Skip to main content
SpringerLink
Log in

An efficient gene disruption method using a positive–negative split-selection marker and Agrobacterium tumefaciens-mediated transformation for Nomuraea rileyi

  • Original Paper
  • Published:
World Journal of Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Targeted gene disruption via Agrobacterium tumefaciens-mediated transformation (ATMT) and homologous recombination is the most common method used to identify and investigate the functions of genes in fungi. However, the gene disruption efficiency of this method is low due to ectopic integration. In this study, a high-efficiency gene disruption strategy based on ATMT and the split-marker method was developed for use in Nomuraea rileyi. The β-glucuronidase (gus) gene was used as a negative selection marker to facilitate the screening of putative transformants. We assessed the efficacy of this gene disruption method using the NrCat1, NrCat4, and NrPex16 genes and found that the targeting efficiency was between 36.2 and 60.7%, whereas the targeting efficiency using linear cassettes was only 1.0–4.2%. The efficiency of negative selection assays was between 64.1 and 82.3%. Randomly selected deletion mutants exhibited a single copy of the hph cassette. Therefore, high-throughput gene disruption could be possible using the split-marker method and the majority of ectopic integration transformants can be eliminated using negative selection markers. This study provides a platform to study the function of genes in N. rileyi.

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

Similar content being viewed by others

References

  • Catlett NL, Lee BN, Yoder OC et al (2003) Split-marker recombination for efficient targeted deletion of fungal genes. Fungal Genet Newslett 50:9–11

    Google Scholar 

  • Chen H, Yin YP, Feng EY et al (2014) Structure and expression of a cysteine proteinase gene from Spodoptera litura and its response to biocontrol fungus Nomuraea rileyi. Insect Mol Biol 23(2):255–268

    Article  CAS  Google Scholar 

  • Fairhead C, Llorente B, Denis F et al (1996) New vectors for combinatorial deletions in yeast chromosomes and for gap-repair cloning using ‘split-marker’ recombination. Yeast 12(14):1439–1457

    Article  CAS  Google Scholar 

  • Gardiner DM, Howlett BJ (2004) Negative selection using thymidine kinase increases the efficiency of recovery of transformants with targeted genes in the filamentous fungus Leptosphaeria maculans. Curr Genet 45(4):249–255

    Article  CAS  Google Scholar 

  • Gauthier GM, Sullivan TD, Gallardo SS et al (2010) SREB, a GATA transcription factor that directs disparate fates in Blastomyces dermatitidis including morphogenesis and siderophore biosynthesis. PLoS Pathog 6(4):e1000846

    Article  Google Scholar 

  • Ho SN, Horton RM (1991) Method for gene splicing by overlap extension using the polymerase chain reaction. US 5023171 A

  • Jeong JS, Mitchell TK, Dean RA (2007) The Magnaporthe grisea snodprot1 homolog MSP1 is required for virulence. FEMS Microbiol Lett 273(2):157–165

    Article  CAS  Google Scholar 

  • Jiang SS, Yin YP, Song ZY et al (2014) RacA and Cdc42 regulate polarized growth and microsclerotium formation in the dimorphic fungus Nomuraea rileyi. Res Microbiol 165(3):233–242

    Article  CAS  Google Scholar 

  • Kim MS, Kim SY, Yoon JK et al (2009) An efficient gene-disruption method in Cryptococcus neoformans by double-joint PCR with NAT-split markers. Biochem Biophys Res Commun 390(3):983–988

    Article  CAS  Google Scholar 

  • Li Y, Wang ZK, Liu XE et al (2016) Siderophore biosynthesis but not reductive iron assimilation is essential for the dimorphic fungus Nomuraea rileyi conidiation dimorphism transition resistance to oxidative stress pigmented microsclerotium formation and virulence. Front Microbiol 7(440)

  • Liang LQ, Li JQ, Cheng L et al (2014) A high efficiency gene disruption strategy using a positive-negative split selection marker and electroporation for Fusarium oxysporum. Microbiol Res 169(11):835–843

    Article  CAS  Google Scholar 

  • Liu JJ, Yin YP, Song ZY et al (2014) NADH: flavin oxidoreductase/NADH oxidase and ROS regulate microsclerotium development in Nomuraea rileyi. World J Microb Biotechnol 30(7):1927–1935

    Article  CAS  Google Scholar 

  • Palma L, Del Valle EE (2015) The fungus Nomuraea rileyi growing on dead larvae of Anticarsia gemmatalis associated with soybean plants (Glycine max) in Esperanza (Argentina). Rev Argent Microbiol 47(3):277–278

    Google Scholar 

  • Rothstein RJ (1983) One-step gene disruption in yeast. Methods Enzymol 101(6):202–211

