Overexpression of the tobacco Tsi1 gene encoding an EREBP/AP2-type transcription factor enhances resistance against pathogen attack and osmotic stress in tobacco

Abstract

Using mRNA differential display analysis, we isolated a salt-induced transcript that showed a significant sequence homology with an EREBP/AP2 DNA binding motif from oilseed rape plants. With this cDNA fragment as a probe, cDNA clone Tsi1 (for Tobacco stress-induced gene1) was isolated from a tobacco cDNA library. RNA gel blot analysis indicated that transcripts homologous with Tsi1 were induced not only in NaCl-treated leaves but also in leaves treated with ethephon or salicylic acid. Transient expression analysis using a Tsi1::smGFP fusion gene in BY-2 cells indicated that the Tsi1 protein was targeted to the nucleus. Fusion protein of Tsi1 with GAL4 DNA binding domain strongly activated transcription in yeast, and the transactivating activity was localized to the 13 C-terminal amino acids of Tsi1. Electrophoretic mobility shift assays revealed that Tsi1 could bind specifically to the GCC and the DRE/CRT sequences, although the binding activity to the former was stronger than that to the latter. Furthermore, Agrobacterium-mediated transient expression and transgenic plants expressing Tsi1 demonstrated that overexpression of the Tsi1 gene induced expression of several pathogenesis-related genes under normal conditions, resulting in improved tolerance to salt and pathogens. These results suggest that Tsi1 might be involved as a positive trans-acting factor in two separate signal transduction pathways under abiotic and biotic stress.

Figure 1.

Analysis of Tobacco cDNA Sequence Encoding a Tsi1 Protein. (A) Nucleotide and deduced amino acid sequences of the Tsi1 gene. The EREBP/AP2 DNA binding domain is underlined. The basic amino acids that potentially act as a nuclear localization signal are shown in boxes, and an acidic C-terminal region that might act as a transcriptional activation domain is shown in italics. The Tsi1 sequence was submitted to GenBank under the accession number AF058827. (B) Comparison of the deduced amino acid sequences of Tsi1, Pti6, and a putative Arabidopsis protein (Ara). The amino acid sequence of Tsi1 was aligned with the amino acid sequences of tomato Pti6 (GenBank accession number O04682) and a putative Arabidopsis protein (GenBank accession number T02896). Boxes represent perfectly conserved amino acids, and dashes indicate gaps introduced to maximize alignment.

Figure 2.

Genomic DNA Gel Blot Analysis and Organ-Specific Expression of Tsi1 in Tobacco. (A) DNA gel blot analysis of the Tsi1 gene. Genomic DNA (10 μg) was digested with BamHI (B), EcoRI (E), HindIII (H), and XbaI (Xb) and gel separated. The DNA gel blots were hybridized with the full-length Tsi1 cDNA (left) or a 3′ UTR fragment probe (right). (B) Organ-specific expression of the Tsi1 gene. In each lane, 20 μg of total RNAs prepared from flowers, roots, leaves, and stems was loaded. The blotted membrane was hybridized with the 3′ UTR fragment probe. Equal amounts of RNA were loaded per lane, as determined by ethidium bromide staining.

Figure 3.

Expression of the Tsi1 Gene in Response to Abiotic Stresses. (A) Induction patterns of the Tsi1 gene after treatment with distilled water (H₂O), NaCl, drought, wounding, SA, ethephon, abscisic acid (ABA), or methyl jasmonate (MeJA). Total RNA was prepared from tobacco leaves treated with distilled water, 200 mM NaCl, drought, wounding, 2 mM SA, 1 mM ethephon, 100 μM abscisic acid, or 100 μM methyl jasmonate for 1 or 24 hr as indicated. (B) Time-course accumulation of Tsi1 transcripts detected upon water, NaCl, SA, or ethephon treatment. Each lane was loaded with 20 μg of total RNA extracted from detached tobacco leaves that had been treated with 200 mM NaCl, 1 mM ethephon, 2 mM SA, or distilled water for the designated times. PR1 and rd29A genes were used as positive controls for the SA, ethephon, or high-salt treatment. Ethidium bromide staining of the RNA gel was used to show equal loading. The RNA gel blots were hybridized with the 3′ flanking sequence of Tsi1 as a specific probe.

Figure 3.

