The cyclic peptide synthetase catalyzing HC-toxin production in the filamentous fungus Cochliobolus carbonum is encoded by a 15.7-kilobase open reading frame.

Race 1 of Cochliobolus carbonum, a fungal plant pathogen, owes its exceptional virulence on certain genotypes of maize to the production of HC-toxin, a cyclic tetrapeptide. Production of HC-toxin is controlled by a single known gene, TOX2. Race 1, but not races that do not make HC-toxin, contains two copies of a 22-kilobase (kb) region of chromosomal DNA that is required for HC-toxin biosynthesis and hence virulence. We have sequenced this 22-kb region and here show that it contains an open reading frame of 15.7 kb that encodes a multifunctional cyclic peptide synthetase of potential M(r)574,620. This gene, called HTS1, apparently contains no introns. The predicted gene product, HC-toxin synthetase (HTS), contains four amino acid-binding (adenylate-forming) domains that are highly similar to those found in other cyclic peptide synthetases and other adenylate-binding enzymes. The DNA sequence encodes tryptic peptides derived from two HC-toxin biosynthetic enzymes, HC-toxin synthetase 1 (HTS-1) and HC-toxin synthetase 2 (HTS-2), indicating that these two enzymes exist in vivo as part of a single polypeptide. Consistent with this, in some enzyme preparations antibodies against the enzyme HTS-2, which was originally purified as a protein with a subunit M(r) of 160,000, recognize a protein with an estimated subunit M(r) greater than 480,000.     

Enzymatic Detoxification of HC-toxin, the Host-Selective Cyclic Peptide from Cochliobolus carbonum

Resistance to the fungal plant pathogen Cochliobolus carbonum race 1 and to its host-selective toxin, HC-toxin, is determined by Hm, a single dominant gene in the host plant maize, (Zea mays L). Radiolabeled HC-toxin of specific activity 70 milliCuries per millimole, prepared by feeding tritiated d,l-alanine to the fungus, was used to study its fate in maize leaf tissues. HC-toxin was converted by resistant leaf segments to a single compound, identified by mass spectrometry and nuclear magnetic resonance as the 8-hydroxy derivative of HC-toxin formed by reduction of the 8-keto group of 2-amino-9, 10-epoxy-8-oxo-decanoic acid, one of the amino acids in HC-toxin. Reduction of HC-toxin occurred in cell-free preparations from etiolated (Hm/hm) maize shoots, and the activity was sensitive to heat and proteolytic digestion, dependent on NADPH, and inhibited by p-hydroxymercuribenzoate and disulfiram. The enzyme (from the Hm/hm genotype) was partially purified by ammonium sulfate precipitation and diethylaminoethyl-ion exchange chromatography. By gel filtration chromatography, the enzyme had a molecular weight of 42,000. NADH was approximately 30% as effective as NADPH as a hydride donor, and flavin-containing cofactors had no effect on activity. When HC-toxin was introduced to maize leaf segments through the transpiration stream, leaf segments from both resistant and susceptible maize inactivated toxin equally well over a time-course of 9 hours. Although these data suggest no relationship between toxin metabolism and host selectivity, we discuss findings in apparent conflict with the current data and describe why the relationship between enzymatic reduction of HC-toxin and Hm remains unresolved.     

A eukaryotic alanine racemase gene involved in cyclic peptide biosynthesis

The cyclic tetrapeptide HC-toxin is an essential virulence determinant for the plant pathogenic fungus Cochliobolus carbonum and an inhibitor of histone deacetylase. The major form of HC-toxin contains the D-isomers of Ala and Pro. The non-ribosomal peptide synthetase that synthesizes HC-toxin has only one epimerizing domain for conversion of L-Pro to D-Pro; the source of D-Ala has remained unknown. Here we present the cloning and characterization of a new gene involved in HC-toxin biosynthesis, TOXG. TOXG is present only in HC-toxin-producing (Tox2(+)) isolates of C. carbonum. TOXG is able to support D-Ala-independent growth of a strain of Escherichia coli defective in D-Ala synthesis. A C. carbonum strain with both of its copies of TOXG mutated grows normally in culture, and although it no longer makes the three forms of HC-toxin that contain D-Ala, it still makes a minor form of HC-toxin that contains Gly in place of D-Ala. The addition of D-Ala to the culture medium restores production of the D-Ala-containing forms of HC-toxin by the toxG mutant. The toxG mutant has only partially reduced virulence. It is concluded that TOXG encodes an alanine racemase whose function is to synthesize D-Ala for incorporation into HC-toxin.     

