Perception of Brassinosteroids by the Extracellular Domain of the Receptor Kinase BRI1
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Li J., Chory J., Cell 90, 92 (1997).
Torii K., et al., Plant Cell 8, 735 (1996).
Rice transformation was as described [
]. Cell suspension lines were established in NB6 medium from transformants. The XA21 cell line was established from homozygous seeds of XA21 transgenic line 106 (7).
Z. He and P. Ronald unpublished data.
M. Yoshikawa and P. Ronald unpublished data.
W. Song and P. Ronald unpublished data.
D. Friedrichsen C. Joazeiro J. Li T. Hunter J. Chory Plant Physiol. in press.
After treatment media were filtered to remove cells with 0.2-μm syringe filters. One hundred–microliter samples were added to 1 ml of reaction buffer [0.25 mM FeSO 4 0.25 mM (NH 4 ) 2 SO 4 25 mM H 2 SO 4 1.25 μM xylenol orange and 1 mM sorbitol] at room temperature for 1 hour. Absorbance was measured at 560 nm and H 2 O 2 levels were calculated by reference to standards [
RNA was extracted from the XA21 cell lines at 0 to 24 hours after inoculation with P6 and K1 (10 7 cells per milliliter) with the TRIzol reagent (Life Technologies Gaithersburg MD). Thirty micrograms of total RNA was used in each lane. A 1.2-kb fragment of rice chitinase RCH10 (13) a 400–base pair fragment of PAL (17) and a cDNA clone of OsCatB (19) were used as probes. The filters were reprobed with 18 S Arabidopsis rDNA for normalization.
A BRI1 DNA fragment encoding the presumed extracellular transmembrane (TM) and juxtamembrane (JM) domains [amino acids 1 to 879 (8)] was fused with the Xa21 fragment encoding the predicted kinase domain [amino acids 708 to 1025 (7)] to make NRG1. NRG2 consisted of amino acids 1 to 769 of BRI1 and the XA21 TM JM and kinase domains with amino acids 625 to 1025. NRG3 consisted of amino acids 1 to 834 of BRI1 and amino acids 684 to 1025 of XA21. NRG1mL contains a mutation (Gly 611 → Glu) corresponding to the allele bri1-113 (8). NRG1mK is a mutation of XA21 (Lys 737 → Glu) obtained by in vitro mutagenesis with the primer (5′-GTTGCAGTGGAGGTACTAA-3′) corresponding to the Xa21 sequence 2197 to 2215 (7).
Cells were homogenized in 50 mM tris-HCl (pH 7.5) 200 mM mannitol 10 mM MgCl 2 and protease inhibitor cocktail (Boehringer Mannheim). After centrifugation at 10 000 g for 20 min the supernatant was centrifuged at 100 000 g for 1 hour to collect membranes. Membrane proteins (80 μg per lane) were resolved on a 3 to 8% gradient SDS-NuPAGE gel (Invitrogen) transferred to nitrocellulose and probed with affinity-purified antibodies raised to BRI1's NH 2 -terminal 106 amino acids.
We thank S. Zhang and L. Chen for providing facilities for biolistic bombardment; L. Klimczak for bioinformatics guidance; T. Murphy for the H 2 O 2 protocol; R. Ruan and T. Dabi for cell line maintenance; Y. Shen for technical help; and R. Larkin D. Weigel Y. Yin and Y. Zhao for helpful comments on the manuscript. This work was supported by grants from the U.S. Department of Agriculture to J.C. and C.L. from NIH to P.R. and the Rockefeller Foundation International Program on Rice Biotechnology (Z.H. P.R. and C.L.). Z.W. is an NSF postdoctoral fellow and J.C. is an Associate Investigator of the Howard Hughes Medical Institute.