Stem Rust Resistance Gene Rpg1 Research Articles

Mapping adult plant stem rust resistance in barley accessions Hietpas-5 and GAW-79.

Key message Major stem rust resistance QTLs proposed to be Rpg2 from Hietpas-5 and Rpg3 from GAW-79 were identified in chromosomes 2H and 5H, respectively, and will enhance the diversity of stem rust resistance in barley improvement programs. Stem rust is a devastating disease of cereal crops worldwide. In barley (Hordeum vulgaressp. vulgare), the disease is caused by two pathogens: Puccinia graminis f. sp. secalis (Pgs) and Puccinia graminis f. sp. tritici (Pgt). In North America, the stem rust resistance gene Rpg1 has protected barley from serious losses for more than 60years; however, widely virulent Pgt races from Africa in the Ug99 group threaten the crop. The accessions Hietpas-5 (CIho 7124) and GAW-79 (PI 382313) both possess moderate-to-high levels of adult plant resistance to stem rust and are the sources of the resistance genes Rpg2 and Rpg3, respectively. To identify quantitative trait loci (QTL) for stem rust resistance in Hietpas-5 and GAW-79, two biparental populations were developed with Hiproly (PI 60693), a stem rust-susceptible accession. Both populations were phenotyped to the North American Pgt races of MCCFC, QCCJB, and HKHJC in St. Paul, Minnesota, and to African Pgt races (predominately TTKSKin the Ug99 group) in Njoro, Kenya. In the Hietpas-5/Hiproly population, a major effect QTL was identified in chromosome 2H, which is proposed as the location for Rpg2. In the GAW-79/Hiproly population, a major effect QTL was identified in chromosome 5H and is the proposed location for Rpg3. These QTLs will enhance the diversity of stem rust resistance in barley improvement programs.

Analysis of Stem Rust Resistance in Australian Barley Cultivars.

Eighty-two Australian and five exotic barley cultivars were evaluated at the seedling stage for resistance to the Australian stem rust pathotype 98-1,2,3,5,6. Although most of these cultivars exhibited mesothetic (mixed infection type) reactions that were associated with a high level of chlorosis, two ('O'Connor' and 'Pacific Ranger') were highly resistant. Marker analysis indicated that four Australian cultivars ('Empress', 'Vlamingh', Pacific Ranger, and 'Yerong') possess the stem rust resistance gene Rpg1. Tests conducted using North American Puccinia graminis f. sp. tritici pathotypes MCCJ and QCCJ supported marker results and indicated that 'Pacific Ranger' and 'Vlamingh' likely carry additional stem rust resistance genes. Based on pedigree information and results from multipathotype tests, these genes are believed to be uncharacterized and, therefore, new. The resistance in Australian barley 'Franklin' conferred resistance against all pathotypes tested in this study. Studies of inheritance to MCCJ revealed that it possessed an unknown seedling resistance, which was independent of and displayed additivity to Rpg1.

A synteny map and disease resistance gene comparison between barley and the model monocotBrachypodium distachyon

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.

Mapping of Wheat Stripe Rust Resistance Gene <I>YrZH84</I> with RGAP Markers and Its Application

Zhou 8425B, with an effective stripe rust resistance gene YrZH84, is a hall-mark parent line of wheat (Triticum aestivum L.) in the Yellow-Huai Rivers wheat growing region. An F2 population derived from the cross Zhou 8425B/Chinese Spring and their parents were used for polymorphism analysis employing the resistance gene-analog polymorphism (RGAP) markers and bulked segregant analysis (BSA) based on marker genotypes and phenotypes selection. An RGAP marker Xrga-1 was identified, which was tightly linked to the stripe rust resistance gene YrZH84, with a genetic distance of 0.8 cM. The fragment amplified by Xrga-1 was 343 bp. BLAST analysis indicated that the rga-1 showed 93% of identical nucleotide sequences to barley stem rust resistance gene Rpg1, and 92% to barley powdery mildew resistance gene Mla homology family. Fifty-eight cultivars from Yel-low-Huai rivers wheat growing region were genotyped by the RGAP marker Xrga-1. From the information of genotypes, pedi-grees and resistance responses, the cultivars Zhoumai 11, Zhoumai 20, Zhoumai 22, Aikang 58, 04 Zhong 36, Yuanyu 3, 05 Zhong 37, Wankang 18, and Yuzhan 10 were identified to carry the resistance gene YrZH84. These cultivars were the derivatives of Zhou 8425B. These results will greatly benefit the marker-assisted selection in wheat and cloning of the resistance gene YrZH84.

