The Screening For Extracellular Ligand Of Receptor-Like Protein Kinase, OsRPK1,from Rice (Oriza Sativa)

Receptor-like protein kinases play an important role in rice, involved in regulating of growth and development, tissue formation, organ development, disease resistance and stress resistance. It’s an important aspect for revealing its biological function and molecular mechanism that finding out corresponding ligands of receptor protein kinases. This research aims to carry out the preliminary screening for possible extracellular ligands of OsRPK1, a new class of receptor-like protein kinases. For this purpose, an artificial chimeric receptor-like protein kinase, OsRPK1-XA21, was constructed. The screening based on the occurrence of hypersensitive response mediated by XA21when the LRR of the OsRPK1co-incubats with the possible ligand. The screening would be determined by three indicators of hypersensitive response, the detection of cell death, reactive oxygen species burst and expression levels of pathogenesis related genes.In previous work, our lab analysised plasma membrane proteome from rice, and identified a series of protein responsing to salt stress, one of which, molecular weight is98kDa and theoretical pi is5.93, the expression level is up regulated remarkably after treatment with salt. It is named OsRPK1. More works found that OsRPK1was participated in the identification and polarity transport of auxin, and its expression increased by2,4-D. An envision that2,4-D may be the extracellular ligand of the OsRPK1should be proved by us.The gene Xa21is the resistance gene family members for bacterial blight Xoo (Xanthomonas oryzae pv. Oryzae).Studies have shown that the rice carrying the Xa21lead to strong resistance against bacterial blight, hypersensitive response stoped the spread of pathogens through local tissue necrosis, when disoperation happening. Hypersensitive response is a programmed cell death, including a series of physiological and biochemical indicators, such as the loss of cell viability, reactive oxygen species burst, resistance gene expression, etc. These indicators are widely used to determine whether the occurrence of hypersensitive response to plant. In this experiment, the LRR and TM of the receptor protein kinase OsRPK1was fused with the JM and kinase of XA21, the LRR、TM and JM of the receptor protein kinase OsRPK1was also fused with the kinase of XA21by means of molecular biology, and then the two of artificial chimeric receptor-like protein kinases were transformed into rice callus through Agrobacterium-mediated genetic transformation system, in the end the risistance callus would cultivate into rice suspension cells. Suspension cell death, reactive oxygen species burst of suspended cells, the H2O2levels, and the semi-quantitative PCR of plant disease resistance genes OsPR1α are detected when suspension cells are co-incubating with different concentrations of2,4-Dichlorophenoxy. Suspension cells added OμM,0.1μM,1μM and20μM of different concentrations of2,4-D as test groups, wild-type Nipponbare suspension cells as a negative control. They were detected of cell death, H2O2content in medium and semi-quantitative analysis of gene OsPR1α. Compared to the negative control, the death ratio was significantly higher, H2O2in medium showed explosive trend in the short term, semi-quantitative analysis of pathogenesis related gene OsPRla also increased within a short time trend. These results indicated that, the XA21kinase activity in intracellular in downstream of the chimeric receptor OsRPK1-XA21were active when the LRR of the OsRPKl in extracellular interacted with2,4-D, which led to the hypersensitive response. Through this research, we basically identified that exogenous auxin2,4-D interacts with OsRPKl as its extracellular ligand, and established the screening model of OsRPK1ligand. It also revealed the signal transduction mechanisms of plant receptor-like protein kinase and the important role of JM in receptor-like protein kinase in signal transmission. As long as the key amino acid in JM does not change, the JM which comes from the other RLKs does not affect signal transduction. These results lay a good foundation for comprehensive analysis of OsRPK1participating in auxin signal transduction.