Generation of the transgene-free canker-resistant Citrus sinensis using Cas12a/crRNA ribonucleoprotein in the T0 generation

Su, Hang; Wang, Yuanchun; Xu, Jin; Omar, Ahmad A.; Grosser, Jude W.; Ćalović, Milica; Zhang, Jintao; Feng, Yue; Vakulskas, Christopher A.; Wang, Nian

doi:10.1038/s41467-023-39714-9

Cited by 28 publications

(24 citation statements)

References 85 publications

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“…While transgenic citrus will offer a complementary strategy that will provide a more sustained solution, this will require significant time for field efficacy studies and regulatory processes before being handed over to the growers (Graham et al, 2020;Su et al, 2023).…”

Section: Discussionmentioning

confidence: 99%

A host‐derived chimeric peptide protects citrus against Huanglongbing without threatening the native microbial community of the phyllosphere

Choi,

Basu,

Thompson

et al. 2023

J of Sust Agri & Env

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IntroductionThe application of host‐derived antibacterial peptides has been highlighted as a potential efficacious and safe tool for the treatment of Huanglongbing (HLB), the most devastating disease of citrus. However, pathogenic bacteria such as HLB‐causing Candidatus liberibacter asiaticus (CLas) often develop resistance against the host antibacterial peptides. We showed that chimeras containing two different host antibacterial peptides not only retain antibacterial activity but also overcome bacterial resistance and enhance plant defence responses. Also, chimeric peptides can have an off‐target impact on the structure and function of plant‐associated microbiomes. However, there is a lack of understanding of the impact of the chimeric peptide therapy on the microbial structure in the citrus phyllosphere while reducing the CLas titre. Here, we aim to evaluate the efficacy of a chimeric peptide (UGK17) to reduce CLas titre, inducing plant defence response and impacting the microbiome associated with the citrus phyllosphere.Material and MethodLeaf samples were collected from orange and grapefruit trees in Texas and identified as old and young leaves according to their maturity. We collected three different types of leaves based on their infection and symptoms: healthy, symptomatic (infected with typical symptoms), and asymptomatic (infected without symptoms). In planta assay was performed by dipping the leaves in the 0, 5 and 25 μM of UGK17 solutions for 48 h. The quantifications of CLas titre and pathogenesis‐related (PR) gene expression were done by quantitative polymerase chain reaction (qPCR) and reverse transcription‐qPCR, respectively. Amplicon sequencing was done to evaluate the impact of UGK17 on individual bacterial community structures. In addition, we performed an ex planta assay to assess the effect of UGK17 on the growth of bacterial isolates including Liberibacter crescens instead of unculturable CLas predominant in the phyllosphere.ResultThe UGK17 treatment reduced the CLas titre in both asymptomatic and symptomatic citrus leaves, regardless of the age of the leaves. The UGK17 application augmented the PR gene expression. In ex planta assay, the growth of L. crescens along with four other strains belonging to the family Rhizobiaceae, was significantly inhibited by the UGK17 while the growth of 74 strains were unaffected. Additionally, there was no statistically significant changes in the microbial community structure with UGK17 treatment.ConclusionOur results suggest that the chimeric peptide therapy is a promising solution to combat HLB by targeting mainly Gram‐negative pathogens and enhancing the plant immune responses without impairing the indigenous microbial community in the citrus phyllosphere.

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Section: Discussionmentioning

confidence: 99%

A host‐derived chimeric peptide protects citrus against Huanglongbing without threatening the native microbial community of the phyllosphere

Choi,

Basu,

Thompson

et al. 2023

J of Sust Agri & Env

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“…These plants exhibit enhanced disease resistance to infection by X. cirti subsp. citri (Su et al 2023).…”

Section: Disadvantages and Reformed Strengths Of Crispr-cas-based Gen...mentioning

confidence: 99%

CRISPR‐Cas9 and beyond: identifying target genes for developing disease‐resistant plants

Park,

Kim,

Lee

et al. 2024

Plant Biol J

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Throughout the history of crop domestication, desirable traits have been selected in agricultural products. However, such selection often leads to crops and vegetables with weaker vitality and viability than their wild ancestors when exposed to adverse environmental conditions. Considering the increasing human population and climate change challenges, it is crucial to enhance crop quality and quantity. Accordingly, the identification and utilization of diverse genetic resources are imperative for developing disease‐resistant plants that can withstand unexpected epidemics of plant diseases. In this review, we provide a brief overview of recent progress in genome‐editing technologies, including zinc‐finger nucleases (ZFNs), transcription activator‐like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)‐associated protein 9 (Cas9) technologies. In particular, we classify disease‐resistant mutants of Arabidopsis thaliana and several crop plants based on the roles or functions of the mutated genes in plant immunity and suggest potential target genes for molecular breeding of genome‐edited disease‐resistant plants. Genome‐editing technologies are resilient tools for sustainable development and promising solutions for coping with climate change and population increases.