Allele Sequencing of the Barley Stem Rust Resistance Gene Rpg1 Identifies Regions Relevant to Disease Resistance

The stem rust resistance gene Rpg1 has protected North American barley cultivars from significant yield losses for over 65 years. The remarkable durability of this gene warrants further study as to its possible origin and allelic variation. Eight Swiss barley (Hordeum vulgare) landraces and eight wild barley (H. vulgare subsp. spontaneum) accessions from diverse geographic regions were analyzed to uncover new alleles of Rpg1 and learn about its possible origin. The two germplasm groups included accessions that were resistant and susceptible to Puccinia graminis f. sp. tritici pathotype MCCF. Allele-specific primers were utilized to amplify 1 kbp overlapping fragments spanning the Rpg1 gene and sequenced if a polymerase chain reaction (PCR) fragment was generated. Variation among the PCR products revealed significant polymorphisms among these Hordeum accessions. Landraces and wild barley accessions susceptible to pathotype MCCF exhibited the highest degree of Rpg1 polymorphism. One resistant landrace (Hv672) and one resistant wild barley accession (WBDC040) yielded all seven Rpg1-specific PCR fragments, but only landrace Hv672 coded for an apparently functional Rpg1 as determined by comparison to previously characterized resistant and susceptible alleles and also resistance to HKHJ, a stem rust pathotype that can specifically detect Rpg1 in the presence of other resistance genes. Accessions resistant to stem rust pathotype MCCF, but completely lacking Rpg1-specific PCR amplification and hybridization with an Rpg1-specific probe, suggested the presence of stem rust resistant gene(s) different from Rpg1 in the Hordeum germplasm pool. Some Rpg1 alleles that retained the ability to autophosphorylate did not confer resistance to Puccinia graminis f. sp. tritici pathotype MCCF, confirming our previous observations that autophosphorylation is essential, but not sufficient for disease resistance. Thus, the RPG1 protein plays a complex role in the stem rust disease resistance-signaling pathway.

Parallel expression profiling of barley–stem rust interactions

The dominant barley stem rust resistance gene Rpg1 confers resistance to many but not all pathotypes of the stem rust fungus Puccinia graminis f. sp. tritici (Pgt). Transformation of Rpg1 into susceptible cultivar Golden Promise rendered the transgenic plants resistant to Pgt pathotype MCC but not to Pgt pathotype QCC. Our objective was to identify genes that are induced/repressed during the early stages of pathogen infection to elucidate the molecular mechanisms and role of Rpg1 in defense. A messenger ribonucleic acid expression analysis using the 22K Barley1 GeneChip was conducted in all pair-wise combinations of two isolines (cv. Golden Promise and Rpg1 transgenic line G02-448F-3R) and two Pgt pathotypes (MCC and QCC) across six time points. Analysis showed that a total of 34 probe sets exhibited expression pattern differences between Golden Promise (susceptible) and G02-448F-3R (resistant) infected with Pgt-MCC. A total of 14 probe sets exhibited expression pattern differences between Pgt-MCC (avirulent) and Pgt-QCC (virulent) inoculated onto G02-448F-3R. These differentially expressed genes were activated during the early infection process, before the hypersensitive response or fungal growth inhibition occurred. Our analysis provides a list of candidate signaling components, which can be analyzed for function in Rpg1-mediated disease resistance.

