Arabidopsis RETICULON-LIKE4 (RTNLB4) Protein Participates in Agrobacterium Infection and VirB2 Peptide-Induced Plant Defense Response

Abstract

Agrobacterium tumefaciens uses the type IV secretion system, which consists of VirB1-B11 and VirD4 proteins, to deliver effectors into plant cells. The effectors manipulate plant proteins to assist in T-DNA transfer, integration, and expression in plant cells. The Arabidopsis reticulon-like (RTNLB) proteins are located in the endoplasmic reticulum and are involved in endomembrane trafficking in plant cells. The rtnlb4 mutants were recalcitrant to A. tumefaciens infection, but overexpression of RTNLB4 in transgenic plants resulted in hypersusceptibility to A. tumefaciens transformation, which suggests the involvement of RTNLB4 in A. tumefaciens infection. The expression of defense-related genes, including FRK1, PR1, WRKY22, and WRKY29, were less induced in RTNLB4 overexpression (O/E) transgenic plants after A. tumefaciens elf18 peptide treatment. Pretreatment with elf18 peptide decreased Agrobacterium-mediated transient expression efficiency more in wild-type seedlings than RTNLB4 O/E transgenic plants, which suggests that the induced defense responses in RTNLB4 O/E transgenic plants might be affected after bacterial elicitor treatments. Similarly, A. tumefaciens VirB2 peptide pretreatment reduced transient T-DNA expression in wild-type seedlings to a greater extent than in RTNLB4 O/E transgenic seedlings. Furthermore, the VirB2 peptides induced FRK1, WRKY22, and WRKY29 gene expression in wild-type seedlings but not efr-1 and bak1 mutants. The induced defense-related gene expression was lower in RTNLB4 O/E transgenic plants than wild-type seedlings after VirB2 peptide treatment. These data suggest that RTNLB4 may participate in elf18 and VirB2 peptide-induced defense responses and may therefore affect the A. tumefaciens infection process.

Keywords: Agrobacterium; RTNLB; VirB2; plant defense response.

Figure 1

The Arabidopsis rtnlb4 T-DNA insertion mutants were recalcitrant to Agrobacterium tumefaciens transformation. (A) Schematic representations of the T-DNA insertion regions around the Arabidopsis RTNLB4 gene. Purple boxes represent exon regions of the RTNLB4 gene. The large open triangle represents T-DNA insertion sites in the RTNLB4 gene. The long and short arrows indicate the locations of primers used in genomic DNA PCR analysis. (B) qPCR results of the RTNLB4 transcript in rtnlb4-1, rtnlb4-2, and rtnlb4-3 mutants. UBQ10 (polyubiquitin 10) transcript level was an internal control. Data are mean ± SE from at least three PCR reactions of each mutant. (C) Transformation efficiencies of three rtnlb4 mutant lines and wild-type plants. Green bars indicate percentages of root segments forming tumors at 1 month after infection with 10⁸ cfu mL⁻¹ tumorigenic A. tumefaciens A208 strain. Blue bars show percentages of root segments with GUS activity 6 days after infection with 10⁸ cfu mL⁻¹ A. tumefaciens At849 strain. Data are mean ± SE from more than 15 plants. At least 80 root segments were examined for each plant. (D) Seedlings from three rtnlb4 mutant lines showed reduced susceptibility to transient transformation. Mutant seedlings were infected with 10⁷ cfu mL⁻¹ acetosyringone (AS)-treated A. tumefaciens strain for 3 days to determine transient transformation efficiencies. Data are mean ± SE. * p < 0.05 compared with the wild-type by pairwise Student’s t test.

Figure 2

RTNLB4 overexpression (O/E) transgenic plants were more susceptible to A. tumefaciens infections. (A) qPCR analysis of RTNLB4 transcript levels in RTNLB4 O/E and wild-type plants. The UBQ10 (polyubiquitin 10) transcript level was an internal control. Data are mean ± SE. (B) Stable and transient transformation efficiencies of RTNLB4 O/E and wild-type plants. Green bars show the percentage of root segments with tumors after infection with 10⁸, 10⁶, or 10⁵ cfu mL⁻¹ A. tumefaciens A208. Blue bars represent the percentage of root segments with GUS activity after infection with 10⁸, 10⁶, or 10⁵ cfu mL⁻¹ A. tumefaciens At849 strain. A. tumefaciens at 10⁸ cfu mL⁻¹ was used to infect wild-type roots as a positive control to indicate successful transformation. Data are mean ± SE from more than 15 plants. At least 80 root segments were examined for each plant. (C) Transient transformation efficiency in seedlings of RTNLB4 O/E and wild-type plants. Seedlings of O/E plants were infected with 10⁵ or 10⁴ cfu mL⁻¹ AS-induced A. tumefaciens strain. Wild-type seedlings were infected with 10⁷ cfu mL⁻¹ 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 3

Induction of defense-related genes by elf18 was significantly impaired in RTNLB4 O/E transgenic plants. Gene expression of RTNLB4 (A), PR1 (B), FRK1 (C), WRKY22 (D), WRKY29 (E), MPK3 (F), and MPK6 (G) in seedlings of RTNLB4 O/E transgenic and wild-type treated with 10 µM elf18 for 0, 10, 30, 60, 90, 120, and 360 min measured by qPCR analysis. The UBQ10 (polyubiquitin 10) transcript level was an internal control. Data are mean ± SE from at least three independent biological experiments. Data were analyzed by Duncan test, and means with the same letter (a–g) were not significantly different (p < 0.05).

