Progress 09/01/05 to 08/31/09
Outputs
OUTPUTS: We have accomplished all the objectives proposed including generating genomic resources for F. oxysporum and F. verticillioides and conducting comparative analyses among the sequenced Fusarium genomes. Through incorporating the existing genomic data from Syngenta, we were able to deliver high quality genome assembly for the genome of F. verticillioides with lower cost. At the same time we increased the amount of sequences generated for F. oxysporum in order to deliver high quality genomic assembly for a 50% increased genome size. To understand the global genome structural variations among these sequenced genomes, special efforts were carried out to create physical maps for all the genome assemblies by incorporating a genetic map of F. verticillioides and creating a new optical map for F. oxysporum. We performed and publicly released automated annotations on both assemblies and created a comprehensive Fusarium gene catalogue.; identified conserved non-coding elements and genes that share the same regulatory elements; and characterized species-specific gene sets. The data generated through this project is released through the Fusarium comparative web site (http://wwwdev.broad.mit.edu/annotation/genome/fusarium_group/). The success of the project is demonstrated by: 1) The web site has been embraced by the research community. Each year, the Fusarium comparative web site has over 17,000 visits for a total of 313,000 times by researchers over 120 countries/territories. 2) Multiple data releases and manuscripts (one is under revision, the other is under reviewer) were produced directly under this project (see publication list below). PARTICIPANTS: Won-Bo Shim: Texas A&M University; Seogchan Kang: Penn State University; Charles Woloshuk, Purdue University; Harold Kistler: University of Minnesota. TARGET AUDIENCES: The novel discovery of HGT and the impact on development of plant pathogenicity achieved from the comparative study has broad applications to both basic scientific researches and applied sciences. We act actively to deliver such science-based knowledge to reach scientists from both groups by anticipating scientific conferences and visiting related Institutes. Over the years, all the participants attended various meetings to share the results. As part of the results, additional collaborations around Fusarium pathogens were formed and many research projects were proposed among the Fusarium research community. As part of the effort, PD Dr. Ma visited the Agricultural Experiment Station in Connecticut and stimulated the discussion to study a novel pathogenic isolate from salt marsh. and Dr. Kistler visited the New Jersey Agricultural Experiment Station. In addtion, Dr. Ma also met the students at University of Massachusetts at Amherst to disseminate the knowledge and to encourage the engagement of students using genomic tools to study applied sciences, particularly in the agricultural practices. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The rich and high quality genomic resources generated through this project have greatly accelerated efforts of scientists worldwide to understand the genetic basis of pathogenicity, virulence, and mycotoxin production in these species. An accurate gene catalogue and species-specific gene sets shortened the time needed for gene discovery and functional characterization. The specific findings resulted from this study include the discoveries of: 1) Horizontal transfer (HT) of lineage specific chromosomes and their impact in pathogenicity. The comparative analysis provides evidence for the HT of four chromosomes accounting for 25% of the genome in an asexual, pathogenic fungal species, Fusarium oxysporum. The direct contribution of the chromosomes to pathogenicity is indicated by the fact that they encode known virulence factors such as effector proteins, necrosis-inducing peptides and a large array of enzymes targeting plant substrates, but lack genes involved in primary metabolism. The mobilization of these TE-rich and pathogenicity related chromosomes contributes to the rapid emergence of new pathogenic lineages of F. oxysporum in otherwise distinct and incompatible genetic backgrounds and provides the genetic basic for the remarkable genetic adaptability of the organism. The result implies that HT may offer previously unrecognized opportunities for genetic exchange and recombination in asexual lineages of lower eukaryotes. Our study deepens the understanding of evolutionary mechanism involving fungal pathogens development, which may have direct impact in developing novel management strategies in agricultural practices. 2) Secondary metabolism gene clusters in Fusarium genomes. Fusarium species are known to produce diverse secondary metabolites, including harmful mycotoxins that contaminate foodstuffs and their levels are stringently regulated in agricultural products in the EU, the US and throughout much of the world. Through our comparative analysis, we also identified many novel gene clusters related to secondary metabolite synthesis pathways. The co-expression of many such clusters, especially the novel clusters that are preferentially expressed in planta, validates their potential functionality and suggests their direct impact on host-pathogen interactions. In the long run, knowledge gain through such studies will lead to novel strategies to control the contamination of these mycotoxins. In addition, the new clusters discovered through this study also provides the opportunities to discover novel anti-microbial components 3) Transcription factor binding sites and potential regulatory network. We have identified 73 candidate regulatory motifs in the promoter region of Fg that are highly enriched in the promoter region of Fg genes that are specific to a functional category. Our analyses showed enrichment of motifs similar to their known binding sites, suggesting a conservation among Fusarium species and the yeast species for TF, their binding site and the target genes regulated by these TFs, revealing the conservation of some functional important regulatory pathways cross wide diverged fungal species.
