Agrobacterium VirE3 Uses Its Two Tandem Domains at the C-Terminus to Retain Its Companion VirE2 on the Cytoplasmic Side of the Host Plasma Membrane

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.

Agrobacterium-delivered VirE2 interacts with host nucleoporin CG1 to facilitate the nuclear import of VirE2-coated T complex

“…Moreover, two additional proteins, VIP1 and VirE3, were reported to facilitate nuclear uptake of VirE2 in host cells (36,37). However, more recent studies indicated that VIP1 is not important for the AMT process (38,39), whereas VirE3 may be involved in T-complex assembly and does not localize to the plant nucleus (22,40). Thus, it is not clear whether and how VirE2 is imported into the host nucleus.…”

Data_Sheet_1.docx

“…A recent report showed that VirE3 is required for full virulence of the agropine/succinamopine strain EHA105, which does not have a close homolog of octopine Ti virF. Deletion of virE3 resulted in a decreased transformation efficiency in the transient transformation assays of Nicotiana benthamiana leaves (Li et al, , 2020. In contrast, another report showed constitutive overexpression of virE2 or virE3 under CaMV 35S promoter in Arabidopsisinduced plant defense responses and conferred resistance to Agrobacterium infection (Duan et al, 2018).…”