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Development of Highly Efficient Resistance to Beet Curly Top Iran Virus (Becurtovirus) in Sugar Beet (B. vulgaris) via CRISPR/Cas9 System

Beet Curly Top Iran Virus (BCTIV, Becurtovirus) is a dominant and widespread pathogen responsible for great damage and yield reduction in sugar beet production in the Mediterranean and Middle East. CRISPR-based gene editing is a versatile tool that has been successfully used in plants to improve res...

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Autores principales: Yıldırım, Kubilay, Kavas, Musa, Küçük, İlkay Sevgen, Seçgin, Zafer, Saraç, Çiğdem Gökcek
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10095410/
https://www.ncbi.nlm.nih.gov/pubmed/37047489
http://dx.doi.org/10.3390/ijms24076515
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author Yıldırım, Kubilay
Kavas, Musa
Küçük, İlkay Sevgen
Seçgin, Zafer
Saraç, Çiğdem Gökcek
author_facet Yıldırım, Kubilay
Kavas, Musa
Küçük, İlkay Sevgen
Seçgin, Zafer
Saraç, Çiğdem Gökcek
author_sort Yıldırım, Kubilay
collection PubMed
description Beet Curly Top Iran Virus (BCTIV, Becurtovirus) is a dominant and widespread pathogen responsible for great damage and yield reduction in sugar beet production in the Mediterranean and Middle East. CRISPR-based gene editing is a versatile tool that has been successfully used in plants to improve resistance against many viral pathogens. In this study, the efficiency of gRNA/Cas9 constructs targeting the expressed genes of BCTIV was assessed in sugar beet leaves by their transient expression. Almost all positive control sugar beets revealed systemic infection and severe disease symptoms (90%), with a great biomass reduction (68%) after BCTIV agroinoculation. On the other hand, sugar beets co-agronioculated with BCTIV and gRNA/Cas9 indicated much lower systemic infection (10–55%), disease symptoms and biomass reduction (13–45%). Viral inactivation was also verified by RCA and qPCR assays for gRNA/Cas9 treated sugar beets. PCR-RE digestion and sequencing assays confirmed the gRNA/Cas9-mediated INDEL mutations at the target sites of the BCTIV genome and represented high efficiencies (53–88%), especially for those targeting BCTIV’s movement gene and its overlapping region between capsid and ssDNA regulator genes. A multiplex CRISPR approach was also tested. The most effective four gRNAs targeting all the genes of BCTIV were cloned into a Cas9-containing vector and agroinoculated into virus-infected sugar beet leaves. The results of this multiplex CRISPR system revealed almost complete viral resistance with inhibition of systemic infection and mutant escape. This is the first report of CRSIPR-mediated broad-spectrum resistance against Becurtovirus in sugar beet.
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spelling pubmed-100954102023-04-13 Development of Highly Efficient Resistance to Beet Curly Top Iran Virus (Becurtovirus) in Sugar Beet (B. vulgaris) via CRISPR/Cas9 System Yıldırım, Kubilay Kavas, Musa Küçük, İlkay Sevgen Seçgin, Zafer Saraç, Çiğdem Gökcek Int J Mol Sci Article Beet Curly Top Iran Virus (BCTIV, Becurtovirus) is a dominant and widespread pathogen responsible for great damage and yield reduction in sugar beet production in the Mediterranean and Middle East. CRISPR-based gene editing is a versatile tool that has been successfully used in plants to improve resistance against many viral pathogens. In this study, the efficiency of gRNA/Cas9 constructs targeting the expressed genes of BCTIV was assessed in sugar beet leaves by their transient expression. Almost all positive control sugar beets revealed systemic infection and severe disease symptoms (90%), with a great biomass reduction (68%) after BCTIV agroinoculation. On the other hand, sugar beets co-agronioculated with BCTIV and gRNA/Cas9 indicated much lower systemic infection (10–55%), disease symptoms and biomass reduction (13–45%). Viral inactivation was also verified by RCA and qPCR assays for gRNA/Cas9 treated sugar beets. PCR-RE digestion and sequencing assays confirmed the gRNA/Cas9-mediated INDEL mutations at the target sites of the BCTIV genome and represented high efficiencies (53–88%), especially for those targeting BCTIV’s movement gene and its overlapping region between capsid and ssDNA regulator genes. A multiplex CRISPR approach was also tested. The most effective four gRNAs targeting all the genes of BCTIV were cloned into a Cas9-containing vector and agroinoculated into virus-infected sugar beet leaves. The results of this multiplex CRISPR system revealed almost complete viral resistance with inhibition of systemic infection and mutant escape. This is the first report of CRSIPR-mediated broad-spectrum resistance against Becurtovirus in sugar beet. MDPI 2023-03-30 /pmc/articles/PMC10095410/ /pubmed/37047489 http://dx.doi.org/10.3390/ijms24076515 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Yıldırım, Kubilay
Kavas, Musa
Küçük, İlkay Sevgen
Seçgin, Zafer
Saraç, Çiğdem Gökcek
Development of Highly Efficient Resistance to Beet Curly Top Iran Virus (Becurtovirus) in Sugar Beet (B. vulgaris) via CRISPR/Cas9 System
title Development of Highly Efficient Resistance to Beet Curly Top Iran Virus (Becurtovirus) in Sugar Beet (B. vulgaris) via CRISPR/Cas9 System
title_full Development of Highly Efficient Resistance to Beet Curly Top Iran Virus (Becurtovirus) in Sugar Beet (B. vulgaris) via CRISPR/Cas9 System
title_fullStr Development of Highly Efficient Resistance to Beet Curly Top Iran Virus (Becurtovirus) in Sugar Beet (B. vulgaris) via CRISPR/Cas9 System
title_full_unstemmed Development of Highly Efficient Resistance to Beet Curly Top Iran Virus (Becurtovirus) in Sugar Beet (B. vulgaris) via CRISPR/Cas9 System
title_short Development of Highly Efficient Resistance to Beet Curly Top Iran Virus (Becurtovirus) in Sugar Beet (B. vulgaris) via CRISPR/Cas9 System
title_sort development of highly efficient resistance to beet curly top iran virus (becurtovirus) in sugar beet (b. vulgaris) via crispr/cas9 system
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10095410/
https://www.ncbi.nlm.nih.gov/pubmed/37047489
http://dx.doi.org/10.3390/ijms24076515
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