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Molecular Biotechnology

, Volume 59, Issue 8, pp 343–352 | Cite as

Host-Induced Silencing of Pathogenicity Genes Enhances Resistance to Fusarium oxysporum Wilt in Tomato

  • Poonam Bharti
  • Poonam Jyoti
  • Priya Kapoor
  • Vandana Sharma
  • V. Shanmugam
  • Sudesh Kumar YadavEmail author
Original Paper

Abstract

This study presents a novel approach of controlling vascular wilt in tomato by RNAi expression directed to pathogenicity genes of Fusarium oxysporum f. sp. lycopersici. Vascular wilt of tomato caused by Fusarium oxysporum f. sp. lycopersici leads to qualitative and quantitative loss of the crop. Limitation in the existing control measures necessitates the development of alternative strategies to increase resistance in the plants against pathogens. Recent findings paved way to RNAi, as a promising method for silencing of pathogenicity genes in fungus and provided effective resistance against fungal pathogens. Here, two important pathogenicity genes FOW2, a Zn(II)2Cys6 family putative transcription regulator, and chsV, a putative myosin motor and a chitin synthase domain, were used for host-induced gene silencing through hairpinRNA cassettes of these genes against Fusarium oxysporum f. sp. lycopersici. HairpinRNAs were assembled in appropriate binary vectors and transformed into tomato plant targeting FOW2 and chsV genes, for two highly pathogenic strains of Fusarium oxysporum viz. TOFOL-IHBT and TOFOL-IVRI. Transgenic tomatoes were analyzed for possible attainment of resistance in transgenic lines against fungal infection. Eight transgenic lines expressing hairpinRNA cassettes showed trivial disease symptoms after 6–8 weeks of infection. Hence, the host-induced posttranscriptional gene silencing of pathogenicity genes in transgenic tomato plants has enhanced their resistance to vascular wilt disease caused by Fusarium oxysporum.

Keywords

Vascular wilt Fusarium oxysporum Gene silencing Hairpin RNA 

Notes

Acknowledgements

We are thankful to the Director, CSIR-Institute of Himalayan Bioresource Technology, Palampur, for support and encouragement during the course of this investigation.

Compliance with Ethical Standards

Funding

This work was financially supported by the Department of Biotechnology, Government of India.

Conflict of interest

Authors declare that they have no conflict of interest.

Supplementary material

12033_2017_22_MOESM1_ESM.pdf (351 kb)
Supplementary material 1 (PDF 350 kb)

