A N. tabacum cultivar, Yunyan87, was used in this study as the wild type (WT-Yunyan87). The surface of the seeds were disinfected with 75% alcohol for 30 s and 10% H₂O₂ for 10 min, then washed five times with sterile water and placed under greenhouse conditions for 2–3 weeks on MS agar medium (Murashige and Skoog, 1962; Dang et al., 2013). Regenerated shoots and healthy resistant shoots were grown on selective shooting medium and rooting medium, respectively, which both contained 500 mg⋅L^-1 carbenicillin and 100 mg⋅L^-1 kanamycin. The transgenic plants for this study were obtained by asexual reproduction. Well-developed rooted plants were transferred to matrix in plastic pots and then placed under greenhouse conditions at 25 ± 2°C with a relative humidity of 80% and 16/8 h light/dark cycle.

Ralstonia solanacearum CQPS-1 (phylotype I, race 1, biovar 3) (Liu et al., 2017a) was maintained in our laboratory and was isolated from Chongqing Municipality, China. R. solanacearum strains were cultured at 180 rpm, 30°C in NB medium (3 g⋅L^-1 beef extract, 1 g⋅L^-1 yeast extract, 5 g⋅L^-1 peptone and 10 g⋅L^-1 glucose), and bacterial suspension was cultured to about 1 × 10⁹ CFU mL^-1.

To study the relative expression levels of NtPR-Q in response to R. solanacearum infections, WT-Yunyan87 plants were inoculated with 10 mL of bacterial suspension (1 × 10⁸ CFU mL^-1). Leaf samples were harvested at 1, 3, 5, and 7 days post inoculation (dpi), frozen and stored at -80°C until further analysis. Control plants were inoculated with sterile water. Three individual plants were mixed to yield one sample.

For the pathogenicity assays, transgenic plants and WT-Yunyan87 plants were root inoculated with a 10 mL bacterial suspension (1 × 10⁸ CFU mL^-1). Assays were conducted with 10 plants and were repeated three times. The disease index of bacterial wilt were surveyed every day for 30 days using a 0 to 4 grading standards, where 0 = asymptomatic plant, 1 = less than 25% leaves wilted, 2 = less than 50% wilted leaves, 3 = more than 50% wilted leaves and 4 = completely wilted leaves (dead plant). The data presented with the average disease index (Wang et al., 2013).

For monitoring a bacterial growth assay in leaf tissues, bacterial suspension (5 × 10⁷ CFU mL^-1) was washed twice with sterile water and resuspended in sterile water. Transgenic plants and WT-Yunyan87 plants were inoculated by infiltrating 20 μL inoculum into the third leaves from the top using a blunt syringe. The inoculated leaves were harvested at the indicated time points (30 min, 1, 3, and 5 days) using a leaf punch 1 cm in diameter and were then grounded, serially diluted, and spread on TTC medium [3 g⋅L^-1 beef extract, 1 g⋅L^-1 yeast extract, 5 g⋅L^-1 peptone, 10 g⋅L^-1 glucose, 20 g⋅L^-1 agar configured to NA medium, 100 mL NA medium supplemented with 500 μL triphenyl tetrazolium chloride (TTC) before use]. The number of colonies was counted after 2 days, and the number of R. solanacearum cells per square centimeter of leaves was calculated. The assay repeated three times.

To test the expression of defense-related genes in transgenic tobacco, we harvested the leaf of transgenic tobacco and WT-Yunyan87 tobacco which were grown on MS medium. To study the relative expression levels of defense-related genes in response to R. solanacearum infection, transgenic plants and WT-Yunyan87 plants were inoculated with 10 mL bacterial suspension (1 × 10⁸ CFU mL^-1). Leaf samples were harvested at 1, 3, and 5 dpi, frozen and stored at -80°C until further analysis. Control plants were inoculated with sterile water. Three individual plants were mixed to yield one sample.

WT-Yunyan87 tobacco plants at the four-leaf stage were sprayed with 0.1 mM MeJA, 2 mM SA, or 7 mM ethephon. SA was dissolved in 10% ethanol, and the others were dissolved in sterile water. Control plants were sprayed with 10% ethanol and sterile water. The disease index of bacterial wilt were surveyed every day for 26 days using 0 to 4 grading standards. Assays were conducted with 10 plants and were repeated three times. Plants treated with SA, MeJA, or ethephon were also harvested as samples at 3, 6, 12, and 24 h post treatment, frozen and stored at -80°C for the quantitative real-time PCR analysis. Three individual plants were mixed to yield one sample.

