Effect of disruption of a cutinase gene (cutA) on virulence and tissue specificity of Fusarium solani f. sp. cucurbitae race 2 toward Cucurbita maxima and C. moschata.

A 3.9-kb genomic DNA fragment from the cucurbit pathogen Fusarium solani f. sp. cucurbitae race 2 was cloned. Sequence analysis revealed an open reading frame of 690 nucleotides interrupted by a single 51-bp intron. The nucleotide and predicted amino acid sequences showed 92 and 98% identity, respectively, to those of the cutA gene of the pea pathogen F. solani f. sp. pisi. A gene replacement vector was constructed and used to generate cutA- mutants that were detected with a polymerase chain reaction (PCR) assay. Seventy-one cutA- mutants were identified among the 416 transformants screened. Vector integration was assessed by Southern analysis in 23 of these mutants. PCR and Southern analysis data showed the level of homologous integration was 14%. Disruption of the cutA locus in mutants was confirmed by RNA gel blot hybridization. Neither virulence on Cucurbita maxima cv. Delica at any of six different inoculum concentrations, nor pathogenicity on intact fruit of four different species or cultivars of cucurbit or hypocotyl tissue of C. maxima cv. Crown, was found to be affected by disruption of the cutA gene.

A detailed linkage map around an apple scab resistance gene demonstrates that two disease resistance classes both carry the V f gene.

A detailed genetic map has been constructed in apple (Malus x domestica Borkh.) in the region of the v f gene. This gene confers resistance to the apple scab fungus Venturia inaequalis (Cooke) Wint. Linkage data on four RAPD (random amplified polymorphic DNA) markers and the isoenzyme marker PGM-1, previously reported to be linked to the v f gene, are integrated using two populations segregating for resistance to apple scab. Two new RAPD markers linked to v f (identified by bulked segregant analysis) and a third marker previously reported as being present in several cultivars containing v f are also placed on the map. The map around v f now contains eight genetic markers spread over approximately 28 cM, with markers on both sides of the resistance gene. The study indicates that RAPD markers in the region of crab apple DNA introgressed with resistance are often transportable between apple clones carrying resistance from the same source. Analysis of co-segregation of the resistance classes 3A (weakly resistant) and 3B (weakly susceptible) with the linked set of genetic markers demonstrates that progeny of both classes carry the resistance gene.

Gene inactivation in the plant pathogen Glomerella cingulata: three strategies for the disruption of the pectin lyase gene pnlA.

The feasibility of performing routine transformation-mediated mutagenesis in Glomerella cingulata was analysed by adopting three one-step gene disruption strategies targeted at the pectin lyase gene pnlA. The efficiencies of disruption following transformation with gene replacement- or gene truncation-disruption vectors were compared. To effect replacement-disruption, G. cingulata was transformed with a vector carrying DNA from the pnlA locus in which the majority of the coding sequence had been replaced by the gene for hygromycin B resistance. Two of the five transformants investigated contained an inactivated pnlA gene (pnlA-); both also contained ectopically integrated vector sequences. The efficacy of gene disruption by transformation with two gene truncation-disruption vectors was also assessed. Both vectors carried at 5' and 3' truncated copy of the pnlA coding sequence, adjacent to the gene for hygromycin B resistance. The promoter sequences controlling the selectable marker differed in the two vectors. In one vector the homologous G. cingulata gpdA promoter controlled hygromycin B phosphotransferase expression (homologous truncation vector), whereas in the second vector promoter elements were from the Aspergillus nidulans gpdA gene (heterologous truncation vector). Following transformation with the homologous truncation vector, nine transformants were analysed by Southern hybridisation; no transformants contained a disrupted pnlA gene. Of nineteen heterologous truncation vector transformants, three contained a disrupted pnlA gene; Southern analysis revealed single integrations of vector sequence at pnlA in two of these transformants. pnlA mRNA was not detected by Northern hybridisation in pnlA- transformants. pnlA- transformants failed to produce a PNLA protein with a pI identical to one normally detected in wild-type isolates by silver and activity staining of isoelectric focussing gels. Pathogenesis on Capsicum and apple was unaffected by disruption of the pnlA gene, indicating that the corresponding gene product, PNLA, is not essential for pathogenicity. Gene disruption is a feasible method for selectively mutating defined loci in G. cingulata for functional analysis of the corresponding gene products.

The ARG4 gene of Candida albicans.

The DNA sequence of a Candida albicans genomic fragment known to complement the arginine mutation designated arg57 in strain 1006 contains an ORF of 1404 nucleotides (nt) predicting a protein of 468 amino acids (aa). Database searches indicated that the deduced protein shares 75% identity and 85% similarity with the ARG4 protein of Saccharomyces cerevisiae. Analysis of the percent aa identity between C. albicans and S. cerevisiae sequences included in available databases suggested these values are within the range expected for biosynthetic enzymes from the two organisms which share similar function. Experiments to isolate C. albicans ARG4 by complementation in an arg4 strain of S. cerevisiae yielded a plasmid (pARG4-1) with a restriction map identical to that of the sequenced clone. From these data, we conclude that the gene previously designated ARG57 is in fact ARG4 encoding the enzyme argininosuccinate lyase (ASL). These results were unexpected, since ARG57 had been localized to chromosome 7, while a mutation causing an ASL deficiency had been linked to ade1, which is on chromosome R. Transformation of C. albicans strains with pARG4-1 indicated it complemented the arginine auxotrophy in strains TMSU221 and 1435, a derivative of 1006. Examination of commonly utilized C. albicans arginine auxotrophs by spheroplast fusion analysis indicated these strains comprise two complementation groups: one consisting of 1006 and TMSU221, which are arg4, and the other of A642, hOG318, hOG357, FC18-6 and WC-5-4, which possess an undefined defect in the arginine biosynthetic pathway which we designate arg100.

