Construction Of A Brassinosteroid Supersensitive Chimeric Receptor And Rice Transformation

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. However, the study of plant receptor-like kinases (RLKs), all of which are serine/threonine kinases, is still in its infancy.Of the various RLKs, the largest group is the leucine-rich repeat receptor kinases (LRR-RLKs), which consists of at least 120 genes in Arabidopsis. A few LRR-RLKs are proved involving in diverse biological processes based on their mutant phenotypes. These processes include the control of meristem development, disease resistance, hormone signaling, and organ elongation and abscission and so on. 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. Likewise, genetic studies suggest that the steroid hormone brassinolide (BL), the most biologically active brassinosteroid, is the ligand for the 5.K/7-encoded LRR-RLK. Thus, the LRR-RLKs might use either small molecules or proteins as ligands.In order to determine if BRI1 plays a direct role in BL perception and to construct a eliciting and signal control system of disease resistance receptor genes, He et al constructed a signal transduction system NRG1 by combining Arabidopsis BRIl's (brassinosteroid (BR) receptor kinase) LRR-JM domain with the rice XA21 (disease resistance receptor kinase) Ser/Thr kinase domain. When treated with BR, the chimic receptor can elicit hypersensitive response (HR) that includes an oxidative burst, defense gene activation, and cell death assame as Xa21.This system strongly confirms the role of BRI1 in BR perception. The chimeric receptor approach, using the XA21 signaling outputs defined here, could 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 resistanceIn order to construct a more sensitive signal transduction system, a chimeric receptor kinase SNRG1 was constructed by the combining of the Arabidopsis SUD1 (brassinosteroid (BR) supersensitive receptor kinase) LRR-JM domain and the rice XA21 (disease resistance receptor kinase) kinase domain with point mutagenisis in vitro. The SUD1 LRR mutant SNRGmL and the XA21 kinase mutant SNRGmK were also constructed as controls respectively. Using particle bombardment, SNRG1, SNRGmL and SNRGmK were successfully transferred into rice embryogenic calli ofjaponica variety Taipei 309. Transgenic rice plants and cell lines were confirmed by PCR x Northern blot and transformant phenotype. Preliminary result showed that rice defense pathways could have been activated by trance amount of endogenous BR in the SNRG1 transgenice plants. These results provided the foundation for the further investigation of XA21 kinase activation upon BR ligand binding.