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.

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.

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.

Transcriptome analyses of the ginseng root rot pathogens Cylindrocarpon destructans and Fusarium solani to identify radicicol resistance mechanisms.

BACKGROUND: The ascomycete fungi Cylindrocarpon destructans (Cd) and Fusarium solani (Fs) cause ginseng root rot and significantly reduce the quality and yield of ginseng. Cd produces the secondary metabolite radicicol, which targets the molecular chaperone Hsp90. Fs is resistant to radicicol, whereas other fungal genera associated with ginseng disease are sensitive to it. Radicicol resistance mechanisms have not yet been elucidated. METHODS: Transcriptome analyses of Fs and Cd mycelia treated with or without radicicol were conducted using RNA-seq. All of the differentially expressed genes (DEGs) were functionally annotated using the Fusarium graminearum transcript database. In addition, deletions of two transporter genes identified by RNA-seq were created to confirm their contributions to radicicol resistance. RESULTS: Treatment with radicicol resulted in upregulation of chitin synthase and cell wall integrity genes in Fs and upregulation of nicotinamide adenine dinucleotide dehydrogenase and sugar transporter genes in Cd. Genes encoding an ATP-binding cassette transporter, an aflatoxin efflux pump, ammonium permease 1 (mep1), and nitrilase were differentially expressed in both Fs and Cd. Among these four genes, only the ABC transporter was upregulated in both Fs and Cd. The aflatoxin efflux pump and mep1 were upregulated in Cd, but downregulated in Fs, whereas nitrilase was downregulated in both Fs and Cd. CONCLUSION: The transcriptome analyses suggested radicicol resistance pathways, and deletions of the transporter genes indicated that they contribute to radicicol resistance.

Survival Factor Gene FgSvf1 Is Required for Normal Growth and Stress Resistance in Fusarium graminearum.

Survival factor 1 (Svf1) is a protein involved in cell survival pathways. In Saccharomyces cerevisiae, Svf1 is required for the diauxic growth shift and survival under stress conditions. In this study, we characterized the role of FgSvf1, the Svf1 homolog in the homothallic ascomycete fungus Fusarium graminearum. In the FgSvf1 deletion mutant, conidial germination was delayed, vegetative growth was reduced, and pathogenicity was completely abolished. Although the FgSvf1 deletion mutant produced perithecia, the normal maturation of ascospore was dismissed in deletion mutant. The FgSvf1 deletion mutant also showed reduced resistance to osmotic, fungicide, and cold stress and reduced sensitivity to oxidative stress when compared to the wild-type strain. In addition, we showed that FgSvf1 affects glycolysis, which results in the abnormal vegetative growth in the FgSvf1 deletion mutant. Further, intracellular reactive oxygen species (ROS) accumulated in the FgSvf1 deletion mutant, and this accumulated ROS might be related to the reduced sensitivity to oxidative stress and the reduced resistance to cold stress and fungicide stress. Overall, understanding the role of FgSvf1 in F. graminearum provides a new target to control F. graminearum infections in fields.

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

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.

Efficacy of Diphenyleneiodonium Chloride (DPIC) Against Diverse Plant Pathogens.

Many of the fungicides and antibiotics currently available against plant pathogens are of limited use due to the emergence of resistant strains. In this study, we examined the effects of diphenyleneiodonium chloride (DPIC), an inhibitor of the superoxide producing enzyme NADPH oxidase, against fungal and bacterial plant pathogens. We found that DPIC inhibits fungal spore germination and bacterial cell proliferation. In addition, we demonstrated the potent antibacterial activity of DPIC using rice heads infected with the bacterial pathogen Burkholderia glumae which causes bacterial panicle blight (BPB). We found that treatment with DPIC reduced BPB when applied during the initial flowering stage of the rice heads. These results suggest that DPIC could serve as a new and useful antimicrobial agent in agriculture.

Functional characterization of a putative DNA methyltransferase, EadM, in Xanthomonas axonopodis pv. glycines by proteomic and phenotypic analyses.

Xanthomonas axonopodis pv. glycines (Xag) is a phytopathogenic bacterium causing bacterial pustule disease in soybean. Functions of DNA methyltransferases have been characterized in animal pathogenic bacteria, but are poorly understood in plant pathogens. Here, we report that functions of a putative DNA methyltransferase, EadM, in Xag. An EadM-overexpressing strain, Xag(EadM), was less virulent than the wild-type carrying an empty vector, Xag(EV). Interestingly, the viable cell numbers of Xag(EadM) were much lower (10-fold) than those of Xag(EV) at the same optical density. Comparative proteomic analysis revealed that proteins involved in cell wall/membrane/envelope and iron-transport were more abundant. Based on proteomic analysis we carried out diverse phenotypic assays. Scanning electron microscopy revealed abnormal bacterial envelopes in Xag(EadM). Additionally, Xag(EadM) showed decreased stress tolerance against ciprofloxacin and sorbitol, but enhanced resistance to desiccation. Exopolysaccharide production in Xag(EadM) was also decreased. Production of siderophores, which are iron-chelators, was much higher in Xag(EadM). As in Xag, Escherichia coli expressing EadM showed significantly reduced (1000-fold) viable cell numbers at the same optical density. Thus, EadM is associated with virulence, envelope biogenesis, stress tolerance, exopolysaccharide production, and siderophore production. Our results provide valuable and fundamental information regarding DNA methyltransferase functions and their related cellular mechanisms in plant pathogenic bacteria.