Publications
- Kistler, H.C.2009 Comparative genomics of plant-pathogenic Fusarium species. Department of Plant Biology and Pathology and the New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick, NJ, April 3, 2009.
- Ma, L.-J.2009 Genetic plasticity and pathogenicity development revealed through Fusarium comparative genomics. August 2009. APS. Portland, Oregon.
- Ma, L.-J. 2009 Lineage-specific chromosomes related to pathogenicity revealed by Fusarium comparative genomics. May 2009. Lockwood Lecture. Agricultural Experiment Station. Connecticut.
- Ma, L.-J. 2009 Lineage-specific chromosomes related to pathogenicity revealed by Fusarium comparative genomics. March 2009. Department seminar. UMASS Amherst. Massachusetts.
- Ma, L.-J.2009 What can you learn from Comparative Genomics. Fusarium workshop. March 2009. The 25th Fungal Genetics Conference, Asilomar, California.
- Ma, L.-J. 2009 Genome Innovation revealed by Fusarium comparative genomics. January 2009. Plant and Animal Genome XVII Conference. San Diego CA.
- Cuomo, C.A., Guldener, U., Xu, J.-R. et al. 2007. The Fusarium graminearum genome reveals a link between localized polymorphism and pathogen specialization. Science 317:1400-1402.
- L.-J. Ma, C. van der Does, K. Borkovich, et al. 2009 Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium oxysporum. Nature (under revision).
- Kumar L., A. Breakspear, J. Menke, C. Kistler, L.-J. Ma, X. Xie. 2009. Systematic Discovery of regulatory motifs in Fusarium by comparison of four Fusarium genomes. BMC Genomics (in review).
- Uma Shankar Sagaram and Won-Bo Shim. 2007. Fusarium verticillioides GBB1, a gene encoding heterotrimeric G protein subunit, is associated with fumonisin B1 biosynthesis and hyphal development but not with fungal virulence. Molecular Plant Pathology 8: 375-384.
- Kistler, H.C.2009 Comparative genomics of plant-pathogenic Fusarium species. Department of Plant Pathology, University of Minnesota, St. Paul, MN, January 26, 2009.
