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A practical random mutagenesis system for Ralstonia solanacearum strains causing bacterial wilt of Pogostemon cablin using Tn5 transposon

  • Yaqin Wang
  • Yuyao Zhang
  • Hua Jin
  • Zhicheng Deng
  • Zhuan Li
  • Yanzhen Mai
  • Guangwei Li
  • Hong HeEmail author
Original Paper
  • 144 Downloads

Abstract

A practical random mutagenesis system of Ralstonia solanacearum by electroporation with Tn5 transposon was established, which may be utilized to provide genetic approach to study virulence genes of R. solanacearum strains and create nonpathogenic mutants for biological control of bacterial wilt in Pogostemon cablin. R. solanacearum strain PRS-84 used in this study was isolated from P. cablin plants infected with bacterial wilt. The bacterial suspension of R. solanacearum strain PRS-84 was mixed with Tn5 transposome complex and the mixture was transformed by electroporation. The electroporated cells were then spread on the 2, 3, 5-triphenyltetrazolium chloride agar plates containing kanamycin to select the kanamycin-resistant colonies. Several factors which determined the bacterial transformation efficiency were optimized. The transformation process was shown to be optimal at the electric field strength of 12.5 kV cm−1. Bacterial cells harvested at mid-exponential phase gave the highest transformation efficiency. 10 µg mL−1 kanamycin was found to be the optimal concentration for transformant selection. Tn5 insertion mutants of R. solanacearum strain PRS-84 were identified by PCR amplification and Southern blot analysis. Mutants subcultured for 100 passages were also detected by PCR amplification and Southern blot analysis. Furthermore, pathogenicity screening test of mutants was performed by inoculating in vitro regenerated patchouli plants. Results revealed that mutants with a single Tn5 insertion in their genomes were obtained from R. solanacearum strain PRS-84, and the Tn5 insertion could be stably inherited in the mutants. Then, mutants with reduced pathogenicity were selected.

Keywords

Electrotransformation Pogostemon cablin (Blanco) Benth Ralstonia solanacearum Tn5 transposon 

Notes

Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (No. 81373901) and Ph.D. Programs Foundation of Ministry of Education of China (No. 20134425110012).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