Cooperative interactions between seed-borne bacterial and air-borne fungal pathogens on rice.

Bacterial-fungal interactions are widely found in distinct environments and contribute to ecosystem processes. Previous studies of these interactions have mostly been performed in soil, and only limited studies of aerial plant tissues have been conducted. Here we show that a seed-borne plant pathogenic bacterium, Burkholderia glumae (Bg), and an air-borne plant pathogenic fungus, Fusarium graminearum (Fg), interact to promote bacterial survival, bacterial and fungal dispersal, and disease progression on rice plants, despite the production of antifungal toxoflavin by Bg. We perform assays of toxoflavin sensitivity, RNA-seq analyses, lipid staining and measures of triacylglyceride content to show that triacylglycerides containing linolenic acid mediate resistance to reactive oxygen species that are generated in response to toxoflavin in Fg. As a result, Bg is able to physically attach to Fg to achieve rapid and expansive dispersal to enhance disease severity.

Molecular mapping of qBK1 WD , a major QTL for bakanae disease resistance in rice.

BACKGROUND: Bakanae or foot rot disease is a prominent disease of rice caused by Gibberella fujikuroi. 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 japonica variety Wonseadaesoo. RESULTS: A primary QTL study using the genotypes/phenotypes of the recombinant inbred lines (RILs) indicated that the locus qBK1 WD 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 qBK1 WD was 8.29, accounting for 20.2% of the total phenotypic variation. We further identified a gene pyramiding effect of two QTLs, qBK WD and previously developed qBK1. The mean proportion of healthy plant for 31 F4 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 qBK WD or qBK1 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 F4 RILs harboring both qBK WD and qBK1 was 80.2%, which was significantly higher than that of the lines with only qBK WD or qBK1. CONCLUSION: Introducing qBK WD or pyramiding the QTLs qBK WD and qBK1 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.

Identification of Genes Related to Fungicide Resistance in Fusarium fujikuroi.

We identified two genes related to fungicide resistance in Fusarium fujikuroi through random mutagenesis. Targeted gene deletions showed that survival factor 1 deletion resulted in higher sensitivity to fungicides, while deletion of the gene encoding F-box/WD-repeat protein increased resistance, suggesting that the genes affect fungicide resistance in different ways.

Chlamydospore Induction from Conidia of Cylindrocarpon destructans Isolated from Ginseng in Korea.

Cylindrocarpon destructans causes root rot disease in ginseng and can survive for a long time, producing chlamydospores. We optimized conditions to induce chlamydospore production from the conidia of C. destructans, isolated from Korean ginseng. This will provide the basis for testing the efficacy of control agents targeting these chlamydospores.

Development of a Selective Medium for the Fungal Pathogen Cylindrocarpon destructans Using Radicicol.

The soil-borne ascomycete fungus Cylindrocarpon destructans causes ginseng root rot disease and produces various secondary metabolites such as brefeldin A and radicicol. The slow growth of this fungus compared with other plant pathogenic and saprophytic fungi in soil disturbs isolation of this fungus from soil and infected ginseng. In this study, we developed a selective medium for C. destructans using radicicol produced by this fungus. Supplementing 50 mg/L of radicicol to medium inhibited the mycelia growth of other fungi including Botrytis cinerea, Rhizoctonia solani and Alternaria panax, but did not affect the growth of C. destructans. In addition, conidia germination of other fungal species except for C. destructans was inhibited in submerged culture supplemented with radicicol. This medium provides a very efficient tool for isolating C. destructans and also can be used as an enrichment medium for this fungus.

Transcriptome analyses to understand effects of the Fusarium deoxynivalenol and nivalenol mycotoxins on Escherichia coli.

Fusarium spp. cause many diseases in farming systems and can produce diverse mycotoxins that can easily impact humans and animals through the ingestion of food and feed. Among these mycotoxins, deoxynivalenol (DON) and nivalenol (NIV) are considered the most important hazards because they can rapidly diffuse into cells and block eukaryotic ribosomes, leading to inhibition of the translation system. Conversely, the effects of DON and NIV mycotoxins on bacteria remain unclear. We employed RNA-seq technology to obtain information regarding the biological responses of bacteria and putative bacterial mechanisms of resistance to DON and NIV mycotoxins. Most differentially expressed genes down-regulated in response to these mycotoxins were commonly involved in phenylalanine metabolism, glyoxylate cycle, and cytochrome o ubiquinol oxidase systems. In addition, we generated an overall network of 1028 up-regulated genes to identify core genes under DON and NIV conditions. The results of our study provide a snapshot view of the transcriptome of Escherichia coli K-12 under DON and NIV conditions. Furthermore, the information provided herein will be useful for development of methods to detect DON and NIV.

