Advertisement

Molecular Genetics and Genomics

, Volume 269, Issue 5, pp 583–591 | Cite as

Virus-induced silencing of WIPK and SIPK genes reduces resistance to a bacterial pathogen, but has no effect on the INF1-induced hypersensitive response (HR) in Nicotiana benthamiana

  • P. C. Sharma
  • A. Ito
  • T. Shimizu
  • R. Terauchi
  • S. Kamoun
  • H. SaitohEmail author
Original Paper

Abstract

Activation of two mitogen-activated protein kinases (MAPKs), wound-induced protein kinase (WIPK) and salicylic acid-induced protein kinase (SIPK), is one of the earliest responses that occur in tobacco plants that have been wounded, treated with pathogen-derived elicitors or challenged with avirulent pathogens. We isolated cDNAs for these MAPKs ( NbWIPK and NbSIPK) from Nicotiana benthamiana. The function of NbWIPK and NbSIPK in mediating the hypersensitive response (HR) triggered by infiltration with INF1 protein (the major elicitin secreted by Phytophthora infestans), and the defense response to an incompatible bacterial pathogen ( Pseudomonas cichorii), was investigated by employing virus-induced gene silencing (VIGS) to inhibit expression of the WIPK and SIPK genes in N. benthamiana. Silencing of WIPK or SIPK, or both genes simultaneously, resulted in reduced resistance to P. cichorii, but no change was observed in the timing or extent of HR development after treatment with INF1.

Keywords

Nicotiana benthamiana WIPK/SIPK INF1  Pseudomonas cichorii Virus-induced gene silencing 

Notes

Acknowledgements

We thank Dr. D. C. Baulcombe (Sainsbury Laboratory, John Innes Centre) for the gift of plasmids pPC2S and pTXS.GFP. We are grateful to Dr. S. Seo, Dr. Y. Ohashi (National Institute of Agrobiological Resources) for the provision of anti-WIPK antibody and for invaluable suggestions. We thank Dr. T. Romeis (Max-Planck-Institute for Plant Breeding Research), Dr. H. Yoshioka (Graduate School of Bioagricultural Sciences, Nagoya University) and Dr. Y. Hikichi (Faculty of Agriculture, Kochi University) for invaluable suggestions. PSC is thanks the JSPS for financial help in the form of an Invitation Research Fellowship. This work was carried out in a containment facility of Iwate Biotechnology Research Center under License No. 13-YokoShoku-965 from the Ministry of Agriculture, Forestry and Fisheries, Japan, and License No. 12-Ken-Kyoku-52 from the Ministry of Education, Culture and Science, Japan. This study was in part supported by the Research for the Future program of the Japan Society for the Promotion of Science