The pectin lyase-encoding gene (pnl) family from Glomerella cingulata: characterization of pnlA and its expression in yeast.

Oligodeoxyribonucleotide primers were designed from conserved amino acid (aa) sequences between pectin lyase D (PNLD) from Aspergillus niger and pectate lyases A and E (PELA/E) from Erwinia chrysanthemi. The polymerase chain reaction (PCR) was used with these primers to amplify genomic DNA from the plant pathogenic fungus Glomerella cingulata. Three different 220-bp fragments with homology to PNL-encoding genes from A. niger, and a 320-bp fragment with homology to PEL-encoding genes from Nicotiana tabacum and E. carotovora were cloned. One of the 220-bp PCR products (designated pnlA) was used as a probe to isolate a PNL-encoding gene from a lambda genomic DNA library prepared from G. cingulata. Nucleotide (nt) sequence data revealed that this gene has seven exons and codes for a putative 380-aa protein. The nt sequence of a cDNA clone, prepared using PCR, confirmed the presence of the six introns. The positions of the introns were different from the sites of the five introns present in the three PNL-encoding genes previously sequenced from A. niger. PNLA was synthesised in yeast by cloning the cDNA into the expression vector, pEMBLYex-4, and enzymatically active protein was secreted into the culture medium. Significantly higher expression was achieved when the context of the start codon, CACCATG, was mutated to CAAAATG, a consensus sequence commonly found in highly expressed yeast genes. The produced protein had an isoelectric point (pI) of 9.4, the same as that for the G. cingulata pnlA product.(ABSTRACT TRUNCATED AT 250 WORDS)

Integration of vectors by homologous recombination in the plant pathogen Glomerella cingulata.

An homologous transformation system has been developed for the plant pathogenic fungus Glomerella cingulata (Colletotrichum gloeosporioides). A transformation vector containing the G. cingulata gpdA promoter fused to the hygromycin phosphotransferase gene was constructed. Southern analyses indicated that this vector integrated at single sites in most transformants. A novel method of PCR amplification across the recombination junction point indicated that the integration event occurred by homologous recombination in more than 95% of the transformants. Deletion studies demonstrated that 505 bp (the minimum length of homologous promoter DNA analysed which was still capable of promoter function) was sufficient to target integration events. Homologous integration of the vector resulted in duplication of the gdpA promoter region. When transformants were grown without selective pressure, a high incidence of vector excision by recombination between the duplicated regions was evident. The significance of these recombination characteristics is discussed with reference to the feasibility of performing gene disruption experiments.

Cloning and molecular characterization of the glyceraldehyde-3-phosphate dehydrogenase-encoding gene and cDNA from the plant pathogenic fungus Glomerella cingulata.

The glyceraldehyde-3-phosphate dehydrogenase gene (gpdA) has been identified from a genomic DNA library prepared from the plant pathogenic fungus Glomerella cingulata. Nucleotide sequence data revealed that this gene codes for a putative 338-amino-acid protein encoded by two exons of 129 and 885 bp, separated by an intron 216 bp long. The 5' leader sequence is also spliced by an intron of 156 bp. A cDNA clone was prepared using the polymerase chain reaction, the sequence of which was used to confirm the presence of the intron in the coding sequence and the splicing of the 5' leader sequence. The transcriptional start point (tsp) was mapped at -253 nt from the site of the initiation of translation by primer extension and is adjacent to a 42-bp pyrimidine-rich region. The general structure of the 5' flanking region shows similarities to gpdA from Aspergillus nidulans. The putative protein product is 71-86% identical at the aa level to GPDs from Aspergillus nidulans, Cryphonectria parasitica, Curvularia lunata, Podospora anserina and Ustilago maydis.

Genetics of the white-opaque transition in Candida albicans: demonstration of switching recessivity and mapping of switching genes.

Spheroplast fusion has been used to analyze the genetics of the reversible phenotypic transition, white-opaque, in Candida albicans WO-1. This transition involves changes in cell shape, permeability, and colony morphology. Fusion of switching with nonswitching cells gave nonswitching fusants, suggesting that the white-opaque phenotype is recessive. Chromosome loss induced by heat shock gave segregants of the fusants which were able to undergo the transition, indicating that the repressor function is genetically defined and probably limited to one or two chromosomes. Chromosomes R, 1, 3, 4, and 7 were eliminated as unique sites for the repressor, leaving 2, 5, and 6 as possible locations. When a ura3 (chromosome 3) nonswitching strain was fused with a switching strain, all ura3 segregants induced by heat shock were incapable of the phenotypic transition. Therefore, some or all of the genes (called SWI genes) essential for the transition are located on chromosome 3. UV irradiation-induced recombination did give rise to Ura- switching progeny, showing that the failure to switch was not due to a side effect of the pyrimidine requirement. The failure to isolate normally switching ura3 progeny generated by UV irradiation suggests a close linkage between the two genes.

