Abstract
Aspergillus flavus often invade many important corps and produce harmful aflatoxins both in preharvest and during storage stages. The regulation mechanism of aflatoxin biosynthesis in this fungus has not been well explored mainly due to the lack of an efficient transformation method for constructing a genome-wide gene mutant library. This challenge was resolved in this study, where a reliable and efficient Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for A. flavus NRRL 3357 was established. The results showed that removal of multinucleate conidia, to collect a homogenous sample of uninucleate conidia for use as the transformation material, is the key step in this procedure. A. tumefaciens strain AGL-1 harboring the ble gene for zeocin resistance under the control of the gpdA promoter from A. nidulans is suitable for genetic transformation of this fungus. We successfully generated A. flavus transformants with an efficiency of ∼ 60 positive transformants per 106 conidia using our protocol. A small-scale insertional mutant library (∼ 1,000 mutants) was constructed using this method and the resulting several mutants lacked both production of conidia and aflatoxin biosynthesis capacity. Southern blotting analysis demonstrated that the majority of the transformants contained a single T-DNA insert on the genome. To the best of our knowledge, this is the first report of genetic transformation of A. flavus via ATMT and our protocol provides an effective tool for construction of genome-wide gene mutant libraries for functional analysis of important genes in A. flavus.
Similar content being viewed by others
References
Affeldt, K.J., Brodhagen, M., and Keller, N.P. 2012. Aspergillus oxylipin signaling and quorum sensing pathways depend on G protein-coupled receptors. Toxins 4, 695–717.
Affeldt, K.J., Carrig, J., Amare, M., and Keller, N.P. 2014. Global survey of canonical Aspergillus flavus G protein-coupled receptors. mBio 5, e01501–14.
Allen, G.C., Flores-Vergara, M.A., Krasnyanski, S., Kumar, S., and Thompson, W.F. 2006. A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide. Nat. Protoc. 1, 2320–2325.
Baird, G.S., Zacharias, D.A., and Tsien, R.Y. 2000. Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc. Natl. Acad. Sci. USA 97, 11984–11989.
Bhatnagar, D., Ehrlich, K.C., and Cleveland, T.E. 2003. Molecular genetic analysis and regulation of aflatoxin biosynthesis. Appl. Microbiol. Biotechnol. 61, 83–93.
Chen, X.L., Yang, J., and Peng, Y.L. 2011. Large-scale insertional mutagenesis in Magnaporthe oryzae by Agrobacterium tumefaciens-mediated transformation. Methods Mol. Biol. 722, 213–224.
Colot, H.V., Park, G., Turner, G.E., Ringelberg, C., Crew, C.M., Litvinkova, L., Weiss, R.L., Borkovich, K.A., and Dunlap, J.C. 2006. A high-throughput gene knockout procedure for neurospora reveals functions for multiple transcription factors. Proc. Natl. Acad. Sci. USA 103, 10352–10357.
Crespo-Sempere, A., Lopez-Perez, M., Martinez-Culebras, P.V., and Gonzalez-Candelas, L. 2011. Development of a green fluorescent tagged strain of Aspergillus carbonarius to monitor fungal colonization in grapes. Int. J. Food Microbiol. 148, 135–140.
de Groot, M.J., Bundock, P., Hooykaas, P.J., and Beijersbergen, A.G. 1998. Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nat. Biotechnol. 16, 839–842.
Foudin, L.L., Papa, K.E., and Hanlin, R.T. 1981. Nuclear behavior during conidiogenesis in Aspergillus flavus. Canadian J. Bot. 59, 2116–2120.
Gouka, R.J., Gerk, C., Hooykaas, P.J., Bundock, P., Musters, W., Verrips, C.T., and de Groot, M.J. 1999. Transformation of Aspergillus awamori by Agrobacterium tumefaciens-mediated homologous recombination. Nat. Biotechnol. 17, 598–601.
Hamer, J.E. and Timberlake, W.E. 1987. Functional organization of the Aspergillus nidulans trpC promoter. Mol. Cell. Biol. 7, 2352–2359.
He, Z.M., Price, M.S., OBrian, G.R., Georgianna, D.R., and Payne, G.A. 2007. Improved protocols for functional analysis in the pathogenic fungus Aspergillus flavus. BMC Microbiol. 7, 104.
