Rpr1, a gene required for Rpg1-dependent resistance to stem rust in barley

Rpg1 is a stem rust resistance gene that has protected barley from severe losses for over 60 years in the US and Canada. It confers resistance to many, but not all, pathotypes of the stem rust fungus Puccinia graminis f. sp. tritici. A fast neutron induced deletion mutant, showing susceptibility to stem rust pathotype Pgt-MCC, was identified in barley cv. Morex, which carries Rpg1. Genetic and Rpg1 mRNA and protein expression level analyses showed that the mutation was a suppressor of Rpg1 and was designated Rpr1 (Required for P. graminis resistance). Genome-wide expression profiling, using the Affymetrix Barley1 GeneChip containing ∼22,840 probe sets, was conducted with Morex and the rpr1 mutant. Of the genes represented on the Barley1 microarray, 20 were up-regulated and 33 were down-regulated by greater than twofold in the mutant, while the Rpg1 mRNA level remained constant. Among the highly down-regulated genes (greater than fourfold), genomic PCR, RT-PCR and Southern analyses identified that three genes (Contig4901_s_at, HU03D17U_s_at, and Contig7061_s_at), were deleted in the rpr1 mutant. These three genes mapped to chromosome 4(4H) bin 5 and co-segregated with the rpr1-mediated susceptible phenotype. The loss of resistance was presumed to be due to a mutation in one or more of these genes. However, the possibility exists that there are other genes within the deletions, which are not represented on the Barley1 GeneChip. The Rpr1 gene was not required for Rpg5- and rpg4-mediated stem rust resistance, indicating that it shows specificity to the Rpg1-mediated resistance pathway.     

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 (∼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.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

GA-20 oxidase as a candidate for the semidwarf gene sdw1/denso in barley

The barley sdw1/denso gene not only controls plant height but also yield and quality. The sdw1/denso gene was mapped to the long arm of chromosome 3H. Comparative genomic analysis revealed that the sdw1/denso gene was located in the syntenic region of the rice semidwarf gene sd1 on chromosome 1. The sd1 gene encodes a gibberellic acid (GA)-20 oxidase enzyme. The gene ortholog of rice sd1 was isolated from barley using polymerase chain reaction. The barley and rice genes showed a similar gene structure consisting of three exons and two introns. Both genes share 88.3% genomic sequence similarity and 89% amino acid sequence identity. A single nucleotide polymorphism was identified in intron 2 between barley varieties Baudin and AC Metcalfe with Baudin known to contain the denso semidwarf gene. The single nucleotide polymorphism (SNP) marker was mapped to chromosome 3H in a doubled haploid population of Baudin × AC Metcalfe with 178 DH lines. Quantitative trait locus analysis revealed that plant height cosegregated with the SNP. The sdw1/denso gene in barley is the most likely ortholog of the sd1 in rice. The result will facilitate understanding of the molecular mechanism controlling semidwarf phenotype and provide a diagnostic marker for selection of semidwarf gene in barley. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Conversion of an RFLP marker for the barley stem rust resistance geneRpg1 to a specific PCR-amplifiable polymorphism

TheRpg1 gene in barley has provided satisfactory levels of stem rust resistance for the last 50 years. The appearance of a new race of stem rust that is virulent toRpg1 has resulted in efforts to incorporate new stem rust resistance genes into barley. Marker-assisted selection may provide the only means of combining this useful gene with resistance genes for which no virulent races have been identified. Several RFLP markers have been identified as linked to theRpg1 locus. One of these, ABG704 was converted into a post-amplification restriction polymorphism. To generate a specific PCR-amplifiable polymorphism the sequence of the ABG704 locus from four barley cultivars was determined. Primers were developed that can detect a single-base difference between resistant and susceptible cultivars. The successful conversion of an RFLP marker to an allele-specific PCR-based marker not only demonstrates that this type of conversion is possible for cereals, but also results in an immediately useful marker for application to plant breeding programmes.     