A Biochemical Phenotype for a Disease Resistance Gene of Maize

In maize, major resistance to the pathogenic fungus Cochliobolus (Helminthosporium) carbonum race 1 is determined by the dominant allele of the nuclear locus hm. The interaction between C. carbonum race 1 and maize is mediated by a pathogen-produced, low molecular weight compound called HC-toxin. We recently described an enzyme from maize, called HC-toxin reductase, that inactivates HC-toxin by pyridine nucleotide-dependent reduction of an essential carbonyl group. We now report that this enzyme activity is detectable only in extracts of maize that are resistant to C. carbonum race 1 (genotype Hm/Hm or Hm/hm). In several genetic analyses, in vitro HC-toxin reductase activity was without exception associated with resistance to C. carbonum race 1. The results indicate that detoxification of HC-toxin is the biochemical basis of Hm-specific resistance of maize to infection by C. carbonum race 1.     

球毛壳菌循环肽HC-毒素基因的序列分析

为了验证Phrap软件是否适合在EST分析中应用,对球毛壳菌循环肽HC-毒素基因进行了序列分析。根据EST分析的结果,从cDNA文库中挑取循环肽HC-毒素基因的克隆进行了测序并序列分析。结果表明cDNA文库中循环肽HC-毒素基因的克隆插入片断大小为1217bp;用Phrap软件拼接出来的循环肽HC-毒素的表达序列标签拼接序列与实际序列不完全一致,因此Phrap软件不适合在EST分析中应用。     

Histone Hyperacetylation in Maize in Response to Treatment with HC-Toxin or Infection by the Filamentous Fungus Cochliobolus carbonum

HC-toxin, the host-selective toxin produced by the filamentous fungus Cochliobolus carbonum, inhibits maize (Zea mays L.) histone deacetylases (HDs) in vitro. Here we show that HDs are also inhibited by HC-toxin in vivo, as demonstrated by the accumulation of hyperacetylated forms of the core (nucleosomal) histones H3.1, H3.2, H3.3, and H4 in both maize embryos and tissue cultures. Hyperacetylation of H4 and all isoforms of H3 in tissue cultures of inbred Pr (genotype hm/hm) occurred at 10 ng/mL (23 nM). The effect was host-selective; acetylation of histones in the near isogenic inbred Pr1 (genotype Hm/Hm) did not occur in tissue cultures or embryos treated with 0.2 [mu]g/mL or 10 [mu]g/mL HC-toxin, respectively. Hyperacetylation of histone H4 in embryos of Pr1 began to occur at 50 [mu]g/mL. HC-toxin, and 200 [mu]g/mL HC-toxin caused equal hyperacetylation in Pr and Pr1 embryos. Hyperacetylated core histones, especially of the isoforms of histone H3, accumulated in leaves of inbred Pr, but not Pr1, after infection by toxin-producing strains of C. carbonum. Accumulation of hyperacetylated histones began at 24 h after inoculation, before the development of visible disease symptoms. Hyperacetylation of H2A or H2B histones were not detected in any of the studies. The results are consistent with HD being a primary site of action of HC-toxin.     