Jeon, J., Park, S.Y., Chi, M.H., Choi, J., Park, J., Rho, H.S., Kim, S., Goh, J., Yoo, S., Choi, J., et al. 2007. Genome-wide functional analysis of pathogenicity genes in the rice blast fungus. Nat. Genet. 39, 561–565.
Kalleda, N., Naorem, A., and Manchikatla, R.V. 2013. Targeting fungal genes by diced siRNAs: A rapid tool to decipher gene function in Aspergillus nidulans. PLoS One 8, e75443.
Klich, M.A. 2007. Aspergillus flavus: The major producer of aflatoxin. Mol. Plant Pathol. 8, 713–722.
Leclerque, A., Wan, H., Abschutz, A., Chen, S., Mitina, G.V., Zimmermann, G., and Schairer, H.U. 2004. Agrobacterium-mediated insertional mutagenesis (aim) of the entomopathogenic fungus Beauveria bassiana. Curr. Genet. 45, 111–119.
Li, G.H., Zhou, Z.Z., Liu, G.F., Zheng, F.C., and He, C.Z. 2007. Characterization of t-DNA insertion patterns in the genome of rice blast fungus Magnaporthe oryzae. Curr. Genet. 51, 233–243.
Liu, N., Chen, G.Q., Ning, G.A., Shi, H.B., Zhang, C.L., Lu, J.P., Mao, L.J., Feng, X.X., Liu, X.H., Su, Z.Z., et al. 2016. Agrobacterium tumefaciens-mediated transformation: An efficient tool for insertional mutagenesis and targeted gene disruption in Harpophora oryzae. Microbiol. Res. 182, 40–48.
Lohmar, J.M., Harris-Coward, P.Y., Cary, J.W., Dhingra, S., and Calvo, A.M. 2016. rTFA, a putative RNA-Pol II transcription elongation factor gene, is necessary for normal morphological and chemical development in Aspergillus flavus. Appl. Microbiol. Biotechnol. 100, 5029–5041.
Lv, Y. 2017. Proteome-wide profiling of protein lysine acetylation in Aspergillus flavus. PLoS One 12, e0178603.
Maniatis, T., Fritsch, E.F., and Sambrook, J. 1982. Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor.
Michielse, C.B., Hooykaas, P.J.J., van den Hondel, C.A.M.J.J., and Ram, A.F.J. 2005. Agrobacterium-mediated transformation as a tool for functional genomics in fungi. Curr. Genet. 48, 1–17.
Michielse, C.B., Hooykaas, P.J., van den Hondel, C.A., and Ram, A.F. 2008. Agrobacterium-mediated transformation of the filamentous fungus Aspergillus awamori. Nat. Protoc. 3, 1671–1678.
Michielse, C.B., Ram, A.F.J., Hooykaas, P.J.J., and van den Hondel, C.A.M.J.J. 2004. Role of bacterial virulence proteins in Agrobacterium-mediated transformation of Aspergillus awamori. Fungal Genet. Biol. 41, 571–578.
Mitchell, N.J., Bowers, E., Hurburgh, C., and Wu, F. 2016. Potential economic losses to the US corn industry from aflatoxin contamination. Food Addit. Contam. A. 33, 540–550.
Mora-Lugo, R., Zimmermann, J., Rizk, A.M., and Fernandez-Lahore, M. 2014. Development of a transformation system for Aspergillus sojae based on the Agrobacterium tumefaciens-mediated approach. BMC Microbiol. 14, 247.
Mullins, E.D., Chen, X., Romaine, P., Raina, R., Geiser, D.M., and Kang, S. 2001. Agrobacterium-mediated transformation of Fusarium oxysporum: An efficient tool for insertional mutagenesis and gene transfer. Phytopathology 91, 173–180.
Nierman, W.C., Yu, J., Fedorova-Abrams, N.D., Losada, L., Cleveland, T.E., Bhatnagar, D., Bennett, J.W., Dean, R., and Payne, G.A. 2015. Genome sequence of Aspergillus flavus NRRL 3357, a strain that causes aflatoxin contamination of food and feed. Genome Announc. 3, e00168–15.
