The fungal genus Cochliobolus and toxin-mediated plant disease.

Several times in this century, new and sometimes devastating diseases of cereal crops, caused by fungi in the genus Cochliobolus, have suddenly appeared. In many fungal diseases of plants the factors required for pathogenicity are unknown; in contrast, the key elements in each of several Cochliobolus diseases are known to be host-selective toxins. Recent research on these systems has given surprising insights into the genetic basis of fungal pathogenicity and plant susceptibility to disease.

Cloning and targeted gene disruption of XYL1, a beta 1,4-xylanase gene from the maize pathogen Cochliobolus carbonum.

The gene, XYL1, encoding the major extracellular endo-beta 1,4-xylanase from the maize pathogen Cochliobolus carbonum was cloned using a synthetic, degenerate oligonucleotide based on a tryptic fragment from the purified enzyme. The deduced product of XYL1 has a M(r) of 20,869 and a predicted pI of 9.1, in good agreement with the measured M(r) and pI of the purified enzyme. The XYL1 product has strong amino acid identity to seven endo-beta 1,4-xylanases from six prokaryotes but no obvious similarity to 10 other prokaryotic endoxylanases or a yeast endoxylanase. An internal fragment of the gene was used to create a specific xylanase mutant by transformation-mediated gene disruption via homologous recombination. Total extracellular xylanase activity in the mutant was reduced by 85-94%. When analyzed by cation exchange HPLC, culture filtrates of the mutant and wild type had identical protein profiles, but the mutant lacked the major peak of UV absorption corresponding to the major xylanase activity. Xylanase II activity was also missing in the mutant, but xylanase III activity was still present. The XYL1 mutant grew as well as the wild type on sucrose, on corn cell walls, and on xylan. The pathogenicity of the mutant was indistinguishable from the wild type, indicating that XYL1 is not required for pathogenicity.

Characterization of two divergent beta-tubulin genes from Colletotrichum graminicola.

We have cloned and sequenced two beta-tubulin genes, TUB1 and TUB2, from the phytopathogenic fungus, Colletotrichum graminicola. The nucleotide sequences of the coding regions of the two genes are only 72.8% homologous. This divergence is reflected in the deduced amino acid (aa) sequences which differ at 94 aa residues. Comparison with the aa sequences of other fungal beta-tubulins indicates that the C. graminicola TUB2 gene encodes a conserved isotype, whereas the C. graminicola TUB1 product is highly divergent. Both genes contain six identically placed introns and the position of each intron is conserved in other fungal beta-tubulin genes. Also typical of other fungal beta-tubulin genes, there is a pronounced bias in codon usage in the C. graminicola TUB2 gene; there is a lesser codon bias in TUB1 from C. graminicola. Both C. graminicola beta-tubulin genes are transcribed and yield similar sized messages.

Identification of peptide synthetase-encoding genes from filamentous fungi producing host-selective phytotoxins or analogs.

Race 1 of Cochliobolus carbonum (Cc) makes a cyclic tetrapeptide, HC-toxin, that is necessary for its virulence on certain genotypes of maize. The synthesis of HC-toxin is catalyzed by a 570-kDa multifunctional enzyme, HC-toxin synthetase (HTS). The gene encoding HTS (HTS1) is absent from other races of Cc and from other species of Cochliobolus. Four other unrelated filamentous fungi make cyclic peptides closely related to HC-toxin, raising the possibility that the corresponding cyclic peptide synthetase (CPS)-encoding genes have moved between these fungi by horizontal gene transfer. Degenerate PCR primers were designed based on several highly conserved amino acid (aa) motifs common to known CPS domains and used to amplify genomic sequences from different fungi. PCR products representing CPS genes from Diheterospora chlamydosporia, which makes the HC-toxin analog chlamydocin, Cylindrocladium macrosporum, which makes the analog Cyl-2, and C. victoriae, which makes the unrelated cyclic pentapeptide victorin, were cloned and analysed. Their sequences are more related to HTS1 than to other cloned CPS, but the percent aa identity is not consistent with very recent horizontal movement of these genes.

