The loss of the inducible Aspergillus carbonarius MFS transporter MfsA leads to ochratoxin A overproduction.

Ochratoxin A (OTA), a nephrotoxic compound produced by certain Aspergillus and Penicillium species, is one of the most abundant mycotoxins in food commodities. Aspergillus carbonarius is the main source of OTA in wine, grape juice and dried vine fruits. Although many studies have focused on OTA production by A. carbonarius, little is known about the genes related to OTA production and transport. We have found a transporter that belongs to the major facilitator superfamily (MfsA) which is highly expressed with a 102-fold induction in an ochratoxigenic A. carbonarius strain compared to a low OTA producer strain. The encoding mfsA gene shows similarity to the multidrug efflux transporter flu1 from Candida albicans. A high number of putative transcription factor binding sites involved in the response to stress were identified within the promoter of mfsA. Phenotypical analysis of ΔmfsA deletion mutants revealed that the loss of mfsA leads to a slight growth reduction and increased OTA production. We therefore hypothesize that MfsA could be a stress response transporter whose disruption could cause an increase in oxidative stress together with a stimulation of mycotoxin production.

Use of GFP-tagged strains of Penicillium digitatum and Penicillium expansum to study host-pathogen interactions in oranges and apples.

Penicillium digitatum and Penicillium expansum are responsible for green and blue molds in citrus and pome fruits, respectively, which result in major monetary losses worldwide. In order to study their infection process in fruits, we successfully introduced a green fluorescent protein (GFP) encoding gene into wild type P. digitatum and P. expansum isolates, using Agrobacterium tumefaciens-mediated transformation (ATMT), with hygromycin B resistance as the selectable marker. To our knowledge, this is the first report describing the transformation of these two important postharvest pathogens with GFP and the use of transformed strains to study compatible and non-host pathogen interactions. Transformation did not affect the pathogenicity or the ecophysiology of either species compared to their respective wild type strains. The GFP-tagged strains were used for in situ analysis of compatible and non-host pathogen interactions on oranges and apples. Knowledge of the infection process of apples and oranges by these pathogens will facilitate the design of novel strategies to control these postharvest diseases and the use of the GFP-tagged strains will help to determine the response of P. digitatum and P. expansum on/in plant surface and tissues to different postharvest treatments.

Development of a green fluorescent tagged strain of Aspergillus carbonarius to monitor fungal colonization in grapes.

An enhanced green fluorescent protein has been used to tag an OTA-producing strain of Aspergillus carbonarius (W04-40) isolated from naturally infected grape berries. Transformation of the fungus was mediated by Agrobacterium tumefaciens. The most efficient transformation occurred when the co-cultivation was done with 10(4) conidia due to higher frequency of resistance colonies (894 per 10(4) conidia) and lower background obtained. To confirm the presence of the hph gene in hygromycin resistant colonies, 20 putative transformants were screened by PCR analysis. The hph gene was identified in all the transformants. Variation on the expression levels of the eGFP was detected among the transformants and 50% of them appeared bright green fluorescent under the microscope. Microscopic analysis of all the bright fluorescent transformants revealed homogeneity of the fluorescent signal, which was clearly visible in the hyphae as well as in the conidia. eGFP expression in A. carbonarius was shown to be stable in all transformants. Confocal Laser scanning microscopy images of grape berries infected with the eGFP transformant demonstrated fungal penetration into the berry tissues. OTA production was importantly increased in the eGFP transformant in comparison with the wild type strain and pathogenicity on grape berries was slightly decreased after four days of inoculation. However, no differences in virulence were found after seven days of inoculation, thus allowing utilization of this eGFP mutant for in situ analysis of A. carbonarius infection of grape berries. To our knowledge, this is the first report describing the construction of a GFP-tagged strain belonging to Aspergillus section Nigri for monitoring Aspergillus rot on grape berries.

Genes differentially expressed by Aspergillus carbonarius strains under ochratoxin A producing conditions.

