Progress 10/01/16 to 09/30/20
Outputs
Target Audience:Target audiences are the researchers in academia, industries, government, and mass media. Specifically, we aimed to distribute and share the findings with fungal molecular geneticists and those who study mycotoxins especially aflatoxins. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two PhD students have been trained by this Hatch support. Dr. Ming-yueh Wu received her PhD in Genetics in 2017, and she has been working as a group leader at Ginkgo BioWorks, Boston, MA. Mr. Heungyun Moon, a fifth-year PhD student in Plant Pathology PhD program has been working on the project since 2017. He successfully completed preliminary examination with proposing this project and became a dissertator in 2019. In 2020, he won the Schreiber Foods Graduate Student Scholarship and the William H. Peterson Fellowship Award that acknowledged outstanding academic and research achievements of this project. As this project required diverse knowledge and experimental techniques in many fields of studies such as genetics, genomics, biochemistry, and bioinformatics, he has been able to be trained with different study approaches robustly and this process is forming him into an independent researcher. How have the results been disseminated to communities of interest?Results generated from this work have been and will be published in various scientific journals and presented at national, international, and regional meetings of relevant associations, i.e., The Genetics Society of America, Gordon Research Conference, the Food Research Institute (FRI) annual meetings, and Fungal Supergroup meeting at the UW-Madison. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
In this project, we aimed to uncover the mechanisms controlling both aflatoxin production and sporulation focusing on the two key regulators WetA and NsdD in the major aflatoxin-producing fungus Aspergillus flavus with a long-term goal of controlling both fungal infestation and aflatoxin contamination in foods and feeds. To accomplish the goals, we have employed an innovative combination of genetic, biochemical, and genomic approaches. Asexual development (conidiation) and sporogenesis occur via the genetically programmed cascade of some essential genes in the filamentous fungi Aspergillus nidulans and A. flavus. For instance, brlA, abaA, and wetA are key regulators of conidiation: the activation of brlA initiates the development of conidiophore while abaA and wetA act during the middle and late stages of conidiation, respectively. To establish proper asexual development, brlA expression must be preceded by the timely release of repressors of conidiation, NsdD and VosA, when vegetative cells become developmentally competent. NsdD is a GATA-type transcription factor with a highly conserved DNA-binding domain among most Aspergillus species. Although the DNA-binding motif of NsdD is well conserved in Aspergilli, NsdD-mediated regulatory gene networks possibly differ in a species-specific manner as they have evolved separately after speciation from a common ancestor. The nsdD deletion mutants across species display common features such as hyper-conidiation and no sexual stage. In contrast, they exhibit distinct characteristics: different morphology of conidiophores and spores as well as mycotoxin production. We carried out a series of genome-wide studies on A. nidulans and A. flavus nsdD to understand the global regulatory differences. To dissect stage-dependent effects of NsdD, we selected three different life stages of wild-type and nsdD null mutants for gene expression profiling: vegetative cell (Veg), asexually developing cell (Asex), and conidia. In A. nidulans, 23%, 43%, and 10% of the total 10,988 genes are significantly up- or down-regulated by NsdD in Veg, Asex, and conidia, respectively. In A. flavus, 3.3%, 9%, and 15% of the total 13,485 genes are controlled by NsdD in Veg, Asex, and conidia, respectively. Depending on the cell types, the number of differentially expressed genes (DEGs) show drastic variations within species. Interestingly, only ~19% and ~10% of total DEGs are shared across cell types in A. nidulans and A. flavus. Further analysis on DEGs revealed that ~70% of total DEGs are core genes which are commonly found in both species and ~30% of them are lineage-specific genes in A. nidulans. In case of A. flavus, core genes form ~45% of total DEGs and ~55% of them are A. flavus-specific genes. These findings suggest that NsdD tends to regulate genes in a cell type-dependent manner and A. flavus NsdD has evolved to control more lineage-specific genes than A. nidulans' one. By combining all data from the project, our results demonstrate that NsdD has diverged and different NsdD-mediated gene regulatory networks are built up in these two Aspergillus species resulting in discrete cellular and chemical development outcomes.