References

  1. Baulcombe DC (1999) Fast forward genetics based on virus-induced gene silencing. Curr Opin Plant Biol 2:109–113PubMedGoogle Scholar
  2. Baulcombe DC, Chapman S, Santa Cruz S (1995) Jellyfish green fluorescent protein as a reporter for virus infections. Plant J 7:1045–1053PubMedGoogle Scholar
  3. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. J Biochem 72:248–254CrossRefGoogle Scholar
  4. Burton RA, Bibeaut DM, Bacic A, Findlay K, Roberts K, Hamilton A, Baulcombe DC, Fincher GB (2000) Virus-induced silencing of a plant cellulose synthase gene. Plant Cell 12:691–705CrossRefPubMedGoogle Scholar
  5. Davis R (2000) Signal transduction by the JNK group of MAP kinases. Cell 103:239-252PubMedGoogle Scholar
  6. Droillard MJ, Thibivilliers S, Cazalé AC, Barbier-Brygoo H, Lauriére C (2000) Protein kinases induced by osmotic stresses and elicitor molecules in tobacco cell suspensions: two cross-road MAP kinases and one osmoregulation-specific protein kinase. FEBS Lett 474:217–222CrossRefPubMedGoogle Scholar
  7. Hikichi Y, Suzuki K, Toyoda K, Horikoshi M, Hirooka T, Okuno T (1998) Successive observation of growth and movement of genetically lux -marked Pseudomonas cichorii and the response of host tissues in the same lettuce leaf. Ann Phytopathol Soc Jpn 64:519–525Google Scholar
  8. Kamoun S, Young M, Glascock C, Tyler BM (1993) Extracellular protein elicitors from Phytophthora: host-specificity and induction of resistance to fungal and bacterial phytopathogens. Mol Plant-Microbe Interact 6:15–25Google Scholar
  9. Kamoun S, Young M, Forster H, Coffey MD, Tyler BM (1994) Potential role of elicitins in the interaction between Pytophthora species and tobacco. Appl Environ Microbiol 60:1593–1598Google Scholar
  10. Kamoun S, van West P, de Jong AJ, de Groot KE, Vleeshouwers VGAA, Govers F (1997) A gene encoding a protein elicitor of Phytophthora infestans. Mol Plant-Microbe Interact 10:13–20Google Scholar
  11. Kamoun S, van West P, Vleeshouwers VGAA, de Groot KE, Govers F (1998) Resistance of Nicotiana benthamiana to Phytophthora infestans is mediated by the recognition of the elicitor protein INF1. Plant Cell 10:1413–1425CrossRefPubMedGoogle Scholar
  12. Keller H, Blein JP, Bonnet P, Ricci P (1996) Physiological and molecular characteristics of elicitin-induced systemic acquired resistance in tobacco. Plant Physiol 110:365–376PubMedGoogle Scholar
  13. Lee J, Klessig DF, Nürnberger T (2001) A harpin binding site in tobacco plasma membranes mediates activation of the pathogenesis-related gene HIN1 independent of extracellular calcium but dependent on mitogen-activated protein kinase activity. Plant Cell 13:1079–1093CrossRefPubMedGoogle Scholar
  14. Matsumura H, Nirasawa S, Kiba A, Urasaki N, Saitoh H, Ito M, Kawai-Yamada M, Uchimiya H, Terauchi R (2003) Overexpression of Bax inhibitor suppresses fungal elicitor-induced cell death in rice ( Oryza sativa L.) cells. Plant J 33:425–434CrossRefPubMedGoogle Scholar
  15. Mikolajczyk M, Awotunde OS, Muszynska G, Klessig DF, Dobrowolska G (2000) Osmotic stress induces rapid activation of a salicylic acid-induced protein kinase and a homolog of protein kinase ASK1 in tobacco cells. Plant Cell 12:165–178PubMedGoogle Scholar
  16. Peart JR, Cook G, Feys BJ, Parker JE, Baulcombe DC (2002a) An EDS1 orthologue is required for N -mediated resistance against tobacco mosaic virus. Plant J 29:569–579CrossRefPubMedGoogle Scholar
  17. Peart JR, Lu R, Sadanandom A, Malcuit I, Moffett P, Brice DC, Schauser L, Jaggard DAW, Xiao S, Coleman M, Dow M, Jones JDG, Shirasu K, Baulcombe DC (2002b) Ubiquitin ligase-associated protein SGT1 is required for host and nonhost disease resistance in plants. Proc Natl Acad Sci USA 99:10865–10869CrossRefPubMedGoogle Scholar
  18. Pernollet JC, Sallantin M, Salle-Tourne M, Huet JC (1993) Elicitin isoforms from seven Phytophthora species: comparison of their physico-chemical properties and toxicity to tobacco and other plant species. Physiol Mol Plant Pathol 42:53–67CrossRefGoogle Scholar
  19. Qutob D, Kamoun S, Gijzen M (2002) Expression of a Phytophthora sojae necrosis-inducing protein occurs during transition from biotrophy to necrotrophy. Plant J 32:361–373CrossRefPubMedGoogle Scholar
  20. Romeis T (2001) Protein kinases in the plant defence response. Curr Opin Plant Biol 4:407–414CrossRefPubMedGoogle Scholar
