Arabidopsis VIRE2 INTERACTING PROTEIN2 is required for Agrobacterium T-DNA integration in plants

Abstract

Agrobacterium tumefaciens-mediated genetic transformation is an efficient tool for genetic engineering of plants. VirE2 is a single-stranded DNA binding Agrobacterium protein that is transported into the plant cell and presumably protects the T-DNA from degradation. Using a yeast two-hybrid system, we identified Arabidopsis thaliana VIRE2-INTERACTING PROTEIN2 (VIP2) with a NOT domain that is conserved in both plants and animals. Furthermore, we provide evidence supporting VIP2 interaction with VIP1, a basic domain/leucine zipper motif-containing protein required for nuclear import and integration of T-DNA. Virus-induced gene silencing of VIP2 in Nicotiana benthamiana and characterization of the Arabidopsis vip2 mutant (At vip2) demonstrate that VIP2 is required for Agrobacterium-mediated stable transformation but not for transient transformation. Assays based upon a promoter-trap vector and quantification of T-DNA integration further confirmed VIP2 involvement in T-DNA integration. Interestingly, VIP2 transcripts were induced to a greater extent over prolonged periods after infection with a T-DNA transfer-competent Agrobacterium strain compared with the transfer-deficient Agrobacterium strain. Transcriptome analyses of At vip2 suggest that VIP2 is likely a transcriptional regulator, and the recalcitrancy to transformation in At vip2 is probably due to the combination of muted gene expression response upon Agrobacterium infection and repression of histone genes resulting in decreased T-DNA integration events.

Figure 1.

At VIP2–VirE2 and At VIP2–At VIP1 interactions in the Two-Hybrid System and Amino Acid Sequences of At VIP2 and Nb VIP2. (A) At VIP2 + VirE2. (B) At VIP2 + At VIP1. (C) At VIP2 + human lamin C. (D) At VIP2 + topoisomerase I. (E) β-Galactosidase assay. From left to right: At VIP2 + VirE2, At VIP2 + At VIP1, At VIP2 + human lamin C, and At VIP2 + topoisomerase I. Cells shown in (A) to (D) were grown in the absence of His, Trp, and Leu, and cells shown in (E) were grown in the absence of Trp and Leu. (F) Multiple sequence alignment by ClustalW (1.81) of amino acid sequences of full-length proteins for At VIP2 and Nb VIP2. The identical amino acids are shown in red, conserved amino acids in blue, semiconserved amino acids in green, and the divergent amino acids in black. The shaded area represents the C-terminal NOT domain between the two proteins.

Figure 2.

Agrobacterium Transformation Assays in Nb VIP2–Silenced Plants. (A) Leaf disk tumorigenesis assay. Leaf disks of the Nb VIP2–silenced plants and TRV:00 (control) plants were inoculated with tumorigenic strain A. tumefaciens A348 and incubated on hormone-free Murashige and Skoog (MS) medium. (B) Quantification of tumors. The number of tumors produced per leaf disk was counted 3 weeks after inoculation. Data represent the mean of two experiments with a minimum of 150 leaf disks each per treatment with their se values shown as error bars. Asterisk denotes significant difference compared with controls using Fisher's least significant difference test at P = 0.05. (C) Stable transformation assay. Leaf disks from the silenced and TRV:00 plants were infected with a nontumorigenic strain A. tumefaciens GV2260 harboring the binary vector pCAS1 and incubated on CIM with GF. (D) Effect of VIP2 gene silencing on cell division. The effect of gene silencing on cell division was evaluated by placing uninoculated leaf disks from the silenced and TRV:00 plants on a nonselective CIM. All the experiments were done with at least five biological replicates and repeated two times, and the results were consistent among the replicates. Photographs shown in (A), (C), and (D) were taken 4 weeks after Agrobacterium inoculation.

Figure 3.

Transient Transformation and T-DNA Integration Assays in Nb VIP2–Silenced Plants. (A) Transient transformation assay. Leaf disks of the Nb VIP2–silenced and TRV:00 plants were inoculated with nontumorigenic strain A. tumefaciens GV2260 carrying pBISN1 (has the uidA-intron gene within the T-DNA). The inoculated leaves were periodically collected and stained with X-Gluc. (B) Quantification of GUS activity. Leaf disks from the experiment in (A) were collected periodically and were used for measuring the fluorescence of 4-methylumbelliferone (4-MU). (C) T-DNA integration assay. Leaf disks from TRV:00 and Nb VIP2–silenced plants were inoculated with Agrobacterium strain carrying a promoterless uidA-intron gene and 35S:luc-intron gene within the T-DNA. Leaf disks were periodically collected, and GUS activity was measured as described above. (D) T-DNA integration in the Nb VIP2–silenced and TRV:00-infected plants. Suspension cells were derived from the calli generated from Nb VIP2–silenced and TRV:00-infected leaf segments infected with the nontumorigenic strain A.tumefaciens GV2260 carrying pBISN1. The suspension cell lines were grown for 8 weeks in nonselective medium. Genomic DNA was isolated from these cells, subjected to electrophoresis through a 0.8% agarose gel, blotted onto a nylon membrane, and hybridized with a uidA gene probe. After autoradiography, the membrane was stripped and rehybridized with the Nb RAR1 gene probe to compare the amount of DNA in each lane. (E) Quantification of T-DNA integration. The amounts of integrated T-DNA molecules in the genomic DNA extracted from calli that were generated from leaf disks transformed with the Agrobacterium strain carrying the uidA-intron gene within the T-DNA were measured by quantitative PCR. The uidA gene transcripts in calli derived from Nb VIP2–silenced plants are represented in relative amounts in comparison to an average T-DNA amount in the calli derived from wild-type and TRV:00 plants. All the experiments were done with at least five biological replicates and repeated two times. Asterisks in (C) and (E) denote value that are significantly different between the two treatments by analysis of variance at P = 0.05. The data represent the average of five biological replicates in two experiments with se values shown as error bars.

