Expression and characterization of necrosis-inducing protein from Streptomyces lavendulae gCLA4

Abstract:

[Background] Streptomyces lavendulae gCLA4 is an actinomycete strain isolated from cucumber. Preliminary researches have showed that the strain was prominent in antagonism against a variety of pathogenic bacteria, and has potential biocontrol value. [Objective] In-depth study of the function of necrosis-inducing protein 4955 in Streptomyces lavendulae gCLA4, and to clarify its mechanism of action in improving plant resistance. [Methods] The necrosis-inducing protein 4955 gene was cloned and expressed in Escherichia coli BL21(DE3). Activity and stability of the protein were tested in tobacco. Then, the basic properties and tertiary structure of the protein were analyzed with Protparam, PredictProtein, NCBI CDD, and SWISS-MODEL. Tobacco defense-related enzymatic activities (CAT, SOD, POD, PAL) were detected after treatment with protein 4955. Additionally, altered gene expression (NPR1, PR1-b, PAL, LOX, PR1-a) was detected by qPCR. [Results] The protein 4955 retained its activity at temperatures up to 40 °C and pH 6.0 to 10.0. The protein has a molecular weight of 24 491.12 Da and consists of 225 amino acids. Its isoelectric point is 5.96, and the amino acid sequence alignment contains a conserved NPP1 domain. The activity of CAT, SOD and PAL increased in tobacco after two days of treatment with 4955, and the activity of POD did not change significantly. The expression of PR1-b and LOX genes was increased 1, 3, and 5 days after treatment with protein 4955, and the expression of PAL was up-regulated on the 4th day. [Conclusion] The necrosis-inducing protein 4955 in Streptomyces lavendulae gCLA4 does induce plant defense response in tobacco.