Molecular mapping of a recessive gene for resistance to stripe rust in barley

Barley stripe rust, caused by Puccinia striiformis f. sp. hordei, is one of the most important barley (Hordeum vulgare) diseases in the United States. The disease is best controlled using resistant cultivars. Barley genotype Grannenlose Zweizeilige (GZ) has a recessive gene (rpsGZ) that is effective against all races of P. striiformis f. sp. hordei identified so far in the USA. To develop a molecular map for mapping the gene, F₈ recombinant inbred lines (RILs) were developed from the Steptoe X GZ cross through single-seed descent. Seedlings of the parents and RILs were evaluated for resistance to races PSH-14 and PSH-54 of P. striiformis f. sp. hordei under controlled greenhouse conditions. Genomic DNA was extracted from the parents and 182 F₈ RILs and used for linkage analysis. The resistance gene analog polymorphism (RGAP) technique was used to identify molecular markers for rpsGZ. A linkage group for the gene was constructed with 12 RGAP markers, of which two markers co-segregated with the resistance locus, and two markers were closely linked to the locus with a genetic distance of 0.9 and 2.0 cM, respectively. These four markers were present only in the susceptible parent. The closest marker to the resistance allele was 11.7 cM away. Analyses of two sets of barley chromosome addition lines of wheat with the two RGAP markers that were cosegregating with the susceptibility allele showed that rpsGZ and the markers were located on the long arm of barley chromosome 4H. Further, tests with four simple sequence repeat (SSR) markers confirmed the chromosomal location of the rpsGZ gene and also integrated the RGAP markers into the known SSR-based linkage map of barley. The closest SSR marker EBmac0679 had a genetic distance of 7.5 cM with the gene in the integrated linkage map constructed with the 12 RGAP markers and 4 SSR markers. The information on chromosomal location and molecular markers for rpsGZ should be useful for incorporating this gene into commercial cultivars and combining it with other resistance genes for durable resistance.     

Evidence that the BONZAI1/COPINE1 protein is a calcium- and pathogen-responsive defense suppressor

Copines are calcium-responsive, phospholipid-binding proteins involved in cellular signaling. The Arabidopsis BONZAI1/COPINE1 (BON1/CPN1) gene is a suppressor of defense responses controlled by the disease resistance (R) gene homolog SNC1. The BON1/CPN1 null mutant cpn1-1 has a recessive, temperature- and humidity-dependent, lesion mimic phenotype that includes activation of Pathogenesis-Related (PR) gene expression. Here, we demonstrated that the accumulation of BON1/CPN1 protein in wild-type plants was up-regulated by bacterial pathogen inoculation and by the activation of defense signaling responses controlled by two R genes, SNC1 and RPS2. Interestingly, however, over-accumulation of BON1/CPN1 in two BON1/CPN1 promoter T-DNA insertion mutants did not affect resistance to a bacterial pathogen. Promoter deletion analysis identified a 280 bp segment of the BON1/CPN1 promoter as being required for pathogen-induced gene expression; the same promoter region was also required for calcium ionophore-induced gene expression. Leaf infiltration with calcium ionophore triggered high-level PR gene expression specifically in cpn1-1 plants grown under permissive conditions, while co-infiltration of the calcium chelator EGTA attenuated this effect. These results explain the conditional nature of the cpn1-1 phenotype and are consistent with BON1/CPN1 being a calcium- and pathogen-responsive plant defense suppressor.     

Interleukin 22 is a candidate gene for Tmevp3, a locus controlling Theiler's virus-induced neurological diseases

After intracerebral inoculation, Theiler's virus induces in its natural host, the mouse, an acute encephalomyelitis followed, in susceptible animals, by chronic inflammation and primary demyelination. Susceptibility to demyelination among strains of laboratory mice is explained by the capacity of the immune system to control viral load during persistence. Also, differences of susceptibility to viral load between the susceptible SJL strain and the resistant B10.S strain are mainly due to two loci, Tmevp2 and Tmevp3, located close to the Ifng locus on chromosome 10. In this article, we show that the Tmevp3 locus controls both mortality during the acute encephalomyelitis and viral load during persistence. Most probably, two genes located in the Tmevp3 interval control these two different phenotypes with efficiencies that depend on the age of the mouse at inoculation. Il22, a member of the IL-10 cytokine family, is a candidate gene for the control of mortality during the acute encephalomyelitis.     

A candidate protein kinase C gene, PKC1, is required for the S. cerevisiae cell cycle

Probes derived from cDNAs encoding isozymes of rat protein kinase C (PKC) were used to screen the genome of the budding yeast S. cerevisiae. A single gene (PKC1) was isolated that encodes a putative protein kinase closely related to the alpha, beta, and gamma subspecies of mammalian PKC. Deletion of PKC1 resulted in recessive lethality. Cells depleted of the PKC1 gene product displayed a uniform phenotype, a characteristic of cell division cycle (cdc) mutants, and arrested cell division at a point subsequent to DNA replication, but prior to mitosis. Unlike most cdc mutants, which continue to grow in the absence of cell division, PKC1-depleted cells arrested growth with small buds. PKC1 may regulate a previously unrecognized checkpoint in the cell cycle.     

