Theresa Lee

Tri13 and Tri7 Determine Deoxynivalenol- and Nivalenol-Producing Chemotypes of Gibberella zeae

ABSTRACT Gibberella zeae, a major cause of cereal scab, can be divided into two chemotypes based on production of the 8-ketotrichothecenes deoxynivalenol (DON) and nivalenol (NIV). We cloned and sequenced a Tri13 homolog from each chemotype. The Tri13 from a NIV chemotype strain (88-1) is located in the trichothecene gene cluster and carries an open reading frame similar to that of Fusarium sporotrichioides, whereas the Tri13 from a DON chemotype strain (H-11) carries several mutations. To confirm the roles of the Tri13 and Tri7 genes in trichothecene production by G. zeae, we genetically altered toxin production in 88-1 and H-11. In transgenic strains, the targeted deletion of Tri13 from the genome of 88-1 caused production of DON rather than NIV. Heterologous expression of the 88-1 Tri13 gene alone or in combination with the 88-1 Tri7 gene conferred on H-11 the ability to synthesize NIV; in the latter case, 4-acetylnivalenol (4-ANIV) also was produced. These results suggest that Tri13 and Tri7 are required for oxygenation and acetylation of the oxygen at C-4 during synthesis of NIV and 4-ANIV in G. zeae. These functional analyses of the Tri13 and Tri7 genes provide the first clear evidence for the genetic basis of the DON and NIV chemotypes in G. zeae.

Two different polyketide synthase genes are required for synthesis of zearalenone in Gibberella zeae

Zearalenone (ZEA) is a polyketide mycotoxin produced by some species of Gibberella/Fusarium and causes hyperestrogenic syndrome in animals. ZEA occurs naturally in cereals infected by Gibberella zeae in temperate regions and threatens animal health. In this study, we report on a set of genes that participate in the biosynthesis of ZEA in G. zeae. Focusing on the non‐reducing polyketide synthase (PKS) genes of the G. zeae genome, we demonstrated that PKS13 is required for ZEA production. Subsequent analyses revealed that a continuous, 50 kb segment of DNA carrying PKS13 consisted of three additional open reading frames that were coexpressed as a cluster during the condition for ZEA biosynthesis. These genes, in addition to PKS13, were essential for the ZEA biosynthesis. They include another PKS gene (PKS4) encoding a fungal reducing PKS; zearalenone biosynthesis gene 1 (ZEB1), which shows a high similarity to putative isoamyl alcohol oxidase genes; and ZEB2 whose deduced product carries a conserved, basic‐region leucine zipper domain. ZEB1 is responsible for the chemical conversion of β‐zearalenonol (β‐ZOL) to ZEA in the biosynthetic pathway, and ZEB2 controls transcription of the cluster members. Transcription of these genes was strongly influenced by different culture conditions such as nutrient starvations and ambient pH. Furthermore, the same set of genes regulated by ZEB2 was dramatically repressed in the transgenic G. zeae strain with the deletion of PKS13 or PKS4 but not in the ZEB1 deletion strain, suggesting that ZEA or β‐ZOL may be involved in transcriptional activation of the gene cluster required for ZEA biosynthesis in G. zeae. This is the first published report on the molecular characterization of genes required for ZEA biosynthesis.

Identification of deoxynivalenol- and nivalenol-producing chemotypes of Gibberella zeae by using PCR.

ABSTRACT Gibberella zeae, a major cause of cereal scab, may be divided into two chemotypes based on production of the trichothecenes deoxynivalenol (DON) and nivalenol (NIV). We cloned and sequenced the gene cluster for trichothecene biosynthesis from each chemotype.G. zeae H-11 is a DON producer isolated from corn, andG. zeae 88-1 is a NIV producer from barley. We sequenced a 23-kb gene cluster from H-11 and a 26-kb cluster from 88-1, along with the unlinked Tri101 genes. Each gene cluster contained 10Tri gene homologues in the same order and transcriptional directions as those of Fusarium sporotrichioides. Between H-11 and 88-1 all of the Tri homologues exceptTri7 were conserved, with identities ranging from 88 to 98% and 82 to 99% at the nucleotide and amino acid levels, respectively. The Tri7 sequences were only 80% identical at the nucleotide level. We aligned the Tri7 genes and found that the Tri7 open reading frame of H-11 carried several mutations and an insertion containing 10 copies of an 11-bp tandem repeat. The Tri7 gene from 88-1 carried neither the repeat nor the mutations. We assayed 100 G. zeae isolates of both chemotypes by PCR amplification with a primer pair derived from the Tri7 gene and could differentiate the chemotypes by polyacrylamide gel electrophoresis. The PCR-based method developed in this study should provide a simple and reliable diagnostic tool for differentiating the two chemotypes of G. zeae.

