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>

GzSNF1 Is Required for Normal Sexual and Asexual Development in the Ascomycete Gibberella zeae

Lee, Seung-Ho,Lee, Jungkwan,Lee, Seunghoon,Park, Eun-Hee,Kim, Ki-Woo,Kim, Myoung-Dong,Yun, Sung-Hwan,Lee, Yin-Won American Society for Microbiology 2009 EUKARYOTIC CELL Vol.8 No.1

<B>ABSTRACT</B><P>The sucrose nonfermenting 1 (<I>SNF1</I>) protein kinase of yeast plays a central role in the transcription of glucose-repressible genes in response to glucose starvation. In this study, we deleted an ortholog of <I>SNF1</I> from <I>Gibberella zeae</I> to characterize its functions by using a gene replacement strategy. The mycelial growth of deletion mutants (ΔGz<I>SNF1</I>) was reduced by 21 to 74% on diverse carbon sources. The virulence of ΔGz<I>SNF1</I> mutants on barley decreased, and the expression of genes encoding cell-wall-degrading enzymes was reduced. The most distinct phenotypic changes were in sexual and asexual development. ΔGz<I>SNF1</I> mutants produced 30% fewer perithecia, which matured more slowly, and asci that contained one to eight abnormally shaped ascospores. Mutants in which only the Gz<I>SNF1</I> catalytic domain was deleted had the same phenotype changes as the ΔGz<I>SNF1</I> strains, but the phenotype was less extreme in the mutants with the regulatory domain deleted. In outcrosses between the ΔGz<I>SNF1</I> mutants, each perithecium contained ∼70% of the abnormal ascospores, and ∼50% of the asci showed unexpected segregation patterns in a single locus tested. The asexual spores of the ΔGz<I>SNF1</I> mutants were shorter and had fewer septa than those of the wild-type strain. The germination and nucleation of both ascospores and conidia were delayed in ΔGz<I>SNF1</I> mutants in comparison with those of the wild-type strain. GzSNF1 expression and localization depended on the developmental stage of the fungus. These results suggest that Gz<I>SNF1</I> is critical for normal sexual and asexual development in addition to virulence and the utilization of alternative carbon sources.</P>

FgVelB globally regulates sexual reproduction, mycotoxin production and pathogenicity in the cereal pathogen Fusarium graminearum

Lee, Jungkwan,Myong, Kilseon,Kim, Jung-Eun,Kim, Hee-Kyoung,Yun, Sung-Hwan,Lee, Yin-Won Microbiology Society 2012 Microbiology Vol.158 No.7

<P>The velvet genes are conserved in ascomycetous fungi and function as global regulators of differentiation and secondary metabolism. Here, we characterized one of the velvet genes, designated FgVelB, in the plant-pathogenic fungus Fusarium graminearum, which causes fusarium head blight in cereals and produces mycotoxins within plants. FgVelB-deleted (δFgVelB) strains produced fewer aerial mycelia with less pigmentation than those of the wild-type (WT) during vegetative growth. Under sexual development conditions, the δFgVelB strains produced no fruiting bodies but retained male fertility, and conidiation was threefold higher compared with the WT strain. Production of trichothecene and zearalenone was dramatically reduced compared with the WT strain. In addition, the δFgVelB strains were incapable of colonizing host plant tissues. Transcript analyses revealed that FgVelB was highly expressed during the sexual development stage, and may be regulated by a mitogen-activated protein kinase cascade. Microarray analysis showed that FgVelB affects regulatory pathways mediated by the mating-type loci and a G-protein alpha subunit, as well as primary and secondary metabolism. These results suggest that FgVelB has diverse biological functions, probably by acting as a member of a possible velvet protein complex, although identification of the FgVelB-FgVeA complex and the determination of its roles require further investigation.</P>

Interaction between Burkholderia glumae and Fusarium graminearum on rice

Jungkwan Lee 한국농약과학회 2018 한국농약과학회 학술발표대회 논문집 Vol.2018 No.10

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>

Stress Tolerance and Virulence-Related Roles of Lipopolysaccharide in Burkholderia glumae

