JOURNAL BROWSE
Search
Advanced SearchSearch Tips
Multi-Homologous Recombination-Based Gene Manipulation in the Rice Pathogen Fusarium fujikuroi
facebook(new window)  Pirnt(new window) E-mail(new window) Excel Download
  • Journal title : The Plant Pathology Journal
  • Volume 32, Issue 3,  2016, pp.173-181
  • Publisher : Korean Society of Plant Pathology
  • DOI : 10.5423/PPJ.OA.12.2015.0263
 Title & Authors
Multi-Homologous Recombination-Based Gene Manipulation in the Rice Pathogen Fusarium fujikuroi
Hwang, In Sun; Ahn, Il-Pyung;
  PDF(new window)
 Abstract
Gene disruption by homologous recombination is widely used to investigate and analyze the function of genes in Fusarium fujikuroi, a fungus that causes bakanae disease and root rot symptoms in rice. To generate gene deletion constructs, the use of conventional cloning methods, which rely on restriction enzymes and ligases, has had limited success due to a lack of unique restriction enzyme sites. Although strategies that avoid the use of restriction enzymes have been employed to overcome this issue, these methods require complicated PCR steps or are frequently inefficient. Here, we introduce a cloning system that utilizes multi-fragment assembly by In-Fusion to generate a gene disruption construct. This method utilizes DNA fragment fusion and requires only one PCR step and one reaction for construction. Using this strategy, a gene disruption construct for Fusarium cyclin C1 (FCC1), which is associated with fumonisin B1 bio-synthesis, was successfully created and used for fungal transformation. In vivo and in vitro experiments using confirmed fcc1 mutants suggest that fumonisin production is closely related to disease symptoms exhibited by F. fujikuroi strain B14. Taken together, this multi-fragment assembly method represents a simpler and a more convenient process for targeted gene disruption in fungi.
 Keywords
FCC1;Fusarium fujikuroi;gene deletion;homologous recombination;multi-fragment assembly;
 Language
English
 Cited by
 References
1.
Aouida, M., Piatek, M. J., Bangarusamy, D. K. and Mahfouz, M. M. 2014. Activities and specificities of homodimeric TALENs in Saccharomyces cerevisiae. Curr. Genet. 60:61-74. crossref(new window)

2.
Bomke, C., Rojas, M. C., Hedden, P. and Tudzynski, B. 2008. Loss of gibberellin production in Fusarium verticillioides (Gibberella fujikuroi MP-A) is due to a deletion in the gibberellic acid gene cluster. Appl. Environ. Microbiol. 74:7790-7801. crossref(new window)

3.
Bundock, P. and Hooykaas, P. J. 1996. Integration of Agrobacterium tumefaciens T-DNA in the Saccharomyces cerevisiae genome by illegitimate recombination. Proc. Natl. Acad. Sci. U. S. A. 93:15272-15275. crossref(new window)

4.
Cruz, A., Marin, P., Gonzalez-Jaen, M. T., Aguilar, K. G. and Cumagun, C. J. 2013. Phylogenetic analysis, fumonisin production and pathogenicity of Fusarium fujikuroi strains isolated from rice in the Philippines. J. Sci. Food Agric. 93:3032-3039. crossref(new window)

5.
Desjardins, A. E., Manandhar, H. K., Plattner, R. D., Manandhar, G. G., Poling, S. M. and Maragos, C. M. 2000. Fusarium species from Nepalese rice and production of mycotoxins and gibberellic acid by selected species. Appl. Environ. Microbiol. 66:1020-1025. crossref(new window)

6.
Frandsen, R. J. 2011. A guide to binary vectors and strategies for targeted genome modification in fungi using Agrobacterium tumefaciens-mediated transformation. J. Microbiol. Methods 87:247-262. crossref(new window)

7.
Fu, C., Donovan, W. P., Shikapwashya-Hasser, O., Ye, X. and Cole, R. H. 2014. Hot fusion: an efficient method to clone multiple DNA fragments as well as inverted repeats without ligase. PLoS One 9:e115318. crossref(new window)

8.
Huang, W., Yang, H. and Qin, H. 2011. Agrobacterium tumefaciens mediated transformation of Aspergillus niger conidiospores and sequence analysis of T-DNA insertion site in conidiation-defective mutant. Wei Sheng Wu Xue Bao 51:270-275.

