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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

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.
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Fig5: 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.

Mentions: The levels of virus in tomato plants inoculated with either TYLCV-OM or TYLCV-OM/ToLCB-OM were determined by a real time quantitative PCR (qPCR) assay on DNA samples extracted from plants at 60 dpi. The efficiency of qPCR reaction was 99.3% and a melt-curve analysis resulted in a single peak, indicative of the amplification of a single product. PCR reactions with DNA extracted from wild type non-inoculated tomato plants did not reach the threshold cycle (Ct), indicative of no viral DNA in these samples. The qPCR analyses showed the presence of the virus in all inoculated tomato plants, including the transgenic plants. However, the level of virus was significantly lower in all transgenic plants than in the non-transgenic control plants (Figure 5). Overall the worst performing line (based on the qPCR results) was line 23 (with an <340 fold lower virus titre than non-transgenic plants) and the best performing lines were 18 and 52 (with a > 290,000 fold reduction). Also, for the majority of plants (both transgenic and non-transgenic) the levels of virus were significantly higher in plants inoculated with TYLCV-OM/ToLCB-OM than in plants inoculated with just the virus.Figure 5


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)

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

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

Fig5: 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.
Mentions: The levels of virus in tomato plants inoculated with either TYLCV-OM or TYLCV-OM/ToLCB-OM were determined by a real time quantitative PCR (qPCR) assay on DNA samples extracted from plants at 60 dpi. The efficiency of qPCR reaction was 99.3% and a melt-curve analysis resulted in a single peak, indicative of the amplification of a single product. PCR reactions with DNA extracted from wild type non-inoculated tomato plants did not reach the threshold cycle (Ct), indicative of no viral DNA in these samples. The qPCR analyses showed the presence of the virus in all inoculated tomato plants, including the transgenic plants. However, the level of virus was significantly lower in all transgenic plants than in the non-transgenic control plants (Figure 5). Overall the worst performing line (based on the qPCR results) was line 23 (with an <340 fold lower virus titre than non-transgenic plants) and the best performing lines were 18 and 52 (with a > 290,000 fold reduction). Also, for the majority of plants (both transgenic and non-transgenic) the levels of virus were significantly higher in plants inoculated with TYLCV-OM/ToLCB-OM than in plants inoculated with just the virus.Figure 5

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