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Fig. 4 | Phytopathology Research

Fig. 4

From: Molecular characterization and pathogenicity of an infectious cDNA clone of tomato brown rugose fruit virus

Fig. 4

Infectivity and pathogenicity of the infectious clone of ToBRFV. a Schematic diagram illustrating the one-step assembly cloning strategy used for the construction of an infectious full-length cDNA clone of ToBRFV. Two overlapping ToBRFV fragments, A and B, were amplified by polymerase chain reaction and infused to the linearized pCB301 vector. The overlapping regions were illustrated by gray rectangles, showing the number of overlapping nucleotides. 35S, the cauliflower mosaic virus promoter; HDRz, hepatitis delta virus ribozyme ribozyme; tNos, Nos terminator. b Symptoms in tomato (Solanum lycopersicum), Nicotiana benthamiana, and N. tabacum cv. Samsun nn plants induced by ToBRFV infection. In the upper panel, plants were mock-inoculated with agrobacteria harboring the pCB301 vector. In the lower panel, plants were agroinoculated with the infectious clone of ToBRFV (pCB301-ToBRFV). Images were taken at 21 dpi for tomato plants, and 14 dpi for N. benthamiana and N. tabacum cv. Samsun nn plants. c RT-PCR analysis of ToBRFV RNA accumulation in systemic leaves of agroinoculated plants using ToBRFV-specific primers. Lane M, GeneRuler 1 kb DNA ladders (Thermo); lanes 1–2, RNA extracted from N. benthamiana plants agroinoculated with pCB301-ToBRFV; lanes 4–5, RNA extracted from tomato plants agroinoculated with pCB301-ToBRFV; lanes 7–8, RNA extracted from N. tabacum cv. Samsun nn plants agroinoculated with pCB301-ToBRFV. Lanes 3, 6, and 9, represent RNA extracted from mock-inoculated N. benthamiana, tomato, and N. tabacum cv. Samsun nn plants, respectively. Lane 10, ddH2O (negative control). d Transmission electron micrograph of viral particles in systemic leaves of tomato and N. benthamiana plants agroinoculated with pCB301-ToBRFV. Bar = 100 nm

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