A mutation in negative regulator of basal resistance WRKY17 of Arabidopsis increases susceptibility to Agrobacterium-mediated genetic transformation

VIP1 represents one of the target genes of WRKY17

A previous microarray analysis of the wrky17 mutant identified a number of upregulated genes², one of which, VIP1, represents a major player in plant genetic transformation by Agrobacterium^7,10,13. However, microarray analyses of gene expression, although commonly used, often yield divergent data^16,17 and, therefore, require direct confirmation by detection of the specific transcripts. Thus, we analyzed the wrky17 mutant for the levels of VIP1 expression.

First, we examined three different lines of Arabidopsis plants derived from the wrky17-1 mutant² for the presence of the WRKY17 transcript using RT-PCR. Figure 1A shows that whereas the wild-type plants produced WRKY17 mRNA, neither of the mutant lines accumulated detectible levels of this transcript. Next, we investigated the effect of the wrky17 mutation on the expression of the VIP1 gene. Using RT-PCR, we analyzed the levels of the VIP1 transcript in plant roots (Figure 1B) and shoots (Figure 1C). The VIP1 transcription activity was substantially higher in the roots of all three wrky17 mutants than in those of wild type plants (Figure 1B). Unexpectedly, we detected no changes in VIP1 expression in the shoots of the same plants, which accumulated VIP1 transcripts in amounts similar to those in the wild-type plants (Figure 1C). Analysis of ACTin2-specific transcripts detected similar amounts of PCR products in all samples, indicating equal efficiencies of the RT-PCR reactions (Figure 1B, C). Collectively, these data suggest that WRKY17 represents one of the transcriptional regulators of the VIP1 gene, but that this regulation is tissue-specific.

Figure 1. RT-PCR analysis of ACTIN2, WRKY17 and VIP1 gene expression in wild-type and wrky17 mutant Arabidopsis plants.

(A) WRKY17 expression in whole plants. (B, C) VIP1 expression in roots and shoots, respectively. WT, wild-type plants; 7, 12, and 13 are the three different lines of the homozygous wrky17-1 mutant.

This is consistent with the previous observations of differential regulation of VIP1 expression during plant development as well as in response to various stimuli. For example, VIP1 transcription is activated upon induction of cell division¹⁸, after osmotic stress, and is differentially expressed in different tissues of Arabidopsis⁵. WRKY17 functions as a transcription inhibitor of several genes involved in plant defense pathways¹. Our results suggest that VIP1 is one of the target genes down-regulated, directly or indirectly, by WRKY17 in tissue-specific fashion. Alternatively, VIP1 expression in the shoot tissue could be regulated by additional factors which mask the effect of the WRKY17 knock-out mutation.

The wrky17 mutant is hypersusceptible to Agrobacterium-mediated genetic transformation

Once we had identified plant tissue showing a clear effect of WRKY17 on VIP1 expression, we investigated whether this effect altered susceptibility to Agrobacterium infection. To this end, we employed the classical Arabidopsis root infection assay¹⁹, in which the efficiency of infection is monitored and quantified by measuring the level of transient T-DNA expression, that is early expression of the invading T-DNA molecules before their stable integration in the host genome. Root segments from the wild-type and wrky17 plants were inoculated with Agrobacterium strain EHA105 harboring the binary plasmid pBISN1 with the β-glucuronidase (GUS) gene expression reporter in its T-DNA region. T-DNA expression was quantified based on the percentage of root segments exhibiting GUS histochemical staining. These experiments revealed that T-DNA expression frequencies in roots of all three wrky17 mutant lines were 30–50% higher than those measured in roots of the wild-type plants (Table 1 and Figure 2).

Figure 2. The effect of wrky17 mutation on susceptibility of Arabidopsis roots to Agrobacterium infection.

Transformation efficiency is expressed as the percent of GUS-stained roots from the total number of roots tested. All data represent average values of three independent experiments with indicated standard deviations. WT, wild-type plants; 7, 12, and 13 are the three different lines of the homozygous wrky17-1 mutant.

The increased susceptibility of the wrky17 roots to Agrobacterium infection correlates with elevated transcription levels of the VIP1 gene in this tissue. Considering the known role of VIP1 as an enhancer of Agrobacterium infectivity^7–15, it is likely that higher VIP1 expression in roots of the wrky17 mutant is responsible for the increased susceptibility to Agrobacterium. This notion is consistent with our earlier observations that overexpression of VIP1 in tobacco further elevates transformation efficiency⁸. That we detected this effect of the wrky17 mutation using a transient T-DNA expression assay indicates that increased VIP1 expression affects the early steps of the infection process, i.e., those that occur prior to T-DNA integration and stable expression.

Transgenic plants

Arabidopsis thaliana plants, wild-type (ecotype Col0) or wrky17-1 T-DNA insertion mutants (obtained from D. Roby, CNRS Montpellier, France), were grown either in soil or on Gamborg’s B5 medium (20 g.L^-1 sucrose, 8 g.L^-1 agar), after seed surface sterilization. All plants were grown in an environment-controlled growth chamber at 22°C under long day (16h light/8h dark) conditions. Three lanes of homozygous plants (lanes 7, 12, 13) were isolated from the original wrky17-1 stock.

RT-PCR

Total RNA was extracted from plant tissues using Trizol (Invitrogen), and cDNA synthesis was performed with a RevertAid cDNA synthesis Kit (Fermentas) according to the manufacturer’s instructions. Transcript levels were then estimated by PCR, with 30 cycles of amplification. The resulting cDNA was PCR-amplified for 30 cycles using primers specific for the tested gene or for ACTIN2 as an internal control of a constitutively expressed gene. The following primer pairs were used: 5´ATGACCGTTGATATTATGCGTTTAC3´/5´TCAAGCCGAACCAAACACCAAAC3´ that amplify the full length 966-bp WRKY17 (At2g24570) cDNA, 5´ATGGAAGGAGGAGGAAGAGG3´/5´TCAGCCTCTCTTGGTGAAATCC3´ that amplify the full length 1,026-bp VIP1 cDNA, and 5´ATGGCTGAGGCTGATGATATT3´/5´TTAGAAACATTTTCTGTGAACGATTCC3´ that amplify the full length 1,134 bp ACTIN2 (At3g18780) cDNA.

Root transformation assay

All infection assays were performed as described by Gelvin (2006)¹⁹ with the Agrobacterium tumefaciens strain EHA105 (from S. Gelvin, Purdue University, USA), harboring a pBISN1 binary plasmid with an intron-containing GUS reporter gene that is not expressed in bacteria²². One-cm long root segments were excised from 3–4 week-old Arabidopsis plants grown on Gamborg’s B5 medium, and bundles of root segments were placed on the MS (Murashige and Skoog) medium. For each experiment, roots were pooled from more than 20 plants and divided into three bundles, each containing more than 100 root segments. Root bundles were overlaid with EHA105 harboring pBISN1 suspension culture at A₆₀₀ = 0.25 in NaCl 0.9%, and excess liquid was removed by pipette aspiration after 15 min of incubation. Root segments were then incubated for two days at 22°C under the long day conditions, rinsed in water containing 100 mg.L^-1 timentine (BioWorld) to eliminate bacteria, and incubated for an additional three days on the MS medium supplemented with timentine. Root segments were then subjected to the GUS histochemical assay²³, with overnight incubation at 37°C, and the number of root segments displaying GUS staining was recorded.