Abstract
Rapid translation of genome sequences into meaningful biological information hinges on the integration of multiple experimental and informatics methods into a cohesive platform. Despite the explosion in the number of genome sequences available1, such a platform does not exist for filamentous fungi. Here we present the development and application of a functional genomics and informatics platform for a model plant pathogenic fungus, Magnaporthe oryzae2. In total, we produced 21,070 mutants through large-scale insertional mutagenesis using Agrobacterium tumefaciens–mediated transformation3. We used a high-throughput phenotype screening pipeline to detect disruption of seven phenotypes encompassing the fungal life cycle and identified the mutated gene and the nature of mutation for each mutant. Comparative analysis of phenotypes and genotypes of the mutants uncovered 202 new pathogenicity loci. Our findings demonstrate the effectiveness of our platform and provide new insights on the molecular basis of fungal pathogenesis. Our approach promises comprehensive functional genomics in filamentous fungi and beyond.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Galagan, J.E., Henn, M.R., Ma, L.J., Cuomo, C.A. & Birren, B. Genomics of the fungal kingdom: insights into eukaryotic biology. Genome Res. 15, 1620–1631 (2005).
Couch, B.C. & Kohn, L.M. A multilocus gene genealogy concordant with host preference indicates segregation of new species, Magnaporthe oryzae from M. grisea. Mycologia 94, 683–693 (2002).
Rho, H.S., Kang, S. & Lee, Y.H. Agrobacterium tumefaciens-mediated transformation of the plant pathogenic fungus, Magnaporthe grisea. Mol. Cells 12, 407–411 (2001).
Giaever, G. et al. Functional profiling of the Saccharomyces cerevisiae genome. Nature 418, 387–391 (2002).
Winzeler, E.A. et al. Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285, 901–906 (1999).
Bevan, M. & Walsh, S. The Arabidopsis genome: a foundation for plant research. Genome Res. 15, 1632–1642 (2005).
Hirochika, H. et al. Rice mutant resources for gene discovery. Plant Mol. Biol. 54, 325–334 (2004).
Andres, A.J. Flying through the genome: a comprehensive study of functional genomics using RNAi in Drosophila. Trends Endocrinol. Metab. 15, 243–247 (2004).
Ashrafi, K. et al. Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory genes. Nature 421, 268–272 (2003).
Berns, K. et al. A large-scale RNAi screen in human cells identifies new components of the p53 pathway. Nature 428, 431–437 (2004).
Talbot, N.J. On the trail of a cereal killer: Exploring the biology of Magnaporthe grisea. Annu. Rev. Microbiol. 57, 177–202 (2003).
Kwon-Chung, K.J. & Bennett, J.E. Medical Mycology (Lea and Febiger, Philadelphia, 1992).
Dean, R.A. et al. The genome sequence of the rice blast fungus Magnaporthe grisea. Nature 434, 980–986 (2005).
Yu, J. et al. A draft sequence of the rice genome (Oryza sativa L. ssp. indica). Science 296, 79–92 (2002).
Goff, S.A. et al. A draft sequence of the rice genome (Oryza sativa L. ssp. japonica). Science 296, 92–100 (2002).
Krysan, P.J., Young, J.C. & Sussman, M.R. T-DNA as an insertional mutagen in Arabidopsis. Plant Cell 11, 2283–2290 (1999).
Idnurm, A. & Howlett, B.J. Pathogenicity genes of phytopathogenic fungi. Mol. Plant Pathol. 2, 241–255 (2001).
Liu, Y.G. & Whittier, R.F. Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 25, 674–681 (1995).
Foster, A.J., Jenkinson, J.M. & Talbot, N.J. Trehalose synthesis and metabolism are required at different stages of plant infection by Magnaporthe grisea. EMBO J. 22, 225–235 (2003).
Xu, J.R., Staiger, C.J. & Hamer, J.E. Inactivation of the mitogen-activated protein kinase Mps1 from the rice blast fungus prevents penetration of host cells but allows activation of plant defense responses. Proc. Natl. Acad. Sci. USA 95, 12713–12718 (1998).
Bhambra, G.K., Wang, Z.Y., Soanes, D.M., Wakley, G.E. & Talbot, N.J. Peroxisomal carnitine acetyl transferase is required for elaboration of penetration hyphae during plant infection by Magnaporthe grisea. Mol. Microbiol. 61, 46–60 (2006).
Chen, C. & Dickman, M.B. Dominant active Rac and dominant negative Rac revert the dominant active Ras phenotype in Colletotrichum trifolii by distinct signalling pathways. Mol. Microbiol. 51, 1493–1507 (2004).