9.
Hur, Y.-J., Lee, S. B., Kim, T. H., Kwon, T. M., Lee, J.-H., Shin, D.-J., Park, S.-K., Hwang, U.-H., Cho, J. H., Yoon, Y.-N., Yeo, U.-S., Song, Y.-C., Kwak, D.-Y., Nam, M.-H. and Park, D.-S. 2015. Mapping of qBK1, a major QTL for bakanae disease resistance in rice. Mol. Breed. 35:78. crossref(new window)

10.
Hwang, I. S., Kang, W. R., Hwang, D. J., Bae, S. C., Yun, S. H. and Ahn I. P. 2013. Evaluation of bakanae disease progression caused by Fusarium fujikuroi in Oryza sativa L. J. Microbiol. 51:858-865. crossref(new window)

11.
Jeon, J., Park, S. Y., Chi, M. H., Choi, J., Park, J., Rho, H. S., Kim, S., Goh, J., Yoo, S., Choi, J., Park, J. Y., Yi, M., Yang, S., Kwon, M. J., Han, S. S., Kim, B. R., Khang, C. H., Park, B., Lim, S. E., Jung, K., Kong, S., Karunakaran, M., Oh, H. S., Kim, H., Kim, S., Park, J., Kang, S., Choi, W. B., Kang, S. and Lee, Y. H. 2007. Genome-wide functional analysis of pathogenicity genes in the rice blast fungus. Nat. Genet. 39:561-565. crossref(new window)

12.
Jeon, Y. A., Yu, S. H., Lee, Y. Y., Park, H. J., Lee, S., Sung, J. S., Kim, Y. G. and Lee, H. S. 2013. Incidence, molecular characteristics and pathogenicity of Gibberella fujikuroi species complex associated with rice seeds from Asian Countries. Mycobiology 41:225-233. crossref(new window)

13.
Jeong, H., Lee, S., Choi, G. J., Lee, T. and Yun, S. H. 2013. Draft genome sequence of Fusarium fujikuroi B14, the causal agent of the bakanae disease of rice. Genome Announc. 1:e00035-13.

14.
Joska, T. M., Mashruwala, A., Boyd, J. M. and Belden, W. J. 2014. A universal cloning method based on yeast homologous recombination that is simple, efficient, and versatile. J. Microbiol. Methods 100:46-51. crossref(new window)

15.
Kim, M. S., Kim, S. Y., Yoon, J. K., Lee, Y. W. and Bahn, Y. S. 2009. An efficient gene-disruption method in Cryptococcus neoformans by double-joint PCR with NAT-split markers. Biochem. Biophys. Res. Commun. 390:983-988. crossref(new window)

16.
Kim, S, Ahn, I. P., Rho. H. S. and Lee, Y. H. 2005. MHP1, a Magnaporthe grisea hydrophobin gene, is required for fungal development and plant colonization. Mol. Microbiol. 57:1224-1237. crossref(new window)

17.
Kuwayama, H., Obara, S., Morio, T., Katoh, M., Urushihara, H. and Tanaka, Y. 2002. PCR-mediated generation of a gene disruption construct without the use of DNA ligase and plasmid vectors. Nucleic Acids Res. 30:e2. crossref(new window)

18.
Laughery, M. F., Hunter, T., Brown, A., Hoopes, J., Ostbye, T., Shumaker, T. and Wyrick, J. J. 2015. New vectors for simple and streamlined CRISPR-Cas9 genome editing in Saccharomyces cerevisiae. Yeast 32:711-720. crossref(new window)

19.
Leslie, J. F. and Summerell, B. A. 2006. The Fusarium laboratory manual. Blackwell, Ames, IA, USA.

20.
Mans, R., van Rossum, H. M., Wijsman, M., Backx, A., Kuijpers, N. G., van den Broek, M., Daran-Lapujade, P., Pronk, J. T., van Maris, A. J. and Daran, J. M. 2015. CRISPR/Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae. FEMS Yeast Res. 15. doi: 10.1093/femsyr/fov004. crossref(new window)

21.
Michielse, C. B., Hooykaas, P. J., van den Hondel, C. A. and Ram, A. F. 2005. Agrobacterium-mediated transformation as a tool for functional genomics in fungi. Curr. Genet. 48:1-17. crossref(new window)

