[Show abstract][Hide abstract] ABSTRACT: The rpg4 gene confers recessive resistance to several races of wheat stem rust (Puccinia graminis f. sp. tritici) and Rpg5 provides dominant resistance against isolates of the rye stem rust (Puccinia graminis f. sp. secalis) in barley. The rpg4 and Rpg5 genes are tightly linked on chromosome 5H and positional cloning using high-resolution populations clearly separated the genes, unambiguously identifying Rpg5 but the identity of rpg4 remained unclear. High-resolution genotyping of critical recombinants at the rpg4/Rpg5 locus, designated here as RMRL (rpg4-mediated resistance locus) delimited two distinct yet tightly linked loci required for resistance, designated as RMRL1 and RMRL2. Utilizing virus-induced gene silencing, each gene at RMRL1, HvRga1 (a nucleotide binding site-leucine rich repeat (NBS-LRR) domain gene), Rpg5 (an NBS-LRR-protein kinase domain gene) and HvAdf3 (an actin depolymerizing factor-like gene), were individually silenced followed by inoculation with Pgt race QCCJ. Silencing each gene changed the reaction type from incompatible to compatible, indicating that all three genes are required for rpg4-mediated resistance. This stem rust resistance mechanism in barley follows the emerging theme of unrelated pairs of genetically linked NBS-LRR genes required for specific pathogen recognition and resistance. It also appears that actin cytoskeleton dynamics may play an important role in determining resistance against several races of stem rust in barley.
[Show abstract][Hide abstract] ABSTRACT: RIN4 is a signaling molecule that plays a key role in disease resistance responses in plants. Barley RIN4 (HvRIN4) was first identified as an interactor of the stem rust resistance protein RPG1 in a yeast two-hybrid system. Therefore, we isolated, characterized and studied HvRIN4 role in barley. Yeast two hybrid analysis, employing several deletion mutants of HvRIN4 revealed that the C-terminal end and a partial deletion of N-terminal end of HvRIN4 are important for interacting with RPG1. HvRIN4 interactions with RPG1 mutated proteins, which inactivate autophosphorylation, suggested that autophosphorylation or the amino acids required for autophosphorylation of RPG1 are essential for interaction.
[Show abstract][Hide abstract] ABSTRACT: Rpg1 is a valuable resistance gene that has protected barley from serious yield losses due to stem rust (Puccinia graminis f. sp. tritici [Pgt]) for over 60 years in the Upper Midwest region of the United States. In previous research, this gene was cloned from cultivar (cv.) Morex and its function validated by transforming the susceptible barley cv. Golden Promise into a resistant cultivar. A single copy of Rpg1 was sufficient to confer stem rust resistance, but we identified high-copy transgenic lines (with four and five copies of Rpg1) that were susceptible at both the seedling and adult plant stages. To elucidate the basis of these susceptible reactions, we selected six transgenic lines with variable copy numbers of Rpg1 and tested them for level of RPG1 protein accumulation, RPG1 protein degradation upon inoculation with the avirulent pathotype Pgt-MCCF, RPG1 phosphorylation in response to the same avirulent pathotype, and elicitation of a hypersensitive response upon infiltration with Pgt effectors RGD-binding protein and VPS9 protein. The stem rust resistant transgenic lines with one and two copies of Rpg1 (G04-271, G04-273 G04-266, and G03-210) reacted very similar to the positive control of cv. Morex with respect to all of the assays. In contrast, the behavior of the stem rust susceptible high-copy lines (G04-287 with four copies and G04-288 with five copies) was different in several respects. First, the level of RPG1 protein was about 1.5–3.0× higher than in the resistant lines and cv. Morex. Second, the protein failed to degrade, unlike the case with the resistant lines and cv. Morex where RPG1 was completely degraded by 28 hpi. Third, the high-copy lines failed to elicit a hypersensitive response after infiltration with the Pgt effectors, whereas the resistant lines did. RPG1 protein from the high-copy transgenic lines appeared to behave normally with respect to its ability to phosphorylate in vivo. However, these two lines over produced apparently functional RPG1 protein, but failed to degrade it normally. Stem rust susceptibility and failure to respond to avirulence factors by the high-copy Rpg1 transgenic lines G04-287 and G04-288 are probably due to the failure to degrade the RPG1 protein.
