Functional Analysis Of PPaWbNy-1-ORF5and PPaWBNy-2-ORF4Genes In The Plasmids Of Paulownia Witches’-broom Phytoplasma

Paulownia witches’-broom (PaWB) disease associated with phytoplasma is a devastatingdisease of paulownia in China. Most species of Chinese paulownia are sensitive to PaWBdisease. PaWB disease could be transmitted by vegetative propagation materials（eg: seedlingand root cutting）and phloem-feeding insects. The vector insects of PaWB phytoplasma wereHalyomorpha halys (St l), Crytopeltis tenuis (Reuter) and Empoasca flavescens (Fabricius). Tobetter understand the function of PaWB phytoplasma plasmids and molecular interactions ofPaWB phytoplasma-vector and insect transmission, we conducted the following researches.We use a clone containing a3.0kb segment of PaWB phytoplasma plasmid pPaWBNy-1and genome DNA extracted from infected paulownia plantlets as DNA templates, PCRamplification of pPaWBNy-1-ORF5and pPaWBNy-2-ORF4hydrophilic peptide codingregions. The amplified DNA fragments were inserted into the prokaryotic expression vectorand the recombinant plasmid was transformed into the Escherichia coli Rosseta(DE3) strain.Under the optimal experimental condition, the15kDa of His-tagged ORF5and38kDa ofGST-tagged ORF4fusion proteins were expressed efficiently in E.coli Rosseta(DE3). Thefusion proteins were purified and injected into white rabbit to raise antiserum. The titers of twoantisera were determined to be1:8100and1:4096by indirect ELISA, respectively.Using the two antisera, we analysed the expression of pPaWBNy-1-ORF5andpPaWBNy-2-ORF4genes in different specimens of host plant and vector insect, the tworesultant antibodies didn’t react with the proteins extracted from infected paulownia plantletsand field-collected symptomed paulownia, but detected17kDa of pPaWBNy-1-ORF5codingprotein and18kDa of pPaWBNy-2-ORF4coding protein in H. halys exposing to PaWBphytoplasma infected paulownia. It was inferred that pPaWBNy-1-ORF5andpPaWBNy-2-ORF4might be involved in the insect transmission of H. halys. The in vivointeraction protein specimens were prepared with H. halys by injecting His-tagged ORF5 fusion protein into the hemolymph of H. halys. Then far Western blot experiments were carriedout on H. halys protein blot with phytoplasma proteins as the overlay to identify the protein ofH. halys involved in the interaction with the pPaWBNy-1-ORF5coding protein. Two proteinsof the thoracoab dominal of H. halys were identified, one with a molecular mass of52kDa andthe second with a mass of36kDa. Moreover, a52kDa protein from the hemolymph of H.halys were identified. Sequencing those insect proteins, will help us to determinepPaWBNy-1-ORF5protein function, therefore, to promote to understand the molecularmechanism of PaWB phytoplasma interacting with vector insect H. halys.H. halys nymphs were collected from paulownia plantations in the yard of Chinese Academyof Froestry. We developed a stable rearing, acquisition method as well as abdominalmicroinjection methods of Beijing population of H. halys. Adults of the stink bugs werecollected by means of sweeping net and beating tray in infested paulownia plantations byPaWB phytoplasma and overwintering sites in Nanyang, Henan province. A total of350stinkbugs were collected:293of H. halys,41of Erhtesina fullo (Thunberg),8of Plautia fimbriata(Fabricus) and8of Cappaea tibialis Hsiao et Cheng. DNA and protein were extracted fromstink bugs collected from Nangyang. Semi-nested PCR and nested PCR amplification of theRepA gene specific to PaWB plasmid was performed using the extracted DNA as templates.The results indicated that E. fullo and C. tibialis didn’t carry PaWB phytoplasma, while thepositive detection rate was29.2%of overwinter H. halys, and16.7%of H. halys collected frompaulownia plantations. Western blot analysis using pPaWBNy-1-ORF5antibody revealed thatthat a specific17kDa protein band in H. halys protein, which indicated thatpPaWBNy-1-ORF5gene expressed in H. halys.In semi-nested PCR using RepA gene primers, from a total of8individual stink bugs tested,1of P. fimbriata samples gave a positive singal. The amplified DNA fragment was cloned andsequenced, the sequence alignment revealed that the sequence was99.4%identical to RepAgene paulownia witches’-broom phytoplasma (EF426472). In Western blot analysis, a18kDapolypeptide was detected in total protein from P. fimbriata. This results indicated that pPaWBNy-2-ORF4gene expressed in P. fimbriata. Whether the P. fimbriata is the vector’insect of PaWB phytoplasma, need to be confirmed through the transmissionassay.