JM47, a cyclic tetrapeptide HC-toxin analogue from a marine Fusarium species

The known metabolite, enniatin B, and a cyclic tetrapeptide, JM47, which is a new natural product, were extracted from brown rice cultures of a marine fungus, identified as a Fusarium species, isolated from the marine alga Codium fragile. NMR studies, including 15N HMQC and 15N HMBC, established the structure of JM47 as cyclo(Ala-Ala-Aoh-Pro), where Aoh is the amino acid, (2S,9S)-2-amino-8-oxo-9-hydroxydecanoic acid. The absolute stereochemistry of the Aoh side chain carbinol centre was determined using Mosher ester methodology. Analysis of NOESY data assisted by molecular modelling revealed an alternating L-, D-, L-, D-configuration for the tetrapeptide core. The absolute stereochemistry of the core was determined by acidic hydrolysis and chiral TLC analysis of the proline residue. JM47 belongs to the HC-toxin family of cyclic tetrapeptides which possess a 2-amino-8-oxo-9,10-epoxydecanoic acid residue in place of the Aoh unit. This is the first report of an analogue of HC-toxin from a marine Fusarium species.     

Reduced virulence caused by meiotic instability of the TOX2 chromosome of the maize pathogen Cochliobolus carbonum

The mechanisms by which pathogenic fungi evolve are poorly understood. Production of the host-selective cyclic peptide HC-toxin is controlled by a complex locus, TOX2, in the plant pathogen Cochliobolus carbonum. Crosses between toxin-producing (Tox2+) and toxin-nonproducing (Tox2-) isolates, as well as crosses between isolates in which the TOX2 genes were on chromosomes of different size, yielded progeny that had lost one or more copies of one or more of the TOX2 genes. Of approximately 200 progeny analyzed, eight (4%) had lost at least one TOX2 gene. All of them still had at least one functional copy of all of the known genes required for HC-toxin production (HTS1, TOXA, TOXC, and TOXE). Most deletion strains could be explained by simple chromosome breaks resulting in the loss of major contiguous portions (0.8 to 1.4 Mb) of the 3.5-Mb TOX2 chromosome, whereas others had more complicated patterns. All deletion strains had normal growth and were fertile, indicating that the 1.4 Mb of DNA contained no essential housekeeping genes. Most strains were also still virulent (Tox2+), but two had a novel phenotype of reduced virulence (RV), characterized by smaller lesions that expanded at a reduced rate and an inability to colonize plants systemically. Although the RV strains made no detectable HC-toxin in culture, the RV phenotype was dependent on the presence of a functional copy of HTS1, which encodes the central enzyme in HC-toxin biosynthesis. We propose that the RV strains still make a low level of HC-toxin, at least in planta, and that this is due to the loss of one or more genes that contribute to, but are not absolutely required for, HC-toxin synthesis.     

Regulation of cyclic peptide biosynthesis and pathogenicity in Cochliobolus carbonum by TOXEp, a novel protein with a bZIP basic DNA-binding motif and four ankyrin repeats

HC-toxin is an epoxide-containing cyclic tetrapeptide that is a critical virulence determinant in the pathogenic interaction between the filamentous fungus Cochliobolus carbonum and maize. HC-toxin exerts a potent cytostatic effect on plant and animal cells by inhibiting histone deacetylase. The biosynthesis of HC-toxin by C. carbonum is controlled by a complex genetic locus, TOX2, that contains multiple, duplicated copies of genes encoding export and biosynthetic enzymes. A new gene in the TOX2 complex, TOXE, has now been isolated. Mutation of TOXE by targeted gene disruption has no effect on growth and sporulation but abolishes HC-toxin production and pathogenicity. TOXE is required for the expression of three genes with a known or putative role in HC-toxin production, but is not required for expression of HTS1, which encodes the large, multifunctional peptide synthetase that is the central enzyme in HC-toxin biosynthesis. At its N-terminus, TOXEp has a bZIP basic DNA binding domain, but it does not contain any discernible leucine zipper or helix-loop-helix. At its carboxy terminus, TOXEp contains four ankyrin repeats. In having these two common regulatory motifs in a single polypeptide, TOXEp appears to represent a novel class of regulatory protein. TOXE is present only in HC-toxin-producing (Tox2⁺) isolates of C. carbonum. Most Tox2⁺ isolates have two copies; in strain SB111, one copy of TOXE is on the same 3.5-Mb chromosome that contains all of the other genes known to be involved in HC-toxin biosynthesis, and the second copy of TOXE is on a 0.7-Mb chromosome. Received: 20 April 1998 / Accepted: 21 September 1998     

