COMPUTATIONAL AND FUNCTIONAL CHARACTERIZATION OF FUSARIUM VERTICILLIOIDES TRANSCRIPTION FACTORS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1012104
• Project Start Date: Feb 21, 2017
• Project End Date: Jan 9, 2022
• Program Code: [(N/A)]- (N/A)

Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001

Performing Department
Plant Pathology & Microbiology

Non Technical Summary
Fusarium verticillioides causes devastating disease of maize, including ear rots and stalk rots. More importantly the pathogen produces fumonisin mycotoxins that accumulate in maize kernels and threaten food safety and human health. The goal of this project is to systematically characterize F. verticillioides transcriptomes to computationally predict transcription factors and signaling pathways that are critical for pathogenesis-related processes, including growth, morphological differentiation, and mycotoxin biosynthesis. A number of fungal pathogens of major human crops have their genomes sequenced, but we are still far from understanding the programming of gene expression. In F. verticillioides and many other plant pathogenic fungi, little is known about the how correlated gene expressions lead to functional outcomes, namely pathogenicity and mycotoxin production. Expansive resources for functional genomics are available in F. verticillioides, including a sequenced genome and transcriptomes, along with techniques for highthroughput gene disruption, making the fungus ideal for computational analyses. In this project, we will explore network-based comparative analysis through probabilistic subnetwork inference using RNA-seq transcriptome data to identify potential F. verticillioides pathogenicity-associated subnetwork modules. Our propject goal is to identify putative hub TF genes, which are critical for regulating the subnetworks associated with pathogenicity.We will then focus on functionally characterizing these TF genes, which will be disrupted via homologous recombination. Phenotypic abnormalities in growth, pathogenesis, and metabolism will be documented for each mutant. All mutants and data generated during this project will be made publicly available to the scientific community.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	1510	1160	100%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1510 - Corn;

Field Of Science
1160 - Pathology;

Keywords

fusarium verticillioides maize interaction

maize ear rot

network biology

subnetwork modules

transcription factors

Goals / Objectives
Fusarium verticillioides causes stalk and ear rots of maize, and produces fumonisin mycotoxins that accumulate in kernels and threaten food safety. The goal of this project is to systematically characterize F. verticillioides transcription factors (TFs) by determining their involvement in pathogenesis-related processes, including growth, morphological differentiation, and secondary metabolism/mycotoxin biosynthesis. Throughout the fungal kingdom, TFs are key intermediaries in reprogramming gene expression. However, in F. verticillioides and many other plant pathogenic fungi, little is known about the function of most TFs. Expansive resources for functional genomics are available in F. verticillioides, including a sequenced genome and techniques for highthroughput gene disruption, making the fungus ideal for genome-wide investigations into TF function.To accomplish the project goal, we are spearheading RNA-Seq experiments and advanced data analytic strategies to gain a better understanding of downstream genes (or pathways) of these TFs in F. verticillioides. We will construct the F. verticillioides co-expression networks from RNA-Seq data and search through these networks to identify subnetwork modules that are differentially activated between the wild type and TF mutant F. verticillioides, which considerably differ in terms of pathogenic potentials. A greedy seed-and-extend approach will be utilized and we anticipate identifying potential pathogenicity-associated subnetwork modules, each of which consists of interacting genes with coordinated expression patterns. When completed, we anticipate identifying key regulators downstream of key TFs that play important roles in plant pathogenesis in F. verticillioides. Key identified genes will be disrupted via homologous recombination. Phenotypic abnormalities in growth, pathogenesis, and metabolism will be documented for each mutant. All mutants and data generated during this project will be made publicly available to the scientific community.

