letters to nature yeast. Liquid culture densities were equalized using absorption at 600 nm, and 10 mlof .............................................................. each dilution was spotted on to histidine minus medium before incubation. Bone recognition mechanism Vesicle analysis Vesicle analysis was performed on leaf segments stained with DAB to detect H2O2 as described15. Leaves of 7-day-old seedlings were inoculated with B. g. hordei, and 24 h later of porcine osteocalcin from they were assessed by differential interference contrast microscopy for vesicles in the short cells of the adaxial epidermis23. Per genotype, 100 sites were scored from each of three crystal structure leaves. Only sites at which penetration had failed were scored. Quyen Q. Hoang1,2, Frank Sicheri3,4, Andrew J. Howard5 See Supplementary Information for barley genotype analysis with B. g. hordei,and & Daniel S. C. Yang1 PEN1 and ROR2 localization. Received 16 June; accepted 15 September 2003; doi:10.1038/nature02076. 1Department of Biochemistry, Faculty of Health Science, McMaster University, 1. Johnson, L. E. B., Bushnell, W. R. & Zeyen, R. J. Defense patterns in nonhost higher plant species Hamilton, Ontario L8N 3Z5, Canada against two powdery mildew fungi. I. Monocotyledonous species. Can. J. Bot. 60, 1068–1083 (1982). 2Microstar Biotech Inc., 7 Innovation Drive, Flamborough, Ontario L9H 7H9, 2. Freialdenhoven, A., Peterha¨nsel, C., Kurth, J., Kreuzaler, F. & Schulze-Lefert, P. Identification of genes Canada required for the function of non-race-specific mlo resistance to powdery mildew in barley. Plant Cell 8, 3Program in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, 5–14 (1996). 3. Hoogkamp, T. J. H., Chen, W. Q. & Niks, R. E. Specificity of prehaustorial resistance to Puccinia hordei Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, and to two inappropriate rust fungi in barley. Phytopathology 88, 856–861 (1998). Canada 4 4. Mellersh, D. G., Foulds, I. V., Higgins, V. J. & Heath, M. C. H2O2 plays different roles in determining Department of Molecular and Medical Genetics, University of Toronto, Toronto, penetration failure in three diverse plant-fungal interactions. Plant J. 29, 257–268 (2002). Ontario M5S 1A8, Canada 5. Hammond-Kosack, K. E. & Parker, J. E. Deciphering plant-pathogen communication: fresh 5Department of Biological, Chemical and Physical Sciences, Illinois Institute perspecitives for molecular resistance breeding. Curr. Opin. Biotechnol. 14, 177–193 (2003). Technology, Chicago, Illinois 60616, USA 6. Peterha¨nsel, C., Freialdenhoven, A., Kurth, J., Kolsch, R. & Schulze-Lefert, P. Interaction analyses of ............................................................................................................................................................................. genes required for resistance responses to powdery mildew in barley reveal distinct pathways leading to leaf cell death. Plant Cell 9, 1397–1409 (1997). Osteocalcin is the most abundant noncollagenous protein in 7. Sanderfoot, A. A., Assaad, F. F. & Raikhel, N. V. The Arabidopsis genome. An abundance of soluble N- bone1, and its concentration in serum is closely linked to bone ethylmaleimide-sensitive factor adaptor protein receptors. Plant Physiol. 124, 1558–1569 (2000). metabolism and serves as a biological marker for the clinical 8. Blatt, M. R. Toward understanding vesicle traffic and the guard cell model. New Phytol. 153, 405–413 2 (2002). assessment of bone disease . Although its precise mechanism of 3,4 9. Jahn, R., Lang, T. & Su¨dhof, T. C. Membrane fusion. Cell 112, 519–533 (2003). action is unclear, osteocalcin influences bone mineralization ,in 10. Fasshauer, D., Sutton, R. B., Brunger, A. T. & Jahn, R. Conserved structural features of the synaptic part through its ability to bind with high affinity to the mineral fusion complex: SNARE proteins reclassified as Q- and R-SNAREs. Proc. Natl Acad. Sci. USA 95, 5 15781–15786 (1998). component of bone, hydroxyapatite . In addition to binding to 11. Shen, Q. H. et al. Recognition specificity and RAR1/SGT1 dependence in barley Mla disease resistance hydroxyapatite, osteocalcin functions in cell signalling and the genes to the powdery mildew fungus. Plant Cell 15, 732–744 (2003). recruitment of osteoclasts6 and osteoblasts7, which have active 12. Lerman, J. C., Robblee, J., Fairman, R. & Hughson, F. M. Structural analsysis of the neuronal SNARE roles in bone resorption and deposition, respectively. Here we protein syntaxin-1A. Biochemistry 39, 8470–8479 (2000). ˚ 13. Munson, M., Chen, X., Cocina, A. E., Schultz, S. M. & Hughson, F. M. Interactions within the yeast t- present the X-ray crystal structure of porcine osteocalcin at 2.0 A SNARE Sso1p that control SNARE complex assembly. Nature Struct. Biol. 7, 894–902 (2000). resolution, which reveals a negatively charged protein surface 14. Misura, K. M. S., Scheller, R. H. & Weis, W. I. Self-association of the H3 region of syntaxin 1A. J. Biol. that coordinates five calcium ions in a spatial orientation that is Chem. 276, 13273–13282 (2001). 