{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,1]],"date-time":"2026-10-01T06:57:30Z","timestamp":1790837850259,"version":"4.1.0"},"reference-count":38,"publisher":"Wiley","license":[{"start":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T00:00:00Z","timestamp":1790726400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T00:00:00Z","timestamp":1790726400000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Plant Biotechnology Journal"],"abstract":"ABSTRACT<\/jats:title>\n \n \n Agrobacterium tumefaciens<\/jats:italic>\n <\/jats:styled-content>\n is a powerful delivery tool for plant genetic transformation and a pivotal platform for agricultural biotechnology. However, laboratory strains of\n Agrobacterium<\/jats:italic>\n have remained genetically unoptimized for decades, with genome engineering hindered by inefficient, endogenous homologous recombination systems. Here, we developed and optimized a lambda (\u03bb) Red recombinase\u2010assisted CRISPR\/Cas9 system that enables efficient and precise introduction of point mutations, insertions, deletions and large fragment replacements into both the chromosome and Ti plasmids of different\n \n A. tumefaciens<\/jats:italic>\n <\/jats:styled-content>\n strains, with minimal off\u2010target editing. Editing efficiencies across eighteen target sites ranged from 26.7% to 100%, demonstrating the versatility of this method. Furthermore, we incorporated a\n SacB<\/jats:italic>\n counter\u2010selection marker into our system that facilitates the plasmids curing after editing. Using this platform, we sequentially deleted four antibiotic\u2010resistance loci in strain EHA105, generating an antibiotic\u2010sensitive derivative, EHA105\u2010S, that maintains wild\u2010type growth kinetics and transformation efficiency. Additionally, we engineered the microbe\u2010associated molecular pattern (MAMPs) of two elongation factor Tu (EF\u2010Tu) genes, allowing the\n \n A. tumefaciens<\/jats:italic>\n <\/jats:styled-content>\n to escape EF\u2010Tu receptor (EFR) perception. The resulting engineered strains displayed enhanced transient expression efficiency in\n \n Arabidopsis thaliana<\/jats:italic>\n <\/jats:styled-content>\n . Our \u03bb Red\u2010assisted CRISPR\/Cas9 system thus provides a robust, scalable and user\u2010friendly tool for\n Agrobacterium<\/jats:italic>\n genome engineering, paving the way for the design of next\u2010generation strains to revolutionize plant transformation and advance synthetic biology applications in plants.\n <\/jats:p>","DOI":"10.1111\/pbi.70768","type":"journal-article","created":{"date-parts":[[2026,10,1]],"date-time":"2026-10-01T06:23:51Z","timestamp":1790835831000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A Lambda Red Recombinase\u2010Assisted\n CRISPR<\/scp>\n \/Cas9 System for Efficient and Precise Genome Editing in\n \n Agrobacterium tumefaciens<\/i>\n <\/scp>"],"prefix":"10.1111","author":[{"given":"Minmin","family":"Zheng","sequence":"first","affiliation":[{"name":"Shanghai Key Laboratory of Plant Functional Genomics and Resources, Shanghai Chenshan Botanical Garden, and Chenshan Science Research Center, CAS Center for Excellence in Molecular Plant Sciences (CEMPS) Chinese Academy of Sciences (CAS) Shanghai China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rui","family":"Liu","sequence":"additional","affiliation":[{"name":"Shanghai Key Laboratory of Plant Functional Genomics and Resources, Shanghai Chenshan Botanical Garden, and Chenshan Science Research