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  • 1
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The bean halo blight pathogen, Pseudomonas syringae pv. phaseolicola (Psph), is differentiated into nine races based on the presence or absence of five avirulence (avr ) genes in the bacterium, which interact with corresponding resistance genes, R1–R5, in Phaseolus vulgaris. The resistance gene R2 is matched by avrPphE, which is located adjacent to the cluster of hrp genes that are required for pathogenicity of Psph. Although only races 2, 4, 5 and 7 are avirulent on cultivars with R2 (inducing the hypersensitive response; HR), homologues of avrPphE are present in all races of Psph. DNA sequencing of avrPphE alleles from races of Psph has demonstrated two routes to virulence: via single basepair changes conferring amino acid substitutions in races 1, 3, 6 and 9 and an insertion of 104 bp in the allele in race 8. We have demonstrated that these base changes are responsible for the difference between virulence and avirulence by generating transconjugants of a virulent race harbouring plasmids expressing the various alleles of avrPphE. Agrobacterium tumefaciens-directed expression of avrPphE from race 4 in bean leaves induced the HR in a resistance gene-specific manner, suggesting that the AvrPphE protein is alone required for HR induction and is recognized within the plant cell. The allele from race 6, which is inactive if expressed in Psph, elicited a weak HR if expressed in planta, whereas the allele from race 1 did not. Our results suggest that the affinity of interaction between AvrPphE homologues and an unknown plant receptor mediates the severity of the plant's response. Mutation of avrPphE alleles did not affect the ability to colonize bean from a low level of inoculum. The avirulence gene avrPphB, which matches the R3 resistance gene, also caused a gene-specific HR following expression in the plant after delivery by A. tumefaciens.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1476-4687
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
    Notes: [Auszug] Galactose oxidase (EC 1.1.3.9) was crystallized from 0.80 M acetate buffer (pH 4.3 to 4.7) with ammonium sulphate as precipitant. The space group is C2 with unit cell parameters a = 98.0, b = 89.4, c = 86.7 A, ft = 117.8á°, and Table 1 summar-izes the crystallographic data. The structure ...
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  • 3
    ISSN: 1432-1327
    Keywords: Key words Galactose oxidase ; Copper ; Tyrosine free radical ; Proton abstraction
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract  The catalytic mechanism of the copper-containing enzyme galactose oxidase involves a protein radical on Tyr272, one of the equatorial copper ligands. The first step in this mechanism has been proposed to be the abstraction of a proton from the alcohol substrate by Tyr495, the axial copper ligand that is weakly co-ordinated to copper. In this study we have generated and studied the properties of a Y495F variant to test this proposal. X-ray crystallography reveals essentially no change from wild-type other than loss of the tyrosyl hydroxyl group. Visible spectroscopy indicates a significant change in the oxidised Y495F compared to wild-type with loss of a broad 810-nm peak, supporting the suggestion that this feature is due to inter-ligand charge transfer via the copper. The presence of a peak at 420 nm indicates that the Y495F variant remains capable of radical formation, a fact supported by EPR measurements. Thus the significantly reduced catalytic efficiency (1100-fold lower k cat / K m) observed for this variant is not due to an inability to generate the Tyr272 radical. By studying azide-induced pH changes, it is clear that the reduced catalytic efficiency is due mainly to the inability of Y495F to accept protons. This provides definitive evidence for the key role of Tyr495 in the initial proton abstraction step of the galactose oxidase catalytic mechanism.
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  • 4
    ISSN: 1573-5028
    Keywords: antifungal proteins ; non-host resitance ; pathogenesis-related protein ; proteinase inhibitor ; Septoria nodorum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Two previously unidentified cDNA clones (bsi1 and bpr1–1) were isolated by differential hybridization from a cDNA library of Stagonospora (Septoria) nodorum (Berk) Castellani & E.G. Germano teleomorph Phaeosphaeria (Leptosphaeria) nodorum (E. Muller) Hedjaroude-challenged barley (Hordeum vulgare L.) coleoptiles. bsi1 encoded a cysteine-rich protein containing 89 amino acids (aa) with a relative molecular mass (M r) of 9405. Protein sequence homologies showed that Bsi1 was very similar to an aluminium-induced protein from wheat and indicated that it was related to the Bowman-Birk-type proteinase inhibitors (BB-PIs). The predicted aa sequence of Bsi1 contained an N-terminal secretory signal sequence which implied that the protein was exported. The other clone, bpr1–1, which was truncated at the 5′ end, encoded a type-1 pathogenesis-related (PR-1) protein. The complete sequence of bpr1–1 was obtained after cloning a barley genomic DNA fragment and was shown to encode a basic protein containing 174 aa with a M r of 18 859. The deduced aa sequence of bpr1–1 contained both an N-terminal secretory signal sequence and a charged C-terminal extension. This latter sequence may represent a vacuolar targeting signal. bsi1 and bpr1–1 and four other defence-related genes (encoding 1,3-β-glucanase, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, a homologue of a putative wheat peroxidase, and barley leaf-specific thionin), showed increased transcription levels in S. nodorum-challenged coleoptiles, although their pattern of accumulation varied after inoculation (a.i.). The potential role of these induced genes in defence against fungal attack is discussed.
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