Skip to main content
SpringerLink
Log in

Efficient Agrobacterium tumefaciens-mediated target gene disruption in the maize pathogen Curvularia lunata

  • Published:
European Journal of Plant Pathology Aims and scope Submit manuscript

Abstract

Curvularia lunata causes Curvularia leaf spot disease of maize, resulting in periodically significant yield losses around the world. To understand the molecular mechanisms of fungal pathogenicity and virulence factors contributed to the host, here we report a knockout transformation system against target genes. The concentration of conidia, Agrobacterium cell density, and method of co-incubation were optimized for deletion of the gene encoding 1, 3, 8-trihydroxynaphthalene reductase (Brn1), a gene in the melanin biosynthesis pathway, as a test case. Transformants contained a single T-DNA copy. The Brn1 mutant strain was reduced in virulence compared with the wild type strain when inoculated on susceptible maize. Transformation efficiency was 130 ± 10 transformants per 1 × 105 germlings and homologous recombination efficiency was 60.0 to 100.0 %.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Amberg, D. C., Botstein, D., & Beasley, E. M. (1995). Precise gene disruption in Saccharomyces cerevisiae by double fusion polymerase chain reaction. Yeast, 11, 1275–1280.

    Article  CAS  PubMed  Google Scholar 

  • Aronson, B. D., Lindgren, K. M., Dunlap, J. C., & Loros, J. J. (1994). An efficient method for gene disruption in Neurospora crassa. Molecular and General Genetics, 242, 490–494.

    Article  CAS  PubMed  Google Scholar 

  • Bird, D., & Bradshaw, R. (1997). Gene targeting is locus dependent in the filamentous fungus Aspergillus nidulans. Molecular and General Genetics, 255, 219–225.

    Article  CAS  PubMed  Google Scholar 

  • Bundock, P., Den Dulk Ras, A., Beijersbergen, A., & Hooykaas, P. J. J. (1995). Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae. EMBO Journal, 14, 3206–3214.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Cooley, R., Shaw, R., Franklin, F., & Caten, C. (1988). Transformation of the phytopathogenic fungus Septoria nodorum to hygromycin B resistance. Current Genetics, 13, 383–389.

    Article  CAS  Google Scholar 

  • Dai, F. C., Gao, W. D., Wu, R. J., & Jin, X. H. (1995). A noticeable corn disease: Curvularia leaf spot. ACTA Phytopathologica Sinica, 25, 330.

    Google Scholar 

  • Dai, F. C., Wang, X. M., Zhu, Z. D., Gao, W. D., & Huo, N. X. (1998). Curvularia leaf spot of maize: pathogens and varietal resistance. ACTA Phytopathologica Sinica, 2, 123–129.

    Google Scholar 

  • de Groot, M. J., Bundock, P., Hooykaas, P. J., & Beijersbergen, A. G. (1998). Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nature Biotechnology, 16, 839–842.

    Article  PubMed  Google Scholar 

  • Gao, S. G., Li, Y. Q., Gao, J. X., Suo, Y. J., & Fu, K. H. (2014). Genome sequence and virulence variation-related transcriptome profiles of Curvularia lunata, an important maize pathogenic fungus. BMC Genomics, 15, 627–645.

    Article  PubMed  PubMed Central  Google Scholar 

  • Henson, J., Blake, N., & Pilgeram, A. (1988). Transformation of Gaeumannomyces graminis to benomyl resistance. Current Genetics, 14, 113–118.

    Article  CAS  Google Scholar 

  • Huang, W. D., Xue, C. S., Zhao, Z. W., & Chen, J. (2010). Agrobacterium tumefaciens-mediated transformation of Curvularia lunata. Hubei Agricultural Sciences, 49, 1537–1539.

    Google Scholar 

  • Kistler, H., & Benny, U. (1988). Genetic transformation of the fungal plant wilt pathogen Fusarium oxysporum. Current Genetics, 13, 145–150.

    Article  CAS  Google Scholar 

  • Li, Y., Dai, F. C., Jing, R. L., Wang, T. Y., Du, J. Y., & Jia, J. Z. (2002). QTL analysis of resistance to Curvularia lunata in maize. Scientia Agricultura Sinica, 35, 1221–1227.

    Google Scholar 

  • Liu, T., Liu, L. X., Jiang, X., Hou, J. M., Fu, K. H., Zhou, F. H., & Chen, J. (2010). Agrobacterium-mediated transformation as a useful tool for the molecular genetic study of the phytopathogen Curvularia lunata. European Journal of Plant Pathology, 126, 363–371.

