Analysis And Identification Of The Essential Domain Of Effector SWP1 From Wheat Blue Dwarf Phytoplasma

Wheat blue dwarf is one of the main important wheat diseases which occur in rid and semi-arid areas of northwest China, including Shaanxi, Shanxi, Gansu, Ningxia and Inner Mongolia. It imperiled the growth of wheat and significantly influenced the production and quality.In recent years, the research about WBD was primarily focused on fully genome sequencing and pathogenicity. Using the bioinformatics software, 525 protein-coding genes were identified in WBD genome. Except 22 WBD-specific genes, most of them were homologous to genes from other phytoplasma. In the meanwhile, 37 genes encoding for WBD secreted proteins were constructed into pGR107. Thses proteins were expressed in Nicotiana benthamiana by Agrobacterium-mediated viral vector assays. When SWP1 infect Nicotiana benthamiana, the base of the plant stem was manifested as proliferation. In this study, the SWP1 gene of WBD was analysed using bioinformatics. On this basis, different mutants of SWP1 genes were amplified and constructed into pGR107. In order to identify the essential domain of secreted protein SWP1 that caused the symptom of N. benthamiana, these different genes were expressed in Nicotiana benthamiana. The major results are listed as follows:1. Molecular structure characteristics of the SWP1 gene: The gene was deduced to encode a peptide of 112 amino acids whose predicted molecular mass was 13.292 kDa and isoelectric point was 9.2. The signal peptide cleavage site was between the 33 th and the 34 th amino acid at N-terminals of the SWP1 protein, so the SWP1 mature protein had only 80 amino acids and its predicted molecular mass was 9.516 kDa. It was predicted that the secondary structure of SWP1 was constructed by four α-helix folds. The coiled coil structure was between the 82 th to 101 th amino acids at C-terminals. There was a monopartite NLS located in the C terminus of SWP1. Otherwise, homologous modeling was predicted using the swiss-model.2. Based on the results of the bioinformatics, a series of truncation mutants were constructed into pGR107, including SWP1△NLS 、 SWP1△NLSCC 、 SWP1△CC 、 SWP1△N、 SWP1△ NC. These different genes were expressed in Nicotiana benthamiana by Agrobacterium-mediated viral vector assays. Analysis the symptoms of different Nicotiana benthamiana, the results indicated that the ten amino acids at C terminus including the monopartite NLS and the fourteen amino acids at N-terminals are not essential for proliferation, and the coiled coil structure is critical for proliferation of SWP1.3. After identification the coiled coil structure is essential for proliferation, another three truncation mutants were constructed, including SWP1△NC1 、 SWP1△NC2、 SWP1△ NNLSCC. These genes were expressed in Nicotiana benthamiana, the results showed that the 11 th to 15 th amino acids of coiled coil structure were critical for proliferation. This indicated that these five amino acids were essential for coiled coil structure and also critical for proliferation of SWP1.