Isolation of a phytoalexin-detoxification gene from the plant pathogenic fungus Nectria haematococca by detecting its expression in Aspergillus nidulans

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.     

Characterization of the gene encoding pisatin demethylase (FoPDA1) in Fusarium oxysporum

The pea pathogen Fusarium oxysporum f. sp. pisi is able to detoxify pisatin produced as a defense response by pea, and the gene encoding this detoxification mechanism, FoPDA1, was 82% identical to the cytochrome P450 pisatin demethylase PDA1 gene in Nectria haematococca. A survey of F. oxysporum f. sp. pisi isolates demonstrated that, as in N. haematococca, the PDA gene of F. oxysporum f. sp. pisi is generally located on a small chromosome. In N. haematococca, PDA1 is in a cluster of pea pathogenicity (PEP) genes. Homologs of these PEP genes also were found in the F. oxysporum f. sp. pisi isolates, and PEP1 and PEP5 were sometimes located on the same small chromosomes as the FoPDA1 homologs. Transforming FoPDA1 into a pda(?) F. oxysporum f. sp. lini isolate conferred pda activity and promoted pathogenicity on pea to some transformants. Different hybridization patterns of FoPDA1 were found in F. oxysporum f. sp. pisi but these did not correlate with the races of the fungus, suggesting that races within this forma specialis arose independently of FoPDA1. FoPDA1 also was present in the formae speciales lini, glycines, and dianthi of F. oxysporum but they had mutations resulting in nonfunctional proteins. However, an active FoPDA1 was present in F. oxysporum f. sp. phaseoli and it was virulent on pea. Despite their evolutionary distance, the amino acid sequences of FoPDA1 of F. oxysporum f. sp. pisi and F. oxysporum f. sp. phaseoli revealed only six amino acid differences, consistent with a horizontal gene transfer event accounting for the origin of these genes.     

Solubilization and partial purification of ATPase from a rose cell plasma membrane fraction

Some isolates of the fungus Nectria haematococca Berk. and Br. can demethylate pisatin, a phytoalexin from pea (Pisum sativum L.). Pisatin demethylation appears to be necessary for tolerance to pisatin and virulence on pea, and is catalyzed by a microsomal cytochrome P-450. We now report solubilization of this enzyme from N. haematococca microsomes. Pisatin demethylase activity was obtained in the high speed supernatant of detergent treated microsomes, if detergent was removed before assay. The CO-binding spectrum of the soluble enzyme preparation indicated the presence of cytochrome P-450. Cholic acids were the most effective of the detergents tested for solubilizing enzyme activity. Loss of enzyme activity during solubilization was reduced by certain protease inhibitors, but not by substrate, reducing agents, antioxidants, or phospholipids. The most effective solubilization medium tested was 1% sodium cholate, 100 millimolar potassium phosphate, 500 millimolar sucrose, 1 millimolar phenylmethylsulfonyl fluoride, pH 7.5, which yielded approximately 30% of the pisatin demethylase and over 95% of the NADPH-cytochrome c reductase in the soluble fraction. Demethylase activity was lost when the reductase was removed by adsorption on 2′,5′-ADP-agarose. The demethylase activity of reductase-free fractions could be restored by adding a reductase preparation purified approximately 100-fold from microsomes of N. haematococca isolate 74-8-1, which does not demethylate pisatin. We conclude that pisatin demethylase requires NADPH-cytochrome c reductase for activity. The inability of some isolates to demethylate pisatin appears to be due to the absence of a suitable cytochrome P-450, rather than to a lack of functional reductase.     

Amplified single-nucleotide polymorphisms and a (GA)(n) microsatellite marker reveal genetic differentiation between populations of Histoplasma capsulatum from the Americas

