Figure 3

(a) Infectivity analysis of wild-type and mutant ToLCPalV on N. benthamiana and S. lycopersicum. Plants were inoculated with agro-infectious clones of respective constructs and photographed at 14 dpi. Plants infiltrated with DNA-A∆AV2; DNA-A alone and DNA-A∆AV2+DNA-B were asymptomatic, whereas DNA-A+DNA-B-infiltrated plants developed severe downward leaf-curling symptoms. (b) Real-time expression for relative quantification of viral DNA in inoculated leaves at 3 dpi. Asterisk on the top of the bars indicate statistical significant difference using Student’s-T test between mock and infected samples (*(P < 0.05), **(P < 0.01) and ***(P < 0.001). EF1α was used as internal control and experiment was repeated twice with three biological and technical replicates. (c) Southern blot for viral DNA accumulation with a CP gene-specific probe. DNA was isolated from newly emerged leaves of N. benthamiana at 21 dpi; higher accumulation of viral single-stranded (ss DNA), super coiled (sc DNA) and open circular (ocDNA) forms was observed in DNA-A+DNA-B-infiltrated leaves. (d) Western blot with AV2 antisera for detection of the AV2 expression in DNA-A+DNA-B- and DNA-A∆AV2+DNA-B-infiltrated N. benthamiana; purified AV2 protein was used as the positive control.