Department of Molecular Biology and Genetics, Faculty of Arts and Sciences, Ondokuz Mayıs University, 55139 Samsun, Turkey.
Department of Agricultural Biotechnology, Faculty of Agriculture, Ondokuz Mayıs University, 55139 Samsun, Turkey.
Int J Mol Sci. 2023 Mar 30;24(7):6515. doi: 10.3390/ijms24076515.
Beet Curly Top Iran Virus (BCTIV, ) 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 in sugar beet.
甜菜曲顶病毒（BCTIV）是一种主要的、广泛存在的病原体，在地中海和中东地区的甜菜生产中造成了巨大的损害和产量降低。基于 CRISPR 的基因编辑是一种多功能工具，已成功用于植物中提高对许多病毒病原体的抗性。在这项研究中，通过瞬时表达评估了针对 BCTIV 表达基因的 gRNA/Cas9 构建体在甜菜叶片中的效率。几乎所有阳性对照甜菜都显示出系统性感染和严重的疾病症状（90%），在 BCTIV agroinoculation 后生物量减少了 68%。另一方面，与 BCTIV 和 gRNA/Cas9 共 agroinoculated 的甜菜显示出较低的系统性感染（10-55%）、疾病症状和生物量减少（13-45%）。还通过 RCA 和 qPCR 分析对用 gRNA/Cas9 处理的甜菜进行了病毒失活验证。PCR-RE 消化和测序分析证实了 gRNA/Cas9 在 BCTIV 基因组靶位点介导的 INDEL 突变，并表现出高效率（53-88%），特别是针对 BCTIV 的运动基因及其衣壳和 ssDNA 调节基因之间的重叠区域。还测试了一种多重 CRISPR 方法。针对 BCTIV 所有基因的最有效四个 gRNA 被克隆到含有 Cas9 的载体中，并 agroinoculated 到感染病毒的甜菜叶片中。该多重 CRISPR 系统的结果显示几乎完全抵抗病毒，抑制系统性感染和突变逃逸。这是首次报道利用 CRISPR 介导的广谱抗性来抵抗甜菜曲顶病毒。
