Identification and Expression of hrpN Gene fromStrain EcbCSL101 and the Activity of Purified HarpinEcbCSL101 Protein

Abstract:

Erwinia carotovora subsp. betavasculorum (Ecb) strain EcbCSL101 produces high levels of extracellular enzymes including pectate lyase (Pel), polygalacturonase (Peh), protease (Prt) and cellulase (Cel) and elicits hypersensitive response (HR) in tobacco leaves. Southern blot analysis using Ecc strain CSDS001 hrpN DNA as probe revealed that EcbCSL101 possesses hrpN gene. A DNA fragment containing entire ORF of EcbCSL101 hrpN was PCR amplified and cloned into vector pET28a(+). Nucleotide sequence analysis reveals that EcbCSL101 hrpN possesses a 1113 bp ORF which encodes a 36.65 kD HarpinEcbCSL101 protein. The deduced amino acid sequence of EcbCSL101 hrpN shares high homology with Harpin proteins of several other Erwinia carotovora strains (GenBank number DQ355519). HarpinEcbCSL101 protein was expressed in E. coli strain JM109 (DE3) by IPTG induction. The purified HarpinEcbCSL101 effectively elicits the HR in tobacco leaves.