Source: USDA ARS Cereal Disease Laboratory submitted to
COMPARATIVE FUNCTIONAL GENOMICS OF PLANT PATHOGENIC FUSARIUM SPECIES.
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
NRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0212906
Grant No.
2008-35604-18800
Cumulative Award Amt.
$771,000.00
Proposal No.
2009-06049
Multistate No.
(N/A)
Project Start Date
Feb 1, 2008
Project End Date
Jan 31, 2013
Grant Year
2008
Program Code
[51.0B]- Microbial Genomics (B): Functional Genomics of Microorganisms
Project Director
Kistler, H. C.
Recipient Organization
USDA ARS Cereal Disease Laboratory
(N/A)
St. Paul,MN 55108
Performing Department
(N/A)
Non Technical Summary
Fusarium species are perhaps the most important fungal plant pathogens worldwide. Together these microbes cause economically important disease on nearly every species of cultivated plant. Many species also pose a threat to human health and food safety by contaminating agricultural products with harmful mycotoxins. Due to their importance to agriculture, whole genome sequences have been developed for the Fusarium pathogens, F. graminearum (cause of scab disease of wheat and barley) F. oxysporum (tomato wilt) and F. verticillioides (corn kernel, root and stalk rot). The research undertaken by this proposal takes advantage of the relatedness of the three fungal genomes and will allow an unprecedented ability to predict genes and how these genes are expressed in plant pathogenic fungi. A database will be constructed allowing public display and analysis of these data. By understanding the genes and gene expression patterns exhibited by these microorganisms while they are causing disease, we may be able to develop novel, stable, and environmentally sensible disease management practices aimed at interfering with the essential processes of pathogenesis. These management strategies may involve chemical or physical disruption of the fungal developmental pathways vital to causing disease.
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
2124020104060%
2124020116040%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
4020 - Fungi;

Field Of Science
1040 - Molecular biology; 1160 - Pathology;
Goals / Objectives
The specific objectives of this proposal are: 1) Development of a multiple-oligonucleotide microarray for the whole-genomes of the fungal plant pathogens Fusarium oxysporum, F. verticillioides, and F. graminearum; 2) Functional analysis of genes required for pathogenicity and host specificity; and 3) Development of an integrated database for comparative genomic studies.
Project Methods
Having DNA sequence data for three pathogenic Fusarium species and the improved accuracy of gene predictions resulting from comparative analysis will allow for the construction of a highly accurate, genome-wide microarray for the three most closely related Fusarium species (Objective 1). Further objectives seek to extend comparative analysis by looking for conserved patterns of gene expression among the three fungi under a variety of environmental conditions including during plant infection (Objective 2). The significance of conserved non-coding regions will be tested by their correlation with genes exhibiting conserved expression patterns. Physical proximity of conserved, co-expressed genes will be used to define functional gene clusters. Because phenotypic similarities and differences exist among the species, comparative gene expression patterns can be placed in a biological context. Thus conserved patterns of gene expression among species will be discovered during, for example, fungal challenge of compatible hosts. We hypothesize that conserved patterns of gene expression may reflect evolutionary constrain based on their functional significance to the developmental process being studied. For plant infection (Objective 2), we intend to test this hypothesis by deleting selected genes such as stage-specific transcription factors, to determine their effect on fungal development. To make results from proposed study and comparative analyses accessible for the community users, a coherent database incorporating comparative structural and functional data (Objective 3) will be constructed.