Many fungi that are pathogenic on pea have the ability to demethylate and thus detoxify the pea phytoalexin pisatin. This detoxification reaction has been studied most thoroughly in Nectria haematococca MP VI where it functions as a virulence trait. The enzyme catalyzing this reaction [pisatin demethylase (pda)] is a cytochrome P450. In the current study, the induction of whole-cell pda activity and the biochemical properties of pda in microsomal preparations from the pea pathogens Ascochyta pisi, Mycosphaerella pinodes, and Phoma pinodella are compared to the pda produced by N. haematococca. Based on cofactor requirements and their inhibition by carbon monoxide, cytochrome P450 inhibitors, and antibodies to NADPH:cytochrome P450 reductase, we conclude that the pdas from the other pea pathogens also are cytochrome P450s. All of the enzymes show a rather selective induction by pisatin, have a low K(m) toward pisatin, and have a fairly high degree of specificity toward pisatin as a substrate, suggesting that each pathogen may have a specific cytochrome P450 for detoxifying this plant antibiotic. Since the pdas in these fungi differ in their pattern of sensitivity to P450 inhibitors and display other minor biochemical differences, we suggest that these fungi may have independently evolved a specialized cytochrome P450 as a virulence trait for a common host.     

An ABC transporter and a cytochrome P450 of Nectria haematococca MPVI are virulence factors on pea and are the major tolerance mechanisms to the phytoalexin pisatin

The fungal plant pathogen Nectria haematococca MPVI produces a cytochrome P450 that is responsible for detoxifying the phytoalexin pisatin, produced as a defense mechanism by its host, garden pea. In this study, we demonstrate that this fungus also produces a specific ATP-binding cassette (ABC) transporter, NhABC1, that enhances its tolerance to pisatin. In addition, although both mechanisms individually contribute to the tolerance of pisatin and act as host-specific virulence factors, mutations in both genes render the fungus even more sensitive to pisatin and essentially nonpathogenic on pea. NhABC1 is rapidly induced after treatment with pisatin in vitro and during infection of pea plants. Furthermore, NhABC1 was able to confer tolerance to the phytoalexin rishitin, produced by potato. NhABC1 appears to be orthologous to GpABC1 of the potato pathogen Gibberella pulicaris and, along with MoABC1 from Magnaporthe oryzae, resides in a phylogenetically related clade enriched with ABC transorters involved in virulence. We propose that NhABC1 and the cytochrome P450 may function in a sequential manner in which the energy expense from pisatin efflux by NhABC1 releases the repression of the cytochrome P450, ultimately allowing pisatin tolerance by two mechanisms. These results demonstrate that a successful pathogen has evolved multiple mechanisms to overcome these plant antimicrobial compounds.     

Biochemical properties of the products of cytochrome P450 genes (PDA) encoding pisatin demethylase activity in Nectria haematococca

Pea plants produce the antibiotic (+)pisatin in response to infection by the fungus Nectria haematococca, which can detoxify pisatin utilizing a cytochrome P450 monooxygenase called pisatin demethylase. Genes (PDA) have been identified that encode different whole-cell Pda phenotypes that can be distinguished by the length of the lag period and the resulting amount of enzyme activity produced: Pda^SH = short lag, high activity; Pda^SM = short lag, moderate activity; and Pda^LL = long lag, low activity. Only the Pda^SH and Pda^SM phenotypes have been correlated with pathogenicity on pea. In this study, we utilize heterologous expression of the PDA ^LL gene PDA6-1 in Aspergillus nidulans to compare the biochemical properties of the product of this gene with the products of the PDA ^SH gene PDA1 expressed in N. haematococca. Preliminary measurements were also done on the PDA ^SM gene PDA5 expressed in N. haematococca. The PDA gene products differed somewhat in their substrate specificity and in their sensitivity to a few cytochrome P450 inhibitors. However, the enzymes produced by PDA6-1 and PDA1 both had low apparent K _m values toward (+)pisatin (< 0.25 μM) and a common high degree of insensitivity to most P450 inhibitors, suggesting similar shared biochemical traits as would be expected of products of a highly homologous gene family. Our results indicate that the different whole-cell phenotypes of N. haematococca are not due to significant differences in the biochemical properties of the gene products and are consistent with recent results that indicate that the phenotypic differences are due to different degrees of expression of the genes. Received: 6 October 1997 / Accepted: 13 May 1998     

