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In Nectria haematococca the MAK1 gene product converts a chick-pea (Cicer arietinum) phytoalexin, maackiain, into a less toxic compound. The presence of MAK1 in this fungal pathogen is also correlated with high virulence on chick-pea. Previous genetic analysis suggested that MAK1 is located on a meiotically unstable, dispensable chromosome. The unstable nature of this chromosome facilitated MAK1 cloning by allowing us to identify a subset of genomic cosmid clones likely to contain MAK1. Truncated forms of the chromosome, generated during meiosis, were isolated from strains either able (Mak+) or unable (Mak-) to metabolize maackiain and used to probe a chromosome-specific cosmid library. Only clones that hybridized exclusively to the chromosome from the Mak+ strain were then screened for their ability to transform a Mak- isolate to the Mak+ phenotype. A 2.7 kb HindIII-PstI fragment was subcloned from a cosmid conferring MAK1 activity, and its nucleotide sequence determined. Because MAK1 transcription is not induced strongly by maackiain, a reverse transcriptase-polymerase chain reaction was required to detect MAK1 transcription in a Mak+ strain, and to isolate MAK1 cDNA fragments. Comparison of the genomic and cDNA sequences of MAK1 revealed the presence of three introns and an open reading frame encoding a protein 460 amino acids in length. Two diagnostic domains in its deduced amino acid sequence suggest MAK1 encodes a flavin-containing mono-oxygenase. MAK1 is the first gene encoding maackiain detoxification to be cloned, and is the second functional gene cloned from this dispensable chromosome. Southern analysis of genomic DNA from ascospore isolates containing MAK2, MAK3, and MAK4 indicated that MAK1 is not homologous to other known maackianin-detoxifying genes.

Lycopersicon species often contain the toxic glycoalkaloid alpha-tomatine, which is proposed to protect these plants from general microbial infection. however, fungal pathogens of tomato often are tolerant to alpha-tomatine and detoxification of alpha-tomatine may be how these pathogens avoid this potential barrier. As an initial step to evaluate this possibility, we have purfied to homogeneity a beta-1,2-D glucosidase from the tomato pathogen Septoria lycopersici that hydrolyzes the beta-1,2-D glucosyl bond on the tetrasaccharide moiety of alpha-tomatine to produce beta2-tomatine. The enzyme is a 110-kDa protein with a pI of 4.5 and a Km for alpha-tomatine of 62 microM. Little or no activity was detected on a variety of other glycosides. The gene encoding this protein was isolated and contains an open reading frame of 803 amino acids that shares sequence homology with several other beta-D-glucosidases. When S. lycopersici was incubated with alpha-tomatine, beta2-tomatinase mRNA accumulated, suggesting that the enzyme is substrate inducible. Aspergillus nidulans expressed ¿beta2-tomatinase¿ activity when transformed with this gene but transformants were still sensitive to alpha-tomatine.

The ability to detoxify pisatin, a phytoalexin produced by garden pea (Pisum sativum), is controlled by a family of PDA (pisatin demethylating ability) genes in the phytopathogenic fungus Nectria haematococa, MP (mating population) VI. Six known PDA genes each encode characteristic levels of inducible enzyme activity and are associated with different degrees of virulence on pea. To elucidate the phenotypic differences associated with these genes, we have cloned and characterized the PDA6-1 gene which encodes a pisatin-detoxifying enzyme and we compare it to another PDA gene, PDAT9. Pisatin demethylation was measured in PDA6-1 transformants of Aspergillus nidulans and shown to be regulated by glucose. The deduced amino acid (aa) sequence of PDA6-1 was 90% identical to that of the cytochrome P-450 encoded by PDAT9, but lacked nine aa at the C terminus, which has been postulated to be a site involved in substrate binding. A 35-bp sequence present upstream of a third PDA gene, PDA1, which appears to be important for induction of PDA1 by pisatin, was conserved in PDAT9, but not in PDA6-1. We conclude that PDA6-1, which does not appear to contribute to the virulence of N. haematococa on pea, differs significantly from PDAT9, which is associated with high virulence.

The gene PDAT9 from the fungus Nectria haematococca encodes pisatin demethylase, an enzyme that detoxifies the phytoalexin pisatin, an antimicrobial compound produced by pea in response to infection by this plant pathogen. PDAT9 was found to contain an open reading frame (ORF) encoding 515 amino acids and four introns of 52-58 nucleotides each within its coding region. The amino acid sequence F-G-A-G-S-R-S-C-I-G, indicative of the "fifth ligand binding site" present in all cytochrome P450s, occurs as residues 446 to 455, confirming that PDAT9 is a cytochrome P450. The deduced amino acid sequence is distinct from all other reported cytochrome P-450s, and PDAT9 has been assigned to a new cytochrome P450 family, CYP57. A 1.3 kb SacI fragment of the PDAT9 ORF that lacked the fifth ligand binding site, hybridized to unique DNA fragments in N. haematococca isolates known to possess PDA genes that encode different whole cell phenotypes for pisatin demethylating activity. These genes were also tentatively identified as cytochrome P450s by the hybridization of the same fragments to separate subclones of PDAT9, one of which contained the fifth ligand sequence. That probe also hybridized to DNA other than that attributed to pisatin demethylase genes; these other DNAs are presumed to represent other cytochrome P450s.

