Gyung Ja Choi

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.

Forest soil metagenome gene cluster involved in antifungal activity expression in Escherichia coli.

ABSTRACT Using two forest soils, we previously constructed two fosmid libraries containing 113,700 members in total. The libraries were screened to select active antifungal clones using Saccharomyces cerevisiae as a target fungus. One clone from the Yuseong pine tree rhizosphere soil library, pEAF66, showed S. cerevisiae growth inhibition. Despite an intensive effort, active chemicals were not isolated. DNA sequence analysis and transposon mutagenesis of pEAF66 revealed 39 open reading frames (ORFs) and indicated that eight ORFs, probably in one transcriptional unit, might be directly involved in the expression of antifungal activity in Escherichia coli. The deduced amino acid sequences of eight ORFs were similar to those of the core genes encoding type II family polyketide synthases, such as the acyl carrier protein (ACP), ACP synthases, aminotransferase, and ACP reductase. The gene cluster involved in antifungal activity was similar in organization to the putative antibiotic production locus of Pseudomonas putida KT2440, although we could not select a similar active clone from the KT2440 genomic DNA library in E. coli. ORFs encoding ATP binding cassette transporters and membrane proteins were located at both ends of the antifungal gene cluster. Upstream ORFs encoding an IclR family response regulator and a LysR family response regulator were involved in the positive regulation of antifungal gene expression. Our results suggested the metagenomic approach as an alternative to search for novel antifungal antibiotics from unculturable soil bacteria. This is the first report of an antifungal gene cluster obtained from a soil metagenome using S. cerevisiae as a target fungus.

Some fungal endophytes from vegetable crops and their anti‐oomycete activities against tomato late blight

Aims:  To isolate endophytic fungi from vegetable plants and examine their in vivo anti‐oomycete activity against Phytophthora infestans in tomato plants.

Production of l-lactic acid from a green microalga, Hydrodictyon reticulum, by Lactobacillus paracasei LA104 isolated from the traditional Korean food, makgeolli

The freshwater microalga, Hydrodictyon reticulum, that contained 47.5% reducing sugars including 35% glucose was used as substrate for the production of l-lactic acid (LA) by LA-producing bacteria. Lactobacillus paracasei LA104 was selected for fermentation in a 5-l fermentor since it was able to grow at pH 3, 60g LA/l, 200g glucose/l, 125g NaCl/l, and 45°C and produced over 97.3% optically pure l-lactic acid with glucose as a substrate. Simultaneous saccharification and cofermentation from H. reticulum to l-LA using LA104 was investigated in a jar fermentor. The yield reached 46g/100g H. reticulum dry material, with a final concentration of 37.11g/l and a productivity of 1.03g/l/h. This is the first report of the production of l-LA from a microalga, and H. reticulum could be a potential feedstock for large-scale production of l-LA by LA104.

Functional Analyses of Two Acetyl Coenzyme A Synthetases in the Ascomycete Gibberella zeae

ABSTRACT Acetyl coenzyme A (acetyl-CoA) is a crucial metabolite for energy metabolism and biosynthetic pathways and is produced in various cellular compartments with spatial and temporal precision. Our previous study on ATP citrate lyase (ACL) in Gibberella zeae revealed that ACL-dependent acetyl-CoA production is important for histone acetylation, especially in sexual development, but is not involved in lipid synthesis. In this study, we deleted additional acetyl-CoA synthetic genes, the acetyl-CoA synthetases (ACS genes ACS1 and ACS2), to identify alternative acetyl-CoA production mechanisms for ACL. The ACS1 deletion resulted in a defect in sexual development that was mainly due to a reduction in 1-palmitoyl-2-oleoyl-3-linoleoyl-rac-glycerol production, which is required for perithecium development and maturation. Another ACS coding gene, ACS2, has accessorial functions for ACS1 and has compensatory functions for ACL as a nuclear acetyl-CoA producer. This study showed that acetate is readily generated during the entire life cycle of G. zeae and has a pivotal role in fungal metabolism. Because ACSs are components of the pyruvate-acetaldehyde-acetate pathway, this fermentation process might have crucial roles in various physiological processes for filamentous fungi.

Functional analyses of the nitrogen regulatory gene areA in Gibberella zeae.

Fusarium head blight caused by Gibberella zeae is a prominent disease of cereal crops that poses serious human health concerns due to the contamination of grains with mycotoxins. In this study, we deleted an orthologue of areA, which is a global nitrogen regulator in filamentous fungi, to characterize its functions in G. zeae. The areA deletion resulted in an inability to use nitrate as a sole nitrogen source, whereas urea utilization was partially available. The virulence of ΔareA strains on wheat heads was markedly reduced compared with the wild-type strain. The areA mutation triggered loss of trichothecene biosynthesis but did not affect zearalenone biosynthesis. The ΔareA strains showed immaturity of asci and did not produce mature ascospores. Chemical complementation by urea restored normal sexual development, whereas the virulence and trichothecene production were not affected by urea addition. GFP-AreA fusion protein was localized to nuclei, and its expression increased in response to nitrogen-limiting conditions. These results suggest that areA-dependent regulation of nitrogen metabolism is required for vegetative growth, sexual development, trichothecene biosynthesis, and virulence in G. zeae.

