24 research outputs found
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A Xenobiotic Detoxification Pathway through Transcriptional Regulation in Filamentous Fungi
Fungi are known to utilize transcriptional regulation of genes that encode efflux transporters to detoxify xenobiotics; however, to date it is unknown how fungi transcriptionally regulate and coordinate different phases of detoxification system (phase I, modification; phase II, conjugation; and phase III, secretion). Here we present evidence of an evolutionary convergence between the fungal and mammalian lineages, whereby xenobiotic detoxification genes (phase I coding for cytochrome P450 monooxygenases [CYP450s] and phase III coding for ATP-binding cassette [ABC] efflux transporters) are transcriptionally regulated by structurally unrelated proteins. Following next-generation RNA sequencing (RNA-seq) analyses of a filamentous fungus, Sclerotinia homoeocarpa, the causal agent of dollar spot on turfgrasses, a multidrug resistant (MDR) field strain was found to overexpress phase I and III genes, coding for CYP450s and ABC transporters for xenobiotic detoxification. Furthermore, there was confirmation of a gain-of-function mutation of the fungus-specific transcription factor S. homoeocarpa XDR1 (ShXDR1), which is responsible for constitutive and induced overexpression of the phase I and III genes, resulting in resistance to multiple classes of fungicidal chemicals. This fungal pathogen detoxifies xenobiotics through coordinated transcriptional control of CYP450s, biotransforming xenobiotics with different substrate specificities and ABC transporters, excreting a broad spectrum of xenobiotics or biotransformed metabolites. A Botrytis cinerea strain harboring the mutated ShXDR1 showed increased expression of phase I (BcCYP65) and III (BcatrD) genes, resulting in resistance to fungicides. This indicates the regulatory system is conserved in filamentous fungi. This molecular genetic mechanism for xenobiotic detoxification in fungi holds potential for facilitating discovery of new antifungal drugs and further studies of convergent and divergent evolution of xenobiotic detoxification in eukaryote lineages. IMPORTANCE Emerging multidrug resistance (MDR) in pathogenic filamentous fungi is a significant threat to human health and agricultural production. Understanding mechanisms of MDR is essential to combating fungal pathogens; however, there is still limited information on MDR mechanisms conferred by xenobiotic detoxification. Here, we report for the first time that overexpression of phase I drug-metabolizing monooxygenases (cytochrome P450s) and phase III ATP-binding cassette efflux transporters is regulated by a gain-of-function mutation in the fungus-specific transcription factor in the MDR strains of the filamentous plant-pathogenic fungus Sclerotinia homoeocarpa. This study establishes a novel molecular mechanism of MDR through the xenobiotic detoxification pathway in filamentous fungi, which may facilitate the discovery of new antifungal drugs to control pathogenic fungi
Characterization of Soybean STAY-GREEN Genes in Susceptibility to Foliar Chlorosis of Sudden Death Syndrome
Fusarium virguliforme causes sudden death syndrome (SDS) of soybean (Glycine max) in the United States. This fungal pathogen inhabits soil and produces multiple phytotoxins, which are translocated from infected roots to leaves, causing SDS foliar chlorosis and necrosis (Hartman et al., 2015). Because SDS foliar symptoms are solely induced by phytotoxins, it represents a unique pathosystem to study plant-phytotoxin interactions (Chang et al., 2016). SDS foliar symptoms typically appear near flowering through late reproductive growth stages, with chlorotic spots that gradually develop into interveinal chlorosis and necrosis (Fig. 1A). The sudden appearance of SDS foliar symptoms not only explains the origin of the disease name, but also reflects the difficulty of early detection in managing this disease. Yield reductions caused by SDS have been documented at 5% to15%, and the economic loss was estimated up to $669 million U.S. dollars in a single year (Navi and Yang, 2016). Seed treatments have been used to manage SDS, but performance differs by year and location. Alternatively, partially resistant soybean cultivars provide a sustainable option for SDS management, but the genetic architecture of SDS resistance is quantitative and complicated. Among more than 80 quantitative trait loci reported for SDS, only a few quantitative trait loci are reproducible due to the complexity of SDS etiology and environmental interactions (Chang et al., 2018)
Population genomic analysis reveals geographic structure and climatic diversification for Macrophomina phaseolina isolated from soybean and dry bean across the United States, Puerto Rico, and Colombia
Macrophomina phaseolina causes charcoal rot, which can significantly reduce yield and seed quality of soybean and dry bean resulting from primarily environmental stressors. Although charcoal rot has been recognized as a warm climate-driven disease of increasing concern under global climate change, knowledge regarding population genetics and climatic variables contributing to the genetic diversity of M. phaseolina is limited. This study conducted genome sequencing for 95Â M. phaseolina isolates from soybean and dry bean across the continental United States, Puerto Rico, and Colombia. Inference on the population structure using 76,981 single nucleotide polymorphisms (SNPs) revealed that the isolates exhibited a discrete genetic clustering at the continental level and a continuous genetic differentiation regionally. A majority of isolates from the United States (96%) grouped in a clade with a predominantly clonal genetic structure, while 88% of Puerto Rican and Colombian isolates from dry bean were assigned to a separate