Agrobacterium Delivers Anchorage Protein VirE3 for Companion VirE2 to Aggregate at Host Entry Sites for T-DNA Protection  Xiaoyang Li, Haitao Tu, Shen Q. Pan  Cell Reports  Volume 25, Issue 2, Pages 302-311.e6 (October 2018) DOI: 10.1016/j.celrep.2018.09.023 Copyright © 2018 The Authors Terms and Conditions

Cell Reports 2018 25, 302-311.e6DOI: (10.1016/j.celrep.2018.09.023) Copyright © 2018 The Authors Terms and Conditions

Figure 1 VirE3 Targets Host Plasma Membranes via an α-Helical-Bundle Domain and Affects VirE2 Accumulation on Host Plasma Membranes (A) Deletion of virE3 abolished VirE2 accumulation on host cellular membranes. Wild-type N. benthamiana leaves were infiltrated with A. tumefaciens EHA105virE2::GFP11 (control) or EHA105virE2::GFP11ΔvirE3 containing both pVBA-RFP ([red fluorescent protein] expressing free DsRed under the virB promoter) and a binary plasmid pGFP1–10 (expressing GFP1–10 under the CaMV 35S promoter on T-DNA). A representative of 30 fields is shown. Arrowheads indicate VirE2 aggregated at bacterial-host contact sites. Scale bars, 20 μm. (B) Deletion of virE3 in A. tumefaciens attenuated the transient transformation efficiency. Wild-type N. benthamiana leaves were infiltrated with A. tumefaciens EHA105 or EHA105ΔvirE3 containing a binary plasmid pQH121-mC (expressing free mCherry under the CaMV 35S promoter on T-DNA). Scale bars, 100 μm. (C) The intensity of transiently expressed mCherry was measured in each image. Data are presented as means ± SDs of n = 30 independent samples. ∗p < 0.01. (D) Detection of Agrobacterium-delivered VirE3 in host cells using a split-GFP approach. Transgenic N. benthamiana (Nb308A) (expressing both GFP1–10 and DsRed) leaves were infiltrated with A. tumefaciens EHA105virE3::GFP11. DsRed indicates cellular structures. The boxed area is enlarged to highlight VirE3-GFPcomp complex. Scale bars, 20 μm. (E) Agrobacterium-delivered VirE3 accumulated at the bacterial-host contact sites. Wild-type N. benthamiana leaves were infiltrated with EHA105virE3::GFP11 containing pVBA-RFP and the binary plasmid pGFP1–10. Scale bars, 20 μm. (F) Accumulation of Agrobacterium-delivered VirE3 on host plasma membranes. Wild-type N. benthamiana leaves were infiltrated with evenly mixed A. tumefaciens, EHA105virE3::GFP11 containing a binary plasmid pGFP1–10, and EHA105virE3::GFP11 containing a binary plasmid pm-rb (expressing a plasma membrane [PM] tracker under the CaMV 35S promoter on T-DNA). Scale bars, 20 μm. (G) VirE3 membrane localization domain (MLD) was important for VirE3 localization on host plasma membranes. Wild-type N. benthamiana leaves were infiltrated with A. tumefaciens EHA105ΔvirE3 containing binary plasmids to express mCherry-labeled full-length or truncated VirE3 constructs. Free mCherry was used as the control. Other types of localizations except plasma membrane are arrowed for VirE3(225-336)-mCherry and VirE3(246-360)-mCherry. Scale bars, 20 μm. (H) mCherry-labeled VirE3 localized to plasma membranes in transgenic Arabidopsis plants. Free mCherry was used as the control. Single optical sections of bright field indicate the cell shape. Scale bars, 20 μm. (I) VirE3 MLD with predicted secondary structures. α-helices shown in red and a β-strand in blue. (J) VirE3 MLD was important for VirE3 function. Wild-type N. benthamiana leaves were infiltrated with A. tumefaciens EHA105, EHA105ΔvirE3, or EHA105ΔVirE3(239–306) containing the binary plasmid pQH121-mC. Scale bars, 100 μm. (K) The intensity of transiently expressed mCherry was measured in each image. Data are presented as means ± SDs of n = 30 independent samples. p < 0.01. See also Figures S1 and Video S1. Cell Reports 2018 25, 302-311.e6DOI: (10.1016/j.celrep.2018.09.023) Copyright © 2018 The Authors Terms and Conditions

