Molecular Cloning And Functional Analysis Of Two Protein Kinase Genes, OsBIMK2 And OsBISERK1, Involved In Rice Disease Resistance

Plants have evolved a series of complicated defense mechanism against pathogens attack during their long-term process of co-evolvement. Plants disease resistance controlled by the resistance gene (R gene) is a resistance with high specificity at the cultivar to race level. In addition, plants have also evolved an adaptive inducible resistance mechanism in response to pathogen invasion. Plant induced resistance interests us due to its non-specificity and wide-spectrum against to pathogens. In previous studies of our lab, differentially expressed cDNA libraries associated with rice (Oryza sativd) induced resistance were constructed by suppression subtractive hybridization (SSH), and over 200 differentially expressed cDNAs were cloned. Of those rice cDNA clones, two clones may encode mitogen-activated protein kinase (MAPK) and somatic embryogenesis receptor kinase (SERK), respectively. In this study, we have cloned and identified two full-length cDNAs of OsBlMK.2 and OsBISERKl, which encoding MAPK and SERK, respectively, and associated with benzothiadiazole (BTH) induced resistance. Their expressions in defense responses induced by BTH as well as in the compatible/incompatible interactions between rice and the blast fungus Magnaporthe grisea were analyzed. Recombinant OsBIMK2 protein was obtained in vitro by expression of fusion OsBIMK2 gene in Escherichia coli and biochemical characteristics of the purified OsBIMK2 protein was analyzed. The promoter sequence of OsBIMK2 gene was cloned and potential as-acting elements of OsBIMK2 promoter sequence involved in gene regulation were identified by Agrobacterium tumefaciens mediated transient expression system. Transgenic tobacco plants of over-expressed OsBIMK2 gene were obtained and showed enhanced disease resistance by constitutive expression of defense gene PR-1.Of rice differentially expressed cDNAs associated with BTH induced resistance by SSH, two cDNAs clone, BIHN-nl (BU18686) and BNHN-4w (BI118743), contained insert fragment of 585 bp and 343 bp, respectively. BLAST similaritysearching against databases revealed that the two clones showed higher homology with MAPK and SERK genes in plants, respectively. Using rapid amplification of cDNA ends (RACE) method, the 5'- and 3'-ends of the two genes sequence were amplified, respectively, with phage DNA prepared from a rice cDNA library as template . Their full-length cDNA clones were amplified by PCR based on information of assembled cDNA sequences. The obtained clones were named as OsBIMK2 (Oryza sativa L. BTH-Induced MAP kinase 2) and OsBISERKl (Oryza sativa L. benzothiadiazole-induced SERK I), respectively.The full-length cDNAof the OsBIMK2 gene was 2132 bp with a predicted 1521 bp open reading frame (ORF), and had 205 bp and 407 bp non-coding sequences at 5'- and 3'-ends, respectively. The ORF of the full-length cDNA predicted to encode a putative MAPK protein with 506 amino acid residues, including 11 conservative serine/threonine domains specialized for MAPK and a special phosphorylation/dephosphorylation TDY motif. Analysis of homology and phylogenic tree indicated that OsBlMK2 protein had 40-71% of homology with known MAPK proteins from other plants and 41.7~78.9% of homology with known MAPK proteins from rice at the level of amino acids sequence, suggesting that OsBIKM2 is a new rice MAPK. Southern blotting demonstrated that OsBIMK2 gene presented as a single-copy in rice genome. In vitro recombinant OsBlMK2 protein was obtained by expression of fusion OsBIMK2 gene in E. coli. The purified OsBIMK2 protein has capacity of self-phosphorylation activity, implying OsBIMK2 encodes a new MAPK protein possessing biochemical activity.The full-length cDNA of the OsBISERKl gene was assembled, which was 2349 bp with a predicted 1875 bp ORF, and had 104 bp and 371 bp non-coding sequences at 5' and 3'-ends, respectively. The 1875 bp ORF of the full-length cDNA predicted to encode a putative SERK protein with 624 amino acid residues. Predicted OsBISERKl protein has 75~85% of homology with known SERK proteins...