T-plastin contributes to epithelial-mesenchymal transition in human lung cancer cells through FAK/AKT/Slug axis signaling pathway.

T-plastin (PLST), a member of the actin-bundling protein family, plays crucial roles in cytoskeletal structure, regulation, and motility. Studies have shown that the plastin family is associated with the malignant characteristics of cancer, such as circulating tumor cells and metastasis, by inducing epithelialmesenchymal transition (EMT) in various cancer cells. However, the role of PLST in the EMT of human lung cancer cells remains unclear. In this study, we observed that PLST overexpression enhanced cell migratory and invasive abilities, whereas its downregulation resulted in their suppression. Moreover, PLST expression levels were associated with the expression patterns of EMT markers, including E-cadherin, vimentin, and Slug. Furthermore, the phosphorylation levels of focal adhesion kinase (FAK) and AKT serine/threonine kinase (AKT) were dependent on PLST expression levels. These findings indicate that PLST induces the migration and invasion of human lung cancer cells by promoting Slug-mediated EMT via the FAK/AKT signaling pathway. [BMB Reports 2024; 57(6): 305-310].

A novel gene, GEA1, is required for ascus cell-wall development in the ascomycete fungus Fusarium graminearum.

The ascomycete fungus Fusarium graminearum 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, GEA1, was found to be required for ascus wall development in F. graminearum. GEA1 deletion mutants produced normal-shaped perithecia and ascospores, yet ascospores were observed to precociously germinate inside the perithecium. Moreover, GEA1 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 F. graminearum.

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.

Mitochondrial carnitine-dependent acetyl coenzyme A transport is required for normal sexual and asexual development of the ascomycete Gibberella zeae.

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 ACS1 deletion mutant. Moreover, deletion of CAT1 and/or CAT2 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.

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

Peroxisomes are organelles that are involved in a number of important cellular metabolic processes, including the ß-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.

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

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.

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.

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

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.

Functional analyses of two acetyl coenzyme A synthetases in the ascomycete Gibberella zeae.

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.

NADPH Oxidase Gene, FgNoxD, Plays a Critical Role in Development and Virulence in Fusarium graminearum.

NADPH oxidase is an enzyme that generates reactive oxygen species from oxygen and NADPH and is highly conserved in eukaryotes. In Fusarium graminearum, a series of different Nox enzymes have been identified. NoxA is involved in sexual development and ascospore production and, like NoxB, also contributes to pathogenicity. Both NoxA and NoxB are regulated by the subunit NoxR, whereas NoxC is usually self-regulated by EF-hand motifs found on the enzyme. In this study, we characterized another NADPH oxidase in F. graminearum, FgNoxD. In the FgNoxD deletion mutant, vegetative growth and conidia production were reduced, while sexual development was totally abolished. The FgNoxD deletion mutant also showed reduced resistance to cell wall perturbing agents; cell membrane inhibitors; and osmotic, fungicide, cold, and extracellular oxidative stress, when compared to the wild type. Moreover, in comparison to the wild type, the FgNoxD deletion mutant exhibited reduced virulence against the host plant. The FgNoxD deletion mutant produced less deoxynivalenol than the wild type, and the Tri5 and Tri6 gene expression was also downregulated. In conclusion, our findings show that FgNoxD is involved in the survival against various stresses, conidiation, sexual development, and virulence, highlighting this enzyme as a new target to control the disease caused by F. graminearum.

Genetic Diversity of Epicoccum nigrum and its Effects on Fusarium graminearum.

Epicoccum nigrum is a saprophytic or endophytic fungus that is found worldwide. Because of the antagonist effects of E. nigrum on many plant pathogens, current studies on E. nigrum have focused on the development of biological control agents and the utilization of its various metabolites. In this study, E. nigrum was collected from a wheat field, and its genetic diversity was analyzed. Phylogenetic analyses identified 63 isolates of E. nigrum divided into seven groups, indicating a wide genetic diversity. Isolates antagonized the wheat pathogen Fusarium graminearum, and reduced disease symptoms caused by F. graminearum in wheat coleoptiles. Moreover, pretreatment of wheat coleoptiles with E. nigrum induced the upregulation of pathogen-related (PR) genes, PR1, PR2, PR3, PR5, PR9, and PR10 in wheat coleoptiles responding to F. graminearum invasion. Overall, this study indicates that E. nigrum isolates can be used as biological pathogen inhibitors applied in wheat fields.

Functional characterization and manipulation of the apicidin biosynthetic pathway in Fusarium semitectum.

