A fungal gene for antibiotic resistance on a dispensable ("B") chromosome.

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 supernumerary chromosome of Nectria haematococca that carries pea-pathogenicity-related genes also carries a trait for pea rhizosphere competitiveness.

Fungi are found in a wide range of environments, and the ecological and host diversity of the fungus Nectria haematococca has been shown to be due in part to unique genes on different supernumerary chromosomes. These chromosomes have been called "conditionally dispensable" (CD) since they are not needed for axenic growth but are important for expanding the host range of individual isolates. From a biological perspective, the CD chromosomes can be compared to bacterial plasmids that carry unique genes that can define the habits of these microorganisms. The current study establishes that the N. haematococca PDA1-CD chromosome, which contains the genes for pea pathogenicity (PEP cluster) on pea roots, also carries a gene(s) for the utilization of homoserine, a compound found in large amounts in pea root exudates. Competition studies demonstrate that an isolate that lacks the PEP cluster but carries a portion of the CD chromosome which includes the homoserine utilization (HUT) gene(s) is more competitive in the pea rhizosphere than an isolate without the CD chromosome.

Characterization of the PDA1 promoter of Nectria haematococca and identification of a region that binds a pisatin-responsive DNA binding factor.

Isolates of Nectria haematococca (anamorph: Fusarium solani) are able to detoxify the pea phytoalexin pisatin through expression of pisatin demethylase (pda). This enzyme is a substrate-inducible cytochrome P450 monooxyenase that is encoded by the PDA gene family. In the current study, PDA1, a highly inducible PDA gene, was cloned and the 5' untranslated region was sequenced. The PDA mRNA levels were measured in pisatin-treated mycelium and found to increase by 20-fold over untreated control. Gel shift assays identified a 35-bp region, -514 to -480 bp relative to the first mRNA start site, that binds a factor found in extracts of pisatin-treated mycelium and absent in untreated mycelium. The function of the binding site in pisatin regulation of the PDA1 gene was tested in an in vivo competition assay by introduction of multiple ectopic copies of the binding site into N. haematococca through transformation. In such transformants, induction of pda activity by pisatin was delayed and reduced, consistent with the titration of a trans-acting factor which responds to pisatin. These results suggest the 35-bp region is functioning as a pisatin-responsive activator binding site for PDA1. Additional controls were characterized that act on PDA1 expression. Induction of pda by pisatin was suppressed by the addition of 0.8% Casamino Acids or 5% glucose to the suspended mycelium. A unique DNA binding factor was detected only in extracts from mycelia treated with the Casamino Acids that bind to the same 35-bp region of the PDA1 gene as the pisatin-responsive factor.

Purification and characterization of beta 2-tomatinase, an enzyme involved in the degradation of alpha-tomatine and isolation of the gene encoding beta 2-tomatinase from Septoria lycopersici.

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.

Cloning and characterization of the PDA6-1 gene encoding a fungal cytochrome P-450 which detoxifies the phytoalexin pisatin from garden pea.

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.

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.

Fungal Sensitivity to and Enzymatic Degradation of the Phytoanticipin alpha-Tomatine.

ABSTRACT alpha-Tomatine, synthesized by Lycopersicon and some Solanum species, is toxic to a broad range of fungi, presumably because it binds to 3beta-hydroxy sterols in fungal membranes. Several fungal pathogens of tomato have previously been shown to be tolerant of this glycoalkaloid and to possess enzymes thought to be involved in its detoxification. In the current study, 23 fungal strains were examined for their ability to degrade alpha-tomatine and for their sensitivity to this compound and two breakdown products, beta(2)-tomatine and tomatidine. Both saprophytes and all five non-pathogens of tomato tested were sensitive, while all but two tomato pathogens (Stemphylium solani and Verticillium dahliae) were tolerant of alpha-to-matine (50% effective dose > 300 muM). Except for an isolate of Botrytis cinerea isolated from grape, no degradation products were detected when saprophytes and nonpathogens were grown in the presence of alpha-tomatine. All tomato pathogens except Phytophthora infestans and Pythium aphani-dermatum degraded alpha-tomatine. There was a strong correlation between tolerance to alpha-tomatine, the ability to degrade this compound, and pathogenicity on tomato. However, while beta(2)-tomatine and tomatidine were less toxic to most tomato pathogens, these breakdown products were inhibitory to some of the saprophytes and nonpathogens of tomato, suggesting that tomato pathogens may have multiple tolerance mechanisms to alpha-tomatine.

Breeding for Highly Fertile Isolates of Nectria haematococca MPVI that are Highly Virulent on Pea and In Planta Selection for Virulent Recombinants.

