The ABC transporter BcatrB affects the sensitivity of Botrytis cinerea to the phytoalexin resveratrol and the fungicide fenpiclonil.

During pathogenesis, fungal pathogens are exposed to a variety of fungitoxic compounds. This may be particularly relevant to Botrytis cinerea, a plant pathogen that has a broad host range and, consequently, is subjected to exposure to many plant defense compounds. In practice, the pathogen is controlled with fungicides belonging to different chemical groups. ATP-binding cassette (ABC) transporters might provide protection against plant defense compounds and fungicides by ATP-driven efflux mechanisms. To test this hypothesis, we cloned BcatrB, an ABC transporter-encoding gene from B. cinerea. This gene encodes a 1,439 amino acid protein with nucleotide binding fold (NBF) and transmembrane (TM) domains in a [NBF-TM6]2 topology. The amino acid sequence has 31 to 67% identity with ABC transporters from various fungi. The expression of BcatrB is up regulated by treatment of B. cinerea germlings with the grapevine phytoalexin resveratrol and the fungicide fenpiclonil. BcatrB replacement mutants are not affected in saprophytic growth on different media but are more sensitive to resveratrol and fenpiclonil than the parental isolate. Furthermore, virulence of deltaBcatrB mutants on grapevine leaves was slightly reduced. These results indicate that BcatrB is a determinant in sensitivity of B. cinerea to plant defense compounds and fungicides.

Fungal transporters involved in efflux of natural toxic compounds and fungicides.

Survival of microorganisms in natural environments is favored by the capacity to produce compounds toxic to competing organisms and the ability to resist the effects of such toxic compounds. Both factors contribute to a competitive advantage of organisms in ecosystems. All organisms have evolved active transport mechanisms by which endogenous and exogenous toxicants can be secreted. Two major classes of transporter proteins are the ATP-binding cassette (ABC) and the major facilitator superfamily (MFS) transporters. Members of both classes can have broad and overlapping substrate specificities for natural toxic compounds and can be regarded as a "first-line defense barrier" in survival mechanisms. In plant pathogens, these transporters can play an essential role in protection against plant defense compounds during pathogenesis. Also, some transporters actively secrete host-specific and non-host-specific toxins. Remarkably, ABC and MFS transporters can also play a major role in fungicide sensitivity and resistance. Their role in multidrug resistance of Aspergillus nidulans, Candida albicans, and Saccharomyces cerevisiae to azoles and other fungitoxic compounds is well established. Knowledge of ABC and MFS transporters opens possibilities of developing novel strategies for controlling plant diseases, either by modulation of transporter activity or by transgenic expression of transporter genes in plants.

Functional expression of the gene cu, encoding the phytotoxic hydrophobin cerato-ulmin, enables Ophiostoma quercus, a nonpathogen on elm, to cause symptoms of Dutch elm disease.

We studied the involvement of the phytotoxic hydrophobin cerato-ulmin (CU) in pathogenesis and virulence of Dutch elm disease (DED) by expressing its encoding gene (cu) in Ophiostoma quercus, a nonpathogenic species on elm closely related to the DED pathogens O. ulmi and O. novo-ulmi. The production of the toxin was quantitatively determined in culture filtrates and in mycelial extracts of the transformants. Production of CU in vitro was associated with the ability to cause typical DED symptoms, consisting of foliar yellow and wilting and vascular tissue discoloration on a moderately resistant elm genotype. The presence of CU was monitored by enzyme-linked immunosorbent assay in symptomatic leaves of plants inoculated with O. quercus transformants expressing CU and found to be associated with wilt symptoms. In general, the virulence of the cu-expressing transformants, as measured in terms of vascular discoloration and percentage of defoliation, was lower than that of the mildly pathogenic isolate E2 of O. ulmi. However, one transformant (C39) displayed a virulence level intermediate between that of E2 and 182, a highly virulent isolate of O. novo-ulmi. Our results indicate that CU production influences virulence in nonaggressive strains of Ophiostoma fungi.

Multidrug resistance in Aspergillus nidulans involves novel ATP-binding cassette transporters.

Two single-copy genes, designated atrA and atrB (ATP-binding cassette transporter A and B), were cloned from the filamentous fungus Aspergillus nidulans and sequenced. Based on the presence of conserved motifs and on hydropathy analysis, the products encoded by atrA and atrB can be regarded as novel members of the ATP-binding cassette (ABC) superfamily of membrane transporters. Both products share the same topology as the ABC transporters PDR5 and SNQ2 from Saccharomyces cerevisiae and CDR1 from Candida albicans, which are involved in multidrug resistance of these yeasts. Significant homology also occurs between the ATP-binding cassettes of AtrA and AtrB, and those of mammalian ABC transporters (P-glycoproteins). The transcription of atrA and, in particular, atrB in mycelium of A. nidulans is strongly enhanced by treatment with several drugs, including antibiotics, azole fungicides and plant defense toxins. The enhanced transcription is detectable within a few minutes after drug treatment and coincides with the beginning of energy-dependent drug efflux activity, reported previously in the fungus for azole fungicides. Transcription of the atr genes has been studied in a wild-type and in a series of isogenic strains carrying the imaA and/or imaB genes, which confer multidrug resistance to various toxic compounds such as the azole fungicide imazalil. atrB is constitutively transcribed at a low level in the wild-type and in strains carrying imaA or imaB. Imazalil treatment enhances transcription of atrB to a similar extent in all strains tested. atrA, unlike atrB, displays a relatively high level of constitutive expression in mutants carrying imaB. Imazalil enhances transcription of atrA more strongly in imaB mutants, suggesting that the imaB locus regulates atrA. Functional analysis demonstrated that cDNA of atrB can complement the drug hypersensitivity associated with PDR5 deficiency in S. cerevisiae.