[Show abstract][Hide abstract] ABSTRACT: The barley stem rust resistance gene Reaction to Puccinia graminis 1 (Rpg1), encoding a receptor-like kinase, confers durable resistance to the stem rust pathogen Puccinia graminis f. sp. tritici. The fungal urediniospores form adhesion structures with the leaf epidermal cells within 1 h of inoculation, followed by hyphae and haustorium formation. The RPG1 protein is constitutively expressed and not phosphorylated. On inoculation with avirulent urediniospores, it is phosphorylated in vivo within 5 min and subsequently degraded. Application of arginine-glycine-aspartic acid peptide loops prevented the formation of adhesion structures for spore attachment, the phosphorylation of RPG1, and germination of the viable spores. Arginine-glycine-aspartic acid affinity chromatography of proteins from the ungerminated avirulent rust spores led to the purification and identification of a protein with fibronectin type III and breast cancer type 1 susceptibility protein domains and a vacuolar protein sorting-associated protein 9 with a coupling of ubiquitin to endoplasmic reticulum degradation domain. Both proteins are required to induce in vivo phosphorylation and degradation of RPG1. Combined application of both proteins caused hypersensitive reaction on the stem rust-resistant cultivar Morex but not on the susceptible cultivar Steptoe. Expression studies indicated that mRNA of both genes are present in ungerminated urediniospores and are constitutively transcribed in sporelings, infected leaves, and haustoria in the investigated avirulent races. Evidence is presented that RPG1, in yeast, interacts with the two protein effectors from the urediniospores that activate cooperatively the stem rust resistance protein RPG1 long before haustoria formation.
Proceedings of the National Academy of Sciences 08/2011; 108(35):14676-81. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Previously, we cloned telomere-associated sequences using Vectorette-PCR and mapped the most telomeric regions of barley chromosome arms 1S (7HS), 2S (2HS), and 4S (4HS) (Kilian and Kleinhofs 1992). Here we report mapping 33 different telomere-associated markers (21 RFLPs and 12 PCR-based) identifying the most telomeric region of 10 out of the 14 barley chromosome arms. Other telomere-associated markers mapped very close, but not at the telomere, or were interstitial. Four markers mapped at centromeric regions. Two markers, generated by PCR using a single primer based on the sequence of the barley telomere repeats, mapped to the end of barley chromosome 7L (5HL) and internally on chromosome 3L (3HL). Cloning and sequencing of PCR products from the 3L (3HL) interstitial location revealed homology to the HvRT family. Several low copy number sequences physically linked to HvRTs were also cloned and used as RFLP probes. These probes often mapped close to the ends of linkage groups. A few RFLPs mapped at centromeric regions in agreement with the result of in situ hybridization of HvRT clones showing positive signal at the ends and around centromeres of barley chromosomes.Key words: Hordeum vulgare, mapping, telomeres, telomere-associated sequences.
[Show abstract][Hide abstract] ABSTRACT: The barley (Hordeum vulgare L.) chromosome 1 centromere region contains two adjacent overlapping quantitative trait loci (QTLs) for malting quality traits, and the chromosome 7L subtelomere region contains the stem rust (causal agent Puccinia graminis f.sp. tritici) resistance gene rpg4. To facilitate the saturation mapping of these two target regions, a synteny-based approach was employed. Syntenic relationships between the barley target regions and the rice (Oryza sativa) genome were established through comparative mapping. The barley chromosome 1 centromere region was found to be syntenic with rice chromosome 8 and parts of rice chromosomes 3 and 10. A 6- to 15-fold difference in genetic distance between barley and rice in the syntenic region was observed, owing to the apparent suppressed recombination in the barley chromosome 1 centromere region. Barley chromosome 7L was found to be syntenic with rice chromosome 3. The establishment of synteny with rice in the two target regions allows well-established and characterized rice resources to be utilized in fine mapping and map-based cloning studies.Key words: genome synteny, quantitative trait loci, QTL, disease resistance gene, Triticeae.
