, Volume 228, Issue 6, pp 977–987 | Cite as

NbLRK1, a lectin-like receptor kinase protein of Nicotiana benthamiana, interacts with Phytophthora infestans INF1 elicitin and mediates INF1-induced cell death

  • Hiroyuki Kanzaki
  • Hiromasa Saitoh
  • Yoshihiro Takahashi
  • Thomas Berberich
  • Akiko Ito
  • Sophien Kamoun
  • Ryohei TerauchiEmail author
Original Article


Phytophthora infestans INF1 elicitin causes the hypersensitive response (HR) in Nicotiana benthamiana (Kamoun et al. in Plant Cell 10:1413–1425, 1998). To identify N. benthamiana proteins that interact with INF1, we carried out a yeast two-hybrid screen. This screen resulted in the isolation of a gene NbLRK1 coding for a novel lectin-like receptor kinase. NbLRK1 interacted with INF1 through its VIb kinase subdomain. Purified INF1 and NbLRK1 proteins also interacted in vitro. INF1 treatment of N. benthamiana leaves induced autophosphorylation of NbLRK1. Most importantly, virus-induced gene silencing (VIGS) of NbLRK1 delayed INF1-mediated HR in N. benthamiana. These data suggest that NbLRK1 is a component of the N. benthamiana protein complex that recognizes INF1 elicitor and transduces the HR signal.


Autophosphorylation Hypersensitive response INF1 NbLRK1 Nicotiana Virus-induced gene silencing Y2H 



Hypersensitive response


Lectin-like receptor kinase


Myelin basic protein


Virus-induced gene silencing


Yeast two-hybrid assay



We acknowledge David Baulcombe, Sainsbury Laboratory, John Innes Center, for pPC2S and PTV:00 and Nam Hai Chua, Rockefeller University, for pTA7001. This work was carried out in part by support from “Program for Promotion of Basic Research Activities for Innovative Biosciences” (Japan), “Iwate University twenty-first Century COE Program: Establishment of Thermo-Biosystem Research Program” and Ministry of Agriculture, Forestry and Fisheries of Japan (Genomics for Agricultural Innovation PMI-0010) to RT. We thank Matt Shenton, IBRC, for the improvement of the manuscript.