Publications
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Progress 10/01/18 to 09/30/19
Outputs
Target Audience:Target audiences are general public and relevant scientists in industry, government sectors, and academia. Results generated from this work have been published in high-profile journals and presented at national, international and regional meetings of relevant associations, i.e., The Genetics Society of America, Gordon Research Conference, and the Food Research Institute (FRI) annual meetings. The PI has been interacting with industry R&D staffs, government regulators, academic researchers, and consumers on food safety issues and provided accurate, useful information and expertise through FRI annual meetings and Newsletters. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Heungyun Moon, a fourth-year PhD student in Plant Pathology program has been working on the project since 2017. He successfully completed preliminary examination with proposing this project and became a dissertator. As this project conducts genetics, genomics, biochemistry, and bioinformatics research, he has been able to be robustly trained with different study approaches. How have the results been disseminated to communities of interest?Results generated from this work have been and will be presented at national, international and regional meetings of relevant associations, i.e., The Genetics Society of America, Gordon Research Conference, and the Food Research Institute (FRI) annual meetings. The work will be submitted for publication in high-profile scientific journals. What do you plan to do during the next reporting period to accomplish the goals?To further understand roles of WetA and NsdD in development and mycotoxin production of A. flavus, we will continue to conduct a combination of genetic, biochemical and genomic researches. Specifically, we will identify direct targets and gene regulatory networks (GRNs) of WetA and NsdD in A. flavus. RNA-seq and ChIP-seq data will be integrated to generate GRNs of WetA and NsdD.
Impacts
What was accomplished under these goals?
In this project, we aimed to uncover the mechanisms controlling both aflatoxin production and sporulation focusing on the WetA and NsdD regulatory factor in the major aflatoxigenic fungus A. flavus with a long-term goal of controlling both fungal infestation and aflatoxin contamination in foods and feeds. To accomplish the goals, we have employed an innovative combination of genetic, biochemical and genomic approaches. Asexual development (conidiation) and sporogenesis occur via the genetically programmed cascade of some essential genes in the filamentous fungi Aspergillus nidulans and A. flavus. For instance, brlA, abaA, and wetA are key regulators of conidiation: the activation of brlA initiates the development of conidiophore while abaA and wetA act during the middle and late stages of conidiation, respectively. To establish proper asexual development, brlA expression must be preceded by the timely release of repressors of conidiation, NsdD and VosA, when vegetative cells become developmentally competent. NsdD is a GATA-type transcription factor with a highly conserved DNA-binding domain among most Aspergillus species. Although the DNA-binding motif of NsdD is well conserved in Aspergilli, NsdD-mediated regulatory gene networks possibly differ in a species-specific manner as they have evolved separately after speciation from a common ancestor. The nsdD deletion mutants across species display common features such as hyper-conidiation and no sexual stage. In contrast, they exhibit distinct characteristics: different morphology of conidiophores and spores as well as mycotoxin production. Of note, the role of NsdD in governing mycotoxin production is just opposite between two aspergillus species. In A. nidulans, NsdD plays an repressive role in production of sterigmatocystin. On the other hand, A. flavus NsdD activates the biosynthesis of aflatoxin even though sterigmatocystin and aflatoxin biosynthetic geneclusters share most of genes. This suggests that mycotoxin-regulatory networks are diffrently formed in these species.We performed genome-wide studies on A. nidulans and A. flavus nsdD to understand transcriptomic differences. To dissect stage-dependent effects of NsdD, we selected three different life stages of wild-type and nsdD null mutants for gene expression profiling: vegetative cell (Veg), asexually developing cell (Asex), and conidia. In A. nidulans, 23%, 43%, and 17% of total 10,988 genes are significantly up- or down-regulated by NsdD in Veg, Asex, and conidia, respectively. In A. flavus, 3.3%, 9%, and 7% of total 13,485 genes are controlled by NsdD in Veg, Asex, and conidia, respectively. Depending on the life stages, the number of differentially expressed genes (DEGs) show drastic variations within species. Interestingly, only 18.6% and 7% of total DEGs are shared across stages in A. nidulans and A. flavus. In addition, there is a significant difference between two species in terms of DEGs indicating the broad influence of NsdD. Our results demonstrate that NsdD-mediated gene regulatory networks and the function of NsdD have diverged in these two Aspergillus species and shed light on howgene regulatory networks in microorganisms control biological processes.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2019
Citation:
HS Park, MK Lee, KH Han, MJ Kim, JH Yu. 2019 Developmental Decisions in Aspergillus nidulans. Biology of the Fungal Cell. The Mycota (A Comprehensive Treatise on Fungi as Experimental Systems for Basic and Applied Research), vol 8. Springer, Cham.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
MJ Kim, WH Jung, YE Son, JH Yu, MK Lee, HS Park. 2019 The velvet repressed vidA gene plays a key role in governing development in Aspergillus nidulans. J Microbiol. 57: 893. 10.1007/s12275-019-9214-4.
