Cargando…

Highly efficient generation of bacterial leaf blight-resistant and transgene-free rice using a genome editing and multiplexed selection system

BACKGROUND: Rice leaf blight, which is a devastating disease worldwide, is caused by the bacterium Xanthomonas oryzae pv. oryzae (Xoo). The upregulated by transcription activator-like 1 (UPT) effector box in the promoter region of the rice Xa13 gene plays a key role in Xoo pathogenicity. Mutation of...

Descripción completa

Detalles Bibliográficos
Autores principales: Yu, Kun, Liu, Zhiqiang, Gui, Huaping, Geng, Lizhao, Wei, Juan, Liang, Dawei, Lv, Jian, Xu, Jianping, Chen, Xi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8066475/
https://www.ncbi.nlm.nih.gov/pubmed/33894749
http://dx.doi.org/10.1186/s12870-021-02979-7
_version_ 1783682579561447424
author Yu, Kun
Liu, Zhiqiang
Gui, Huaping
Geng, Lizhao
Wei, Juan
Liang, Dawei
Lv, Jian
Xu, Jianping
Chen, Xi
author_facet Yu, Kun
Liu, Zhiqiang
Gui, Huaping
Geng, Lizhao
Wei, Juan
Liang, Dawei
Lv, Jian
Xu, Jianping
Chen, Xi
author_sort Yu, Kun
collection PubMed
description BACKGROUND: Rice leaf blight, which is a devastating disease worldwide, is caused by the bacterium Xanthomonas oryzae pv. oryzae (Xoo). The upregulated by transcription activator-like 1 (UPT) effector box in the promoter region of the rice Xa13 gene plays a key role in Xoo pathogenicity. Mutation of a key bacterial protein-binding site in the UPT box of Xa13 to abolish PXO99-induced Xa13 expression is a way to improve rice resistance to bacteria. Highly efficient generation and selection of transgene-free edited plants are helpful to shorten and simplify the gene editing-based breeding process. Selective elimination of transgenic pollen of T0 plants can enrich the proportion of T1 transgene-free offspring, and expression of a color marker gene in seeds makes the selection of T2 plants very convenient and efficient. In this study, a genome editing and multiplexed selection system was used to generate bacterial leaf blight-resistant and transgene-free rice plants. RESULTS: We introduced site-specific mutations into the UPT box using CRISPR/Cas12a technology to hamper with transcription-activator-like effector (TAL) protein binding and gene activation and generated genome-edited rice with improved bacterial blight resistance. Transgenic pollen of T0 plants was eliminated by pollen-specific expression of the α-amylase gene Zmaa1, and the proportion of transgene-free plants increased from 25 to 50% among single T-DNA insertion events in the T1 generation. Transgenic seeds were visually identified and discarded by specific aleuronic expression of DsRed, which reduced the cost by 50% and led to up to 98.64% accuracy for the selection of transgene-free edited plants. CONCLUSION: We demonstrated that core nucleotide deletion in the UPT box of the Xa13 promoter conferred resistance to rice blight, and selection of transgene-free plants was boosted by introducing multiplexed selection. The combination of genome editing and transgene-free selection is an efficient strategy to accelerate functional genomic research and plant breeding. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12870-021-02979-7.
format Online
Article
Text
id pubmed-8066475
institution National Center for Biotechnology Information
language English
publishDate 2021
publisher BioMed Central
record_format MEDLINE/PubMed
spelling pubmed-80664752021-04-26 Highly efficient generation of bacterial leaf blight-resistant and transgene-free rice using a genome editing and multiplexed selection system Yu, Kun Liu, Zhiqiang Gui, Huaping Geng, Lizhao Wei, Juan Liang, Dawei Lv, Jian Xu, Jianping Chen, Xi BMC Plant Biol Research BACKGROUND: Rice leaf blight, which is a devastating disease worldwide, is caused by the bacterium Xanthomonas oryzae pv. oryzae (Xoo). The upregulated by transcription activator-like 1 (UPT) effector box in the promoter region of the rice Xa13 gene plays a key role in Xoo pathogenicity. Mutation of a key bacterial protein-binding site in the UPT box of Xa13 to abolish PXO99-induced Xa13 expression is a way to improve rice resistance to bacteria. Highly efficient generation and selection of transgene-free edited plants are helpful to shorten and simplify the gene editing-based breeding process. Selective