Hye-Seon Kim

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.

Polymorphism of trichothecene biosynthesis genes in deoxynivalenol- and nivalenol-producing Fusarium graminearum isolates

Diversity in trichothecene mycotoxin production by 167 isolates of Fusarium graminearum was examined by chemical and molecular methods. Isolates from barley, corn, and wheat grown in Korea produced either deoxynivalenol (DON) or nivalenol (NIV), whereas isolates from corn grown in the United States produced DON only. Southern blotting of MseI-digested genomic DNAs from these isolates was performed using a 0.6-kb fragment of Tri5, a key enzyme for trichothecene production, as a probe. This technique revealed a single-band polymorphism between these isolates, with 1.8- and 2.2-kb bands arising from DON and NIV producers, respectively. The same set of isolates was subjected to previously developed PCR assays using primers derived from Tri7 or Tri13. These assays also revealed a single-band polymorphism between NIV- and DON-producing chemotypes. The polymorphisms at Tri5, Tri7, or Tri13 in all of the US isolates were consistent with their chemotypes as identified by GC-MS. However, for seven Korean isolates, chemical and molecular analyses yielded seemingly inconsistent results. This issue was resolved by Southern blot analysis with the Tri5 probe using two other restriction enzymes and sequence comparison of a 3.8-kb region spanning Tri5. In addition, one of these exceptional isolates was found to carry both DON and NIV chemotype-specific regions, possibly resulting from recombination between the two chemotypes.

Population structure of and mycotoxin production by Fusarium graminearum from maize in South Korea.

ABSTRACT Fusarium graminearum (Gibberella zeae) is an important pathogen of wheat, maize, barley, and rice in South Korea, and harvested grain often is contaminated with trichothecenes such as deoxynivalenol and nivalenol. In this study, we examined 568 isolates of F. graminearum collected from maize at eight locations in South Korea. We used amplified fragment length polymorphisms (AFLPs) to identify four lineages (2, 3, 6, and 7); lineage 7 was the most common (75%), followed by lineage 6 (12%), lineage 3 (12%), and lineage 2 (1%). The genetic identity among populations was high (>0.98), and the effective migration rate between locations was higher than that between lineages. Female fertility varied by lineage: all lineage 7 isolates were fertile, while 70%, 26%, and 14% of the isolates in lineages 6, 3, and 2, respectively, were fertile. All lineage 3 and lineage 7 isolates produced deoxynivalenol, whereas most lineage 2 and 6 isolates produced nivalenol. Genotypic diversity in lineage 3 and lineage 6 populations is similar to that found in previously described Korean rice populations, but genotypic diversity in lineage 7 is much lower, even though similar levels of gene flow occur between lineage 7 populations. We conclude that lineage 7 was relatively recently introduced into South Korea, perhaps accompanying imported maize seeds.

Evolution of structural diversity of trichothecenes, a family of toxins produced by plant pathogenic and entomopathogenic fungi

Trichothecenes are a family of terpenoid toxins produced by multiple genera of fungi, including plant and insect pathogens. Some trichothecenes produced by the fungus Fusarium are among the mycotoxins of greatest concern to food and feed safety because of their toxicity and frequent occurrence in cereal crops, and trichothecene production contributes to pathogenesis of some Fusarium species on plants. Collectively, fungi produce over 150 trichothecene analogs: i.e., molecules that share the same core structure but differ in patterns of substituents attached to the core structure. Here, we carried out genomic, phylogenetic, gene-function, and analytical chemistry studies of strains from nine fungal genera to identify genetic variation responsible for trichothecene structural diversity and to gain insight into evolutionary processes that have contributed to the variation. The results indicate that structural diversity has resulted from gain, loss, and functional changes of trichothecene biosynthetic (TRI) genes. The results also indicate that the presence of some substituents has arisen independently in different fungi by gain of different genes with the same function. Variation in TRI gene duplication and number of TRI loci was also observed among the fungi examined, but there was no evidence that such genetic differences have contributed to trichothecene structural variation. We also inferred ancestral states of the TRI cluster and trichothecene biosynthetic pathway, and proposed scenarios for changes in trichothecene structures during divergence of TRI cluster homologs. Together, our findings provide insight into evolutionary processes responsible for structural diversification of toxins produced by pathogenic fungi.

Protective Effect of Tat PTD-Hsp27 Fusion Protein on Tau Hyperphosphorylation Induced by Okadaic Acid in the Human Neuroblastoma Cell Line SH-SY5Y

Alzheimer’s disease (AD) is an age-related disorder that causes a loss of brain function. Hyperphosphorylation of tau and the subsequent formation of intracellular neurofibrillary tangles (NFTs) are implicated in the pathogenesis of AD. Hyperphosphorylated tau accumulates into insoluble paired helical filaments that aggregate into NFTs; therefore, regulation of tau phosphorylation represents an important treatment approach for AD. Heat shock protein 27 (Hsp27) plays a specific role in human neurodegenerative diseases; however, few studies have examined its therapeutic effect. In this study, we induced tau hyperphosphorylation using okadaic acid, which is a protein phosphatase inhibitor, and generated a fusion protein of Hsp27 and the protein transduction domain of the HIV Tat protein (Tat-Hsp27) to enhance the delivery of Hsp27. We treated Tat-Hsp27 to SH-SY5Y neuroblastoma cells for 2xa0h; the transduction level was proportional to the Tat-hsp27 concentration. Additionally, Tat-Hsp27 reduced the level of hyperphosphorylated tau and protected cells from apoptotic cell death caused by abnormal tau aggregates. These results reveal that Hsp27 represents a valuable protein therapeutic for AD.