Center, CAS Center for Excellence in Molecular Plant Sciences (CEMPS) Chinese Academy of Sciences (CAS) Shanghai China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wei\u2010Chang","family":"Huang","sequence":"additional","affiliation":[{"name":"Shanghai Key Laboratory of Plant Functional Genomics and Resources, Shanghai Chenshan Botanical Garden, and Chenshan Science Research Center, CAS Center for Excellence in Molecular Plant Sciences (CEMPS) Chinese Academy of Sciences (CAS) Shanghai China"},{"name":"Eastern China Conservation Centre for Wild Endangered Plant Resources Shanghai Chenshan Botanical Garden Shanghai China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6877-6564","authenticated-orcid":false,"given":"Zuren","family":"Li","sequence":"additional","affiliation":[{"name":"Yuelushan Laboratory Changsha China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4007-2589","authenticated-orcid":false,"given":"Kai","family":"Hua","sequence":"additional","affiliation":[{"name":"Shanghai Key Laboratory of Plant Functional Genomics and Resources, Shanghai Chenshan Botanical Garden, and Chenshan Science Research Center, CAS Center for Excellence in Molecular Plant Sciences (CEMPS) Chinese Academy of Sciences (CAS) Shanghai China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,9,30]]},"reference":[{"key":"e_1_2_10_2_1","doi-asserted-by":"crossref","first-page":"1916","DOI":"10.1111\/pbi.13872","article-title":"CRISPR RNA\u2010Guided Integrase Enables High\u2010Efficiency Targeted Genome Engineering in \n Agrobacterium tumefaciens","volume":"20","author":"Aliu E.","year":"2022","journal-title":"Plant Biotechnology Journal"},{"key":"e_1_2_10_3_1","doi-asserted-by":"crossref","DOI":"10.1128\/aem.02499-21","article-title":"Development of a New Recombineering System for Agrobacterium Species","volume":"88","author":"Bian Z.","year":"2022","journal-title":"Applied and Environmental Microbiology"},{"key":"e_1_2_10_4_1","doi-asserted-by":"crossref","first-page":"1209","DOI":"10.1111\/tpj.16234","article-title":"A Split GAL4 RUBY Assay for Visual In Planta Detection of Protein\u2013Protein Interactions","volume":"114","author":"Chen J.","year":"2023","journal-title":"Plant Journal"},{"key":"e_1_2_10_5_1","doi-asserted-by":"crossref","DOI":"10.1038\/s41467-023-36283-9","article-title":"Systematically Attenuating DNA Targeting Enables CRISPR\u2010Driven Editing in Bacteria","volume":"14","author":"Collias D.","year":"2023","journal-title":"Nature Communications"},{"key":"e_1_2_10_6_1","doi-asserted-by":"crossref","DOI":"10.1002\/cpz1.605","article-title":"Recombineering in Non\u2010Model Bacteria","volume":"2","author":"Corts A.","year":"2022","journal-title":"Current Protocols"},{"key":"e_1_2_10_7_1","doi-asserted-by":"crossref","first-page":"6640","DOI":"10.1073\/pnas.120163297","article-title":"One\u2010Step Inactivation of Chromosomal Genes in. \n Escherichia coli\n K\u201012 Using PCR Products","volume":"97","author":"Datsenko K. A.","year":"2000","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"e_1_2_10_8_1","doi-asserted-by":"crossref","DOI":"10.1016\/j.biotechadv.2020.107677","article-title":"Agrobacterium Strains and Strain Improvement: Present and Outlook","volume":"53","author":"De Saeger J.","year":"2021","journal-title":"Biotechnology Advances"},{"key":"e_1_2_10_9_1","doi-asserted-by":"crossref","DOI":"10.1128\/aem.02250-23","article-title":"Genomic Editing in \n Burkholderia multivorans\n by CRISPR\/Cas9","volume":"90","author":"Ferreira