Use of green fluorescent protein to detect expression of an endopolygalacturonase gene of Colletotrichum lindemuthianum during bean infection.
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The 5' noncoding region of clpg2, an endopolygalacturonase gene of the bean pathogen Colletotrichum lindemuthianum, was fused to the coding sequence of a gene encoding a green fluorescent protein (GFP), and the construct was introduced into the fungal genome. Detection of GFP accumulation by fluorescence microscopy examination revealed that clpg2 was expressed at the early stages of germination of the conidia and during appressorium formation both in vitro and on the host plant. (+info)
Colletotrichum trifolii mutants disrupted in the catalytic subunit of cAMP-dependent protein kinase are nonpathogenic.
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Colletotrichum trifolii is the fungal pathogen of alfalfa that causes anthracnose disease. For successful plant infection, this fungus must undergo a series of morphological transitions following conidial attachment, including germination and subsequent differentiation, resulting in appressorium formation. Our previous studies with pharmacological effectors of signaling pathways have suggested the involvement of cyclic AMP (cAMP)-dependent protein kinase (PKA) during these processes. To more precisely evaluate the role of PKA in C. trifolii morphogenesis, the gene encoding the catalytic (C) subunit of PKA (Ct-PKAC) was isolated, sequenced, and inactivated by gene replacement. Southern blot analysis with C. trifolii genomic DNA suggested that Ct-PKAC is a single-copy gene. Northern (RNA) blot analysis with total RNA from different fungal growth stages indicated that the expression of this gene was developmentally regulated. When Ct-PKAC was insertionally inactivated by gene replacement, the transformants showed a small reduction in growth relative to the wild type and conidiation patterns were altered. Importantly, PKA-deficient strains were unable to infect intact alfalfa (host) plants, though only a slight delay was observed in the timing for conidial germination and appressorial formation in the Ct-PKAC disruption mutants. Moreover, these mutants were able to colonize host tissues following artificial wounding, resulting in typical anthracnose disease lesions. Coupled with microscopy, these data suggest that the defect in pathogenicity is likely due to a failure in penetration. Our results demonstrate that PKA has an important role in regulating the transition between vegetative growth and conidiation, and is essential for pathogenic development in C. trifolii. (+info)
Processing, targeting, and antifungal activity of stinging nettle agglutinin in transgenic tobacco.
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The gene encoding the precursor to stinging nettle (Urtica dioica L. ) isolectin I was introduced into tobacco (Nicotiana tabacum). In transgenic plants this precursor was processed to mature-sized lectin. The mature isolectin is deposited intracellularly, most likely in the vacuoles. A gene construct lacking the C-terminal 25 amino acids was also introduced in tobacco to study the role of the C terminus in subcellular trafficking. In tobacco plants that expressed this construct, the mutant precursor was correctly processed and the mature isolectin was targeted to the intercellular space. These results indicate the presence of a C-terminal signal for intracellular retention of stinging nettle lectin and most likely for sorting of the lectin to the vacuoles. In addition, correct processing of this lectin did not depend on vacuolar deposition. Isolectin I purified from tobacco displayed identical biological activities as isolectin I isolated from stinging nettle. In vitro antifungal assays on germinated spores of the fungi Botrytis cinerea, Trichoderma viride, and Colletotrichum lindemuthianum revealed that growth inhibition by stinging nettle isolectin I occurs at a specific phase of fungal growth and is temporal, suggesting that the fungi had an adaptation mechanism. (+info)
Spore surface glycoproteins of Colletotrichum lindemuthianum are recognized by a monoclonal antibody which inhibits adhesion to polystyrene.
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Conidia (spores) of Colletotrichum lindemuthianum, a fungal plant pathogen causing bean anthracnose, adhere to the aerial parts of host plants to initiate the infection process. These spores possess a fibrillar 'spore coat' as well as a cell wall. In a previous study a mAb, UB20, was raised that recognized glycoproteins on the spore surface. In this study UB20 was used to localize and characterize these glycoproteins and to investigate their possible role in adhesion. Glycoproteins recognized by UB20 were concentrated on the outer surface of the spore coat and, to a lesser extent, at the plasma membrane/cell wall interface. Extraction of spores with hot water or 0.2% SDS resulted in removal of the spore coat. Western blotting with UB20 showed that a relatively small number of glycoproteins were extracted by these procedures, including a major component at 110 kDa. Biotinylation of carbohydrate moieties, together with cell fractionation, confirmed that these glycoproteins were exposed at the surface of the spores. In adhesion assays, > 90% of ungerminated conidia attached to polystyrene Petri dishes within 30 min. UB20 IgG at low concentrations inhibited attachment in an antigen-specific manner. This suggests that the glycoproteins recognized by this mAb may function in the initial rapid attachment of conidia to hydrophobic substrata. Polystyrene microspheres bound selectively to the 110 kDa glycoprotein in Western blots, providing further evidence that this component could mediate interactions with hydrophobic substrata. (+info)
WF14861, a new cathepsins B and L inhibitor produced by Colletotrichum sp. I. Taxonomy, production, purification and structure elucidation.