    Article  CAS  Google Scholar 

  • Michielse, C. B., Arentshorst, M., Ram, A. F., & Van Den Hondel, C. A. (2005). Agrobacterium-mediated transformation leads to improved gene replacement efficiency in Aspergillus awamori. Fungal Genetics and Biology, 42, 9–19.

    Article  CAS  PubMed  Google Scholar 

  • Mullins, E. D., & Kang, S. (2001). Transformation: a tool for studying fungal pathogens of plants. Cellular and Molecular Life Sciences, 58, 2043–2052.

    Article  CAS  PubMed  Google Scholar 

  • Oide, S., Moeder, W., Krasnoff, S., Gibson, D., Haas, H., Yoshioka, K., & Turgeon, B. G. (2006). NPS6, encoding a nonribosomal peptide synthetase involved in siderophore-mediated iron metabolism, is a conserved virulence determinant of plant pathogenic Ascomycetes. Plant Cell, 18, 2836–2853.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Parsons, K. A., Chumley, F. G., & Valent, B. (1987). Genetic transformation of the fungal pathogen responsible for rice blast disease. Proceedings of the National Academy of Sciences USA, 84, 4161–4165.

    Article  CAS  Google Scholar 

  • Rodriguez, R. J., & Yoder, O. C. (1987). Selectable genes for transformation of the fungal plant pathogen Glomerella cingulata f. sp. phaseoli (Colletotrichum lindemuthianum). Gene, 54, 73–81.

    Article  CAS  PubMed  Google Scholar 

  • Sambrook, J., & Russell, D. W. (Eds.). (2001). Molecular cloning: A laboratory manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.

    Google Scholar 

  • Sugui, J. A., Chang, Y. C., & Kwon-Chung, K. J. (2005). Agrobacterium tumefaciens-mediated transformation of Aspergillus fumigatus: an efficient tool for insertional mutagenesis and targeted gene disruption. Applied and Environment Microbiology, 71, 1798–1802.

    Article  CAS  Google Scholar 

  • Thompson, J. E., Fahnestock, S., Farrall, L., Liao, D. I., Valent, B., & Jordan, D. B. (2000). The second naphthol reductase of fungal melanin biosynthesis in Magnaporthe grisea: tetrahydroxynaphthalene reductase. Journal of Biological Chemistry, 275, 34867–34872.

    Article  CAS  PubMed  Google Scholar 

  • Turgeon, B. G., Garber, R. C., & Yoder, O. C. (1985). Transformation of the fungal maize pathogen Cochliobolus heterostrophus using the Aspergillus nidulans amdS gene. Molecular Genetics and Genomics, 201, 450–453.

    Article  CAS  Google Scholar 

  • Xue, C. S., Xiao, S. Q., Zhai, Y. H., Gao, Y., Gao, Z. G., & Chen, J. (2008). Pathogenicity differentiation of Curvularia lunata. Acta Phytopathologica Sinica, 38, 6–12.

    Google Scholar 

  • Xue, C. S., Wu, D. L., Condon, B. J., Bi, Q., Wang, W. W., & Turgeon, B. G. (2013). Efficient gene knockout in the maize pathogen Setosphaeria turcica using Agrobacterium tumefaciens-mediated transformation. Phytopathology, 103, 641–647.

    Article  CAS  PubMed  Google Scholar 

  • Zhao, J., Wang, G. Y., Hu, J., Zhang, X. H., & Dai, J. R. (2002). Genetic analysis of maize resistance to Curvularia leaf spot by ADAA model. Acta Agronomica Sinica, 28, 127–130.

    Google Scholar 

Download references

Acknowledgments

This study was supported by the National Natural Science Foundation of China (grant number 31271992). We gratefully thank Dr. Gillian Turgeon, Department of Plant Pathology & Plant-Microbe Biology, Cornell University, for providing the plasmid pPZP100.

Author information

Authors and Affiliations

  1. College of Plant Protection, Shenyang Agricultural University, Shenyang, Liaoning Province, 110866, China

    Li Zhang, Hui Li, Shuqin Xiao, Yuanyuan Lu, Guofu Li & Chunsheng Xue

  2. Department of Resources and Environmental Sciences, School of Agriculture and Biology, Shanghai Jiaotong University, Shanghai, 200240, China

    Jie Chen

Authors
  1. Li Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hui Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shuqin Xiao
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yuanyuan Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Guofu Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Chunsheng Xue
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jie Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jie Chen.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Table 1

(DOC 35 kb)

Supplementary Figure 1

(DOC 193 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhang, L., Li, H., Xiao, S. et al. Efficient Agrobacterium tumefaciens-mediated target gene disruption in the maize pathogen Curvularia lunata . Eur J Plant Pathol 145, 155–165 (2016). https://doi.org/10.1007/s10658-015-0825-2

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10658-015-0825-2

Keywords

Search

Navigation