Agrobacterium-Mediated Cucumber Mosaic Virus Movement-Deficient Reporter

Microinjection, bombardment and tobamovirus or potexvirus based complementation assay have been developed to identify the putative movement protein (MP) and to study 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 48-60 hr post infiltration (hpi), whereas the optimal incubation temperature was between 25-28 ℃. 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 a powerful and alternative tool for identifying putative movement protein and for characterizing macromolecular trafficking.