References

  1. 1.
    Ajit, N. S., Verma, R., & Shanmugam, V. (2006). Extracellular chitinases of fluorescent pseudomonads antifungal to Fusarium oxysporum f. sp. dianthi causing carnation wilt. Current Microbiology, 52, 310–316.CrossRefGoogle Scholar
  2. 2.
    Arie, T., Takahashi, H., Kodama, M., & Teraoka, T. (2007). Tomato as a model plant for plant-pathogen interactions. Plant Biotechnology, 24, 135–147.CrossRefGoogle Scholar
  3. 3.
    Beckman, C. H. (1990). Host responses to the pathogen. In F. W. Banan & R. C. Ploetz (Eds.), Fusarium wilt of banana (pp. 93–105). St. Paul: APS Press.Google Scholar
  4. 4.
    Bhai, R. S., Remya, B., Danesh, J., & Eapen, S. J. (2009). In vitro and in planta assays for biological control of Fusarium root rot disease of vanilla. Biological Control, 23, 83–86.Google Scholar
  5. 5.
    Bonfim, K., Faria, J. C., Nogueira, E. O., Mendes, E. A., & Aragao, F. J. (2007). RNAi-mediated resistance to Bean golden mosaic virus in genetically engineered common bean (Phaseolus vulgaris). Molecular Plant Microbe Interaction, 20, 717–726.CrossRefGoogle Scholar
  6. 6.
    Carlier, J., De Waele, D., Escalant, J-V., Vézina, A., & Picq, C. (2002). Global evaluation of Musa germplasm for resistance to Fusarium wilt, Mycosphaerella leaf spot diseases and nematodes: performance evaluation. INIBAP Technical Guidelines, Montpellier, France, 7, p. 58.Google Scholar
  7. 7.
    Ghag, S. B., Shekhawat, U. K. S., & Ganapathi, T. R. (2014). Host-induced post-transcriptional hairpin RNA-mediated gene silencing of vital fungal genes confers efficient resistance against Fusarium wilt in banana. Plant Biotechnology Journal, 12, 541–553.CrossRefGoogle Scholar
  8. 8.
    Gnanasekaran, A., & Vijayalakshmi, S. (2012). An economic analysis of tomato cultivation in Dindigul district of Tamil Nadu. International Journal of Science and Research, 3, 995–997.Google Scholar
  9. 9.
    Goswami, R. S., & Kistler, H. C. (2004). Heading for disaster: Fusarium graminearum on cereal crops. Molecular Plant Pathology, 5, 515–525.CrossRefGoogle Scholar
  10. 10.
    Grover, A., & Gowthaman, R. (2003). Strategies for development of fungus-resistant transgenic plants. Current Science, 84, 330–340.Google Scholar
  11. 11.
    Guenther, J. C., & Trail, F. (2005). The development and differentiation of Gibberella zeae (anamorph: Fusarium graminearum) during colonization of wheat. Mycologia, 97, 229–237.CrossRefGoogle Scholar
  12. 12.
    Haroldsen, V. M., Szczerba, M. W., Aktas, H., Lopez-Baltazar, J., Odias, M. J., Chi-Ham, C. L., et al. (2012). Mobility of transgenic nucleic acids and proteins within grafted rootstocks for agricultural improvement. Frontiers in Plant Science, 3, 39.CrossRefGoogle Scholar
  13. 13.
    Idnurm, A., & Howlett, B. J. (2001). Pathogenicity genes of phytopathogenic fungi. Molecular Plant Pathology, 2, 241–255.CrossRefGoogle Scholar
  14. 14.
    Imazaki, I., Kurahashi, M., Iida, Y., & Tsuge, T. (2007). Fow2, a Zn(II)2Cys6-type transcription regulator, controls plant infection of the vascular wilt fungus Fusarium oxysporum. Molecular Microbiology, 63, 737–753.CrossRefGoogle Scholar
  15. 15.
    Jain, S., Akiyama, K., Mae, K., Ohguchi, T., & Takata, R. (2002). Targeted disruption of a G protein alpha subunit gene results in reduced pathogenicity in Fusarium oxysporum. Current Genetics, 41, 407–413.CrossRefGoogle Scholar
  16. 16.
    Kadotani, N., Nakayashiki, H., Tosa, Y., & Mayama, S. (2003). RNA silencing in the phytopathogenic fungus Magnaporthe oryzae. Molecular Plant Microbe Interaction, 16, 769–776.CrossRefGoogle Scholar
  17. 17.
