title: An Improved Single-Step Cloning Strategy Simplifies the Agrobacterium tumefaciens-Mediated Transformation (ATMT)-Based Gene-Disruption Method for Verticillium dahliae.
abstract: The soilborne fungal pathogen Verticillium dahliae infects a broad range of plant species to cause severe diseases. The availability of Verticillium genome sequences has provided opportunities for large-scale investigations of individual gene function in Verticillium strains using Agrobacterium tumefaciens-mediated transformation (ATMT)-based gene-disruption strategies. Traditional ATMT vectors require multiple cloning steps and elaborate characterization procedures to achieve successful gene replacement; thus, these vectors are not suitable for high-throughput ATMT-based gene deletion. Several advancements have been made that either involve simplification of the steps required for gene-deletion vector construction or increase the efficiency of the technique for rapid recombinant characterization. However, an ATMT binary vector that is both simple and efficient is still lacking. Here, we generated a USER-ATMT dual-selection (DS) binary vector, which combines both the advantages of the USER single-step cloning technique and the efficiency of the herpes simplex virus thymidine kinase negative-selection marker. Highly efficient deletion of three different genes in V. dahliae using the USER-ATMT-DS vector enabled verification that this newly-generated vector not only facilitates the cloning process but also simplifies the subsequent identification of fungal homologous recombinants. The results suggest that the USER-ATMT-DS vector is applicable for efficient gene deletion and suitable for large-scale gene deletion in V. dahliae.
authors.affiliation: All authors: State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101; and first and second authors: University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Sheng
authors.initials: S
authors.lastname: Wang
authors.affiliation: All authors: State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101; and first and second authors: University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Haiying
authors.initials: H
authors.lastname: Xing
authors.affiliation: All authors: State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101; and first and second authors: University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Chenlei
authors.initials: C
authors.lastname: Hua
authors.affiliation: All authors: State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101; and first and second authors: University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Hui-Shan
authors.initials: HS
authors.lastname: Guo
authors.affiliation: All authors: State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101; and first and second authors: University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Zhang
mesh_terms: D016960:Agrobacterium tumefaciens / Q000502:physiology*; D003001:Cloning, Molecular; D017353:Gene Deletion; D014170:Transformation, Genetic; D020172:Verticillium / Q000235:genetics*
keywords: HSVtk