A Putative Transcription Factor pcs1 Positively Regulates Both Conidiation and Sexual Reproduction in the Cereal Pathogen Fusarium graminearum.

The plant pathogen Fusarium graminearum causes Fusarium head blight in cereal crops and produces mycotoxins that are harmful to animals and humans. For the initiation and spread of disease, asexual and sexual reproduction is required. Therefore, studies on fungal reproduction contribute to the development of new methods to control and maintain the fungal population. Screening a previously generated transcription factor mutant collection, we identified one putative C2H2 zinc-finger transcription factor, pcs1, which is required for both sexual and asexual reproduction. Deleting pcs1 in F. graminearum resulted in a dramatic reduction in conidial production and a complete loss of sexual reproduction. The pathways and gene ontology of pcs1-dependent genes from microarray experiments showed that several G-protein related pathways, oxidase activity, ribosome biogenesis, and RNA binding and processing were highly enriched, suggesting that pcs1 is involved in several different biological processes. Further, overexpression of pcs1 increased conidial production and resulted in earlier maturation of ascospores compared to the wild-type strain. Additionally, the vegetative growth of the overexpression mutants was decreased in nutrient-rich conditions but was not different from the wild-type strain in nutrient-poor conditions. Overall, we discovered that the pcs1 transcription factor positively regulates both conidiation and sexual reproduction and confers nutrient condition-dependent vegetative growth.

Microcyle conidiation in filamentous fungi.

The typical life cycle of filamentous fungi commonly involves asexual sporulation after vegetative growth in response to environmental factors. The production of asexual spores is critical in the life cycle of most filamentous fungi. Normally, conidia are produced from vegetative hyphae (termed mycelia). However, fungal species subjected to stress conditions exhibit an extremely simplified asexual life cycle, in which the conidia that germinate directly generate further conidia, without forming mycelia. This phenomenon has been termed as microcycle conidiation, and to date has been reported in more than 100 fungal species. In this review, first, we present the morphological properties of fungi during microcycle conidiation, and divide microcycle conidiation into four simple categories, even though fungal species exhibit a wide variety of morphological differences during microcycle conidiogenesis. Second, we describe the factors that influence microcycle conidiation in various fungal species, and present recent genetic studies that have identified the genes responsible for this process. Finally, we discuss the biological meaning and application of microcycle conidiation.

Entomopathogenicity of Simplicillium lanosoniveum Isolated in Korea.

Fruiting bodies similar to those of the ascomycete fungi Podostroma cornu-damae and Cordyceps militaris were collected from Mt. Seunghak in Busan, Korea on August 21, 2012. The fruiting bodies were cylindrical, with tapered ends and golden red in color. The fruiting bodies contained abundant conidiophores bearing single-celled conidia, but no perithecia or asci. Pure culture of the fungal isolates was obtained through single-spore isolation. Analyses of morphological characteristics, including conidia shape, and phylogenetic traits, using internal transcribed spacer sequences, showed that these isolates belonged to the species Simplicillium lanosoniveum. Although this fungal species is known to be mycoparasitic, the isolates obtained in this study were unable to infect fungi. However, silkworms (Bombyx mori) inoculated with the fungal isolates died during the larval or pupal stages, as has been shown for the strongly entomopathogenic fungus Beauveria bassiana. This study is the first report of the entomopathogenicity of S. lanosoniveum and indicates its potential for use in biological control of insects.

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.

Biological Efficacy of Streptomyces sp. Strain BN1 against the Cereal Head Blight Pathogen Fusarium graminearum.

Fusarium head blight (FHB) caused by the filamentous fungus Fusarium graminearum is one of the most severe diseases threatening the production of small grains. Infected grains are often contaminated with mycotoxins such as zearalenone and trichothecences. During survey of contamination by FHB in rice grains, we found a bacterial isolate, designated as BN1, antagonistic to F. graminearum. The strain BN1 had branching vegetative hyphae and spores, and its aerial hyphae often had long, straight filaments bearing spores. The 16S rRNA gene of BN1 had 100% sequence identity with those found in several Streptomyces species. Phylogenetic analysis of ITS regions showed that BN1 grouped with S. sampsonii with 77% bootstrap value, suggesting that BN1 was not a known Streptomyces species. In addition, the efficacy of the BN1 strain against F. graminearum strains was tested both in vitro and in vivo. Wheat seedling length was significantly decreased by F. graminearum infection. However, this effect was mitigated when wheat seeds were treated with BN1 spore suspension prior to F. graminearum infection. BN1 also significantly decreased FHB severity when it was sprayed onto wheat heads, whereas BN1 was not effective when wheat heads were point inoculated. These results suggest that spraying of BN1 spores onto wheat heads during the wheat flowering season can be efficient for plant protection. Mechanistic studies on the antagonistic effect of BN1 against F. graminearum remain to be analyzed.