Detoxification of the phytoalexin pisatin by a fungal cytochrome P-450

The fungus Nectria haematococca, a pathogen of garden pea (Pisum sativum), can demethylate pisatin, an antimicrobial compound synthesized by infected pea tissue. The phenolic product is less toxic than pisatin to many microorganisms. Cell extracts catalyzing pisatin demethylation were obtained from N. haematococca, and the properties of the reaction were examined. The enzyme activity was greatest in the high-speed pellet fraction, in which rates up to 20 nmol/min/mg protein were observed. The K_m for pisatin was relatively low, less than 5 μm. The reaction was dependent on NADPH, which could not be replaced by any other cofactor tested. However, in the presence of NADPH, NADH increased the rate of demethylation. Oxygen uptake by the enzyme was stimulated by addition of pisatin, the increment of oxygen utilization being approximately equimolar with pisatin added. Formaldehyde was a product of the reaction. The effects of various inhibitors were tested to determine whether this reaction is mediated by cytochrome P-450. The respiratory inhibitors KCN (1 mm) and antimycin A strongly inhibited the demethylation of pisatin by intact cells of the fungus, but not by the NADPH-supplemented enzyme. The cytochrome P-450 inhibitors SKF 525-A and 1-(2-isopropylphenyl)imidazole inhibited demethylation both in whole cells and in the enzyme preparation, though the latter compound was effective only at high concentrations. Most other cytochrome P-450 inhibitors tested had little effect. However the reaction was quite sensitive to CO, and this inhibition was readily reversed by light at wavelengths near 450 nm. It is concluded that pisatin demethylase is a cytochrome P-450 monooxygenase.     

Duplication of a conditionally dispensable chromosome carrying pea pathogenicity (PEP) gene clusters in Nectria haematococca

A supernumerary chromosome called a conditionally dispensable chromosome (CDC) is essential for pathogenicity of Nectria haematococca on pea. Among several CDCs discovered in N. haematococca, the PDA1 CDC that harbors the pisatin demethylation gene PDA1 is one of the best-studied CDCs and serves as a model for plant-pathogenic fungi. Although the presence of multiple copies is usual for supernumerary chromosomes in other eukaryotes, this possibility has not been examined well for any CDCs in N. haematococca. In this study, we produced strains with multiple copies of the PDA1 CDC by protoplast fusion and analyzed dosage effects of this chromosome. Using multiple methods, including cytological chromosome counting and fluorescence in situ hybridization, the fusion products between two transformants derived from the same strain that bears a single PDA1 CDC were shown to contain two PDA1 CDCs from both transformants and estimated to be haploid resulting from the deletion of an extra set or sets of A chromosomes in the fused nuclei. In phenotype assays, dosage effects of PDA1 CDC in the fusion products were evident as increased virulence and homoserine-utilizing ability compared with the parents. In a separate fusion experiment, PDA1 CDC accumulated up to four copies in a haploid genome.     

A gene from the fungal plant pathogen Nectria haematococca that encodes the phytoalexin-detoxifying enzyme pisatin demethylase defines a new cytochrome P450 family

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 P454s, 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.     

Pisatin demethylase genes are on dispensable chromosomes while genes for pathogenicity on carrot and ripe tomato are on other chromosomes in Nectria haematococca

Studies on the wide-host-range fungus Nectria haematococca MP VI have shown a linkage between virulence on pea and five of nine PDA genes that encode the ability to detoxify the pea phytoalexin, pisatin. Most of the PDA genes are on chromosomes of approximately 1.6 megabases (Mb) and two of these genes, PDA1-2 and PDA6-1, have been demonstrated to reside on approximately 1.6-Mb chromosomes that can be lost during meiosis. Prior studies also have shown that the dispensable chromosome carrying PDA6-1 contains a gene (MAK1) necessary for maximum virulence on chickpea. The present study evaluated whether the other approximately 1.6-Mb chromosomes that carry PDA genes also are dispensable, their relationship to each other, and whether they contain genes for pathogenicity on hosts other than pea or chickpea. DNA from the PDA1-1 chromosome (associated with virulence on pea) and the PDA6-1 chromosome (associated with virulence on chickpea) were used to probe blots of contour-clamped homogeneous electric field (CHEF) gels of isolates carrying different PDA genes and genetically related Pda- isolates. All of the approximately 1.6-Mb PDA-bearing chromosomes hybridized with both probes, indicating that they share significant similarity. Genetically related Pda-progeny lacked chromosomes of approximately 1.6 Mb and there was no significant hybridization of any chromosomes to the PDA1-1 and PDA6-1 chromosome probes. When isolates carrying different PDA genes and related Pda- isolates were tested for virulence on carrot and ripe tomato, there was no significant difference in lesion sizes produced by Pda+ and Pda- isolates, indicating that genes for pathogenicity on these hosts are not on the PDA-containing chromosomes. These results support the hypothesis that the chromosomes carrying PDA genes are dispensable and carry host-specific virulence genes while genes for pathogenicity on other hosts are carried on other chromosomes.     