Figure 4.

Differential Gene Expression of Nb VIP2 upon Infection with Agrobacterium. Individual leaves of two separate N. benthamiana plants were syringe (needleless) infiltrated with either an avirulent strain Agrobacterium A136 (lacks Ti plasmid; cannot transfer T-DNA) or a T-DNA transfer-competent strain A. tumefaciens GV2260 carrying pBISN1. Leaf samples from the infiltrated area were collected at different times after inoculation, and total RNA was isolated for real-time quantitative PCR. RNA from the buffer-infiltrated N. benthamiana leaves collected at 12 HAI was used as a calibrator to determine the relative amount of Nb VIP2 transcripts. Samples were pooled together from two independent experiments, and the average of two technical replicates is shown.

Figure 5.

In Planta Interaction of Nb VIP2 with VirE2. The top panels depict the YFP fluorescence, and the bottom panels represent the epifluorescence images of epidermal leaf cells from the same leaf infiltrated with Agrobacterium suspension cultures harboring the indicated proteins. Individual leaves of N. benthamiana plants were syringe (needleless) infiltrated with Agrobacterium suspension cultures singly or in the following combinations: pCAMBIA1390-35S:YFP, pSPYNE:VIP2, pSPYCE:VirE2, pSPYNE:VIP2/pSPYCE:VirE2, pSPYNE:VIP2/pSPYCE:TGA2, and pSPYNE:VIP2/pSPYCE:VirE2*. Wild-type 35S:YFP and fusion protein pSPYNE:VIP2/pSPYCE:VirE2 are both localized to the nucleus of plant cells, while pSPYNE:VIP2/pSPYCE:VirE2* carrying the VirE2 stop codon and pSPYNE:VIP2/pSPYCE:TGA2 did not produce any fluorescence. All the images are from a single confocal section. Bars = 10 μm.

Figure 6.

Identification of At vip2 and Transformation Assays in the Mutant. (A) The full-length genomic sequence of the At VIP2 gene (exons are shaded) showing the T-DNA insertion in the second exon of the Arabidopsis T-DNA mutant line (At vip2; GABI_676A06). (B) RNA gel blot analysis confirms the absence of At VIP2 transcripts in At vip2. Five micrograms of RNA extracted from leaves was fractionated on a formaldehyde-agarose gel, blotted onto a nylon membrane, and probed with ³²P-labeled At VIP2 gene (top panel). Ethidium bromide–stained gel showing rRNA suggests equal amounts of total RNA were loaded in each lane (bottom panel). (C) Roots of wild-type and vip2 mutant plants were infected with a tumorigenic strain A. tumefaciens A208 (nopaline strain), and tumors incited on the roots were visualized and scored 4 weeks after infection. (D) Transient and stable GUS expression. Roots of the wild-type and At vip2 plants were inoculated with a strain A. tumefaciens GV3101 carrying the uidA-intron gene within the T-DNA. The inoculated roots were periodically collected and stained with X-Gluc. All the experiments were repeated two times.

Figure 7.

Expression Profile for the 52 Differentially Expressed Histone or Histone-Associated Genes Represented in the ATH1 Gene Chips in Col-0 and At vip2. Color code represents expression values of ratio between At vip2 and Col-0, wherein red and green indicate up- and downregulation of genes, respectively. Each horizontal line displays the expression data for one gene. Data were clustered with correlation using the TIGR Multiple Experiment Viewer.

Figure 8.

Validation of the Microarray Data by Real-Time qRT-PCR. Eight different histone genes that had less transcript abundance in At vip2 compared with Col-0, based on microarray experiments, were selected for validation. Total RNA was extracted from leaves of wild-type Col-0 and At vip2 following agroinfiltration at 0, 48, and 72 h. The first-strand cDNA was synthesized and used for qRT-PCR using gene-specific primers (see Methods). The amount of elongation factor-1-α transcripts was determined and used for normalization. cDNA extracted from Col-0 and At vip2 at 0 HAI was used as calibrator to obtain the relative transcript levels for each gene following agroinfiltration. The data represent the average of three biological replicates, including three technical replicates for each biological replicate with se values shown as error bars.