Genome-wide functional analysis of pathogenicity genes in the rice blast fungus

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.

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Figure 1: Schematic diagram of the high-throughput screening system and the life cycle of Magnaporthe oryzae.
Figure 2: Composition and pairing analysis of phenotypes among pathogenicity-defective mutants.
Figure 3: Distribution of T-DNA insertions over the chromosomes and functional categorization of the T-DNA–tagged genes.
Figure 4: Representative phenotypes of the mutants.

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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).

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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.

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Correspondence to Yong-Hwan Lee.

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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)

Supplementary Methods (PDF 74 kb)

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Jeon, J., Park, S., Chi, M. 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

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