Semenza, J.C., Hardwick, K.G., Dean, N. & Pelham, H.R. ERD2, a yeast gene required for the receptor-mediated retrieval of luminal ER proteins from the secretory pathway. Cell 61, 1349–1357 (1990).
Tani, S. et al. Characterization of the amyR gene encoding a transcriptional activator for the amylase genes in Aspergillus nidulans. Curr. Genet. 39, 10–15 (2001).
Lee, K.S. & Levin, D.E. Dominant mutations in a gene encoding a putative protein kinase (BCK1) bypass the requirement for a Saccharomyces cerevisiae protein kinase C homolog. Mol. Cell. Biol. 12, 172–182 (1992).
Donofrio, N. et al. 'PACLIMS': a component LIM system for high-throughput functional genomic analysis. BMC Bioinformatics 10.1186/1471–2105–6-94 (2005).
Baker, E.J., Galloway, L., Jackson, B., Schmoyer, D. & Snoddy, J. MuTrack: a genome analysis system for large-scale mutagenesis in the mouse. BMC Bioinformatics 10.1186/1471–2105–5-11 (2004).
Mullins, E.D. & Kang, S. Transformation: a tool for studying fungal pathogens of plants. Cell. Mol. Life Sci. 58, 2043–2052 (2001).
Rogers, S.O. & Bendich, A.J. Extraction of DNA from milligram amount of fresh, herbarium, and mummified plant tissue. Plant Mol. Biol. 5, 69–76 (1985).
Yu, J.H. et al. Double-joint PCR: a PCR-based molecular tool for gene manipulations in filamentous fungi. Fungal Genet. Biol. 41, 973–981 (2004).
Acknowledgements
We are grateful to K. Lee and N.J. Talbot for their comments and suggestions on the manuscript. This research was partially supported by a grant from the Biogreen21 project funded by the Rural Development Administration, by grants from the Crop Functional Genomics Center (CG1421) and the Microbial Genomics and Applications Center (0462-20060021) of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology, and by Korean Research Foundation Grant (KRF-2004-005-F00013) to Y.H.L. Requests for materials should be addressed to Y.H.L. (yonglee@snu.ac.kr).
Author information
Authors and Affiliations
Contributions
S.-Y.P., M.-H.C., J.J., H.-S.R., S.K., J.G. and S.Y. generated the mutants and performed high-throughput phenotype screening. J.C., J.-Y.P., M.Y., S.Y., S.-E.L. and M.-J.K. assisted in phenotype assessment. J.P., K.J., S.K., S.K., J.P., B.P. and S.K. developed the ATMT database. J.J., M.-H.C., S.Y., J.G., M.K. and W.-B.C. performed targeted knockout of the selected ORFs. S.-S.H. and B.R.K. performed pathogenicity tests on pot-grown rice plants. J.C., J.J., J.G., S.Y. and M.-H.C. performed TAIL PCR and sequence analysis. J.J., C.H.K., H.-S.O., H.K., S.K., S.K. and Y.-H.L. contributed to the writing of this paper. Y.-H.L. designed and directed this study.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Supplementary information
Supplementary Table 1
Summary of high-throughput phenotype screening and selection of mutants. (PDF 43 kb)
Supplementary Table 2
Phenotypes and genes affected by T-DNA insertions in ATMT mutants. (PDF 384 kb)
Supplementary Table 3
Predicted protein and genome sequence source for 48 fungal species. (PDF 55 kb)
Supplementary Table 4
In-depth phenptype analysis and T-DNA-tagged locations of pathogenicity-defective mutations. (PDF 836 kb)
Supplementary Table 5
Analysis of the linkage between T-DNA insertion and pathogenicity defects by targeted disruption. (PDF 268 kb)
Rights and permissions
About this article
Cite this article
Jeon, J., Park, SY., Chi, MH. et al. Genome-wide functional analysis of pathogenicity genes in the rice blast fungus. Nat Genet 39, 561–565 (2007). https://doi.org/10.1038/ng2002
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/ng2002
This article is cited by
-
Current perspective on production and applications of microbial cellulases: a review
Bioresources and Bioprocessing (2021)
-
Genfunktionen effizient identifizieren mit iPool-seq
BIOspektrum (2020)
-
Genome-wide study of saprotrophy-related genes in the basal fungus Conidiobolus heterosporus
Applied Microbiology and Biotechnology (2020)
-
Genetic diversity and pathogenicity dynamics of Magnaporthe oryzae in the Wuling Mountain area of China
European Journal of Plant Pathology (2019)
-
Comparative genomic analysis revealed rapid differentiation in the pathogenicity-related gene repertoires between Pyricularia oryzae and Pyricularia penniseti isolated from a Pennisetum grass
BMC Genomics (2018)