Southern blot hybridization analysis of McrBC-treated and control non-treated DNA from susceptible (S) and Ty-1 resistant (R) plants mock-inoculated or infected with IL, IS76 or IL+IS76 at 10 and 30 days post-inoculation (dpi).

Total DNA extracted from tomato plants was digested with McrBC or incubated in digestion buffer without McrBC and then separated on 1% agarose gel (4 separate gels for S and R plants at 10 and 30 dpi, respectively). As control, plasmid DNA with one methylated cytosine site was spiked into total DNA from the R plant infected with IS76 at 30 dpi and loaded on one of the 4 gels. Following electrophoresis, the gels were stained with ethidium bromide and then DNA was transferred to nylon membranes by blotting and denatured. The membranes were successively hybridized with ³²P-labelled DNA oligonucleotide probes specific for the complementary and virion strands of viral DNA and, following each hybridization, exposed together to a phosphor screen for 1 hour to 2 weeks and scanned on a PosphorImager. Note that after the first hybridization, the membranes were stripped to remove the first probe and then hybridized with the second probe. Pictures of EtBr-stained gels of the samples from 10 dpi and 30 dpi are shown in panels (A) and (C), respectively, while the respective membrane scans are shown in panels (B) and (D). Positions of plant genomic DNA (gDNA), undigested and digested plasmid DNA, viral circular double-stranded DNA (dsDNA) and viral circular single-stranded DNA (ss) are indicated.

(PDF)