Apicidin is a cyclic tetrapeptide produced by certain isolates of Fusarium semitectum and has been shown to inhibit Apicomplexan histone deacetylase. An apicidin-producing strain (KCTC16676) of the filamentous fungus was mutated using an Agrobacterium tumefaciens-mediated transformation, resulting in 24 apicidin-deficient mutants. Three of the mutants had a T-DNA insertion in a gene that encodes a non-ribosomal peptide synthetase (NRPS). Results of sequence, expression, and gene deletion analyses defined an apicidin biosynthetic gene cluster, and the NRPS gene was named as apicidin synthetase gene 1 (APS1). A 63 kb region surrounding APS1 was sequenced and analysis revealed the presence of 19 genes. All of the genes including APS1 were individually deleted to determine their roles in apicidin biosynthesis. Chemical analyses of the mutant strains showed that eight genes are required for apicidin production and were used to propose an apicidin biosynthetic pathway. The apicidin analogues apicidin E, apicidin D(2) and apicidin B were identified from chemical analysis of the mutants. The cluster gene APS2, a putative transcription factor, was shown to regulate expression of the genes in the cluster and overexpression of APS2 increased apicidin production. This study establishes the apicidin biosynthetic pathway and provides new opportunities to improve the production of apicidin and produce new analogues.

A putative ABC transporter gene, ZRA1, is required for zearalenone production in Gibberella zeae.

Zearalenone (ZEA) is a secondary metabolite produced by various Fusarium species and causes estrogenic disorders in humans and animals. Recent studies have identified the ZEA biosynthesis gene cluster in F. graminearum, but other genes such as transporters responsible for ZEA export have not been identified in the cluster. In this study, we performed microarray analyses from the wild-type strain with and without ZEA supplementation and ZEA-nonproducing strain zeb2 to discover other genes responsible for ZEA biosynthesis. Three putative ABC transporters were significantly down-regulated in the zeb2 and were under positive regulation of the ZEB2 gene, which functions as a transcriptional activator for ZEA production in this fungus. However, only one gene (ZRA1) was found to be up-regulated by 20-fold in the wild-type strain supplemented with ZEA, and deletion of ZRA1 resulted in reduced ZEA production. Deletions of the other two genes showed similar ZEA productions as the wild-type strain. ZRA1 localized to the plasma membrane and vacuoles indicating possible roles of ZRA1 as a transporter. This study indicated that ZRA1 is involved in ZEA production and shares a common regulatory mode with ZEA cluster genes by ZEB2.

ATP citrate lyase is required for normal sexual and asexual development in Gibberella zeae.

Adenosine triphosphate (ATP) citrate lyase (ACL) is a key enzyme in the production of cytosolic acetyl-CoA, which is crucial for de novo lipid synthesis and histone acetylation in mammalian cells. In this study, we characterized the mechanistic roles of ACL in the homothallic ascomycete fungus Gibberella zeae, which causes Fusarium head blight in major cereal crops. Deletion of ACL in the fungus resulted in a complete loss of self and female fertility as well as a reduction in asexual reproduction, virulence, and trichothecene production. When the wild-type strain was spermatized with the ACL deletion mutants, they produced viable ascospores, however ascospore delimitation was not properly regulated. Although lipid synthesis was not affected by ACL deletion, histone acetylation was dramatically reduced in the ACL deletion mutants during sexual development, suggesting that the defects in sexual reproduction were caused by the reduction in histone acetylation. This study is the first report demonstrating a link between sexual development and ACL-mediated histone acetylation in fungi.

A novel gene, ROA, is required for normal morphogenesis and discharge of ascospores in Gibberella zeae.

Head blight, caused by Gibberella zeae, is a significant disease among cereal crops, including wheat, barley, and rice, due to contamination of grain with mycotoxins. G. zeae is spread by ascospores forcibly discharged from sexual fruiting bodies forming on crop residues. In this study, we characterized a novel gene, ROA, which is required for normal sexual development. Deletion of ROA (Δroa) resulted in an abnormal size and shape of asci and ascospores but did not affect vegetative growth. The Δroa mutation triggered round ascospores and insufficient cell division after spore delimitation. The asci of the Δroa strain discharged fewer ascospores from the perithecia but achieved a greater dispersal distance than those of the wild-type strain. Turgor pressure within the asci was calculated through the analysis of osmolytes in the epiplasmic fluid. Deletion of the ROA gene appeared to increase turgor pressure in the mutant asci. The higher turgor pressure of the Δroa mutant asci and the mutant spore shape contributed to the longer distance dispersal. When the Δroa mutant was outcrossed with a Δmat1-2 mutant, a strain that contains a green fluorescence protein (GFP) marker in place of the MAT1-2 gene, unusual phenotypic segregation occurred. The ratio of GFP to non-GFP segregation was 1:1; however, all eight spores had the same shape. Taken together, the results of this study suggest that ROA plays multiple roles in maintaining the proper morphology and discharge of ascospores in G. zeae.

Mapping of a Major QTL, qBK1Z, for Bakanae Disease Resistance in Rice.