  1. Adams JA (2003) Activation loop phosphorylation and catalysis in protein kinases: is there functional evidence for the autoinhibitor model? Biochemistry 42:601–607PubMedCrossRefGoogle Scholar
  2. Aoyama T, Chua NH (1997) A glucocorticoid-mediated transcriptional induction system in transgenic plants. Plant J 11:605–612PubMedCrossRefGoogle Scholar
  3. Barre A, Herve C, Lescure B, Rouge P (2002) Lectin receptor kinases in plants. Crit Rev Plant Sci 21:379–399CrossRefGoogle Scholar
  4. Baulcombe DC, Chapman S, Santa CS (1995) Jellyfish green fluorescent protein as a reporter for virus infections. Plant J 8:1045–1053CrossRefGoogle Scholar
  5. Baulcombe DC (1999) Fast forward genetics based on virus-induced gene silencing. Curr Opin Plant Biol 2:109–113PubMedCrossRefGoogle Scholar
  6. Belkhadir Y, Subramaniam R, Dangl JL (2004) Plant disease resistance protein signaling: NBS–LRR proteins and their partners. Curr Opin Plant Biol 7:391–399PubMedCrossRefGoogle Scholar
  7. Berberich T, Sano H, Kusano T (1999) Involvement of a MAP kinase, ZmMPK5, in senescence and recovery from low-temperature stress in maize. Mol Gen Genet 262:534–542PubMedCrossRefGoogle Scholar
  8. Bonnet P, Bourdon E, Ponchet M, Blein JP, Ricci P (1996) Acquired resistance triggered by elicitins in tobacco and other plants. Eur J Plant Pathol 102:181–192CrossRefGoogle Scholar
  9. Brummer M, Arend M, Fromm J, Schlenzig A, Osswald WF (2002) Ultrastructural changes and immunocytochemical localization of the elicitin quercinin in Quercus robur L. roots infected with Phytophthora quecrina. Physiol Mol Plant Pathol 61:109–120CrossRefGoogle Scholar
  10. Catanzariti AM, Dodds PN, Lawrence GJ, Ayliffe MA, Elllis JG (2006) Haustorially expressed secreted proteins from flax rust are highly enriched for avirulence elicitors. Plant Cell 18:243–256PubMedCrossRefGoogle Scholar
  11. Cock JM, Vanoosthuyse V, Gaude T (2002) Receptor kinase signaling in plants and animals: distinct molecular systems with mechanistic similarities. Curr Opin Cell Biol 14:230–236PubMedCrossRefGoogle Scholar
  12. Coemans B, Takahashi Y, Berberich T, Ito A, Kanzaki H, Matsumura H, Saitoh H, Tsuda S, Kamoun S, Sagi L, Swennen R, Terauchi R (2008) High-throughput in planta expression screening identifies an ADP-ribosylation factor (ARF1) involved in non-host resistance and R gene-mediated resistance. Mol Plant Pathol 9:25–36PubMedGoogle Scholar
  13. Dangl JL, Jones JD (2001) Plant pathogens and integrated defence responses to infection. Nature 411:826–833PubMedCrossRefGoogle Scholar
  14. Dardick C, Ronald P (2006) Plant and animal pathogen recognition receptors signal through non-RD kinases. PLOS Pathogens 2:14–28CrossRefGoogle Scholar
  15. Dayhoff MO (1978) Survey of new data and computer methods of analysis. In: Dayhoff MO (ed) Atlas of protein sequence and structure, vol 5, suppl 3. National Biomedical Research Foundation, Georgetown University, Washington, DCGoogle Scholar
  16. Ellis J, Dodds P, Pryor T (2000) Structure, function and evolution of plant disease resistance genes. Curr Opin Plant Biol 3:278–284PubMedCrossRefGoogle Scholar
  17. Ellis J, Catanzariti AM, Dodds P (2006) The problem of how fungal and oomycete avirulence proteins enter plant cells. Trends Plant Sci 11:61–63PubMedCrossRefGoogle Scholar
  18. Felix G, Duran JD, Volko S, Boller T (1999) Plants have a sensitive perception system for the most conserved domain of bacterial flagellin. Plant J 18:265–276PubMedCrossRefGoogle Scholar
  19. Fields S, Song O (1989) A novel genetic system to detect protein–protein interactions. Nature 340:245–246PubMedCrossRefGoogle Scholar
  20. Florentino LH, Santos AA, Fontenelle MA, Pinheiro GL, Zerbini FM, Baracat-Pereira MC, Fontes EPB (2006) A PERK-like receptor kinase interacts with the genimivirus nuclear shuttle protein and potentiates viral infection. J Virol 80:6648–6656PubMedCrossRefGoogle Scholar
  21. Fontes EPB, Santos AA, Luz DF, Waclawovsky AJ, Chory J (2004) The genimivirus nuclear shuttle protein is a virulence factor that suppresses transmembrane receptor kinase activity. Genes Dev 18:2545–2556PubMedCrossRefGoogle Scholar
  22. Gouget A, Senchou V, Govers F, Sanson A, Barre A, Rouge P, Pont-Lezica R, Canut H (2006) Lectin receptor kinases participate in protein-protein interactions to mediate plasma membrane–cell wall adhesions in Arabidopsis. Plant Physiol 140:81–90PubMedCrossRefGoogle Scholar
  23. Hanks SK, Quinn AM (1991) Protein kinase catalytic domain sequence database: identification of conserved features of primary structure and classification of family members. Methods Enzymol 200:38–61PubMedCrossRefGoogle Scholar
  24. Hanks SK, Hunter T (1995) Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification. FASEB J 9:576–596PubMedGoogle Scholar
  25. He XJ, Zhang ZG, Yan DQ, Zhang JS, Chen SY (2004) A salt-responsive receptor-like kinase gene regulated by the ethylene signaling pathway encodes a plasma membrane serine/threonine kinase. Theor Appl Genet 109:377–383PubMedCrossRefGoogle Scholar
  26. Hervé C, Serres J, Dabos P, Canut H, Barre A, Rougé P, Lescure B (1996) Characterization of the Arabidopsis lecRK-a genes: members of a superfamily encoding putative receptors with an extracellular domain homologous to legume lectins. Plant Mol Biol 39:671–682CrossRefGoogle Scholar
