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

Son, Hokyoung,Seo, Young-Su,Min, Kyunghun,Park, Ae Ran,Lee, Jungkwan,Jin, Jian-Ming,Lin, Yang,Cao, Peijian,Hong, Sae-Yeon,Kim, Eun-Kyung,Lee, Seung-Ho,Cho, Aram,Lee, Seunghoon,Kim, Myung-Gu,Kim, Yongs Public Library of Science 2011 PLoS pathogens Vol.7 No.10

<▼1><P><I>Fusarium graminearum</I> 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 <I>Fusarium graminearum</I> 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 <I>F. graminearum</I> 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.</P></▼1><▼2><P><B>Author Summary</B></P><P>Large collections of mutant lines allow for identification of gene functions. Here we constructed a mutant library of 657 putative transcription factors (TFs) through homologous recombination in the head blight fungus, <I>Fusarium graminearum</I>, providing a resource for understanding gene regulation in fungus. By screening these mutants in 17 phenotypic categories, we constructed a dataset of over 11,000 phenotypes. This study provides new insight into understanding multiple phenotypes caused by single TF as well as regulation of gene expression at the transcription level in <I>F. graminearum</I>. Furthermore, our TF mutant library will be a valuable resource for fungal studies through the distribution of mutants and easy access to our phenotypic and genetic data.</P></▼2>

WetA Is Required for Conidiogenesis and Conidium Maturation in the Ascomycete Fungus <i>Fusarium graminearum</i>

Son, Hokyoung,Kim, Myung-Gu,Min, Kyunghun,Lim, Jae Yun,Choi, Gyung Ja,Kim, Jin-Cheol,Chae, Suhn-Kee,Lee, Yin-Won American Society for Microbiology 2014 EUKARYOTIC CELL Vol.13 No.1

<P><I>Fusarium graminearum</I>, a prominent fungal pathogen that infects major cereal crops, primarily utilizes asexual spores to spread disease. To understand the molecular mechanisms underlying conidiogenesis in <I>F. graminearum</I>, we functionally characterized the <I>F. graminearum</I> ortholog of <I>Aspergillus nidulans</I> <I>wetA</I>, which has been shown to be involved in conidiogenesis and conidium maturation. Deletion of <I>F. graminearum</I> <I>wetA</I> did not alter mycelial growth, sexual development, or virulence, but the <I>wetA</I> 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 <I>F. graminearum</I> <I>wetA</I> deletion mutants was reduced. The <I>wetA</I> 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 <I>F. graminearum</I>. Transcriptome analyses demonstrated that most of the putative conidiation-related genes are expressed constitutively and that only a few genes are specifically involved in <I>F. graminearum</I> conidiogenesis. The conserved and distinct roles identified for WetA in <I>F. graminearum</I> provide new insights into the genetics of conidiation in filamentous fungi.</P>

Mitochondrial Carnitine-Dependent Acetyl Coenzyme A Transport Is Required for Normal Sexual and Asexual Development of the Ascomycete Gibberella zeae

Son, Hokyoung,Min, Kyunghun,Lee, Jungkwan,Choi, Gyung Ja,Kim, Jin-Cheol,Lee, Yin-Won American Society for Microbiology 2012 EUKARYOTIC CELL Vol.11 No.9

<B>ABSTRACT</B><P> Fungi have evolved efficient metabolic mechanisms for the exact temporal (developmental stages) and spatial (organelles) production of acetyl coenzyme A (acetyl-CoA). We previously demonstrated mechanistic roles of several acetyl-CoA synthetic enzymes, namely, ATP citrate lyase and acetyl-CoA synthetases (ACSs), in the plant-pathogenic fungus Gibberella zeae . In this study, we characterized two carnitine acetyltransferases (CATs; CAT1 and CAT2) to obtain a better understanding of the metabolic processes occurring in G. zeae . We found that CAT1 functioned as an alternative source of acetyl-CoA required for lipid accumulation in an <I>ACS1</I> deletion mutant. Moreover, deletion of <I>CAT1</I> and/or <I>CAT2</I> resulted in various defects, including changes to vegetative growth, asexual/sexual development, trichothecene production, and virulence. Although CAT1 is associated primarily with peroxisomal CAT function, mislocalization experiments showed that the role of CAT1 in acetyl-CoA transport between the mitochondria and cytosol is important for sexual and asexual development in G. zeae . Taking these data together, we concluded that G. zeae CATs are responsible for facilitating the exchange of acetyl-CoA across intracellular membranes, particularly between the mitochondria and the cytosol, during various developmental stages. </P>

A novel gene, <i>GEA1</i>, is required for ascus cell-wall development in the ascomycete fungus <i>Fusarium graminearum</i>

Son, Hokyoung,Lee, Jungkwan,Lee, Yin-Won Society for General Microbiology 2013 Microbiology Vol.159 No.6