Transposon-like sequences at the TOX2 locus of the plant-pathogenic fungus Cochliobolus carbonum.

The ascomycete fungus Cochliobolus carbonum race 1 is pathogenic on certain genotypes of maize due to the production of HC-toxin, a host-specific cyclic peptide. HC-toxin production is controlled, at least in part, by a duplicated 22-kb region of DNA that is found only in toxin-producing isolates of the fungus. This 22-kb region of DNA is flanked by a repetitive element. We have sequenced the element and found an interrupted reading frame that would encode a product similar to transposases from the fungal transposons Fot1 of Fusarium oxysporum and Pot2 of Magnaporthe grisea. The individual element cloned from C. carbonum is likely to function neither in cis nor trans, as it had a nonsense mutation in frame and several substitutions in its terminal inverted repeats. However, similar elements in the C. carbonum genome may be active, as the putative transposase-encoding region hybridized to mRNA of the size predicted by the reading frame. The element was found in varying copy number in the genomes of all Cochliobolus spp. examined, giving a distinct fingerprint in each species and race tested. The sequence similarity of the C. carbonum repetitive element to other fungal transposons, along with its presence in multiple copies per genome, strongly suggest that the C. carbonum repetitive element is a member of the Fot1 family of fungal transposons.

Presence of peptide synthetase gene transcripts and accumulation of ergopeptines in Claviceps purpurea and Neotyphodium coenophialum.

The production of toxic ergopeptine alkaloids by the fungi Claviceps purpurea and Neotyphodium coenophialum involves the activity of one or more nonribosomal peptide synthetases. Claviceps purpurea and N. coenophialum each have several different peptide synthetase genes, fragments of which have been cloned previously. An additional Claviceps purpurea peptide synthetase gene was cloned by hydridization with one of the N. coenophialum peptide synthetase gene fragments. We detected the presence of mRNA from the peptide synthetase genes in cultures of different ages grown under conditions favorable or unfavorable for ergopeptine production. All four peptide synthetase genes from Claviceps purpurea were transcribed under at least some of the experimental conditions. Transcripts from three of the four genes were detected under conditions consistent with their potential involvement in ergopeptine biosynthesis. All three peptide synthetase genes previously identified in N. coenophialum were transcribed during symbiotic growth of this fungus with tall fescue, as well as ergopeptine-producing cultures. The data show that all of the peptide synthetase genes are transcribed, that one of the peptide synthetase genes is dissociated from ergopeptine biosynthesis, and, as a result, prioritize the remaining genes for functional analyses by transformation-mediated gene disruption.

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.

Endopolygalacturonase is not required for pathogenicity of Cochliobolus carbonum on maize.

A gene (PGN1) encoding extracellular endopolygalacturonase was isolated from the fungal maize pathogen Cochliobolus carbonum race 1. A probe was synthesized by polymerase chain reaction using oligonucleotides based on the endopolygalacturonase amino acid sequence. Genomic and cDNA copies of the gene were isolated and sequenced. The corresponding mRNA was present in C. carbonum grown on pectin but not on sucrose as carbon source. The single copy of PGN1 in C. carbonum was disrupted by homologous integration of a plasmid containing an internal fragment of the gene. Polygalacturonase activity in one transformant chosen for further analysis was 10% or 35% of the wild-type activity based on viscometric or reducing sugar assays, respectively. End product analysis indicated that the residual activity in the mutant was due to an exopolygalacturonase. Pathogenicity on maize of the mutant lacking endopolygalacturonase activity was qualitatively indistinguishable from the wild-type strain, indicating that in this disease interaction endopolygalacturonase is not required. Either pectin degradation is not critical to this interaction or exopolygalacturonase alone is sufficient.