  27. Kaku H, Nishizawa Y, Ihii-Minami N, Akimoto-Tomiyama C, Dohmae N, Tako K, Minami E, Shibuya N (2006) Plant cells recognize chitin fragments for defense signaling through a plasma membrane receptor. Proc Natl Acad Sci USA 103:11086–91PubMedCrossRefGoogle Scholar
  28. Kamoun S, Young M, Glascok C, Tyler BM (1993) Extracellular protein elicitors from Phytophthora: host-specificity and induction of resistance to fungal and bacterial phytopathogens. Mol Plant Microbe Interact 10:13–20CrossRefGoogle Scholar
  29. 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 is down-regulated during infection of potato. Mol Plant Microbe Interact 10:13–20PubMedCrossRefGoogle Scholar
  30. Kamoun S, van West P, Vleeshouwers GAA, 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 10:1413–1425PubMedCrossRefGoogle Scholar
  31. Kamoun S, Honee G, Weide R, Lauge R, Kooman-Gersmann M, de Groot K, Govers F, de Wit PJGM (1999a) The fungal gene Avr9 and the oomycete gene inf1 confer avirulence to potato virus X on tobacco. Mol Plant Microbe Interact 12:459–462CrossRefGoogle Scholar
  32. Kamoun S, Huitema E, Vleeshouwers VGAA (1999b) Resistance to oomycetes: a general role for hypersensitive response? Trends Plant Sci 4:196–200PubMedCrossRefGoogle Scholar
  33. Kamoun S (2001) Nonhost resistance to Phytophthora: novel prospects for a classical problem. Curr Opin Plant Biol 4:295–300PubMedCrossRefGoogle Scholar
  34. Kanzaki H, Saitoh H, Ito A, Fujisawa S, Kamoun S, Katou S, Yoshioka H, Terauchi R (2003) Cytosolic HSP90 and HSP70 are essential components of INF1-mediated hypersensitive response and non-host resistance to Pseudomonas cichorii in Nicotiana benthamiana. Mol Plant Pathol 4:383–391CrossRefGoogle Scholar
  35. Krogh A, Larsson B, von Heijne G, Sonnhammer ELL (2001) Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 305:567–580PubMedCrossRefGoogle Scholar
  36. Martin GB (1999) Functional analysis of plant disease resistance genes and their downstream effectors. Curr Opin Plant Biol 2:273–279PubMedCrossRefGoogle Scholar
  37. Martin GB, Bogdanove AJ, Sessa G (2003) Understanding the functions of plant disease resistance proteins. Annu Rev Plant Biol 54:23–61PubMedCrossRefGoogle Scholar
  38. Navarro-Gochicoa MT, Camut S, Timmers ACJ, Niebel A, Herve C, Boutet E, Bono JJ, Imberty A, Cullimore JV (2003) Characterization of four lectin-like receptor kinases expressed in roots of Medicago truncatula. Structure, location, regulation of expression, and potential role in the symbiosis with Sinorhizobium meliloti. Plant Physiol 133:1893–1910PubMedCrossRefGoogle Scholar
  39. Nishiguchi M, Yoshida K, Sumizono T, Tazaki K (2002) A receptor-like protein kinase with a lectin-like domain from lombardy poplar: gene expressions in response to wounding and characterization of phosphorylation activity. Mol Genet Genomics 267:506–514PubMedCrossRefGoogle Scholar
  40. Ouaked F, Rozhon W, Lecoureux D, Hirt H (2003) A MAPK pathway mediates ethylene signaling in plants. EMBO J 22:1282–1288PubMedCrossRefGoogle Scholar
  41. Peart JR, Lu R, Sadanandom A, Malcuit I, Moffett P, Brice DC, Shauser L, Jaggard DA, Xiao S, Coleman MJ, Dow M, Jones JD, Shirasu K, Baulcombe DC (2002) Ubiquitin ligase-associated protein SGT1 is required for host and nonhost disease resistance in plants. Proc Natl Acad Sci USA 99:1085–1089CrossRefGoogle Scholar
  42. 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
  43. Ratcliff F, Martin-Hernandez AM, Baulcombe DC (2001) Tobacco Rattle Virus for analysis of gene function by silencing. Plant J 25:237–245PubMedCrossRefGoogle Scholar
  44. Saitoh N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425Google Scholar
  45. 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 14:111–115PubMedCrossRefGoogle Scholar
  46. Thordal-Christensen H, Zhang Z, Wei Y, Collinge DB (1997) Subcellular localization of H2O2 in plants: H2O2 accumulation in papillae and hypersensitive response during the barley-powdery mildew interaction. Plant J 11:1187–1194CrossRefGoogle Scholar
  47. Tyler BM (2002) Molecular basis of recognition between Phytophthora pathogens and their hosts. Annu Rev Phytopathol 40:137–167PubMedCrossRefGoogle Scholar
  48. van Damme EJM, Peumans WJ, Barre A, Rouge P (1998) Plant lectins: a composite of several distinct families of structurally and evolutionary related proteins with diverse biological roles. Crit Rev Plant Sci 17:575–692CrossRefGoogle Scholar
  49. Wendehenne D, Binet MN, Blein JP, Ricci P, Pugin A (1995) Evidence for specific, high-affinity binding sites for a proteinaceous elicitor in tobacco plasma membrane. FEBS Lett 374:203–207PubMedCrossRefGoogle Scholar
  50. Yoshioka H, Numata N, Nakajima K, Katou S, Kawakita K, Rowland O, Jones JD, Doke N (2003) Nicotiana benthamiana gp91 phox homologs NbrbohA and NbrbohB participate in H2O2 accumulation and resistance to Phytophthora infestans. Plant Cell 15:706–718PubMedCrossRefGoogle Scholar
  51. Zipfel C, Felix G (2005) Plants and animals: a different taste for microbes? Curr Opin Plant Biol 8:353–60PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Hiroyuki Kanzaki
    • 1
  • Hiromasa Saitoh
    • 1
  • Yoshihiro Takahashi
    • 1
  • Thomas Berberich
    • 1
  • Akiko Ito
    • 1
  • Sophien Kamoun
    • 3
  • Ryohei Terauchi
    • 1
    • 2
    Email author
  1. 1.Iwate Biotechnology Research CenterKitakamiJapan
  2. 2.The United Graduate School of Agricultural SciencesIwate UniversityMoriokaJapan
  3. 3.The Sainsbury LaboratoryJohn Innes CentreNorwichUK

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