Thomas M. Hohn

Evidence for a gene cluster involving trichothecene-pathway biosynthetic genes in Fusarium sporotrichioides

Two overlapping cosmid clones (Cos1-1 and Cos9-1) carrying the Tox5 gene were isolated from a library of F. sporotrichioides strain NRRL 3299 genomic DNA. These cosmids were used to transform three T-2 toxin-deficient mutants that are blocked at different steps in the trichothecene pathway. Both cosmids restored T-2 toxin production to Tox3-1− or Tox4-1− mutants but neither restored T-2 toxin production to a Tox1–2− mutant. The production of T-2 toxin by the complemented Tox3-1− and Tox4-1− mutants, as well as the production of diacetoxyscirpenol by the cosmid-transformed Tox1-2− mutant, were 2- to 10- fold higher than in strain NRRL 3299. In addition, those transformants carrying Cos9-1 produced significantly higher levels of trichothecenes than transformants carrying Cos1-1. Two different DNA fragments (FSC13-9 and FSC14-5), representing the region of overlap between the cosmid clones, were isolated. These fragments specifically complemented either the Tox3-1− mutant (FSC14-5) or the Tox4-1− mutant (FSC13-9). The trichothecene-production phenotype of these transformants was similar to NRRL 3299. These results suggest that two or more genes involved in the biosynthesis of trichothecenes are closely linked to Tox5.

The Tri4 gene of Fusarium sporotrichioides encodes a cytochrome P450 monooxygenase involved in trichothecene biosynthesis.

TheTri4 gene ofFusarium sporotrichioides was isolated from a cloned DNA fragment carrying theTri5 gene by complementation of aTri4− mutant. The nucleotide sequence ofTri4 was determined and the locations of three introns were identified. Analysis ofTri4 mRNA levels revealed that transcription reached maximum levels coincidently with the onset of trichothecene biosynthesis, and then declined 20-fold over the next 8 h. Disruption ofTri4 resulted in the loss of production of both trichothecenes and apotrichodiol and the accumulation of the unoxygenated pathway intermediate trichodiene. Transformants lacking a functionalTri4 gene were able to convert isotrichotriol, an early pathway intermediate, to T-2 toxin suggesting that most pathway enzymes are present inTri4− mutants. These data suggest that the enzyme encoded byTri4 catalyzes the first oxygenation step in the trichothecene pathway and participates in apotrichodiol biosynthesis.Tri4 encodes a protein of 520 residues (Mr=59 056) that shows significant homology with members of the superfamily of cytochromes P450. It appears most similar to the CYP3A subfamily (24.6% amino acid identity). Because it contains less than 40% positional identity with other cytochromes P450, theTri4 gene has been placed in a new cytochrome P450 gene family designatedCY P58.

Restoration of wild-type virulence to Tri5 disruption mutants of Gibberella zeae via gene reversion and mutant complementation

Gibberella zeae is a pathogen of small grain crops and produces trichothecene mycotoxins in infected host tissue. The role of trichothecenes in the virulence of G. zeae was previously investigated using trichothecene-non-producing mutants that were generated via transformation-mediated disruption of a gene (Tri5) that encodes the first enzyme in the trichothecene biosynthetic pathway. The mutants were less virulent on some hosts than the wild-type strain from which they were derived. Here, we used two approaches to determine whether the reduced virulence of mutants was due specifically to Tri5 disruption or to non-target effects caused by the transformation process. First, we generated a revertant from a Tri5 disruption mutant by allowing the mutant to pass through the sexual phase of its life cycle. In approximately 2% of the resulting progeny the disrupted Tri5 had reverted to wild-type; however, only one of three revertant progeny also regained the ability to produce trichothecenes. In the second approach, we complemented the Tri5 mutation in a disruption mutant by transforming the mutant with a plasmid carrying a functional copy of Tri5. In all transformants examined, the ability to produce trichothecenes was restored. The restoration of trichothecene production in the revertant progeny and in the complemented mutant was accompanied by restoration of wild-type or near wild-type levels of virulence on wheat seedlings (cultivar Wheaton). The results indicate that the reduced virulence of the mutants was caused by disruption of Tri5 rather than non-target effects resulting from the transformation process. The results also provide further evidence that trichothecenes contribute to the virulence of plant-pathogenic fungi.