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 December 2009-31 January 2010

This article documents the addition of 220 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Allanblackia floribunda, Amblyraja radiata, Bactrocera cucurbitae, Brachycaudus helichrysi, Calopogonium mucunoides, Dissodactylus primitivus, Elodea canadensis, Ephydatia fluviatilis, Galapaganus howdenae howdenae, Hoplostethus atlanticus, Ischnura elegans, Larimichthys polyactis, Opheodrys vernalis, Pelteobagrus fulvidraco, Phragmidium violaceum, Pistacia vera, and Thunnus thynnus. These loci were cross‐tested on the following species: Allanblackia gabonensis, Allanblackia stanerana, Neoceratitis cyanescens, Dacus ciliatus, Dacus demmerezi, Bactrocera zonata, Ceratitis capitata, Ceratitis rosa, Ceratits catoirii, Dacus punctatifrons, Ephydatia mülleri, Spongilla lacustris, Geodia cydonium, Axinella sp., Ischnura graellsii, Ischnura ramburii, Ischnura pumilio, Pistacia integerrima and Pistacia terebinthus.

Deletion and complementation of the mating type (MAT) locus of the wheat head blight pathogen Gibberella zeae.

ABSTRACT Gibberella zeae, a self-fertile, haploid filamentous ascomycete, causes serious epidemics of wheat (Triticum aestivum) head blight worldwide and contaminates grain with trichothecene mycotoxins. Anecdotal evidence dating back to the late 19th century indicates that G. zeae ascospores (sexual spores) are a more important inoculum source than are macroconidia (asexual spores), although the fungus can produce both during wheat head blight epidemics. To develop fungal strains to test this hypothesis, the entire mating type (MAT1) locus was deleted from a self-fertile (MAT1-1/MAT1-2), virulent, trichothecene-producing wild-type strain of G. zeae. The resulting MAT deletion (mat1-1/mat1-2) strains were unable to produce perithecia or ascospores and appeared to be unable to mate with the fertile strain from which they were derived. Complementation of a MAT deletion strain by transformation with a copy of the entire MAT locus resulted in recovery of production of perithecia and ascospores. MAT deletion strains and MAT-complemented strains retained the ability to produce macroconidia that could cause head blight, as assessed by direct injection into wheat heads in greenhouse tests. Availability of MAT-null and MAT-complemented strains provides a means to determine the importance of ascospores in the biology of G. zeae and perhaps to identify novel approaches to control wheat head blight.

A putative pheromone signaling pathway is dispensable for self-fertility in the homothallic ascomycete Gibberella zeae.

Gibberella zeae, a homothallic ascomycetous fungus, does not seek a partner for mating. Here, we focused on the role(s) of putative pheromone and receptor genes during sexual development in G. zeae. Orthologs of two pheromone precursor genes (GzPPG1 and GzPPG2), and their cognate receptor genes (GzPRE2 and GzPRE1) were transcribed during sexual development. The expression of these genes was controlled by the mating-type (MAT) locus and a MAP kinase gene, but not in a MAT-specific manner. Targeted gene deletion and subsequent outcrosses generated G. zeae strains lacking these putative pheromone/receptor genes in various combinations (from single to quadruple deletions). All G. zeae deletion strains were similar to the self-fertile progenitor in both male- and female fertility and other traits. Sometimes, the deletions including DeltaGzPPG1;DeltaGzPRE2 caused increased numbers of immature perithecia. Taken together, it is clear that these putative pheromones/receptors play a non-essential role in the sexual development of G. zeae.