AbaA Regulates Conidiogenesis in the Ascomycete Fungus Fusarium graminearum

Fusarium graminearum (teleomorph Gibberella zeae) is a prominent pathogen that infects major cereal crops such as wheat, barley, and maize. Both sexual (ascospores) and asexual (conidia) spores are produced in F. graminearum. Since conidia are responsible for secondary infection in disease development, our objective of the present study was to reveal the molecular mechanisms underlying conidiogenesis in F. graminearum based on the framework previously described in Aspergillus nidulans. In this study, we firstly identified and functionally characterized the ortholog of AbaA, which is involved in differentiation from vegetative hyphae to conidia and known to be absent in F. graminearum. Deletion of abaA did not affect vegetative growth, sexual development, or virulence, but conidium production was completely abolished and thin hyphae grew from abnormally shaped phialides in abaA deletion mutants. Overexpression of abaA resulted in pleiotropic defects such as impaired sexual and asexual development, retarded conidium germination, and reduced trichothecene production. AbaA localized to the nuclei of phialides and terminal cells of mature conidia. Successful interspecies complementation using A. nidulans AbaA and the conserved AbaA-WetA pathway demonstrated that the molecular mechanisms responsible for AbaA activity are conserved in F. graminearum as they are in A. nidulans. Results from RNA-sequencing analysis suggest that AbaA plays a pivotal role in conidiation by regulating cell cycle pathways and other conidiation-related genes. Thus, the conserved roles of the AbaA ortholog in both A. nidulans and F. graminearum give new insight into the genetics of conidiation in filamentous fungi.

Functional analyses of regulators of G protein signaling in Gibberella zeae

Regulators of G protein signaling (RGS) proteins make up a highly diverse and multifunctional protein family that plays a critical role in controlling heterotrimeric G protein signaling. In this study, seven RGS genes (FgFlbA, FgFlbB, FgRgsA, FgRgsB, FgRgsB2, FgRgsC, and FgGprK) were functionally characterized in the plant pathogenic fungus, Gibberella zeae. Mutant phenotypes were observed for deletion mutants of FgRgsA and FgRgsB in vegetative growth, FgFlbB and FgRgsB in conidia morphology, FgFlbA in conidia production, FgFlbA, FgRgsB, and FgRgsC in sexual development, FgFlbA and FgRgsA in spore germination and mycotoxin production, and FgFlbA, FgRgsA, and FgRgsB in virulence. Furthermore, FgFlbA, FgRgsA, and FgRgsB acted pleiotropically, while FgFlbB and FgRgsC deletion mutants exhibited a specific defect in conidia morphology and sexual development, respectively. Amino acid substitutions in Gα subunits and overexpression of the FgFlbA gene revealed that deletion of FgFlbA and dominant active GzGPA2 mutant, gzgpa2(Q207L), had similar phenotypes in cell wall integrity, perithecia formation, mycotoxin production, and virulence, suggesting that FgFlbA may regulate asexual/sexual development, mycotoxin biosynthesis, and virulence through GzGPA2-dependent signaling in G. zeae.

Suppression of pine wilt disease by an antibacterial agent, oxolinic acid

BACKGROUND Pine wilt disease (PWD) is very complex and has been reported to be caused by pine wood nematode, Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle, and its accompanying bacteria. However, there is no report on the control of PWD by antibacterial agent. The present study was performed to investigate disease control efficacy of antibacterial agents against PWD. RESULTS Among six antibacterial antibiotics tested, oxolinic acid (OA) showed the strongest antibacterial activity against five bacteria isolated from three strains of pine wood nematode. In in vivo assay, it effectively suppressed the development of PWD in three-year-old seedlings of Pinus densiflora Sieb. & Zucc.; it showed 71% control when injected at 3 mg per seedling. A mixture of OA and the nematicidal agent abamectin (Ab) showed higher disease control efficacy against PWD than either OA or Ab alone. In addition, OA alone and a mixture of OA and Ab also controlled PWD in approximately 20-year-old pine trees under field conditions. CONCLUSION This is the first report on the suppression of PWD by OA. The result strongly indicates that PWD could be controlled by antibacterial antibiotic alone and a combination of antibacterial and nematicidal agents.

WetA Is Required for Conidiogenesis and Conidium Maturation in the Ascomycete Fungus Fusarium graminearum

ABSTRACT Fusarium graminearum, a prominent fungal pathogen that infects major cereal crops, primarily utilizes asexual spores to spread disease. To understand the molecular mechanisms underlying conidiogenesis in F. graminearum, we functionally characterized the F. graminearum ortholog of Aspergillus nidulans wetA, which has been shown to be involved in conidiogenesis and conidium maturation. Deletion of F. graminearum wetA did not alter mycelial growth, sexual development, or virulence, but the wetA deletion mutants produced longer conidia with fewer septa, and the conidia were sensitive to acute stresses, such as oxidative stress and heat stress. Furthermore, the survival rate of aged conidia from the F. graminearum wetA deletion mutants was reduced. The wetA deletion resulted in vigorous generation of single-celled conidia through autophagy-dependent microcycle conidiation, indicating that WetA functions to maintain conidial dormancy by suppressing microcycle conidiation in F. graminearum. Transcriptome analyses demonstrated that most of the putative conidiation-related genes are expressed constitutively and that only a few genes are specifically involved in F. graminearum conidiogenesis. The conserved and distinct roles identified for WetA in F. graminearum provide new insights into the genetics of conidiation in filamentous fungi.