M. R.","year":"2024","journal-title":"Applied and Environmental Microbiology"},{"key":"e_1_2_10_10_1","doi-asserted-by":"crossref","DOI":"10.1128\/aem.00333-22","article-title":"Induction of AmpC\u2010Mediated b\u2010Lactam Resistance Requires a Single Lytic Transglycosylase in \n Agrobacterium tumefaciens","volume":"88","author":"Figueroa\u2010Cuilan W. M.","year":"2022","journal-title":"Applied and Environmental Microbiology"},{"key":"e_1_2_10_11_1","doi-asserted-by":"crossref","DOI":"10.3389\/fpls.2012.00052","article-title":"Traversing the Cell: Agrobacterium T\u2010DNA's Journey to the Host Genome","volume":"3","author":"Gelvin S. B.","year":"2012","journal-title":"Frontiers in Plant Science"},{"key":"e_1_2_10_12_1","doi-asserted-by":"crossref","first-page":"195","DOI":"10.1146\/annurev-genet-120215-035320","article-title":"Integration of Agrobacterium T\u2010DNA Into the Plant Genome","volume":"51","author":"Gelvin S. B.","year":"2017","journal-title":"Annual Review of Genetics"},{"key":"e_1_2_10_13_1","doi-asserted-by":"crossref","DOI":"10.1111\/tpj.70015","article-title":"Engineering Agrobacterium for Improved. Plant Transformation","volume":"121","author":"Goralogia G. S.","year":"2025","journal-title":"Plant journal: for cell and molecular biology"},{"key":"e_1_2_10_14_1","doi-asserted-by":"crossref","DOI":"10.1128\/jb.00165-23","article-title":"Three Genes Controlling Streptomycin Susceptibility in Agrobacterium fabrum","volume":"205","author":"Howarth R. E.","year":"2023","journal-title":"Journal of Bacteriology"},{"key":"e_1_2_10_15_1","doi-asserted-by":"crossref","first-page":"2165","DOI":"10.1007\/s00253-013-5412-x","article-title":"Genome Engineering. Of \n Agrobacterium tumefaciens\n Using the Lambda Red Recombination System","volume":"98","author":"Hu S.","year":"2014","journal-title":"Applied Microbiology and Biotechnology"},{"key":"e_1_2_10_16_1","doi-asserted-by":"crossref","first-page":"3901","DOI":"10.1002\/j.1460-2075.1987.tb02730.x","article-title":"GUS Fusions: Beta\u2010Glucuronidase as a Sensitive and Versatile Gene Fusion Marker in Higher Plants","volume":"6","author":"Jefferson R. A.","year":"1987","journal-title":"EMBO Journal"},{"key":"e_1_2_10_17_1","doi-asserted-by":"crossref","first-page":"233","DOI":"10.1038\/nbt.2508","article-title":"RNA\u2010Guided Editing of Bacterial Genomes Using CRISPR\u2010Cas Systems","volume":"31","author":"Jiang W.","year":"2013","journal-title":"Nature Biotechnology"},{"key":"e_1_2_10_18_1","doi-asserted-by":"crossref","first-page":"2506","DOI":"10.1128\/AEM.04023-14","article-title":"Multigene Editing in the \n Escherichia coli\n Genome via the CRISPR\u2010Cas9 System","volume":"81","author":"Jiang Y.","year":"2015","journal-title":"Applied and Environmental Microbiology"},{"key":"e_1_2_10_19_1","doi-asserted-by":"crossref","first-page":"129","DOI":"10.1128\/jb.141.1.129-136.1980","article-title":"Transposition of Tn904 Encoding Streptomycin Resistance Into the Octopine Ti Plasmid of \n Agrobacterium tumefaciens","volume":"141","author":"Klapwijk P. M.","year":"1980","journal-title":"Journal of Bacteriology"},{"key":"e_1_2_10_20_1","doi-asserted-by":"crossref","first-page":"231","DOI":"10.1146\/annurev-phyto-082718-100101","article-title":"Pathways of DNA Transfer to Plants From Agrobacterium tumefaciens and Related Bacterial Species","volume":"57","author":"Lacroix B.","year":"2019","journal-title":"Annual Review of Phytopathology"},{"key":"e_1_2_10_21_1","doi-asserted-by":"crossref","first-page":"963","DOI":"10.1038\/nbt1091-963","article-title":"A