Project Methods
We will build co-expression networks based on the transcriptomic data. In a co-expression network, the molecules of interest (e.g., genes) are represented as nodes, and the edges are used to indicate the similarity between the expression patterns of the connected molecules. Two different types of measures, correlation and mutual information, will be used to estimate expression similarity between molecules. The planned network analysis can be broadly divided into the following research activities: (i) identification of functional modules; (ii) prediction of regulatory mechanisms; (iii) analysis of F. verticillioides-maize interactions at different time points. The planned experiment will analyze immense transcriptomic data from F. verticillioides - maize interactions. The structure of the resulting network reflects the overall gene expression patterns across various stages of pathogenesis. We will identify the functional modules embedded in this stationary co-expression network using latest module detection. This will unveil novel components and pathways in the F. verticillioides-maize interaction.The predicted functional modules will be further investigated to elucidate the regulatory mechanisms within and across the modules. An information-theoretic concept called "synergy" will be used to identify synergistically interacting genes (within a given module) that contribute to the progression of the infection-resistance process. The molecular logic governing the behavior of these genes will be inferred subsequently. Regulatory relationships among the modules can be predicted in a similar manner after we estimate their activity levels using the probabilistic pathway activity inference method. This inference method is highly effective in predicting the pathway activity based on the individual activity of the member genes.Analysis of the time-course transcriptomic data will enable us to find modules that are activated at different stages of fungal pathogenesis. Through this analysis, we can predict the genetic interactions that are active (or inactive) at different stages of F. verticillioides pathogenesis. Furthermore, we will compare the co-expression networks to identify differences in molecular interaction patterns and dynamics, and ultimately unveil hidden mechanisms that lead to critical differences in pathogenicity.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience: During the project period our research team disseminated our outcomes to our national and international peers in plant pathology, plant science, and fungal genetics research area through peer-reviewed publications. Unfortunatelt,this yearwe were not able to participate in any national and international conferences due to the disruptions caused by theCOVID-19 pandemic. Changes/Problems:The COVID-19 pandemic has caused critical disruptions in research and teaching throughout 2020. Fortunately, we were able to conduct in silico research along with limited in-lab experiments while focusing on preparingscientific publications to overcome these disruptions. What opportunities for training and professional development has the project provided? The project provided research and training opportunities for a Ph.D. graduate student. How have the results been disseminated to communities of interest? Project participants presented their research outcomes through three peer-reviewed journal articles. What do you plan to do during the next reporting period to accomplish the goals?We will continue to investigate regulatory mechanisms associated with fumonisin biosynthesis and plant pathogenesis in Fusarium verticillioides. One of the research topics we are spearheading is how cellular trafficking impacts mycotoxin and pathogenesis, particularly microtubule-assisted mechanisms. We are also interested in the role peroxisome biogenesis proteins in mycotoxin regulation.

Impacts
What was accomplished under these goals? We were able to complete our research on characterizingFusarium verticillioides FvSYN1gene, which was demonstrated to play an important role in maize stalk rot virulence. We investigated the cellular features of the FvSyn1 protein, namely on how different motifs contribute to development and virulence inF. verticillioidesby generating motif-specific deletion mutants.Characterization of the FvSyn1 domains indicated that both the syntaxin N-terminus (SynN) domain and the SNARE C-terminus domain play distinct roles in fungal development, but also function collectively in the context of virulence.We also determined that two domains in FvSyn1 are not required for fumonisin production.In addition, as proposed last year, wefurther investigated the role of G proteins and Regulators of G proteins Fusarium mycotoxin regulation and plant pathogenesis.Rab GTPases are responsible for a variety of membrane trafficking and vesicular transportation in fungi. These Small GTPases in fungi, particularly those homologous toSaccharomyces cerevisiaeSec4, are known to be associated with protein secretion, vesicular trafficking, secondary metabolism and pathogenicity. We investigated the molecular functions of FvSec4 inF. verticillioidesassociated with physiology and virulence. Our data indicate that FvSec4 is critical inF. verticillioideshyphal development, virulence, mycotoxin production and stress responses.Our recent effort was to investigate the functional role of a G beta-like protein FvGbb2, a putative receptor for activated C kinase, in maize ear rot pathogen Fusarium verticillioides.The gene deletion mutant exhibited severe defects not only in fumonisin biosynthesis but also vegetative growth and conidiation. FvGbb2 was positively associated with carbon source utilization and stress agents but negatively regulated general amino acid control. While FvGbb2 does not interact with canonical G protein subunits, it may associate with diverse proteins in the cytoplasm to regulate vegetative growth, virulence, fumonisin biosynthesis and stress response in F. verticillioides.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Huan Zhang, Huijuan Yan, Won Bo Shim. 2019. Fusarium verticillioides SNARE protein FvSyn1 harbors two key functional motifs that play selective roles in fungal development and virulence. Microbiology 165: 1075-1085.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Huijuan Yan, Jun Huang, Huan Zhang, Won Bo Shim. 2020. A Rab GTPase protein FvSec4 is necessary for fumonisin B1 biosynthesis and virulence in Fusarium verticillioides. Current Genetics 66: 205-216
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Huijuan Yan and Won Bo Shim. 2020. Characterization of non-canonical G beta-like protein FvGbb2 and its relationship with heterotrimeric G proteins in Fusarium verticillioides. Environmental Microbiology 22: 615-628

