Yin-Won Lee

Jasmonic acid carboxyl methyltransferase: A key enzyme for jasmonate-regulated plant responses

Methyl jasmonate is a plant volatile that acts as an important cellular regulator mediating diverse developmental processes and defense responses. We have cloned the novel gene JMT encoding an S-adenosyl-l-methionine:jasmonic acid carboxyl methyltransferase (JMT) from Arabidopsis thaliana. Recombinant JMT protein expressed in Escherichia coli catalyzed the formation of methyl jasmonate from jasmonic acid with Km value of 38.5 μM. JMT RNA was not detected in young seedlings but was detected in rosettes, cauline leaves, and developing flowers. In addition, expression of the gene was induced both locally and systemically by wounding or methyl jasmonate treatment. This result suggests that JMT can perceive and respond to local and systemic signals generated by external stimuli, and that the signals may include methyl jasmonate itself. Transgenic Arabidopsis overexpressing JMT had a 3-fold elevated level of endogenous methyl jasmonate without altering jasmonic acid content. The transgenic plants exhibited constitutive expression of jasmonate-responsive genes, including VSP and PDF1.2. Furthermore, the transgenic plants showed enhanced level of resistance against the virulent fungus Botrytis cinerea. Thus, our data suggest that the jasmonic acid carboxyl methyltransferase is a key enzyme for jasmonate-regulated plant responses. Activation of JMT expression leads to production of methyl jasmonate that could act as an intracellular regulator, a diffusible intercellular signal transducer, and an airborne signal mediating intra- and interplant communications.

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.

A Phenome-Based Functional Analysis of Transcription Factors in the Cereal Head Blight Fungus, Fusarium graminearum

Fusarium graminearum is an important plant pathogen that causes head blight of major cereal crops. The fungus produces mycotoxins that are harmful to animal and human. In this study, a systematic analysis of 17 phenotypes of the mutants in 657 Fusarium graminearum genes encoding putative transcription factors (TFs) resulted in a database of over 11,000 phenotypes (phenome). This database provides comprehensive insights into how this cereal pathogen of global significance regulates traits important for growth, development, stress response, pathogenesis, and toxin production and how transcriptional regulations of these traits are interconnected. In-depth analysis of TFs involved in sexual development revealed that mutations causing defects in perithecia development frequently affect multiple other phenotypes, and the TFs associated with sexual development tend to be highly conserved in the fungal kingdom. Besides providing many new insights into understanding the function of F. graminearum TFs, this mutant library and phenome will be a valuable resource for characterizing the gene expression network in this fungus and serve as a reference for studying how different fungi have evolved to control various cellular processes at the transcriptional level.

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.

Shifting fungal reproductive mode by manipulation of mating type genes: obligatory heterothallism of Gibberella zeae.

Fungi capable of sexual reproduction use heterothallic (self‐sterile) or homothallic (self‐fertile) mating strategies. In most ascomycetes, a single mating type locus, MAT, with two alternative forms (MAT1‐1 and MAT1‐2) called idiomorphs, controls mating ability. In heterothallic ascomycetes, these alternative idiomorphs reside in different nuclei. In contrast, most homothallic ascomycetes carry both MAT1‐1 and MAT1‐2 in a single nucleus, usually closely linked. An example of the latter is Gibberella zeae, a species that is capable of both selfing and outcrossing. G. zeae is a devastating cereal pathogen of ubiquitous geographical distribution, and also a producer of mycotoxins that threaten human and animal health. We asked whether G. zeae could be made strictly heterothallic by manipulation of MAT. Targeted gene replacement was used to differentially delete MAT1‐1 or MAT1‐2 from a wild‐type haploid MAT1‐1; MAT1‐2 strain, resulting in MAT1‐1; mat1‐2, mat1‐1; MAT1‐2 strains that were self‐sterile, yet able to cross to wild‐type testers and, more importantly, to each other. These results indicated that differential deletion of MAT idiomorphs eliminates selfing ability of G. zeae, but the ability to outcross is retained. They also indicated that both MAT idiomorphs are required for self‐fertility. To our knowledge, this is the first report of complete conversion of fungal reproductive strategy from homothallic to heterothallic by targeted manipulation of MAT. Practically, this approach opens the door to simple and efficient procedures for obtaining sexual recombinants of G. zeae that will be useful for genetic analyses of pathogenicity and other traits, such as the ability to produce mycotoxins.

Double-stranded RNA mycovirus from Fusarium graminearum

ABSTRACT Double-stranded RNA (dsRNA) viruses in some fungi are associated with hypovirulence and have been used or proposed as biological control agents. We isolated 7.5-kb dsRNAs from 13 of 286 field strains of Fusarium graminearum isolated from maize in Korea. One of these strains, DK21, was examined in more detail. This strain had pronounced morphological changes, including reduction in mycelial growth, increased pigmentation, reduced virulence towards wheat, and decreased (60-fold) production of trichothecene mycotoxins. The presence or absence of the 7.5-kb dsRNA was correlated with the changes in pathogenicity and morphology. The dsRNA could be transferred to virus-free strains by hyphal fusion, and the recipient strain acquired the virus-associated phenotype of the donor strain. The dsRNA was transmitted to approximately 50% of the conidia, and only colonies resulting from conidia carrying the mycovirus had the virus-associated phenotype. Partial nucleotide sequences of the purified dsRNA identify an RNA-dependent RNA polymerase sequence and an ATP-dependent helicase that are closely related to those of Cryphonectria hypovirus and Barley yellow mosaic virus. Collectively, these results suggest that this dsRNA isolated from F. graminearum encodes traits for hypovirulence.