DNA Transformation\u2013Competent Arabidopsis Genomic Library in Agrobacterium","volume":"9","author":"Lazo G. R.","year":"1991","journal-title":"Bio\/Technology"},{"key":"e_1_2_10_22_1","doi-asserted-by":"crossref","first-page":"620","DOI":"10.1093\/abbs\/gmab036","article-title":"A Modified pCas\/pTargetF System for CRISPR\u2010Cas9\u2010Assisted Genome Editing in \n Escherichia coli","volume":"53","author":"Li Q.","year":"2021","journal-title":"Acta Biochimica et Biophysica Sinica"},{"key":"e_1_2_10_23_1","doi-asserted-by":"crossref","first-page":"672","DOI":"10.1021\/acssynbio.2c00619","article-title":"Improving the Editing. Efficiency of CRISPR\u2010Cas9 by Reducing the Generation of Escapers Based on the Surviving Mechanism","volume":"12","author":"Li Q.","year":"2023","journal-title":"ACS Synthetic Biology"},{"key":"e_1_2_10_24_1","doi-asserted-by":"crossref","DOI":"10.1016\/j.engmic.2023.100097","article-title":"The Emerging Role of Recombineering in. Microbiology","volume":"3","author":"Li R.","year":"2023","journal-title":"Engineering Microbiology"},{"key":"e_1_2_10_25_1","doi-asserted-by":"crossref","first-page":"2125","DOI":"10.1111\/nph.70795","article-title":"Floral Stage Optimization and Immune Evasion Enhance Agrobacterium\u2010Mediated Genome Editing in Arabidopsis","volume":"249","author":"Liu M. S.","year":"2026","journal-title":"New Phytologist"},{"key":"e_1_2_10_26_1","doi-asserted-by":"crossref","first-page":"618","DOI":"10.1128\/JB.181.2.618-626.1999","article-title":"Cloning and Characterization of a Tetracycline. Resistance Determinant Present in \n Agrobacterium tumefaciens\n C58","volume":"181","author":"Luo Z.\u2010Q.","year":"1999","journal-title":"Journal of Bacteriology"},{"key":"e_1_2_10_27_1","doi-asserted-by":"crossref","first-page":"668","DOI":"10.1094\/MPMI.1998.11.7.668","article-title":"Role of the \n Agrobacterium tumefaciens\n VirD2 Protein in T\u2010DNA Transfer and Integration","volume":"11","author":"Mysore K. S.","year":"1998","journal-title":"Molecular Plant\u2010Microbe Interactions"},{"key":"e_1_2_10_28_1","doi-asserted-by":"crossref","first-page":"5103","DOI":"10.1128\/AEM.01248-15","article-title":"Coupling the CRISPR\/Cas9 System With Lambda Red Recombineering Enables Simplified Chromosomal Gene Replacement in \n Escherichia coli","volume":"81","author":"Pyne M. E.","year":"2015","journal-title":"Applied and Environmental Microbiology"},{"key":"e_1_2_10_29_1","doi-asserted-by":"crossref","first-page":"2581","DOI":"10.1038\/s41467-022-30180-3","article-title":"Agrobacterium Expressing a Type III Secretion System Delivers Pseudomonas Effectors Into Plant Cells to Enhance Transformation","volume":"13","author":"Raman V.","year":"2022","journal-title":"Nature Communications"},{"key":"e_1_2_10_30_1","doi-asserted-by":"crossref","DOI":"10.1073\/pnas.2013338118","article-title":"Efficient CRISPR\u2010Mediated Base Editing in Agrobacterium spp","volume":"118","author":"Rodrigues S. D.","year":"2021","journal-title":"Proceedings of the National Academy of Sciences of the United States of America"},{"key":"e_1_2_10_31_1","doi-asserted-by":"crossref","first-page":"685","DOI":"10.1111\/tpj.14060","article-title":"Application of phiLOV2.1 as a Fluorescent Marker for Visualization of Agrobacterium Effector Protein Translocation","volume":"96","author":"Roushan M. R.","year":"2018","journal-title":"Plant Journal"},{"key":"e_1_2_10_32_1","doi-asserted-by":"crossref","first-page":"1708","DOI":"10.1038\/s41587-024-02462-2","article-title":"Binary