Aspergillus carbonarius is an important ochratoxin A (OTA)-producing fungus that is responsible for toxin contamination of grapes and wine, coffee and cocoa. A suppression subtractive hybridization (SSH) approach was performed with two strains of A. carbonarius, antagonistic in their OTA-production ability, to identify genes whose expression is linked with the ability to produce OTA. BlastX analysis identified 109 differentially-expressed sequences putatively involved in the production of OTA, with significant similarities (E(value)<10(-5)) to sequences deposited in the NCBI non-redundant protein database. Of the 109 ESTs, 26% were involved in regulation processes, 15% corresponded to hypothetical proteins, 12% were involved in stress response and detoxification, 9% corresponded to transport and secretion processes, 7% corresponded to amino acid metabolism, 7% were involved in hydrolysis of energy reserves and 5% involved in secondary metabolism. Other unisequences showed homology to genes involved in protein synthesis and general metabolism. According to their sequence similarities to genes in the NCBI database, the possible functional roles they might play in the production and regulation of OTA are discussed. Worth noting is the high percentage of genes involved in regulation, including specific and global regulators. It is also important to note the high percentage of genes involved in the response to stress and detoxification.

Development of a citrus genome-wide EST collection and cDNA microarray as resources for genomic studies.

A functional genomics project has been initiated to approach the molecular characterization of the main biological and agronomical traits of citrus. As a key part of this project, a citrus EST collection has been generated from 25 cDNA libraries covering different tissues, developmental stages and stress conditions. The collection includes a total of 22,635 high-quality ESTs, grouped in 11,836 putative unigenes, which represent at least one third of the estimated number of genes in the citrus genome. Functional annotation of unigenes which have Arabidopsis orthologues (68% of all unigenes) revealed gene representation in every major functional category, suggesting that a genome-wide EST collection was obtained. A Citrus clementina Hort. ex Tan. cv. Clemenules genomic library, that will contribute to further characterization of relevant genes, has also been constructed. To initiate the analysis of citrus transcriptome, we have developed a cDNA microarray containing 12,672 probes corresponding to 6875 putative unigenes of the collection. Technical characterization of the microarray showed high intra- and inter-array reproducibility, as well as a good range of sensitivity. We have also validated gene expression data achieved with this microarray through an independent technique such as RNA gel blot analysis.

The use of transgenic yeasts expressing a gene encoding a glycosyl-hydrolase as a tool to increase resveratrol content in wine.

Resveratrol, a phenolic compound produced in grapes, exhibits properties that may contribute to the reduction of the incidence of coronary heart disease and other human health related processes. Recombinant yeast strains expressing the Aspergillus niger abfB gene encoding an alpha-L-arabinofuranosidase or the Candida molischiana bgIN gene encoding a beta-glucosidase have been used in vinifications as tools to increase the resveratrol content of white wine. Glycosylated resveratrol isomers (trans- and cis-piceid) and free resveratrol isomers (trans- and cis-resveratrol) were detected and quantified in white wines. Wines fermented with the strain expressing BgiN showed an increased amount of total resveratrol derivatives, particularly the non-glycosylated forms.

Requirement for either a host- or pectin-induced pectate lyase for infection of Pisum sativum by Nectria hematococca.

Fungal pathogens usually have multiple genes that encode extracellular hydrolytic enzymes that may degrade the physical barriers in their hosts during the invasion process. Nectria hematococca, a plant pathogen, has two inducible pectate lyase (PL) genes (pel) encoding PL that can help degrade the carbohydrate barrier in the host. pelA is induced by pectin, whereas pelD is induced only in planta. We show that the disruption of either the pelA or pelD genes alone causes no detectable decrease in virulence. Disruption of both pelA and pelD drastically reduces virulence. Complementation of the double disruptant with pelD gene, or supplementation of the infection droplets of the double disruptant with either purified enzyme, PLA, or PLD, caused a recovery in virulence. These results show that PL is a virulence factor. Thus, we demonstrate that disruption of all functionally redundant genes is required to demonstrate the role of host barrier-degrading enzymes in pathogenesis and that dismissal of the role of such enzymes based on the effects of single-gene disruption may be premature.

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.

Heterologous Expression of a Candida molischiana Anthocyanin-beta-glucosidase in a Wine Yeast Strain.