    Kang, Z., & Buchenauer, H. (2000). Cytology and ultrastructure of the infection of wheat spikes by Fusarium culmorum. Mycology Research, 104, 1083–1093.CrossRefGoogle Scholar
  18. 18.
    Kawabe, M., Mizutani, K., Yoshida, T., Teraoka, T., Yoneyama, K., Yamaguchi, I., et al. (2004). Cloning of the pathogenicity-related gene FPD1 in Fusarium oxysporum f. sp. lycopersici. Journal of General Plant Pathology, 70, 16–20.CrossRefGoogle Scholar
  19. 19.
    Koch, A., & Kogel, K. H. (2014). New wind in the sails: improving the agronomic value of crop plants through RNAi-mediated gene silencing. Plant Biotechnology Journal, 12, 821–831.CrossRefGoogle Scholar
  20. 20.
    Koch, A., Kumar, N., Weber, L., Keller, H., Imani, J., & Kogel, K.-H. (2013). Host-induced gene silencing of cytochrome P450 lanosterol C14α-demethylase-encoding genes confers strong resistance to Fusarium species. Proceedings of National Academy of Science USA, 110, 19324–19329.CrossRefGoogle Scholar
  21. 21.
    Madrid, M. P., Di Pietro, A., & Roncero, M. I. G. (2003). Class V chitin synthase determines pathogenesis in the vascular wilt fungus Fusarium oxysporum and mediates resistance to plant defence compounds. Molecular Microbiology, 47, 257–266.CrossRefGoogle Scholar
  22. 22.
    McDonald, B. A., & Linde, C. (2002). The population genetics of plant pathogens and breeding strategies for durable resistance. Euphytica, 124, 163–180.CrossRefGoogle Scholar
  23. 23.
    McGovern, R. J. (2015). Management of tomato diseases caused by Fusarium oxysporum. Crop Protection, 73, 78–92.CrossRefGoogle Scholar
  24. 24.
    Md Ali, E., Kobayashi, K., Yamaoka, N., Ishikawa, M., & Nishiguchi, M. (2013). Graft transmission of RNA silencing to non-transgenic scions for conferring virus resistance in tobacco. PLoS ONE, 8, e63257.CrossRefGoogle Scholar
  25. 25.
    Michielse, C. B., van Wijk, R., Reijnen, L., Manders, E. M. M., Boas, S., Olivain, C., et al. (2009). The nuclear protein Sge1 of Fusarium oxysporum is required for parasitic growth. PLoS Pathogens, 5, e1000637.CrossRefGoogle Scholar
  26. 26.
    Moss, M. O., & Smith, J. E. (1984). The applied mycology of Fusarium. Cambridge: Cambridge University Press.Google Scholar
  27. 27.
    Murray, M. G., & Thompson, W. F. (1980). Rapid isolation of high molecular weight plant DNA. Nucleic Acids Research, 8, 4321–4326.CrossRefGoogle Scholar
  28. 28.
    Nakayashiki, H., Hanada, S., Quoc, N. B., Kadotani, N., Tosa, Y., & Mayama, S. (2005). RNA silencing as a tool for exploring gene function in ascomycete fungi. Fungal Genetics and Biology, 42, 275–283.CrossRefGoogle Scholar
  29. 29.
    Nowara, D., Gay, A., Lacomme, C., Shaw, J., Ridout, C., Douchkov, D., et al. (2010). HIGS: Host-induced gene silencing in the obligate biotrophic fungal pathogen Blumeria graminis. Plant Cell, 22, 3130–3141.CrossRefGoogle Scholar
  30. 30.
    Nunes, A. C. S., Vianna, G. R., Cuneo, F., Amaya-Farfán, J., de Capdeville, G., Rech, E. L., et al. (2006). RNAi-mediated silencing of the myo-inositol-1-phosphate synthase gene (GmMIPS1) in transgenic soybean inhibited seed development and reduced phytate content. Planta, 224, 125–132.CrossRefGoogle Scholar
  31. 31.
    Odonkor, S. T., & Addo, K. K. (2011). Bacteria resistance to antibiotics: recent trends and challenges. International Journal of Biological and Medical Research, 2, 1204–1210.Google Scholar
  32. 32.
    Shanmugam, V., Atri, K., Gupta, S., Kanoujia, N., & Naruka, D. S. (2011). Selection and differentiation of Bacillus spp. Antagonistic to Fusarium oxysporum f.sp. lycopersici and Alternaria solani infecting Tomato. Folia Microbiology (Praha), 56, 170–177.CrossRefGoogle Scholar
  33. 33.
