1,720,994 research outputs found
The rpg4/Rpg5 stem rust resistance locus in barley; resistance genes and cytoskeleton dynamics
Full text linkTwo 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.Brueggeman, Robert; Steffenson, Brian; Kleinhofs, Andris. (2009). The rpg4/Rpg5 stem rust resistance locus in barley; resistance genes and cytoskeleton dynamics. Retrieved from the University Digital Conservancy, http://dx.doi.org/10.4161/cc.8.7.8079
Rice-barley synteny and its applications to saturation mapping of the barley Rpg1 region
Full text linkIn order to facilitate the map-based cloning of the barley stem rust resistance gene Rpg1, we have demonstrated a high degree of synteny at a micro level between the telomeric region of barley chromosome 1P and rice chromosome 6. We have also developed and applied a simple and efficient method for selecting useful probes from large insert genomic YAC and cosmid clones. The gene order within the most terminal 6.5 cM of barley chromosome 1P was compared with the most terminal 2.7 cM of rice chromosome 6. Nine rice probes, previously mapped in rice or isolated from YAC or cosmid clones from this region, were mapped in barley. All, except one, were in synteny with the rice gene order. The exception, probe Y617R, was duplicated in barley. One copy was located on a different chromosome and the other in a non-syntenic position on barley chromosome 1P. The barley probes from this region could not be mapped to rice, but two of them were inferred to be in a syntenic location based on their position on a rice YAC. This work demonstrates the utility of applying the results of genetic and physical mapping of the small genome cereal rice to map-based cloning of interesting genes from large genome relatives.Steffenson, Brian; Kilian, Andrzej; Kudrna, David A.; Kleinhofs, Andris; Yano, Masahiro; Kurata, Nori; Sasaki, Takuji. (1995). Rice-barley synteny and its applications to saturation mapping of the barley Rpg1 region. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/188629
Genetically engineered stem rust resistance in barley using the Rpg1 gene
Full text linkThe stem-rust-susceptible barley cv. Golden Promise was transformed by Agrobacterium-mediated transformation of immature zygotic embryos with the Rpg1 genomic clone of cv. Morex containing a 520-bp 5′ promoter region, 4,919-bp gene region, and 547-bp 3′ nontranscribed sequence. Representatives of 42 transgenic barley lines obtained were characterized for their seedling infection response to pathotype Pgt-MCC of the stem rust fungus Puccinia graminis f. sp. tritici. Golden Promise was converted from a highly susceptible cultivar into a highly resistant one by transformation with the dominant Rpg1 gene. A single copy of the gene was sufficient to confer resistance against stem rust, and progenies from several transformants segregated in a 3:1 ratio for resistance/susceptibility as expected for Mendelian inheritance. These results unequivocally demonstrate that the DNA segment isolated by map-based cloning is the functional Rpg1 gene for stem rust, resistance. One of the remarkable aspects about the transformants is that they exhibit a higher level of resistance than the original sources of Rpg1 (cvs. Chevron and Peatland). In most cases, the Golden Promise transformants exhibited a highly resistant reaction where no visible sign of infection was evident. Hypersensitive necrotic “fleck” reactions were also observed, but less frequently. With both infection types, pathogen sporulation was prevented. Southern blot and RT-PCR analysis revealed that neither Rpg1 gene copy number nor expression levels could account for the increased resistance observed in Golden Promise transformants. Nevertheless, this research demonstrates that stem-rust-susceptible barley can be made resistant by transformation with the cloned Rpg1 gene.Steffenson, Brian; Horvath, Henriette; Rostoks, Nils; Brueggeman, Robert; Wettstein, Diter von; Kleinhofs, Andris. (2003). Genetically engineered stem rust resistance in barley using the Rpg1 gene. Retrieved from the University Digital Conservancy, 10.1073/pnas.0136911100
Regeneration of plants from crown gall cells: a step in the T-DNA mediated genetic engineering of plants
No full textSub-cellular localization and functions of the barley stem rust resistance receptor-like serine/threonine-specific protein kinase Rpg1
Full text linkThe Rpg1 gene confers resistance to many pathotypes of the stem rust fungus Puccinia graminis f. sp. tritici and has protected barley from serious disease losses for over 60 years. Rpg1 encodes a constitutively expressed protein with two tandem kinase domains. Fractionation by differential centrifugation and aqueous two-phase separation of the microsome proteins located Rpg1 mainly in the cytosol but also in the plasma membrane and intracellular membranes. Recombinant Rpg1 autophosphorylates in vitro intramolecularly only serine and threonine amino acids with a preference for Mn2+ cations and a Km of 0.15 and a Vmax of 0.47 nmol·min−1·mg−1 protein. The inability of wild-type Rpg1 to transphosphorylate a recombinant Rpg1 inactivated by site-directed mutation confirmed that Rpg1 autophosphorylation proceeds exclusively via an intramolecular mechanism. Site-directed mutagenesis of the two adjacent lysine residues in the ATP anchor of the two-kinase domains established that the first of the two tandem kinase domains is nonfunctional and that lysine 461 of the second domain is the catalytically active residue. Transgenic barley, expressing Rpg1 mutated in either the kinase 1 or 2 domains, were fully susceptible to P. graminis f. sp. tritici revealing requirement of both kinase domains for resistance. In planta-expressed Rpg1 mutant protein confirmed that mutation in domain 2, but not 1, rendered the protein incapable of autophosphorylation.Steffenson, Brian; Nirmala, Jayaveeramuthu; Brueggeman, Robert; Maier, Christina; Clay, Christine; Rostoks, Nils; Kannangara, C.Gamini; Wettstein, Diter von; Kleinhofs, Andris. (2006). Sub-cellular localization and functions of the barley stem rust resistance receptor-like serine/threonine-specific protein kinase Rpg1. Retrieved from the University Digital Conservancy, 10.1073/pnas.0602379103
Studies Concerning the Effect of the Cu/cu Alleles on Coumarin Biosynthesis in \u3ci\u3eMelilotus alba\u3c/i\u3e
No full textPhenylalanine deaminase activity was examined in the isogenic sweet-clover (Melilotus alba Decr.) lines differing in the Cu/cu alleles. No definite differences in the activity of this enzyme were demonstrated in the two genotypes. The fact that this enzyme is active in the plants of the cucu genotype provides some support for the existence of a block in the ortho-hydroxylation of trans-cinnamic acid in this genotype.