Regulation of cyclic peptide biosynthesis and pathogenicity in Cochliobolus carbonum by TOXEp, a novel protein with a bZIP basic DNA-binding motif and four ankyrin repeats

HC-toxin is an epoxide-containing cyclic tetrapeptide that is a critical virulence determinant in the pathogenic interaction between the filamentous fungus Cochliobolus carbonum and maize. HC-toxin exerts a potent cytostatic effect on plant and animal cells by inhibiting histone deacetylase. The biosynthesis of HC-toxin by C. carbonum is controlled by a complex genetic locus, TOX2, that contains multiple, duplicated copies of genes encoding export and biosynthetic enzymes. A new gene in the TOX2 complex, TOXE, has now been isolated. Mutation of TOXE by targeted gene disruption has no effect on growth and sporulation but abolishes HC-toxin production and pathogenicity. TOXE is required for the expression of three genes with a known or putative role in HC-toxin production, but is not required for expression of HTS1, which encodes the large, multifunctional peptide synthetase that is the central enzyme in HC-toxin biosynthesis. At its N-terminus, TOXEp has a bZIP basic DNA binding domain, but it does not contain any discernible leucine zipper or helix-loop-helix. At its carboxy terminus, TOXEp contains four ankyrin repeats. In having these two common regulatory motifs in a single polypeptide, TOXEp appears to represent a novel class of regulatory protein. TOXE is present only in HC-toxin-producing (Tox2⁺) isolates of C. carbonum. Most Tox2⁺ isolates have two copies; in strain SB111, one copy of TOXE is on the same 3.5-Mb chromosome that contains all of the other genes known to be involved in HC-toxin biosynthesis, and the second copy of TOXE is on a 0.7-Mb chromosome.     

An inhibitor-resistant histone deacetylase in the plant pathogenic fungus Cochliobolus carbonum.

We have partially purified and characterized histone deacetylases of the plant pathogenic fungus Cochliobolus carbonum. Depending on growth conditions, this fungus produces HC-toxin, a specific histone deacetylase inhibitor. Purified enzymes were analyzed by immunoblotting, by immunoprecipitation, and for toxin sensitivity. The results demonstrate the existence of at least two distinct histone deacetylase activities. A high molecular weight complex (430,000) is sensitive to HC-toxin and trichostatin A and shows immunoreactivity with an antibody against Cochliobolus HDC2, an enzyme homologous to yeast RPD3. The second activity, a 60,000 molecular weight protein, which is resistant even to high concentrations of well-known deacetylase inhibitors, such as HC-toxin and trichostatin A, is not recognized by antibodies against Cochliobolus HDC1 (homologous to yeast HOS2) or HDC2 and represents a different and/or modified histone deacetylase which is enzymatically active in its monomeric form. This enzyme activity is not present in the related filamentous fungus Aspergillus nidulans. Furthermore, in vivo treatment of Cochliobolus mycelia with trichostatin A and analysis of HDACs during the transition from non-toxin-producing to toxin-producing stages support an HC-toxin-dependent enzyme activity profile.     

Structure-Based Computational Study of Two Disease Resistance Gene Homologues (Hm1 and Hm2) in Maize (Zea mays L.) with Implications in Plant-Pathogen Interactions