Progress 10/01/18 to 09/30/19

Outputs
Target Audience: During the project period our research team disseminated our outcomes to our national and international peers in plant pathology, plant science, and fungal genetics research area through peer-reviewed publications, conference abstracts and presentations. For international dissemination, in addition to publications I presentedscientific discoveries through invited conference presentations and seminars. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The project provided research and training opportunities for a Ph.D. graduate student and an undergraduate research intern. How have the results been disseminated to communities of interest? Project participants presented their research outcomes through peer-reviewed journals and at international meetings during this reporting period. We gave two oral presentations as well as one poster presentation. What do you plan to do during the next reporting period to accomplish the goals?We will be furtherinvestigating the role of heterotrimeric G proteins and Regulators of G proteins Fusarium mycotoxin regulation and plant pathogenesis. Our recent effort is to investigate the functional role of aG beta-like protein FvGbb2, a putative receptor for activated C kinase,in maize ear rot pathogenFusarium verticillioides. Through a series of mutagenesis and physiological experiments, we will demonstratehowthis non-canonicalG beta-likeprotein can regulate various important physiological functions in this fungal pathogen. We will also investigatewhether FvGbb2 directly interacts with canonical heterotrimeric G proteins.

Impacts
What was accomplished under these goals? In an effort to further investigate the role of a striatin-like protein Fsr1 that plays an important role in stalk rot, we characterized transcriptome networks downstream of Fsr1. We performed next-generation sequencing (NGS) to investigate relative read abundance and also to infer co-expression networks utilizing the preprocessed expression data through partial correlation. We used a probabilistic pathway activity inference strategy to identify functional subnetwork modules likely involved in virulence. Each subnetwork modules consisted of multiple correlated genes with coordinated expression patterns, but the collective activation levels were significantly different in F. verticillioides wild type versus fsr1 mutant. We also identified putative hub genes from predicted subnetworks for functional validation and network robustness studies through mutagenesis, virulence and qPCR assays. Our results suggested that these genes are important virulence genes that regulate the expression of closely correlated genes, demonstrating that these are important hubs of their respective subnetworks. In addition to this research activities, our team spearheaded how game theory can be applied to improve our understanding of host-pathogen association in phytobiomes. In this effort, we described theoretical comparison between original game theory and plant-pathogen application, and provided examples of how this concept can be applied to phytobiomes research. Our proposed strategy may not completely explain underlying principles of association between host and pathogens, but it can lead to predictive interpretation of "games" played between multiple organisms in plant microbiome and agroecosystem. In our publication, we proposed applying game theory to explore transcriptomic dynamics in host-pathogen associations and plant microbiome.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Man S. Kim, Huan Zhang, Huijuan Yan, Byung-Jun Yoon, and Won-Bo Shim. 2018. Characterizing co-expression networks underpinning maize stalk rot virulence in Fusarium verticillioides through functional subnetwork module analyses. Scientific Reports 8:8310.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Man S. Kim, Huan Zhang, and Won-Bo Shim. 2018. Application of game theory to explore the dynamics of host-pathogen association in phytobiomes. Phytobiomes 2: 111-116.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Yingzi Yun, Xin Zhou, Shuai Yang, Ya Wen, Haixia You, Yuru Zheng, Justice Norvienyeku, Won Bo Shim and Zhonghua Wang. 2019. Fusarium oxysporum f. sp. lycopersici C2H2 transcription factor FolCzf1 is required for conidiation, fusaric acid production, and early host infection. Current Genetics 65: 773-783.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Huijuan Yan, and Won-Bo Shim. 2019. G beta-like protein FvGbb2, a putative receptor for activated C kinase, plays important roles in Fusarium verticillioides hyphal development and secondary metabolism. Fusarium workshop at 30th Fungal Genetics Conference, Asilomar, CA
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Shim, W. B. 2019. Spearheading mycotoxin prevention in food and feed through genomics and big data analytics. 3rd International Conference of Mycotoxicology and Food Security. Bangkok, Thailand