A recombinant wine yeast strain expressing the Candidamolischiana bgln gene encoding a beta-glucosidase/anthocyanase under the control of the Saccharomyces cerevisiae actin gene promoter has been constructed. The corresponding protein, BGLN, was mainly located on the cell wall. BGLN was purified in a single chromotagraphic step, and different physicochemical and kinetic properties have been determined. BGLN showed maximum activity against the artificial substrate p-nitrophenyl beta-D-glucopyranoside. It also hydrolyzed salicin, p-nitrophenyl beta-D-xyloside, cellobiose, and arbutin to a lesser extent. Fructose and SO(2) did not affect enzyme activity, which was activated by ethanol, while glucose was a strong competitive inhibitor. The purified BGLN showed a novel anthocyanase decolorizing capability on red wines. This anthocyanase activity was readily observed during microvinification experiments. However, the physicochemical characteristics of the wines obtained with the recombinant wine yeast strain were indistinguishable from those obtained with the parental strain.

Transformants of Trichoderma longibrachiatum Overexpressing the beta-1,4-Endoglucanase Gene egl1 Show Enhanced Biocontrol of Pythium ultimum on Cucumber.

ABSTRACT Nine transformants of Trichoderma longibrachiatum with extra copies of the egl1 gene were studied for mitotic stability, endoglucanase production, and biocontrol activity against Pythium ultimum on cucumber seedlings. The transformants showed a significantly higher level of expression of the egl1 gene in comparison to the wild type under both inducing and noninducing growth conditions. Transformants with the egl1 gene under the control of a constitutive promoter had the highest enzymatic activity. Both the endoglucanase activity and the transforming sequences were stable under nonselective conditions. When applied to cucumber seeds sown in P. ultimum-infested soil, T. longibrachiatum transformants with increased inducible or constitutive egl1 expression generally were more suppressive than the wild-type strain.

Expression in a wine yeast strain of the Aspergillus niger abfB gene.

A recombinant wine yeast strain has been constructed expressing the gene coding for alpha-L-arabinofuranosidase B from Aspergillus niger under the control of the yeast actin gene promoter. The protein is efficiently secreted by the recombinant yeast, allowing its purification and characterisation. The heterologous alpha-L-arabinofuranosidase B shows similar physico-chemical properties to the native enzyme. The wine produced in microvinification experiments using the recombinant yeast presents the same oenological characteristics as obtained with the untransformed strain. The alpha-L-arabinofuranosidase B protein is detected throughout the fermentation.

Identification of a novel pelD gene expressed uniquely in planta by Fusarium solani f. sp. pisi (Nectria haematococca, mating type VI) and characterization of its protein product as an endo-pectate lyase.

Antibodies prepared against a pectin-inducible pectate lyase (PLA) produced by a phytopathogenic fungus Fusarium solani f. sp. pisi (Nectria haematococca, mating type VI) were previously found to protect the host from infection. The gene (pelA) and two of its homologs were cloned and sequenced. Here we report the isolation of a new pectate lyase gene, pelD, from a genomic library of F. solani pisi. A 1.5-kb DNA fragment containing pelD and its flanking regions was sequenced. The nucleotide sequence of pelD would encode a protein of 24.5 kDa which shares 49, 44, and 65% amino acid sequence identity with PLA, PLB, and PLC, respectively, from the same fungus. Because the first 19 amino acid residues appeared to be a signal peptide, the mature enzyme could be a 22.7-kDa protein. pelD transcripts and PLD protein could not be detected in fungus cultured in glucose, pectin, pea epicotyl extract, or a pea cell wall preparation. However, pelD transcripts were readily found by RT-PCR with RNA isolated from infected pea tissues. The cDNA of pelD, thus obtained, was expressed in Pichia pastoris with the putative pelD signal sequence. The secreted PLD was purified and characterized to be an endopectate lyase, and its lyase activity could be inhibited by anti-PLA IgG. Thus, protection of the host observed with the anti-PLA antibodies could reflect inhibition of immunologically related pectate lyases including PLD which is expressed uniquely in planta.

Cloning of a novel constitutively expressed pectate lyase gene pelB from Fusarium solani f. sp. pisi (Nectria haematococca, mating type VI) and characterization of the gene product expressed in Pichia pastoris.

