Genetic diversity and genomic distribution of homologs encoding NBS-LRR disease resistance proteins in sunflower

Three-fourths of the recognition-dependent disease resistance genes (R-genes) identified in plants encode nucleotide binding site (NBS) leucine-rich repeat (LRR) proteins. NBS-LRR homologs have only been isolated on a limited scale from sunflower (Helianthus annuus L.), and most of the previously identified homologs are members of two large NBS-LRR clusters harboring downy mildew R-genes. We mined the sunflower EST database and used comparative genomics approaches to develop a deeper understanding of the diversity and distribution of NBS-LRR homologs in the sunflower genome. Collectively, 630 NBS-LRR homologs were identified, 88 by mining a database of 284,241 sunflower ESTs and 542 by sequencing 1,248 genomic DNA amplicons isolated from common and wild sunflower species. DNA markers were developed from 196 unique NBS-LRR sequences and facilitated genetic mapping of 167 NBS-LRR loci. The latter were distributed throughout the sunflower genome in 44 clusters or singletons. Wild species ESTs were a particularly rich source of novel NBS-LRR homologs, many of which were tightly linked to previously mapped downy mildew, rust, and broomrape R-genes. The DNA sequence and mapping resources described here should facilitate the discovery and isolation of recognition-dependent R-genes guarding sunflower from a broad spectrum of economically important diseases.     

Genome-wide analysis of defense-responsive genes in bacterial blight resistance of rice mediated by the recessive<Emphasis Type="Italic"> R</Emphasis> gene<Emphasis Type="Italic"> xa13</Emphasis>

Defense responses triggered by dominant and recessive disease resistance ( R) genes are presumed to be regulated by different molecular mechanisms. In order to characterize the genes activated in defense responses against bacterial blight mediated by the recessive R gene xa13, two pathogen-induced subtraction cDNA libraries were constructed using the resistant rice line IRBB13—which carries xa13 —and its susceptible, near-isogenic, parental line IR24. Clustering analysis of expressed sequence tags (ESTs) identified 702 unique expressed sequences as being involved in the defense responses triggered by xa13; 16% of these are new rice ESTs. These sequences define 702 genes, putatively encoding a wide range of products, including defense-responsive genes commonly involved in different host-pathogen interactions, genes that have not previously been reported to be associated with pathogen-induced defense responses, and genes (38%) with no homology to previously described functional genes. In addition, R -like genes putatively encoding nucleotide-binding site/leucine rich repeat (NBS-LRR) and LRR receptor kinase proteins were observed to be induced in the disease resistance activated by xa13. A total of 568 defense-responsive ESTs were mapped to 588 loci on the rice molecular linkage map through bioinformatic analysis. About 48% of the mapped ESTs co-localized with quantitative trait loci (QTLs) for resistance to various rice diseases, including bacterial blight, rice blast, sheath blight and yellow mottle virus. Furthermore, some defense-responsive sequences were conserved at similar locations on different chromosomes. These results reveal the complexity of xa13 -mediated resistance. The information obtained in this study provides a large source of candidate genes for understanding the molecular bases of defense responses activated by recessive R genes and of quantitative disease resistance.Electronic Supplementary Material Supplementary material is available in the online version of this article at The first two authors contributed equally to this workCommunicated by R. Hagemann     

Survey and characterization of NBS-LRR (R) genes in Curcuma longa transcriptome

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.     

Characterization of disease resistance gene candidates of the nucleotide binding site (NBS) type from banana and correlation of a transcriptional polymorphism with resistance to <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f.sp. <Emphasis Type="Italic">cubense</Emphasis> race 4

Most plant disease resistance (R) genes encode proteins with a nucleotide binding site and leucine-rich repeat structure (NBS-LRR). In this study, degenerate primers were used to amplify genomic NBS-type sequences from wild banana (Musa acuminata ssp. malaccensis) plants resistant to the fungal pathogen Fusarium oxysporum formae specialis (f. sp.) cubense (FOC) race 4. Five different classes of NBS-type sequences were identified and designated as resistance gene candidates (RGCs). The deduced amino acid sequences of the RGCs revealed the presence of motifs characteristic of the majority of known plant NBS-LRR resistance genes. Structural and phylogenetic analyses grouped the banana RGCs within the non-TIR (homology to Toll/interleukin-1 receptors) subclass of NBS sequences. Southern hybridization showed that each banana RGC is present in low copy number. The expression of the RGCs was assessed by RT-PCR in leaf and root tissues of plants resistant or susceptible to FOC race 4. RGC1, 3 and 5 showed a constitutive expression profile in both resistant and susceptible plants whereas no expression was detected for RGC4. Interestingly, RGC2 expression was found to be associated only to FOC race 4 resistant lines. This finding could assist in the identification of a FOC race 4 resistance gene.     