Nectria haematococca MPVI can be found in many different biological habitats but has been most studied as a pathogen of pea (Pisum sativum). Genetic analyses of isolates obtained from a variety of biological sources has indicated that a number of genes control pathogenicity on pea but that one important PEa Pathogenicity (PEP) gene is PDA, which confers the ability to detoxify the pea phytoalexin pisatin. In these studies, all naturally occurring isolates that lacked PDA (i.e. Pda- isolates) and all Pda- progeny were essentially non-pathogenic on pea. However, we have demonstrated recently that Pda- mutants created by transformation-mediated gene disruptions, while having a modest reduction in virulence, and more virulent than any naturally occurring Pda- isolates. In addition we know that PDA genes are on dispensable (DS) chromosomes in this fungus. We believed that the gene disruption mutants have allowed the detection of other PEP genes that are present on the DS chromosomes along with PDA and that naturally occurring Pda- isolates usually lack this DS chromosome. This would explain why naturally occurring Pda- isolates are always low in virulence. We propose that the DS chromosomes in fungi are analogous to bacterial plasmids which allow those microorganisms to colonise different habitats, i.e. the DS chromosomes of Nectria haematococca contain genes that allow individual isolates of this broad host range pathogen to occupy different biological niches.

A family of cytochrome P-450 (Pda) genes in the pathogenic fungus Nectria haematococca is responsible for the detoxification of the phytoalexin pisatin, an antimicrobial compound produced by garden pea (Pisum sativum L.). The Pda6 gene was mapped by electrophoretic karyotype analysis to a small meiotically unstable chromosome that is dispensable for normal growth. Such traits are typical of B chromosomes. The strains of Nectria studied here have no sequences that are homologous to the Pda family other than Pda6 and therefore demonstrate that unique, functional genes can be found on B chromosomes. Unstable B chromosomes may be one mechanism for generating pathogenic variation in fungi.

The ability to detoxify the phytoalexin, pisatin, an antimicrobial compound produced by pea (Pisum sativum L.), is one requirement for pathogenicity of the fungus Nectria haematococca on this plant. Detoxification is mediated by a cytochrome P-450, pisatin demethylase, encoded by any one of six Pda genes, which differ with respect to the inducibility and level of pisatin demethylase activity they confer, and which are associated with different levels of virulence on pea. A previously cloned Pda gene (PdaT9) was used in this study to characterize further the known genes and to identify additional members of the Pda family in this fungus by Southern analysis. DNA from all isolates which demethylate pisatin (Pda+ isolates) hybridized to PdaT9, while only one Pda- isolate possessed DNA homologous to the probe. Hybridization intensity and, in some cases, restriction fragment size, were correlated with enzyme inducibility. XhoI/BamHI restricted DNA from reference strains with a single active Pda allele had only one fragment with homology to PdaT9; no homology attributable to alleles associated with the Pda- phenotype was found. Homology to this probe was also limited to one or two restriction fragments in most of the 31 field isolates examined. Some unusual progeny from laboratory crosses that failed to inherit demethylase activity also lost the single restriction fragment homologous to PdaT9. At the chromosome level, N. haematococca is highly variable, each isolate having a unique electrophoretic karyotype. In most instances, PdaT9 hybridized to one or two chromosomes containing 1.6-2 million bases of DNA, while many Pda- isolates lacked chromosomes in this size class. The results from this study of the Pda family support the hypothesis that deletion of large amounts of genomic DNA is one mechanism that reduces the frequency of Pda genes in N. haematococca, while simultaneously increasing its karyotypic variation.

Detoxification of the pea phytoalexin pisatin via demethylation, mediated by a cytochrome P-450 monooxygenase, is thought to be important for pathogenicity of the fungus Nectria haematococca on pea. To isolate a fungal gene encoding pisatin demethylating activity (pda), we transformed Aspergillus nidulans with a genomic library of N. haematococca DNA constructed in a cosmid which carried the A. nidulans trpC gene. Transformants were selected for Trp+ and then screened for pda. One transformant among 1250 tested was Pda+ and was less sensitive to pisatin in culture than Pda- A. nidulans. The cosmid containing the gene (PDA) conferring this activity was recovered by phage lambda packaging of transformant genomic DNA. When A. nidulans was transformed with the cloned cosmid, 98% of the Trp+ transformants were Pda+. RNA blots probed with a 3.35 kb subclone carrying PDA indicated that the gene is expressed constitutively in A. nidulans but is inducible by pisatin in N. haematococca.