Since plant-pathogenic fungi must penetrate through pectinaceous layers of the host cell wall, pectin-degrading enzymes are thought to be important for pathogenesis. Antibodies prepared against a pectin-inducible pectate lyase (pectate lyase A [PLA]) produced by a phytopathogenic fungus, Fusarium solani f. sp. pisi (Nectria haematococca, mating type VI), was previously found to protect the host from infection. The gene (pelA) and its cDNA were cloned and sequenced. Here we report the isolation of a new pectate lyase gene, pelB, from a genomic library of F. solani f. sp. pisi with the pelA cDNA as the probe. A 2.6-kb DNA fragment containing pelB and its flanking regions was sequenced. The coding region of pelB was amplified by reverse transcription-mediated PCR, using total RNA isolated from F. solani pisi culture grown in the presence of glucose as the sole carbon source. The predicted open reading frame of pelB would encode a 25.6-kDa protein of 244 amino acids which has 65% amino acid sequence identity with PLA from F. solani f. sp. pisi but no significant homology with other pectinolytic enzymes. The first 16 amino acid residues at the N terminus appeared to be a signal peptide. The pelB cDNA was expressed in Pichia pastoris, yielding a pectate lyase B (PLB) which was found to be a glycoprotein of 29 kDa. PLB was purified to homogeneity by using a two-step procedure involving ammonium sulfate precipitation followed by Superdex G75 gel filtration chromatography. Purified PLB showed optimal lyase activity at pH 10.0. A rapid drop in the viscosity of the substrate and Mono Q anion-exchange chromatography of the products generated by the lyase showed that PLB cleaved polygalacturonate chains in an endo fashion. Western blotting (immunoblotting) with antibodies raised against PLA showed that PLB and PLA are immunologically related to each other. The 5' flanking regions of both pelA and pelB were translationally fused to the beta-glucuronidase gene and introduced into F. solani f. sp. pisi, and beta-glucuronidase activities of the transformants were measured. Expression of the marker gene by the transformants showed that pelA expression is induced by pectin and repressed by glucose, whereas expression of pelB is constitutive and is not subject to glucose repression. Reverse transcription-mediated PCR showed that both pelA and pelB are expressed when F. solani f. sp. pisi infects pea epicotyl.

Cloning of a new pectate lyase gene pelC from Fusarium solani f. sp. pisi (Nectria haematococca, mating type VI) and characterization of the gene product expressed in Pichia pastoris.

Antibodies prepared against a pectate lyase (PLA) produced by a phytopathogenic fungus Fusarium solani f. sp. pisi (Nectria haematococca, mating type VI) were previously found to protect the host against infection. The cDNA and gene (pelA) for PLA were cloned and sequenced. A new pectate lyase gene, pelC, was isolated from a genomic library of F. solani pisi with pelA cDNA as a probe. A 1.3-kb DNA fragment containing the pelC gene and its flanking regions was identified and sequenced. The coding region of pelC was amplified by reverse transcription-polymerase chain reaction using total RNA isolated from a pectin-induced F. solani pisi culture as template. The open reading frame of pelC was predicted to encode a 23.3-kDa protein of 219 amino acid residues, which shares 51% identity with PLA from F. solani pisi. No typical fungal leader peptide sequence could be identified at the N-terminus of the predicted protein sequence. The amplified pelC cDNA was expressed in Pichia pastoris yielding a pectate lyase C (PLC) with a molecular mass of 26.0 kDa and containing carbohydrates. PLC was purified to homogeneity using Mono Q anion-exchange chromatography. Purified PLC required Ca2+ for its activity and showed optimal lyase activity at pH 9.5 and 55 degrees C. Rapid drop in the viscosity of the substrate and Mono Q anion-exchange chromatography of the products generated by the lyase showed that PLC cleaved polygalacturonate chains in an endo fashion. Western blot using antibodies raised against PLA and PLC showed that PLC and PLA are immunologically related to each other.

[Applications of molecular biology in the wine industry]. / Aplicaciones de la biología molecular en enología.

Population dynamics of natural and inoculated industrial wine fermentations have been studied by using a simple molecular biology technique based on mitochondrial DNA restriction analysis profile. The predominance of the inoculated strain in the inoculated fermentations is obvious. A genetic transformation system has been developed for an industrial wine yeast strain named T73. By using this technique, different fungal hydrolases in this industrial strain have been expressed. Problems and benefits of the application of recombinant DNA techniques in wine yeast strains are also discussed here.

Construction of a recombinant wine yeast strain expressing a fungal pectate lyase gene.