Vector Copy Number Engineering Improves Agrobacterium\u2010Mediated Transformation","volume":"43","author":"Szarzanowicz M. J.","year":"2025","journal-title":"Nature Biotechnology"},{"key":"e_1_2_10_33_1","doi-asserted-by":"crossref","first-page":"113","DOI":"10.1016\/0378-1119(91)90112-O","article-title":"Nucleotide Sequence Analysis of a Chloramphenicol\u2010Resistance Determinant From Agrobacterium tumefaciens and Identification of Its Gene Product","volume":"98","author":"Tennigkeit J.","year":"1991","journal-title":"Gene"},{"key":"e_1_2_10_34_1","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pone.0200972","article-title":"RepB C\u2010Terminus Mutation of a pRi\u2010repABC Binary Vector Affects Plasmid Copy Number in Agrobacterium and Transgene Copy Number in Plants","volume":"13","author":"Vaghchhipawala Z.","year":"2018","journal-title":"PLoS One"},{"key":"e_1_2_10_35_1","doi-asserted-by":"crossref","first-page":"2034","DOI":"10.1111\/pbi.70028","article-title":"Plant Genetic Transformation: Achievements, Current Status and Future Prospects","volume":"23","author":"Wang P.","year":"2025","journal-title":"Plant Biotechnology Journal"},{"key":"e_1_2_10_36_1","doi-asserted-by":"crossref","DOI":"10.1186\/1746-4811-10-19","article-title":"AGROBEST: An Efficient Agrobacterium\u2010Mediated Transient Expression Method for Versatile Gene Function Analyses in Arabidopsis Seedlings","volume":"10","author":"Wu H.\u2010Y.","year":"2014","journal-title":"Plant Methods"},{"key":"e_1_2_10_37_1","doi-asserted-by":"crossref","first-page":"2493","DOI":"10.1111\/nph.18694","article-title":"Engineered Agrobacterium Improves. Transformation by Mitigating Plant Immunity Detection","volume":"237","author":"Yang F.","year":"2023","journal-title":"New Phytologist"},{"key":"e_1_2_10_38_1","doi-asserted-by":"crossref","first-page":"269","DOI":"10.1016\/j.synbio.2020.08.001","article-title":"Expanding Application of CRISPR\u2010Cas9 System in. Microorganisms","volume":"5","author":"Zhao J.","year":"2020","journal-title":"Synthetic and Systems Biotechnology"},{"key":"e_1_2_10_39_1","doi-asserted-by":"crossref","first-page":"749","DOI":"10.1016\/j.cell.2006.03.037","article-title":"Perception of the Bacterial PAMP EF\u2010Tu by the Receptor EFR Restricts Agrobacterium\u2010Mediated Transformation","volume":"125","author":"Zipfel C.","year":"2006","journal-title":"Cell"}],"container-title":["Plant Biotechnology Journal"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1111\/pbi.70768","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/full-xml\/10.1111\/pbi.70768","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1111\/pbi.70768","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,1]],"date-time":"2026-10-01T06:23:57Z","timestamp":1790835837000},"score":1,"resource":{"primary":{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/10.1111\/pbi.70768"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,30]]},"references-count":38,"alternative-id":["10.1111\/pbi.70768"],"URL":"https:\/\/doi.org\/10.1111\/pbi.70768","archive":["Portico"],"relation":{},"ISSN":["1467-7644","1467-7652"],"issn-type":[{"value":"1467-7644","type":"print"},{"value":"1467-7652","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,30]]},"assertion":[{"value":"2026-06-25","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-09-22","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-09-30","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"pbi.70768"}}