Expression of the Tsi1 Gene in Response to Abiotic Stresses. (A) Induction patterns of the Tsi1 gene after treatment with distilled water (H₂O), NaCl, drought, wounding, SA, ethephon, abscisic acid (ABA), or methyl jasmonate (MeJA). Total RNA was prepared from tobacco leaves treated with distilled water, 200 mM NaCl, drought, wounding, 2 mM SA, 1 mM ethephon, 100 μM abscisic acid, or 100 μM methyl jasmonate for 1 or 24 hr as indicated. (B) Time-course accumulation of Tsi1 transcripts detected upon water, NaCl, SA, or ethephon treatment. Each lane was loaded with 20 μg of total RNA extracted from detached tobacco leaves that had been treated with 200 mM NaCl, 1 mM ethephon, 2 mM SA, or distilled water for the designated times. PR1 and rd29A genes were used as positive controls for the SA, ethephon, or high-salt treatment. Ethidium bromide staining of the RNA gel was used to show equal loading. The RNA gel blots were hybridized with the 3′ flanking sequence of Tsi1 as a specific probe.

Figure 4.

Subcellular Localization of the Tsi1 Gene Product. The Tsi1 coding region was fused in frame to the smGFP coding region in the plant transformation vector pMBP2. Constructs were introduced into tobacco BY-2 cell protoplasts by polyethylene glycol–mediated transformation. Expression of the introduced genes was examined after 12 hr by fluorescence and light microscopy.

Figure 5.

Transcriptional Activation Activity of Tsi1 in Yeast Cells. The Tsi1 and Tsi1 deletion constructs were fused in frame to the GAL4 DNA binding domain (DB) expression vector and then transformed into yeast strain Y190. The transformants were selected by growth on Trp⁻ synthetic dropout medium at 30°C for 3 days. The colony lift filter assay and liquid culture assay using o-nitrophenyl β-d-galactopyranoside as a substrate were subsequently performed to determine the ability of each translation product to activate transcription. Hatched boxes represent the EREBP/AP2 DNA binding domain. a.a., amino acids.

Figure 6.

Characterization of the DNA Binding Affinity of the Recombinant Tsi1 Protein. (A) Sequence of the oligonucleotides used in the DNA binding studies. (B) Gel retardation assay showing sequence-specific binding of the recombinant Tsi1 protein. The radiolabeled probes were incubated in the presence or absence of the recombinant Tsi1 protein. Lane 1 contained only the free probes, and lane 2 contained GST only. Lane 3 contained 1 μg of bacterial extracts, and lane 2 and lanes 4 to 10 contained 2 μg of bacterial extracts. For competition assays, 2 μg of E. coli extract containing the Tsi1 protein was incubated with either the DRE/CRT or GCC oligonucleotide before the addition of the GCC or DRE/CRT probe. The amount of competitor DNA is indicated at the top of each lane.

Figure 7.

Transient and Stable Overexpression of the Tsi1 Gene. (A) Induction of the PR1 and SAR8.2 genes in tobacco by Tsi1 expression. Seven-week-old soil-grown wild-type tobacco (cv Samsun NN) was infiltrated with Agrobacterium (strain LBA4404) bearing the expression plasmids pMBP2 and 35S::Tsi1 or treated with TMV and 1 mM SA. Plants were harvested at 0 or 24 hr after treatment, and RNA gel blots were hybridized with the probes indicated at left. Mock-inoculated plants were rubbed with phosphate buffer and carborundum only. (B) Expression of Tsi1 target genes in 35S::Tsi1 transgenic plants and in a control plant under normal conditions. Total RNA was isolated from the plants and analyzed by RNA gel blot hybridization using the 3′ UTR Tsi1 cDNA as a specific probe; numbers indicate independent lines of transgenic T0 plants. RNA gel blotting also was conducted to measure the amount of osmotin, SAR8.2, PR1, PR2, PR3, PR4, or rd29A mRNA in control and transgenic tobacco plants carrying 35S::Tsi1. C indicates a pMBP2-transformed tobacco T0 plant; 1 to 9 indicate Tsi1-transformed independent tobacco T0 lines.

Figure 7.