Lee, Chaeyeong,Mannaa, Mohamed,Kim, Namgyu,Kim, Juyun,Choi, Yeounju,Kim, Soo Hyun,Jung, Boknam,Lee, Hyun-Hee,Lee, Jungkwan,Seo, Young-Su The Korean Society of Plant Pathology 2019 Plant Pathology Journal Vol.35 No.5

The lipopolysaccharide (LPS) composed of lipid A, core, and O-antigen is the fundamental constituent of the outer membrane in gram-negative bacteria. This study was conducted to investigate the roles of LPS in Burkholderia glumae, the phytopathogen causing bacterial panicle blight and seedling rot in rice. To study the roles of the core oligosaccharide (OS) and the O-antigen region, mutant strains targeting the waaC and the wbiFGHI genes were generated. The LPS profile was greatly affected by disruption of the waaC gene and slight reductions were observed in the O-antigen region following wbiFGHI deletions. The results indicated that disruption in the core OS biosynthesis-related gene, waaC, was associated with increased sensitivity to environmental stress conditions including acidic, osmotic, saline, and detergent stress, and to polymyxin B. Moreover, significant impairment in the swimming and swarming motility and attenuation of bacterial virulence to rice were also observed in the waaC-defective mutant. The motility and virulence of O-antigen mutants defective in any gene of the wbiFGHI operon, were not significantly different from the wild-type except in slight decrease in swimming and swarming motility with wbiH deletion. Altogether, the results of present study indicated that the LPS, particularly the core OS region, is required for tolerance to environmental stress and full virulence in B. glumae. To our knowledge, this is the first functional study of LPS in a plant pathogenic Burkholderia sp. and presents a step forward toward full understanding of B. glumae pathogenesis.

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

Lee, Jungkwan,Chang, In-Young,Kim, Hun,Yun, Sung-Hwan,Leslie, John F.,Lee, Yin-Won American Society for Microbiology 2009 Applied and environmental microbiology Vol.75 No.10

<B>ABSTRACT</B><P><I>Fusarium graminearum</I> 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 <I>F. graminearum</I> 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 <I>F. graminearum</I> 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.</P>

Molecular mapping of <i>qBK1</i> ^<i>WD</i> , a major QTL for bakanae disease resistance in rice

Lee, Sais-Beul,Hur, Yeon-Jae,Cho, Jun-Hyeon,Lee, Jong-Hee,Kim, Tae-Heon,Cho, Soo-Min,Song, You-Chun,Seo, Young-Su,Lee, Jungkwan,Kim, Tae-sung,Park, Yong-Jin,Oh, Myung-Kyu,Park, Dong-Soo Springer US 2018 Rice Vol.11 No.-

<P><B>Background</B></P><P>Bakanae or foot rot disease is a prominent disease of rice caused by <I>Gibberella fujikuroi</I>. This disease may infect rice plants from the pre-emergence stage to the mature stage. In recent years, raising rice seedlings in seed boxes for mechanical transplanting has increased the incidence of many seedling diseases; only a few rice varieties have been reported to exhibit resistance to bakanae disease. In this study, we attempted to identify quantitative trait loci (QTLs) conferring bakanae disease resistance from the highly resistant <I>japonica</I> variety Wonseadaesoo.</P><P><B>Results</B></P><P>A primary QTL study using the genotypes/phenotypes of the recombinant inbred lines (RILs) indicated that the locus <I>qBK1</I><SUP><I>WD</I></SUP> conferring resistance to bakanae disease from Wonseadaesoo was located in a 1.59 Mb interval delimited on the physical map between chr01_13542347 (13.54 Mb) and chr01_15132528 (15.13 Mb). The log of odds (LOD) score of <I>qBK1</I><SUP><I>WD</I></SUP> was 8.29, accounting for 20.2% of the total phenotypic variation. We further identified a gene pyramiding effect of two QTLs, <I>qBK</I><SUP><I>WD</I></SUP> and previously developed <I>qBK1</I>. The mean proportion of healthy plant for 31 F<SUB>4</SUB> RILs that had no resistance genes was 35.3%, which was similar to that of the susceptible check variety Ilpum. The proportion of healthy plants for the lines with only <I>qBK</I><SUP><I>WD</I></SUP> or <I>qBK1</I> was 66.1% and 55.5%, respectively, which was significantly higher than that of the lines without resistance genes and that of Ilpum. The mean proportion of the healthy plant for 15 F<SUB>4</SUB> RILs harboring both <I>qBK</I><SUP><I>WD</I></SUP> and <I>qBK1</I> was 80.2%, which was significantly higher than that of the lines with only <I>qBK</I><SUP><I>WD</I></SUP> or <I>qBK1</I>.</P><P><B>Conclusion</B></P><P>Introducing <I>qBK</I><SUP><I>WD</I></SUP> or pyramiding the QTLs <I>qBK</I><SUP><I>WD</I></SUP> and <I>qBK1</I> could provide effective tools for breeding rice with bakanae disease resistance. To our knowledge, this is the first report on a gene pyramiding effect that provides higher resistance against bakanae disease.</P><P><B>Electronic supplementary material</B></P><P>The online version of this article (10.1186/s12284-017-0197-7) contains supplementary material, which is available to authorized users.</P>

