Purification, Gene Cloning And Function Characterization Of An Elicitor From Verticillium Dahliae

Elicitors play an important role in the induction of plant defense responses and enhanced plant disease resistance againse pathogences. In this paper, a protein elicitor from the culture filtrate of cotton Verticillium wilt pathogenic fungus, Verticillium dahliae, was isolated and purified. This elicitor induced a hypersensitive response and resistance substances production and accumulation in tobacco leaves. The elicitor protein was expressed in Escherichia coli. Recombinant protein induced the up-expression of disease-related gene in tobacco with RT-PCR technique, This study revealed that the elicitor could induce systemic acquired resistance in tobacco and provide a base for further study in mechanisms of the protein elicitor.1. The protein elicitor from the culture filtrate of cotton Verticillium wilt pathogenic fungus, Verticillium dahliae, was isolated and purified through the procedures of ammonium sulfate precipitation, ?KTA Explorer 10 Protein Purification System, native-PAGE cut and electroelution.The proein showed a single band on SDS-PAGE gel with molecular mass 17 kD, pI=4.34. We designated the protein as PevD1 (Protein elicitor from Verticillium dahliae). Infiltrated with this elicitor induced a hypersensitive response in tobacco leaves.2. The protein elicitor PevD1 triggered plant defense responses and activated early signaling events (ROS burst, H₂O₂ production, Extracellular medium alkalization, NO production and accumulate) in tobacco, induced secondary metabolite generation (callose deposition, phenolics metabolism and lignin synthesis). The activity of Phenylalanine ammonia lyase (PAL), Polypheol oxidase (PPO) and Peroxides (POD) increased after elicitor inducement compared with control in tobacco leaves, the maximum activity of PAL appeared at 144 h and was 3.3 times of untreatment. The activity of PPO and POD increased by 236.8% and 204.6% at 120 h, respectively. Cell membrane permeability increased, programmed cell death, cell DNA fragmentation, and hypersensitive response marker genes hrs203J, hin1 were induced in PevD1–treated tobacco cell.3. Three peptide segments of the purified protein were obtained by analysis of ESI-MS/MS and de novo sequencing. Protein similarities by a protein BLAST search in NCBI indicated that the protein matched with only one conserved hypothetical protein from Verticillium albo-atrum VaMs.102. A pair of primers was designed for the amplification of the full-length fragment of the elicitor-encoding gene. The pevD1 gene contained 468 bp, encoding a protein of 155 amino acids, and a signal peptide containing 18 amino acids residues with theoretical molecular weight of 16.23 kDa. The pevD1 gene was constructed in the pET-28a-c (+) vector and transferred into Escherichia coli. Recombinant protein was expressed and purified.4. Recombinant protein PevD1 induced systemic acquired resistance(SAR)in tobacco against tobaco mosaic virus (TMV). The inhibition in lesion number and size was 53.85% and 32.06% respectively at 6th day post PevD1 treatment. The Semi-quantitative RT-PCR revealed that resistance-related genes-PR1-a, PR1-b, PDF1.2, NPR1, MAPK were upregulated expression after PevD1 treatment in tobacco, which was in a time-dependent. The result showed it is one of the important mechanisms of systemic acquired resistance activated by protein elicitor-PevD1 in tobacco.This study laid a foundation for future research in disease signal transduction pathway.