The leaves of plants were grounded into powder in liquid nitrogen using mortars. RNA was extracted using an RNAprep Pure Plant Kit (Tiangen, DP432, Beijing, China). The RNA sample was then reverse transcribed with an iScript^TM cDNA Synthesis Kit (BIO-RAD, 1708891, Hercules, CA, United States) in a 20 μL reaction.

To analyze the relative transcript levels of various genes, quantitative real-time PCR (RT-qPCR) was performed with specific primers, NtUBI3 as the reference gene (Zhao et al., 2009) (see Table 1 for the gene-specific primers) using SsoFast^TM EvaGreen^® Supermix (BIO-RAD, 1725201, Hercules, CA, United States) and the CFX96 real-time system (BIO-RAD, United States). The 20 μL reaction mixtures contained 1 μL cDNA, 10 μL SsoFast^TM EvaGreen^® Supermix, 1 μL of each gene-specific primer (10 μM) and 7 μL ddH₂O. The amplifications were performed at 95°C for 3 min, followed by 40 cycles of denaturation at 95°C for 15 s, 55°C for 20 s and elongation at 56°C for 30 s. Melt curve analyses at 95°C were included at the end to ensure the consistency of the amplified products. The expression level and data analyses were performed by using Bio-Rad CFX Manager 3.0 software with the ^ΔΔCq method (Li et al., 2017). Each assay was performed with three independent technical repeats and the expression level of the genes was calculated from three biological replicates.

The full-length cDNA of NtPR-Q with a downstream in-frame His-Tag was cloned using BglII and BstEII restriction sites. The specific primers of NtPR-Q vector used in this study are listed in Table 1. The plant expression vector pVCT2024 was provided by Dr. Xingguo Zhang (College of Horticulture and Landscape Architecture, Southwest University). After digestion with BglII and BstEII, the NtPR-Q cDNA was inserted into pVCT2024 under the control of the Cauliflower mosaic virus (CaMV) 35S promoter. The positive recombinant plasmid pVCT2024-NtPR-Q was transformed into Agrobacterium tumefaciens strain EHA 105, which was then transformed into WT-Yunyan87 plants using the leaf disk method described by Oh et al. (2005). Transgenic plants were further confirmed to have T-DNA insertions by standard PCR and RT-qPCR.

To analyze the T-DNA insertion in transgenic tobacco, standard PCR was performed with the primers of NtPR-Q insertion and NtACTIN (see Table 1), WT-Yunyan87 tobacco plants as the blank control, NtACTIN served as an endogenous control (Dang et al., 2013). The 20 μL reaction mixtures contained 1 μL cDNA, 2 μL 10 × buffer (TaKaRa, Dalian, China), 1 μL of each gene-specific primer (10 μM), 1.6 μL dNTP (TIANGEN, Beijing, China), 0.1 μL Taq DNA polymerase (TIANGEN, Beijing, China), and 13.3 μL ddH₂O. The amplifications were performed at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, 59°C for 30 s and elongation at 72°C for 2.5 min, 72°C for 5 min. PCR product was electrophoresed on 1% agarose gel and observed by Bio-Rad gel imager.

The SPSS version 17.0 software was used for statistical analysis. The data between two treatments were compared and statistically analyzed through the Student t-test. The data among multiple treatments were compared and statistically analyzed though one-way analysis of variance and Student–Newman–Keuls test.

To test whether NtPR-Q is involved in the plant immune response against R. solanacearum, the transcriptional expression levels of NtPR-Q in WT-Yunyan87 was determined by RT-qPCR after inoculation with R. solanacearum strain CQPS-1 (Figure 1A). Compared with 0 hpi, NtPR-Q transcripts levels were enhanced in tobacco leaves starting at 1 dpi, and reached the maximum at 3 dpi. Subsequently, at 5 dpi, the NtPR-Q transcript levels decreased to the level of 1 dpi and maintained until 7 dpi. These results showed that transcripts of NtPR-Q accumulate in response to inoculation with R. solanacearum, suggesting NtPR-Q might be involved in defense reactions of tobacco to R. solanacearum.

Phytohormones are important signaling molecules that play an important role in the regulation of plant defense reactions against external stresses and mediating the expression of downstream defense genes (Fujita et al., 2006). To evaluate the possible involvement of NtPR-Q in the hormone signaling pathways, NtPR-Q transcript levels were determined by RT-qPCR in tobacco leaves after exogenous treatments with SA, MeJA, and ethephon. SA application enhanced the NtPR-Q transcript levels between 0 and 24 h, reaching the maximum at approximately 6 h (Figure 1B). In response to MeJA, the transcript levels of NtPR-Q was reduced at 3 h, but increased to the maximal level at 6 h and then returned to the basal level at 24 h (Figure 1C). After ethephon treatment, the NtPR-Q transcript levels increased between 0 and 24 h (Figure 1D). The transcript levels of NtPR-Q after the application of ethephon were the highest of the three treatments.