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WF14861, a novel cathepsins B and L inhibitor, was obtained from the culture mycelium of a fungus strain Colletotrichum sp. No. 14861. Spectroscopic analysis showed that WF14861 consisted of trans-epoxysuccinic acid, L-tyrosine and spermidine, WF14861 inhibited cathepsins B and L selectively. (+info)
WF14861, a new cathepsins B and L inhibitor produced by Colletotrichum sp. II. Biological properties.
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WF14861, 3-(N-(1-(N-(4-aminobutyl)-N-(3-aminopropyl)carbamoyl)-2-(4-hydroxyphenyl )ethyl)carbamoyl)oxirane-2-carboxylic acid, was obtained from the culture mycelium of Colletotrichum sp. as a novel cathepsins B and L inhibitor. WF14861 also showed inhibitory activities against bone derived crude protease and other cysteine proteases in vitro. The compound ameliorated the tissue damage and the bone destruction models of low-calcium-diet-fed mouse and adjuvant arthritis rat model. (+info)
Identification of an ancestral resistance gene cluster involved in the coevolution process between Phaseolus vulgaris and its fungal pathogen Colletotrichum lindemuthianum.
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The recent cloning of plant resistance (R) genes and the sequencing of resistance gene clusters have shed light on the molecular evolution of R genes. However, up to now, no attempt has been made to correlate this molecular evolution with the host-pathogen coevolution process at the population level. Cross-inoculations were carried out between 26 strains of the fungal pathogen Colletotrichum lindemuthianum and 48 Phaseolus vulgaris plants collected in the three centers of diversity of the host species. A high level of diversity for resistance against the pathogen was revealed. Most of the resistance specificities were overcome in sympatric situations, indicating an adaptation of the pathogen to the local host. In contrast, plants were generally resistant to allopatric strains, suggesting that R genes that were efficient against exotic strains but had been overcome locally were maintained in the plant genome. These results indicated that coevolution processes between the two protagonists led to a differentiation for resistance in the three centers of diversity of the host. To improve our understanding of the molecular evolution of these different specificities, a recombinant inbred (RI) population derived from two representative genotypes of the Andean (JaloEEP558) and Mesoamerican (BAT93) gene pools was used to map anthracnose specificities. A gene cluster comprising both Andean (Co-y; Co-z) and Mesoamerican (Co-9) host resistance specificities was identified, suggesting that this locus existed prior to the separation of the two major gene pools of P. vulgaris. Molecular analysis revealed a high level of complexity at this locus. It harbors 11 restriction fragment length polymorphisms when R gene analog (RGA) clones are used. The relationship between the coevolution process and diversification of resistance specificities at resistance gene clusters is discussed. (+info)
Heterologous expression and product identification of Colletotrichum lagenarium polyketide synthase encoded by the PKS1 gene involved in melanin biosynthesis.
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The Colletotrichum lagenarium PKS1 gene was expressed in the heterologous fungal host, Aspergillus oryzae, under the starch-inducible alpha-amylase promoter to identify the direct product of polyketide synthase (PKS) encoded by the PKS1 gene. The main compound produced by an A. oryzae transformant was isolated and characterized to be 1,3,6,8-tetrahydroxynaphthalene (T4HN) as its tetraacetate. Since the PKS1 gene was cloned from C. lagenarium to complement the nonmelanizing albino mutant, T4HN was assumed to be an initial biosynthetic intermediate, and thus the product of the PKS reaction, but had not been isolated from the fungus. The production of T4HN by the PKS1 transformant unambiguously identified the gene to encode a PKS of pentaketide T4HN. In addition, tetraketide orsellinic acid and pentaketide isocoumarin were isolated, the latter being derived from a pentaketide monocyclic carboxylic acid, as by-products of the PKS1 PKS reaction. Production of the pentaketide carboxylic acid provided insights into the mechanism for the PKS1 polyketide synthase reaction to form T4HN. (+info)