Progress 02/01/08 to 01/31/13

Outputs
Target Audience: During this reporting period we reached target audiences of other plant scientists, plant pathologistsand plant improvement professionals through publications and web accessable databases. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Training: Lokesh Kumar (master student): developed a computational program to identify potential transcription factor binding sites based on the aligned sequence of F. graminearum with F. oxysporum, F. verticillioides and F. solani species and generated a list of genes associated with each potential binding site. Combining the expression data that will be generated through this study, the information can be used to investigate the potential functional association of the binding sites and genes have the binding sites in their promoter regions (see publication Kumar et al., 2010). Wilfried Jonkers (postdoctoral): created deletion mutations in orthologs of the SGE1, ZIF1 and ZCF1 genes in F. graminearum and/or F. oxysporum and is conducting functional analysis of these transcription factors by comparing their expression profiles in mutant and wild type and by analyzing the differences in expression patterns between species. He has taken the lead in writing the first manuscript describing the design and performance of the new, multi-fungal genome microarray. Wende Liu (postdoctoral) Xiaoying Zhou (graduate student) Undergraduate students: Cynallyhia Indriago (Coker College), Bryan Gmusungu (Coker College), Sam Egel (Purdue), Samantha Strangeland (University of Minnesota). Microarray analysis with the cpkA and Fac1 mutants was conducted by Cynallyhia Indriago and Bryan Gmusungu. How have the results been disseminated to communities of interest? http://www.broadinstitute.org/annotation/genome/fusarium_graminearum/MultiHome.html http://www.plexdb.org/plex.php?database=Fusarium What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Accomplishments: Goal 1. A custom Affymetrix GeneChip has been designed and manufactured incorporating all genes from four Fusarium genomes (F. graminearum, F. oxysporum, F. verticillioides, and F. solani) in an exon-based expression platform as well as a tiling array for the F. graminearum and F. oxysporum genomes. The array design has been validated to establish baseline expression patterns for the Fusarium species in vitro. Goal 2. Mutants deficient in genes involved in signaling (adenyl cyclase, MAP kinase and cAMP-PKA) and genes for transcription factors regulating pathogenicity (Sge1, Zcf1, Zif1) have been obtained in two or more of the targeted Fusarium species. Manuscripts comparing expression of the wild type and mutants using the microarray data are under development. Goal 3. The microarray probe information had integrated into the PLEXdb http://www.plexdb.org/, a computational software prototype to reconstruct regulatory network using expression data was generated, and database upgrades have been made to the Broad Institute’s Fusarium comparative website http://www.broadinstitute.org/annotation/genome/fusarium_graminearum/MultiHome.html. The Fusarium comparative studies shed lights into the genome evolution among this group of agricultural important fungal pathogens and identified more than half of the genes encoded in these genomes as orthologs. However, there is no clear understanding whether these genes conserved at sequence level are equally conserved functionally. The functional comparative genomics designed through this project allowed an objective comparison among three phylogenetically closely related with distinctive function divergence. For instance, we generated the fmk1, cpkA, and fac1 mutants in Fusarium verticillioides. Surprisingly, we found that the cAMP-PKA pathway does not play a role in regulating the biosynthesis of fumonisins. In F. graminearum, cAMP signaling is involved in DON production. These observations indicate that this well conserved signal transduction pathway plays species specific roles in regulating secondary metabolism. Nevertheless, this pathway is important for pathogenesis in F. oxysporum, F. verticillioides, and F. graminearum. Microarray data have been generated to determine genes that are commonly regulated in these three Fusarium species. Some of these genes may be general pathogenicity factors common to Fusarium pathogens.