Punt, P.J., Zegers, N.D., Busscher, M., Pouwels, P.H., and van den Hondel, C.A. 1991. Intracellular and extracellular production of proteins in Aspergillus under the control of expression signals of the highly expressed Aspergillus nidulans gpdA gene. J. Biotechnol. 17, 19–33.
Rho, H.S., Kang, S., and Lee, Y.H. 2001. Agrobacterium tumefaciens mediated transformation of the plant pathogenic fungus, Magnaporthe grisea. Mol. Cells 12, 407–411.
Runa, F., Carbone, I., Bhatnagar, D., and Payne, G.A. 2015. Nuclear heterogeneity in conidial populations of Aspergillus flavus. Fungal Genet. Biol. 84, 62–72.
Schmidt-Heydt, M., Abdel-Hadi, A., Magan, N., and Geisen, R. 2009. Complex regulation of the aflatoxin biosynthesis gene cluster of Aspergillus flavus in relation to various combinations of water activity and temperature. Int. J. Food Microbiol. 135, 231–237.
Sorensen, L.Q., Lysoe, E., Larsen, J.E., Khorsand-Jamal, P., Nielsen, K.F., and Frandsen, R.J. 2014. Genetic transformation of Fusarium avenaceum by Agrobacterium tumefaciens mediated transformation and the development of a user-brick vector construction system. BMC Mol. Biol. 15, 15.
Sugui, J.A., Chang, Y.C., and Kwon-Chung, K.J. 2005. Agrobacterium tumefaciens-mediated transformation of Aspergillus fumigatus: An efficient tool for insertional mutagenesis and targeted gene disruption. Appl. Environ. Microbiol. 71, 1798–1802.
Wang, D.Y., He, D., Li, G.Q., Gao, S., Lv, H.Y., Shan, Q.S., and Wang, L. 2014. An efficient tool for random insertional mutagenesis: Agrobacterium tumefaciens-mediated transformation of the filamentous fungus Aspergillus terreus. J. Microbiol. Methods 98, 114–118.
Woloshuk, C.P., Seip, E.R., Payne, G.A., and Adkins, C.R. 1989. Genetic transformation system for the aflatoxin-producing fungus Aspergillus flavus. Appl. Environ. Microbiol. 55, 86–90.
Yang, K.L., Liang, L.L., Ran, F.L., Liu, Y.H., Li, Z.G., Lan, H.H., Gao, P.L., Zhuang, Z.H., Zhang, F., Nie, X.Y., et al. 2016. The DmtA methyltransferase contributes to Aspergillus flavus conidiation, sclerotial production, aflatoxin biosynthesis and viru lence. Sci. Rep. 6, 23259.
Yang, K., Liu, Y., Liang, L., Li, Z., Qin, Q., Nie, X., and Wang, S. 2017. The high-affinity phosphodiesterase PdeH regulates development and aflatoxin biosynthesis in Aspergillus flavus. Fungal Genet. Biol. 101, 7–19.
Zhang, S., Yue, Y., Sheng, L., Wu, Y., Fan, G., Li, A., Hu, X., Shangguan, M., and Wei, C. 2013. Pasmir: A literature-curated database for miRNA molecular regulation in plant response to abiotic stress. BMC Plant Biol. 13, 33.
Zhi, Q.Q., Li, J.Y., Liu, Q.Y., and He, Z.M. 2017. A cytosine methyltransferase ortholog dmtA is involved in the sensitivity of Aspergillus flavus to environmental stresses. Fungal Biol. 121, 501–514.
Zhu, K.J., Zhou, X., Yan, Y.P., Mo, H.M., Xie, Y.F., Cheng, B.J., and Fan, J. 2017. Cleavage of fusion proteins on the affinity resins using the TEV protease variant. Protein Expr. Purif. 131, 27–33.
Author information
Authors and Affiliations
Corresponding author
Additional information
Supplemental material for this article may be found at http://www.springerlink.com/content/120956.
Electronic supplementary material
Rights and permissions
About this article
Cite this article
Han, G., Shao, Q., Li, C. et al. An efficient Agrobacterium-mediated transformation method for aflatoxin generation fungus Aspergillus flavus. J Microbiol. 56, 356–364 (2018). https://doi.org/10.1007/s12275-018-7349-3
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12275-018-7349-3