ABSTRACT The heterothallic ascomycete Nectria haematococca mating population VI (anamorph Fusarium solani) is a broad host range pathogen. Field isolates of this fungus that are pathogenic on pea tend to be female sterile, of low fertility, and the same mating type (MAT-1), whereas female fertile isolates of either mating type that are highly fertile tend to be nonpathogenic on this plant. To facilitate genetic analysis of traits that may be important in the ability of N. haematococca to parasitize peas, a breeding project was undertaken to produce hermaphroditic isolates of each mating type that are highly fertile and highly virulent on peas. Although the association of high virulence on peas with female sterility and the MAT-1 mating type was not completely broken, isolates with high fertility and high virulence on peas were bred within two generations. Highly virulent progeny were also isolated by an alternative method in which pea plants were inoculated with a mixture of ascospores from a cross between two moderately virulent parents. Whereas all ascospores isolated without selection in planta had lower virulence than the parents, many isolates recovered from diseased tissue were more virulent than the parental isolates. Some of the recovered isolates were shown by restriction fragment length polymorphism analysis to be genetic recombinants of the parents, demonstrating that the pea tissue selected virulent recombinants. All highly virulent isolates tested had the ability to detoxify the pea phytoalexin pisatin, again showing a link between this trait and pathogenicity on the pea.

Spatial Analysis of Phytophthora infestans Genotypes and Late Blight Severity on Tomato and Potato in the Del Fuerte Valley Using Geostatistics and Geographic Information Systems.

ABSTRACT Genetic structure of Phytophthora infestans, the causal agent of potato and tomato late blight, was analyzed spatially in a mixed potato and tomato production area in the Del Fuerte Valley, Sinaloa, Mexico. Isolates of P. infestans were characterized by mating type, allozyme analysis at the glucose-6-phosphate isomerase and peptidase loci, restriction fragment length polymorphism with probe RG57, metalaxyl sensitivity, and aggressiveness to tomato and potato. Spatial patterns of P. infestans genotypes were analyzed by geographical information systems and geo-statistics during the seasons of 1994-95, 1995-96, and 1996-97. Spatial analysis of the genetic structure of P. infestans indicates that geographic substructuring of this pathogen occurs in this area. Maps displaying the probabilities of occurrence of mating types and genotypes of P. infestans, and of disease severity at a regional scale, were presented. Some genotypes that exhibited differences in epidemiologically important features such as metalaxyl sensitivity and aggressiveness to tomato and potato had a restricted spread and were localized in isolated areas. Analysis of late blight severity showed recurring patterns, such as the earliest onset of the disease in the area where both potato and tomato were growing, strengthening the hypothesis that infected potato tubers are the main source of primary inoculum. The information that geostatistical analysis provides might help improve management programs for late blight in the Del Fuerte Valley.

Three Genes for Metabolism of the Phytoalexin Maackiain in the Plant Pathogen Nectria haematococca: Meiotic Instability and Relationship to a New Gene for Pisatin Demethylase.

Some isolates of the plant-pathogenic fungus Nectria haematococca mating population (MP) VI metabolize maackiain and medicarpin, two antimicrobial compounds (phytoalexins) synthesized by chickpea (Cicer arietinum L.). The enzymatic modifications by the fungus convert the phytoalexins to less toxic derivatives, and this detoxification has been proposed to be important for pathogenesis on chickpea. In the present study, loci controlling maackiain metabolism (Mak genes) were identified by crosses among isolates of N. haematococca MP VI that differed in their ability to metabolize the phytoalexin. Strains carrying Mak1 or Mak2 converted maackiain to 1a-hydroxymaackiain, while those with Mak3 converted it to 6a-hydroxymaackiain. Mak1 and Mak2 were unusual in that they often failed to be inherited by progeny. Mak1 was closely linked to Pda6, a new member in a family of genes in N. haematococca MP VI that encode enzymes for detoxification of pisatin, the phytoalexin synthesized by garden pea. Like Mak1, Pda6 was also transmitted irregularly to progeny. Although the unusual meiotic behaviors of some Mak genes complicate genetic analysis, identification of these genes should afford a more through evaluation of the role of phytoalexin detoxification in the pathogenesis of N. haematococca MP VI on chickpea.

Genetic Analysis of the Role of Phytoalexin Detoxification in Virulence of the Fungus Nectria haematococca on Chickpea (Cicer arietinum).

Chickpea (Cicer arietium L.) produces the antimicrobial compounds (phytoalexins) medicarpin and maackiain in response to infection by microorganisms. Nectria haematococca mating population (MP) VI, a fungus pathogenic on chickpea, can metabolize maackiain and medicarpin to less toxic products. These reactions are thought to be detoxification mechanisms in N. haematococca MP VI and required for pathogenesis by this fungus on chickpea. In the present study, these hypotheses were tested by examining the phenotypes of progeny from crosses of the fungus that segregated for genes (Mak genes) controlling phytoalexin metabolism. Mak1 and Mak2, two genes that individually confer the ability to convert maackiain to its 1a-hydroxydienone derivative, were linked to higher tolerance of the phytoalexins and high virulence on chickpea. These results indicate that this metabolic reaction is a mechanism for increased phytoalexin tolerance in the fungus, which thereby allows a higher virulence on chickpea. Mak3, a gene conferring the ability to convert maackiain to its 6a-hydroxypterocarpan derivative, also increased tolerance to maackiain in strains which carried it; however, the contribution of Mak3 to the overall level of pathogenesis could not be evaluated because most progeny from the cross segregating for this gene were low in virulence. Thus, metabolic detoxification of phytoalexins appeared to be necessary, as demonstrated in the Mak1 and Mak2 crosses, but not sufficient by itself, as in the Mak3 cross, for high virulence of N. haematococca MP VI on chickpea.