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Progress 10/01/17 to 09/30/18
Outputs
Target Audience:Target audiences are general public and relevant scientists in industry, government sectors, and academia. Results generated from this work have been published in high-profile journals and presented at national, international and regional meetings of relevant associations, i.e., The Genetics Society of America, Gordon Research Conference, and the Food Research Institute (FRI) annual meetings. The PI has been interacting with industry R&D staffs, government regulators, academic researchers, and consumers on food safety issues and provided accurate, useful information and expertise through FRI annual meetings and Newsletters. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Through this project, a former Genetics PhD student, Ming-yuehWu, obtained her PhDat the University of Wisconsin-Madison. Dr. Wu is now working as a scientist in a Biotech company. Dr. Wu worked with Drs. Mead and Rokas at Vanderbilt University, and completed the publicationof her PhD dissertation work in a high-profile journal mBio. How have the results been disseminated to communities of interest?The work has been published in one of the best Microbiology journal, mBio. Dueto the importance of the work, the paper entitled "Systematic Dissection of the Evolutionarily Conserved WetA Developmental Regulator across a Genus of Filamentous Fungi" was recommended by F-1000 Prime. The results were also presented at several international meetings- (ex.Cellular and Molecular Fungal Biology meeting). What do you plan to do during the next reporting period to accomplish the goals?We will continue to test the central hypothesis a combination of genetic, biochemical and genomic approaches. Specifically, we will identify direct targets and consensus binding sequence(s) of WetA and NsdD in A. flavus. We will also reveal genome-wide expression changes resulting from the absence of WetA or NsdD action.
Impacts
What was accomplished under these goals?
In this project, we aimed to uncover the mechanisms controlling both aflatoxin production and sporulation focusing on the novel WetA regulatory factorin the major aflatoxigenic fungus A. flavus with a long-term goal of controlling both fungal infestation and aflatoxin contamination in foods and feeds. To accomplish the goals, we have employed an innovative combination of genetic, biochemical and genomic approaches. Asexual sporulation is fundamental to the ecology and lifestyle of filamentous fungi and can facilitate both plant and human infection. InAspergillus, the production of asexual spores is primarily governed by the BrlA→AbaA→WetA regulatory cascade. The final step in this cascade is controlled by the WetA protein and governs not only the morphological differentiation of spores but also the production and deposition of diverse metabolites into spores. Previously, we reported that WetA is a multi-functional regulator that couples spore differentiation and survival, and governs proper chemical development in the aflatoxigenic fungusAspergillusflavus. The deletion ofwetAresults in the formation of conidia with defective cell walls and no intra-cellular trehalose, leading to reduced stress tolerance, a rapid loss of viability, and disintegration of spores.WetAis also required for normal vegetative growth, hyphal branching, and production of aflatoxins.While WetA is conserved across the genusAspergillus, the structure and degree of conservation of thewetAgene regulatory network (GRN) remain largely unknown. We carried out comparative transcriptome analyses of comparisons betweenwetAnull mutant and wild-type asexual spores in three representative species spanning the diversity of the genusAspergillus:A.nidulans,A.flavus, andA.fumigatus. We discovered that WetA regulates asexual sporulation in all three species via a negative-feedback loop that represses BrlA, the cascade's first step. Furthermore, data from chromatin immunoprecipitation sequencing (ChIP-seq) experiments inA.nidulansasexual spores suggest that WetA is a DNA-binding protein that interacts with a novel regulatory motif. Several global regulators known to bridge spore production and the production of secondary metabolites show species-specific regulatory patterns in our data. These results suggest that the BrlA→AbaA→WetA cascade's regulatory role in cellular and chemical asexual spore development is functionally conserved but that thewetA-associated GRN has diverged duringAspergillusevolution. IMPORTANCE: The formation of resilient spores is a key factor contributing to the survival and fitness of many microorganisms, including fungi. In the fungal genusAspergillus, spore formation is controlled by a complex gene regulatory network that also impacts a variety of other processes, including secondary metabolism. To gain mechanistic insights into how fungal spore formation is controlled acrossAspergillus, we dissected the gene regulatory network downstream of a major regulator of spore maturation (WetA) in three species that span the diversity of the genus: the genetic modelA.nidulans, the human pathogenA.fumigatus, and the aflatoxin producerA.flavus. Our data show that WetA regulates asexual sporulation in all three species via a negative-feedback loop and likely binds a novel regulatory element that we term the WetA response element (WRE). These results shed light on how gene regulatory networks in microorganisms control important biological processes and evolve across diverse species.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Wu, MY., Mead, ME, Lee, MK., Kim, SC., Rokas, A., Yu, J-H. 2018 Systematic dissection of the evolutionarily conserved WetA developmental regulator across a genus of lilamentous fungi. mBio 9: 4 e01130-18.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Eom, TJ, Moon, H, Yu, JH, Park HS. 2018. Characterization of the velvet regulators in Aspergillus flavus. J. Microbiol. 56:893-901 10.1007/s12275-018-8417-4 (underlined co-corresponding authors).