[Show abstract][Hide abstract] ABSTRACT: The chomosomal location of a glutathione S-transferase (GST) gene was determined in both hexaploid wheat (Triticum aestivum) and barley (Hordeum vulgare). The GST cDNA used to map the gene was cloned from the diploid wheat Triticum tauschii. GST loci were located on the short arms of chromosomes 6A, 6B, and 6D in T. aestivum and also on the short arm of chromosome 6H in H. vulgare. The GST locus in barley was absolutely linked to the RFLP marker E148A and was located 0.8 cM proximal to the RFLP marker ABC169B on barley chromosome 6H. At least two copies of the GST gene were present in each of the T. aestivum A, B, and D genomes, and a homologous GST gene was present as a single-copy gene in the barley genome. GST mRNA transcripts were not detected in RNA isolated from shoots of control (unsafened) seedlings of T. tauschii or T. aestivum. It was determined that the expression of the GST gene was regulated by herbicide safener treatment in T. tauschii and T. aestivum by detecting safener-increased GST mRNA transcript levels.Key words: Triticum aestivum, Triticum tauschii, Hordeum vulgare, herbicide safener, glutathione S-transferase, genetic mapping.
[Show abstract][Hide abstract] ABSTRACT: Stem rust threatens cereal production worldwide. Understanding the mechanism by which durable resistance genes, such as Rpg1, function is critical. We show that the RPG1 protein is phosphorylated within 5 min by exposure to spores from avirulent but not virulent races of stem rust. Transgenic mutants encoding an RPG1 protein with an in vitro inactive kinase domain fail to phosphorylate RPG1 in vivo and are susceptible to stem rust, demonstrating that phosphorylation is a prerequisite for disease resistance. Protein kinase inhibitors prevent RPG1 phosphorylation and result in susceptibility to stem rust, providing further evidence for the importance of phosphorylation in disease resistance. We conclude that phosphorylation of the RPG1 protein by the kinase activity of the pK2 domain induced by the interaction with an unknown pathogen spore product is required for resistance to the avirulent stem rust races. The pseudokinase pK1 domain is required for disease resistance but not phosphorylation. The very rapid phosphorylation of RPG1 suggests that an effector is already present in or on the stem rust urediniospores when they are placed on the leaf surface. However, spores must be alive, as determined by their ability to germinate, in order to elicit RPG1 phosphorylation.
[Show abstract][Hide abstract] ABSTRACT: Loose smut of barley is a common disease which can be controlled using resistant varieties. Information on the chromosome location of loci controlling loose smut resistance and the development of molecular markers to aid in selection for these genes can be beneficial in the resistant variety development process. The objectives of this work were to determine the resistance or susceptibility of doubled haploid barley lines arising from a cross of the varieties ‘Steptoe’ and ‘Morex’ to Ustilago nuda, the causal agent of loose smut of barley, and map the chromosome location of the loose smut resistance locus in ‘Morex’. The reaction to Ustilago nuda of the doubled-haploid barley plants was determined by inoculating spikelets of each line at anthesis by injection of a teliospore suspension using a needle inoculation method. Mature seeds from the inoculated spikelets were grown to determine the percentage of plants that developed with smutted heads. The lines were classified as susceptible if greater than 10% of the plants were smutted. The loose smut resistance locus from the resistant source ‘Morex’ was mapped using an existing DNA marker map of the ‘Steptoe’/‘Morex’ population. The distribution of the resistant and susceptible progeny from the loose smut testing fit a single gene model. The resistance gene was mapped to chromosome 3 (3H).
