Arabidopsis RETICULON-LIKE3 (RTNLB3) and RTNLB8 Participate in Agrobacterium-Mediated Plant Transformation

Abstract

Agrobacterium tumefaciens can genetically transform various eukaryotic cells because of the presence of a resident tumor-inducing (Ti) plasmid. During infection, a defined region of the Ti plasmid, transfer DNA (T-DNA), is transferred from bacteria into plant cells and causes plant cells to abnormally synthesize auxin and cytokinin, which results in crown gall disease. T-DNA and several virulence (Vir) proteins are secreted through a type IV secretion system (T4SS) composed of T-pilus and a transmembrane protein complex. Three members of Arabidopsis reticulon-like B (RTNLB) proteins, RTNLB1, 2, and 4, interact with VirB2, the major component of T-pilus. Here, we have identified that other RTNLB proteins, RTNLB3 and 8, interact with VirB2 in vitro. Root-based A. tumefaciens transformation assays with Arabidopsis rtnlb3, or rtnlb5-10 single mutants showed that the rtnlb8 mutant was resistant to A. tumefaciens infection. In addition, rtnlb3 and rtnlb8 mutants showed reduced transient transformation efficiency in seedlings. RTNLB3- or 8 overexpression transgenic plants showed increased susceptibility to A. tumefaciens and Pseudomonas syringae infection. RTNLB1-4 and 8 transcript levels differed in roots, rosette leaves, cauline leaves, inflorescence, flowers, and siliques of wild-type plants. Taken together, RTNLB3 and 8 may participate in A. tumefaciens infection but may have different roles in plants.

Keywords: Agrobacterium; RTNLB.

Figure 1

RTNLB8, not 3, and 5–7 proteins, interacted with the processed VirB2 in yeast. RTNLB1-8 proteins were tested for interactions with VirB2 or the RTNLB1-8 using a yeast two-hybrid assay. The RTNLB5-7 proteins showed no interactions with RTNLB1-8 proteins in yeast. The unrelated Lamin C bait protein was the negative control.

Figure 2

The GST-VirB2 fusion protein interacted with RTNLB1-4 and 8 proteins in vitro. The GST-fusion and GST only proteins were linked with glutathione-sepharose beads and incubated with T7-tagged proteins to test their interactions in vitro. Bound proteins were eluted with glutathione and analyzed by protein gel blot using anti-T7 tag and anti-GST antibodies. Panel A, interactions between VirB2 and RTNLB1-4 and 8 were determined by using the GST-VirB2 fusion protein and T7-tagged-RTNLB1-4 and 8 or the GST-fusion of RTNLB1-4 and 8 and the T7-tagged-VirB2 protein. Panel B, the GST-only protein was used as a negative control in GST pull-down assays. The GST fusions of RTNLB1 (Panel C), RTNLB2 (Panel D), RTNLB3 (Panel E), RTNLB4 (Panel F), and RTNLB8 (Panel G) were used to investigate their interactions with T7-tagged-RTNLB1-4 and 8 proteins.