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Progress 10/01/16 to 09/30/17
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Ming-yueh Wu, a sixth-year PhD student in Genetics programhas been working on the project since 2012. Heungyun Moon, a first-year PhD student in Pathology program has joined this project since September, 2017. This projectconducts genetics, genomics, biochemistry, and bioinformatics research, and therefore provides a great education opportunity to training a next generation scientist. This project demonstrates outreach science education. The different phenotypes of A. flavus mutants which are created in this project will be an attractive target for nearly all age groups and delight their interestsin science. Students will be exposed to the beauty of genetics and the most up-to-date research concepts. How have the results been disseminated to communities of interest?Results generated from this work have been and will be presented at national, international and regional meetings of relevant associations, i.e., The Genetics Society of America, Gordon Research Conference, and the Food Research Institute (FRI) annual meetings. The work will be submitted for publication in high-profile scientific journals. What do you plan to do during the next reporting period to accomplish the goals?To further understand molecular functions of WetA in A. flavus, we will carry out more integrated genetic, genomic and physiological studies. We are planning to do ChIP-seq analyses of WT and the WetA mutants in A. flavus. The RNA-seq data will be analyzed to find more mycotocin- and conidiation-regulatory genesfor further study.
Impacts
What was accomplished under these goals?
Filamentous fungi have been used by humans for benefits, their metabolites and enzymes, including antibiotics, organicacids, pigments, and food additives. However, some are serious pathogens, which result in the agricultural loss, environmentaldamage, and adverse health effects on humans and animals. Because of the importance of filamentous fungi in human dailylife, molecular tools have been developed to enable scientists to understand these microorganisms. The main reproductivemode of filamentous fungi is the formation of asexual spores. In some cases, the fungal secondary metabolites are highlyrelated to development. Aspergillus flavus, an opportunistic pathogen of plant and human, produces numerous secondarymetabolites, including the most notorious aflatoxin. Among mycotoxins, aflatoxin B1 is one of the most potent carcinogensand can contaminate oil-seed crops, such as corn, cereals, sorghum, and peanuts. Due to the carcinogenicity and toxicity,aflatoxins have been regulated by the USFDA since 1965. In 2003, mycotoxins, including aflatoxin, were estimated to cause acrop loss of $932 million per year in the United States. The cost of aflatoxin regulation and testing averages $466 million peryear. Besides of economic loss, aflatoxins are also a threat to life. Acute aflatoxicosis, associated with extremely high dosesof aflatoxin, can lead to death in humans.Therefore, controlling both fungal dissemination and aflatoxin production isvery important. Bridging cellular reproduction and survival is essential for all life forms.Aspergillusfungi primarily reproduce by forming asexual spores called conidia, whose formation and maturation is governed by the central genetic regulatory circuit BrlA→AbaA→WetA. Here, we report that WetA is a multi-functional regulator that couples spore differentiation and survival, and governs proper chemical development inAspergillus flavus. The deletion ofwetAresults in the formation of conidia with defective cell walls and no intra-cellular trehalose, leading to reduced stress tolerance, a rapid loss of viability, and disintegration of spores. WetA is also required for normal vegetative growth, hyphal branching, and production of aflatoxins. Targeted and genome-wide expression analyses reveal that WetA exerts feedback control ofbrlAand that 5,700 genes show altered mRNA levels in the mutant conidia. Functional category analyses of differentially expressed genes in ΔwetARNA-seq data indicate that WetA contributes to spore integrity and maturity by properly regulating the metabolic pathways of trehalose, chitin, α-(1,3)-glucan, β-(1,3)-glucan, melanin, hydrophobins, and secondary metabolism more generally. Moreover, 160 genes predicted to encode transcription factors are differentially expressed by the absence ofwetA, suggesting that WetA may play a global regulatory role in conidial development. Collectively, we present a comprehensive model for developmental control that bridges spore differentiation and survival inA.flavus. Moreover, we have generated the WetA-specific polyclonal antibody for further WetA protein qualification and quantification. We used the A. nidulans as the model system and carried out ChIP-seq to identify the WetA binding motif. The result shows that AniWetA is a DNA-binding protein. Based on the high similarity of the AniWetA and AflWetA, we proposed that AflWetA also directly binds to its target DNA sequence in A. flavus genome. The A. flavus WetA ChIP-seq is in progress now.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Wu, M. Y., Mead, M. E., Kim, S. C., Rokas, A., & Yu, J. H. (2017). WetA bridges cellular and chemical development in Aspergillus flavus. PloS one, 12(6), e0179571.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
M-Y Wu, M E Mead, S-C Kim, A Rokas, and J-H Yu (2017) WetA is a DNA-binding protein governing cellular and chemical development. 14th International Aspergillus Meeting. Pacific Grove, CA
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
M-Y Wu and J-H Yu (2016) The Global Regulator WetA Governs Fungal Development and Secondary Metabolism. 2016 American Society for Microbiology General Meeting, Boston, MA
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