, a chromosomal structural variation encompassing two novel TIR-NLR-class disease resistance (R) protein-coding genes was identified, and the expressed NLR gene (NLR1) was identified as a candidate for M. persicae resistance. Consistent with its proposed role in controlling GPA resistance, NLR1 was only expressed in the leaves of resistant peach phenotypes. A molecular marker that was designed based on the NLR1 sequence co-segregated with the GPA-resistant phenotype in four segregating populations, 162 peach cultivars, and 14 wild relatives, demonstrating the dominant inheritance of the Rm3 locus. Our findings can be exploited to facilitate future breeding for GPA-resistance in peach.

In-Silico Characterization and Expression Analysis of NB-ARC Genes in Response to Erwinia mallotivora in Carica papaya

Disease resistance in plants is commonly associated with resistance (R) genes that encode nucleotide binding site-leucine rich repeat (NBS-LRR) domains that are essential for pathogen recognition and defence signalling. In this study, we identified and analyzed the sequence of putative pathogen-responsive NB-ARC transcripts from Carica papaya transcriptome database, carried out the structural and phylogenetic analysis, and determined the expression profile of the transcripts in C. papaya challenged with Erwinia mallotivora. The findings indicate CpNBS1, the only pathogen-responsive NB-ARC protein identified in this study belongs to the CC-NBS-LRR group. Semi-quantitative PCR showed CpNBS1 was differentially expressed in response to E. mallotivora. Structural analysis of the 4993-Eksotika and 4993-Viorica translated proteins showed striking differences in terms of the number of β-sheets and α-helixes as well their ligand-binding surface, suggesting the role of the LRR domain in determining the specificity of recognition of E. mallotivora effector. Collectively, this study provides new insights into the role of NBS-LRR genes in C. papaya and its implications for enhancing of plant disease resistance through genetic engineering.

High-quality chromosome-level genomes of Cucumis metuliferus and Cucumis melo provide insight into Cucumis genome evolution.

Cucumis metuliferus (African horned cucumber), a wild relative of Cucumis sativus (cucumber) and Cucumis melo (melon), displays high-level resistance to several important plant pathogens (e.g., root-knot nematodes and several viruses). Here, we report a chromosome-level genome assembly for C. metuliferus, with a 316Mb genome sequence comprising 29039 genes. Phylogenetic analysis of related species in family Cucurbitaceae indicated that the divergence time between C. metuliferus and melon was 17.8 million years ago. Comparisons between the C. metuliferus and melon genomes revealed large structural variations (inversions and translocations >1Mb) in eight chromosomes of these two species. Gene family comparison showed that C. metuliferus has the largest number of resistance-related nucleotide-binding site leucine-rich repeat (NBS-LRR) genes in Cucurbitaceae. The loss of NBS-LRR loci caused by large insertions or deletions (indels) and pseudogenization caused by small indels explained the loss of NBS-LRR genes in Cucurbitaceae. Population structure analysis suggested that C. metuliferus originated in Zimbabwe, then spread to other southern African regions where it likely underwent similar domestic selection as melon. This C. metuliferus reference sequence will accelerate the understanding of the molecular evolution of resistance-related genes and enhance cucurbit crop improvement efforts.

Building a cluster of NLR genes conferring resistance to pests and pathogens: the story of the Vat gene cluster in cucurbits

Most molecularly characterized plant resistance genes (R genes) belong to the nucleotide-binding-site-leucine-rich-repeat (NLR) receptor family and are prone to duplication and transposition with high sequence diversity. In this family, the Vat gene in melon is one of the few R genes known for conferring resistance to insect, i.e., Aphis gossypii, but it has been misassembled and/or mispredicted in the whole genomes of Cucurbits. We examined 14 genomic regions (about 400 kb) derived from long-read assemblies spanning Vat-related genes in Cucumis melo, Cucumis sativus, Citrullus lanatus, Benincasa hispida, Cucurbita argyrosperma, and Momordica charantia. We built the phylogeny of those genes. Investigating the paleohistory of the Vat gene cluster, we revealed a step by step process beginning from a common ancestry in cucurbits older than 50 my. We highlighted Vat exclusively in the Cucumis genera, which diverged about 20 my ago. We then focused on melon, evaluating a minimum duplication rate of Vat in 80 wild and cultivated melon lines using generalist primers; our results suggested that duplication started before melon domestication. The phylogeny of 44 Vat-CDS obtained from 21 melon lines revealed gain and loss of leucine-rich-repeat domains along diversification. Altogether, we revealed the high putative recognition scale offered in melon based on a combination of SNPs, number of leucine-rich-repeat domains within each homolog and number of homologs within each cluster that might jointly confer resistance to a large pest and pathogen spectrum. Based on our findings, we propose possible avenues for breeding programs.