Functional analysis of the homoserine O -acetyltransferase gene and its identification as a selectable marker in Gibberella zeae

We used restriction enzyme-mediated integration (REMI) to identify a methionine auxotrophic mutant of Gibberella zeae, an important cereal pathogen. In addition to its methionine requirement, the G. zeae REMI mutant designated Z43R3912 showed pleiotropic phenotypes, including reduced virulence on host plants and lack of sexual development. Outcrossing of Z43R3912 with a mat1-1 deletion strain confirmed that the mutation of Z43R3912 was tagged with the hygromycin B resistance marker. The vector insertion site in Z43R3912 was identified within the ORF designated GzmetE, encoding a putative homoserine O-acetyltrasferase (HOA). Gene disruption analyses confirmed that GzmetE was responsible for the pleiotropic phenotypes of Z43R3912. Genetic complementation of the G. zeae methionine auxotroph with an intact copy of the Aspergillus nidulans metE and GzmetE genes suggests that the HOA gene can be used as a selectable marker for transformation of G. zeae.

Identification of a 12-Gene Fusaric Acid Biosynthetic Gene Cluster in Fusarium Species Through Comparative and Functional Genomics

In fungi, genes involved in biosynthesis of a secondary metabolite (SM) are often located adjacent to one another in the genome and are coordinately regulated. These SM biosynthetic gene clusters typically encode enzymes, one or more transcription factors, and a transport protein. Fusaric acid is a polyketide-derived SM produced by multiple species of the fungal genus Fusarium. This SM is of concern because it is toxic to animals and, therefore, is considered a mycotoxin and may contribute to plant pathogenesis. Preliminary descriptions of the fusaric acid (FA) biosynthetic gene (FUB) cluster have been reported in two Fusarium species, the maize pathogen F. verticillioides and the rice pathogen F. fujikuroi. The cluster consisted of five genes and did not include a transcription factor or transporter gene. Here, analysis of the FUB region in F. verticillioides, F. fujikuroi, and F. oxysporum, a plant pathogen with multiple hosts, indicates the FUB cluster consists of at least 12 genes (FUB1 to FUB12). Deletion analysis confirmed that nine FUB genes, including two Zn(II)2Cys6 transcription factor genes, are required for production of wild-type levels of FA. Comparisons of FUB cluster homologs across multiple Fusarium isolates and species revealed insertion of non-FUB genes at one or two locations in some homologs. Although the ability to produce FA contributed to the phytotoxicity of F. oxysporum culture extracts, lack of production did not affect virulence of F. oxysporum on cactus or F. verticillioides on maize seedlings. These findings provide new insights into the genetic and biochemical processes required for FA production.

Occurrence of Fusarium mycotoxins in rice and its milling by-products in Korea.

A total of 201 samples of brown rice, polished rice, and two types of by-products, blue-tinged rice and discolored rice, were collected from rice stores maintained at 51 rice processing complexes in Korea. These samples were analyzed for the presence of Fusarium mycotoxins such as deoxynivalenol (DON), nivalenol (NIV), and zearalenone (ZEA). Contaminants (and their ranges) found in discolored rice samples were DON (59 to 1,355 ng g(-1)), NIV (66 to 4,180 ng g(-1)), and ZEA (25 to 3,305 ng g(-1)); those found in blue-tinged (less-ripe) rice were DON (86 to 630 ng g(-1)), NIV (50 to 3,607 ng g(-1)), and ZEA (26 to 3,156 ng g(-1)). Brown rice samples were contaminated mostly with NIV and ZEA (52 to 569 ng g(-1) and 47 to 235 ng g(-1), respectively). Polished rice samples were largely free from mycotoxins, although one sample was contaminated with NIV (77 ng g(-1)). When the fungal flora associated with each rice sample was investigated, blue-tinged rice was the most often contaminated with Fusarium graminearum (3.8%), followed by the discolored rice (2.4%) and brown rice (1.6%) samples. Using PCR, toxin genotyping of 266 isolates of F. graminearum revealed that most isolates (96%) were NIV producers. In conclusion, this survey is the first report of the cocontamination of Korean rice and its by-products with trichothecenes and ZEA. Importantly, it also provides new information on the natural contamination of rice by Fusarium mycotoxins.

Draft Genome Sequence of Fusarium fujikuroi B14, the Causal Agent of the Bakanae Disease of Rice.

ABSTRACT Here, we present the genome sequence of a Korean strain (B14) of Fusarium fujikuroi, a fungal rice pathogen. The final assembly consists of 455 contigs with 43,810,516 bp and 14,017 predicted genes. Comparison with the F. verticillioides 7600 genome revealed a reference coverage of 83% (66.3% of reads mapped).