Stress Tolerance and Virulence-Related Roles of Lipopolysaccharide in Burkholderia glumae

Chaeyeong Lee,Mohamed Mannaa,Namgyu Kim,Juyun Kim,Yeounju Choi,Soo Hyun Kim,Boknam Jung,Hyun-Hee Lee,Jungkwan Lee,Young-Su Seo 한국식물병리학회 2019 Plant Pathology Journal Vol.35 No.5

The lipopolysaccharide (LPS) composed of lipid A, core, and O-antigen is the fundamental constituent of the outer membrane in gram-negative bacteria. This study was conducted to investigate the roles of LPS in Burkholderia glumae, the phytopathogen causing bacterial panicle blight and seedling rot in rice. To study the roles of the core oligosaccharide (OS) and the Oantigen region, mutant strains targeting the waaC and the wbiFGHI genes were generated. The LPS profile was greatly affected by disruption of the waaC gene and slight reductions were observed in the O-antigen region following wbiFGHI deletions. The results indicated that disruption in the core OS biosynthesis-related gene, waaC, was associated with increased sensitivity to environmental stress conditions including acidic, osmotic, saline, and detergent stress, and to polymyxin B. Moreover, significant impairment in the swimming and swarming motility and attenuation of bacterial virulence to rice were also observed in the waaC-defective mutant. The motility and virulence of O-antigen mutants defective in any gene of the wbiFGHI operon, were not significantly different from the wild-type except in slight decrease in swimming and swarming motility with wbiH deletion. Altogether, the results of present study indicated that the LPS, particularly the core OS region, is required for tolerance to environmental stress and full virulence in B. glumae. To our knowledge, this is the first functional study of LPS in a plant pathogenic Burkholderia sp. and presents a step forward toward full understanding of B. glumae pathogenesis.

Development of a Selective Medium for the Fungal Pathogen Fusarium graminearum Using Toxoflavin Produced by the Bacterial Pathogen Burkholderia glumae

Jung, Boknam,Lee, Sehee,Ha, Jiran,Park, Jong-Chul,Han, Sung-Sook,Hwang, Ingyu,Lee, Yin-Won,Lee, Jungkwan The Korean Society of Plant Pathology 2013 Plant Pathology Journal Vol.29 No.4

The ascomycete fungus Fusarium graminearum is a major causal agent for Fusarium head blight in cereals and produces mycotoxins such as trichothecenes and zearalenone. Isolation of the fungal strains from air or cereals can be hampered by various other airborne fungal pathogens and saprophytic fungi. In this study, we developed a selective medium specific to F. graminearum using toxoflavin produced by the bacterial pathogen Burkholderia glumae. F. graminearum was resistant to toxoflavin, while other fungi were sensitive to this toxin. Supplementing toxoflavin into medium enhanced the isolation of F. graminearum from rice grains by suppressing the growth of saprophytic fungal species. In addition, a medium with or without toxoflavin exposed to wheat fields for 1 h had 84% or 25%, respectively, of colonies identified as F. graminearum. This selection medium provides an efficient tool for isolating F. graminearum, and can be adopted by research groups working on genetics and disease forecasting.