GENOME DYNAMICS OF HOST SPECIFICITY IN THE FUSARIM OXYSPORUM SPECIES COMPLEX

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: NRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 0225721
• Grant No.: 2008-35600-30379
• Project No.: MASW-2011-01922
• Proposal No.: 2011-01922
• Program Code: 51.0A
• Project Start Date: Jan 15, 2010
• Project End Date: Aug 31, 2013
• Grant Year: 2011

Project Director
Ma, L.

Recipient Organization
BROAD INSTITUTE, INC.
7 CAMBRIDGE CENTER
CAMBRIDGE,MA 02142

Performing Department
(N/A)

Non Technical Summary
The genus Fusarium collectively represents the most important group of fungal plant pathogens, causing various diseases on nearly every economically important plant species. Of equal concern is the health hazard posed to humans and livestock by the plethora of Fusarium mycotoxins. Besides their economic importance, species of Fusarium also serve as key model organisms for biological and evolutionary research. The significance of Fusaria as model systems is growing rapidly with increasing genomic resources, including the publicly accessible Fusarium graminearum genomic sequence. Previous work has demonstrated the power of comparative genomics to improve and correct errors in gene annotation and to identify functional non-coding elements. As a widely used model plant pathogen, F. graminearum is a prime candidate for such efforts, because the genes and functional elements identified through comparative analysis will be immediately put to use in on going functional studies. Therefore, we propose to sequence two additional Fusarium species, F. oxysporum and F. verticillioides. These genomes are appropriately diverged from F. graminearum to fulfill the objectives of a highly informative comparative project, while being important research platforms in their own right. F. oxysporum is a broad host range pathogen that has caused some of the worlds most dramatic and economically devastating plant disease epidemics. Recently F. oxysporum has also emerged as a model for soil-borne fungal diseases with Arabidopsis and tomato as hosts. F. verticillioides is a cosmopolitan pathogen of maize and sorghum, and produces carcinogenic mycotoxins known as fumonisins. A threeway comparison of F. oxysporum, F. verticillioides and F. graminearum offers powerful synergy in studies of pathogenicity and virulence factors, and their evolution within this genus.

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	2410	1040	100%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
2410 - Cross-commodity research--multiple crops;

Field Of Science
1040 - Molecular biology;

Keywords

f. oxysporum

plant pathogen

Goals / Objectives
Our specific objectives are to: 1) Generate genomic resources of F. oxysporum and F. verticillioides. We will Produce 7X and 4X sequences for F. oxysporum and F. verticillioides, respectively. Generate and publicly release high quality whole-genome shotgun assemblies for both. The F. verticillioides assembly will be augmented by the existing ~4X sequence of Syngenta. Perform and publicly release automated annotations on both assemblies. 2) Conduct comparative analyses among three Fusaria to: Create a comprehensive Fusarium gene catalogue. Identify conserved non-coding elements. Characterize species-specific gene sets. 3) Broaden the impact of the project by integrating educational and mentoring activities through established outreach programs as well as initiating outreach to growers and producers to enhance their understanding of the biology and control of Fusarium diseases.

Project Methods
Whole-genome shotgun sequence will be obtained as paired reads from both plasmid and Fosmid clones obtained by random shearing of genomic DNA. With standardized sequencing approaches, the highquality draft (HQD) assembly has become a highly predictable sequence product at the Broad with 7X sequence coverage. Validation of the fungal assemblies generated at the Broad, using genetic maps and independently generated genomic sequences, has shown that these HQD genome assemblies offer excellent genome coverage, high base pair accuracy and long-range continuity (http://www.broad.mit.edu/annotation/fungi/fgi/). The proposed sequence coverage for F. oxysporum is 7X generated from three whole-genome shotgun libraries (Table 2). Because we will combine our sequence of F. verticillioides with that from Syngenta, we will produce only 4X coverage from two libraries (Table 3). The number of Fosmid reads has been calculated to yield 10% sequence contribution to the final assembly with more than 7X coverage.

