CRISPR/Cas9-mediated resurrection of tobacco NB-LRR class virus resistance gene from a susceptible allele with partial duplication

IF 1 4区 农林科学 Q3 PLANT SCIENCES Journal of General Plant Pathology Pub Date : 2024-07-22 DOI:10.1007/s10327-024-01189-x
Saki Miyoshi, Okon Odiong Unung, Hidetaka Kaya, Takashi Yaeno, Kappei Kobayashi
{"title":"CRISPR/Cas9-mediated resurrection of tobacco NB-LRR class virus resistance gene from a susceptible allele with partial duplication","authors":"Saki Miyoshi, Okon Odiong Unung, Hidetaka Kaya, Takashi Yaeno, Kappei Kobayashi","doi":"10.1007/s10327-024-01189-x","DOIUrl":null,"url":null,"abstract":"<p>It is still difficult to manipulate the nucleotide-binding site-leucine-rich repeat (NB-LRR) class disease resistance genes because of their large multigenic family. Here, we report the successful application of CRISPR/Cas9 to resurrect a functional allele from a susceptible allele of the <i>N′</i> tobamovirus resistance gene. The susceptible alleles of <i>N′</i> from some <i>Nicotiana tabacum</i> cultivars (<i>Nt-n′</i>) have a partial duplication of the <i>N′</i> coding sequence upstream of a complete coding sequence of the <i>N′</i> gene, which likely abolished the <i>N′</i>-mediated resistance. We first established a transgenic tobacco line expressing Cas9 under the control of a chemically inducible promoter. The plant line was retransformed with a construct expressing a guide RNA targeting the sequences common to the duplicated partial sequence in the upstream and the complete sequence in the downstream. The T<sub>0</sub> transformants had different ratios of the sequences devoid of the duplicated partial sequence. Sequencing proved that some of them had sequences identical to that of the functional <i>N′</i> gene, suggesting the successful resurrection of the functional <i>N′</i> gene. The resurrected allele, <i>N′</i><sup><i>-R</i></sup>, was inherited by a few T<sub>1</sub> progenies and subsequent generations with the least mutation at the target site under Cas9-uninduced conditions. The plants homozygous for <i>N′</i><sup><i>-R</i></sup> showed resistance to a tobamovirus, indicating that the resurrected <i>N′</i><sup><i>-R</i></sup> allele is functional.</p>","PeriodicalId":15825,"journal":{"name":"Journal of General Plant Pathology","volume":null,"pages":null},"PeriodicalIF":1.0000,"publicationDate":"2024-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of General Plant Pathology","FirstCategoryId":"97","ListUrlMain":"https://doi.org/10.1007/s10327-024-01189-x","RegionNum":4,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"PLANT SCIENCES","Score":null,"Total":0}
引用次数: 0

Abstract

It is still difficult to manipulate the nucleotide-binding site-leucine-rich repeat (NB-LRR) class disease resistance genes because of their large multigenic family. Here, we report the successful application of CRISPR/Cas9 to resurrect a functional allele from a susceptible allele of the N′ tobamovirus resistance gene. The susceptible alleles of N′ from some Nicotiana tabacum cultivars (Nt-n′) have a partial duplication of the N′ coding sequence upstream of a complete coding sequence of the N′ gene, which likely abolished the N′-mediated resistance. We first established a transgenic tobacco line expressing Cas9 under the control of a chemically inducible promoter. The plant line was retransformed with a construct expressing a guide RNA targeting the sequences common to the duplicated partial sequence in the upstream and the complete sequence in the downstream. The T0 transformants had different ratios of the sequences devoid of the duplicated partial sequence. Sequencing proved that some of them had sequences identical to that of the functional N′ gene, suggesting the successful resurrection of the functional N′ gene. The resurrected allele, N′-R, was inherited by a few T1 progenies and subsequent generations with the least mutation at the target site under Cas9-uninduced conditions. The plants homozygous for N′-R showed resistance to a tobamovirus, indicating that the resurrected N′-R allele is functional.

Abstract Image

查看原文
分享 分享
微信好友 朋友圈 QQ好友 复制链接
本刊更多论文
CRISPR/Cas9 介导的烟草 NB-LRR 类病毒抗性基因从部分重复的易感等位基因中复活
由于核苷酸结合位点-富亮氨酸重复(NB-LRR)类抗病基因具有庞大的多基因家族,因此对其进行操作仍然很困难。在此,我们报告了成功应用 CRISPR/Cas9 从 N′ tobamovirus 抗病基因的易感等位基因中复活一个功能等位基因的过程。一些烟草栽培品种的 N′易感等位基因(Nt-n′)在 N′基因完整编码序列的上游有部分 N′编码序列的重复,这很可能取消了 N′介导的抗性。我们首先建立了一个在化学诱导启动子控制下表达 Cas9 的转基因烟草品系。用表达引导 RNA 的构建质粒对该植物品系进行再转化,引导 RNA 的靶标是上游重复的部分序列和下游完整序列的共同序列。T0 转化子中没有重复的部分序列的序列比例各不相同。测序结果表明,其中一些转化子的序列与功能 N′基因的序列相同,表明功能 N′基因成功复活。复活的等位基因 N′-R,在 Cas9 未诱导的条件下,由少数 T1 后代和后代继承,目标位点的突变最小。N′-R基因同源的植株表现出对托布病毒的抗性,表明复活的N′-R等位基因是有功能的。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 去求助
来源期刊
CiteScore
2.40
自引率
8.30%
发文量
60
审稿时长
6 months
期刊介绍: The Journal of General Plant Pathology welcomes all manuscripts dealing with plant diseases or their control, including pathogen characterization, identification of pathogens, disease physiology and biochemistry, molecular biology, morphology and ultrastructure, genetics, disease transmission, ecology and epidemiology, chemical and biological control, disease assessment, and other topics relevant to plant pathological disorders.
期刊最新文献
Development of a real-time loop-mediated isothermal amplification method with toothpick sampling for non-destructive detection of Ustilago esculenta in Zizania latifolia Polymorphic microsatellite markers for genetic analysis of Berkeleyomyces rouxiae, a causal agent of black root rot WRKY transcription factors identified in the transcriptome of Citrus latifolia Tan. and their expression in response to Huanglongbing disease Possible roles of immunity-related response in modulating chlorosis induced by the silencing of chloroplast HSP90C in tobacco models CRISPR/Cas9-mediated resurrection of tobacco NB-LRR class virus resistance gene from a susceptible allele with partial duplication
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
现在去查看 取消
×
提示
确定
0
微信
客服QQ
Book学术公众号 扫码关注我们
反馈
×
意见反馈
请填写您的意见或建议
请填写您的手机或邮箱
已复制链接
已复制链接
快去分享给好友吧!
我知道了
×
扫码分享
扫码分享
Book学术官方微信
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术
文献互助 智能选刊 最新文献 互助须知 联系我们:info@booksci.cn
Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。
Copyright © 2023 Book学术 All rights reserved.
ghs 京公网安备 11010802042870号 京ICP备2023020795号-1