References

  1. Bunrathep S, Lockwood GB, Songsak T et al (2006) Chemical constituents from leaves and cell cultures of Pogostemon cablin and use of precursor feeding to improve patchouli alcohol level. Sci Asia 32:293–296.  https://doi.org/10.2306/scienceasia1513-1874.2006.32.293 CrossRefGoogle Scholar
  2. Chaudhry V, Bhatia A, Bharti SK et al (2015) Metabolite profiling reveals abiotic stress tolerance in Tn5 mutant of Pseudomonas putida. PLoS ONE 10(1):e0113487CrossRefGoogle Scholar
  3. Couteaudier Y (1992) Competition for carbon in soil and rhizosphere, a mechanism involved in biological control of Fusarium wilts. In: Tjamos EC, Papavizas GC, Cook RJ (eds) Biological control of plant diseases. Springer, Boston, pp 99–104CrossRefGoogle Scholar
  4. Hayward AC (1991) Biology and epidemiology of bacterial wilt caused by Pseudomonas solanacearum. Annu Rev Phytopathol 29:65–87.  https://doi.org/10.1146/annurev.py.29.090191.000433 CrossRefPubMedGoogle Scholar
  5. Hoffman LM, Jendrisak JJ, Meis RJ et al (2000) Transposome insertional mutagenesis and direct sequencing of microbial genomes. Genetica 108(1):19–24.  https://doi.org/10.1023/A:1004083307819 CrossRefPubMedGoogle Scholar
  6. Jin H, Deng ZC, He H (2014) Effect of explant types and plant growth regulators on direct regeneration in medicinal plant Pogostemon cablin. Plant Omics 7(5):322–327Google Scholar
  7. Kelman A (1954) The relationship of pathogenicity of Pseudomonas solanacearum to colony appearance in a tetrazolium medium. Phytopathol 44(12):693–695Google Scholar
  8. Li JG, Dong YH (2013) Effect of a rock dust amendment on disease severity of tomato bacterial wilt. Antonie Van Leeuwenhoek 103(1):11–22.  https://doi.org/10.1007/s10482-012-9781-4 CrossRefPubMedGoogle Scholar
  9. Li YR, Che YZ, Zou HS et al (2011) Hpa2 is required by hrpF to translocate Xanthomonas oryzae TAL effectors into rice for pathogenicity. Appl Environ Microbiol 77(11):3809–3818CrossRefGoogle Scholar
  10. Lin YM, Chou IC, Wang JF et al (2008) Transposon mutagenesis reveals differential pathogenesis of Ralstonia solanacearum on tomato and Arabidopsis. Mol Plant Microbe Interact 21(9):1261–1270.  https://doi.org/10.1094/MPMI-21-9-1261 CrossRefPubMedGoogle Scholar
  11. Liu D, He H, Huang HB, Xie JH, Chai TT (2011) Isolation of pathogenic Ralstonia solanacearum from Pogostemon cablin and determination of its pathogenicity. Chin Tradit Herbal Drugs 42(8):1596–1599. (In Chinese)Google Scholar
  12. Luo JY, Qiu W, Chen L et al (2015) Identification of pathogenicity-related genes in biofilm-defective Acidovorax citrulli by transposon Tn5 mutagenesis. Int J Mol Sci 16(12):28050–28062CrossRefGoogle Scholar
  13. Mahanta JJ, Chutia M, Sarma TC (2007) Study on weed flora and their influence on patchouli (Pogostemon cablin Benth.) oil and patchoulol. J Plant Sci 2(1):96–101CrossRefGoogle Scholar
  14. Nakaune M, Tsukazawa K, Uga H et al (2012) Low sodium chloride priming increases seedling vigor and stress tolerance to Ralstonia solanacearum in tomato. Plant Biotechnol 29(29):9–18CrossRefGoogle Scholar
  15. Ramya HG, Palanimuthu V, Rachna S (2013) An introduction to patchouli (Pogostemon cablin Benth.)-A medicinal and aromatic plant: it’s importance to mankind. Agric Eng Int 15(2):243–250Google Scholar
  16. Ray SK, Kumar R, Peeters N et al (2015) rpoN1, but not rpoN2, is required for twitching motility, natural competence, growth on nitrate, and virulence of Ralstonia solanacearum. Front Microbiol 6:229.  https://doi.org/10.3389/fmicb.2015.00229 CrossRefPubMedPubMedCentralGoogle Scholar
  17. Rygulla W, Snowdon RJ, Eynck C et al (2007) Broadening the genetic basis of Verticillium longisporum resistance in Brassica napus by interspecific hybridization. Phytopathology 97(11):1391–1396.  https://doi.org/10.1094/PHYTO-97-11-1391 CrossRefPubMedGoogle Scholar
  18. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  19. Scherf JM, Milling A, Allen C (2010) Moderate temperature fluctuations rapidly reduce the viability of Ralstonia solanacearum race 3, biovar 2, in infected geranium, tomato, and potato plants. Appl Environ Microbiol 76(21):7061–7067.  https://doi.org/10.1128/AEM.01580-10 CrossRefPubMedPubMedCentralGoogle Scholar
  20. Singh M, Rao RSG (2009) Influence of sources and doses of N and K on herbage, oil yield and nutrient uptake of patchouli [Pogostemon cablin (Blanco) Benth.] in semi-arid tropics. Ind Crop Prod 29(1):229–234.  https://doi.org/10.1016/j.indcrop.2008.05.005 CrossRefGoogle Scholar
  21. Song X, Guo J, Ma WX et al (2015) Identification of seven novel virulence genes from Xanthomonas citri subsp. citri by Tn5-based random mutagenesis. J Microbiol 53(5):330–336.  https://doi.org/10.1007/s12275-015-4589-3 CrossRefPubMedGoogle Scholar
  22. Titarenko E, López-solanilla E, García-olmedo F et al (1997) Mutants of Ralstonia (Pseudomonas) solanacearum sensitive to antimicrobial peptides are altered in their lipopolysaccharide structure and are avirulent in tobacco. J Bacteriol 179(21):6699–6704CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  • Yaqin Wang
    • 1
  • Yuyao Zhang
    • 1
  • Hua Jin
    • 1
  • Zhicheng Deng
    • 1
  • Zhuan Li
    • 1
  • Yanzhen Mai
    • 1
  • Guangwei Li
    • 1
  • Hong He
    • 1
    Email author
  1. 1.School of Pharmaceutical SciencesGuangzhou University of Chinese MedicineGuangzhouPeople’s Republic of China

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