Discontinuous distribution of fumonisin biosynthetic genes in the Gibberella fujikuroi species complex.

Production of the carcinogenic mycotoxins fumonisins has been reported in several Fusarium species, most of which are members of the Gibberella fujikuroi (Gf) complex. In this study, we examined 15 Fusarium species in the Gf complex and 12 other species for fumonisin production and the presence of fumonisin biosynthetic genes (FUM). Among the species within the Gf complex, fumonisin production was detected only in F. fujikuroi, F. globosum, F. proliferatum, F. nygamai, F. oxysporum and F. verticillioides. These five species include members of two of the three major clades delineated in the Gf complex. The FUM genes were detected in these same five species and in F. anthophilum, a member of the third clade. Among the species outside the Gf complex, fumonisin production and FUM genes were detected only in F. oxysporum. Phylogenetic analyses of nucleotide sequences from two FUM gene fragments inferred relationships similar but not identical to those inferred from previous analyses of other genes. The results indicate the FUM genes are discontinuously distributed in the Gf complex and that this distribution gives rise to the differences in the abilities of closely related Fusarium species to produce fumonisins.

Genetic Diversity and Fitness of Fusarium graminearum Populations from Rice in Korea

ABSTRACT Fusarium graminearum is an important fungal pathogen of cereal crops and produces mycotoxins, such as the trichothecenes nivalenol and deoxynivalenol. This species may be subdivided into a series of genetic lineages or phylogenetic species. We identified strains of F. graminearum from the Republic of Korea to lineage, tested their ability to produce nivalenol and deoxynivalenol, and determined the genetic composition and structure of the populations from which they were recovered. Based on amplified fragment length polymorphism (AFLP), PCR genotyping, and chemical analyses of trichothecenes, all 249 isolates from southern provinces belonged to lineage 6, with 241 having the nivalenol genotype and 8 having the deoxynivalenol genotype. In the eastern Korea province, we recovered 84 lineage 6 isolates with the nivalenol genotype and 23 lineage 7 isolates with the deoxynivalenol genotype. Among 333 lineage 6 isolates, 36% of the AFLP bands were polymorphic, and there were 270 multilocus haplotypes. Genetic identity among populations was high (>0.972), and genotype diversity was low (30 to 58%). To test the adaptation of lineage 6 to rice, conidial mixtures of strains from lineages 3, 6, and 7 were inoculated onto rice plants and then recovered from the rice grains produced. Strains representing lineages 6 and 7 were recovered from inoculated spikelets at similar frequencies that were much higher than those for the strain representing lineage 3. Abundant perithecia were produced on rice straw, and 247 single-ascospore isolates were recovered from 247 perithecia. Perithecia representing lineage 6 (87%) were the most common, followed by those representing lineage 7 (13%), with perithecia representing lineage 3 not detected. These results suggest that F. graminearum lineage 6 may have a host preference for rice and that it may be more fit in a rice agroecosystem than are the other lineages present in Korea.

Putative Polyketide Synthase and Laccase Genes for Biosynthesis of Aurofusarin in Gibberella zeae

ABSTRACT Mycelia of Gibberella zeae (anamorph, Fusarium graminearum), an important pathogen of cereal crops, are yellow to tan with white to carmine red margins. We isolated genes encoding the following two proteins that are required for aurofusarin biosynthesis from G. zeae: a type I polyketide synthase (PKS) and a putative laccase. Screening of insertional mutants of G. zeae, which were generated by using a restriction enzyme-mediated integration procedure, resulted in the isolation of mutant S4B3076, which is a pigment mutant. In a sexual cross of the mutant with a strain with normal pigmentation, the pigment mutation was linked to the inserted vector. The vector insertion site in S4B3076 was a HindIII site 38 bp upstream from an open reading frame (ORF) on contig 1.116 in the F. graminearum genome database. The ORF, designated Gip1 (for Gibberella zeae pigment mutation 1), encodes a putative laccase. A 30-kb region surrounding the insertion site and Gip1 contains 10 additional ORFs, including a putative ORF identified as PKS12 whose product exhibits about 40% amino acid identity to the products of type I fungal PKS genes, which are involved in pigment biosynthesis. Targeted gene deletion and complementation analyses confirmed that both Gip1 and PKS12 are required for aurofusarin production in G. zeae. This information is the first information concerning the biosynthesis of these pigments by G. zeae and could help in studies of their toxicity in domesticated animals.