Progress 10/01/17 to 09/30/18

Outputs
Target Audience: During the project period our research team disseminated our outcomes to our national and international peers in plant pathology, plant science, and fungal genetics research area through peer-reviewed publications, conference abstracts and presentations. For international dissemination, in addition to publications I presented and share scientific discoveries through invited conference presentations and seminars. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The project provided research and training opportunities for a Ph.D. graduate student and a postdoc. How have the results been disseminated to communities of interest? Project participants presented their research outcomes through peer-reviewed jounrals andatinternational meetings during this reporting period. We gave oral presentations as well as posters. What do you plan to do during the next reporting period to accomplish the goals?We are continuing our effort to characterize histidine kinase gene subnetworksthat are transcriptionally active during pathogenesis. In addition, we are currently targeting G-protein-mediated gene subnetworks in F. verticillioides to gain deeper understanding of how they regulate F. verticillioides virulence.G protein-coupled receptors (GPCRs) are 7 transmembrane (TM) receptors activate the heterotrimeric G protein complex, consisting of G alpha, G beta and G gamma, to transduce signals from external environmental stimuli. Our previous study characterized the function of the G beta protein Gbb1 inFusarium verticilloides. However, recent studies suggested that a G beta-like protein are found in filamentous fungi, but role of these proteins in G protein-mediated signaling remains largely unknown.We identified FvGbb2, a G beta - like protein inF. verticilloides, and generated a null mutant to investigate its role inF. verticilllioides.TheFvgbb2mutant will be tested for its impact on vegetative growth and conidiation. Moreover, we will test whether FvGbb2 is required for proper expression of key conidia regulation genes, includingFvBrlA,FvWetA,FvAbaA, andFvStuA. In addition, we will test whetherFvgbb2mutation impacts fumonisin B1 production in synthetic and maize cultures along with testing expression of key biosynthesis genes, such asFUM1andFUM8. We are currently investigating the relationship between FvGbb2 and heterotrimeric G protein components, especially with G beta protein FvGbb1, through cellular localization an protein-protein interaction studies.?

Impacts
What was accomplished under these goals? We continued our investigation onourlarge-scaleF. verticillioidesRNA-seq datasets by a systematic network-based computational analyses to identify potential genetic modules associated with virulence and fumonisin regulation. We identified a putative hub gene (FRN1), encoding a putative GPCR with 7 transmembrane regions, in a subnetwork module exhibiting a strong discriminative power. We hypothesized thatFRN1operates collectively with other linked genes in subnetwork to regulate fumonisin biosynthesis and pathogenesis. Knockout mutantfrn1showed little effect on morphology and fertility but have significant defects on fumonisin B1 production. Notably,FRN1demonstrated polymorphism in different Texas field isolates. Protein alignment revealed significant similarity withinFusariumspecies, while predominantly absent in other fungal species.We arealso studying the function of 5 histidine kinase (HK) genes in F. verticillioides. Gene knockout mutants are generated by split marker method we use in the lab. To elucidate the impact of the knockout mutants in growth and development, we will perform standard growth assays on various synthetic media. In addition, 5 HKs mutants and WT will be tested on different stress inducing ingredients amended to media. These media will contain NaCl (salt stress), Congo Red (cell stress), sorbitol (osmotic stress), NaNO2 (hypoxia mimicking), and CoCl2 (hypoxia stress), CuSO4 (ion stress), and H2O2 (reactive oxygen species) with various concentrations. Radial growth will be measured. A statistical quantification and analysis will also be followed.?

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Angelyn Hilton, Huanming Zhang, Wenying Yu, and Won Bo Shim. 2017. Identification and characterization of pathogenic and endophytic fungal species associated with Pokkah Boeng disease of sugarcane. The Plant Pathology Journal 33 (3): 238-248.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Won Bo Shim. 2018. Discovery of functional gene subnetwork modules associated with Fusarium verticillioides virulence and fumonisin regulation. Proceedings of the 2nd Mycokey International Conference. Wuhan. People's Republic of China (P18-19).
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Mansuck Kim, Huan Zhang, Charles Woloshuk, Won-Bo Shim and Byung-Jun Yoon. 2018. Computational Approach for Identifying Pathogenic Modules of Fusarium verticillioides. IEEE/ACM Transactions on Computational Biology and Bioinformatics 15 (2): 506-515.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Huan Zhang, Mala Mukherjee, Jung-Eun Kim, Wenying Yu, and Won Bo Shim. 2018. Fsr1, a striatin homolog, forms an endomembrane-associated complex that regulates virulence in the maize pathogen Fusarium verticillioides. Molecular Plant Pathology 19 (4); 812  826.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Huijuan Yan, Man Kim, and Won-Bo Shim. 2018. FRN1, a gene encoding putative GPCR protein, is critical for ear rot and fumonisin biosynthesis in maize pathogen Fusarium verticillioides. 2018 Corn Utilization and Technology Conference. St. Louis, MO.

