Tomato chlorotic mottle virus (ToCMoV) is a begomovirus found widespread in tomato fields in Brazil.ToCMoV isolate BA-Se1 (ToCMoV-[BA-Se1]) was shown to trigger the plant RNA silencing surveillance in different host plants and, coinciding with a decrease in viral DNA levels, small interfering RNAs (siRNAs) specific to ToCMoV-[BA-Se1] accumulated in infected plants. Although not homogeneously distributed, the siRNA population in both infected Nicotiana benthamiana and tomato plants represented the entire DNA-A and DNA-B genomes. We determined that in N. benthamiana, the primary targets corresponded to the 5 end of AC1 and the embedded AC4, the intergenic region and 5 end of AV1 and overlapping central part of AC5. Subsequently, transgenic N. benthamiana plants were generated that were preprogrammed to express double-stranded RNA corresponding to this most targeted portion of the virus genome by using an intron-hairpin construct. These plants were shown to indeed produce ToCMoVspecific siRNAs. When challenge inoculated, most transgenic lines showed significant delays in symptom development, and two lines had immune plants. Interestingly, the levels of transgene-produced siRNAs were similar in resistant and susceptible siblings of the same line. This indicates that, in contrast to RNA viruses, the mere presence of transgene siRNAs corresponding to DNA virus sequences does not guarantee virus resistance and that other factors may play a role in determining RNA-mediated resistance to DNA viruses.

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The biochemical and functional properties of the movement protein (MP) of brome mosaic virus (BMV) were investigated. Expression and purification of the BMV MP from Escherichia coli resulted in a pure and soluble protein preparation. Sucrose gradient centrifugation revealed that BMV MP forms oligomers consisting of two or more copies but no higher order multimers even when different ionic strengths and pHs were applied. Nitro-cellulose filter binding and gel retardation studies showed that in vitro the BMV MP preferentially bound to ss nucleic acids (RNA and DNA); the affinity to ssRNA was lower compared to BMV coat protein. The binding to ss nucleic acid was cooperative and not sequence specific and the hypothetical binding site was calculated to be around three to six nucleotides per MP monomer. The nucleic acid binding properties of the BMV MP are discussed in relation to the recent finding that this protein is also able to form tubular structures in infected protoplasts.