Identification of new pisatin demethylase genes (PDA5 and PDA7) in Nectria haematococca and non-Mendelian segregation of pisatin demethylating ability and virulence on pea due to loss of chromosomal elements

Previous studies have shown that high virulence on pea in Nectria haematococca Mating Population VI is linked to the ability to detoxify the pea phytoalexin, pisatin, via demethylation (Pda). To test this linkage further, a highly virulent Pda(+) isolate (34-18) was used as the recurrent parent in backcrosses to Pda(-) isolates, but most of the progeny were low in virulence on pea, and tetrad analysis gave conflicting ratios for the genetic control of Pda. Southern analysis of 34-18 and progeny showed that 34-18 carries a gene similar to PDA1 (PDA1-2), two new PDA genes, PDA5 and PDA7, and that all three genes can be lost during meiosis. Southern analysis of electrophoretic karyotypes showed that PDA1-2 is on a 1.5-Mb dispensable chromosome in 34-18 and that PDA5 and PDA7 are on a 4.9-Mb chromosome in 34-18 but are found on variably sized chromosomes in progeny. Loss of PDA5 or PDA7 in progeny was not generally associated with morphological phenotypes, except in progeny from some crosses between PDA5 parents. Loss of PDA5 was associated with growth abnormalities in these crosses, suggesting that in some genetic backgrounds at least a portion of the PDA5/PDA7 chromosome is essential for normal growth. All highly virulent progeny had PDA1-2 or a combination of PDA5 and PDA7 while isolates that lacked the three genes were low in virulence, supporting the hypothesis that Pda, or genes linked to PDA genes, are necessary for virulence on pea. However, low virulence isolates with PDA genes were also identified, suggesting that there are pathogenicity genes that can segregate independently of PDA genes.     

Identification and chromosomal locations of a family of cytochrome P-450 genes for pisatin detoxification in the fungus Nectria haematococca

Summary 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.     

Isolation and identification of an allelopathic substance in Pisum sativum

The residue of peas (Pisum sativum L.) has allelopathic activity and the putative compound causing this inhibitory effect was isolated from a methanol extract of pea shoots. Chemical structure of this compound was determined by high-resolution MS, IR and 1H NMR spectral data as pisatin. Pisatin inhibited growth of cress (Lepidium sativum L.) and lettuce (Lactuca sativa L.) seedlings at concentrations greater than 10 and 30 microM, respectively. The doses required for 50% growth inhibition of roots and hypocotyls of cress were 61 and 91 microM, respectively, and those of lettuce were 78 and 115 microM, respectively. The concentration of pisatin in the pea shoots was 32.7 nmol x g(-1) fresh weight. The effectiveness of pisatin on growth inhibition in cress and lettuce, and its occurrence in pea shoots suggest that it may contribute to the growth inhibitory effect of pea residue, and may play an important role in pea allelopathy.     

Partial purification of pisatin demethylase,a cytochrome P-450 from the pathogenic fungus Nectria haematococca

The plant pathogen Nectria haematococca can demethylate pisatin, a phytoalexin from pea. Demethylation is apparently necessary for virulence on pea and is catalyzed by a microsomal cytochrome P-450 monooxygenase system. The cytochrome P-450 and NADPH-cytochrome P-450 reductase of this system were solubilized with sodium cholate and partially purified by chromatography on blue A-agarose and -aminohexyl-agarose. The reductase was further purified by chromatography on 2,5-ADP-agarose to a specific activity of about 16 moles cytochrome c reduced per min per mg protein. Upon sodium dodecyl sulfatepolyacrylamide gel electrophoresis, the reductase fraction contained one major band of molecular weight 84,000. The partially purified cytochrome P-450 fraction contained a number of minor bands and three major bands of molecular weights 52,000, 56,000 and 58,000. This fraction lost all demethylase activity during concentration after -aminohexyl-agarose chromatography, so it could not be purified further. The purified reductase could reconstitute demethylase activity of cytochrome P-450 fractions and appeared to be rate-limiting for demethylase activity in microsomal extracts.