Differential proteomic analysis of Trichoplusia ni cells after continuous selection with activated Cry1Ac toxin

Development of insect resistance to Bacillus thuringiensis (Bt) toxins threatens the sustained successful application of Bt-based biological control tactics. Multi-mechanisms of resistance have been proposed, such as alteration of toxin-binding proteins, changes of proteases in midgut and so on. The other responses of the Cry1Ac-selected insects might also contribute to the evolution of resistance. Here, the Cry1Ac-selected Trichoplusia ni TnH5 cells with high resistance were subjected to analysis of proteome and the differentially expressed proteins were identified using mass spectrometry. The differential proteins included transporter, molecular chaperon, structural molecules and many other molecules involved in protein metabolism, signal transduction, nucleotide binding, lipid biosynthesis, carbohydrates metabolism and energy production, suggesting that a complex mechanisms involved in the development of insect resistance to Bt Cry1Ac toxins at cellular levels. The decrease of protein synthesis, changes of signal transduction, more rapid energy production, the enhanced lipid synthesis and the decline of possible Cry1Ac-binding proteins in cytoplasm and other events might contribute to the development of resistance in the selected cells. Our results provide some new cues for understanding the mechanism of Bt resistance.     

Barley disease resistance gene analogs of the NBS-LRR class: identification and mapping

The majority of verified plant disease resistance genes isolated to date are of the NBS-LRR class, encoding proteins with a predicted nucleotide binding site (NBS) and a leucine-rich repeat (LRR) region. We took advantage of the sequence conservation in the NBS motif to clone, by PCR, gene fragments from barley representing putative disease resistance genes of this class. Over 30 different resistance gene analogs (RGAs) were isolated from the barley cultivar Regatta. These were grouped into 13 classes based on DNA sequence similarity. Actively transcribed genes were identified from all classes but one, and cDNA clones were isolated to derive the complete NBS-LRR protein sequences. Some of the NBS-LRR genes exhibited variation with respect to whether and where particular introns were spliced, as well as frequent premature polyadenylation. DNA sequences related to the majority of the barley RGAs were identified in the recently expanded public rice genomic sequence database, indicating that the rice sequence can be used to extract a large proportion of the RGAs from barley and other cereals. Using a combination of RFLP and PCR marker techniques, representatives of all barley RGA gene classes were mapped in the barley genome, to all chromosomes except 4H. A number of the RGA loci map in the vicinity of known disease resistance loci, and the association between RGA S-120 and the nematode resistance locus Ha2 on chromosome 2H was further tested by co-segregation analysis. Most of the RGA sequences reported here have not been described previously, and represent a useful resource as candidates or molecular markers for disease resistance genes in barley and other cereals.     

The Absence of TIR-Type Resistance Gene Analogues in the Sugar Beet (Beta vulgaris L.) Genome

The majority of known plant resistance genes encode proteins with conserved nucleotide-binding sites and leucine-rich repeats (NBS-LRR). Degenerate primers based on conserved NBS-LRR motifs were used to amplify analogues of resistance genes from the dicot sugar beet. Along with a cDNA library screen, the PCR screen identified 27 genomic and 12 expressed NBS-LRR RGAs (nlRGAs) sugar beet clones. The clones were classified into three subfamilies based on nucleotide sequence identity. Sequence analyses suggested that point mutations, such as nucleotide substitutions and insertion/deletions, are probably the primary source of diversity of sugar beet nlRGAs. A phylogenetic analysis revealed an ancestral relationship among sugar beet nlRGAs and resistance genes from various angiosperm species. One group appeared to share the same common ancestor as Prf, Rx, RPP8, and Mi, whereas the second group originated from the ancestral gene from which 12C1, Xa1, and Cre3 arose. The predicted protein products of the nlRGAs isolated in this study are all members of the non-TIR-type resistance gene subfamily and share strong sequence and structural similarities with non-TIR-type resistance proteins. No representatives of the TIR-type RGAs were detected either by PCR amplification using TIR type-specific primers or by in silico screening of more than 16,000 sugar beet ESTs. These findings suggest that TIR type of RGAs is absent from the sugar beet genome. The possible evolutionary loss of TIR type RGAs in the sugar beet is discussed. These authors (Yanyan Tian, Longjiang Fan) contributed equally to this work.     

