Development of Cotton leaf curl virus resistant transgenic cotton using antisense ßC1 gene

Sohrab, Sayed Sartaj; Kamal, Mohammad Amjad; Ilah, Abdul; Husen, Azamal; Bhattacharya, Pinaki; Rana, Debashis

doi:10.1016/j.sjbs.2014.11.013

Cited by 22 publications

(11 citation statements)

References 27 publications

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“…Finally, a positive correlation was observed between virus accumulation and disease severity ( Figure 5 and Figure 6 ) in T 0 and T 1 generation plants. These results are comparable to a report by Asad et al [ 10 ] in which siRNA sequences homologous to viral AC1 , AC2 and AC3 coding regions were expressed in transgenic tobacco plants, resulting in ameliorated foliar symptoms, and another involving G. hirsutum “Coker 310” transgenic cotton plants expressing an antisense βC1 , which showed reduced symptom severity [ 13 ]. Using the cotton leaf curl disease severity rating system of Akhtar and Khan 2002 [ 34 ], scores of 4 to 6 were assigned to non-transgenic cotton plants showing severe leaf curling, vein-thickening, enations, and stunting of plants, while transgenic plants were scored as 0 to 1, and developed mild or no symptoms.…”

Section: Discussionsupporting

confidence: 86%

“…Despite the widespread use of transgenic solutions to pest control in cotton—for example, Bt-toxin expression to reduce feeding damage by lepidopteran pests—and the robust evidence showing that pathogen-derived resistance based on gene silencing is effective for combatting diseases caused by plant viruses [ 5 , 6 , 7 ], the commercialization of transgenic technology for virus disease control has been limited [ 3 ]. In particular, RNA-interference (RNAi) [ 8 ], induced by double-stranded (ds) RNA, has been shown highly effective for silencing viral gene expression in plants to achieve disease resistance, including for geminiviruses [ 9 , 10 , 11 , 12 , 13 ]. Stable knockdown of viral gene expression has been demonstrated using small interfering RNA (siRNA) technology by targeting viral coding and non-coding regions involved in viral replication and movement, which are crucial for establishing system infection of the host plant [ 8 , 11 ].…”

Section: Introductionmentioning

confidence: 99%

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Engineered Disease Resistance in Cotton Using RNA-Interference to Knock down Cotton leaf curl Kokhran virus-Burewala and Cotton leaf curl Multan betasatellite Expression

Ahmad

Zia-ur-Rehman

Hameed

et al. 2017

Viruses

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Cotton leaf curl virus disease (CLCuD) is caused by a suite of whitefly-transmitted begomovirus species and strains, resulting in extensive losses annually in India and Pakistan. RNA-interference (RNAi) is a proven technology used for knockdown of gene expression in higher organisms and viruses. In this study, a small interfering RNA (siRNA) construct was designed to target the AC1 gene of Cotton leaf curl Kokhran virus-Burewala (CLCuKoV-Bu) and the βC1 gene and satellite conserved region of the Cotton leaf curl Multan betasatellite (CLCuMB). The AC1 gene and CLCuMB coding and non-coding regions function in replication initiation and suppression of the plant host defense pathway, respectively. The construct, Vβ, was transformed into cotton plants using the Agrobacterium-mediated embryo shoot apex cut method. Results from fluorescence in situ hybridization and karyotyping assays indicated that six of the 11 T1 plants harbored a single copy of the Vβ transgene. Transgenic cotton plants and non-transgenic (susceptible) test plants included as the positive control were challenge-inoculated using the viruliferous whitefly vector to transmit the CLCuKoV-Bu/CLCuMB complex. Among the test plants, plant Vβ-6 was asymptomatic, had the lowest amount of detectable virus, and harbored a single copy of the transgene on chromosome six. Absence of characteristic leaf curl symptom development in transgenic Vβ-6 cotton plants, and significantly reduced begomoviral-betasatellite accumulation based on real-time polymerase chain reaction, indicated the successful knockdown of CLCuKoV-Bu and CLCuMB expression, resulting in leaf curl resistant plants.

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Section: Discussionsupporting

confidence: 86%

Section: Introductionmentioning

confidence: 99%

Engineered Disease Resistance in Cotton Using RNA-Interference to Knock down Cotton leaf curl Kokhran virus-Burewala and Cotton leaf curl Multan betasatellite Expression

Ahmad

Zia-ur-Rehman

Hameed

et al. 2017

Viruses

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“…Whereas, the transgenic plants harboring sgRNA multiplex cassette would be symptom free depending upon the level of expression of each sgRNA ( Figure 5E ). Once proof-of-concept established in N. benthamiana plants then whole system could be tested in the local cotton germplasm such as ‘Cocker’, which is not only susceptible to CLCuD but also easy to transform ( Ikram-Ul-Haq, 2004 ; Sohrab et al, 2014 ). The plant transformation will be done by Agrobacterium -mediated transformation and the fully established transgenic plants will be challenged by viruliferous whiteflies either in the cages or directly exposed to the viral inoculum in the field.…”