22.
Piers, K. L., Heath, J. D., Liang, X., Stephens, K. M. and Nester, E. W. 1996. Agrobacterium tumefaciens-mediated transformation of yeast. Proc. Natl. Acad. Sci. U. S. A. 93:1613-1618. crossref(new window)

23.
Raman, M. and Martin, K. 2014. One solution for cloning and mutagenesis: In-Fusion HD Cloning Plus. Nat. Methods 11.

24.
Sahoo, K. K., Tripathi, A. K., Pareek, A., Sopory, S. K. and Singla-Pareek, S. L. 2011. An improved protocol for efficient transformation and regeneration of diverse indica rice cultivars. Plant Methods 7:49. crossref(new window)

25.
Schirawski, J., Mannhaupt, G., Munch, K., Brefort, T., Schipper, K., Doehlemann, G., Di Stasio, M., Rossel, N., Mendoza-Mendoza, A., Pester, D., Muller, O., Winterberg, B., Meyer, E., Ghareeb, H., Wollenberg, T., Munsterkotter, M., Wong, P., Walter, M., Stukenbrock, E., Guldener, U. and Kahmann, R. 2010. Pathogenicity determinants in smut fungi revealed by genome comparison. Science 330:1546-1548. crossref(new window)

26.
Shim, W. B. and Woloshuk, C. P. 2001. Regulation of fumonisin B1 biosynthesis and conidiation in Fusarium verticillioides by a cyclin-like (C-type) gene, FCC1. Appl. Environ. Microbiol. 67:1607-1612. crossref(new window)

27.
Takahara, H., Tsuji, G., Kubo, Y., Yamamoto, M., Toyoda, K., Inagaki, Y., Ichinose, Y. and Shiraishi, T. 2004. Agrobacterium tumefaciens-mediated transformation as a tool for random mutagenesis of Colletotrichum trifolii. J. Gen. Plant Pathol. 70:93-96. crossref(new window)

28.
Tudzynski, B. 1999. Biosynthesis of gibberellins in Gibberella fujikuroi: biomolecular aspects. Appl. Microbiol. Biotechnol. 52:298-310. crossref(new window)

29.
Tudzynski, B. 2005. Gibberellin biosynthesis in fungi: genes, enzymes, evolution, and impact on biotechnology. Appl. Microbiol. Biotechnol. 66:597-611. crossref(new window)

30.
Tudzynski, B., Kawaide, H. and Kamiya, Y. 1998. Gibberellin biosynthesis in Gibberella fujikuroi: cloning and characterization of the copalyl diphosphate synthase gene. Curr. Genet. 34:234-240. crossref(new window)

31.
Tudzynski, B., Mihlan, M., Rojas, M. C., Linnemannstons, P., Gaskin, P. and Hedden, P. 2003. Characterization of the final two genes of the gibberellin biosynthesis gene cluster of Gibberella fujikuroi: des and P450-3 encode GA4 desaturase and the 13-hydroxylase, respectively. J. Biol. Chem. 278:28635-28643. crossref(new window)

32.
Wiemann, P., Sieber, C. M., von Bargen, K. W., Studt, L., Niehaus, E. M., Espino, J. J., Huss, K., Michielse, C. B., Albermann, S., Wagner, D., Bergner, S. V., Connolly, L. R., Fischer, A., Reuter, G., Kleigrewe, K., Bald, T., Wingfield, B. D., Ophir, R., Freeman, S., Hippler, M., Smith, K. M., Brown, D. W., Proctor, R. H., Münsterkötter, M., Freitag, M., Humpf, H. U., Guldener, U. and Tudzynski, B. 2013. Deciphering the cryptic genome: genome-wide analyses of the rice pathogen Fusarium fujikuroi reveal complex regulation of secondary metabolism and novel metabolites. PLoS Pathog. 9:e1003475. crossref(new window)

33.
Yu, J. H., Hamari, Z., Han, K. H., Seo, J. A., Reyes-Dominguez, Y. and Scazzocchio, C. 2004. Double-joint PCR: a PCRbased molecular tool for gene manipulations in filamentous fungi. Fungal Genet. Biol. 41:973-981. crossref(new window)

34.
Zhu, B., Cai, G., Hall, E. O. and Freeman, G. J. 2007. In-Fusion assembly: seamless engineering of multidomain fusion proteins, modular vectors, and mutations. Biotechniques 43:354-359. crossref(new window)