A gene fusion between the Saccharomyces cerevisiae actin gene promoter and the cDNA of the Fusarium solani f. sp. pisi pelA gene has been constructed. This expression cassette has been introduced into the industrial wine yeast strain T73. The resulting recombinant strain is able to secrete active PELA enzyme into the culture medium. In preliminary microvinification experiments the wine produced by this pectinolytic strain is indistinguishable from wine produced using the non-transformed strain on the basis of the chemical analyses. Large scale fermentations need to be carried out in order to assess the effects on filtrability.

Molecular cloning and transcriptional analysis of the Aspergillus terreus gla1 gene encoding a glucoamylase.

The Aspergillus terreus gla1 gene, coding for a glucoamylase, has been cloned by heterologous hybridization. The gene is interrupted by four introns and encodes a protein with an N-terminal catalytic domain and a C-terminal starch-binding domain. The expression of the gene is induced by starch and maltose and repressed by glucose.

Transcriptional regulation of the Trichoderma longibrachiatum egl1 gene.

Transcription of the Trichoderma longibrachiatum egl1 gene is induced in the presence of lactose and beta-methylglucoside and repressed by glucose. A DNA fragment containing 722 bp upstream of the ATG codon has been sequenced. The gene has two major transcription start points (20 and 24 nucleotides upstream from the ATG codon) and several transcription termination points (located in a region around 130 nt downstream of the stop codon). Two 6-mer sequences (5'-CTGGAG-3') separated by 16 bp are present in the egl1 gene promoter. These sequences match the Aspergillus nidulans consensus CreA binding site and might be implicated in carbon catabolite repression of egl1 transcription.

Development of a transformation system for Trichoderma longibrachiatum and its use for constructing multicopy transformants for the egl1 gene.

An efficient transformation system for the fungus Trichoderma longibrachiatum has been developed. Transformation was obtained both by electroporation and polyethyleneglycol treatment, using a plasmid carrying the Escherichia coli hygromycin B phosphotransferase gene as a dominant selectable marker. The transformation frequency was 0.5 to 5 transformants/micrograms plasmid DNA. Transformation normally occurred by tandem integration of the transforming DNA. A high percentage of the transformants were mitotically unstable. The efficiency of co-transformation was very high (around 90%), and several co-transformants containing multiple copies of the egl1 gene encoding a beta-(1,4)-endoglucanase were obtained. Some of them secrete increased levels of endoglucanase to the culture medium. In addition, the E. coli lacZ gene was expressed in an active form under control of the Aspergillus nidulans gpdA gene promoter.

Isolation and analysis of a novel inducible pectate lyase gene from the phytopathogenic fungus Fusarium solani f. sp. pisi (Nectria haematococca, mating population VI).

A pectate lyase produced by Fusarium solani f. sp. pisi (Nectria haematococca, mating population VI) was previously shown to be essential for host infection (M. S. Crawford and P. E. Kolattukudy, Arch. Biochem. Biophys. 258:196-205, 1987). Pectate lyase genes have not been cloned from any phytopathogenic fungi. A gene, designated pelA, encoding an inducible pectate lyase was isolated from F. solani f. sp. pisi. A probe was synthesized by polymerase chain reaction with oligonucleotide primers based on the known amino acid sequences of two regions of the mature protein and first-strand cDNA as template. Both cDNA and the gene were isolated and sequenced. That the cloned cDNA represents the previously purified pectate lyase is shown by the complete match of the sequences of the N-terminal 38 amino acid residues and the 20 amino acid residues of an internal peptide with the sequence deduced from the cDNA sequence. This lyase sequence shows little homology to those of other pectolytic enzymes. The pelA gene shows standard characteristics with respect to promoter, intron, and polyadenylation sequences. As determined by primer extension and nuclease S1 analysis of the origin of the transcription, there are multiple initiation sites clustered in a region of 12 nucleotides located about 55 bp upstream of the start codon. Northern (RNA) blot analysis showed a single band of mRNA at about 1 kb. The pelA gene mRNA was detected only when F. solani f. sp. pisi was grown with pectin, and there was no detectable transcript accumulation when the fungus was grown with glucose as the sole carbon source. When both carbon sources were present, the pelA gene was transcribed only after glucose was completely depleted, indicating carbon catabolite repression. Moreover, the levels of transcription decreased rapidly prior to maximal enzyme accumulation, suggesting a mechanism of self catabolite repression.