Section: Proposed Methodology For Designing Multiplex Sgrna To Circummentioning

confidence: 99%

CRISPR/Cas9: A Tool to Circumscribe Cotton Leaf Curl Disease

2016

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The begomoviruses (family Geminiviridae ) associated with cotton leaf curl disease (CLCuD) pose a major threat to cotton productivity in South–East Asia including Pakistan and India. These viruses have single-stranded, circular DNA genome, of ∼2800 nt in size, encapsidated in twinned icosa-hedera, transmitted by ubiquitous whitefly and are associated with satellite molecules referred to as alpha- and betasatellite. To circumvent the proliferation of these viruses numerous techniques, ranging from conventional breeding to molecular approaches have been applied. Such devised strategies worked perfectly well for a short time period and then viruses relapse due to various reasons including multiple infections, where related viruses synergistically interact with each other, virus proliferation and evolution. Another shortcoming is, until now, that all molecular biology approaches are devised to control only helper begomoviruses but not to control associated satellites. Despite the fact that satellites could add various functions to helper begomoviruses, they remain ignored. Such conditions necessitate a very comprehensive technique that can offer best controlling strategy not only against helper begomoviruses but also their associated DNA-satellites. In the current scenario clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR associated nuclease 9 (Cas9) has proved to be versatile technique that has very recently been deployed successfully to control different geminiviruses. The CRISPR/Cas9 system has been proved to be a comprehensive technique to control different geminiviruses, however, like previously used techniques, only a single virus is targeted and hitherto it has not been deployed to control begomovirus complexes associated with DNA-satellites. Here in this article, we proposed an inimitable, unique, and broad spectrum controlling method based on multiplexed CRISPR/Cas9 system where a cassette of sgRNA is designed to target not only the whole CLCuD-associated begomovirus complex but also the associated satellite molecules.

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“…The presence of antisense viral transcripts is suggested to lead to the formation of dsRNAs with the complementary sense transcripts produced by plant viruses upon infection. The dsRNAs, in turn, elicit the RNA silencing machinery and provide substantial resistance against most geminiviruses (Bejarano and Lichtenstein 1994, Yang et al 2004, Haq et al 2010, Amudha et al 2011, Sohrab et al 2016. Thirdly, a hairpin RNA encoding sense and antisense sequences in a single transcript is expressed producing a dsRNA in transgenic plants conferring resistance to the cognate virus upon viral infection (Hu et al 2011, Leibman et al 2011, Aslam et al 2018.…”

Section: Introductionmentioning

confidence: 99%

Sense- and antisense-mediated resistance against Sri Lankan cassava mosaic virus (SLCMV) in Nicotiana benthamiana

Gogoi¹,

Kaldis²,

Dasgupta³

et al. 2019

Biol. plant.

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Sri Lankan cassava mosaic virus (SLCMV) is the principal causal agent of cassava mosaic disease in the Indian subcontinent. To gain resistance against the virus, the coat protein (CP) gene, namely the AV1 of SLCMV-Adivaram isolate, was cloned in either sense or antisense orientation under the Cauliflower mosaic virus 35S promoter, and transgenic Nicotiana benthamiana plants were obtained through Agrobacterium-mediated transformation. A total of eight T1 transgenic lines, four harboring the CP-sense construct and four harboring the CP-antisense construct were challenged with agro-infectious clones of SLCMV DNA-A and DNA-B. Based on symptom exhibition at 20 days post inoculation, 3 out of the 4 CP-sense transgenic lines and all 4 CP-antisense transgenic lines showed a high level of resistance against SLCMV. In addition, a delay in symptom initiation was observed in all the transgenic lines inoculated with a high viral load at agro-dilution 1:625 from an absorbance (A600) of 1. However, the resistance was more prominent at a lower viral load of 1:1000 agro-dilution. The viral titer was lower in the SLCMV-challenged transgenic lines compared to the non-transgenic N. benthamiana plants as confirmed by quantitative PCR and dot blot analysis. Furthermore, small RNA Northern blot analysis revealed lowered amounts of virus-specific small interfering RNAs in the resistant transgenic lines as compared to the non-transgenic plants upon SLCMV infection, which correlates to lower virus titers due to resistance against the virus.

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Development of Cotton leaf curl virus resistant transgenic cotton using antisense ßC1 gene

Cited by 22 publications

References 27 publications

Engineered Disease Resistance in Cotton Using RNA-Interference to Knock down Cotton leaf curl Kokhran virus-Burewala and Cotton leaf curl Multan betasatellite Expression

Engineered Disease Resistance in Cotton Using RNA-Interference to Knock down Cotton leaf curl Kokhran virus-Burewala and Cotton leaf curl Multan betasatellite Expression

CRISPR/Cas9: A Tool to Circumscribe Cotton Leaf Curl Disease

Sense- and antisense-mediated resistance against Sri Lankan cassava mosaic virus (SLCMV) in Nicotiana benthamiana

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