Perception of Brassinosteroids by the Extracellular Domain of the Receptor Kinase BRI1

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Science  30 Jun 2000:
Vol. 288, Issue 5475, pp. 2360-2363
DOI: 10.1126/science.288.5475.2360


An assay was developed to study plant receptor kinase activation and signaling mechanisms. The extracellular leucine-rich repeat (LRR) and transmembrane domains of the Arabidopsis receptor kinase BRI1, which is implicated in brassinosteroid signaling, were fused to the serine/threonine kinase domain of XA21, the rice disease resistance receptor. The chimeric receptor initiates plant defense responses in rice cells upon treatment with brassinosteroids. These results, which indicate that the extracellular domain of BRI1 perceives brassinosteroids, suggest a general signaling mechanism for the LRR receptor kinases of plants. This system should allow the discovery of ligands for the LRR kinases, the largest group of plant receptor kinases.

Receptor kinases mediate extracellular signals for diverse processes in plants and animals. Detailed mechanistic studies of both receptor tyrosine kinases and receptor serine/threonine kinases have been well documented in animal cells, where it has been shown that ligand binding to the extracellular domains of receptors induces receptor dimerization and stimulates receptor phosphorylation, resulting in the activation of intracellular signaling cascades (1–3). In contrast, the study of plant receptor-like kinases (RLKs), all of which are serine/threonine kinases, is still in its infancy (4,5). Despite the large numbers of putative RLKs encoded in the genomes of plants, how these receptors carry out signal transduction has yet to be determined.

Of the various RLKs, the largest group is the leucine-rich repeat receptor kinases (LRR- RLKs). This class consists of at least 120 genes in Arabidopsis. A few LRR-RLKs are involved in diverse biological processes based on their mutant phenotypes. These processes include the control of meristem development (6), disease resistance (7), hormone signaling (8), and organ elongation and abscission (9, 10). However, in no case is there biochemical evidence for the identity of a ligand, although genetic studies have provided some clues. On the basis of the similarity of mutant phenotypes and their adjacent expression domains within the meristem, CLAVATA3, a putative extracellular protein of 96 amino acids, has been proposed as the ligand of the LRR-RLK CLAVATA1 (11). Likewise, genetic studies suggest that the steroid hormone brassinolide (BL), the most biologically active brassinosteroid, is the ligand for the BRI1-encoded LRR-RLK (8). Thus, the LRR-RLKs might use either small molecules or proteins as ligands.

To determine if BRI1 plays a direct role in BL perception, we developed a cell-based assay using the XA21 LRR-RLK from rice. XA21 confers resistance to Xanthomonas oryzaepv. oryzae (Xoo) (7). Most incompatible plant/pathogen interactions lead to a hypersensitive response (HR) that includes an oxidative burst, defense gene activation, and cell death (12,13). Thus, XA21 signaling outputs may provide a facile assay for determining the mechanism of LRR-RLK signaling. Figure 1 shows that stably transformed O. sativa ssp. Japonica var. Taipei 309 cells expressing full-length XA21 from its native promoter exhibit race-specific defense responses (14). XA21 expression initiated cell death in lines that were inoculated with the incompatible Xoo Phillipine Race 6 (P6) strain PXO99A, but not when inoculated with the compatible Korean Race 1 (K1) strain DY89031 (Fig. 1A).

Figure 1

Cell death, oxidative burst, and defense pathways are initiated in the XA21 cell lines inoculated withXoo. (A) XA21 and Taipei 309 cell lines were inoculated with 107 cells/ml of the incompatible Phillipine Race 6 strain (P6) (gray bars) and the compatible Korean Race 1 strain (K1) (open bars) 5 days after transfer to fresh medium and grown for an additional 24 hours. Cells were stained with Evans blue, and dye binding increases over uninoculated controls were quantified as an indicator of cell death. The experiment was repeated five times. (B) Cells were inoculated for 1 hour with P6 and K1 (107 cells/ml). H2O2 levels in media were assayed (23) with at least three repeats. (C) Northern blotting shows changes in the expression of defense genes RCH10, PAL, and OsCatBover a time course of 0 to 24 hours after Xoo inoculation (24). (D) mRNA levels were estimated with a PhosphoImager System (Molecular Dynamics, Sunnyvale, California). Levels at time 0 were set as onefold for RCH10 andPAL and 10-fold for OsCatB. Curves indicate incompatible (▪) and compatible (□) interactions.