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Progress 09/01/07 to 08/31/08
Outputs
OUTPUTS: In March 2007, we have Publicly Released the Fusarium comparative web site (http://wwwdev.broad.mit.edu/annotation/genome/fusarium_group/), which provides the research community windows to access: 1). The physical maps and high quality assemblies for all three Fusarium genomes. 2). Automated gene annotation for all three genomes and improved gene models utilizing the information of sequence conservation. 3). Comparative searches among three Fusarium genomes. The web site had been embraced by the research community. Since it is public release, the page was viewed more than 180,000 times. In the past year, we have focus on understanding the genome conservation and variation among the three genomes. The three selected Fusarium species, F. oxysporum (FO), F. verticillioides (FV) and their out-group F. graminearum (FG), have short evolutionary divergent time, but have adapted to different ecological niches with distinct biological and pathological phenotypes. The careful design of the project allowed us to unambiguously identify orthologous genomic regions, therefore highlights species-specific genomic features that may hold answers to why and how these species are different physiologically and ecologically. The comparative analysis clearly differentiates the F. oxysporum (FO), the species with very wide host range, into highly conserved regions and species-specific regions. These Species-specific regions are located on a few lineage-specific chromosomes that are enriched for transposable elements and encode genes related to virulence and host specificity. While the conserved genomics regions share over 90% homologous sequences at over 90% sequence identity with its closely relative F. verticillioides (FV), the genes reside in the LS chromosomes have either no homolog or very low similarity to the FV sequence. Computationally, the genes encoded in these LS chromosomes have different genetic property as codon usage and discontinuous phylogenetic distribution. We hypothesize that these LS-chromosomes contribute ditectly to the pathogenicity and wide host range of F. oxysporum. In addition, we have been collaborating with Dr. Xiaohui Xie at the UC Irvine to identify the conserved non-coding sequences as potential regulatory elements following three steps. 1) discover those 7-mers in the Fusarium genomes which are highly conserved. 2), expand these highly conserved motifs using degenerate characters, and 3) cluster degenerated motifs in 5' and 3' regions respectively using the hierarchical clustering method. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Fusarium is perhaps the most important genus of plant pathogenic fungi. Together these fungal species cause economically important disease on nearly every species of cultivated plants. Many species also pose a threat to human health and food safety by contaminating agricultural products with harmful mycotoxins. The three Fusarium species to be studied are important plant pathogens to U.S. agriculture and pose threats to human health. The rich and high quality genomic resources generated in the past year will greatly accelerate efforts of scientists worldwide to understand the genetic basis of pathogenicity, virulence, and mycotoxin production in these species. An accurate gene catalogue and species-specific gene sets will shorten the time needed for gene discovery and functional characterization. Many Fusarium species also are notorious for producing mycotoxins, such as trichothecenes or fumonisins, which are harmful to both human and animals. Employing the power of comparative genomics, we have identified potential secondary metabolite producing gene clusters in these three sequenced Fusarium genomes. In the long run, knowledge gain through such studies will lead to novel strategies to control the contamination of these mycotoxins.
Publications
- No publications reported this period
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Progress 09/01/06 to 08/31/07
Outputs
In March 2007, we have Publicly Released the Fusarium comparative web site (http://wwwdev.broad.mit.edu/annotation/genome/fusarium_group/), which provides the research community windows to access: 1). The physical maps and high quality assemblies for all three Fusarium genomes including: an 8X F. verticllioides assembly; A 6.8X genome assembly and an optical map for F. oxysporum; and an improved 10X F. graminearum genome assembly. An optical map with ~55X physical coverage and consists of 15 linkage groups was created or F. oxysporum. All three high quality assemblies have been anchored to the genetic/optical maps. Using improved assembly algorithm, the newly created F. graminearum assembly incorporated more sequence reads (from 90% to 96%) and provided 200,000 additional assembled bases with much improved assembly quality. 2). Automated gene annotation for all three genomes and improved gene models utilizing the information of sequence conservation. We have predicted
and publicly released the F. oxysporum protein gene set of 17,735 genes, F. verticillioides protein gene set of 14,206 genes, and the re-annotated F. graminearum protein gene set of 13,332 genes. ESTs from F. graminearum (68,000) and F. verticillioides (87,000) were analyzed with FindORFs, a EST based gene finding program developed at Broad, to identify mis-annotated gene locus and improve gene structure predictions. In addition, the final gene set of F. verticillioides was mapped onto the F. graminearum and F. oxysporum genomes by FindORFs as additional lines of evidence to improve the automated gene prediction in these two fungi. Furthermore, targeted manual annotation was carried out for loci where automated gene predictions were inconsistent or conflict with EST evidence or BlastX analysis. The re-annotation of F. graminearum greatly improved the gene structures and greater than 90% accuracy was reached when comparing this annotation against a set of 4000 high confidence genes.
3). Comparative searches among three Fusarium genomes. The comparative feature search tool allows the users to comparing the presentation of features such as PFAM domain, Blast homolog, and tRNAs among three Fusarium genomes. We have also developed the computational program to automatically identify potential secondary metabolic gene clusters. Over 30 such clusters have been identified in each of the three Fusarium genomes. The data was presented at the Society of Industrial Microbiology annual meeting.