elimination of transgenic pollen of T0 plants can enrich the proportion of T1 transgene-free offspring, and expression of a color marker gene in seeds makes the selection of T2 plants very convenient and efficient. In this study, a genome editing and multiplexed selection system was used to generate bacterial leaf blight-resistant and transgene-free rice plants. RESULTS: We introduced site-specific mutations into the UPT box using CRISPR/Cas12a technology to hamper with transcription-activator-like effector (TAL) protein binding and gene activation and generated genome-edited rice with improved bacterial blight resistance. Transgenic pollen of T0 plants was eliminated by pollen-specific expression of the α-amylase gene Zmaa1, and the proportion of transgene-free plants increased from 25 to 50% among single T-DNA insertion events in the T1 generation. Transgenic seeds were visually identified and discarded by specific aleuronic expression of DsRed, which reduced the cost by 50% and led to up to 98.64% accuracy for the selection of transgene-free edited plants. CONCLUSION: We demonstrated that core nucleotide deletion in the UPT box of the Xa13 promoter conferred resistance to rice blight, and selection of transgene-free plants was boosted by introducing multiplexed selection. The combination of genome editing and transgene-free selection is an efficient strategy to accelerate functional genomic research and plant breeding. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12870-021-02979-7. BioMed Central 2021-04-24 /pmc/articles/PMC8066475/ /pubmed/33894749 http://dx.doi.org/10.1186/s12870-021-02979-7 Text en © The Author(s) 2021 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
spellingShingle Research
Yu, Kun
Liu, Zhiqiang
Gui, Huaping
Geng, Lizhao
Wei, Juan
Liang, Dawei
Lv, Jian
Xu, Jianping
Chen, Xi
Highly efficient generation of bacterial leaf blight-resistant and transgene-free rice using a genome editing and multiplexed selection system
title Highly efficient generation of bacterial leaf blight-resistant and transgene-free rice using a genome editing and multiplexed selection system
title_full Highly efficient generation of bacterial leaf blight-resistant and transgene-free rice using a genome editing and multiplexed selection system
title_fullStr Highly efficient generation of bacterial leaf blight-resistant and transgene-free rice using a genome editing and multiplexed selection system
title_full_unstemmed Highly efficient generation of bacterial leaf blight-resistant and transgene-free rice using a genome editing and multiplexed selection system
title_short Highly efficient generation of bacterial leaf blight-resistant and transgene-free rice using a genome editing and multiplexed selection system
title_sort highly efficient generation of bacterial leaf blight-resistant and transgene-free rice using a genome editing and multiplexed selection system
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8066475/
https://www.ncbi.nlm.nih.gov/pubmed/33894749
http://dx.doi.org/10.1186/s12870-021-02979-7
work_keys_str_mv AT yukun highlyefficientgenerationofbacterialleafblightresistantandtransgenefreericeusingagenomeeditingandmultiplexedselectionsystem
AT liuzhiqiang highlyefficientgenerationofbacterialleafblightresistantandtransgenefreericeusingagenomeeditingandmultiplexedselectionsystem
AT guihuaping highlyefficientgenerationofbacterialleafblightresistantandtransgenefreericeusingagenomeeditingandmultiplexedselectionsystem
AT genglizhao highlyefficientgenerationofbacterialleafblightresistantandtransgenefreericeusingagenomeeditingandmultiplexedselectionsystem
AT weijuan highlyefficientgenerationofbacterialleafblightresistantandtransgenefreericeusingagenomeeditingandmultiplexedselectionsystem
AT liangdawei highlyefficientgenerationofbacterialleafblightresistantandtransgenefreericeusingagenomeeditingandmultiplexedselectionsystem
AT lvjian highlyefficientgenerationofbacterialleafblightresistantandtransgenefreericeusingagenomeeditingandmultiplexedselectionsystem
AT xujianping highlyefficientgenerationofbacterialleafblightresistantandtransgenefreericeusingagenomeeditingandmultiplexedselectionsystem
AT chenxi highlyefficientgenerationofbacterialleafblightresistantandtransgenefreericeusingagenomeeditingandmultiplexedselectionsystem