Effect Of PopW From Ralstonia Solanacearum On Inducing Plants Disease Resistance And Obtaining Of Transgenic Tobacco Expressing PopW

Harpins are extra cellular proteins encoded by Gram-negative bacterial hrp (hypersensitive response and pathogenity) gene cluster. They are a major class of proteins that travel the pathway, elicit the HR (hypersensitive reaction) when infiltrated into the apoplast of leaf tissue (hypersensitive cell death.HCD) drought tolerance, defense responses against pathogens and insects in plants, and they can enhance Plant growth as well. They are heat stable, rich in Gly and/or Ser, lack Cys, and differ in their primary sequences.In previoue study, we had identified a new extra cellular harpin protein, PopW, from Ralstonia solanacearum ZJ3721, likes other known harpins, is acidic, rich in glycine and serine, and lacks cysteine. When infiltrated into plants, PopW induced rapid tissue collapse on tobacco (non-host).This hypersensitive reaction (HR)-eliciting activity of PopW is heat stable and protease sensitive. The expression of recombinant protein PopW was induced by different concentration IPTG, and was analyzed to find an optimal concentration level for its peak expression. IPTG concentration was examined from 0.0002 to 1 mM and the expression of soluble product was analyzed by SDS-PAGE. The results showed that the protein production was increased with the added IPTG concentration up to 0.03 mM. But compared with the concentration of the soluble at 0.01 mM, it was not significant difference.So all of other studies were carried out with the IPTG concentration at 0.01 mM.PopW can induce plant disease resistance, called systemic acquired resistance, meditaed by salicylic acid. Tobacco leaves(Nicotiana tabacum cv. Xanthi NN) were treated with PopW followed by the inoculation of TMV onto these leaves. The Lesion number infected by TMV was significantly less than blank control inoculated with TMV. The efficacy of PopW at the concentration of 2.5 mg/L,25 mg/L and 250 mg/L was about 36.24%,64.53% and 85.39%, respectively. The Biocontrol efficacy increased with the increase in PopW concentration from 2.5 to 250 mg/L. In general, the resistance induced by PopW (2.5 mg/L) against plant disease can last more than 8 days.We also found that the PopW (2.5 mg/L) inhibited tomato leaf mold caused by Cladosporium fulvum and rice false smut caused by Ustilaginoidea virens, and the biocontrol efficacy were about 80.19% and 86.84%, respectively.When the pepper were treated with PopW (2.5 mg/L), the yield was increased by about 16.89% in comparasion to the mock.In this study, we cloned pop W gene from Ralstonia solanacearum ZJ3721, and inserted it into the transformation vector pBI121 digestion with restriction enzymes BamH1 and Xba I. The resulting recombinant pBI121-popW was transformed into E. coli TOP 10. After verifying correct orientation of genes, these transgenic constructs were transformed into Agribacterium tumefaciens EHA105 by the freeze-thaw method.Transformation was conducted by infection of tobacco leaf discs with recombinant A. tumefaciens, and the TO generation transgenic tobacco plants were obtained by screening with kanamycin. Genomic integration and expression of the transferred genes were determined by PCR, the results indicated that popW has been transformed into tobacco plant.