    Article  CAS  Google Scholar 

  • Shang YF, Xiao GH, Zheng P et al (2016) Divergent and convergent evolution of fungal pathogenicity. Genome Biol Evol 8(5):1374–1387

    Article  Google Scholar 

  • Shao CW, Yin YP, Qi ZR et al (2015) Agrobacterium tumefaciens-mediated transformation of the entomopathogenic fungus Nomuraea rileyi. Fungal Genet Biol 83:19–25

    Article  CAS  Google Scholar 

  • Song ZY, Yin YP, Jiang SS et al (2013) Comparative transcriptome analysis of microsclerotia development in Nomuraea rileyi. BMC Genom 14(1):411

    Article  CAS  Google Scholar 

  • Song ZY, Zhong Q, Yin YP et al (2016) The high osmotic response and cell wall integrity pathways cooperate to regulate morphology microsclerotia development and virulence in Metarhizium rileyi. Sci Rep-UK 6:38765

    Article  CAS  Google Scholar 

  • St. Leger RJ, Shimizu S, Joshi L et al (1995) Co-transformation of Metarhizium anisopliae by electroporation or using the gene gun to produce stable GUS transformants. FEMS Microbiol Lett 131(3):289–294

    Article  CAS  Google Scholar 

  • Thakre M, Thakur M, Malik N et al (2011) Mass scale cultivation of entomopathogenic fungus Nomuraea rileyi using agricultural products and agro wastes. J Biopestic 4:176–179

    CAS  Google Scholar 

  • Vega-Aquino P, Blanco CA, Sanchez-Pea SR (2010) Activity of oil-formulated conidia of Nomuraea rileyi and Paecilomyces tenuipes against Spodoptera Heliothis and Helicoverpa larvae and pupae. J Invertebr Pathol 103:145–149

    Article  Google Scholar 

  • Wang JY, Zhang Z, Du XF et al (2009) Dual screening for targeted gene replacement mutant in Magnaporthe oryzae with GUS as negative marker. Chin J Biotechnol 25(1):129–138 (Chinese)

    Google Scholar 

  • Wang Y, Diguistini S, Wang T et al (2010) Agrobacterium-meditated gene disruption using split-marker in Grosmannia clavigera a mountain pine beetle associated pathogen. Curr Genet 56(3):297–307

    Article  Google Scholar 

  • Wang Z, Ye SF, Li JJ et al (2011) Fusion primer and nested integrated PCR (FPNI-PCR): a new high-efficiency strategy for rapid chromosome walking or flanking sequence cloning. BMC Biotechnol 11(1):109

    Article  Google Scholar 

  • Weld RJ, Plummer KM, Carpenter MA et al (2006) Approaches to functional genomics in filamentous fungi. Cell Res 16:31–44

    Article  CAS  Google Scholar 

  • Wendland J (2003) PCR-based methods facilitate targeted gene manipulations and cloning procedures. Curr Genet 44:115–123

    Article  CAS  Google Scholar 

  • Xu C, Zhang X, Qian Y et al (2014) A high-throughput gene disruption methodology for the entomopathogenic fungus Metarhizium robertsii. PLoS ONE 9(9):e107657

    Article  Google Scholar 

  • You BJ, Lee MH, Chung KR (2009) Gene-specific disruption in the filamentous fungus Cercospora nicotianae using a split-marker approach. Arch Microbiol 191(7):615–622

    Article  CAS  Google Scholar 

  • Zhou GL, Song ZY, Yin YP et al (2015) Involvement of alternative oxidase in the regulation of hypha growth and microsclerotia formation in Nomuraea rileyi CQNr01. World J Microb Biotechnol 311:1343–1352

    Article  Google Scholar 

Download references

Acknowledgements

This research was financially supported by the National Natural Science Foundation of China (Grant No. 31570073).

Author information

Authors and Affiliations

  1. School of Life Science, Chongqing University, Chongqing, 400030, China

    Yu Su, Zhongkang Wang, Li Wang & Youping Yin

  2. College of Agriculture and Forestry, Linyi University, Linyi, 276000, China

    Changwen Shao

  3. Southeast Chongqing Academy of Agricultural Sciences, Chongqing, 408000, China

    Yu Su & Yuanli Luo

Authors
  1. Yu Su
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zhongkang Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Changwen Shao
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yuanli Luo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Li Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Youping Yin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Youping Yin.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Su, Y., Wang, Z., Shao, C. et al. An efficient gene disruption method using a positive–negative split-selection marker and Agrobacterium tumefaciens-mediated transformation for Nomuraea rileyi. World J Microbiol Biotechnol 34, 26 (2018). https://doi.org/10.1007/s11274-018-2409-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s11274-018-2409-8

Keywords

Search

Navigation