  21. Romeis T, Piedras P, Zhang S, Klessig DF, Hirt H, Jones JDG (1999) Rapid Avr9- and Cf9-dependent activation of MAP kinases in tobacco cell cultures and leaves: convergence of resistance gene, elicitor, wound, and salicylate responses. Plant Cell 11:273–287PubMedGoogle Scholar
  22. Romeis T, Piedras P, Jones JDG (2000) Resistance gene-dependent activation of a calcium-dependent protein kinase in the plant defense response. Plant Cell 12:803–815PubMedGoogle Scholar
  23. Romeis T, Ludwig AA, Martin R, Jones JDG (2001) Calcium-dependent protein kinases play an essential role in a plant defence response. EMBO J 20:5556–5567Google Scholar
  24. Saitoh H, Terauchi R (2002) Virus-induced silencing of FtsH gene in Nicotiana benthamiana causes a striking bleached leaf phenotype. Genes Genet Syst 77:335–340CrossRefPubMedGoogle Scholar
  25. Saitoh H, Kiba A, Nishihara M, Yamamura S, Suzuki K, Terauchi R (2001) Production of antimicrobial defensin in Nicotiana benthamiana with a potato virus X vector. Mol Plant-Microbe Interact 14:111–115Google Scholar
  26. Samuel MA, Ellis BE (2002) Double jeopardy: both overexpression and suppression of a redox-activated plant mitogen-activated protein kinase render tobacco plants ozone sensitive. Plant Cell 14:2059–2069CrossRefPubMedGoogle Scholar
  27. Samuel MA, Miles GP, Ellis BE (2000) Ozone treatment rapidly activates MAP kinase signalling in plants. Plant J 22:367–376PubMedGoogle Scholar
  28. Seo S, Okamoto M, Seto H, Ishizuka K, Sano H, Ohashi Y (1995) Tobacco MAP kinase: a possible mediator in wound signal transduction pathways. Science 270:1988–1992PubMedGoogle Scholar
  29. Seo S, Sano H, Ohashi Y (1999) Jasmonate-based wound signal transduction requires activation of WIPK, a tobacco mitogen-activated protein kinase. Plant Cell 11:289–298PubMedGoogle Scholar
  30. Voinnet O (2001) RNA silencing as a plant immune system against viruses. Trends Genet 17:449–459CrossRefPubMedGoogle Scholar
  31. Widman C, Gibson S, Jarpe MB, Johnson GL (1999) Mitogen-activated protein kinase: conservation of a three-kinase module from yeast to human. Physiol Rev 79:143–180PubMedGoogle Scholar
  32. Xing T, Quellet T, Miki BL (2002) Towards genomic and proteomic studies of protein phosphorylation in plant-pathogen interactions. Trends Plant Sci 7:224–230CrossRefPubMedGoogle Scholar
  33. Yang K-Y, Liu Y, Zhang S (2001) Activation of a mitogen-activated protein kinase pathway is involved in disease resistance in tobacco. Proc Natl Acad Sci USA 98:741–746PubMedGoogle Scholar
  34. Zhang S, Klessig DF (1997) Salicylic acid activates a 48 kD MAP kinase in tobacco. Plant Cell 9:809–824PubMedGoogle Scholar
  35. Zhang S, Klessig DF (1998) Resistance gene N-mediated de novo synthesis and activation of a tobacco mitogen-activated protein kinase by tobacco mosaic virus infection. Proc Natl Acad Sci USA 95:7433–7438PubMedGoogle Scholar
  36. Zhang S, Klessig DF (2001) MAPK cascades in plant defense signaling. Trends Plant Sci 6:520–527PubMedGoogle Scholar
  37. Zhang S, Liu Y (2001) Activation of salicylic acid-induced protein kinase, a mitogen-activated protein kinase, induces multiple defense responses in tobacco. Plant Cell 13:1877–1889PubMedGoogle Scholar
  38. Zhang S, Du H, Klessig DF (1998) Activation of the tobacco SIP kinase by both a cell wall-derived carbohydrate elicitor and purified proteinaceous elicitins from Phytophthora spp. Plant Cell 10:435–449CrossRefPubMedGoogle Scholar
  39. Zhang S, Liu Y, Klessig DF (2000) Multiple levels of tobacco WIPK activation during the induction of cell death by fungal elicitins. Plant J 23:339–347CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  • P. C. Sharma
    • 1
    • 5
  • A. Ito
    • 2
  • T. Shimizu
    • 2
  • R. Terauchi
    • 2
  • S. Kamoun
    • 3
  • H. Saitoh
    • 2
    • 4
    • 6
    Email author
  1. 1.Department of Agricultural BotanyCh. Charan Singh UniversityMeerutIndia
  2. 2.Iwate Biotechnology Research CenterKitakamiJapan
  3. 3.Department of Plant Pathology, Ohio Agricultural Research and Development CenterOhio State UniversityWoosterUSA
  4. 4.Japan Society for the Promotion of ScienceTokyo 102-8471Japan
  5. 5.School of BiotechnologyGuru Gobind Singh Indraprastha UniversityDelhiIndia
  6. 6.Department of Plant Microbe InteractionsMax-Planck-Institute for Plant Breeding ResearchCologneGermany

Personalised recommendations