Impacts
Fusarium is perhaps the most important genus of plant pathogenic fungi. Together these fungal species cause economically important disease on nearly every species of cultivated plants. Many species also pose a threat to human health and food safety by contaminating agricultural products with harmful mycotoxins. The three Fusarium species to be studied are important plant pathogens to U.S. agriculture and pose threats to human health. The rich and high quality genomic resources generated in the past year will greatly accelerate efforts of scientists worldwide to understand the genetic basis of pathogenicity, virulence, and mycotoxin production in these species. An accurate gene catalogue and species-specific gene sets will shorten the time needed for gene discovery and functional characterization. Many Fusarium species also are notorious for producing mycotoxins, such as trichothecenes or fumonisins, which are harmful to both human and animals. Employing the power of
comparative genomics, we have identified potential secondary metabolite producing gene clusters in these three sequenced Fusarium genomes. In the long run, knowledge gain through such studies will lead to novel strategies to control the contamination of these mycotoxins.
Publications
- Whole genome NCBI release. 2006. F. verticillioides AAIM02000000, F. oxysporum AAXH01000000, and F. graminearum AACM02000000
- Cuomo, C.A., Guldener, U., Xu, J.-R., Trail, F.,Turgeon, B.G., Di Pietro, A.,Walton, J.D., Ma, L.-J., Baker, S.E., Rep, M., Adam, G., Antoniw, J., Baldwin, T., Calvo, S., Chang, Y.-L., DeCaprio, D., Gale, L., Gnerre, S., Goswami, R.S., Hammond-Kosack, K., Harris, L.J., Hilburn, K., Kennell, J.C., Kroken, S., Magnuson, J.K., Mannhaupt, G., Mauceli, E., Mewes, H.-W., Mitterbauer, R., Muehlbauer, G. J., Munsterkotter, M., Nelson, D., O'Donnell, K., Ouellet, T., Qi, W., Quesneville, H., Roncero, M.I.G., Seong, K.-Y., Tetko, I.V., Urban, M., Waalwijk, C., Ward, T.J., Yao, J., Birren, B.W., Kistler, H.C. 2007. The Fusarium graminearum genome reveals a link between localized polymorphism and pathogen specialization. Science 317:1400-1402.
- Uma Shankar Sagaram and Won-Bo Shim. 2007. Fusarium verticillioides GBB1, a gene encoding heterotrimeric G protein subunit, is associated with fumonisin B1 biosynthesis and hyphal development but not with fungal virulence. Molecular Plant Pathology 8: 375-384.
- Ma, L.-J. 2007 (July). Comparative Study of Fusarium Secondary Metabolite Gene Clusters. Society of Industrial Microbiology, Denver Colorado
- Ma, L.-J. W.B. Shim, S. Kang, C. Woloshuk, C.H. Kistler. 2007 (March). Fusarium Comparative Genomics. Fungal Genetic Conference, Asilomar, California
- Shim, W. B., C. Woloshuk, and L.-J. Ma. 2007 (January). Functional Genomics of Fusarium vertcillioides Secondary Metabolism. Fungal Genomics Workshop. Plant & Animal Genome XV. The International Conference on the Status of Plant and Animal Genome Research. San Diego, CA
- Ma, L.-J. 2006 (November). Comparative genomics of Fusarium verticillioids and F. graminearum. 10th International Symposium on Toxic Microorganisms. College Park, MD, USA
- Ma, L.-J., W.B. Shim, S. Kang, C. Woloshuk, C.H. Kistler. 2006 (September). Fusarium comparative genomics. International conference on Advances on genomics, biodiversity and rapid systems for detection of toxigenic fungi and mycotoxins. Monopoli (Bari) Italy.
