Planta

, Volume 226, Issue 6, pp 1525–1533

Host-delivered RNAi: an effective strategy to silence genes in plant parasitic nematodes

  • David J. Fairbairn
  • Antonino S. Cavallaro
  • Margaret Bernard
  • Janani Mahalinga-Iyer
  • Michael W. Graham
  • José R. Botella
Original Article

Abstract

Root-knot nematodes (Meloidogyne spp.) are obligate, sedentary endoparasites that infect many plant species causing large economic losses worldwide. Available nematicides are being banned due to their toxicity or ozone-depleting properties and alternative control strategies are urgently required. We have produced transgenic tobacco (Nicotiana tabacum) plants expressing different dsRNA hairpin structures targeting a root-knot nematode (Meloidogyne javanica) putative transcription factor, MjTis11. We provide evidence that MjTis11 was consistently silenced in nematodes feeding on the roots of transgenic plants. The observed silencing was specific for MjTis11, with other sequence-unrelated genes being unaffected in the nematodes. Those transgenic plants able to induce silencing of MjTis11, also showed the presence of small interfering RNAs. Even though down-regulation of MjTis11 did not result in a lethal phenotype, this study demonstrates the feasibility of silencing root-knot nematode genes by expressing dsRNA in the host plant. Host-delivered RNA interference-triggered (HD-RNAi) silencing of parasite genes provides a novel disease resistance strategy with wide biotechnological applications. The potential of HD-RNAi is not restricted to parasitic nematodes but could be adapted to control other plant-feeding pests.