clade with higher genetic diversity. A redundancy analysis (RDA) was used to estimate the contributions of climate and spatial structure to genomic variation (11,421 unlinked SNPs). Climate significantly contributed to genomic variation at a continental level with temperature seasonality explaining the most variation while precipitation of warmest quarter explaining the most when spatial structure was accounted for. The loci significantly associated with multivariate climate were found closely to the genes related to fungal stress responses, including transmembrane transport, glycoside hydrolase activity and a heat-shock protein, which may mediate climatic adaptation for M. phaseolina. On the contrary, limited genome-wide differentiation among populations by hosts was observed. These findings highlight the importance of population genetics and identify candidate genes of M. phaseolina that can be used to elucidate the molecular mechanisms that underly climatic adaptation to the changing climate
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Investigation of Fungicide Resistance Mechanisms and Dynamics of the Multiple Fungicide Resistant Population in Sclerotinia homoeocarpa
A filamentous ascomycete fungus Sclerotinia homoeocarpa causes dollar spot, which is the most important disease of turfgrasses in the United States. Despite the increased number of reports of site-specific fungicide resistance and a recent report of multidrug resistance (MDR) in S. homoeocarpa field populations, the genetic mechanisms behind resistance or reduced sensitivity to fungicides remain poorly explained in the fungus. In order to prevent further development of fungicide resistance in the dollar spot pathosystem, a detailed elucidation of mechanisms of site-specific fungicide resistance and MDR is needed. In addition, the previous studies of MDR in fungi mostly focused on efflux transporter mediated drug/xenobiotic detoxification. However, the recent release of fungal genome sequences has revealed that ascomycete filamentous fungi including S. homoeocarpa possess a large number of cytochrome p450s (CYP450s) that are involved in xenobiotic metabolism. Chapters 2 and 3 of this dissertation describe demethylation inhibitor (DMI) fungicide/MDR mechanisms in S. homoeocarpa through Phase I xenobiotic metabolizing enzymes (CYP450s) and Phase III efflux transporters using functional genomic and genetic techniques. We identified a fungal specific transcription factor (Shxdr1) that regulates the Phase I and III genes and a novel gain of function mutation of the transcription factor found from a MDR field strain is responsible for constitutive and induced overexpression of the Phase I and III genes, resulting in MDR. In Chapter 4, the mechanisms of qualitative and quantitative resistance of a dicarboximide fungicide have been determined using field and lab mutant S. homoeocarpa strains. We confirmed that S. homoeocarpa field isolates gained qualitative and quantitative dicarboximide resistance through the polymorphism in the histidine kinase gene Shos1 and the overexpression of ABC efflux transporter ShPDR1, respectively. Chapter 5 studies the management of a S. homoeocarpa population with different combinations of resistance to dicarboximide, DMI, and benzimidazole fungicides to find the best fungicide options for controlling the S. homoeocarpa population and further understanding the dynamics of how the population responds to fungicide applications, and to long-term lack of exposure to fungicides during winter. Succinate dehydrogenase inhibitors (SDHI; fluxapyroxad and boscalid (high rate)), multi-site fungicide (fluazinam), and the fungicide mixture (chlorothalonil, iprodione, thiophanate methyl, and tebuconazole) controlled the S. homoeocarpa population very well. The isolates with resistance to DMI and dicarboximide were most frequently selected by iprodione or propiconazole applications and the isolates with resistance to DMI and benzimidazole were selected by boscalid applications, but these multiple fungicide resistant isolates decreased after overwintering
A Sclerotinia sclerotiorum Transcription Factor Involved in Sclerotial Development and Virulence on Pea
White mold, caused by Sclerotinia sclerotiorum, is a destructive disease on important legume species such as soybean, dry bean, and pea. This study investigated expression levels of transcription factors in S. sclerotiorumin planta (pea lines) and in vitro (culture medium). One transcription factor displaying high expression in planta was found to be involved in sclerotial development and virulence on pea. This report provides a new understanding regarding transcription factors of S. sclerotiorum in development and virulence.Sclerotinia sclerotiorum is a plant-pathogenic ascomycete fungus and infects over 400 host plants, including pea (Pisum sativum L.). The fungus causes white mold on pea, and substantial yield loss is attributed to the disease. To improve white mold management, further understanding of S. sclerotiorum pathogenicity is crucial. In this study, 389 transcription factors (TFs) were mined from the complete genome sequence of S. sclerotiorum and their in planta expression patterns were determined in susceptible and partially resistant pea lines and compared to in vitro expression patterns on culture medium. One of the transcription factors was significantly induced in planta at 24 and 48 h postinfection compared to the expression in vitro. This putative C6 transcription factor of S. sclerotiorum (SsC6TF1) was knocked down using a gene-silencing approach to investigate its functions in vegetative growth and sclerotial development as well as its virulence and pathogenicity in pea. While the SsC6TF1 knockdown mutants had hyphal growth rates identical to those of the wild-type strain and were capable of infection, the knockdown mutants produced no sclerotia or significantly fewer and smaller sclerotia on the culture medium and exhibited reduced virulence on both pea lines. This study profiled genome-wide expression for S. sclerotiorum transcription factors in planta and in vitro and functionally characterized a novel transcription factor, SsC6TF1, which positively regulates sclerotial development and virulence on pea. The finding provides molecular insights into S. sclerotiorum biology and interaction with pea and other economically important crops
