title: A versatile complementation assay for cell-to-cell and long distance movements by cucumber mosaic virus based agro-infiltration.
abstract: Microinjection, bombardment or tobamovirus and potexvirus based assay has been developed to identify the putative movement protein (MP) or to characterize plasmodesma-mediated macromolecular transport. In this study, we developed a versatile complementation assay for the cell-to-cell and long distance movements of macromolecules by agro-infiltration based on the infectious clones of cucumber mosaic virus (CMV). The movement-deficient CMV reporter was constructed by replacing the MP on RNA 3 with ER targeted GFP. The ectopic expression of CMV MP was able to efficiently move the RNA3-MP::erGFP reporter from the original cell to neighboring cells, whereas CMV MP-M5 mutant was unable to initiate the movement. Importantly, the presence of CMV RNA1 and RNA2 can dramatically amplify the movement signals once the RNA3-MP::erGFP reporter moves out of the original cell. The appropriate observation time for this movement complementation assay was at 48-72 hours post infiltration (hpi), whereas the optimal incubation temperature was between 25 and 28 °C. The ectopic co-expression of MPs from other virus genera, NSm from tomato spotted wilt tospovirus (TSWV) or NSvc4 from rice stripe tenuivirus (RSV), could also facilitate the movement of the RNA3::erGFP reporter from the original cell into other cells. The chimeric mutant virus created by substituting the MP of CMV RNA3 with NSm from TSWV or NSvc4 from RSV move systemically in Nicotiana benthamiana plants by agro-infiltration. This agro-infiltration complementation assay is simple, efficient and reliable. Our approach provides an alternative and powerful tool with great potentials in identifying putative movement protein and characterizing macromolecular trafficking.
authors.affiliation: Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Department of Plant Pathology, Nanjing Agricultural University, Nanjing 210095, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Shen
authors.affiliation: Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Department of Plant Pathology, Nanjing Agricultural University, Nanjing 210095, China.
authors.forename: Xiaohui
authors.initials: X
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Department of Plant Pathology, Nanjing Agricultural University, Nanjing 210095, China.
authors.forename: Min
authors.initials: M
authors.lastname: Yao
authors.affiliation: Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Department of Plant Pathology, Nanjing Agricultural University, Nanjing 210095, China.
authors.forename: Chun
authors.initials: C
authors.lastname: Li
authors.affiliation: Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Department of Plant Pathology, Nanjing Agricultural University, Nanjing 210095, China.
authors.forename: Karwitha
authors.initials: K
authors.lastname: Miriam
authors.affiliation: Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Department of Plant Pathology, Nanjing Agricultural University, Nanjing 210095, China.
authors.forename: Xue
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Department of Plant Pathology, Nanjing Agricultural University, Nanjing 210095, China. Electronic address: taoxiaorong@njau.edu.cn.
authors.forename: Xiaorong
authors.initials: X
authors.lastname: Tao
mesh_terms: D058948:Cell Tracking / Q000379:methods*; D017799:Cucumovirus / Q000235:genetics / Q000502:physiology*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D010935:Plant Diseases / Q000821:virology*; D053830:Plant Viral Movement Proteins / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000821:virology; D014773:Virology / Q000379:methods*
chemical_list: D053830:Plant Viral Movement Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins
keywords: Agro-infiltration assay; Cell-to-cell movement; Complementation; Cucumber mosaic virus; Long distance movement; Macromolecular transport