Abstract
Agrobacterium tumefaciens is the causal agent of crown gall disease in nature; in the laboratory the bacterium is widely used for plant genetic modification. The bacterium delivers a single-stranded transferred DNA (T-DNA) and a group of crucial virulence proteins into host cells. A putative T-complex is formed inside host cells that is composed of T-DNA and virulence proteins VirD2 and VirE2, which protect the foreign DNA from degradation and guide its way into the host nucleus. However, little is known about how the T-complex is assembled inside host cells. We combined the split-GFP and split-sfCherry labeling systems to study the interaction of Agrobacterium-delivered VirE2 and VirE3 in host cells. Our results indicated that VirE2 co-localized with VirE3 on the cytoplasmic side of the host cellular membrane upon the delivery. We identified and characterized two tandem domains at the VirE3 C-terminus that interacted with VirE2 in vitro. Deletion of these two domains abolished the VirE2 accumulation on the host plasma membrane and affected the transformation. Furthermore, the two VirE2-interacting domains of VirE3 exhibited different affinities with VirE2. Collectively, this study demonstrates that the anchorage protein VirE3 uses the two tandem VirE2-interacting domains to facilitate VirE2 protection for T-DNA at the cytoplasmic side of the host cell entrance.
Highlights
Agrobacterium tumefaciens is a soil-borne phytopathogen that causes crown gall disease in a variety of plant species in nature (Nester, 2014)
The 16 amino-acid GFP11 was used to label the effector proteins inside bacterial cells and the GFP1–10 was expressed in host cells; spontaneous complementation of the two fragments occurred upon effector translocation and resulted in fluorescent labeling of the effectors in the host cell
As shown in (Figure 1), Agrobacterium-delivered VirE2 (Figure 1A) and VirE3 (Figure 1B) could co-localize with the plant plasma membrane, which was indicated by transient expression of a plant plasma membrane tracker (Nelson et al, 2007)
Summary
Agrobacterium tumefaciens is a soil-borne phytopathogen that causes crown gall disease in a variety of plant species in nature (Nester, 2014). This bacterium is well-known for its unique ability for inter–kingdom DNA transfer (Chilton et al, 1977; Zambryski et al, 1980; Albright et al, 1987) and has been widely used in plant biotechnology for decades (Tzfira and Citovsky, 2006; Hwang et al, 2017). VirB1-11 are the main structure components of this membrane-associated export apparatus while VirD4 is a type IV coupling protein located at the entrance of the secretion channel, which mainly acts to deliver substrate proteins into the channel (Cabezon et al, 1997)
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