Canadian Journal of Plant Pathology 06/2010; 32(2):247-251. · 0.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Grass species have coevolved with current economically important crop pathogens over millions of years. During this time, speciation of current domestic crops has occurred, resulting in related yet divergent genomes. Here, we present a synteny map between the crop species Hordeum vulgare and the recently sequenced Brachypodium distachyon genome, focusing on regions known to harbor important barley disease resistance genes. The resistance genes have orthologous genes in Brachypodium that show conservation of the form and likely the function of the genes. The level of colinearity between the genomes is highly dependent on the region of interest and, at the DNA level or protein level, the gene of interest. The stem rust resistance gene Rpg1 has an ortholog with a high level of identity at the amino acid level, while the stem rust resistance gene Rpg5 has two orthologs with a high level of identity, one corresponding to the NBS-LRR domain and the other to the serine/threonine protein kinase domain, on different contigs. Interestingly, the predicted product of the Brachypodium Rpg1 ortholog contained a WD40 domain at the C-terminal end. The stem rust resistance gene rpg4 (actin depolymerizing factor 2) also has an ortholog with a high level of identity, in which one of the three residues indicated by allele sequencing in barley cultivars to be important in disease resistance is conserved. The syntenous region of the seedling spot blotch resistance locus, Rcs5, has a high level of colinearity that may prove useful in efforts to identify and clone this gene. A synteny map and orthologous resistance gene comparisons are presented.
[Show abstract][Hide abstract] ABSTRACT: The transcriptome of two fast neutron induced allelic barley mutants, FN362 and FN363, was analyzed with the Affymetrix Barley1 GeneChip microarray in order to characterize the necrotic leaf spot 3 (nec3) gene and its function. Twenty one genes, at least two-fold down-regulated in the mutants compared to the wild-type, were detected, but PCR analyses failed to identify a candidate Nec3 gene. It is possible that the probe set for the Nec3 gene is not on the Barley1 GeneChip, or that it is expressed at very low levels or the expression is confined to specific developmental stage or tissue type. Comparison of the genes differentially expressed in FN362 and FN363 mutants with publicly available Affymetrix Barley1 GeneChip expression data sets revealed significant overlap with barley abiotic stress transcriptome. The highest similarity was observed with the transcriptome of barley under drought and freezing stress. These results imply a possible involvement of the wild-type Nec3 in signaling pathways regulating abiotic stress response in barley.
Environmental and Experimental Biology. 01/2010; 8:1-16.
[Show abstract][Hide abstract] ABSTRACT: High density genetic maps of plants have, nearly without exception, made use of marker datasets containing missing or questionable genotype calls derived from a variety of genic and non-genic or anonymous markers, and been presented as a single linear order of genetic loci for each linkage group. The consequences of missing or erroneous data include falsely separated markers, expansion of cM distances and incorrect marker order. These imperfections are amplified in consensus maps and problematic when fine resolution is critical including comparative genome analyses and map-based cloning. Here we provide a new paradigm, a high-density consensus genetic map of barley based only on complete and error-free datasets and genic markers, represented accurately by graphs and approximately by a best-fit linear order, and supported by a readily available SNP genotyping resource.
Approximately 22,000 SNPs were identified from barley ESTs and sequenced amplicons; 4,596 of them were tested for performance in three pilot phase Illumina GoldenGate assays. Data from three barley doubled haploid mapping populations supported the production of an initial consensus map. Over 200 germplasm selections, principally European and US breeding material, were used to estimate minor allele frequency (MAF) for each SNP. We selected 3,072 of these tested SNPs based on technical performance, map location, MAF and biological interest to fill two 1536-SNP "production" assays (BOPA1 and BOPA2), which were made available to the barley genetics community. Data were added using BOPA1 from a fourth mapping population to yield a consensus map containing 2,943 SNP loci in 975 marker bins covering a genetic distance of 1099 cM.