15. Hu¨ckelhoven, R., Fodor, J., Preis, C. & Kogel, K. H. Hypersensitive cell death and papilla formation in complementary to calcium ions in a hydroxyapatite crystal barley attacked by the powdery mildew fungus are associated with hydrogen peroxide but not with lattice. On the basis of our findings, we propose a model of salicylic acid accumulation. Plant Physiol. 119, 1251–1260 (1999). osteocalcin binding to hydroxyapatite and draw parallels with 16. Lauber, M. H. et al. The Arabidopsis KNOLLE protein is a cytokinesis-specific syntaxin. J. Cell Biol. 139, 1485–1493 (1997). other proteins that engage crystal lattices. 17. Betz, W. J. & Richards, D. A. What goes out must come in. Nature Neurosci. 3, 636–637 (2000). The primary structure of osteocalcin (OC) is highly conserved 18. Emans, N., Zimmerman, S. & Fischer, R. Uptake of fluorescent marker in plant cells is sensitive to among vertebrates and contains three vitamin-K-dependent g- brefeldin A and wortmannin. Plant Cell 14, 71–86 (2002). carboxylated glutamic acid (Gla) residues at positions 17, 21 and 19. Lamb, C. & Dixon, R. A. The oxidative burst in plant disease resistance. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48, 251–275 (1997). 24 in porcine OC (pOC; Fig. 1a and Supplementary Fig. 1). Solution 20. Snyder, B. A. & Nicholson, R. L. Synthesis of phytoalexins in sorghum as a site-specific response to studies have shown that mature OC is largely unstructured in the fungal ingress. Science 248, 1637–1639 (1990). absence of calcium and undergoes a transition to a folded state on 21. Adam, L. & Somerville, S. C. Genetic characterization of five powdery mildew disease resistance loci in 8 Arabidopsis thaliana. Plant J. 9, 341–356 (1996). the addition of physiological concentrations of calcium . NMR 22. Yu, Y. et al. A bacterial artificial chromosome library for barley (Hordeum vulgare L.) and the analysis has shown that OC is a globular protein consisting of identification of clones containing putative resistance genes. Theor. Appl. Genet. 101, 1093–1099 (2000). a-helical secondary structure in its folded state8,9, but the detailed 23. Koga, H., Bushnell, W. R. & Zeyen, R. J. Specificity of cell type and timing of events associated with three-dimensional structure of OC has not been forthcoming. papilla formation and the hypersensitive reaction in leaves of Hordeum vulgare attacked by Erysiphe graminis f. sp hordei. Can. J. Bot. 68, 2344–2352 (1990). To gain further insight into the structure of OC and its ability to 24. Sutton, R. B., Fasshauer, D., Jahn, R. & Brunger, A. T. Crystal structure of a SNARE complex involved recognize the hydroxyapatite (HA) mineral component of bone, we in syntaptic exocytosis at 2.4 A˚ resolution. Nature 395, 347–353 (1998). have determined the crystal structure of pOC at 2.0 A˚ using the Iterative Single Anomalous Scattering method10. Bijvoet difference Supplementary Information accompanies the paper on www.nature.com/nature. Patterson map analysis detected the presence of three tightly bound 2þ Acknowledgements This work was supported by Syngenta, the Max Planck Society, the Gatsby Ca ions and two S atoms corresponding to a disulphide bridge Charitable Foundation, the Danish Agricultural and Veterinary Research Council, the GABI Non- between Cys 23 and Cys 29, which together were used to phase the host Resistance Consortium (Bundesministerium fu¨r Bildung und Forschung), the Carnegie pOC structure. An atomic model corresponding to residues Pro 13 Institution of Washington, and the US Department of Energy. M.S. was supported in part by a Stanford Graduate Fellowship. N.C.C. thanks the host group of K. Shirasu in the Sainsbury to Ala 49 was built into well-defined electron density (Supplemen- Laboratory. We thank R. Bradbourne and H. Tippmann for technical assistance; M. Gale for tary Fig. 2) and refined to an R work and R free of 25.5% and 28.3%, microsatellite primers; M. Miklis and J. Uhrig for silencing and yeast two-hybrid vectors; respectively. Data collection and structure refinement statistics are þ R. Serrano and R. Napier for H -ATPase and CALRETICULIN antisera; and R. Oliver for the summarized in Supplementary Table 1. GUS reporter construct. pOC forms a tight globular structure comprising a previously 11 Competing interests statement The authors declare that they have no competing financial unknown fold (no matches in the DALI database ) with a topology interests. consisting, from its amino