Proteolysis of the barley receptor-like protein kinase RPG1 by a proteasome pathway is correlated with Rpg1 -mediated stem rust resistance

In plants, disease resistance mediated by the gene-for-gene mechanism involves the recognition of specific effector molecules produced by the pathogen either directly or indirectly by the resistance-gene products. This recognition triggers a series of signals, thereby serving as a molecular switch in regulating defense mechanisms by the plants. To understand the mechanism of action of the barley stem rust resistance gene Rpg1, we investigated the fate of the RPG1 protein in response to infection with the stem rust fungus, Puccinia graminis f. sp. tritici. The investigations revealed that RPG1 disappears to undetectable limits only in the infected tissues in response to avirulent, but not virulent pathotypes. The RPG1 protein disappearance is rapid and appears to be due to specific protein degradation via the proteasome-mediated pathway as indicated by inhibition with the proteasomal inhibitor MG132, but not by other protease inhibitors.

The barley serine/threonine kinase gene Rpg1 providing resistance to stem rust belongs to a gene family with five other members encoding kinase domains

The barley (Hordeum vulgare L.) stem rust (Puccinia graminis f. sp. tritici) resistance gene Rpg1 encodes a serine/threonine protein kinase with two tandem kinase domains. The Rpg1 gene family was identified from the cv. Morex and consists of five additional members with divergent homology to Rpg1. All family members encode serine/threonine kinase-like proteins with at least one predicted catalytically active kinase domain. The five family members were sequenced from cDNA and genomic DNA and genetically mapped. The family member most closely related to Rpg1, ABC1037, is located on chromosome 1(7H) bin 01, very near (approximately 50 kb) but not co-segregating with Rpg1. Two others, ABC1036 and ABC1040, are closely related to each other and tightly linked on chromosome 7(5H) bin 07. ABC1041 mapped to chromosome 7(5H) bin 13, tightly linked to the rust resistance genes rpg4 and Rpg5 providing resistance to barley stem rust pathotype QCC and rye stem rust pathotype 92-MN-90, respectively, but segregated away in a high-resolution population. ABC1063 was localized to chromosome 4(4H) bin 6. An interesting Rpg1 allele that appears to be the result of unequal recombination between Rpg1 and ABC1037 was characterized. No known resistance loci cosegregated with any family members, however characterization of the Rpg1 family has provided insight into the evolution of this novel gene family and may present tools for understanding the functional domains of Rpg1. The genetic mapping, gene structures, and analysis of amino-acid sequences of the Rpg1 gene family members are presented.

Structure and expression of the barley stem rust resistance gene Rpg1 messenger RNA

The barley stem rust resistance gene Rpg1 confers resistance to many pathotypes of the stem rust fungus Puccinia graminis f. sp. tritici. Rpg1 was recently cloned by a map-based approach from the barley cultivar Morex. Haplotype sequencing and transformation of a susceptible barley cultivar were employed to prove its identity. As a first step in understanding the molecular mechanism of action of Rpg1, we characterized the structure and transcription of the Rpg1 messenger RNA. Several alternative splice forms were identified, some of which could be translated into a predicted full-length protein product. The expression of the gene occurred in all plant organs and developmental stages analyzed. However, leaf epidermis exhibited ca. 30-fold higher Rpg1 expression than the whole leaf. Expression of Rpg1 was not induced by infection of barley cultivar Morex seedlings with the incompatible P. graminis f. sp. tritici pathotype MCC, while some defense-related genes, such as barley β-1,3-glucanases and PR1a, were strongly induced.

A barley gene family homologous to the maize rust resistance gene Rp1-D

Many characterized plant disease resistance genes encode proteins which have conserved motifs such as the nucleotide binding site. Conservation extends across different species, therefore resistance genes from one species can be used to isolate homologous regions from another by employing DNA sequences encoding conserved protein motifs as probes. Here we report the isolation and characterization of a barley ( Hordeum vulgare L.) resistance gene analog family consisting of nine members homologous to the maize rust resistance gene Rp1-D. Five barley Rp1-D homologues are clustered within approximately 400 kb on chromosome 1(7H), near, but not co-segregating with, the barley stem rust resistance gene Rpg1; while others are localized on chromosomes 3(3H), 5(1H), 6(6H) and 7(5H). Analyses of predicted amino-acid sequences of the barley Rp1-D homologues and comparison with known plant disease resistance genes are presented.