Figure 3

The Arabidopsis rtnlb3 and rtnlb8 T-DNA insertion mutant seedlings were resistant to A. tumefaciens infection. Panel A, schematic representations of the T-DNA insertion regions around the Arabidopsis RTNLB3 (Panel A-1), RTNLB5 (Panel A-2), RTNLB6 (Panel A-3), RTNLB7 (Panel A-4), RTNLB8 (Panel A-5), RTNLB9 (Panel A-6), and RTNLB10 (Panel A-7) genes. Blue boxes represented exon regions of each RTNLB gene. The large open triangle represents T-DNA insertion sites in each RTNLB gene. The long and short arrows indicate the locations of primers used in genomic DNA PCR analysis. Panel B, RT-PCR results of target RTNLB transcripts in rtnlb3 and rtnlb5-10 single mutants. The α-tubulin was an internal control. Panel C, transcript levels of each RTNLB gene in rtnlb single mutants shown as a relative percentage of wild-type plants. Data are mean ± SE from at least 3 RT-PCR reactions of each mutant. Panel D, transformation efficiencies of rtnlb8-1 and rtnlb8-2 and wild-type plants. Black bars indicate the percentage of root segments forming tumors 1 month after infection with 10⁸ cfu·mL⁻¹ tumorigenic A. tumefaciens A208 strain. Grey bars show the percentage of root segments with GUS activity 6 days after infection with 10⁸ cfu·mL⁻¹ A. tumefaciens At849 strain. Panel E, rtnlb3 and rtnlb8 mutant seedlings showed decreased susceptibility to transient transformation. Transient transformation efficiency in mutant seedlings infected with 10⁷ cfu·mL⁻¹ acetosyringone (AS)-induced A. tumefaciens strain for 3 days. Data are mean ± SE. * p < 0.05 compared with the wild-type by pairwise Student’s t test.

Figure 4

RTNLB3 and RTNLB8 overexpression (O/E) transgenic plants were hypersusceptible to A. tumefaciens infections. Panel A, RT-PCR analysis of RTNLB transcript levels in RTNLB3 (Panel A-1) and RTNLB8 (Panel A-2) O/E plants and the wild type. The α-tubulin was used as an internal control. Panel B, transcript levels of RTNLB3 (Panel B-1) or RTNLB8 (Panel B-2) in O/E plants relative to wild-type expression. Data are mean ± SE from at least 3 RT-PCR reactions of each mutant. Panel C, the T7-tagged-RTNLB3 (Panel C-1) and RTNLB8 (Panel C-2) O/E plants accumulated T7-tagged RTNLB proteins. Ponceau S (PS) staining was used to show equivalent loading of total protein in each lane. Panel D, Transient transformation efficiency of RTNLB3 and 8 O/E and wild-type plants. Green bars represent the percentage of root segments developing tumors after infection with 10⁸, 10⁶, or 10⁵ cfu·mL⁻¹ of A. tumefaciens A208. Blue bars indicate the percentage of root segments with GUS activity after infection with 10⁸, 10⁶, or 10⁵ cfu·mL⁻¹ of A. tumefaciens At849 strain. The 10⁸ cfu mL⁻¹ of A. tumefaciens was used to infect wild-type roots as a positive control to indicate successful transformation. Panel E, Enhanced transient transformation efficiency in seedlings of RTNLB3 and 8 O/E plants. Seedlings of O/E plants were infected with 10⁵ cfu·mL⁻¹ of AS-induced A. tumefaciens strain. Wild-type seedlings were infected with 10⁷ cfu·mL⁻¹ of A. tumefaciens strain as a positive control. Data are mean ± SE. * p < 0.05 compared with the wild-type by pairwise Student’s t test.

Figure 5

RTNLB3 and 8 O/E plants were more sensitive to Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) infection. Panel A, leaves of wild-type and RTNLB O/E plants were syringe-infiltrated with Pst DC3000 (Panel A-1) and hrcC mutant (Panel A-2). Bacterial numbers in infected leaves were quantified at 0, 1, 3, 5, and 7 days post-infection. Data are mean ± SE. * p < 0.05 compared with the wild-type by pairwise Student’s t test. Panel B, disease symptoms of wild-type and RTNLB O/E plant leaves 5 days after infection with Pst DC3000 or hrcC mutant. Panel C, trypan blue staining of infected leaves of wild-type and RTNLB O/E plants 5 days after infection with Pst DC3000. Yellow bar = 1 cm.

Figure 6

Levels of RTNLB1-4 and 8 in various tissues of wild-type Arabidopsis (ecotype: Columbia) plants. RNA from root, rosette leaf, cauline leaf, inflorescence, flower, and silique of wild-type plants were isolated, reverse-transcribed, and used for quantitative real-time PCR. UBQ10 (polyubiquitin 10) transcript level was an internal control. Data are mean ± SE.