Progress 01/15/10 to 08/31/13

Outputs
Target Audience: This comparative genomics project takes systems biology approach to understand the development and pathogenicity in the Fusarium genomes. This project has two groups of targeted audiences. 1) Fungal biologiests who are interested in using genomic information to understand pathogenicity of F. oxysporum: Prior to the publication of the data geneated through this support, all the genome information was released to public through the Fusarium comparative database. PI Ma presented the knowledge gained through this project at regional, national and international meeting, including a plenary talk at the 10th International Congress of Plant Pathology. 2) Students who are interested in learning genomics and familiarize with the analytic tools: PI Ma designed and taught two new courses "Applications of Genomics" and "Comparative Genomics using the data generated through this project and a previous NIFA funded project. Students responded enthusiastically and felt that the courses provided them with a very good grasp on many concepts of genomics and equipped them to independently perform bioinformatics analysis. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? 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? Both PI and co-PI of this project were sharing the prepulished data to the interesting research communities without any resovation. Since the public release of the genomic data generated through this support, there are at least 10 manuscripts have cited the website. More collaborations were established among PIs' labs and national and internatinal experts. Collaborations: Gerhard Adam(BOKU, Vienna) andUlrich Güldener(Helmholtz Zentrum, München) are collaborating to provide additional genome annotation and to display microarray data through the MIPS FGDBhttp://mips.helmholtz-muenchen.de/genre/proj/FGDB/. They have incorporated features and links to the gene expression display database PLEXdb. A Fusariumcomparative genomics database featuring the addition ofFusarium oxysporumgenome sequences is planned. Roger Wiseand his colleagues at Iowa State University are working to display data from our second generationFusariummicroarray on the Plant Expression database PLEXdbhttp://www.plexdb.org/with annotation links to the Broad Institute and MIPS FGDB websites. Joseph FlahertyCoker College, South Carolina. Martijn Rep, University of Amsterdam, the Netherlands. Antonio DiPietro, University of Cordoba, Spain. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? we have accomplished all three objectives. For objective 1, we have: 1) deposited all the strains selected for the sequencing project at ARS Culture Collection (NRRL accession listed); 2) generated the Illumina data (>25X coverage) for 10 proposed strains 3) produced the Illumina sequence for the reference strain for the comparison 4) developed and tested a pipeline using MAQ to align the short Illumina reads to the reference genome assembly for SNP discovery and coverage analysis. For objective 2, we have: 1) constructed optical maps for all four strains; 2) conducted the pair-wise comparison using the map data, which confirmed our project plan to study genome structural polymorphisms and provide guidance for preparing samples for deep sequencing of the LS chromosomes. In accomplishing objective 3, we encountered difficulty in isolating sufficient DNA for the LS chromosomes to generate sequence. The revolutionary of new sequencing technologies makes the cost of generating sequence data so much lower. With that, we decided to generate whole genome sequence for these isolates instead. For objective 3, we have: 1) produced whole genome shotgun sequence for all 10 strains using the newest Illumina sequencing platform HiC. All the Illumina sequence data sets were deposited at NCBI trace repository for public access (http://www.ncbi.nlm.nih.gov/sites/entrez) under the “Study Accession” number SRP002087”. 2) generated whole genome assemblies for all. The strategies to combine Illumina fragment reads (180 bp) and large insert libraries (4 kb and 8 kb) produced high quality assemblies for all the genomes with an average N50 scaffold over 2 Mb in size. All the assemblies are released through the Fusarium comparative genomics website (http://www.broadinstitute.org/annotation/genome/fusarium_group) in spring 2012.

