Functional Analysis Of Protein SWP6 Inhabit Death In Wheat Blue Dwarf Phytoplasma

Wheat blue dwarf?WBD?disease is one of the important phytoplasma disease that cause severe losses for wheat production in northwestern China.In the 1950s,the disease was first reported in Shaanxi and then spreads to Gansu,Ningxia and Shanxi.Wheat blue dwarf disease is transmitted by the vector insect Psammotettix alienus L.With the promotion of wheat straw covering technology,the number of mediator insect increased rapidly.The disease diffused gradually to the arid areas of the middle and lower reaches of the Yellow River,and caused severe losses of wheat production.In recent years,the genomic map of WBD phytoplasmas was completed by firstly in our laboratory using Solexa DNA sequencing platform.In the meanwhile,37 genes encoded WBD secreted proteins were predicted by computerized algorithm and then constructed into pGR107 vector.These proteins were expressed in N benthamiana by Agrobacterium-mediated viral vector assays.In this study,SWP6 can inhibit the hypersensitive response of N benthamiana induced by BAX.Whereafter,the characteristic analysis of the SWP6 was carried out via using bioinformatics.In the meanwhile,identification of key functional domain?subcellular localization and RNA-seq were proceeded to explicit the function of SWP6 and its functional mechanism.The main results are described below:1.The analysis and prediction of SWP6 gene encoding effector physicochemical and properties and spatial structure indicated that SWP6 gene is 618bp in length,encodes 206amino acids,coding protein molecular weight size is about 23.8 kDa,isoelectric point pI is6.98.polypeptide of atoms as the C₁₀₆₃H₁₇₂₁N₂₆₉O₃₁₅S₁₁,hydrophobicity coefficient is 0.120,belong to the hydrophobic protein.The cleavage site of the signal peptide is 30-31 amino acids,and the mature secreted protein secreted to the extracellular is 174 amino acids.Its molecular weight size is about 19.6 kDa,the SWP6 encoded protein contains all common 20amino acids,with the highest aspartate content,the proportion is 13.6%.There is a transmembrane region existed in the 10th and 30th amino acids,and the secondary structure prediction that the SWP6 has 3 alpha helices and multiple irregular coiled coil structures.2.Based on the results of the bioinformatics analysis and prediction,a series of truncation mutants were constructed into pGR107,including pGR107-SWP6DH1?pGR107-SWP6DH2?pGR107-SWP6DH3?pGR107-SWP6DH4?pGR107-SWP6DH5.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 84-113 amino acids in the coiled-coil of SWP6 encoded protein play a key role in its function.A subcellular localization expression vector pCAMBIA1302-SWP6 was constructed,which transiently expressed SWP6 and eGFP fusion proteins in A.nicotianae by Agrobacterium tumefaciens.The result confirmed that SWP6 was mainly localized in the cell membrane.3.In this study,the transcriptome of N.benthamiana in response to SWP6 transiently expressed was profiled,total 1,093 differentially expressed genes were identified,of which540 genes were up-regulated,553 genes were down-regulated,and the expression of key node genes of the salicylic acid pathway were significant difference compared with the control.The accuracy of the RNA-seq data was verified by qRT-PCR.Different levels of down-regulation were observed,which was in agreement with the RNA-seq results to a large extent,indicating that the RNA-seq results were accurate and reliable,providing statistical analysis and verification for large amounts of data in the later period.