The unprecedented density of genic markers and marker bins enabled a high resolution comparison of the genomes of barley and rice. Low recombination in pericentric regions is evident from bins containing many more than the average number of markers, meaning that a large number of genes are recombinationally locked into the genetic centromeric regions of several barley chromosomes. Examination of US breeding germplasm illustrated the usefulness of BOPA1 and BOPA2 in that they provide excellent marker density and sensitivity for detection of minor alleles in this genetically narrow material.
[Show abstract][Hide abstract] ABSTRACT: Race TTKSK (Ug99) of the wheat stem rust pathogen (Puccinia graminis f. sp. tritici) is a serious threat to both wheat and barley production worldwide because of its wide virulence on many cultivars and rapid spread from eastern Africa. Line Q21861 is one of the most resistant barleys known to this race. To elucidate the genetics of resistance in this line, we evaluated the Q21861/SM89010 (Q/SM) doubled-haploid population for reaction to race TTKSK at the seedling stage. Segregation for resistance:susceptibility in Q/SM doubled-haploid lines fit a 1:1 ratio (58:71 with chi2=1.31 and P=0.25), indicating that a single gene in Q21861 confers resistance to race TTKSK. In previous studies, a recessive gene (rpg4) and a partially dominant gene (Rpg5) were reported to control resistance to P. graminis f. sp. tritici race QCCJ and P. graminis f. sp. secalis isolate 92-MN-90, respectively, in Q21861. These resistance genes co-segregate with each other in the Q/SM population and were mapped to the long arm of chromosome 5H. Resistance to race TTKSK also co-segregated with resistance to both rusts, indicating that the gene conferring resistance to race TTKSK also lies at the rpg4/Rpg5 locus. This result was confirmed through the molecular analysis of recombinants previously used to characterize loci conferring resistance to race QCCJ and isolate 92-MN-90. The 70-kb region contains Rpg5 (a nucleotide-binding site leucine-rich repeat serine/threonine-protein kinase gene), rpg4 (an actin depolymerizing factor-like gene), and two other genes of unidentified function. Research is underway to resolve which of the genes are required for conferring resistance to race TTKSK. Regardless, the simple inheritance should make Q21861 a valuable source of TTKSK resistance in barley breeding programs.
[Show abstract][Hide abstract] ABSTRACT: Preharvest sprouting (PHS) and dormancy (DOR) can be problems in barley production and end use quality, especially for barley
used for seed and malting. Three crosses previously analyzed for DOR inheritance, were reanalyzed for PHS and DOR inheritance
using artificial rain to calculate sprout score (SSc) and measure alpha-amylase activity (AA). Germination percentage of untreated
grain for DOR was also measured. The crosses are ‘Steptoe’/’Morex’ (previously published), ‘Harrington’/TR306, and ‘Triumph’/Morex.
Among the three crosses, DOR QTLs were located to six and PHS QTLs to five chromosomes, respectively. Chromosome 6H was never
implicated. Previously identified DOR QTLs were confirmed in each cross, and most PHS QTLs coincided with DOR QTLs, but not
all. Unique PHS QTLs were identified on chromosomes 1H (AA), 2H (SSc, AA), 3H (SSc, AA), and 7H (SSc, AA) and unique DOR QTLs
on 1H, 2H, and 7H. Results indicate that PHS susceptibility and DOR are not always represented by opposite alleles at a locus.
Some QTL regions for a given trait are conserved across crosses and some are not. Several QTLs are suitable for marker-assisted
selection to balance PHS and DOR in breeding new cultivars.