Publications

• Type: Journal Articles Status: Published Year Published: 2010 Citation: Ma L.-J. , H. Charlotte van der Does, et al. 2010. Fusarium comparative genomics reveals lineage-specific chromosomes related to pathogenicity. Nature 465:367-373
• Type: Journal Articles Status: Published Year Published: 2010 Citation: Kumar, L., Breakspear, A., Kistler, C., Ma, L.-J. and Xie, X. 2010. Systematic discovery of regulatory motifs in Fusarium graminearum by comparing four Fusarium genomes. BMC Genomics 2010, 11:208 DOI:10.1186/1471-2164-11-208.
• Type: Book Chapters 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. DOI 10.1016/j.pbi.2010.04.004.
• 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: Book Chapters Status: Published Year Published: 2012 Citation: Ma L.-J, M. Rep and H. C. Kistler. 2012. Evolution of plant pathogenicity in Fusarium species. In Sibley, L. D., B. J. Howlett, et al. (2012). Evolution of virulence in eukaryotic microbes. Hoboken, N.J., Wiley-Blackwell. 485-500
• Type: Conference Papers and Presentations Status: Published Year Published: 2010 Citation: Corby Kistler. March 28, 2010 Fusarium comparative genomics reveals lineage-specific chromosomes related to pathogenicity. Fusarium Satellite Meeting, Amsterdam, the Netherlands.
• Type: Conference Papers and Presentations Status: Published Year Published: 2010 Citation: Corby Kistler. March 31, 2010 Fusarium graminearum as a Model for Human Niemann-Pick Type C Disease, 10th European Conference on Fungal Genetics, Noordwijker-hout, The Netherlands.
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Li-Jun Ma. Feb. 2013. Horizontal gene transfer and pathogenicity Keynote speaker at Midwestern Universities Filamentous Fungal Symposium University of Missouri- Kansas City
• Type: Conference Papers and Presentations Status: Published Year Published: 2011 Citation: Li-Jun Ma. October 2011. Strategies for moving fungal genomics in China. Second Chinas Fungal Genome Initiative. Kunming, Yunnan China
• Type: Conference Papers and Presentations Status: Published Year Published: 2011 Citation: Li-Jun Ma. August 2011. Genomic dynamics and host specificity in Fusarium oxysorum species complex. American Phytopathologial Society annual Conference. Honolulu, Hawaii.
• Type: Conference Papers and Presentations Status: Published Year Published: 2011 Citation: Li-Jun Ma. November 7, 2011. Genomic dynamics and host specificity in Fusarium oxysorum species complex. North Carolina State University, Plant Pathology Department. Raleigh, NC
• Type: Conference Papers and Presentations Status: Published Year Published: 2011 Citation: Li-Jun Ma. July 2011. Genomic plasticity and niche adaptation -- comparative genomics of wilt pathogens. Mycological Society of America. Fairbanks, Alaska.
• Type: Other Status: Published Year Published: 2012 Citation: Li-Jun Ma.November 2012. Genomic dynamics and host specificity in Fusarium oxysporum species complex. Department of Plant Pathology. University of Kentucky, Lexington KY
• Type: Other Status: Published Year Published: 2012 Citation: Li-Jun Ma. October 2012. Genomic dynamics and host specificity in Fusarium oxysporum species complex. Keynote speaker at MassMyco. Clark University, Worcester MA
• Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Li-Jun Ma. June 2012. Combating Panama Disease through Comparative Genomics. 5th International Symposium on Tropical and Subtropical Fruits Guangzhou China
• Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Li-Jun Ma. April 2012. Understanding fungal genome evolution and niche adaptation using comparative genomics. Plenary section speaker at European Conference of Fungal Genetics. Marburg Germany.
• Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Li-Jun Ma. March 2012. Exploring LS chromosomes in Fusarium oxysporum species complex using next-gen sequencing technology. 11th European Conference on Fungal Genetics. Fusarium Satellite meeting. Marburg Germany
• Type: Other Status: Published Year Published: 2012 Citation: Li-Jun Ma. March 2012. Horizontal Transfer of Pathogenicity Chromosomes Determines Host Specificity in Fusarium oxysorum species complex (FOSC). SciLifeLab, University Uppsala.
• Type: Conference Papers and Presentations Status: Published Year Published: 2011 Citation: Li-Jun Ma. January 2011. Understanding fungal biology through comparative genomics. International Plant Pathogenomics Conference. Shenzhen
• Type: Conference Papers and Presentations Status: Published Year Published: 2010 Citation: Li-Jun Ma. March 31, 2010.  Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium oxysporum, 10th European Conference on Fungal Genetics, Noordwijker-hout, The Netherlands.
• Type: Conference Papers and Presentations Status: Published Year Published: 2010 Citation: Li-Jun Ma. March 28, 2010 Exploring Lineage-specific (LS) chromosomes in F. oxysporum species complex (FOSC) Fusarium Satellite Meeting, Amsterdam, the Netherlands.
• Type: Conference Papers and Presentations Status: Published Year Published: 2010 Citation: Li-Jun Ma. January 18, 2010  Pathogenicity of wilt pathogens  Verticillium comparative genomics Microbial Genome Sequencing Program Awardee Workshop. San Diego, USA