Characterization of the gene cluster for biosynthesis of macrocyclic trichothecenes in Myrothecium roridum

Abstract Macrocyclic trichothecenes are toxic sesquiterpenoids that are produced by certain fungi and plants. The unique structural features of macrocyclic trichothecenes result in increased toxicity relative to other trichothecene structural types. Here we report the sequences and relative locations of the MRTRI5, MRTRI6, and MRTRI4 genes in the biosynthetic pathway for macrocyclic trichothecenes in Myrothecium roridum. The deduced sequences of the products of MRTRI5 and MRTRI4 display overall identities of 75 and 63%, respectively, with the corresponding proteins in Fusarium sporotrichioides. Based on sequence comparisons, MRTRI5 encodes the enzyme trichodiene synthase, which has been shown to catalyze the first step in the trichothecene pathways of Fusarium and Trichothecium species. MRTRI6 encodes a transcription factor (392 amino acids) required for pathway gene expression, and the predicted MRTRI4 product (533 amino acids) is a cytochrome P450 monooxygenase responsible for the initial oxygenation step in the pathway. The sizes of the predicted products of MRTRI5 and MRTRI4 show good agreement with their apparent counterparts in the Fusarium pathway; however, the protein specified by MRTRI6 is almost twice the size of its putative homolog in F. sporotrichioides. Only the C-terminal 124 residues of MRTRI6, containing the proposed Cys2His2 zinc finger motifs, show significant similarity (65% identity) to the TRI6 sequence in F. sporotrichioides. MRTRI4 can successfully complement a TRI4− mutant in F. sporotrichioides, although the resulting trichothecene profile differed from that observed in wild-type strains. Complemented mutants accumulated low levels of T-2 toxin, in addition to sambucinol, deoxysambucinol, and the pathway intermediates trichothecene and isotrichodiol. Mapping data indicate that the genes of the macrocyclic trichothecene pathway in M. roridum are clustered, but that their organization and orientation differ markedly from those of the trichothecene gene cluster found in F. sporotrichioides. These results show that the biosynthetic pathways for macrocyclic trichothecenes are closely related to other trichothecene pathways and that the evolution of gene clusters for the biosynthesis of natural products in fungi can involve significant rearrangements.

Transgenic expression of the TRI101 or PDR5 gene increases resistance of tobacco to the phytotoxic effects of the trichothecene 4,15-diacetoxyscirpenol

Mycotoxins are fungal secondary compounds that are toxic to vertebrates. Their presence in food and feeds, as the result of fungal disease in crops, can present a danger to animal or human health. Many mycotoxins have also been shown to be phytotoxic and in some cases, such as with trichothecenes produced by the wheat head blight fungus Fusarium graminearum, mycotoxins may act as virulence factors. Antibiotic-producing organisms, including fungi, protect themselves from their own toxins by metabolic alteration of the compound, modification of the target site of action or by exporting the compound to the extracellular space. We have tested the effectiveness of adapting two of these strategies, metabolic alteration and extracellular transport, to protect plant cells from the deleterious effects of the trichothecene 4,15-diacetoxyscirpenol (DAS). Tobacco plants were transformed with either the Saccharomyces cerevisiae gene PDR5, which encodes a multi-drug transporter, or with the Fusarium sporotrichioides gene TRI101, which encodes a trichothecene 3-O-acetyltransferase. Both genes conferred significant increased tolerance to DAS as measured by a sensitive seed germination assay. Expression of PDR5 or TRI101 in a seed-specific manner in crop plants such as wheat could lower the incidence of head blight as well as reduce mycotoxin levels within the seed.

Characterization of Novel Sesquiterpenoid Biosynthesis in Tobacco Expressing a Fungal Sesquiterpene Synthase

The gene encoding trichodiene synthase (Tri5), a sesquiterpene synthase from the fungus Fusarium sporotrichioides, was used to transform tobacco (Nicotiana tabacum). Trichodiene was the sole sesquiterpene synthase product in enzyme reaction mixtures derived from unelicited transformant cell-suspension cultures, and both trichodiene and 5-epi-aristolochene were observed as reaction products following elicitor treatment. Immunoblot analysis of protein extracts revealed the presence of trichodiene synthase only in transformant cell lines producing trichodiene. In vivo labeling with [3H]mevalonate revealed the presence of a novel trichodiene metabolite, 15-hydroxytrichodiene, that accumulated in the transformant cell-suspension cultures. In a trichodiene-producing transformant, the level of 15-hydroxytrichodiene accumulation increased after elicitor treatment. In vivo labeling with [14C]acetate showed that the biosynthetic rate of trichodiene and 15-hydroxytrichodiene also increased after elicitor treatment. Incorporation of radioactivity from [14C]acetate into capsidiol was reduced following elicitor treatment of a trichodiene-producing transformant as compared with wild type. These results demonstrate that sesquiterpenoid accumulation resulting from the constitutive expression of a foreign sesquiterpene synthase is responsive to elicitation and that the farnesyl pyrophosphate present in elicited cells can be utilized by a foreign sesquiterpene synthase to produce high levels of novel sesquiterpenoids.