Pathogen-induced cell death is often accompanied by an oxidative burst (13). A small, but highly reproducible oxidative burst was observed in the XA21 cell line inoculated for 1 hour with the incompatible P6, compared with inoculation with the compatible K1 strain (Fig. 1B). This small increase of H2O2levels, although not as large as those reported for other plants, is consistent with the levels of H2O2 that we have seen in rice (15).

Activation of XA21 signaling leads to rapid and strong induction of transcription of the rice defense genes chitinaseRCH10 (16) and phenylalanine ammonia-lyase (PAL) (17) in the incompatible interaction with Xoo (Fig. 1, C and D), whereas the compatible interaction shows a weaker and slower accumulation of these transcripts. This race-specific difference correlates to whole plant assays (18). The expression of a rice catalase B gene (OsCatB) (19) was strongly down-regulated in the XA21 cell line inoculated with the incompatible strain (Fig. 1, C and D), as seen in whole plants (20). Taken together, these results establish the rice cell culture system as an excellent reporter of the signaling output of LRR-RLKs.

To test the mechanism by which BRI1 signals, we constructed several chimeric receptors between BRI1 and XA21 (Fig. 2; NRG1, NRG2, and NRG3). Of the three receptors, only one, NRG1, consisting of BRI1's extracellular and transmembrane domains and 65 amino acids of the intracellular domain (juxtamembrane domain) fused to the kinase domain of XA21, was able to elicit the HR (Figs. 2 and 3, discussed below). As controls, we also constructed mutant versions of the NRG1 chimeric receptor. Previous studies have implicated the importance of a 70–amino acid island embedded between the 21st and 22nd LRR of BRI1's extracellular domain for BRI1 function (8). One naturally occurring allele of BRI1,bri1-113, is a mutation of glycine at position 611 in this domain to glutamate (Gly611 → Glu). The mutant chimeric receptor, NRG1mL, incorporates this change into the NRG1 construct. We also constructed a kinase domain mutant of XA21 (Lys737 → Glu) in the chimeric receptor NRG1mK, which lacks kinase activity in vitro (19). Transgenic cell lines were established by transforming the rice line Taipei 309 (14). Northern and Western blotting confirmed that two of the NRG1-expressing lines, NRG1-30 and NRG1-34, and the mutant receptor, NRG1mK, were expressed at comparable levels in the cell lines (Fig. 2, B and C). NRG1mL accumulated to higher levels (Fig. 2, B and C). Regenerated NRG1 transgenic plants were dwarfed and sterile and exhibited partial resistance to Xoo after BL treatment as compared with controls (15).

Figure 2

(A) Schematic diagram of chimeric receptor kinases NRG1, NRG2, and NRG3 and mutant controls NRG1mL and NRG1mK. The XA21 and BRI1 protein structures are labeled in white and gray, respectively, with signal peptides indicated in dark gray. These chimeras were constructed by in vitro mutagenesis (25) and driven by the cauliflower mosaic virus 35S promoter in rice cells (9) (B) Northern hybridization shows mRNA accumulation of each chimeric gene, with a 1.3-kb DNA fragment of the Xa21 kinase domain as a probe. (C) Western blot shows the expression of BRI1-XA21 chimeric proteins (26).

Figure 3

BL induces cell death, oxidative burst, and defense pathway activation in NRG1 cell lines. (A) Cell suspensions (14): NRG1-30, NRG1-34, NRG1mL, NRG1mK, and wild-type Taipei 309 were treated with 2 μM BL for 24 hours. Cell death was assayed as described in Fig. 1A. (B) NRG1 and control cell lines were treated for 30 min with 2 μM BL for H2O2 production assay (23), with gray bars for treatment and open bars for nontreatment. (C) Cell lines were treated with 2 μM BL for 0 to 24 hours. Transcript accumulation of defense-related genesRCH10, PAL, and OsCatB was determined by Northern blotting (24). (D) RNA levels were estimated as in Fig. 1D. Cell lines are NRG1-30 (▪), NRG1-34 (•), NRG1mL (□), NRG1mK (○), and Taipei 309 (▵).