[Show abstract][Hide abstract] ABSTRACT: Two closely linked resistance genes, rpg4 and Rpg5, conferring resistance to several races of Puccinia graminis, were cloned and characterized. The Rpg5 gene confers resistance to an isolate of Puccinia graminis f. sp. secalis (Pgs), while rpg4 confers resistance to Puccinia graminis f. sp. tritici (Pgt). Rpg5 is a novel gene containing nucleotide binding site-leucine rich repeat domains in combination with a serine threonine protein kinase domain. High-resolution mapping plus allele and recombinant sequencing identified the rpg4 gene, which encodes an actin depolymerizing factor-like protein (ADF2). Resistance against the Pgt races QCCJ, MCCF, TTKSK (aka Ug99) and RCRS requires both Rpg5 and rpg4, while Rpg5 alone confers resistance to Pgs isolate 92-MN-90. The dependency on the actin modifying protein ADF2 indicates cytoskeleton reorganization or redirection plays a role in pathogen-host interactions. Rpg5 may interact with ADF2 to activate or deactivate its function in the resistance response. Alternatively, Rpg5 could initiate signal transduction leading to resistance in response to detecting ADF2 protein modification. Pgt may redirect the actin cytoskeleton by inducing modifications of ADF2. The redirection of actin could possibly enable the pathogen to develop a haustoria-plant cell cytoskeleton interface for acquisition of nutrients.
[Show abstract][Hide abstract] ABSTRACT: Approaches utilizing microlinearity between related species allow for the identification of syntenous regions and orthologous genes. Within the barley Chromosome 7H(1) is a region of high recombination flanked by molecular markers cMWG703 and MWG836. We present the constructed physical contigs linked to molecular markers across this region using bacterial artificial chromosomes (BAC) from the cultivar Morex. Barley expressed sequence tags (EST), identified by homology to rice chromosome 6 between the rice molecular markers C425A and S1434, corresponded to the barley syntenous region of Chromosome 7H(1) Bins 2-5 between molecular markers cMWG703-MWG836. Two hundred and thirteen ESTs were genetically mapped yielding 267 loci of which 101 were within the target high recombination region while 166 loci mapped elsewhere. The 101 loci were joined by 43 other genetic markers resulting in a highly saturated genetic map. In order to develop a physical map of the region, ESTs and all other molecular markers were used to identify Morex BAC clones. Seventy-four BAC contigs were formed containing 2-102 clones each with an average of 19 and a median of 13 BAC clones per contig. Comparison of the BAC contigs, generated here, with the Barley Physical Mapping Database contigs, resulted in additional overlaps and a reduction of the contig number to 56. Within cMWG703-MWG836 are 24 agriculturally important traits including the seedling spot blotch resistance locus, Rcs5. Genetic and physical analysis of this region and comparison to rice indicated an inversion distal of the Rcs5 locus. Three BAC clone contigs spanning the Rcs5 locus were identified.
Theoretical and Applied Genetics 02/2009; 118(4):811-20. · 3.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rusts are biotrophic pathogens that attack many plant species but are particularly destructive on cereal crops. The stem rusts (caused by Puccinia graminis) have historically caused severe crop losses and continue to threaten production today. Barley (Hordeum vulgare L.) breeders have controlled major stem rust epidemics since the 1940s with a single durable resistance gene Rpg1. As new epidemics have threatened, additional resistance genes were identifi ed to counter new rust races, such as the rpg4/Rpg5 complex locus against races QCCJ and TTKSK. To understand how these genes work, we initiated research to clone and characterize them. The Rpg1 gene encodes a unique protein kinase with dual kinase domains, an active kinase, and a pseudokinase. Function of both domains is essential to confer resistance. The rpg4 and Rpg5 genes are closely linked and function coordinately to confer resistance to several wheat (Triticum aestivum L.) stem rust races, including the race TTKSK (also called Ug99) that threatens the world's barley and wheat crops. The Rpg5 gene encodes typical resistance gene domains NBS, LRR, and protein kinase but is unique in that all three domains reside in a single gene, a previously unknown structure among plant disease resistance genes. The rpg4 gene encodes an actin depolymerizing factor that functions in cytoskeleton rearrangement. R usts are biotrophic fungal pathogens (phylum:
The Plant Genome 01/2009; 2(2):109-120. · 3.88 Impact Factor