The NADPH-dependent HC-toxin reductases (HCTR1 and 2) encoded by enzymatic class of disease resistance homologous genes (Hm1 and Hm2) protect maize by detoxifying a cyclic tetrapeptide, HC-toxin, secreted by the fungus Cochliobolus carbonum race 1(CCR1). Unlike the other classes'' resistance (R) genes, HCTR-mediated disease resistance is an inimitable mechanism where the avirulence (Avr) component from CCR1 is not involved in toxin degradation. In this study, we attempted to decipher cofactor (NADPH) recognition and mode of HC-toxin binding to HCTRs through molecular docking, molecular dynamics (MD) simulations and binding free energy calculation methods. The rationality and the stability of docked complexes were validated by 30-ns MD simulation. The binding free energy decomposition of enzyme-cofactor complex was calculated to find the driving force behind cofactor recognition. The overall binding free energies of HCTR1-NADPH and HCTR2-NADPH were found to be −616.989 and −16.9749 kJ mol⁻¹ respectively. The binding free energy decomposition revealed that the binding of NADPH to the HCTR1 is mainly governed by van der Waals and nonpolar interactions, whereas electrostatic terms play dominant role in stabilizing the binding mode between HCTR2 and NADPH. Further, docking analysis of HC-toxin with HCTR-NADPH complexes showed a distinct mode of binding and the complexes were stabilized by a strong network of hydrogen bond and hydrophobic interactions. This study is the first in silico attempt to unravel the biophysical and biochemical basis of cofactor recognition in enzymatic class of R genes in cereal crop maize.     

An extended physical map of the TOX2 locus of Cochliobolus carbonum required for biosynthesis of HC-toxin

In genetic crosses, HC-toxin production in the filamentous fungus Cochliobolus carbonum appears to be controlled by a single locus, TOX2. At the molecular level, TOX2 is composed of at least seven duplicated and coregulated genes involved in HC-toxin biosynthesis, export, and regulation. All copies of four of the TOX2 genes were previously mapped within a 540-kb stretch of DNA in strain SB111. Subsequently, an additional three TOX2 genes, TOXE, TOXF, and TOXG, have been discovered. In this paper we have mapped all copies of the new genes, a total of seven, and show that except for one of the two copies of TOXE, which was previously shown to be on a chromosome of 0.7 Mb in strain SB111, they are all linked to the previously known TOX2 genes within approximately 600 kb of each other on a chromosome of 3.5 Mb. We show here that this chromosome also contains at least one non-TOX2 gene, EXG2, which encodes an exo-beta1,3-glucanase. EXG2 is still present in strains that have undergone spontaneous deletion of up to approximately 1.4 Mb of the 3.5-Mb chromosome. The results contribute to our understanding of the complex organization of the genes involved in HC-toxin biosynthesis and are consistent with the hypothesis that a reciprocal chromosomal translocation accounts for the pattern of distribution of the TOX2 genes in different C. carbonum isolates.     

Chromosomal organization of TOX2, a complex locus controlling host-selective toxin biosynthesis in Cochliobolus carbonum.

Race 1 isolates of the filamentous fungus Cochliobolus carbonum are exceptionally virulent on certain genotypes of maize due to production of a cyclic tetrapeptide, HC-toxin. In crosses between toxin-producing (Tox2+) and toxin-nonproducing (Tox2-) isolates, toxin production segregates in a simple 1:1 pattern, suggesting the involvement of a single genetic locus, which has been named TOX2. Earlier work had shown that in isolate SB111, TOX2 consists in part of two copies of a gene, HTS1, that encodes a 570-kD cyclic peptide synthetase and is lacking in Tox2- isolates. The genomic structure of TOX2 and the relationship between the two copies of HTS1 have now been clarified by using pulsedfield gel electrophoresis and physical mapping. In isolate SB111, both copies of HTS1 are on the largest chromosome (3.5 Mb), which is not present in the related Tox2- strain SB114. Two other genes known or thought to be important for HC-toxin biosynthesis, TOXA and TOXC, are also on the same chromosome in multiple copies. Other independent Tox2+ isolates also have two linked copies of HTS1, but in some isolates the size of the chromosome containing HTS1 is 2.2 Mb. Evidence obtained with Tox2+ -unique and with random probes is consistent with a reciprocal translocation as the cause of the difference in the size of the HTS1-containing chromosome among the Tox2+ isolates studied here. Physical mapping of the 3.5-Mb chromosome of SB111 that contains HTS1 using rare-cutting restriction enzymes and engineered restriction sites was used to map the chromosome location of the two copies of HTS1 and the three copies of TOXC. The results indicate that TOX2 is a complex locus that extends over more than 500 kb. The capacity to produce HC-toxin did not evolve by any single, simple mechanism.     