We found that NRG1 could initiate the HR upon addition of BL using two different cell lines, NRG1-30 and NRG1-34 (Fig. 3). Cell death was observed after treatment for 24 hours with 2 μM BL (Fig. 3A), whereas very little cell death occurred in the Taipei 309 control, NRG1mL (Gly611 → Glu), or NRG1mK (Lys737 → Glu) cells. The magnitude of increase in cell death was comparable to that seen in the incompatible pathogen-XA21 interaction (Fig. 1A). Likewise, we observed a detectable oxidative burst in the NRG1 cell lines within 30 min of BL treatment (Fig. 3B). Changes in expression of defense genes were monitored in the wild-type and mutant receptor lines (Fig. 3, C and D). We observed an accumulation of both RCH10 and PAL mRNAs in response to 2 μM BL in both NRG1 lines, with peak levels (five- to eightfold) occurring 4 to 8 hours after BL treatment (Fig. 3, C and D). In contrast, neither NRG1mL, NRG1mK, nor the Taipei 309 control cells showed an induction of RCH10 or PAL mRNAs (Fig. 3, C and D). Accumulation of OsCatB RNA was inhibited in NRG1-30 and NRG1-34 cell lines 2 to 12 hours after BL treatment (Fig. 3, C and D). We did not detect cell death in transgenic cell lines carrying the XA21 wild-type protein or overexpressing the XA21 kinase domain after BL treatment (15).

A BL dose-response curve was constructed with RCH10 RNA accumulation as a reporter. We saw RCH10 induction using concentrations of BL as low as 10 nM; the response began to saturate at about 2 μM BL (Fig. 4). These BL concentrations are physiologically relevant, being consistent with those for rescue of the Arabidopsis BL biosynthetic mutant,det2 (21). These three assays indicate that the BRI1-XA21 chimeric receptor can recognize BL to activate cell death, the oxidative burst, and defense gene induction. Moreover, both the extracellular/transmembrane/juxtamembrane domains of BRI1 and the XA21 kinase domain are required for these responses.

Figure 4

BL dose response for RCH10 induction inNRG1 cell lines. Cells were treated with 0 to 4 μM BL. RNA was extracted 6 hours after treatment, and transcript levels were determined (24). Cell lines are NRG1-30 (▪), NRG1-34 (•), NRG1mL (□), NRG1mK (○), and Taipei 309 (▵).

Our studies indicate that BRI1 plays a direct role in brassinosteroid perception and that the response is cell autonomous. We have recently shown that BRI1 is a ubiquitously expressed, plasma membrane–localized protein (22). Thus, our data provide strong evidence that plant steroids are perceived at the cell surface. Moreover, the observation that a mutation in the 70–amino acid island region of the extracellular domain of BRI1 results in a receptor that cannot perceive BL reinforces the notion that this region is important for steroid binding or proper folding of the extracellular domain for BL recognition.

These results suggest a mechanism of signaling conserved between BRI1 and XA21 that may be extrapolated to the large number of LRR-RLKs found in plant genomes. The model would include ligand perception through the extracellular/transmembrane domain, whereas the intracellular kinase domain determines the downstream signaling response. There are greater than 120 LRR-RLKs predicted in the Arabidopsisgenome sequencing project. The chimeric receptor approach, using the XA21 signaling outputs defined here, should provide an assay system that is applicable to the discovery of ligands for the LRR-RLKs, as well as aid in the design of novel signaling genes for controlling plant development and disease resistance.

  • * Present address: Department of Biology, University of Michigan, MI 48190–1048, USA.

  • Present address: Dupont Company, Wilmington, DE 19880–0402, USA.

  • Present address: John Innes Centre, Norwich, NR4 7UH, UK.

  • § To whom correspondence should be addressed. E-mail: chory{at}


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