Transcriptomic analysis of Paulownia infected by Paulownia witches'-broom Phytoplasma.

Phytoplasmas are plant pathogenic bacteria that have no cell wall and are responsible for major crop losses throughout the world. Phytoplasma-infected plants show a variety of symptoms and the mechanisms they use to physiologically alter the host plants are of considerable interest, but poorly understood. In this study we undertook a detailed analysis of Paulownia infected by Paulownia witches'-broom (PaWB) Phytoplasma using high-throughput mRNA sequencing (RNA-Seq) and digital gene expression (DGE). RNA-Seq analysis identified 74,831 unigenes, which were subsequently used as reference sequences for DGE analysis of diseased and healthy Paulownia in field grown and tissue cultured plants. Our study revealed that dramatic changes occurred in the gene expression profile of Paulownia after PaWB Phytoplasma infection. Genes encoding key enzymes in cytokinin biosynthesis, such as isopentenyl diphosphate isomerase and isopentenyltransferase, were significantly induced in the infected Paulownia. Genes involved in cell wall biosynthesis and degradation were largely up-regulated and genes related to photosynthesis were down-regulated after PaWB Phytoplasma infection. Our systematic analysis provides comprehensive transcriptomic data about plants infected by Phytoplasma. This information will help further our understanding of the detailed interaction mechanisms between plants and Phytoplasma.

[Complete sequence of a full-length DNA and molecular characterization of one plasmid from chinaberry (Melia azedarach Z) witches'-broom phytoplasma].

OBJECTIVE: To clone plasmid from chinaberry witches'-broom phytoplasma and analyse its molecular characterization. METHODS: Fragments of one plasmid (pCWBFq) in chinaberry witches'-broom phytoplasma-Fuqing strain (CWBFq) were amplified with primer pairs which were designed according to plasmid sequences published on NCBI. Transmembrane domain and subcellular localization predictions of proteins encoded by the plasmid pCWBFq as well as phylogenetic analysis among the plasmid sequences were completed by using bioinformatic softwares. Southern blot analysis was performed to detect the plasmids existed in CWBFq and several other phytoplasmas with the pCWBFq repA probe. RESULTS: One complete plasmid was sequenced from CWBFq. pCWBFq comprised 4446 bp and had a nucleotide content of 73.5% A + T and encoded six proteins. Protein P2, P3, P4 and P5 of pCWBFq contained 3, 2, 1 and 2 tranmembrane domains respectively, and their predicted signal peptide values were 0.989, 0.505, 0.918 and 0.914 respectively. Homologous comparison showed that RepA homology between pCWBFq and other phytoplasmas was between 9.6% -85.6% , however, the homology of different SSB proteins was between 74.0% - 89.4%. Southern blotting with pCWBFq repA probe confirmed the existence of the plasmids in CWBFq. In addition, The hybridizations occurred with paulownia witches'-broom phytoplasma-Nanyang strain (PaWBNy), periwinkle virescence phytoplasma-Hainan stanin (PeVHn), chinaberry witches'-broom phytoplasma-Fuzhou strain (CWBFz) and mulberry dwarf phytoplasma - Puyang strain (MDPy), whereas, no hybridizarions occurred with jujube witches'-broom phytoplasma-Beijing strain (JWBBj), cherry lethal yellows phytoplasma-Xichang strain (CLYXc) and Bischofia polycarpa witches'-broom phytoplasma-Nanchang strain (BiWBNc). CONCLUSION: The plasmid encoded a replication associated protein (RepA) and a single-stranded DNA binding protein (SSB), which were for the replication of plasmid. Four putative proteins encoded by the plasmid were predicted to contain one or more hydrophobic transmembrane domains, respectively, and presumably to be localized to the membrane. The alignment and homology analysis as well as phylogenetic analysis to the DNA and encoded protein amino acid sequences of the whole plasmids and single ORFs on the known phytoplasmal plasmids showed that the different homologous sequences have distinct variation, among which the repA gene with the largest diversity appeared in all the known plasmids while ssb with less variation were only found in 16SrI plasmids. CWBFq, PaWBNy, PeVHn, CWBFz and MDPy possessed distinct plasmids in terms of number and size, whereas there was no plasmid detected in JWBBj, CLYXc and BiWBNc, perhaps as a result of low homology among repA genes in plasmids of JWBBj, CLYXc and BiWBNc.

[Cloning and analysis of tuf and rp gene of the phytoplasma associated with jujube witches' -broom].

OBJECTIVE: Jujube witches' -broom (JWB) is an important plant disease caused by phytoplasma. The major objective of our research was to classify JWB in Beijing and Hebei districts and to provide reference for classification in subgroup level. METHODS: By use of PCR, the elongation factor Tu (tuf gene) and ribosomal protein (rp) gene of phytoplasma associated with JWB in Beijing and Hebei districts were amplified separately with universal primer pairs fTufu/rTufu and rp(v)F1A/rp(v) R1A. Partial tuf gene and rp gene were sequenced and similarity analysed with other phytoplasmas. RESULTS: We obtained partial tuf gene sequence (824bp) and complete rp gene (1196bp) from the diseased sample. In tuf gene, JWB in Beijing shared most similarity (92.84%) with Flavescence dorée (FD) phytoplasma (Candidatus Phytoplasma vitis), however, shared a low similarity (57.29%) with JWB in Shaanxi district which had been already reported. The similarity analysis for sequences of rp gene showed a high identity (> 96%) with members of the 16SrV group phytoplasmas. It shared most identity (99.83%) with JWB strain Taishan and Hemp fiber witches' -broom phytoplasma (HFWB) of the 16SrV group. CONCLUSION: The JWB strains in Beijing and Hebei are members of 16Sr V; JWB in Beijing and Hebei share high similarity, and show a diversity with JWB in Shaanxi.