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Progress 09/01/05 to 08/31/06
Outputs
A major accomplishment at this point is the public release of the genome assembly of Fusarium verticillioides. The data can be accessed at: http://www.broad.mit.edu/annotation/genome/fusarium_verticillioides.2 /Home.html. The genome was sequenced using the Whole Genome Shotgun approach to 8X sequence coverage and over 60X physical coverage. This assembly combines 4X sequence reads generated by the Broad using this grant and 4X sequence reads contributed by Syngenta. The traces of all reads can be downloaded from the NCBI trace repository. This assembly has been deposited at GenBank under the project accession AAIM02000000. This high quality assembly contains 41.7 million assembled bases and offers high base pair accuracy and long-range continuity. Specifically, 99.5% of the assembled bases have the quality score greater or equal to q40. More than 50% of all assembled bases are contained in scaffolds of 1.96 Mb or longer, and about 99% of the assembly exists in the
scaffold greater than 200 kb. We also obtained the plasmid library that contains the random fragment length polymorphism markers used to create a F. verticillioides genetic map by Xu and Leslie. By sequencing these markers, we were able to anchor more than 90% of the genome assembly on the genetic map. On average, each mapped linkage group contains only two scaffolds. In addition, we have mapped 87,000 F. verticillioides ESTs generated by scientists at National Center for Agricultural Utilization Research of USDA. The public release of the F. verticillioides genome allows users to download the consensus sequence and additional files for the genome assembly; to conduct BLAST searches against the genomic and mitochondrial assemblies; to search and visualize features including alignments of BLAST, ESTs, and pfam domains; gene predictions based on the programs FGGENSH, GENEID, and GENEWISE; and to access the combined genetic and physical map.
Impacts
Fusarium verticillioides is the causal agent of kernel and ear rot of maize, a destructive disease where ever maize is grown. The most detrimental economic impact of F. verticillioides is due to its ability to produce fumonisins that can cause various illnesses in humans and animals. The availability of the genome sequence data of this pathogen will allow the Fusarium research community to take functional genomics approaches for studying the genes and signaling pathways involved in the synthesis and regulation of this and other mycotoxins. In the long run, knowledge gain through such studies will lead to novel strategies to control the contamination of these toxins in maize. In addition, completion of this F. verticillioides genome project is the initial step toward Fusarium comparative genomic analyses. The high quality assembly and the large amount of ESTs will aid us in improving the annotation of other Fusarium genomes that have been sequenced (F. graminearum and
F. solani) or currently being sequenced (F. oxysporum). Their genome sequences will allow us to study genome dynamics among these phylogentically closely related, but organizationally significantly different genomes, and to understand the evolutionary mechanisms underlying these processes.
Publications
- Ma, L.-J., W.B. Shim, S. Kang, C. Woloshuk, C.H. Kistler. Fusarium comparative genomics. International conference on Advances on genomics, biodiversity and rapid systems for detection of toxigenic fungi and mycotoxins. Monopoli (Bari) Italy. September 2006.
- Ma, L.-J., W.B. Shim, S. Kang, C. Woloshuk, C.H. Kistler. Comparative genomics of plant pathogenic Funsarium species. Microbial Genome Sequencing Program Awardee Workshop. San Diego, January 2006.
- Birren,B., Lander,E., Galagan,J., Nusbaum,C., Devon,K., Ma,L.-J., Jaffe,D., Butler,J., Alvarez,P., Gnerre,S., Grabherr,M., Kleber,M., Mauceli,E., Brockman,W., MacCallum,I.A., Young,S., LaButti,K., DeCaprio,D., Crawford,M., Koehrsen,M., Engels,R., Montgomery,P., Pearson,M., Howarth,C., Larson,L., White,J., O'Leary,S., Kodira,C., Zeng,Q., Yandava,C., Alvarado,L., Amedeo,P., Briggs,S., Baker,S., Goff,S., Hohn,T., Hutchison,D., Lam,S., Martin,C., Miguel,T., Rose,M., Turgeon,B.G., Wu,G., Yoder,O., Zheng,L., Kistler,C., Shim,W.-B., Kang,S. and Woloshuk,C. 2006, NCBI Direct Submission AAIM02000000.
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