Attempts to demonstrate the enzymatic conversion of trans-cinnamic acid to o-coumaryl glucoside have been unsuccessful. Since no positive results were obtained, it appears that the postulated glucosidation of trans-cinnamic acid probably does not occur under the conditions used.
The results obtained indicate that crude enzyme preparations of CuCuBB plants are not capable of converting o-tyrosine to o-coumaric acid.
Advisor: Francis A. Haskins
Proteolysis of the barley receptor-like protein kinase RPG1 by a proteasome pathway is correlated with Rpg1-mediated stem rust resistance
Full text linkIn 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.Steffenson, Brian; Nirmala, Jayaveeramuthu; Dahl, Stephanie; Kannangara, C.Gamini; Wettstein, Diter von; Chen, Xianming; Kleinhofs, Andris. (2007). Proteolysis of the barley receptor-like protein kinase RPG1 by a proteasome pathway is correlated with Rpg1-mediated stem rust resistance. Retrieved from the University Digital Conservancy, 10.1073/pnas.0703758104
I. EFFECT OF THE CU/RECESSIVE CU ALLELES ON ENZYMES INVOLVED IN ORTHO-HYDROXYCINNAMIC ACID METABOLISM IN MELILOTUS ALBA. II. CHEMICAL MUTAGENESIS IN MELILOTUS ALBA ANNUA
No full textAbstract not availabl
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CLONING OF THE SEEDLING SPOT BLOTCH RESISTANCE GENE Rcs5
Full text linkThe focus of my thesis was to investigate and clone the seedling spot blotch resistance gene, Rcs5. Using a positional cloning approach and molecular markers derived by using synteny with related sequenced genomes of rice and Brachypodium, I was able to saturate the Rcs5 region, identify a small interval that is likely to contain Rcs5, and sequence six BACs from the resistant cultivar Morex that should contain Rcs5. Allele sequencing and recombinant analysis limited the number of candidate genes to two Wall-associated kinases, ctg19 and ctg37. These genes represent a novel class of resistance genes in which only WAK1 and WAKL22 from Arabidopsis have been shown to be involved in disease resistance. This research will add to the understanding of disease resistance to hemibiotrophic organisms in crop species
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ROLE OF BRASSINOSTEROID CATABOLISM IN ARABIDOPSIS DEVELOPMENT
Full text linkBAS1 (phyB-4 ACTIVATION TAGGED SUPPRESSOR 1) and SOB7 (SUPPRESSOR OF phyB-4 7) are brassinosteroid-catabolizing P450s in Arabidopsis thaliana that synergistically/redundantly modulate photomorphogenic traits such as flowering time. This study investigates the role of BAS1 and SOB7 in photomorphogenesis by studying null-mutant genetic interactions with the photoreceptors phyA, phyB and cry1 with regard to seed-germination and flowering time. The removal of BAS1 and/or SOB7 rescued the low germination rate of the phyA-211 phyB-9 double-null mutant. With regard to floral induction, bas1-2 and sob7-1 showed a complex set of genetic interactions with photoreceptor-null mutants. Histochemical analysis of transgenic plants harboring BAS1:BAS1-GUS and SOB7:SOB7-GUS translational fusions revealed overlapping and distinct expression patterns. BAS1's expression in the shoot apex increases during the phase transition from short-to-long-day growth conditions and requires phyB in red light.Application of this kind of genetic analysis approach to study other BR catabolic genes requires availability of the loss-of-function mutants. Our analysis shows that the ben1-1 (bri1 enhanced 1-1) mutant (T-DNA intronic insertion mutant of BEN1) is not suitable for multiple mutant analyses which involve combining two or more T-DNA insertion mutations. We generated a genetic triple-mutant from a cross between the bas1-2 sob7-1 double-null (T-DNA exonic insertion mutants) and ben1-1. The single ben1-1 line behaves as a transcript null. However, in the triple-mutant background ben1-1 was reverted to a partial loss-of-function allele. The enhanced expression of BEN1 remained stable when the ben1-1 single-mutant was re-isolated from a cross with the wild-type. In addition, the two genetically identical pre-triple and post-triple ben1-1 mutants also differed phenotypically. Restriction endonuclease analysis with methylation sensitive enzymes demonstrates that the recovered ben1-1 mutant is epigenetically different from the original ben1-1 allele
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