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

Ammara Ue, Mansoor S, Saeed M, Amin I, Briddon RW, Al-Sadi AM - Virol. J. (2015)

Bottom Line: Unusually, TYLCV infections in Oman are sometimes associated with a betasatellite (Tomato leaf curl betasatellite [ToLCB]; a symptom modulating satellite).These results show that RNAi can be used to develop resistance against geminiviruses in tomato.Also, the betasatellite may compromise resistance, increasing the proportion of plants which ultimately show symptoms.

View Article: PubMed Central - PubMed

Affiliation: Department of Crop Sciences, College of Agriculture and Marine Sciences, Sultan Qaboos University, P.O. Box-34, 123, Al-Khod, Oman. ammarajawad@gmail.com.

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.

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Related in: MedlinePlus

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.
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Fig1: 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.

Mentions: N. benthamiana plants inoculated with TYLCV-OM or TYLCV-OM/ToLCB-OM showed the first symptoms of infection at15 days post inoculation (dpi) and all plants were ultimately symptomatic by 30 dpi (Table 1). The symptoms consisted of mild leaf curling which gradually increased in severity. By 30 dpi plants showed severe stunting, leaf curling, vein swelling and foliar yellowing (Figure 1, panels I and J). Overall the symptoms for TYLCV-OM/ToLCB-OM infected plants were more severe than those of TYLCV-OM inoculated plants, with leaves being smaller with more pronounced leaf curling, yellowing and vein swelling.Table 1


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

Ammara Ue, Mansoor S, Saeed M, Amin I, Briddon RW, Al-Sadi AM - Virol. J. (2015)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4359554&req=5

Fig1: 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.
Mentions: N. benthamiana plants inoculated with TYLCV-OM or TYLCV-OM/ToLCB-OM showed the first symptoms of infection at15 days post inoculation (dpi) and all plants were ultimately symptomatic by 30 dpi (Table 1). The symptoms consisted of mild leaf curling which gradually increased in severity. By 30 dpi plants showed severe stunting, leaf curling, vein swelling and foliar yellowing (Figure 1, panels I and J). Overall the symptoms for TYLCV-OM/ToLCB-OM infected plants were more severe than those of TYLCV-OM inoculated plants, with leaves being smaller with more pronounced leaf curling, yellowing and vein swelling.Table 1

Bottom Line: Unusually, TYLCV infections in Oman are sometimes associated with a betasatellite (Tomato leaf curl betasatellite [ToLCB]; a symptom modulating satellite).These results show that RNAi can be used to develop resistance against geminiviruses in tomato.Also, the betasatellite may compromise resistance, increasing the proportion of plants which ultimately show symptoms.

View Article: PubMed Central - PubMed

Affiliation: Department of Crop Sciences, College of Agriculture and Marine Sciences, Sultan Qaboos University, P.O. Box-34, 123, Al-Khod, Oman. ammarajawad@gmail.com.

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.

Show MeSH
Related in: MedlinePlus