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“…CRISPR/Cas mediated genome editing is deemed the most promising approach to breed new citrus cultivars, owing to its short time requirement, precise genetic improvement and predictable results ( Chen et al., 2019 ; Gao, 2021 ; Cao et al., 2022 ; Huang et al., 2022a ). To date, SpCas9/gRNA from Streptococcus pyogenes , SaCas9/gRNA from Staphylococcus aureus , LbCas12a/crRNA from Lachnospiraceae bacterium and base editor derived from SpCas9/gRNA have been successfully adapted to modify citrus genomes for gene function study and new citrus cultivar breeding ( Jia and Wang, 2014a ; Jia et al., 2016 ; Peng et al., 2017 ; Zhang et al., 2017 ; Jia et al., 2017a , Jia et al., 2017b ; LeBlanc et al., 2018 ; Zhu et al., 2019 ; Jia et al., 2019a ; Dutt et al., 2020 ; Huang et al., 2020 ; Jia and Wang, 2020 ; Huang and Wang, 2021 ; Jia et al., 2021 ; Alquezar et al., 2022 ; Jia et al., 2022 ; Mahmoud et al., 2022 ; Parajuli et al., 2022 ; Yang et al., 2022 ; Huang et al., 2022b , 2023 ; Su et al., 2023 ).…”

Section: Introductionmentioning

confidence: 99%

“…Multiple strategies have been developed to produce transgene-free plants after target-gene editing with CRISPR/Cas ( Kocsisova and Coneva, 2023 ). Some examples include delivering DNA-free gene editing reagents such as ribonucleoproteins ( Woo et al., 2015 ; Malnoy et al., 2016 ; Subburaj et al., 2016 ; Svitashev et al., 2016 ; Liang et al., 2017 ; Andersson et al., 2018 ; Su et al., 2023 ), novel delivery vectors such as viruses ( Mei et al., 2019 ; Ma et al., 2020 ; Liu et al., 2023 ), unconventional selection methods to bypass integration of transgenes ( Veillet et al., 2019 ; Alquezar et al., 2022 ; Huang et al., 2022b , 2023 ; Wei et al., 2023 ), graft-mobile editing systems ( Yang et al., 2023 ), and so on. Initially, Alquezar et al.…”

Section: Introductionmentioning

confidence: 99%

“…Su et al. took advantage of Cas12a/crRNA ribonucleoprotein to disrupt the coding region of LOB1 ( Su et al., 2023 ), and another study employed Cas12/CBE co-editing method to edit EBE PthA4 -LOBP of pummelo, which is highly homozygous and relatively easy to work with ( Huang et al., 2023 ). Notably, the latter took advantage of green fluorescent protein for selecting transgene-free transformants, Agrobacterium -mediated transient expression of cytosine base editor (CBE) to edit ALS encoding acetolactate synthase to confer herbicide chlorsulfuron resistance as a selection marker, and Cas12a/CRISPR RNA for editing gene(s) of interest ( Huang et al., 2023 ).…”

Section: Introductionmentioning

confidence: 99%

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Generation of transgene-free canker-resistant Citrus sinensis cv. Hamlin in the T0 generation through Cas12a/CBE co-editing

Jia,

Omar,

et al. 2024

Front. Plant Sci.

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Citrus canker disease affects citrus production. This disease is caused by Xanthomonas citri subsp. citri (Xcc). Previous studies confirmed that during Xcc infection, PthA4, a transcriptional activator like effector (TALE), is translocated from the pathogen to host plant cells. PthA4 binds to the effector binding elements (EBEs) in the promoter region of canker susceptibility gene LOB1 (EBEPthA4-LOBP) to activate its expression and subsequently cause canker symptoms. Previously, the Cas12a/CBE co-editing method was employed to disrupt EBEPthA4-LOBP of pummelo, which is highly homozygous. However, most commercial citrus cultivars are heterozygous hybrids and more difficult to generate homozygous/biallelic mutants. Here, we employed Cas12a/CBE co-editing method to edit EBEPthA4-LOBP of Hamlin (Citrus sinensis), a commercial heterozygous hybrid citrus cultivar grown worldwide. Binary vector GFP-p1380N-ttLbCas12a:LOBP1-mPBE:ALS2:ALS1 was constructed and shown to be functional via Xcc-facilitated agroinfiltration in Hamlin leaves. This construct allows the selection of transgene-free regenerants via GFP, edits ALS to generate chlorsulfuron-resistant regenerants as a selection marker for genome editing resulting from transient expression of the T-DNA via nCas9-mPBE:ALS2:ALS1, and edits gene(s) of interest (i.e., EBEPthA4-LOBP in this study) through ttLbCas12a, thus creating transgene-free citrus. Totally, 77 plantlets were produced. Among them, 8 plantlets were transgenic plants (#HamGFP1 - #HamGFP8), 4 plantlets were transgene-free (#HamNoGFP1 - #HamNoGFP4), and the rest were wild type. Among 4 transgene-free plantlets, three lines (#HamNoGFP1, #HamNoGFP2 and #HamNoGFP3) contained biallelic mutations in EBEpthA4, and one line (#HamNoGFP4) had homozygous mutations in EBEpthA4. We achieved 5.2% transgene-free homozygous/biallelic mutation efficiency for EBEPthA4–LOBP in C. sinensis cv. Hamlin, compared to 1.9% mutation efficiency for pummelo in a previous study. Importantly, the four transgene-free plantlets and 3 transgenic plantlets that survived were resistant against citrus canker. Taken together, Cas12a/CBE co-editing method has been successfully used to generate transgene-free canker‐resistant C. sinensis cv. Hamlin in the T0 generation via biallelic/homozygous editing of EBEpthA4 of the canker susceptibility gene LOB1.

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Generation of the transgene-free canker-resistant Citrus sinensis using Cas12a/crRNA ribonucleoprotein in the T0 generation

Cited by 28 publications

References 85 publications

A host‐derived chimeric peptide protects citrus against Huanglongbing without threatening the native microbial community of the phyllosphere

A host‐derived chimeric peptide protects citrus against Huanglongbing without threatening the native microbial community of the phyllosphere

CRISPR‐Cas9 and beyond: identifying target genes for developing disease‐resistant plants

Generation of transgene-free canker-resistant Citrus sinensis cv. Hamlin in the T0 generation through Cas12a/CBE co-editing

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