| Harpins are a class of helper proteins secreted by the type III secretion systems of many Gram-negative phytopathogenic bacteria and a class of non-specific elicitors that elicit a hypersensitive response (HR) in nonhost tobacco, and induce disease resistance in plants. HR and disease resistance signaling pathway activated by harpins were closely related to its acting positions and targets in plant. Therefore, screening, isolation and identification of receptor molecules will be helpful for us to understand the structure of harpins and the interaction mechanism between plants and harpins. According to the forming CC structural characteristics of Hpalxoo N-terminal peptide N21 from Xanthomonas oryzae pv. oryzae, the affinity chromatography (AC) was selected to screen the receptor molecules from the tobacco plants, and the mass spectrometry (MS) and circular dichroism (CD) spectroscopy methods were selected to identify and characterize the receptor.The AC column was filled with CNBr Bio-sep 4FF carrier coupled with chemically synthesized peptide N21. Concentrated apoplastic fluids, cell wall and plasma membrane sample liquids from tobacco (Nicotiana tabacum L. "Xanthi") leaves were prepared and injected on an AC column, respectively, and eluted with non-specific running buffer containing 0.1 mol/LNaCl, 0.2 mol/L NaHCO3 and pH9.0. A single SDS-PAGE protein band with a molecular weight about 20 kDa was obtained only from apoplastic fluid eluent. The protein was certified as a 19.4 KDa photosystem II oxygen-evolving proteins PsbP (also known as the 23 kDa protein) by analytical in gel digestion, matrix-assisted laser desorption ionization-time of flight MS-MS (MALDI-TOF/MS-MS) and Mascot Ions Search analysis. The amino acid sequences of the receptor protein PsbP harbors multiple a-helical regions using Biogem online S2Spred program (http://www.biogemorg/tool/S2Spred/). The COILS program (http://www.ch.embnet.org/ software/COILS_form.html) predicted that the 35-55 amino acid residues (AR1),97-117 amino acid residues (AR2) and 156-176 amino acid residues (AR3) had the potential to form CC, and among which the possibility of 35-55 amino acid residues was more higher. Based on the principle of CC interactions, the region of the protein might be combined with N21 peptide in CC manner.CD analysis results showed that AR1, AR2 and AR3 had the a-helix structure, and the content of a-helices was AR1>AR2>AR3. Oligomeric state prediction (LOGICOIL andSCORER 2.0 program) showed that the receptor peptide itself and which with N21 peptides can form dimers or trimer throuth CC structure. CD spectra data fitting showed that the ARl and N21 equimolar mixture (AR1+N21) had the highest content of α-helix with 31.2%, followed by AR2+N21 and AR3+N21, the a-helix content were 26.6%and 25.7%respectively. Variational temperature (VT) CD spectra revealed that ARl and N21 peptide equimolar aqueous solution presented two-phase structure process, and the TM was 56+2 ℃. And the variable temperature curves of AR2+N21 and AR3+N21 equimolar aqueous solution were approximate straight-line, lacking a two-phase process, only AR2+N21 existed a weak unfolding process. The variable temperature curve of VT-CD spectra proved that the receptor and N21 can form oligomerization structure, with high credibility. Most FAM fluorescent labeled N21 peptide (FAM-N21) located in tobacco chloroplasts, only a few FAM-N21 located in the apoplasts space by laser scanning confocal microscope, indicating that the receptors of Hpalxoo-N21 peptide maybe located in the chloroplast and apoplasts space of tobacco.Bioinformatics analysis showed that the receptor proteins are widely existed in high plants, some algae and photosynthetic bacteria, and located in chloroplasts. In addition, the PsbP protein does not contain a signal peptide by SignalP program, indicating that PsbP protein was not a transportation protein. |
PDF Full Text Request