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. Author manuscript; available in PMC: 2016 Dec 1.
Published in final edited form as: Plant Biotechnol J. 2015 Feb 16;13(9):1300–1311. doi: 10.1111/pbi.12349

Figure 4.

Figure 4

Ectopic expression of T474D in tomato confers tolerance to ToYSV infection. (a) Ectopic expression of T474D in tomato plants attenuates the development of symptoms upon ToYSV infection. Tandemly repeated ToYSV DNA-A and DNA-B sequences were introduced into the indicated lines by biolistic inoculation. Photographs were taken at 21 days postinoculation (DPI). (b) Symptoms associated with ToYSV infection in the 35S-T474D-5 line. Photographs were taken at 21 DPI. (c) Viral DNA accumulation in the infected leaves of the T474-5 line. Total DNA was isolated from infected plants at 21 DPI, and PCR was performed with viral DNA-B-specific primers and actin-specific primers (as an internal control) in the same reaction. Lo indicates inoculated and Sys denotes systemic leaves. ‘Un’ indicates mock-inoculated leaves. The gel shows representative samples of WT and the 35S::T474D-5 transgenic line. The upper band (1.2 kb, arrow) is the amplified genomic fragment from actin, and the lower band (0.5 kb, arrowhead) is the viral DNA fragment. (d) and (e) The line 35S-T474D-6 displayed tolerance to ToYSV infection. The T474D-6 line was infected with ToYSV, and photographs were taken at 21 DPI. Viral DNA accumulation was detected by PCR in inoculated (‘Lo’) and systemic (‘Sys’) leaves. (f) The course of infection was delayed in T474D lines. The indicated lines were infected with ToYSV by the biolistic method, and the course of infection was monitored by PCR amplification of viral DNA. In each experiment (three biological replicates), 20 plants of each line were inoculated. Values represent the percentage of systemically infected plants at different DPI. (g) Infection efficiency in T474D-overexpressing lines. The infection efficiency is expressed as the DPI required to infect 50% of the plants (mean ± SD of three replicates). Asterisks indicate significantly different means (P < 0.05, Student’s t-test). (h) Viral DNA accumulation in T474D-overexpressing lines, as determined by quantitative PCR at 28 DPI. The fold variation (±SD, n = 3 biological replicates) is shown as log2-scaled copy units of the viral genome. Viral DNA accumulation was determined in systemic leaves at 28 DPI.