<P>The ascomycete fungus <I>Fusarium graminearum</I> is a devastating plant pathogen for major cereal crops. Ascospores are produced via sexual reproduction and forcibly discharged from mature perithecia, which function as the primary inocula. Perithecium development involves complex cellular processes and is under polygenic control. In this study, a novel gene, <I>GEA1</I>, was found to be required for ascus wall development in <I>F. graminearum</I>. <I>GEA1</I> deletion mutants produced normal-shaped perithecia and ascospores, yet ascospores were observed to precociously germinate inside the perithecium. Moreover, <I>GEA1</I> deletions resulted in abnormal ascus walls that collapsed prior to ascospore discharge. Based on localization of GEA1 to plasma membrane, GEA1 may be directly involved in ascus wall biogenesis. This is the first report to identify a unique gene required for ascus wall development in <I>F. graminearum</I>.</P>

Genome-wide exonic small interference RNA-mediated gene silencing regulates sexual reproduction in the homothallic fungus <i>Fusarium graminearum</i>

Son, Hokyoung,Park, Ae Ran,Lim, Jae Yun,Shin, Chanseok,Lee, Yin-Won Public Library of Science 2017 PLoS genetics Vol.13 No.2

<▼1><P>Various ascomycete fungi possess sex-specific molecular mechanisms, such as repeat-induced point mutations, meiotic silencing by unpaired DNA, and unusual adenosine-to-inosine RNA editing, for genome defense or gene regulation. Using a combined analysis of functional genetics and deep sequencing of small noncoding RNA (sRNA), mRNA, and the degradome, we found that the sex-specifically induced exonic small interference RNA (ex-siRNA)-mediated RNA interference (RNAi) mechanism has an important role in fine-tuning the transcriptome during ascospore formation in the head blight fungus <I>Fusarium graminearum</I>. Approximately one-third of the total sRNAs were produced from the gene region, and sRNAs with an antisense direction or 5′-U were involved in post-transcriptional gene regulation by reducing the stability of the corresponding gene transcripts. Although both Dicers and Argonautes partially share their functions, the sex-specific RNAi pathway is primarily mediated by <I>Fg</I>Dicer1 and <I>Fg</I>Ago2, while the constitutively expressed RNAi components <I>Fg</I>Dicer2 and <I>Fg</I>Ago1 are responsible for hairpin-induced RNAi. Based on our results, we concluded that <I>F</I>. <I>graminearum</I> primarily utilizes ex-siRNA-mediated RNAi for ascosporogenesis but not for genome defenses and other developmental stages. Each fungal species appears to have evolved RNAi-based gene regulation for specific developmental stages or stress responses. This study provides new insights into the regulatory role of sRNAs in fungi and other lower eukaryotes.</P></▼1><▼2><P><B>Author summary</B></P><P>Control of gene expression by small noncoding RNA (sRNA) has recently been highlighted as a significant post-transcriptional regulatory mechanism. To date, researchers have predominantly focused on the identification of microRNA-like RNAs (milRNAs) in fungi because microRNAs (miRNAs) are key regulators in animals and plants. In this study, we discovered that the sex-induced RNA interference (RNAi) mechanism had important roles in sexual reproduction in the head blight fungus <I>Fusarium graminearum</I>. In the late stages of sexual reproduction, small interference RNAs that were produced from gene regions (ex-siRNAs) were involved in post-transcriptional gene regulation at a genome-wide level. Based on our results, we concluded that <I>F</I>. <I>graminearum</I> specifically utilizes ex-siRNA-mediated RNAi for sexual development but not for other biological processes. This is the first genome-wide characterization of the sRNAs involved in fungal development.</P></▼2>

FgFlbD regulates hyphal differentiation required for sexual and asexual reproduction in the ascomycete fungus Fusarium graminearum.

Son, Hokyoung,Kim, Myung-Gu,Chae, Suhn-Kee,Lee, Yin-Won Microbiological Society of Korea 2014 The journal of microbiology Vol.52 No.11

<P>Fusarium graminearum is a filamentous fungal plant pathogen that infects major cereal crops. The fungus produces both sexual and asexual spores in order to endure unfavorable environmental conditions and increase their numbers and distribution across plants. In a model filamentous fungus, Aspergillus nidulans, early induction of conidiogenesis is orchestrated by the fluffy genes. The objectives of this study were to characterize fluffy gene homologs involved in conidiogenesis and their mechanism of action in F. graminearum. We characterized five fluffy gene homologs in F. graminearum and found that FlbD is the only conserved regulator for conidiogenesis in A. nidulans and F. graminearum. Deletion of fgflbD prevented hyphal differentiation and the formation of perithecia. Successful interspecies complementation using A. nidulans flbD demonstrated that the molecular mechanisms responsible for FlbD functions are conserved in F. graminearum. Moreover, abaA-wetA pathway is positively regulated by FgFlbD during conidiogenesis in F. graminearum. Deleting fiebD abolished morphological effects of abaA overexpression, which suggests that additional factors for FgFlbD or an AbaA-independent pathway for conidiogenesis are required for F. graminearum conidialion. Importantly, this study led to the construction of a genetic pathway of F. graminearum conidiogenesis and provides new insights into the genetics of conidiogenesis in fungi.</P>