The axon guidance receptor gene ROBO1 is a candidate gene for developmental dyslexia

Dyslexia, or specific reading disability, is the most common learning disorder with a complex, partially genetic basis, but its biochemical mechanisms remain poorly understood. A locus on Chromosome 3, DYX5, has been linked to dyslexia in one large family and speech-sound disorder in a subset of small families. We found that the axon guidance receptor gene ROBO1, orthologous to the Drosophila roundabout gene, is disrupted by a chromosome translocation in a dyslexic individual. In a large pedigree with 21 dyslexic individuals genetically linked to a specific haplotype of ROBO1 (not found in any other chromosomes in our samples), the expression of ROBO1 from this haplotype was absent or attenuated in affected individuals. Sequencing of ROBO1 in apes revealed multiple coding differences, and the selection pressure was significantly different between the human, chimpanzee, and gorilla branch as compared to orangutan. We also identified novel exons and splice variants of ROBO1 that may explain the apparent phenotypic differences between human and mouse in heterozygous loss of ROBO1. We conclude that dyslexia may be caused by partial haplo-insufficiency for ROBO1 in rare families. Thus, our data suggest that a slight disturbance in neuronal axon crossing across the midline between brain hemispheres, dendrite guidance, or another function of ROBO1 may manifest as a specific reading disability in humans.     

OsGA20ox1, a candidate gene for a major QTL controlling seedling vigor in rice

Seedling vigor is among the major determinants of stable stand establishment in direct-seeded rice (Oryza sativa L.) in temperate regions. Quantitative trait loci (QTL) for seedling vigor were identified using 250 recombinant inbred lines (RILs) derived from a cross between two japonica rice cultivars Kakehashi and Dunghan Shali. Seedling heights measured at 14 days after sowing were 20.3 and 29.4 cm for Kakehashi and Dunghan Shali, respectively. For the RILs, the height ranged from 14.1 to 31.7 cm. Four putative QTLs associated with seedling height were detected. qPHS3-2, the major QTL that was located on the long arm of chromosome 3, accounted for 26.2 % of the phenotypic variance. Using progeny of the near isogenic lines (NILs) produced by the backcross introduction of a chromosome segment carrying this major QTL into an elite cultivar Iwatekko, we fine-mapped qPHS3-2 to a 81-kb interval between two markers, ID_CAPS_01 and RM16227. Within this mapped region, we identified the gene OsGA20ox1, which is related to gibberellin (GA) biosynthesis. The relative expression levels of GA20ox1 in seedlings of Dunghan Shali and NILs were higher than that of Iwatekko. Concomitantly, the amount of endogenous active GA was higher in Dunghan Shali and the NILs compared to the level detected in Iwatekko. These results indicate that OsGA20ox1 is a strong candidate gene for major QTL controlling seedling vigor in rice.     

A cDNA clone for a pathogenesis-related protein 1 from barley

A barley cDNA clone (PRb-1) corresponding to an mRNA differentially induced in resistant compared to susceptible barley cultivars by powdery mildew infection was isolated and characterised. The deduced amino acid sequence revealed 24 amino acids comprising the signal peptide and 140 amino acids of the mature peptide (15 kDa). This showed close homology to PR-1-like proteins, which have been isolated from maize, tobacco, tomato and Arabidopsis thaliana. Northern blot analysis showed accumulation of the corresponding mRNA 12 h after inoculation of resistant barley cultivars with Erysiphe graminis. Increased expression of the PRb-1 gene was also observed in resistant compared with near-isogenic susceptible barley plants following treatment with ethylene, salicylic acid, methyl jasmonate and 2,6-dichloro-isonicotinic acid.     

The novel MMS-inducible gene Mif1/KIAA0025 is a target of the unfolded protein response pathway

In a search for genes induced by DNA-damaging agents, we identified two genes that are activated by methyl methanesulfonate (MMS). Expression of both genes is regulated after endoplasmic reticulum (ER) stress via the unfolded protein response (UPR) pathway. The first gene of those identified is the molecular chaperone BiP/GRP78. The second gene, Mif1, is identical to the anonymous cDNA KIAA0025. Treatment with the glycosylation inhibitor tunicamycin both enhances the synthesis of Mif1 mRNA and protein. The Mif1 5' flanking region contains a functional ER stress-responsive element which is sufficient for induction by tunicamycin. MMS, on the other hand, activates Mif1 via an UPR-independent pathway. The gene encodes a 52 kDa protein with homology to the human DNA repair protein HHR23A and contains an ubiquitin-like domain. Overexpressed Mif1 protein is localized in the ER.