Recombinant expression and functional characterization of a novel toll interleukin receptor nucleotide-binding site leucine-rich repeat protein from arabidopsis thaliana

Abstract

The presence of adenylate cyclases in higher plants has generally been questioned. A BLAST search of the Arabidopsis genome using a 14-amino acid motif with specificity for ATP binding bioinformatically identified the toll interleukin-like receptor nucleotide binding site leucine rich repeat (TIR-NBS-LRR) protein encoded by the At3g04220 gene from Arabidopsis thaliana to have a putative adenylate cyclase catalytic centre. To test whether TIR-NBS-LRR possesses adenylate cyclase activity, total mRNA was extracted from the leaf material of 6-week old A. thaliana plants and used as template for complementary DNA synthesis and amplification of a 114 amino acid-long adenylate cyclase catalytic domain fragment of the At3g04220 gene using Reverse Transcriptase Polymerase Chain Reaction in conjunction with sequence-specific primers. The amplified fragment was then cloned into a pCR®T7/NT-TOPO® vector and the recombinant vector was transformed into the expression host, Escherichia coli BL21 (DE3) pLysS. Positive transformants were determined using Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis; and confirmed by sequence-specific colony PCR and restriction double digestion. The His-tagged recombinant protein was over-expressed following induction with isopropyl-β-D-1-thiogalactopyranoside and purified using a nickel affinity gel system. The endogenous and in vitro adenylate cyclase activities of the recombinant TIR-NBS-LRR were then tested via a cAMP-specific enzyme immunoassaying system and the in vivo adenylate cyclase activity was tested through a complementation test using the Escherichia coli cyaA SP850 mutant. The results of all these three assays indicated that the TIR-NBS-LRR protein encoded by the At3g04220 gene from A. thaliana possessed endogenous, in vitro and in vivo adenylate cyclase activities, and thus, confirmed TIR-NBS-LRR as a higher plant adenylate cyclase with a possible cAMP-mediated signaling system.