Transient and Stable Overexpression of the Tsi1 Gene. (A) Induction of the PR1 and SAR8.2 genes in tobacco by Tsi1 expression. Seven-week-old soil-grown wild-type tobacco (cv Samsun NN) was infiltrated with Agrobacterium (strain LBA4404) bearing the expression plasmids pMBP2 and 35S::Tsi1 or treated with TMV and 1 mM SA. Plants were harvested at 0 or 24 hr after treatment, and RNA gel blots were hybridized with the probes indicated at left. Mock-inoculated plants were rubbed with phosphate buffer and carborundum only. (B) Expression of Tsi1 target genes in 35S::Tsi1 transgenic plants and in a control plant under normal conditions. Total RNA was isolated from the plants and analyzed by RNA gel blot hybridization using the 3′ UTR Tsi1 cDNA as a specific probe; numbers indicate independent lines of transgenic T0 plants. RNA gel blotting also was conducted to measure the amount of osmotin, SAR8.2, PR1, PR2, PR3, PR4, or rd29A mRNA in control and transgenic tobacco plants carrying 35S::Tsi1. C indicates a pMBP2-transformed tobacco T0 plant; 1 to 9 indicate Tsi1-transformed independent tobacco T0 lines.

Figure 8.

Analysis of Salt-Induced Senescence and Disease Resistance of Tsi1 Transgenic Plants. (A) Retardation of salt-induced senescence in transgenic tobacco plants. Leaf discs from the transgenic plants carrying the Tsi1 gene in the sense orientation (bars 1 to 7) and the wild-type tobacco plants (bars N1 to N3) were floated in 400 mM NaCl solution for 48 or 72 hr under continuous white light at 25°C. As a control, the wild-type leaf discs were floated in water (H₂O). Phenotypic differences were observed (72 hr), and chlorophyll contents (mg/g fresh weight) were measured (48 hr) from NaCl-treated leaf discs of 35S::Tsi1 transgenic plants and wild-type tobacco. The experiments were repeated three times, each with seven leaf discs. (B) Analysis of disease resistance against the bacterial pathogen P. s. tabaci in wild-type and 35S::Tsi1 transgenic plants. Disease symptoms caused by P. s. tabaci in wild-type and Tsi1 transgenic plants are shown in the inset. The photograph was taken 7 days after inoculation. The growth of P. s. tabaci strains in wild-type and 35S::Tsi1 transgenic plants was compared as follows. Fully expanded leaves of 7-week-old tobacco plants were inoculated with 10⁷ colony-forming units (cfu)/mL of a P. s. tabaci strain. At 7 days after inoculation, the infected leaves were collected and the bacterial populations were determined. Bars N1 and N2 indicate wild-type tobacco, and bars 2 and 4 to 7 indicate Tsi1-transformed independent tobacco T0 lines. Values are means of three different experiments. Error bars indicate ±se.

Figure 8.

Analysis of Salt-Induced Senescence and Disease Resistance of Tsi1 Transgenic Plants. (A) Retardation of salt-induced senescence in transgenic tobacco plants. Leaf discs from the transgenic plants carrying the Tsi1 gene in the sense orientation (bars 1 to 7) and the wild-type tobacco plants (bars N1 to N3) were floated in 400 mM NaCl solution for 48 or 72 hr under continuous white light at 25°C. As a control, the wild-type leaf discs were floated in water (H₂O). Phenotypic differences were observed (72 hr), and chlorophyll contents (mg/g fresh weight) were measured (48 hr) from NaCl-treated leaf discs of 35S::Tsi1 transgenic plants and wild-type tobacco. The experiments were repeated three times, each with seven leaf discs. (B) Analysis of disease resistance against the bacterial pathogen P. s. tabaci in wild-type and 35S::Tsi1 transgenic plants. Disease symptoms caused by P. s. tabaci in wild-type and Tsi1 transgenic plants are shown in the inset. The photograph was taken 7 days after inoculation. The growth of P. s. tabaci strains in wild-type and 35S::Tsi1 transgenic plants was compared as follows. Fully expanded leaves of 7-week-old tobacco plants were inoculated with 10⁷ colony-forming units (cfu)/mL of a P. s. tabaci strain. At 7 days after inoculation, the infected leaves were collected and the bacterial populations were determined. Bars N1 and N2 indicate wild-type tobacco, and bars 2 and 4 to 7 indicate Tsi1-transformed independent tobacco T0 lines. Values are means of three different experiments. Error bars indicate ±se.