Bakanae disease is a fungal disease of rice (Oryza sativa L.) caused by the pathogen Gibberella fujikuroi (also known as Fusarium fujikuroi). This study was carried out to identify novel quantitative trait loci (QTLs) from an indica variety Zenith. We performed a QTL mapping using 180 F2:9 recombinant inbred lines (RILs) derived from a cross between the resistant variety, Zenith, and the susceptible variety, Ilpum. A primary QTL study using the genotypes and phenotypes of the RILs indicated that the locus qBK1z conferring bakanae disease resistance from the Zenith was located in a 2.8 Mb region bordered by the two RM (Rice Microsatellite) markers, RM1331 and RM3530 on chromosome 1. The log of odds (LOD) score of qBK1z was 13.43, accounting for 30.9% of the total phenotypic variation. A finer localization of qBK1z was delimited at an approximate 730 kb interval in the physical map between Chr01_1435908 (1.43 Mbp) and RM10116 (2.16 Mbp). Introducing qBK1z or pyramiding with other previously identified QTLs could provide effective genetic control of bakanae disease in rice.

Identification and functional characterization of genes involved in the sexual reproduction of the ascomycete fungus Gibberella zeae.

We previously reported that G protein alpha subunit 1 (GPA1) is essential for sexual reproduction in the homothallic ascomycete fungus Gibberella zeae. In this study we performed microarray analyses on a GPA1 deletion mutant of G. zeae (Δgpa1) to identify genes involved in the sexual reproduction of this fungus. In the Δgpa1 strain, 645 genes were down-regulated and 550 genes were up-regulated during sexual reproduction when compared to the wild-type strain. One hundred of the down-regulated genes were selected for further investigation based on orthologous group clusters and differences in transcript levels. Quantitative real time-PCR was used to determine transcriptional profiles of these genes at various sexual and vegetative stages. We observed that transcript levels of 78 of these genes were dramatically increased in the wild-type strain during sexual reproduction compared to levels observed during vegetative growth, and were down-regulated in Δgpa1 compared to the wild-type strain. We deleted 57 of these genes and found that four of the deletion mutants lost self-fertility and five produced fewer perithecia compared to the wild-type strain. Two mutants produced wild-type numbers of perithecia, but maturation of perithecia and ascospores was delayed. In all we identified 11 genes that are involved in sexual reproduction of G. zeae and present evidence that some of these genes function at distinct stages during sexual reproduction in the fungus.

Functional analyses of two syntaxin-like SNARE genes, GzSYN1 and GzSYN2, in the ascomycete Gibberella zeae.

We identified two syntaxin-like SNARE genes, named GzSYN1 and GzSYN2, from the plant pathogenic ascomycete Gibberella zeae, and characterized the functions and cellular localization of these genes. The GzSYN1 deletion mutant (Deltagzsyn1) had 71% reduced hyphal growth compared to the wild-type strain, but produced perithecia with normal ascospores. Deltagzsyn2 had the same hyphal growth rate as the wild-type, but completely lost both self and female fertility. When Deltagzsyn2 was spermatized for Deltamat1-1 or Deltamat1-2 strains, it retained its male fertility, but the ascus shape was abnormal and ascospore delimitation was delayed. The Deltagzsyn1 and Deltagzsyn2 virulence on barley was reduced by 67% and 75%, respectively, compared to the wild-type. The GFP::GzSYN1 fusion protein was localized in vesicles, vacuoles, plasma membranes, and septa, whereas GFP::GzSYN2 was found only in plasma membranes and septa. These results suggest that syntaxins have key roles in fungal development and virulence in G. zeae.

Development of a conditional gene expression system using a zearalenone-inducible promoter for the ascomycete fungus Gibberella zeae.

The ascomycete fungus Gibberella zeae 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 G. zeae. Through microarray analysis, we isolated one zearalenone response gene (ZEAR) whose expression was increased more than 50 times after ZEA treatment. Northern blot analysis showed that the ZEAR transcript dramatically increased after 1 h of ZEA treatment. To determine the utility of the ZEAR promoter, called Pzear, in a conditional expression system, we transformed a Pzear::GFP fusion construct into G. zeae. Our data showed a ZEA concentration-dependent increase in GFP expression. We also replaced the promoter of G. zeae metE (GzmetE), an essential gene for methionine biosynthesis, with the Pzear promoter. The growth of the Pzear-GzmetE 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 G. zeae. 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.

GzSNF1 is required for normal sexual and asexual development in the ascomycete Gibberella zeae.

The sucrose nonfermenting 1 (SNF1) 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 SNF1 from Gibberella zeae to characterize its functions by using a gene replacement strategy. The mycelial growth of deletion mutants (DeltaGzSNF1) was reduced by 21 to 74% on diverse carbon sources. The virulence of DeltaGzSNF1 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. DeltaGzSNF1 mutants produced 30% fewer perithecia, which matured more slowly, and asci that contained one to eight abnormally shaped ascospores. Mutants in which only the GzSNF1 catalytic domain was deleted had the same phenotype changes as the DeltaGzSNF1 strains, but the phenotype was less extreme in the mutants with the regulatory domain deleted. In outcrosses between the DeltaGzSNF1 mutants, each perithecium contained approximately 70% of the abnormal ascospores, and approximately 50% of the asci showed unexpected segregation patterns in a single locus tested. The asexual spores of the DeltaGzSNF1 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 DeltaGzSNF1 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 GzSNF1 is critical for normal sexual and asexual development in addition to virulence and the utilization of alternative carbon sources.