Simultaneous detection of cucumber mosaic virus, tomato mosaic virus and potato virus Y by flow cytometry.

The simultaneous detection is described of cucumber mosaic virus (CMV), potato virus Y (PVY) and tomato mosaic virus (ToMV) by flow cytometry. Extracts from leaves of healthy and CMV or PVY infected plants were incubated with latex particles, each with a diameter of 3 microm. Extracts from ToMV infected or uninfected plants, however, were incubated with particles, each with a diameter of 6 microm. Beads were washed and incubated in succession with primary and secondary antibodies, the latter labeled with phycoerythrin (PE) or fluorescein (FITC). CMV and PVY were distinguished on the basis of the fluorescence emitted by FITC and PE; ToMV was distinguished from CMV and PVY on the basis of the different diameter (6 microm) of the particles on which it was adsorbed. The three viruses were detected also by another approach. Latex particles with a diameter of 3, 6 and 10 microm were separately sensitized with antibodies specific for CMV, PVY and ToMV. An equal number of sensitized particles was mixed and incubated with the plant extracts containing the three viruses and then with anti-CMV, anti-PVY and anti-ToMV antibodies labeled with FITC. The study describes also a virus purification method based on the use of antibody coated latex particles. The method is simple technically and applicable to the purification of large as well as minute amounts of different viruses (CMV, PVY and ToMV).

Potential of genes and gene products from Trichoderma sp. and Gliocladium sp. for the development of biological pesticides.

Fungal cell wall degrading enzymes produced by the biocontrol fungi Trichoderma harzianum and Gliocladium virens are strong inhibitors of spore germination and hyphal elongation of a number of phytopathogenic fungi. The purified enzymes include chitinolytic enzymes with different modes of action or different substrate specificity and glucanolytic enzymes with exo-activity. A variety of synergistic interactions were found when different enzymes were combined or associated with biotic or abiotic antifungal agents. The levels of inhibition obtained by using enzyme combinations were, in some cases, comparable with commercial fungicides. Moreover, the antifungal interaction between enzymes and common fungicides allowed the reduction of the chemical doses up to 200-fold. Chitinolytic and glucanolytic enzymes from T. harzianum were able to improve substantially the antifungal ability of a biocontrol strain of Enterobacter cloacae. DNA fragments containing genes encoding for different chitinolytic enzymes were isolated from a cDNA library of T. harzianum and cloned for mechanistic studies and biocontrol purposes. Our results provide additional information on the role of lytic enzymes in processes of biocontrol and strongly suggest the use of lytic enzymes and their genes for biological control of plant diseases.

Production of polyclonal antibodies for cyclopaldic acid, a major phytotoxic metabolite produced by the plant pathogen Seiridium cupressi.

An antiserum against cyclopaldic acid (CA), a phytotoxic metabolite produced by Seiridium cupressi, the fungal pathogen of a canker disease of cypress, was prepared by immunization of rabbits with a CA-bovine serum albumin conjugate (CA-BSA). Antibodies recognizing CA were purified by affinity chromatography through an immunoadsorbent prepared by conjugating CA to lysine-Sepharose 4B. The specificity of antibodies was assayed against CA and some of its derivatives by competitive indirect enzyme-linked immunoassay. The homologous antigen CA-BSA and CA showed the highest binding activity with antibodies. The derivatives of CA modified in 1 of the 2 aldehyde groups reacted with antibodies to a lesser extent compared with CA-BSA and CA. Isocyclopaldic acid and the tetraacetylderivative of CA, which have more than one modified functional group, showed a further decrease in their reactivity. Other derivatives, which have more substantial structural modifications of the carbon skeleton, did not react. The possible use of these antibodies in studies concerning the involvement of CA in the canker disease of cypress is discussed.

Goat-mouse hybridomas secreting goat immunoglobulins.

Monoclonal antibodies specific for an allotypic marker of goat IgG2 were used to select goat-mouse hybrid cells secreting goat IgG2. Four of these hybrid cell clones continued to synthesize goat IgG2 (5-15 micrograms/ml) for over eight months. They will be used to study goat IgG gene regulation.

Identification of goat allotypic determinants and generation of goat-mouse hybridomas secreting goat IgG2.

Five allotypic determinants controlled by independent genes have been identified in goat. Of these determinants, four have been detected with alloimmune antisera and one with monoclonal antibodies. The specificities A1, C1 and D1 are lipoproteins; B1 is possibly an alpha 2 macroglobulin and E1 and IgG2. The specificity B1 is not expressed until the age of 3-4 months. The gene controlling the specificity E1 is present at about the same frequency (0.38-0.41) in goat, sheep, cattle and water buffalo. Stable hybridomas secreting goat IgG2 have been obtained by the fusion of goat peripheral lymphocytes with mouse myeloma cells.