Figure 2 VirE3 Interacts with Itself and VirE2 to Facilitate VirE2 Accumulation on Host Plasma Membranes (A) VirE3 interacted with VirE2 through its C-terminal domain, based on the yeast two-hybrid assay. (B) Partial deletion of the VirE2-interacting domain on VirE3 decreased the VirE2 accumulation at host cell borders. N. benthamiana (Nb308A) leaves were infiltrated with A. tumefaciens EHA105virE2::GFP11 (control) or EHA105 virE2::GFP11ΔVirE3(598–644). White lines are added to indicate borders between leaf epidermal cells. Arrowheads point to VirE2 aggregates at cell borders. Scale bars, 20 μm. (C) The intensity of VirE2-GFPcomp signals associated with host cell borders was measured in each image. Data are presented as means ± SDs of n = 20 independent samples. ∗p < 0.01. (D) Partial deletion of the VirE2-interacting domain on VirE3 attenuated the transient transformation efficiency. Wild-type N. benthamiana leaves were infiltrated with A. tumefaciens EHA105 (control) or EHA105ΔVirE3(598–644) containing the binary plasmid pQH121-mC. Scale bars, 100 μm. (E) The intensity of transiently expressed mCherry was measured in each image. Data are presented as means ± SDs of n = 30 independent samples. ∗p < 0.01. (F) VirE3 interacted with itself based on the yeast two-hybrid assay. (G) Deletion of VirE3 N terminus (amino acids 2–224) altered the VirE3 distribution on host plasma membranes from punctate structures to a continuous pattern. N. benthamiana (Nb308A) leaves were infiltrated with A. tumefaciens EHA105virE3::GFP11 (control) or EHA105virE3::GFP11ΔVirE3(2–224). The boxed area is enlarged to highlight VirE3-GFPcomp complex. Scale bars, 20 μm. (H) Deletion of VirE3 N terminus attenuated the transient transformation efficiency. Wild-type N. benthamiana leaves were infiltrated with A. tumefaciens EHA105(control) or EHA105ΔVirE3(2–224) containing the binary plasmid pQH121-mC. Scale bars, 100 μm. (I) The intensity of transiently expressed mCherry was measured in each image. Data are presented as means ± SDs of n = 30 independent samples. ∗p < 0.01. See also Figure S2. Cell Reports 2018 25, 302-311.e6DOI: (10.1016/j.celrep.2018.09.023) Copyright © 2018 The Authors Terms and Conditions

Figure 3 VirE3 Functions at Its Entry Sites to Promote Efficient Protection of T-DNA by VirE2 (A) VirE3 could not be extracellularly complemented, based on the transient transformation assay. T-DNA donor cells containing the binary plasmid pQH121-mC and VirE3 donor cells were mixed at a 1:10 ratio and infiltrated into wild-type N. benthamiana leaves. (A1) EHA105(pQH121-mC) + EHA105ΔvirE3-VirE2sec−. (A2) EHA105ΔvirE3(pQH121-mC) + EHA105ΔvirE3-VirE2sec−. (A3) EHA105ΔvirE3(pQH121-mC) + EHA105-VirE2sec−. Scale bars, 100 μm. (B) The intensity of transiently expressed mCherry was measured in each image. Data are presented as means ± SDs of n = 30 independent samples. p < 0.01. (C) Deletion of virE3 could be extracellularly complemented by an excess amount of VirE2 in the transient transformation assay. T-DNA donor cells containing the binary plasmid pQH121-mC and VirE2 donor cells were mixed at a 1:10 ratio and infiltrated into wild-type N. benthamiana leaves. (C1) EHA105(pQH121-mC) + EHA105ΔvirE2ΔvirE3ΔvirD2ΔvirD5ΔvirF. (C2) EHA105(pQH121-mC) + EHA105ΔvirE3ΔvirD2ΔvirD5ΔvirF. (C3) EHA105ΔvirE3(pQH121-mC) + EHA105ΔvirE2ΔvirE3ΔvirD2ΔvirD5ΔvirF. (C4) EHA105ΔvirE3(pQH121-mC) + EHA105ΔvirE3ΔvirD2ΔvirD5vΔirF. Scale bars, 100 μm. (D) The intensity of transiently expressed mCherry was measured in each image. Data are presented as means ± SDs of n = 30 independent samples. p < 0.01. (E) Deletion of virE3 may cause more T-DNA truncations at the left border. Wild-type N. benthamiana leaves were infiltrated with A. tumefaciens EHA105 or EHA105ΔvirE3 containing the binary plasmid pmC13 or pmC13-Reverse, respectively. Scale bars, 100 μm. (F) The intensity of transiently expressed mCherry was measured in each image. Data are presented as means ± SDs of n = 30 independent samples. p < 0.01. (G) Analysis of T-DNA/plant DNA junctions for T-DNA truncations at the left border. The sequence of the 25-bp T-DNA left border is indicated. The arrow indicates the site of VirD2 cleavage between bases 3 and 4. The numbers indicate the number of nucleotides of T-DNA deleted in each T-DNA/plant DNA junction region. Data are presented as means ± SDs of n = 25 independent samples. ∗p < 0.01. See also Figure S3. Cell Reports 2018 25, 302-311.e6DOI: (10.1016/j.celrep.2018.09.023) Copyright © 2018 The Authors Terms and Conditions

Figure 4 Schematic Representation of VirE3 Functional Domains and Their Role in Transformation (A) Schematic representation of the functional domains in VirE3 and their homologs in Agrobacterium and rhizobia species. The relevant amino acid positions are indicated. (B) A hypothetical model for VirE3 functions during AMT. Agrobacterium-delivered VirE3 interacts with itself and forms an anchorage structure at the host cellular membranes to enable VirE2 aggregation at the host entry sites for T-DNA protection. See also Figure S4 and Table S1. Cell Reports 2018 25, 302-311.e6DOI: (10.1016/j.celrep.2018.09.023) Copyright © 2018 The Authors Terms and Conditions