Cloning and partial characterization of endopolygalacturonase genes from Botrytis cinerea.
Botrytis cinerea is a plant-pathogenic fungus infecting over 200 different plant species. We use a molecular genetic approach to study the process of pectin degradation by the fungus. Recently, we described the cloning and characterization of an endopolygalacturonase (endoPG) gene from B. cinerea (Bcpg1) which is required for full virulence. Here we describe the cloning and characterization of five additional endoPG-encoding genes from B. cinerea SAS56. The identity at the amino acid level between the six endoPGs of B. cinerea varied from 34 to 73%. Phylogenetic analysis, by using a group of 35 related fungal endoPGs and as an outgroup one plant PG, resulted in the identification of five monophyletic groups of closely related proteins. The endoPG proteins from B. cinerea SAS56 could be assigned to three different monophyletic groups. DNA blot analysis revealed the presence of the complete endoPG gene family in other strains of B. cinerea, as well as in other Botrytis species. Differential gene expression of the gene family members was found in mycelium grown in liquid culture with either glucose or polygalacturonic acid as the carbon source. (+info)
Processing, targeting, and antifungal activity of stinging nettle agglutinin in transgenic tobacco.
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)
Requirement of functional ethylene-insensitive 2 gene for efficient resistance of Arabidopsis to infection by Botrytis cinerea.
Inoculation of wild-type Arabidopsis plants with the fungus Alternaria brassicicola results in systemic induction of genes encoding a plant defensin (PDF1.2), a basic chitinase (PR-3), and an acidic hevein-like protein (PR-4). Pathogen-induced induction of these three genes is almost completely abolished in the ethylene-insensitive Arabidopsis mutant ein2-1. This indicates that a functional ethylene signal transduction component (EIN2) is required in this response. The ein2-1 mutants were found to be markedly more susceptible than wild-type plants to infection by two different strains of the gray mold fungus Botrytis cinerea. In contrast, no increased fungal colonization of ein2-1 mutants was observed after challenge with avirulent strains of either Peronospora parasitica or A. brassicicola. Our data support the conclusion that ethylene-controlled responses play a role in resistance of Arabidopsis to some but not all types of pathogens. (+info)
Redox chemistry in laccase-catalyzed oxidation of N-hydroxy compounds.
1-Hydroxybenzotriazole, violuric acid, and N-hydroxyacetanilide are three N-OH compounds capable of mediating a range of laccase-catalyzed biotransformations, such as paper pulp delignification and degradation of polycyclic hydrocarbons. The mechanism of their enzymatic oxidation was studied with seven fungal laccases. The oxidation had a bell-shaped pH-activity profile with an optimal pH ranging from 4 to 7. The oxidation rate was found to be dependent on the redox potential difference between the N-OH substrate and laccase. A laccase with a higher redox potential or an N-OH compound with a lower redox potential tended to have a higher oxidation rate. Similar to the enzymatic oxidation of phenols, phenoxazines, phenothiazines, and other redox-active compounds, an "outer-sphere" type of single-electron transfer from the substrate to laccase and proton release are speculated to be involved in the rate-limiting step for N-OH oxidation. (+info)
The BMP1 gene is essential for pathogenicity in the gray mold fungus Botrytis cinerea.
In Magnaporthe grisea, a well-conserved mitogen-activated protein (MAP) kinase gene, PMK1, is essential for fungal pathogenesis. In this study, we tested whether the same MAP kinase is essential for plant infection in the gray mold fungus Botrytis cinerea, a necrotrophic pathogen that employs infection mechanisms different from those of M. grisea. We used a polymerase chain reaction-based approach to isolate MAP kinase homologues from B. cinerea. The Botrytis MAP kinase required for pathogenesis (BMP) MAP kinase gene is highly homologous to the M. grisea PMK1. BMP1 is a single-copy gene. bmp1 gene replacement mutants produced normal conidia and mycelia but were reduced in growth rate on nutrient-rich medium. bmp1 mutants were nonpathogenic on carnation flowers and tomato leaves. Re-introduction of the wild-type BMP1 allele into the bmp1 mutant restored both normal growth rate and pathogenicity. Further studies indicated that conidia from bmp1 mutants germinated on plant surfaces but failed to penetrate and macerate plant tissues. bmp1 mutants also appeared to be defective in infecting through wounds. These results indicated that BMP1 is essential for plant infection in B. cinerea, and this MAP kinase pathway may be widely conserved in pathogenic fungi for regulating infection processes. (+info)
A new method to monitor airborne inoculum of the fungal plant pathogens Mycosphaerella brassicicola and Botrytis cinerea.
