RNA interference-based resistance in transgenic tomato plants against Tomato yellow leaf curl virus-Oman (TYLCV-OM) and its associated betasatellite

Abstract

Background: Tomato yellow leaf curl virus (TYLCV), a monopartite begomovirus (family Geminiviridae) is responsible for heavy yield losses for tomato production around the globe. In Oman at least five distinct begomoviruses cause disease in tomato, including TYLCV. Unusually, TYLCV infections in Oman are sometimes associated with a betasatellite (Tomato leaf curl betasatellite [ToLCB]; a symptom modulating satellite). RNA interference (RNAi) can be used to develop resistance against begomoviruses at either the transcriptional or post-transcriptional levels.

Results: A hairpin RNAi (hpRNAi) construct to express double-stranded RNA homologous to sequences of the intergenic region, coat protein gene, V2 gene and replication-associated gene of Tomato yellow leaf curl virus-Oman (TYLCV-OM) was produced. Initially, transient expression of the hpRNAi construct at the site of virus inoculation was shown to reduce the number of plants developing symptoms when inoculated with either TYLCV-OM or TYLCV-OM with ToLCB-OM to Nicotiana benthamiana or tomato. Solanum lycopersicum L. cv. Pusa Ruby was transformed with the hpRNAi construct and nine confirmed transgenic lines were obtained and challenged with TYLCV-OM and ToLCB-OM by Agrobacterium-mediated inoculation. For all but one line, for which all plants remained symptomless, inoculation with TYLCV-OM led to a proportion (≤25%) of tomato plants developing symptoms of infection. For inoculation with TYLCV-OM and ToLCB-OM all lines showed a proportion of plants (≤45%) symptomatic. However, for all infected transgenic plants the symptoms were milder and virus titre in plants was lower than in infected non-transgenic tomato plants.

Conclusions: These results show that RNAi can be used to develop resistance against geminiviruses in tomato. The resistance in this case is not immunity but does reduce the severity of infections and virus titer. Also, the betasatellite may compromise resistance, increasing the proportion of plants which ultimately show symptoms.

Figure 1

Resistance to infection by TYLCV-OM and TYLCV-OM/ToLCB-OM imparted by the transient expression of the hpRNAi construct. Tomato plants inoculated with TYLCV-OM and the hpRNAi construct (A and B), TYLCV-OM/ToLCB-OM and the hpRNAi construct (C and D), TYLCV-OM (E) or TYLCV-OM/ToLCB-OM (F). N. benthamiana plants inoculated with TYLCV-OM and the hpRNAi construct (G), TYLCV-OM/ToLCB-OM and the hpRNAi construct (H), TYLCV-OM/ToLCB-OM (I) or TYLCV-OM (J). Photographs of plants were taken at 30 dpi. Southern blot probed for the presence of TYLCV-OM sequences (K). The DNA samples run on the gel were extracted from a healthy, non-inoculated N. benthamiana plant (lane 2), N. benthamiana plants inoculated with TYLCV-OM (lane 3), hpRNAi and TYLCV-OM (lanes 7 and 8) and tomato plants inoculated with TYLCV-OM (lane 4) or hpRNAi and TYLCV-OM (lanes 5 and 6). The sample in lane 1 consisted of 50 ng of TYLCV-OM plasmid.With the exception of lane 1, approx. equal amounts (10 μg) of total DNA extract was loaded in each case. Southern blot probed for the presence of ToLCBV-OM sequences (L). The DNA samples run on the gel were extracted from a healthy, non-inoculated N. benthamiana plant (lane 1) and N. benthamiana plants inoculated with TYLCV-OM/ToLCB-OM (lane 2) or hpRNAi and TYLCV-OM/ToLCB-OM (lanes 6 and 7) and tomato plants inoculated with TYLCV-OM/ToLCB-OM (lane 3) or hpRNAi, TYLCV-OM/ToLCB-OM (lanes 4 and 5). Approx. equal amounts (10 μg) of total DNA extract were loaded in each case. The positions of the viral single-stranded (ss) and supercoiled (sc) replicative DNA forms are indicated.

Figure 2

Analysis of the resistance imparted by the hpRNAi construct in transgenic tomato to infection by TYLCV-OM. Plants of transgenic tomato cv. Pusa Ruby lines 11, 12, 13, 18, 21, 23, 41, 51 and 52 inoculated with TYLCV-OM (A to I). A TYLCV-OM inoculated wild-type Pusa Ruby plant is shown for comparison (J). Photographs were taken at 60 dpi.

Figure 3

Analysis of the resistance imparted by the hpRNAi construct in transgenic tomato to infection by TYLCV-OM/ToLCB-OM. Plants of transgenic tomato cv. Pusa Ruby lines 11, 12, 13, 18, 21, 23, 41, 51 and 52 inoculated with TYLCV-OM/ToLCB-OM (A to I). A TYLCV-OM/ToLCB-OM inoculated wild-type Pusa Ruby plant is shown for comparison (J). Photographs were taken at 60 dpi.

Figure 4

Southern blot analysis of transgenic tomato plants harboring the hpRNAi construct inoculated with TYLCV-OM probed for the presence of TYLCV-OM (A) and inoculated with TYLCV-OM/ToLCB-OM probed for the presence of ToLCB-OM (B). The DNA samples run on the gel were extracted from a non-transgenic non-inoculated tomato cv. Pusa Ruby plant (N), symptomatic TYLCV-OM (panel A) or TYLCV-OM/ToLCB-OM (panel B) inoculated non-transgenic Pusa Ruby plants (C) and two plants each of 9 transgenic Pusa Ruby lines (as indicated on the figure) inoculated with either TYLCV-OM (panel A) or TYLCV-OM/ToLCB-OM (panel B). Approx. 10 μg of total DNA was loaded on each case and the samples were extracted at 60 dpi. The positions of the viral single-stranded (ss) and supercoiled (sc) replicative DNA forms are indicated.

Figure 5

Real-time quantitative PCR assay of TYLCV-OM in transgenic and non-transgenic tomato cv. Pusa Ruby plants inoculated with TYLCV-OM and TYLCV-OM/ToLCB-OM. The bars indicate calculated virus titre (genome copies per 25 ng total DNA) in each case. The results are from qPCR reactions with DNA extracted from inoculated non-transgenic tomato cv. Pusa Ruby plants (C), a non-inoculated non-transgenic plant (N) and four plants each of 9 transgenic Pusa Ruby lines (as indicated on the figure) that were inoculated. For all the inoculated plants the left two bars (indicated by a yellow block at the base) are plants inoculated with only TYLCV-OM and the right two bars (indicated by a green block at the base) are plants inoculated with TYLCV-OM and ToLCB-OM. Each bar is the mean of three replicates of the qPCR reaction and the error bars indicate standard deviation. The leaf samples from which DNA was extracted were collected at 60 dpi.