Differences in expression of NtPR-Q in response to R. solanacearum inoculation and the treatment of hormones suggested a role for this gene in plant immunity. To test this possibility, we established transgenic tobacco NtPR-Q-OE under the control of the 35S promoter in the pVCT2024 vector (Figure 2A). PCR analysis was used to verify the T-DNA insertion in transgenic tobacco lines (Figure 2B). The NtPR-Q transcript levels in NtPR-Q-OE and WT-Yunyan87 plants were determined in 10 independent transgenic T1 tobacco lines via RT-qPCR. The transgenic lines 8 and 9 exhibited higher levels of NtPR-Q mRNA than other NtPR-Q-OE lines and were therefore used for subsequent experiments (Figure 2C). We did not observe any differences in the growth phenotype between the NtPR-Q-OE transgenic plants and WT-Yunyan87 plants (Figure 2D).

NtPR-Q-OE and WT-Yunyan87 tobacco plants were infected by highly virulent R. solanacearum strain CQPS-1 via root irrigation. At 15 dpi, we observed clear wilting symptoms on a majority of WT-Yunyan87 plants, whereas only a few NtPR-Q-OE tobacco plants exhibited slight disease symptom (Figure 3A). At 10, 14, 18, and 22 dpi, the disease indexes of NtPR-Q-OE tobacco plants were approximately 0.06, 0.28, 0.55, and 1.11, respectively, but more intense wilting symptoms were observed in the WT-Yunyan87 plants, the disease indexes were approximately 1.56, 2.61, 3.33, and 3.89, respectively (Figure 3B). To measure the bacterial growth, WT-Yunyan87 plants and NtPR-Q-OE tobacco were inoculated by 20 μL inoculum (1 × 10⁸ CFU mL^-1) into the third leaves. The results showed that the amount of R. solanacearum in the leaves of WT-Yunyan87 plants was significantly higher than NtPR-Q-OE tobacco plants at 0, 1, 3, and 5 dpi (Figure 3C).

To further affirm the role of NtPR-Q in disease resistance and explore the possible mechanism that how NtPR-Q overexpression enhanced tobacco resistance to R. solanacearum, the transcriptional expression of defense-related genes in NtPR-Q-OE plants were investigated by RT-qPCR. We examined the transcript levels of the SA-responsive genes NtPR1a/c, NtPR2, and NtCHN50 (Brogue et al., 1991; Ward et al., 1991a), the JA-responsive gene NtPR1b (Sohn et al., 2007), ET production-associated genes NtACC Oxidase and NtEFE26 (Chen et al., 2003), the HR-associated genes NtHSR201 and NtHIN1 (Sohn et al., 2007) and the reactive oxygen species (ROS) detoxification-associated genes NtGST1 and NtCAT1 (Takahashi and Nagata, 1992; Takahashi et al., 1997). All the genes were shown to be up-regulated in response to pathogen infection (Rizhsky et al., 2002; Sohn et al., 2007; Chen et al., 2012). The results of RT-qPCR showed that the transcript levels of these genes were significantly increased in NtPR-Q-OE plants except for NtCAT1 (Figure 4).

The transcriptional profiles of defense-related genes in R. solanacearum-infected tobacco NtPR-Q-OE8, NtPR-Q-OE9, and WT-Yunyan87 were investigated by RT-qPCR (Figure 5). After R. solanacearum infection, the transcript levels of HR- (NtHIN1), SA- (NtPR1a/c, NtPR2 and NtCHN50), ET- (NtEFE26, NtACC Oxidase), JA- (NtPR1b) and ROS- (NtGST1) associated genes in NtPR-Q-OE plants were higher than in WT-Yunyan87 plants at least at three of the four tested timepoints. While, expression levels of NtHIN1 in WT-Yunyan87 and NtPR-Q-OE plants were similar.

Salicylic acid, MeJA, and ethephon application led to an increase in NtPR-Q transcript levels (Figure 1). To assess the capacity of them in the resistance of tobacco to R. solanacearum, the disease index was surveyed after treatment with 2 mM SA, 0.1 mM MeJA, and 7 mM ethephon. The results showed that the disease index after MeJA treatment was lower than control but the difference was not significant. Tobacco plants treated with SA were significantly more resistant than the control. Ethephon application also obvious enhanced the plants resistance at 10–22 days but had no significant effect at final stage. We found that the application of these hormones enhanced the resistance of tobacco to R. solanacearum, with the effect of SA application being the best (Table 2).