Direct visualization of Agrobacterium‐delivered VirE2 in recipient cells. (16th January 2014)

• Record Type: Journal Article
• Authors: Li, Xiaoyang; Yang, Qinghua; Tu, Haitao; Lim, Zijie; Pan, Shen Q.
• Is Part Of: Plant journal. Volume 77:Number 3(2014:Feb.)
• Page Start: 487
• Page End: 495
• Publication Date: 2014-01-16
• Subjects: Plant molecular biology -- Periodicals; Plant cells and tissues -- Periodicals; Botany -- Periodicals; 580
• DOI: 10.1111/tpj.12397
• Languages: English
• ISSNs: 0960-7412

Abstract

<abstract abstract-type="main" id="tpj12397-abs-0001"> <title>Summary</title> <p> <italic>Agrobacterium tumefaciens</italic> 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 <italic>A. tumefaciens</italic>; 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 <italic>Agrobacterium</italic>‐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<sup>−1</sup> 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 <italic>Agrobacterium</italic>‐delivered transgene, but only 0.2% yeast cells expressed the transgene. This indicates that <italic>Agrobacterium</italic> 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.</p> </abstract>