First Report of Root Rot of Dendropanax trifidus Caused by Fusarium oxysporum in Korea
Dendropanax trifidus belonging to the family Araliaceae, is a warm-temperate evergreen tree distributed in Jeju Island, Bogil Island, Geomun Island, Geoje Island, Wando, and Haenam in Korea. In June 2021, a root rot disease in which branches of Dendropanax trifidus seedlings turned brown and shrunk was discovered at the seedling cultivation facility in Naju-si, Republic of Korea. To identify the root rot fungus, three strains were isolated from the diseased tissues of seedlings and their mycological characteristics were investigated on potato dextrose agar. In addition, a molecular phylogenetic analysis was performed using sequences of the internal transcribed spacer (ITS) region and translation elongation factor 1-α (EF1-α) gene. The fungus was identified as Fusarium oxysporum. For pathogenicity test, the roots of seedlings were immersed in the conidia suspension of the strains and planted. After 20 days inoculation, root rot and browning symptoms were confirmed in the inoculated plants. This is the first report of F. oxysporum on D. trifidus in Korea
Annotation Resource of Tandem Repeat-containing Secretory Proteins in Sixty Fungi
Annotation of fungal secretory proteins mainly relies on outward comparison methods such as BLAST or hidden Markov model. These approaches are reaching a bottle neck for annotating fungal effectors which rarely contain conserved motif. In desire of more sequence features for annotating and prioritizing fungal effectors of interest, this study develops a pipeline to identify tandem repeat (TR) within each sequence of putative secretory proteins and search if any type of TR in the non-orthologous secretory proteins remains evolutionarily conserved for plant pathogenicity. There were 2,804 types of TR units and a total of 2,925 TR-containing secretory proteins were found from 60 fungi. There was rare conserved type of TR shared by plant pathogenic fungi, indicating functional divergence for different types TR and TR-containing secretory proteins. The annotation resource of fungal TR-containing secretory proteins provides new sequence features that will be useful for the community interested in the fungal effector biology
Binding Affinities and Thermodynamics of Noncovalent Functionalization of Carbon Nanotubes with Surfactants
Binding affinity and thermodynamic
understanding between a surfactant
and carbon nanotube is essential to develop various carbon nanotube
applications. Flavin mononucleotide-wrapped carbon nanotubes showing
a large redshift in optical signature were utilized to determine the
binding affinity and related thermodynamic parameters of 12 different
nanotube chiralities upon exchange with other surfactants. Determined
from the midpoint of sigmoidal transition, the equilibrium constant
(<i>K</i>), which is inversely proportional to the binding
affinity of the initial surfactant-carbon nanotube, provided quantitative
binding strengths of surfactants as SDBS > SC ≈ FMN >
SDS,
irrespective of electronic types of SWNTs. Binding affinity of metallic
tubes is weaker than that of semiconducting tubes. The complex <i>K</i> patterns from semiconducting tubes show preference to
certain SWNT chiralities and surfactant-specific cooperativity according
to nanotube chirality. Controlling temperature was effective to modulate <i>K</i> values by 30% and enables us to probe thermodynamic parameters.
Equally signed enthalpy and entropy changes produce Gibbs energy changes
with a magnitude of a few kJ/mol. A greater negative Gibbs energy
upon exchange of surfactant produces an enhanced nanotube photoluminescence,
implying the importance of understanding thermodynamics for designing
nanotube separation and supramolecular assembly of surfactant
Binding Affinities and Thermodynamics of Noncovalent Functionalization of Carbon Nanotubes with Surfactants
Binding affinity and thermodynamic
understanding between a surfactant
and carbon nanotube is essential to develop various carbon nanotube
applications. Flavin mononucleotide-wrapped carbon nanotubes showing
a large redshift in optical signature were utilized to determine the
binding affinity and related thermodynamic parameters of 12 different
nanotube chiralities upon exchange with other surfactants. Determined
from the midpoint of sigmoidal transition, the equilibrium constant
(<i>K</i>), which is inversely proportional to the binding
affinity of the initial surfactant-carbon nanotube, provided quantitative
binding strengths of surfactants as SDBS > SC ≈ FMN >
SDS,
irrespective of electronic types of SWNTs. Binding affinity of metallic
tubes is weaker than that of semiconducting tubes. The complex <i>K</i> patterns from semiconducting tubes show preference to
certain SWNT chiralities and surfactant-specific cooperativity according
to nanotube chirality. Controlling temperature was effective to modulate <i>K</i> values by 30% and enables us to probe thermodynamic parameters.
Equally signed enthalpy and entropy changes produce Gibbs energy changes
with a magnitude of a few kJ/mol. A greater negative Gibbs energy
upon exchange of surfactant produces an enhanced nanotube photoluminescence,
implying the importance of understanding thermodynamics for designing
nanotube separation and supramolecular assembly of surfactant