Increasing plant susceptibility to Agrobacterium infection by overexpression of the Arabidopsis nuclear protein VIP1

Abstract

Agrobacterium is a unique model system as well as a major biotechnological tool for genetic manipulation of plant cells. It is still unknown, however, whether host cellular factors exist that are limiting for infection, and whether their overexpression in plant cells can increase the efficiency of the infection. Here, we examined the effect of overexpression in tobacco plants of an Arabidopsis gene, VIP1, which encodes a recently discovered cellular protein required for Agrobacterium infection. Our results indicate that VIP1 is imported into the plant cell nucleus via the karyopherin alphadependent pathway and that elevated intracellular levels of VIP1 render the host plants significantly more susceptible to transient and stable genetic transformation by Agrobacterium, probably because of the increased nuclear import of the transferred-DNA.

Fig 1.

Karyopherin α-dependent nuclear import of VIP1. Specific interaction between VIP1 and AtKAPα in the two-hybrid assay. (A) Cell growth on a histidine-deficient medium. (B) Cell growth in the presence of histidine. Lane 1, VIP1 + AtKAPα; lane 2, VIP1 + VirE2; lane 3, VIP1 + lamin C; lane 4, AtKAPα + VirE2. VIP1 nuclear import in srp1–31 yeast cells. (C–F) GFP-VIP1 expressed in wild-type cells grown at 25°C. (G–J) GFP-VIP1 expressed in srp1–31 cells grown at 25°C. (K–N) GFP-VIP1 expressed in wild-type cells grown at 37°C. (O–R) GFP-VIP1 expressed in srp1–31 cells grown at 37°C. (C, G, K, and O) Phase-contrast images; (D, H, L, and P) images of GFP fluorescence; (E, I, M, and Q) images of DAPI fluorescence, presented in red to facilitate image merging); (F, J, N, and R) merged GFP–DAPI images. (Bar = 5 μm.)

Fig 2.

Northern blot analysis of VIP1 expression and in wild-type and VIP1 plants and increased Agrobacterium-mediated transient and stable genetic transformation of VIP1 plants. VIP1 S1 and VIP1 S2 represent two independent VIP1-transgenic plant lines. (A) VIP1 expression as detected by Northern blot hybridization (Upper) and amounts of rRNA in each lane as detected by the ethidium bromide staining (Lower). (B) Quantification of transient T-DNA expression. Black, gray, and white bars indicate the numbers of disks that developed 1–50, 51–99, and 100–150 GUS-stained areas per disk, respectively. Total number of GUS-stained disks for each experimental condition was defined as 100%. (C) Quantification of stable T-DNA expression. Black, gray, and white bars indicate the numbers of disks that developed 1–4, 5–9, and 10–20 shoots per disk, respectively. Total number of disks with regenerated shoots for each experimental condition was defined as 100%. All data represent three independent experiments with at least 20 disks for each experimental condition; all of these transformed disks were used for data collection.

Fig 3.

Increased Agrobacterium-mediated stable genetic transformation of VIP1 plants. Regeneration of stably transformed shoots: (A–C) Agrobacterium-infected disks from the VIP1 S1 line, VIP1 S2 line, or wild-type plants, respectively, grown on hygromycin-containing selective medium. Left to right: disks inoculated with Agrobacterium cultures at OD₆₀₀ = 0.1, 0.5, and 1.0, respectively. (D–F) Uninfected disks from the wild-type plants, VIP1 S1 line, or VIP1 S2 line, respectively, grown on hygromycin-containing selective medium. (G–I) Uninfected disks from the wild-type plants, VIP1 S1 line, or VIP1 S2 line, respectively, grown in the absence of selection. GUS-staining of transgenic shoots regenerated from Agrobacterium-infected VIP1 plants: (J) Shoots from Agrobacterium-infected VIP1 S1 and VIP1 S2 lines regenerated on hygromycin-containing medium. (K) Shoots from uninfected VIP1 S1 and VIP1 S2 lines regenerated without selection.

Fig 4.

RT-PCR analysis of early T-DNA expression in VIP1 plants. (A) Wild-type plants. (B) VIP1 S1 line. (C) VIP1 S2 line. (Upper) T-DNA-specific products. (Lower) Actin-specific products. Lanes 1–5, RT-PCR analysis of leaf samples collected 0, 4, 8, 12, and 24 hr after inoculation, respectively; lane 6, PCR analysis of Agrobacterium alone control.