Cargando…
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...
Autores principales: | , , , , |
---|---|
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 |
_version_ | 1785024076769656832 |
---|---|
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. |
format | Online Article Text |
id | pubmed-10095410 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
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 |
work_keys_str_mv | AT yıldırımkubilay developmentofhighlyefficientresistancetobeetcurlytopiranvirusbecurtovirusinsugarbeetbvulgarisviacrisprcas9system AT kavasmusa developmentofhighlyefficientresistancetobeetcurlytopiranvirusbecurtovirusinsugarbeetbvulgarisviacrisprcas9system AT kucukilkaysevgen developmentofhighlyefficientresistancetobeetcurlytopiranvirusbecurtovirusinsugarbeetbvulgarisviacrisprcas9system AT secginzafer developmentofhighlyefficientresistancetobeetcurlytopiranvirusbecurtovirusinsugarbeetbvulgarisviacrisprcas9system AT saraccigdemgokcek developmentofhighlyefficientresistancetobeetcurlytopiranvirusbecurtovirusinsugarbeetbvulgarisviacrisprcas9system |