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

Lee, Seunghoon,Son, Hokyoung,Lee, Jungkwan,Min, Kyunghun,Choi, Gyung Ja,Kim, Jin-Cheol,Lee, Yin-Won American Society for Microbiology 2011 EUKARYOTIC CELL Vol.10 No.8

<B>ABSTRACT</B><P> 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 ( <I>ACS</I> genes <I>ACS1</I> and <I>ACS2</I> ), to identify alternative acetyl-CoA production mechanisms for ACL. The <I>ACS1</I> 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, <I>ACS2</I> , has accessorial functions for <I>ACS1</I> and has compensatory functions for <I>ACL</I> 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. </P>

Functional characterization of sucrose non-fermenting 1 protein kinase complex genes in the Ascomycete Fusarium graminearum

Yu, Jungheon,Son, Hokyoung,Park, Ae Ran,Lee, Seung-Ho,Choi, Gyung Ja,Kim, Jin-Cheol,Lee, Yin-Won Springer-Verlag 2014 Current genetics Vol.60 No.1

Peroxisome function is required for virulence and survival of Fusarium graminearum.

Min, Kyunghun,Son, Hokyoung,Lee, Jungkwan,Choi, Gyung Ja,Kim, Jin-Cheol,Lee, Yin-Won APS Press 2012 Molecular plant-microbe interactions Vol.25 No.12

<P>Peroxisomes are organelles that are involved in a number of important cellular metabolic processes, including the β-oxidation of fatty acids, biosynthesis of secondary metabolites, and detoxification of reactive oxygen species (ROS). In this study, the role of peroxisomes was examined in Fusarium graminearum by targeted deletion of three genes (PEX5, PEX6, and PEX7) encoding peroxin (PEX) proteins required for peroxisomal protein import. PEX5 and PEX7 deletion mutants were unable to localize the fluorescently tagged peroxisomal targeting signal type 1 (PTS1)- and PTS2-containing proteins to peroxisomes, respectively, whereas the PEX6 mutant failed to localize both fluorescent proteins. Deletion of PEX5 and PEX6 resulted in retarded growth on long-chain fatty acids and butyrate, while the PEX7 deletion mutants utilized fatty acids other than butyrate. Virulence on wheat heads was greatly reduced in the PEX5 and PEX6 deletion mutants, and they were defective in spreading from inoculated florets to the adjacent spikelets through rachis. Deletion of PEX5 and PEX6 dropped survivability of aged cells in planta and in vitro due to the accumulation of ROS followed by necrotic cell death. These results demonstrate that PTS1-dependent peroxisomal protein import mediated by PEX5 and PEX6 are critical to virulence and survival of F. graminearum.</P>

Development of a Conditional Gene Expression System Using a Zearalenone-Inducible Promoter for the Ascomycete Fungus Gibberella zeae

Lee, Jungkwan,Son, Hokyoung,Lee, Seunghoon,Park, Ae Ran,Lee, Yin-Won American Society for Microbiology 2010 Applied and environmental microbiology Vol.76 No.10

<B>ABSTRACT</B><P>The ascomycete fungus <I>Gibberella zeae</I> is an important plant pathogen that causes fusarium head blight on small grains. Molecular studies of this fungus have been performed extensively to uncover the biological mechanisms related to pathogenicity, toxin production, and sexual reproduction. Molecular methods, such as targeted gene deletion, gene overexpression, and gene fusion to green fluorescent protein (GFP), are relatively easy to perform with this fungus; however, conditional expression systems have not been developed. The purpose of this study was to identify a promoter that could be induced by zearalenone (ZEA) for the development of a conditional expression system in <I>G. zeae</I>. Through microarray analysis, we isolated one zearalenone response gene (<I>ZEAR</I>) whose expression was increased more than 50 times after ZEA treatment. Northern blot analysis showed that the <I>ZEAR</I> transcript dramatically increased after 1 h of ZEA treatment. To determine the utility of the <I>ZEAR</I> promoter, called Pzear, in a conditional expression system, we transformed a <I>Pzear</I>::<I>GFP</I> fusion construct into <I>G. zeae</I>. Our data showed a ZEA concentration-dependent increase in GFP expression. We also replaced the promoter of <I>G. zeae metE</I> (<I>GzmetE)</I>, an essential gene for methionine biosynthesis, with the Pzear promoter. The growth of the <I>Pzear-GzmetE</I> mutant on minimal medium was dependent on the ZEA concentration supplemented in the medium and showed that GzMetE expression was induced by ZEA. This study is the first report of an inducible promoter in <I>G. zeae.</I> Our system will be useful for the characterization of essential gene functions in this fungus through differential and ZEA-dependent gene expression. In addition, the Pzear promoter may be applicable as a biosensor for the detection of ZEA contamination in agricultural products.</P>