Figure 4

Pretreatment with elf18 peptide reduced transient transformation efficiency in wild-type, RTNLB4 O/E transgenic, and three rtnlb4 mutant seedlings. (A) Transient transformation efficiencies of wild-type seedlings pretreated with 10 µM elf18 peptide for 0, 6, or 24 hr before infection with A. tumefaciens. Transient transformation rates of wild-type, efr-1, fls2, and bak1 mutants (B), RTNLB4 O/E transgenic plants (C), and three rtnlb4 mutant plants (D) pretreated with 10 µM elf18 peptide for 6 h before infection with A. tumefaciens. Distilled H₂O (dH₂O) was used as the mock control in seedling transient transformation assays. Data are mean ± SE from at least three independent transformation assays. Data were analyzed by Duncan tests and means with different letters were significantly different (p < 0.05).

Figure 5

Transient transformation rates of wild-type, RTNLB4 O/E transgenic and three rtnlb4 mutant seedlings were decreased with VirB2 peptide pretreatments. (A) Transient transformation rates of wild-type seedlings pretreated with 1, 5, 10, 20, or 50 µM of five VirB2 peptides, S111-T58, I63-I80, I80-V101, G95-F112, or I104-G121, for 6 hr before infection with A. tumefaciens. (B) Transient transformation efficiency of wild-type seedlings pretreated with 10 µM of two VirB2 peptides, S111-T58 or I63-I80, at 0, 6, or 24 hr before infection with A. tumefaciens. Agrobacterium-mediated transient transformation rates of wild-type, efr-1, fls2, bak1 mutants (C), RTNLB4 O/E transgenic plants (D), and three rtnlb4 mutant plants (E) pretreated with 10 µM of the two VirB2 peptides for 6 hrs before infection with A. tumefaciens. DMSO solution was used as the mock control in seedling transient transformation assays. Data are mean ± SE from at least three independent transformation assays. Data were analyzed by Duncan tests and means with different letters were significantly different (p < 0.05).

Figure 6

The VirB2 peptide, S111-T58, induced the expression of defense-related genes in wild-type plants but to a lesser extent in RTNLB4 O/E transgenic plants. Gene expression of FRK1 (A), CYP81F2 (B), At2g17740 (C), WRKY22 (D), WRKY29 (E), MPK3 (F), and MPK6 (G) in seedlings of wild-type and RTNLB4 O/E transgenic plants treated with 10 µM VirB2 peptide for 0, 10, 30, 60, 90, 120, and 360 min measured by qPCR analysis. The UBQ10 (polyubiquitin 10) transcript level was an internal control. Data are mean ± SE from at least three independent biological experiments. Data were analyzed by Duncan tests and means with different letters were significantly different (p < 0.05).

Figure 7

Arabidopsis seedling growth was inhibited by elf18 and VirB2 peptide treatments. Plant widths of wild-type, RTNLB4 O/E transgenic (A), and rtnlb4 mutant plants (B) were determined after treatment with 20 μM elf18 peptide or the mock control (dH₂O) for 2 weeks. Plant widths of wild-type and efr-1 and fls2 mutants (C) were also measured after treatment with 20 μM elf18, Agro-flg22 or the mock control (dH₂O). Five VirB2 peptides and the mock control (DMSO) were used to treat wild-type and efr-1 and fls2 mutants (D). Seedlings of wild-type, RTNLB4 O/E transgenic (E) and rtnlb4 mutant plants (F) were treated with two VirB2 peptides, S111-T58, or I63-I80, and the mock control (DMSO). Plant widths of these plants were determined after 2-week treatments. Data are mean ± SE from three independent experiments. More than 15 seedlings of each type plant for each treatment were examined in each independent experiment. Data were analyzed by Duncan tests and means with different letters were significantly different (p < 0.05).

Figure 8

Induced H₂O₂ amounts were lower in RTNLB4 O/E transgenic plants and rtnlb4, efr-1, and fls2 mutants than in wild-type plants after with elf18 and VirB2 peptides. The H₂O₂ amount in wild-type, RTNLB4 O/E transgenic plants (A) and three rtnlb4, efr-1, and fls2 mutant plants (B) was determined at 0, 0.5, 2, 5, and 10 min after the addition of elf18. The H₂O₂ amount at each time was normalized to the H₂O₂ amount at 0 min. H₂O₂ amount in wild-type, RTNLB4 O/E transgenic (C,E) and three rtnlb4, efr-1, and fls2 mutant plants (D,F) was determined after adding two VirB2 peptides S111-T58 (C,D) or I63-I80 (E,F). Data are mean ± SE from more than 10 plants. Data were analyzed by Duncan tests and means with different letters were significantly different (p < 0.05).