The PYR1 gene of the plant pathogenic fungus Colletotrichum graminicola: selection by intraspecific complementation and sequence analysis.

A spontaneous uridine-requiring auxotroph of Colletotrichum graminicola was recovered by selection for resistance to 5-fluoro-orotic acid. The auxotroph lacked orotate phosphoribosyl transferase (OPRTase) and was complemented with a clone from a cosmid library of C. graminicola DNA. A 3.1 kb HindIII-SalI fragment was subcloned from the cosmid and it could efficiently transform the auxotrophic strain to uridine prototrophy and integrate by site-specific recombination. This DNA fragment contains an open reading frame that is similar to OPRTase genes of the fungi Sordaria macrospora, Trichoderma reesei, Podospora anserina, and Saccharomyces cerevisiae. Based on the sequence similarities and the ability to restore uridine prototrophy, we conclude that the fragment contains the C. graminicola gene for OPRTase, which we have named PYR1. Our results demonstrate that cloning by complementation is feasible in C. graminicola, that the gene for OPRTase from C. graminicola can be useful as a selectable marker in transformation of the fungus, and that the OPRTase gene product is similar to OPRTase from other fungi.

A cyclic peptide synthetase gene required for pathogenicity of the fungus Cochliobolus carbonum on maize.

Specificity in many plant-pathogen interactions is determined by single genes in pathogen and host. The single locus for host-selective pathogenicity (TOX2) in the fungus Cochliobolus carbonum governs production of a cyclic tetrapeptide named HC-toxin. We have isolated a chromosomal region, 22 kilobases (kb) long, that contains a 15.7-kb open reading frame (HTS1) encoding a multifunctional cyclic peptide synthetase. The 22-kb chromosomal region is duplicated in toxin-producing isolates of the fungus but is completely absent from the genomes of toxin-nonproducing isolates. Mutants of the fungus with disruptions in both copies of HTS1, at either of two different sites within HTS1, were engineered by DNA-mediated transformation. Disruption of both copies at either site resulted in loss of ability to produce HC-toxin and loss of host-selective pathogenicity, but the mutants displayed different biochemical phenotypes depending on the site of disruption. The results demonstrate that TOX2 encodes, at least in part, a large, multifunctional biosynthetic enzyme and that the evolution of host range in C. carbonum involved the insertion or deletion of a large piece of chromosomal DNA.

Elimination of ergovaline from a grass-Neotyphodium endophyte symbiosis by genetic modification of the endophyte.

The fungal endophytes Neotyphodium lolii and Neotyphodium sp. Lp1 from perennial ryegrass (Lolium perenne), and related endophytes in other grasses, produce the ergopeptine toxin ergovaline, among other alkaloids, while also increasing plant fitness and resistance to biotic and abiotic stress. In the related fungus, Claviceps purpurea, the biosynthesis of ergopeptines requires the activities of two peptide synthetases, LPS1 and LPS2. A peptide synthetase gene hypothesized to be important for ergopeptine biosynthesis was identified in C. purpurea by its clustering with another ergot alkaloid biosynthetic gene, dmaW. Sequence analysis conducted independently of the research presented here indicates that this gene encodes LPS1 [Tudzynski, P., Holter, K., Correia, T., Arntz, C., Grammel, N. & Keller, U. (1999) Mol. Gen. Genet. 261, 133-141]. We have cloned a similar peptide synthetase gene from Neotyphodium lolii and inactivated it by gene knockout in Neotyphodium sp. Lp1. The resulting strain retained full compatibility with its perennial ryegrass host plant as assessed by immunoblotting of tillers and quantitative PCR. However, grass-endophyte associations containing the knockout strain did not produce detectable quantities of ergovaline as analyzed by HPLC with fluorescence detection. Disruption of this gene provides a means to manipulate the accumulation of ergovaline in endophyte-infected grasses for the purpose of determining the roles of ergovaline in endophyte-associated traits and, potentially, for ameliorating toxicoses in livestock.