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 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.

Cloning and properties of a cyanide hydratase gene from the phytopathogenic fungus Gloeocercospora sorghi.

The Cht gene encoding cyanide hydratase (CHT, EC 4.2.1.66), which detoxifies HCN and is thought to be important in fungal infection of cyanogenic plants, has been cloned from the phytopathogenic fungus Gloeocercospora sorghi. The gene was isolated by screening an expression library of G. sorghi using a CHT-specific antibody and using one of the positive cDNA clones as a probe in Southern hybridization to identify a 3.1 kb PstI genomic fragment. This PstI fragment expressed CHT activity when transformed into Aspergillus nidulans, a fungus that normally lacks CHT activity. Sequence analysis identified a single open reading frame of 1,107 base pairs which encodes a polypeptide of 40,904 daltons. The deduced amino acid sequence of CHT shares 36.5% identity to a nitrilase from the bacterium Klebsiella pneumoniae subsp. ozaenae.

Characterization of pisatin-inducible cytochrome p450s in fungal pathogens of pea that detoxify the pea phytoalexin pisatin.

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.

Purification and Characterization of S-Adenosyl-l-methionine:6a-Hydroxymaackiain 3-O-Methyltransferase from Pisum sativum.

The isoflavonoid phytoalexin pisatin is synthesized by Pisum sativum in response to microbial infection and certain other forms of stress. An enzyme which synthesizes pisatin by methylating the 3-hydroxyl of (+)6a-hydroxymaackiain (HMK) was extracted from CuCl(2)-stressed pea seedlings. The enzyme was enriched 370-fold by (NH(4))(2)SO(4) precipitation, DEAE chromatography, chromatofocusing, and hydrophobic interaction chromatography (HIC), to a specific activity of 8.2 microkatals per gram protein. Enzyme activity profiles from chromatofocusing and HIC columns suggested the presence of two isozymes, of pl 5.2 and 4.9. Nondenaturing gel filtration of the HIC-purified enzyme gave a single peak of activity at the same elution volume as BSA (66 kilodaltons); the active fractions showed two proteins upon SDS-PAGE, of M(r) 66,000 and 43,000. The smaller protein was most abundant in chromatographic fractions containing peak enzyme activity throughout purification. In a partially purified preparation, this 43 kilodalton protein was the only one photoaffinity labelled by [(3)H]S-adenosyl-l-methionine. The purified enzyme preferred the (+) over the (-) stereoisomer of HMK and other pterocarpans; overall, (+)HMK was the best substrate. K(m) values were 2.3 micromolar for (+)HMK and 35 micromolar for S-adenosyl-l-methionine. The methyltransferase had a pH optimum of 7.9 and no apparent divalent cation requirement.

Solubilization and Reconstitution of Pisatin Demethylase, a Cytochrome P-450 from the Pathogenic Fungus Nectria haematococca.

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.

Purification and characterization of cyanide hydratase from the phytopathogenic fungus Gloeocercospora sorghi.

Previous studies have demonstrated that fungal pathogens of cyanogenic plants produce cyanide hydratase (CHT, EC 4.2.1.66), which converts HCN to formamide. Production of CHT in these fungi is thought to be a means of circumventing cyanide toxicity, and CHT is thus believed to be an important pathogenicity trait. In the present study, 13 species of fungi were assayed for CHT production, and all 7 species that were pathogens of sorghum, a cyanogenic plant, produced this enzyme. CHT was purified to apparent homogeneity from one of these sorghum pathogens, Gloeocercospora sorghi. The enzyme had a Km of 12 mM for KCN. Enzymatically functional CHT was obtained only as a large molecular entity of greater than 300 kDa. However, a polypeptide of approximately 45 kDa was identified as the only component of purified CHT detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 45-kDa polypeptide band could be resolved into three isozymes of pI 6.1, 6.3, and 6.5. Antibodies raised against the 45-kDa polypeptide inhibited the G. sorghi CHT activity and showed high specificity in Western blots to a polypeptide of approximately the same size. The evidence suggests that functional G. sorghi CHT is an aggregated protein that consists of 45-kDa polypeptides. A CHT with similar properties was also found in the fungus Colletotrichum graminicola, another pathogen of sorghum.