Keywords

Nematode resistance RNA interference Root knot nematodes 

Abbreviations

dsRNA

Double-stranded RNA

HD-RNAi

Host-delivered RNA interference

RKN

Root knot nematode

RNAi

RNA interference

qRT-PCR

Quantitative real time PCR

siRNA

Small interfering RNA

References

  1. Abad P, Favery B, Rosso MN, Castagnone-Sereno P (2003) Root-knot nematode parasitism and host response: molecular basis of a sophisticated interaction. Mol Plant Pathol 4:217–224CrossRefGoogle Scholar
  2. Bakhetia M, Charlton W, Atkinson HJ, McPherson MJ (2005a) RNA interference of dual oxidase in the plant nematode Meloidogyne incognita. Mol Plant Microbe Interact 18:1099–1106PubMedCrossRefGoogle Scholar
  3. Bakhetia M, Charlton WL, Urwin PE, McPherson MJ, Atkinson HJ (2005b) RNA interference and plant parasitic nematodes. Trends Plant Sci 10:362–367PubMedCrossRefGoogle Scholar
  4. Bockenhoff A, Grundler FMW (1994) Studies on nutrient uptake by the beet cyst nematode Heterodera schachtii by in situ microinjection of fluorescent probes into the feeding structures in Arabidopsis thaliana. Parasitology 109:249–254CrossRefGoogle Scholar
  5. Chakravorty D, Botella JR (2007) Over-expression of a truncated Arabidopsis thaliana heterotrimeric G protein gamma subunit results in a phenotype similar to alpha and beta subunit knockouts. Gene 393:163–170PubMedCrossRefGoogle Scholar
  6. Chen Q, Rehman S, Smant G, Jones JT (2005) Functional analysis of pathogenicity proteins of the potato cyst nematode Globodera rostochiensis using RNAi. Mol Plant Microbe Interact 18:621–625PubMedCrossRefGoogle Scholar
  7. Davis EL, Hussey RS, Baum TJ (2004) Getting to the roots of parasitism by nematodes. Trends Parasitol 20:134–141PubMedCrossRefGoogle Scholar
  8. Devereux J, Haeberli P, Smithies O (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12:387–395PubMedCrossRefGoogle Scholar
  9. Fire A, Xu SQ, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806–811PubMedCrossRefGoogle Scholar
  10. Fraser AG, Kamath RS, Zipperlen P, Martinez-Campos M, Sohrmann M, Ahringer J (2000) Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature 408:325–330PubMedCrossRefGoogle Scholar
  11. Gheysen G, Fenoll C (2002) Gene expression in nematode feeding sites. Ann Rev Phytopathol 40:191–219CrossRefGoogle Scholar
  12. Gheysen G, Vanholme B (2007) RNAi from plants to nematodes. Trends Biotechnol 25:89–92PubMedCrossRefGoogle Scholar
  13. Hannon GJ (2002) RNA interference. Nature 418:244–251PubMedCrossRefGoogle Scholar
  14. Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA (1983) A binary plant vector strategy based on separation of Vir-region and T-region of the Agrobacterium-tumefaciens Ti-plasmid. Nature 303:179–180CrossRefGoogle Scholar
  15. Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT (1985) A simple and general method for transferring genes into plants. Science 227:1229–1231CrossRefGoogle Scholar
  16. Huang G, Allen R, Davis EL, Baum TJ, Hussey RS (2006) Engineering broad root-knot resistance in transgenic plants by RNAi silencing of a conserved and essential root-knot nematode parasitism gene. Proc Natl Acad Sci USA 103:14302–14306PubMedCrossRefGoogle Scholar
  17. Hussey RS, Mims CW (1991) Ultrastructure of feeding tubes formed in giant cells induced in plants by the root-knot nematode Meloidogyne incognita. Protoplasma 162:99–107CrossRefGoogle Scholar
  18. Jung C, Wyss U (1999) New approaches to control plant parasitic nematodes. Appl Microbiol Biotechnol 51:439–446CrossRefGoogle Scholar
  19. Lipardi C, Wei Q, Paterson BM (2001) RNAi as random-degradative PCR: siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs. Cell 107:297–307PubMedCrossRefGoogle Scholar
  20. McCarter J, Bird D, Clifton S, Waterston R (2000) Nematode gene sequences, December 2000 Update. J Nematol 32:331–333PubMedGoogle Scholar
  21. McDaniel CN, Hartnett LK, Sangrey KA (1996) Regulation of node number in day-neutral Nicotiana tabacum: a factor in plant size. Plant J 9:55–61CrossRefGoogle Scholar
  22. Moyle R, Fairbairn DJ, Ripi J, Crowe M, Botella JR (2005) Developing pineapple fruit has a small transcriptome dominated by metallothionein. J Exp Bot 56:101–112PubMedGoogle Scholar
  23. Nykanen A, Haley B, Zamore PD (2001) ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell 107:309–321PubMedCrossRefGoogle Scholar
  24. Opperman C, Taylor C, Conkling M (1994) Root-knot nematode-directed expression of a plant root-specific gene. Science 263:221–223PubMedCrossRefGoogle Scholar
  25. Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:2002–2007CrossRefGoogle Scholar
  26. Purnell MP, Skopelitis DS, Roubelakis-Angelakis KA, Botella JR (2005) Modulation of higher-plant NAD(H)-dependent glutamate dehydrogenase activity in transgenic tobacco via alteration of beta subunit levels. Planta 222:167–180PubMedGoogle Scholar
  27. Rosso MN, Dubrana MP, Cimbolini N, Jaubert S, Abad P (2005) Application of RNA interference to root-knot nematode genes encoding esophageal gland proteins. Mol Plant Microbe Interact 18:615–620PubMedCrossRefGoogle Scholar
  28. Sijen T, Fleenor J, Simmer F, Thijssen KL, Parrish S, Timmons L, Plasterk RHA, Fire A (2001) On the role of RNA amplification in dsRNA-triggered gene silencing. Cell 107:465–476PubMedCrossRefGoogle Scholar
  29. Smith NA, Singh SP, Wang M-B, Stoutjesdijk PA, Green AM, Waterhouse PM (2000) Total silencing by intron-spliced hairpin RNAs. Nature 407:319–320PubMedCrossRefGoogle Scholar
  30. Steeves RM, Todd TC, Essig JS, Trick HN (2006) Transgenic soybeans expressing siRNAs specific to a major sperm protein gene suppress Heterodera glycines reproduction. Funct Plant Biol 33:991–999CrossRefGoogle Scholar
  31. Timmons L, Fire A (1998) Specific interference by ingested dsRNA. Nature 395:854PubMedCrossRefGoogle Scholar
  32. Timmons L, Court DL, Fire A (2001) Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans. Gene 263:103–112PubMedCrossRefGoogle Scholar
  33. Trusov Y, Rookes JE, Chakravorty D, Armour D, Schenk PM, Botella JR (2006) Heterotrimeric G proteins facilitate Arabidopsis resistance to necrotrophic pathogens and are involved in jasmonate signaling. Plant Physiol 140:210–220PubMedCrossRefGoogle Scholar
  34. Urwin P, Lilley C, Atkinson H (2002) Ingestion of double-stranded RNA by preparasitic juvenile cyst nematodes leads to RNA interference. Mol Plant Microbe Interact 15:747–752PubMedCrossRefGoogle Scholar
  35. Urwin PE, Moller SG, Lilley CJ, McPherson MJ (1997) Continual green-fluorescent protein monitoring of Cauliflower Mosaic Virus 35S promoter activity in nematode-induced feeding cells in Arabidopsis thaliana. Mol Plant Microbe Interact 10:394–400PubMedCrossRefGoogle Scholar
  36. Vaistij FE, Jones L, Baulcombe DC (2002) Spreading of RNA targeting and DNA methylation in RNA silencing requires transcription of the target gene and a putative RNA-dependent RNA polymerase. Plant Cell 14:857–867PubMedCrossRefGoogle Scholar
  37. Wesley SV, Helliwell CA, Smith NA, Wang M, Rouse DT, Liu Q, Gooding PS, Singh SP, Abbott D, Stoutjesdijk PA, Robinson SP, Gleave AP, Green AG, Waterhouse PM (2001) Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J 27:581–590PubMedCrossRefGoogle Scholar
  38. Williamson VM, Gleason CA (2003) Plant–nematode interactions. Curr Opin Plant Biol 6:327–333PubMedCrossRefGoogle Scholar
  39. Yadav BC, Veluthambi K, Subramaniam K (2006) Host-generated double-stranded RNA induces RNAi in plant-parasitic nematodes and protects the host from infection. Mol Biochem Parasitol 148:219–222PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • David J. Fairbairn
    • 1
  • Antonino S. Cavallaro
    • 1
  • Margaret Bernard
    • 2
  • Janani Mahalinga-Iyer
    • 1
  • Michael W. Graham
    • 2
  • José R. Botella
    • 1
  1. 1.Department of Botany, School of Integrative BiologyUniversity of QueenslandBrisbaneAustralia
  2. 2.Emerging Technologies Delivery, Department of Primary Industries and FisheriesUniversity of QueenslandBrisbaneAustralia

Personalised recommendations