Publications

  • Type: Book Chapters Status: Published Year Published: 2010 Citation: Zhou, X., Li, G., and Xu, J. R. 2010. Efficient approaches for generating GFP fusion and epitope-tagging constructs in filamentous fungi, In: Fungal Genomics: Methods and Protocols (eds. Jin-Rong Xu and Burt Bluhm). Humana Press, Springer Science+Business Media, LLC.
  • Type: Book Chapters Status: Published Year Published: 2010 Citation: Leslie, J.F. and Xu, J.-R. 2010. Fusarium genetics and pathogenicity, pp. 607-621. In: Cellular and Molecular Biology of Filamentous Fungi (eds. K. A. Borkovich & D. J. Ebbole). ASM Press, Washington, D.C.
  • Type: Book Chapters Status: Published Year Published: 2013 Citation: Kistler H.C., Rep M. L.-J. Ma. Structural dynamics of Fusarium genomes In: Fusarium: genomics, molecular and cellular biology. Eds. Brown, D.W. and Proctor, R.H., Horizon Scientific Press, Norwich, United Kingdom. 2013.
  • Type: Journal Articles Status: Published Year Published: 2010 Citation: Choi, Y. E. and Xu, J. -R. 2010. The cAMP signaling pathway in Fusarium verticillioides is important for conidiation, plant infection, and stress responses but not fumonisin production. Molecular Plant-Microbe Interactions. 23: 522-533.
  • Type: Journal Articles Status: Published Year Published: 2010 Citation: Liu, W., Xie, S., Zhao, X., Chen, X., Yi, Y., Liu, S., Lu, G., Xu, J. R, Wang, Z. 2010. A homeodomain transcription factor is essential for asexual reproduction in a filamentous ascomycete. Molecular Plant-Microbe Interactions. 23: 366375.
  • Type: Journal Articles Status: Published Year Published: 2010 Citation: Ma L.-J., van der Does, C., Borkovich, K., Coleman, J., Daboussi, M.-J., Di Pietro, A., Dufresne, M., Freitag, M., Grabherr, M., Henrissat, B., Houterman, P.M., Kang, S., Shim, W.-B., Woloshuk, C., Xie, X., Xu, J.-R., Antoniw, J., Baker, S.E., Bluhm, B.H., Breakspear, A., Brown, D., Butchko, R., Chapman, S., Coulson, R., Coutinho, P.M., Danchin, E., Diener, A., Gale, L.R., Gardiner, D., Goff , S., Hammond-Kosack, K., Hilburn, K., Hua-Van, A., Jonkers, W., Kazan, K., Kodira, C., Koehrsen, M., Kumar, L., Lee, Y.-H., Li, L., Manners, J., Miranda-Saavedra, D., Mukherjee, M., Park, G., Park, J., Park, S.-Y., Proctor, R., Regev, A., Ruiz-Roldan, C., Sain, D., Sakthikumar, S., Sykes, S., Schwartz, D., Turgeon, B.G., Wapinski, I., Yoder, O.., Young, S., Zeng, Q., Zhou, S., Galagan, J., Cuomo, C., Kistler, H.C. and Rep, M. 2010. Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium. Nature 464: 367-373
  • Type: Journal Articles Status: Published Year Published: 2010 Citation: Rep, M. and Kistler H.C. 2010. The genomic organization of plant pathogenicity in Fusarium species. Current Opinion in Plant Biology. 13:420-426.
  • Type: Journal Articles Status: Published Year Published: 2011 Citation: Wang, Y., Liu, W., Hou, Z., Wang, C., Zhou, X., Jonkers, W., Ding, S., Kistler, H. C. and Xu, J.-R.. 2011. A novel transcriptional factor important for pathogenesis and ascosporogenesis in Fusarium graminearum. Molecular Plant-Microbe Interactions 24:118-128.
  • Type: Journal Articles Status: Published Year Published: 2010 Citation: Zhou, X., Heyer, C., Choi, Y., Mehrabi, R., and Xu, J. R. 2010. CID1 is important for plant infection in Fusarium verticillioides. Fungal Genetics and Biology. 47: 143151.
  • Type: Journal Articles Status: Accepted Year Published: 2013 Citation: Jonkers, W., Xayamongkhon, H., Haas, M., Olivain, C., van der Does, H.C., Broz, K., Rep, M., Alabouvette, C., Steinberg, C. and Kistler, H.C. 2013. EBR1 genomic expansion and its role in virulence of Fusarium species. Environmental Microbiology (In press).


Progress 02/01/08 to 01/31/09

Outputs
OUTPUTS: A custom Affymetrix GeneChip microarray has been designed and manufactured containing all gene sequences from the three fungal genomes, Fusarium graminearum, F. verticillioides and F. oxysporum. Through the interactions with the Affymetrix supporting teams and the Broad Institute Expression profiling platform, the PIs have developed a detailed project plan including sample collection, experimental conditions, imagine scanning, data processing and analysis. DNAs from all organisms and RNAs collected in vitro to validate the chip have been collected. Mutants of F. graminearum and F. verticillioides that are deleted for genes encoding the FMK1 MAP kinase, adenylate cyclase, and catalytic subunits of PKA have been generated and phenotypically characterized. We also have requested and received the Gbb1 (mutants deleted for the G-beta subunit of trimeric G-protein gene) deletion mutants of F. graminearum and F. verticillioides from Dr. Yin-Won Lee at Seoul National University and Dr. Won-Bo Shim at Texas A&M University, respectively. RNA samples have been isolated from these mutants and are ready for microarray analysis. PARTICIPANTS: Dr. Kistler has isolated RNAs and DNAs to be used to validate the microarray and has had contributed to the design of the array. Dr. Ma has been the team leader on bioinformatics input on array design. Dr. Jin-Rong Xu has contributed to array design and has overseen generation of fungal mutants. Graduate student Xiaoying Zhou has conducted mutagenesis experiments and gained laboratory training in molecular biology and genomics. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The design of the custom microarray is in the public domain and will be available to all scientists conducting research on these important plant pathogenic fungi. The design can be used for manufacturing third party arrays or to purchase as ready-made arrays directly from the primary manufacturer. This will be the first whole genome tiling array for a filamentous fungus and likely will be very useful for additional gene discovery and analysis of small RNAs.

Publications

  • No publications reported this period