We describe a new microtiter immunospore trapping device (MTIST device) that uses a suction system to directly trap air particulates by impaction in microtiter wells. This device can be used for rapid detection and immunoquantification of ascospores of Mycosphaerella brassicicola and conidia of Botrytis cinerea by an enzyme-linked immunosorbent assay (ELISA) under controlled environmental conditions. For ascospores of M. brassicicola correlation coefficients (r(2)) of 0.943 and 0.9514 were observed for the number of MTIST device-impacted ascospores per microtiter well and the absorbance values determined by ELISA, respectively. These values were not affected when a mixed fungal spore population was used. There was a relationship between the number of MTIST device-trapped ascospores of M. brassicicola per liter of air sampled and the amount of disease expressed on exposed plants of Brassica oleracea (Brussels sprouts). Similarly, when the MTIST device was used to trap conidia of B. cinerea, a correlation coefficient of 0.8797 was obtained for the absorbance values generated by the ELISA and the observed number of conidia per microtiter well. The relative collection efficiency of the MTIST device in controlled plant growth chambers with limited airflow was 1.7 times greater than the relative collection efficiency of a Burkard 7-day volumetric spore trap for collection of M. brassicicola ascospores. The MTIST device can be used to rapidly differentiate, determine, and accurately quantify target organisms in a microflora. The MTIST device is a portable, robust, inexpensive system that can be used to perform multiple tests in a single sampling period, and it should be useful for monitoring airborne particulates and microorganisms in a range of environments. (+info)
The hypersensitive response facilitates plant infection by the necrotrophic pathogen Botrytis cinerea.
BACKGROUND: Plants have evolved efficient mechanisms to combat pathogen attack. One of the earliest responses to attempted pathogen attack is the generation of oxidative burst that can trigger hypersensitive cell death. This is called the hypersensitive response (HR) and is considered to be a major element of plant disease resistance. The HR is thought to deprive the pathogens of a supply of food and confine them to initial infection site. Necrotrophic pathogens, such as the fungi Botrytis cinerea and Sclerotinia sclerotiorum, however, can utilize dead tissue. RESULTS: Inoculation of B. cinerea induced an oxidative burst and hypersensitive cell death in Arabidopsis. The degree of B. cinerea and S. sclerotiorum pathogenicity was directly dependent on the level of generation and accumulation of superoxide or hydrogen peroxide. Plant cells exhibited markers of HR death, such as nuclear condensation and induction of the HR-specific gene HSR203J. Growth of B. cinerea was suppressed in the HR-deficient mutant dnd1, and enhanced by HR caused by simultaneous infection with an avirulent strain of the bacterium Pseudomonas syringae. HR had an opposite (inhibitory) effect on a virulent (biotrophic) strain of P. syringae. Moreover, H(2)O(2) levels during HR correlated positively with B. cinerea growth but negatively with growth of virulent P. syringae. CONCLUSIONS: We show that, although hypersensitive cell death is efficient against biotrophic pathogens, it does not protect plants against infection by the necrotrophic pathogens B. cinerea and S. sclerotiorum. By contrast, B. cinerea triggers HR, which facilitates its colonization of plants. Hence, these fungi can exploit a host defense mechanism for their pathogenicity. (+info)
Identification and characterization of a hexapeptide with activity against phytopathogenic fungi that cause postharvest decay in fruits.
A hexapeptide of amino acid sequence Ac-Arg-Lys-Thr-Trp-Phe-Trp-NH2 was demonstrated to have antimicrobial activity against selected phytopathogenic fungi that cause postharvest decay in fruits. The peptide synthesized with either all D- or all L-amino acids inhibited the in vitro growth of strains of Penicilium italicum, P. digitatum, and Botrytis cinerea, with MICs of 60 to 80 microM and 50% inhibitory concentration (IC50) of 30 to 40 microM. The inhibitory activity of the peptide was both sequence- and fungus-specific since (i) sequence-related peptides lacked activity (including one with five residues identical to the active sequence), (ii) other filamentous fungi (including some that belong to the genus Penicllium) were insensitive to the peptide's antifungal action, and (iii) the peptide did not inhibit the growth of several yeast and bacterial strains assayed. Experiments on P. digitatum identified conidial germination as particularly sensitive to inhibition although mycelial growth was also affected. Our findings suggest that the inhibitory effect is initially driven by the electrostatic interaction of the peptide with fungal components. The antifungal peptide retarded the blue and green mold diseases of citrus fruits and the gray mold of tomato fruits under controlled inoculation conditions, thus providing evidence for the feasibility of using very short peptides in plant protection. This and previous studies with related peptides indicate some degree of peptide amino acid sequence and structure conservation associated with the antimicrobial activity, and suggest a general sequence layout for short antifungal peptides, consisting of one or two positively charged residues combined with aromatic amino acid residues. (+info)