| Wheat blue dwarf（WBD） disease is one of the important phytoplasma disease that cause severe losses for wheat production in northwestern China. In the past three decades, a series of studies were carried out, including pathogen identification and classification, characterization of vector insects, the investigation of host range and the identification of disease-resistance varieties et al. Because WBD phytoplasma（WBD） cannot be cultured in vitro, there are less effective methods for the further research. With the development of high-through output sequencing technology, genome sequencing has been utilized as a powerful tool to study phytoplasmas. The genome sequencing of WBD and functional characterization of genes from WBD reveal the mechanism of interaction between WBD phytoplasma and its host（plant and insect） to some extent.In this study, using WBD-infected periwinkle as materials, WBD phytoplasma plasmids were cloned and their copy numbers were quantified. The chromosome DNA of WBD was isolated and enriched. Then the genome of WBD was sequenced and the bioinformatic analysis was carried out. At last, WBD secreted proteins were characterized. The main results are described as follows:1. One hundred and ninety four suspected WBD-infected wheats were collected from Hancheng and Heyang of Shaanxi province in 2011. The total DNA was extracted for the detection of WBD and WDV. The result showed 10 wheats were infected with WBD. The result of PCR using specific primers from WDV suggested that 91.6% wheats were infected with WDV and the 10 WBD-infected wheats also infected with WDV. WBD was transmitted to healthy periwinkle（Catharanthus roseus） from infected wheat seedlings by Psammotettix striatus. It was then maintained and propagated in an insect-proof greenhouse by periodic grafting. The periwinkles infected with WBD showed virescence, phyllody, proliferation and leaves yellowing. PCR confirmed these periwinkles were infected with WBD phytoplasma. And the result of phytoplasma classification tool iphy Classifier showed the WBD phytoplasma in this study belonging to the 16Srâ… -B subgroup.2. According to the published phytoplasma plasmid sequences, several primers was designed. Three WBD plasmids（p WBD1, p WBD2 and p WBD3） were cloned from WBD-infected periwinkles, and confirmed by southern blot. They are 3449 bp, 3601 bp and 3844 bp in length, respectively, and all encode 15 proteins, which included five secreted proteins and five membrane proteins, as well as the replication-associated protein（Rep）, copy number control protein（Cop） and single-stranded DNA-binding protein（SSB）. Phylogenetic analysis based on the sequences of Rep proteins from 16 phytoplasma type II extrachromosomal DNA molecules（EC-DNAs） and geminiviruses indicated that p WBD1 and p WBD2 were most closely related to p Pa WBNy-1 from paulownia witches’ broom phytoplasma Nanyang strain, while p WBD3 was most closely related to p Pa WBNy-2. Real-time PCR analysis revealed that the copy numbers of WBD plasmids had tissue specificity in infected periwinkle and changed during infection. The copy numbers of plasmids were higher in the tissues below the graft union, especially in the roots. The copy number of p WBD1 had no significant changes, whilst the copy numbers of p WBD2 and p WBD3 peaked three months after symptom appearance.3. Using WBD-infected periwinkles as materials, a band about 650 kb in size was isolated by differential centrifugation and plus-filed gel electrophoresis（PFGE）, and then was proved to be WBD chromosome DNA by PCR and southern blot analysis. WBD chromosome DNA was extracted by dialysis bag electroelution, and concentrated by ultrafiltration. The concentrated DNA was amplified by whole genome amplification（WGA）. The ratio of phytoplasma DNA in WGA production and sample from each step was quantified by real-time PCR. WBD chromosome DNA was gradually purified by differential centrifugation, PFGE and WGA. Finally, thirty-seven microgram WBD chromosome DNA with purity of 92.9% was obtained.4. Using WGA production, two WBD genomic libraries in different size were constructed and sequenced. Eleven Gb of raw date was generated. These read-pairs were assembled by de novo assembly of CLC Genomic Workbench, and then further assembled by SSPACE-BASIC v2.0. A large number of contigs were emerged, after screening by TBLASTN and PCR, the contigs from phytoplasma were connected by outward PCR. Finally, the WBD phytoplasma draft genome was obtained. It was comprised of six contigs with a total size of 611,462 bp, covering 94% of WBD chromosome. Five-hundred-twenty-five protein-coding genes, two operons for r RNA genes and 32 t RNA genes were identified. Comparative genome analyses between WBD and other phytoplasmas were subsequently carried out. The results showed that extensive arrangements and inversions existed among the WBD, OY-M and AY-WB phytoplasma genomes. Most protein-coding genes in WBD were found to be homologous to genes from other phytoplasmas; only 22 WBD-specific genes were identified. KEGG pathway analysis indicated that WBD-had strongly reduced metabolic capabilities. However, 46 transporters were identified, which were involved with dipeptides/oligopeptides, spermidine/putrescine, cobalt and Mn/Zn transport, and so on. A total of 37 potential secreted proteins were identified in the WBD chromosome and plasmids. Of these, three secreted proteins were similar to the reported phytoplasma virulence factors: TENGU, SAP11 and SAP54. In addition, WBD contained several proteins that were predicted to play a role in its adaptation to diverse environments.5. Thirty-seven genes encoding for WBD secreted proteins were amplified and cloned into p GR107, a binary potato virus X（PVX） vector. WBD secreted proteins were expressed in Nicotiana benthamiana by Agrobacterium-mediated viral vector assays, and a virulence factor SWP1 for proliferation was identified. SWP1 was coded by WBD0004. Amino acid sequence analysis showed that SWP1 had 42.22% amino acid sequence identity with SAP11, but inconsistent part of sequence was found in the critical region（nuclear localization signal domain（NLS） and target binding domain） of these two proteins. Although monopartite NLS located in the C terminus of SWP1, the location and function of SWP1 and SWP1 mutant indicated that the NLS of SWP1 had no function on nuclear localization, and it was not critical for proliferation of SWP1. Real-time PCR analysis showed the expressions of SWP1 were varied during the infection period and in different tissues. The expressions of SWP1 were highest in flowers. During the infection period, expression level of SWP1 was the highest at 40 d after grafting in periwinkle leaves.6. SWP11, a secreted protein encoded by WBD0236, was identified as an elicitor. This protein could induce the hypersensitive response and cause a point necrosis in Nicotiana benthamiana. No fluoresce were found in Nicotiana benthamiana l6 c leaf expressing SWP11 and s GFP, suggesting that SWP11 had no function on gene sciencing suppressor. The results of histochemistry staining showed that H2O2 accumulation and callose deposition could be detected in the Nicotiana benthamiana leaves expressing SWP11. The expression of two hypersensitive response marker genes（HIN1 and HSR203J）, the pivotal defense regulatory gene NPR1, and three pathogenesis-related genes PR1, PR2 and PR3 were up-regulated in Nicotiana benthamiana leaves expressing SWP11. Subcellular localization studies showed that the localization of SWP11 was same with the movement protein of tobacco mosaic virus, which localizes to in plasmodesmata. Further research showed that the N-terminus was the core region necessary for the SWP11-induced hypersensitive response. Real-time PCR analysis revealed that the expression level of WBD0236 was the highest at 30 d after WBD grafting. At 90 d after grafting, WBD0236 was most highly expressed in root.7 Co-expression WBD secreted proteins and elicitors in Nicotiana benthamiana found that several WBD secreted proteins could inhabit the program cell death inducing by SWP11, Bax and INF1. Four WBD secreted proteins SWP6, SWP12, SWP21 and SWP31 could inhabit the program cell death inducing by SWP11 and Bax; two WBD secreted proteins SWP6 and SWP31 could inhabit the program cell death inducing by INF1. |