    Shanmugam, V., Sharma, V., Bharti, P., Jyoti, P., Yadav, S. K., Aggarwal, R., et al. (2017). RNAi induced silencing of pathogenicity genes of Fusarium spp. for vascular wilt management in tomato. Annals of Microbiology, 67, 359–369.CrossRefGoogle Scholar
  34. 34.
    Shekhawat, U. K. S., Ganapathi, T. R., & Hadapad, A. B. (2012). Transgenic banana plants expressing small interfering RNAs targeted against viral replication initiation gene display high-level resistance to banana bunchy top virus infection. Journal of General Virology, 93, 1804–1813.CrossRefGoogle Scholar
  35. 35.
    Spanò, R., Mascia, T., Kormelink, R., & Gallitelli, D. (2015). Grafting on a non-transgenic tolerant tomato variety confers resistance to the infection of a Sw5-breaking strain of tomato spotted wilt virus via RNA silencing. PLoS ONE, 10, e0141319.CrossRefGoogle Scholar
  36. 36.
    Sutherland, R., Viljoen, A., Myburg, A. A., & van den Berg, N. (2013). Pathogenicity associated genes in Fusarium oxysporum f. sp. cubense race 4. South African Journal of Science, 109, 1–10.CrossRefGoogle Scholar
  37. 37.
    Szczechura, W., Staniaszek, M., & Habdas, H. (2013). Fusarium oxysporum f. sp. radicis-lycopersici-The cause of Fusarium crown and root rot in tomato cultivation. Journal of Plant Protection Research, 53, 172–176.CrossRefGoogle Scholar
  38. 38.
    Tinoco, M. L. P., Dias, B. B. A., Dall’Astta, R. C., Pamphile, J. A., & Aragão, F. J. L. (2010). In vivo trans-specific gene silencing in fungal cells by in planta expression of a double-stranded RNA. BMC Biology, 8, 27.CrossRefGoogle Scholar
  39. 39.
    Tomilov, A. A., Tomilova, N. B., Wroblewski, T., Michelmore, R., & Yoder, J. I. (2008). Trans-specific gene silencing between host and parasitic plants. Plant Journal, 56, 389–397.CrossRefGoogle Scholar
  40. 40.
    Vakalounakis, D. J., Doulis, A. G., & Klironomou, E. (2005). Characterization of Fusarium oxysporum f. sp. radicis-cucumerinum attacking melon under natural conditions in Greece. Plant Pathology, 54, 339–346.CrossRefGoogle Scholar
  41. 41.
    Wesley, S. V., Helliwell, C. A., Smith, N. A., Wang, M. B., Rouse, D. T., Liu, Q., et al. (2001). Construct design for efficient, effective and high-throughput gene silencing in plants. Plant Journal, 27, 581–590.CrossRefGoogle Scholar
  42. 42.
    White, T. J., Bruns, S., Lee, S., & Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protocols–A Guide to Methods Application, 18, 315–322.Google Scholar
  43. 43.
    Yoo, A. B., Kragler, F., Varkonyi-gasic, E., Haywood, V., Lee, Y. M., & Lough, T. J., et al. (2013). A systemic small RNA signaling system in plants. In A.-E. Young, M. Lee, T. J. Lough, & W. J. Lucas (Eds.) Reviewed work (Vol. 16, pp. 1979–2000). Rockville: American Society of Plant Biologists (ASPB).Google Scholar
  44. 44.
    Zhang, W., Kollwig, G., Stecyk, E., Apelt, F., Dirks, R., & Kragler, F. (2014). Graft-transmissible movement of inverted-repeat-induced siRNA signals into flowers. Plant Journal, 80, 106–121.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2017

Authors and Affiliations

  • Poonam Bharti
    • 1
    • 2
  • Poonam Jyoti
    • 1
  • Priya Kapoor
    • 1
  • Vandana Sharma
    • 1
  • V. Shanmugam
    • 3
  • Sudesh Kumar Yadav
    • 1
    • 2
    • 4
    Email author
  1. 1.Plant Metabolic Engineering Laboratory, Biotechnology DivisionCSIR-Institute of Himalayan Bioresource TechnologyPalampurIndia
  2. 2.Academy of Scientific and Innovative Research (AcSIR)New DelhiIndia
  3. 3.Division of Plant PathologyICAR-Indian Agricultural Research InstituteNew DelhiIndia
  4. 4.Center of Innovative and Applied Bioprocessing (CIAB)MohaliIndia

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