Molecular Characterization And Protein Function Of Pathogenesis-related Genes From Paulownia Witches’-broom Phytoplasma

Paulownia sp. is a kind of important broad leaf species in China. Paulownia witches’broom disease, is infectious disease and caused by Paulownia witches’ broom phytoplasma, itstypical symptoms includes abnormal adventitious bud and axillary bud germination to formcluster, even changed from flowers to leaves and can not form normal flowers and fruits. Inorder to understand the pathogenic mechanism of phytoplasma, the related function researchand analysis were explored. Based on the related gene cloning and prokaryotic expression,antiserum preparation, protein activity determination, instantaneous expression and diseasesand symptoms analysis etc, it was found that the tRNA-ipt gene may influence plant celldivision and growth, which provides a new clue for the study of phytoplasma pathogenicmolecular mechanism.Four kinds of full-length sequences of tRNA-ipts from aster yellows group(16SrI)phytoplasmas were amplified, sequenced and analyzed in-silico. It was found that the length ofopen reading frame of tRNA-ipt genes from paulownia witches’-broom，mulberry dwarf，periwinkle virescence and Chinaberry witches’-broom phytoplasmas is876bp, their G+Ccontents are30%,29.5%,29.5%and29.3%,respectively. All these genes encode291aminoacids, predicting the molecular weight is33kda. They contain an identical motif (GPTASGKT）at N-terminal region related to the ATP or GTP binding sites. It was predicted that the proteinsare without transmembrane region, distributed in the cytoplasm. Amino acid sequencehomology of four phytoplasma tRNA-IPTs was99.1-99.5%, sequence homology with thesame group phytoplasmas was95.4-99.3%, whereas with16SrX apple proliferation and16SrXII Australia grapevine yellows phytoplasmas was less than70%.Phylogenetic analysisrevealed that all nine known tRNA-IPTs of phytoplasmas were clustered into a common clade，and they had very close relationship with that of Acholeplasma sp. The paulownia witches’-broom phytoplasma (PaWB) tRNA-ipt gene was expressed in E.coli, specific antibody was prepared and used for the gene expression analyses by Western blotand FITC indirect immunofluroscence microscopy. Then the growth curve and cytokinincontents of E. coli with PaWB tRNA-ipt were measured by photodensitometry and ELISA,respectively. The pET expression vector containing the PaWB tRNA-ipt gene was constructedand the induced fusion protein was expressed and detected by SDS-PAGE. By Western blottingand immunofluorescence detection, the expression and localization of the phytoplasmastRNA-IPT protein in the phloem of infected paulownia were confirmed. According to thedetermination of growth curve and zeatin content, we found that the gene can promote thegrowth of E. coli in the late stage and zeatin nucleotide accumulation. The determination ofgrowth curve suggested that the growth rate increase of E. coli was effected by thetransformation of exogenous tRNA-ipt gene, which might be associated with the cytokininaccumulation. This protein was assumed to involve in the cytokinin synthesis in phytoplasmasas well as other bacteria, which may play an important role in pathogenic processes ofphytoplasmas and symptom development.The virus expression vector of pCAPE-ipt were constructed to transform into healthypaulownia plantlets leaf cells by agroinfiltrayion. Results showed that the detached paulownialeaves which infiltrated with GV3101-pCAPE-ipt could maintain a longer growing andsurvived period; When the PaWB tRNA-IPT was expressed in paulownia plants two weekslater, these treated plants showed symptoms of witches’ broom and dwarfism, which weretypical ones of phytoplasma infection. At the same time, Western blotting confirmed that theprotein tRNA-IPT was expressed in treated plants but not in control plants. According to thisresult, we speculateed that the phytoplasma tRNA-IPT protein may influence the host planthormone balance, and then lead to the symptom display of witches broom disease, whichprovides a new clue for the study of the phytoplasma pathogenic molecular mechanism.On the basis of the known phytoplasma plasmid sequences, two pairs of specific primerswere designed and used for PCR. Two section of DNA amplified fragments with different sizes covering whole plasmid were obtained through two round of PCR amplification. Aftersequenced and spliced,four phytoplasmal full-length circular plasmid DNA sequences wereobtained, including the plasmids of mulberry dwarf(pMDPy) in Puyang of Henan province,periwinkle（Catharanthus roseus）virescence (pPEVHn) in Hainan province, paulowniawitches’-broom (pPaWBG33D) in Yanzhou of Shandong province and chinaberrywitches’-broom phytoplasma strains (pCWBFqHu) in Fuqing of Fujian province,with thesizes of3833bp,3943bp,3843bp and3913bp, respectively. They are predicted to encode fiveproteins,open reading frame ORF1encoding replication associated protein（RepA）,ORF5encoding binding protein（SSB）and ORF2-4encoding unknown function proteinss. pMDPy isthe first plasmid determined from mulberry dwarf phytoplasma.Sequence analysis ofnon-coding regions (NCR) of the four kinds of plasmids revealed that the NCR sequencesbetween ORF1and ORF2exist promoters and ribosome binding sites.The four phytoplasmaplasmid sequences were compared with the phytoplasma plasmid sequences found in GenBankthrough multiple alignment and phylogenetic analysis, the results showed that these fourplasmids and other16SrI group plasmid grouped into a large branch,which was consistent withthe phylogenetic tree created from16S rDNA comparisons. This research may not only providefurther insight into the structure and function, host specificity and plasmid variation andevolution within different phytoplasma species or strains,but also offers new theoretical basisfor multiple genes and index based system for phytoplasmal classification.Four different thymidylate kinase(tmk) genes were also identified in different paulowniacultivars of various regions. It was found that the length of tmk genes from PaWB-G33D、PaWB-JAN、PaWB-PS and PaWB-BJ phytoplasmas is591、600、639and588bp respected.Four tmks share the90-98%nucleotide sequence homology with the same groupphytoplasmas.The tmks of PaWB from diferent regions were different, which suggested thatthere might exist abundant genetic variation and population diversity in paulowniawitches’-broom phytoplasmas.