Polyphenoloxidase activity in coffee leaves and its role in resistance against the coffee leaf miner and coffee leaf rust

In plants, PPO has been related to defense mechanism against pathogens and insects and this role was investigated in coffee trees regarding resistance against a leaf miner and coffee leaf rust disease. PPO activity was evaluated in different genotypes and in relation to methyl-jasmonate (Meja) treatment and mechanical damage. Evaluations were also performed using compatible and incompatible interactions of coffee with the fungus Hemileia vastatrix (causal agent of the leaf orange rust disease) and the insect Leucoptera coffeella (coffee leaf miner). The constitutive level of PPO activity observed for the 15 genotypes ranged from 3.8 to 88 units of activity/mg protein. However, no direct relationship was found with resistance of coffee to the fungus or insect. Chlorogenic acid (5-caffeoylquinic acid), the best substrate for coffee leaf PPO, was not related to resistance, suggesting that oxidation of other phenolics by PPO might play a role, as indicated by HPLC profiles. Mechanical damage, Meja treatment, H. vastatrix fungus inoculation and L. coffeella infestation caused different responses in PPO activity. These results suggest that coffee resistance may be related to the oxidative potential of the tissue regarding the phenolic composition rather than simply to a higher PPO activity.     

The tomato R gene products I-2 and MI-1 are functional ATP binding proteins with ATPase activity

Most plant disease resistance (R) genes known today encode proteins with a central nucleotide binding site (NBS) and a C-terminal Leu-rich repeat (LRR) domain. The NBS contains three ATP/GTP binding motifs known as the kinase-1a or P-loop, kinase-2, and kinase-3a motifs. In this article, we show that the NBS of R proteins forms a functional nucleotide binding pocket. The N-terminal halves of two tomato R proteins, I-2 conferring resistance to Fusarium oxysporum and Mi-1 conferring resistance to root-knot nematodes and potato aphids, were produced as glutathione S-transferase fusions in Escherichia coli. In a filter binding assay, purified I-2 was found to bind ATP rather than other nucleoside triphosphates. ATP binding appeared to be fully dependent on the presence of a divalent cation. A mutant I-2 protein containing a mutation in the P-loop showed a strongly reduced ATP binding capacity. Thin layer chromatography revealed that both I-2 and Mi-1 exerted ATPase activity. Based on the strong conservation of NBS domains in R proteins of the NBS-LRR class, we propose that they all are capable of binding and hydrolyzing ATP.     

植物TIR-NB-LRR类型抗病基因各结构域的研究进展

植物抗病反应是一个多基因调控的复杂过程,在这个过程中R基因发挥了非常重要的作用。根据其氨基酸基序组成以及跨膜结构域的不同,R基因可以分为多种类型,其中NBS-LRR类型是植物基因组中最大的基因家族之一。TIR-NB-LRR类型的抗病基因又是NB-LRR类型中的一大类,也是目前抗病基因研究的热点。该文总结了TIR-NB-LRR类型抗病基因各个结构域的功能和相关的研究进展。相关研究表明,TIR结构域主要通过自身或异源的二聚体化介导抗性信号的转导,但也有部分研究表明,该结构域可能参与病原菌的特异性识别。NBS结构域常被认为具有"分子开关"的功能,它可以通过结合ADP或ATP来调节植物抗病蛋白的构象变化,从而调节下游抗病信号的传导。LRR结构域在植物与病原菌互作的过程中可以通过与病原菌的无毒蛋白直接或间接互作来特异识别病原菌。也有研究发现,LRR结构域具有调节信号传导的功能。这些信息将为研究植物抗病机理提供理论依据,也为将来通过基因编辑技术对作物进行定向抗病育种提供思路。     

Perspectives of genomic diversification and molecular recombination towards R-gene evolution in plants

Plants are under strong evolutionary pressure in developing new and noble R genes to recognize pathogen avirulence (avr) determinants and bring about stable defense for generation after generations. Duplication, sequence variation by mutation, disparity in the length and structure of leucine rich repeats etc., causes tremendous variations within and among R genes in a plant thereby developing diverse recognitional specificity suitable enough for defense against new pathogens. Recent studies on genome sequencing, diversity and population genetics in different plants have thrown new insights on the molecular evolution of these genes. Tandem and segmental duplication are important factors in R gene abundance as inferred from the distribution of major nucleotide binding site-leucine rich repeats (NBS-LRRs) type R-genes in plant genomes. Likewise, R-gene evolution is also thought to be facilitated by cluster formation thereby causing recombination and sequence exchange and resulting in haplotypic diversity. Population studies have further proven that balancing selection is responsible for the maintenance of allelic diversity in R genes. In this review, we emphasize and discuss on improved perspectives towards the molecular mechanisms and selection pressure responsible for the evolution of NBS-LRR class resistance genes in plants.     

