Tomato chlorotic mottle virus is a target of RNA silencing but the presence of specific short interfering RNAs does not guarantee resistance in transgenic plants

Abstract

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 ToCMoV-specific 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.

FIG. 1.

Symptoms of ToCMoV-[BA-Se1] infection in petunia, tomato, and N. benthamiana, and Northern detection of virus-derived siRNAs in these hosts using a viral DNA-A specific probe. Low molecular RNAs were extracted from virus-infected plant (I) or mock inoculated control (C) plants. Synthetic siRNA molecules were used as size references (M).

FIG. 2.

Time course analysis of ToCMoV-[BA-Se1] DNA and virus-derived siRNA accumulation in infected N. benthamiana plants. (a) Viral DNA was extracted at different days postinoculation and detected by Southern hybridization using either DNA-A (left panel) or DNA-B-derived probe (right panel). ToCMoV-[BA-Se1] single-stranded (SS) and supercoiled double-stranded (SC) DNA forms are indicated. (b) Northern blot of ToCMoV-[BA-Se1]-specific siRNAs at different time points after inoculation. C, mock-inoculated control.

FIG. 3.

Origin and distribution of siRNAs from ToCMoV-[BA-Se1]-infected N. benthamiana and tomato plants. The bipartite genome of ToCMoV-[BA-Se1], consisting of DNA-A (a) and DNA-B (b), is schematically depicted. PCR fragments covering the viral genome were obtained and probed with 5′-labeled small RNAs extracted from inoculated N. benthamiana and tomato plants. As controls, the same blots were also hybridized with 5′-labeled small RNAs extracted from noninoculated N. benthamiana and with probes derived from the complete DNA-A or DNA-B components of ToCMoV-[BA-Se1].

FIG. 4.

Polarity of siRNAs accumulating in infected tomato plants corresponding to different ToCMoV-[BA-Se1] DNA-A-derived transcripts. Fragments A3, A4, and A6 were transcribed and 100 μg of transcripts in viral (V) and complementary sense (C) were blotted and probed with 5′-labeled siRNAs extracted from ToCMoV-[BA-Se1]-infected tomato plants. The blot was quantified by using Gene Tools software, and the numbers below each lane corresponds to the relative amounts of siRNAs for the respective polarity of a fragment. As a control, the same blot was hybridized with a ToCMoV-[BA-Se1] DNA-A-labeled probe.

FIG. 5.

Genetic map of ToCMoV-[BA-Se1] DNA-A showing the origin of the fragment amplified and cloned into the plant expression vector pIR-RC used for N. benthamiana transformation.

FIG. 6.

Systemic symptoms and virus accumulation in transgenic N. benthamiana plant lines challenge-inoculated with ToCMoV-[BA-Se1]. (a) Percentage of plants with symptoms evaluated visually in relation to time postinoculation (dpi). (b) Percentage of plants containing virus at 20 and 45 dpi as evaluated by tissue blot hybridization. (c) Symptoms on plants from transgenic line RC-19.3 at 20 dpi: plants 1, resistant inoculated transgenic plants; plants 2, susceptible inoculated transgenic plants; plants 3, transgenic mock-inoculated controls; and plants 4, wild-type inoculated controls.

FIG. 7.

Analysis of resistant and susceptible T1 siblings of N. benthamiana lines RC-19-3 and RC-24-2. (a) Presence of siRNAs corresponding to the RC probe at 0, 20, and 45 dpi; (b) accumulation of ToCMoV-[BA-Se1] DNA-A and DNA-B at 45 dpi. I, inoculated wild-type control; C, mock-inoculated wild-type control. The arrow indicates the higher-molecular-mass RNA species discussed in the text.