Sequence analysis of a rice BAC covering the syntenous barley Rpg1 region

In the course of map-based cloning of the barley stem rust resistance gene Rpg1, we identified a rice bacterial artificial chromosome (BAC) containing the Rpg1 flanking markers. Based on the excellent gene order colinearity between barley and rice in this region, we expected that this rice BAC would contain the barley Rpg1 homologue. In order to identify the putative rice homologue, we sequenced ca. 35 kb of the rice BAC at random and then an additional 33 kb of contiguous sequence between the two most closely spaced Rpg1 flanking markers. Sequence analysis revealed a total of 15 putative genes, 5 within the 33-kb contiguous region. A rice Rpg1 homologue was not identified, although a gene encoding a hypothetical polypeptide with similarity to a membrane protein could not be eliminated as a candidate. Surprisingly, four of the genes identified in the 33-kb contiguous rice sequence showed a high degree of similarity with genes on Arabidopsis chromosome 4. The genome regions harboring these genes showed some relatedness, but many rearrangements were also evident. These data suggest that some genes have remained linked even over the long evolutionary separation of Arabidopsis and rice, as has also been reported for mammals and invertebrates.

Resistance to stem rust in barley and inheritance of resistance to race QCC

Twenty-two barley (Hordeum vulgare L.) cultivars and lines were evaluated for resistance to stem rust (Puccinia graminis Pers. f. sp. tritici Eriks. & Henn.) race QCC in field trials over a 3-yr period, and the inheritance of resistance of several genotypes to race QCC was investigated using replicated F3 lines. Rust severities ranged from 17 to 58% between 14 different cultivars and lines which carried the stem rust resistance gene Rpg1, indicating that factors other than Rpg1 conferred resistance. Hietpas-5 (Rpg2) had moderate resistance while PI 382313 (Rpg3) had good resistance to race QCC. In the cross Hiproly-15/Peatland-9, F2 adult plants segregated 3R: 1S to race MCC, confirming the presence of gene Rpg1 in Peatland-9. Some F3 lines derived from F2 plants with Rpg1 were susceptible to race QCC, however, indicating that Rpg1 was ineffective against race QCC. A second gene in Peatland-9, designated for the interim as RpgU and independent of Rpg1, was shown to confer a moderate level of resistance to race QCC. Similar segregation patterns in the presence of race QCC and pedigree analysis suggested that the cultivars Husky and Diamond contain this gene. This gene is likely present in other related cultivars such as Vantage and Bonanza. Segregation in progeny of the cross Hiproly-15/Q21861 indicated that resistance to race QCC was controlled by two recessive genes in Q21861. Gene Rpg2, tested in F3 lines, was found to provide a moderate level of resistance to race QCC, similar to RpgU, but was less effective in the heterozygous condition. The phenotypic expression of RpgU differed from Rpg1, Rpg2, and Rpg3, suggesting that RpgU is a previously unreported gene for stem rust resistance in cultivated barley. Key words: Barley, Hordeum vulgare, stem rust, Puccinia graminis, resistance

Detection of the Stem Rust Resistance GeneRpg1 in Barley Seedlings

The stem rust resistance gene Rpg1 has conferred durable resistance in barley (Hordeum vulgare) to Puccinia graminis f. sp. tritici (wheat stem rust pathogen) for nearly 50 yr in the northern Great Plains. Detection of Rpg1 at the seedling stage can be difficult because most barley genotypes show mesothetic reactions to the stem rust pathogen. Genotypes with and without genes for stem rust resistance were inoculated with pathotypes Pgt-TPM, -MCC, and -HPH of P. g. tritici in the seedling stage. After 12 days of incubation at 25-28 C, infection types were assessed on plants. With pathotype TPM, differences between barley genotypes with and without resistance genes were not distinct, since similar infection types were frequently observed on genotypes in both groups (.)