The backbone and side chain conformations of the cyclic tetrapeptide HC-toxin

A study of the conformational parameters of HC-toxin and its diacetyl derivative in chloroform solution has been carried out. Two-dimensional NMR spectroscopy and the nuclear Overhauser effect have been used in order to determine connectivities (assignments and sequence) and approximate torsion angles and interproton distances. The results are consistent with a bis-gamma-turn conformation previously reported for dihydrochlamydocin. Model building based upon NMR data supports a D configuration for Ala2 and Pro4 residues.     

Bicyclic tetrapeptides as potent HDAC inhibitors: Effect of aliphatic loop position and hydrophobicity on inhibitory activity

Several histone deacetylase (HDAC) inhibiting bicyclic tetrapeptides have been designed and synthesized through intramolecular ring-closing metathesis (RCM) reaction and peptide cyclization. We designed bicyclic tetrapeptides based on CHAP31, trapoxin B and HC-toxin I. The HDAC inhibitory and p21 promoter assay results showed that the aliphatic loop position as well as the hydrophobicity plays an important role toward the activity of the bicyclic tetrapeptide HDAC inhibitors.     

Mutational analysis of beta-glucanase genes from the plant-pathogenic fungus Cochliobolus carbonum

Two new beta-glucanase-encoding genes, EXG2 and MLG2, were isolated from the plant-pathogenic fungus Cochliobolus carbonum using polymerase chain reaction based on amino acid sequences from the purified proteins. EXG2 encodes a 46.6-kDa exo-beta1,3-glucanase and is located on the same 3.5-Mb chromosome that contains the genes of HC-toxin biosynthesis. MLG2 encodes a 26.8-kDa mixed-linked (beta1,3-beta1,4) glucanase with low activity against beta1,4-glucan and no activity against beta1,3-glucan. Specific mutants of EXG2 and MLG2 were constructed by targeted gene replacement. Strains with multiple mutations (genotypes exg1/mlg1, exg2/mlg1, mlg1/mlg2, and exg1/exg2/mlg1/mlg2) were also constructed by sequential disruption and by crossing. Total mixed-linked glucanase activity in culture filtrates of mlg1/mlg2 and exg1/exg2/mlg1/mlg2 mutants was reduced by approximately 73%. Total beta1,3-glucanase activity was reduced by 10, 54, and 96% in exg2, mlg1, and exg1/exg2/mlg1/mlg2 mutants, respectively. The quadruple mutant showed only a modest decrease in growth on beta1,3-glucan or mixed-linked glucan. None of the mutants showed any decrease in virulence.     

A Gene Related to Yeast HOS2 Histone Deacetylase Affects Extracellular Depolymerase Expression and Virulence in a Plant Pathogenic Fungus

A gene, HDC1, related to the Saccharomyces cerevisiae histone deacetylase (HDAC) gene HOS2, was isolated from the filamentous fungus Cochliobolus carbonum, a pathogen of maize that makes the HDAC inhibitor HC-toxin. Engineered mutants of HDC1 had smaller and less septate conidia and exhibited an approximately 50% reduction in total HDAC activity. Mutants were strongly reduced in virulence as a result of reduced penetration efficiency. Growth of hdc1 mutants in vitro was normal on glucose, slightly decreased on sucrose, and reduced by 30 to 73% on other simple and complex carbohydrates. Extracellular depolymerase activities and expression of the corresponding genes were downregulated in hdc1 mutant strains. Except for altered conidial morphology, the phenotypes of hdc1 mutants were similar to those of C. carbonum strains mutated in ccSNF1 encoding a protein kinase necessary for expression of glucose-repressed genes. These results show that HDC1 has multiple functions in a filamentous fungus and is required for full virulence of C. carbonum on maize.