Progress 02/21/17 to 09/30/17

Outputs
Target Audience:During the project period our research teamdisseminated ouroutcomes to our national and international peers in plant pathologyand fungal genetics research area through peer-reviewed publications, conference abstracts and presentations. For international dissemination, in addition to publications I presented and share scientific discoveries through invited seminars. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provided research and training opportunities for a Ph.D. graduate student and a postdoc. How have the results been disseminated to communities of interest?Project participants presented their research outcomes at an international meeting during this reporting period. They gave oral presentations as well as posters. What do you plan to do during the next reporting period to accomplish the goals?While we pursue functional, biological experiments associated with fungal genes in secondary metabolism, we also want to study F. verticillioides RNA-Seq datasets and computationally identify histidine kinase (HK)-related functional subnetwork modules that are transcriptionally active during pathogenesis. HK are important group of two-component signaling system in eukaryotes and have been linked to the regulation of mycotoxin biosynthesis in fungi.We will apply our computational modeling to identify potential functional subnetwork modules associated with F. verticillioides HK activities that are significantly differentially activated during pathogenesis and mycotoxin biosynthesis. We have identified 17 genes that encodes HK in F. vrticillioides genome. We will perform an updated robust systematic network-based comparative analysis approach with our RNA-Seq data. Specifically, we will focus on improving our previous analysis by first collecting and combining candidate subnetwork modules through adaptive branching-out technique and performing post-pruning process to eliminate relatively insignificant components of the modules. Through our systematic analysis approach, we will identify potential subnetwork modules associated in HK activities of F. verticillioides, where the member genes were harmoniously coordinated and relevant, and significantly differentially activated during kernel rot pathogenesis and fumonisin biosynthesis.

Impacts
What was accomplished under these goals? Continuing the effort to characterize MADS-box TF regulatory pathways in F. verticillioides, a next-generation sequencing (NGS) experiment was performed to compare the expression pattern of genetic networks downstream of MADS-box TFs and as a result, identify novel genes with an impact on secondary metabolite production in F. verticillioides. Through our proposed computational analyses, we identified two co-expression subnetwork modules down stream of MADS-Box TFs with strong correlation with mycotoxin biosynthesis. The two proposed subnetworks were analyzed in silico, and five genes were selected as candidates for functional characterization. Hub gene prediction was based on five criteria, including putative function and homology to other known fungal genes in secondary metabolism. FVNOG1 (FVEG_00035), FVCUP9 (FVEG_07056), FVRSR1 (FVEG_02390), and FVCYN1 (FVEG_07804) were selected from subnetwork A and are all upregulated in the WT compared to Mads1 gene-deletion mutant. FVCYN1 (FVEG_07804), FVCUP9 (FVEG_07056), and FVEFM3 (FVEG_11168), a constitutively expressed gene, were selected from subnetwork B. While the majority of the genes identified are differentially regulated downstream of MADS-box TFs, the putative CArg-box binding motif, CC[A/T]6GG, was not identified in the 1 kb promoter regions by SCOPE algorithms, suggesting that MADS-box TFs do not directly bind to these genes in F. verticillioides. We will be functionally characterizing these genes in the next reporting period.

Publications

• Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2017 Citation: Yan, H., Kim, M.S. Huang, J. and Shim, W. B. 2017. FRN1, a gene encoding putative GPCR protein, is critical for ear rot and fumonisin biosynthesis in maize pathogen Fusarium verticillioides. Phytopathology 107: (In Press)
• Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2017 Citation: Zhang, H., Kim, M.S., Huang, J., and Shim, W. B. 2017. FvLcp1, a novel LysM/Chitin-binding protein, is important for ear rot virulence and fumonisin biosynthesis in�Fusarium verticillioides. Phytopathology 107: (In Press)
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Yan, H., Zhang, H., Kim, M.S., Huang, J., and Shim, W. B. 2017. Characterization of functional subnetwork modules associated with ear rot and fumonisin biosynthesis in maize pathogen Fusarium verticillioides. Phytopathology 107: S3.14
• Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2017 Citation: Ridenour, J.B., Hawkins, W. D., Dhillon, B., Smith, J. E., Ramegowda, Y., Shim, W. B., Woloshuk, C.P.,�and Bluhm, B.H. 2017. Casting a new die: Integrating novel biological insights and translational approaches to combat mycotoxin problems in maize. Phytopathology 107: (In Press)
• Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2017 Citation: Kim, M.S. and Shim, W. B. 2017. Computational prediction of time-course subnetwork modules associated with histidine kinase activities in maize pathogen Fusarium verticillioides. Phytopathology 107: (In Press)