title: Direct visualization of Agrobacterium-delivered VirE2 in recipient cells.
abstract: Agrobacterium tumefaciens is a natural genetic engineer widely used to deliver DNA into various recipients, including plant, yeast and fungal cells. The bacterium can transfer single-stranded DNA molecules (T-DNAs) and bacterial virulence proteins, including VirE2. However, neither the DNA nor the protein molecules have ever been directly visualized after the delivery. In this report, we adopted a split-GFP approach: the small GFP fragment (GFP11) was inserted into VirE2 at a permissive site to create the VirE2-GFP11 fusion, which was expressed in A. tumefaciens; and the large fragment (GFP1-10) was expressed in recipient cells. Upon delivery of VirE2-GFP11 into the recipient cells, GFP fluorescence signals were visualized. VirE2-GFP11 was functional like VirE2; the GFP fusion movement could indicate the trafficking of Agrobacterium-delivered VirE2. As the natural host, all plant cells seen under a microscope received the VirE2 protein in a leaf-infiltration assay; most of VirE2 moved at a speed of 1.3-3.1 μm sec⁻¹ in a nearly linear direction, suggesting an active trafficking process. Inside plant cells, VirE2-GFP formed filamentous structures of different lengths, even in the absence of T-DNA. As a non-natural host recipient, 51% of yeast cells received VirE2, which did not move inside yeast. All plant cells seen under a microscope transiently expressed the Agrobacterium-delivered transgene, but only 0.2% yeast cells expressed the transgene. This indicates that Agrobacterium is a more efficient vector for protein delivery than T-DNA transformation for a non-natural host recipient: VirE2 trafficking is a limiting factor for the genetic transformation of a non-natural host recipient. The split-GFP approach could enable the real-time visualization of VirE2 trafficking inside recipient cells.
authors.affiliation: Department of Biological Sciences, National University of Singapore, Singapore, 117543, Singapore.
authors.forename: Xiaoyang
authors.initials: X
authors.lastname: Li
authors.forename: Qinghua
authors.initials: Q
authors.lastname: Yang
authors.forename: Haitao
authors.initials: H
authors.lastname: Tu
authors.forename: Zijie
authors.initials: Z
authors.lastname: Lim
authors.forename: Shen Q
authors.initials: SQ
authors.lastname: Pan
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics*; D017360:Arabidopsis / Q000166:cytology* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics; D004277:DNA, Single-Stranded / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D007473:Ion Channels / Q000235:genetics / Q000378:metabolism*; D018515:Plant Leaves / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D021381:Protein Transport; D011993:Recombinant Fusion Proteins; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000166:cytology* / Q000235:genetics / Q000378:metabolism; D019076:Transgenes; D037521:Virulence Factors
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D007473:Ion Channels; D011993:Recombinant Fusion Proteins; C036483:T-DNA; D037521:Virulence Factors; C051583:virE2 protein, Agrobacterium; D049452:Green Fluorescent Proteins
keywords: Agrobacterium; Nicotiana benthamiana; Saccharomyces cerevisiae; T-DNA; VirE2; protein delivery; technical advance; visualization