Isolation,genetic variation and expression of TIR-NBS-LRR resistance gene analogs from western white pine (<Emphasis Type="Italic">Pinus monticola </Emphasis>Dougl. ex. D. Don.)

Western white pine (Pinus monticola Dougl. ex. D. Don., WWP) shows genetic variation in disease resistance to white pine blister rust (Cronartium ribicola). Most plant disease resistance (R) genes encode proteins that belong to a superfamily with nucleotide-binding site domains (NBS) and C-terminal leucine-rich repeats (LRR). In this work a PCR strategy was used to clone R gene analogs (RGAs) from WWP using oligonucleotide primers based on the conserved sequence motifs in the NBS domain of angiosperm NBS-LRR genes. Sixty-seven NBS sequences were cloned from disease-resistant trees. BLAST searches in GenBank revealed that they shared significant identity to well-characterized R genes from angiosperms, including L and M genes from flax, the tobacco N gene and the soybean gene LM6. Sequence alignments revealed that the RGAs from WWP contained the conserved motifs identified in angiosperm NBS domains, especially those motifs specific for TIR-NBS-LRR proteins. Phylogenic analysis of plant R genes and RGAs indicated that all cloned WWP RGAs can be grouped into one major branch together with well-known R proteins carrying a TIR domain, suggesting they belong to the subfamily of TIR-NBS-LRR genes. In one phylogenic tree, WWP RGAs were further subdivided into fourteen clusters with an amino acid sequence identity threshold of 75%. cDNA cloning and RT-PCR analysis with gene-specific primers demonstrated that members of 10 of the 14 RGA classes were expressed in foliage tissues, suggesting that a large and diverse NBS-LRR gene family may be functional in conifers. These results provide evidence for the hypothesis that conifer RGAs share a common origin with R genes from angiosperms, and some of them may play important roles in defense mechanisms that confer disease resistance in western white pine. Ratios of non-synonymous to synonymous nucleotide substitutions (Ka/Ks) in the WWP NBS domains were greater than 1 or close to 1, indicating that diversifying selection and/or neutral selection operate on the NBS domains of the WWP RGA family.Communicated by R. Hagemann     

Genome-wide association mapping of quantitative resistance to sudden death syndrome in soybean

Background

Sudden death syndrome (SDS) is a serious threat to soybean production that can be managed with host plant resistance. To dissect the genetic architecture of quantitative resistance to the disease in soybean, two independent association panels of elite soybean cultivars, consisting of 392 and 300 unique accessions, respectively, were evaluated for SDS resistance in multiple environments and years. The two association panels were genotyped with 52,041 and 5,361 single nucleotide polymorphisms (SNPs), respectively. Genome-wide association mapping was carried out using a mixed linear model that accounted for population structure and cryptic relatedness.

Result

A total of 20 loci underlying SDS resistance were identified in the two independent studies, including 7 loci localized in previously mapped QTL intervals and 13 novel loci. One strong peak of association on chromosome 18, associated with all disease assessment criteria across the two panels, spanned a physical region of 1.2 Mb around a previously cloned SDS resistance gene (GmRLK18-1) in locus Rfs2. An additional variant independently associated with SDS resistance was also found in this genomic region. Other peaks were within, or close to, sequences annotated as homologous to genes previously shown to be involved in plant disease resistance. The identified loci explained an average of 54.5% of the phenotypic variance measured by different disease assessment criteria.

Conclusions

This study identified multiple novel loci and refined the map locations of known loci related to SDS resistance. These insights into the genetic basis of SDS resistance can now be used to further enhance durable resistance to SDS in soybean. Additionally, the associations identified here provide a basis for further efforts to pinpoint causal variants and to clarify how the implicated genes affect SDS resistance in soybean.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-809) contains supplementary material, which is available to authorized users.