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Direct visualization of A grobacterium ‐delivered V ir E 2 in recipient cells
Author(s) -
Li Xiaoyang,
Yang Qinghua,
Tu Haitao,
Lim Zijie,
Pan Shen Q.
Publication year - 2014
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/tpj.12397
Subject(s) - green fluorescent protein , agrobacterium tumefaciens , agrobacterium , yeast , fusion protein , biology , microbiology and biotechnology , plant cell , dna , saccharomyces cerevisiae , transgene , gene , genetics , recombinant dna
Summary A grobacterium 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 – DNA s) and bacterial virulence proteins, including V ir E 2. 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 ( GFP 11) was inserted into V ir E 2 at a permissive site to create the V ir E 2‐ GFP 11 fusion, which was expressed in A . tumefaciens ; and the large fragment (GFP1–10) was expressed in recipient cells. Upon delivery of V ir E 2‐ GFP 11 into the recipient cells, GFP fluorescence signals were visualized. V ir E 2‐ GFP 11 was functional like V ir E 2; the GFP fusion movement could indicate the trafficking of A grobacterium ‐delivered V ir E 2. As the natural host, all plant cells seen under a microscope received the V ir E 2 protein in a leaf‐infiltration assay; most of V ir E 2 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, V ir E 2‐ 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 V ir E 2, which did not move inside yeast. All plant cells seen under a microscope transiently expressed the A grobacterium ‐delivered transgene, but only 0.2% yeast cells expressed the transgene. This indicates that A grobacterium is a more efficient vector for protein delivery than T ‐ DNA transformation for a non‐natural host recipient: V ir E 2 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 V ir E 2 trafficking inside recipient cells.