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Engineering resistance against Tomato yellow leaf curl virus via the CRISPR/Cas9 system in tomato.

ABSTRACT: CRISPR/Cas systems confer molecular immunity against phages and conjugative plasmids in prokaryotes. Recently, CRISPR/Cas9 systems have been used to confer interference against eukaryotic viruses. Here, we engineered Nicotiana benthamiana and tomato (Solanum lycopersicum) plants with the CRISPR/Cas9 system to confer immunity against the Tomato yellow leaf curl virus (TYLCV). Targeting the TYLCV genome with Cas9-single guide RNA at the sequences encoding the coat protein (CP) or replicase (Rep) resulted in efficient virus interference, as evidenced by low accumulation of the TYLCV DNA genome in the transgenic plants. The CRISPR/Cas9-based immunity remained active across multiple generations in the N. benthamiana and tomato plants. Together, our results confirmed the efficiency of the CRISPR/Cas9 system for stable engineering of TYLCV resistance in N. benthamiana and tomato, and opens the possibilities of engineering virus resistance against single and multiple infectious viruses in other crops.

SUBMITTER: Tashkandi M

PROVIDER: S-EPMC6204811 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Engineering resistance against Tomato yellow leaf curl virus via the CRISPR/Cas9 system in tomato.

Tashkandi Manal M Ali Zahir Z Aljedaani Fatimah F Shami Ashwag A Mahfouz Magdy M MM Mahfouz Magdy M MM

Plant signaling & behavior 20181005 10

CRISPR/Cas systems confer molecular immunity against phages and conjugative plasmids in prokaryotes. Recently, CRISPR/Cas9 systems have been used to confer interference against eukaryotic viruses. Here, we engineered Nicotiana benthamiana and tomato (Solanum lycopersicum) plants with the CRISPR/Cas9 system to confer immunity against the Tomato yellow leaf curl virus (TYLCV). Targeting the TYLCV genome with Cas9-single guide RNA at the sequences encoding the coat protein (CP) or replicase (Rep) r ...[more]

PMID: 30289378

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Project description:The sweetpotato whitefly, Bemisia tabaci, is a major pest of cultivated tomato. Whitefly feeding-related injuries and transmission of viruses including tomato yellow leaf curl virus (TYLCV) cause serious losses. Management strategy includes planting resistant cultivars/hybrids. However, TYLCV resistance is incomplete and whiteflies on TYLCV-resistant cultivars/hybrids are managed by insecticides. Acylsugars'-mediated resistance against whiteflies has been introgressed from wild solanums into cultivated tomato. This study evaluated acylsugar-producing tomato lines with quantitative trait loci (QTL) containing introgressions from Solanum pennellii LA716, known to alter acylsugars' levels or chemistry. Evaluated acylsugar-producing lines were the benchmark line CU071026, QTL6/CU071026-a CU071026 sister line with QTL6, and three other CU071026 sister lines with varying QTLs-FA2/CU71026, FA7/CU071026, and FA2/FA7/CU071026. Non-acylsugar tomato hybrid Florida 47 (FL47) was also evaluated. Acylsugars' amounts in FA7/CU071026 and FA2/FA7/CU071026 were 1.4 to 2.2 times greater than in other acylsugar-producing lines. Short chain fatty acid, i-C5, was dominant in all acylsugar-producing lines. Long chain fatty acids, n-C10 and n-C12, were more abundant in FA7/CU071026 and FA2/FA7/CU071026 than in other acylsugar-producing lines. Whiteflies preferentially settled on non-acylsugar hybrid FL47 leaves over three out of five acylsugar-producing lines, and whiteflies settled 5 to 85 times more on abaxial than adaxial leaf surface of FL47 than on acylsugar-producing lines. Whiteflies' survival was 1.5 to 1.9 times lower on acylsugar-producing lines than in FL47. Nevertheless, whiteflies' developmental time was up to 12.5% shorter on acylsugar-producing lines than on FL47. TYLCV infection following whitefly-mediated transmission to acylsugar-producing lines was 1.4 to 2.8 times lower than FL47, and TYLCV acquisition by whiteflies from acylsugar-producing lines was up to 77% lower than from FL47. However, TYLCV accumulation in acylsugar-producing lines following infection and TYLCV loads in whiteflies upon acquisition from acylsugar-producing lines were not different from FL47. Combining TYLCV resistance with acylsugars'-mediated whitefly resistance in cultivated tomato could substantially benefit whiteflies and TYLCV management.

Project description:BackgroundTomato 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.ResultsA 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.ConclusionsThese 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.

Dataset Information

Engineering resistance against Tomato yellow leaf curl virus via the CRISPR/Cas9 system in tomato.

Publications

Engineering resistance against Tomato yellow leaf curl virus via the CRISPR/Cas9 system in tomato.

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