Function and Biosynthesis of Trichothecenes Produced by Fusarium Species

Trichothecene toxins are produced by Fusarium species in food and feed grains worldwide and represent an important economic problem for agriculture. F. graminearum is the major source of trichothecene contamination in the U.S. and the causative agent of Wheat Head Scab (WHS). Recently, it was shown that trichothecene-deficient mutants of F. graminearum display significantly reduced levels of virulence on wheat. Based on these results, trichothecenes appear to be examples of fungal toxins that can function as virulence factors without strong host selectivity. Biosynthesis of trichothecenes is complex, and at least ten pathway genes have been identified within a gene cluster spanning a 23 Kb region of DNA in F. sporotrichioides. Recent investigations of the trichothecene pathway gene cluster have provided new information concerning the transcriptional regulation of pathway gene expression (TRI6) and the transport of pathway products (TRI12). A trichothecene resistance gene (TRI) has also been identified in F. sporotrichioides. Expression of TRI or other microbial trichothecene resistance genes in wheat may provide a means for further investigating the importance of trichothecenes in Fusarium WHS.

Altered Regulation of 15-Acetyldeoxynivalenol Production in Fusarium graminearum

ABSTRACT Most Fusarium graminearum isolates produce low or undetectable levels of trichothecenes in liquid shake cultures, making it difficult to perform biochemical studies of trichothecene biosynthesis. To develop strains with higher levels of trichothecene production under liquid shake conditions we transformed F. graminearum with both a reporter gene containing a homologous trichothecene pathway gene promoter (TRI5) and a gene encoding a heterologous trichothecene pathway transcription factor (TRI6). The TRI5 and TRI6 genes are part of the trichothecene pathway gene clusters of both Fusarium sporotrichioides and F. graminearum. These genes encode trichodiene synthase (encoded by TRI5), the first enzyme in the trichothecene pathway, and a transcription factor (encoded by TRI6) required for pathway gene expression. Transformation of F. graminearum with plasmids containing either an F. graminearum TRI5 promoter fragment (FGTRI5P) or FGTRI5Pcoupled with the β-d-glucuronidase (GUS) reporter gene resulted in the identification of several transformants capable of producing 45 to 200 mg of 15-acetyldeoxynivalenol (15-ADON)/liter in liquid shake culture after 7 days. Increased 15-ADON production was only observed in transformants where plasmid integration occurred through the FGTRI5P sequence and was not accompanied by increased GUS expression. 15-ADON production was further increased in liquid culture up to 1,200 mg/liter following introduction of the F. sporotrichioides TRI6 gene (FSTRI16) into F. graminearum. The effects of FSTRI6 on 15-ADON production also depended on plasmid integration via homologous recombination of the FGTRI5P fragment and resulted in a 100-fold increase in GUS expression. High-level production of 15-ADON in liquid shake cultures provides a convenient method for large-scale trichothecene preparation. The results suggest that targeting transformation vector integration toFGTRI5P alters pathway gene expression and are consistent with the proposed conservation of TRI6 function betweenFusarium species.

E. coli spheroplast-mediated transfer of cloned cauliflower mosaic virus DNA into plant protoplasts

SummarySaishin (Brassica chinensis L.) mesophyll protoplasts and E. coli spheroplasts harbouring hybrid plasmid with tandemly dimerized cauliflower mosaic virus DNA were mixed in ratios of 1:1,000 and incubated for 20 min at 30° C in the presence of 20% polyvinyl alcohol. Subsequently, protoplasts/spheroplasts mixture was washed with high pH-high Ca buffer. After 3 days of culture, 8% of Saishin protoplasts were transfected as monitored by immunofluorescence technique. When plant protoplasts and bacterial spheroplasts were mixed in ratios of 1:100 or 1:2,000, 1% or 5% of protoplasts were transfected, respectively.

Structural characterization of 15-hydroxytrichodiene, a sesquiterpenoid produced by transformed tobacco cell suspension cultures expressing a trichodiene synthase gene from Fusarium sporotrichioides

Tobacco (Nicotiana tabaccum) cell suspension cultures transformed with a gene encoding trichodiene synthase, a sesquiterpene synthase from the fungus Fusarium sporotrichioides, produced a novel sesquiterpenoid derived from the in vivo production of trichodiene. Mass and nuclear magnetic resonance spectroscopic analyses identified the new compound as 15-hydroxytrichodiene. The in vivo hydroxylation of trichodiene by transformant tobacco cell suspension cultures demonstrates that the introduction of a foreign sesquiterpene synthase gene can result in the production of novel sesquiterpenoid metabolites.