High efficiency transformation of Fusarium solani f. sp. cucurbitae race 2 (mating population V).

A cosmid vector, suitable for library construction and DNA transformation in filamentous fungi, has been constructed and a reliable and highly efficient PEG-mediated DNA transformation system for F. solani f. sp. cucurbitae, based on resistance to hygromycin B, has been developed for use with this vector. This transformation system yielded 10(4) transformants per micrograms of DNA when using 10(7) protoplasts. Factors important in achieving high efficiency included: the maintenance of an osmoticum in all transformation steps, PEG 4000 concentration, and the ratio of transforming vector DNA to protoplasts. Approximately 60% of transformants stably integrated vector DNA. Molecular analysis revealed multiple copies of the plasmid integrated into the genome at one or more sites. The frequency of transformation achieved will facilitate the isolation of genes from this fungus by complementation.

Differentiation of Fusarium solani f. sp. cucurbitae races 1 and 2 by random amplification of polymorphic DNA.

We have used a PCR-based technique, involving the random amplification of polymorphic DNA (RAPD), to assess genome variability between 21 isolates from F. solani f. sp. cucurbitae races 1 and 2. Based on RAPD marker patterns the isolates fell into two distinct groups corresponding to mating populations MPI and MPV. Four isolates that could not be assigned to one or other mating population by traditional means were distinguished by RAPD patterns. Seven polymorphic RAPD products were used to probe Southern blots of MPI and MPV genomic DNA. Six of the seven probes hybridized to single-copy sequences and five of the seven probes showed specificity for one or other mating population. We suggest that not only is the technique a rapid and reliable tool for isolate-typing of fungi but it also provides a rapid method for obtaining species- or race-specific hybridization probes.

Genomic structure of Candida stellatoidea: extra chromosomes and gene duplication.

Candida albicans and Candida stellatoidea are two closely related imperfect yeasts. Some isolates characterized as C. stellatoidea are in fact C. albicans, while others differ with respect to virulence and to karyotype, containing extra small chromosomes. Experiments in this study allowed us to infer that a typical C. stellatoidea isolate, Y2360, has 12 chromosomes rather than the 7 previously shown for C. albicans. The majority of cloned sequences tested hybridized to analogous chromosomes in C. albicans and in C. stellatoidea, although there were exceptions, and a repeated element isolated as specific for C. albicans hybridized to most of the chromosomes of C. stellatoidea. Several genes tested hybridized to one of the smaller, C. stellatoidea-specific chromosomes as well as to a larger one. The arrangement of restriction enzyme sites around the gene was the same in both the large and small chromosomes. For ADE2 and LYS2, the arrangements were identical to those of a typical C. albicans strain, FC18, suggesting a high degree of sequence conservation between the two species. Spheroplast fusion and segregation experiments showed that the ADE2 genes on both the large and small chromosomes of C. stellatoidea are active, implying that the organism is functionally at least triploid for this gene and probably for any others duplicated on the smaller chromosomes.

Opaque-white phenotype transition: a programmed morphological transition in Candida albicans.

This paper reports that the opaque and white phenotypes of Candida albicans constitute a true high-frequency reversible transition system. The rDNA restriction fragment and orthogonal field alternating gel electrophoresis profiles of opaque and white phenotypes are indistinguishable, and a genetic marker introduced into a white strain is present in all opaque derivatives of this strain. Opaque and white derivatives appear markedly different on a bismuth indicator medium and differ in a number of other respects. We have used bismuth medium to examine the spontaneous and temperature-induced frequencies of transition from opaque to white. The temperature-induced transition from opaque to white does not occur when opaque cells are held in water.

Genetic analysis of red, adenine-requiring mutants of Candida albicans.

A number of investigators have described the isolation of red, adenine-requiring mutants of Candida albicans. Other fungi have been shown to give rise to two phenotypically similar, but genetically distinct, types of red, adenine-requiring mutants. This paper is the first indication that the red adenine mutants of C. albicans can similarly be resolved into two distinct classes. It is also believed to be the first report of such a resolution in an imperfect fungus. The resolution of these two classes was achieved by applying three distinct parasexual, analytical methods to this imperfect, naturally diploid yeast. The methods employed were complementation analysis of fused protoplasts and two methods of recombination analysis, induced mitotic crossing over in heterozygous revertants and induced mitotic crossing over in the heterozygous tetraploid products of protoplast fusion. The recombination methods depended on linkage analysis between the ade loci and two loci, met1 (methionine) and arg1 (arginine). The three analytical methods supported the same resolution. The results support the generally accepted view that C. albicans is diploid since they indicate disomic inheritance at the ade1, ade2, and met1 loci.