Genome-Wide Identification of the NPR1-like Gene Family in Solanum tuberosum and Functional Characterization of StNPR1 in Resistance to Ralstonia solanacearum

The NPR1 (nonexpressor of pathogenesis-related genes 1) gene is an activator of the systemic acquisition of resistance (SAR) in plants and is one of the central factors in their response to pathogenic bacterial infestation, playing an important role in plant disease resistance. Potato (Solanum tuberosum) is a crucial non-grain crop that has been extensively studied. However, the identification and analysis of the NPR1-like gene within potato have not been understood well. In this study, a total of six NPR1-like proteins were identified in potato, and phylogenetic analysis showed that the six NPR1-like proteins in Solanum tuberosum could be divided into three major groups with NPR1-related proteins from Arabidopsis thaliana and other plants. Analysis of the exon–intron patterns and protein domains of the six NPR1-like genes from potato showed that the exon–intron patterns and protein domains of the NPR1-like genes belonging to the same Arabidopsis thaliana subfamily were similar. By performing quantitative real-time PCR (qRT-PCR) analysis, we found that six NPR1-like proteins have different expression patterns in different potato tissues. In addition, the expression of three StNPR1 genes was significantly downregulated after being infected by Ralstonia solanacearum (RS), while the difference in the expression of StNPR2/3 was insignificant. We also established potato StNPR1 overexpression lines that showed a significantly increased resistance to R. solanacearum and elevated activities of chitinase, β-1,3-glucanase, and phenylalanine deaminase. Increased peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) activities, as well as decreased hydrogen peroxide, regulated the dynamic balance of reactive oxygen species (ROS) in the StNPR1 overexpression lines. The transgenic plants activated the expression of the genes associated with the Salicylic acid (SA) defense response but suppressed the expression of the genes associated with Jasmonic acid (JA) signaling. This resulted in resistance to Ralstonia solanacearum.


Introduction
Potato is a crop of the Solanum genus and is considered a vegetable and food crop in many countries because of its high nutritional content [1]. In recent years, China has become one of the leading potato producers in the world in terms of its production and planting area [2]. Bacterial wilt (BW) caused by Ralstonia solanacearum (RS) is one of the major diseases that occurs in potato production, and can cause massive yield losses in potato, up to 80% in severe cases [3]. R. solanacearum is a pathogen that damages the vascular tissue system of plants by invading the root xylem and spreading to the stem potato resistance to R. solanacearum stress, which can provide a new method for the control of R. solanacearum.

Bioinformatics Analysis of StNPR1
The potato reference genome was from the Spud DB database (http://spuddb.uga. edu/, accessed on 13 October 2022). A hidden Markov model (HMM) of the NPR1 family members was built using the conserved domains of BTB/POZ (PF00651) and ankyrin repeat (PF00023) derived from the Pfam database (https://pfam.xfam.org/, accessed on 6 December 2022) and searched against the genome of potato. NPR1 family members were then screened and identified using the NCBI-CDD website. Using MEGA7.0 software, the amino acid evolutionary tree was created via the neighbor-joining process [23].

Plant Growth and Disease Treatment
The potato (Atlantic variety) used in this study was saved and provided by the College of Life Sciences, Northeast Agricultural University. The potato culture condition was 28 • C, and the light cycle was 16/8 h. Potato roots, stems, leaves, flowers, and tubers were collected as samples, quick-frozen in liquid nitrogen, and stored in a −80 • C refrigerator. The pathogenic strain of the bacterial treatment was the Ralstonia solanacearum (RS) strain PO41 (minor species 3) (biovar 2). The R. solanacearum bacterial suspension (OD 600 = 0.8) was used to treat potato seedlings at the 8-9-leaf stage and inoculated using root inoculation bacteria [10]. We used water as the control. Potato leaves were sampled 0, 3, 6, 12, and 24 h after pathogen treatment, quick-frozen in liquid nitrogen, and stored in a −80 • C refrigerator for later use.

qRT-PCR
The transcription levels of six StNPR1-like genes in different potato tissues and their responses to biological stress were determined via qRT-PCR. In order to perform qRT-PCR, total RNA was extracted using Trizol according to the instructions of the plant RNA extraction kit (Takara, Dalian, China). The removal of genomic DNA contamination was performed using the Ambion Turbo DNase kit (Life Technology, Carlsbad, CA, USA). A Reverse Transcription System (Promega, Madison, WI, USA) was used to reverse transcribe the extracted total RNA to synthesize cDNA. The PCR reaction was performed using the actin gene as the internal reference gene (GenBank accession number: X55747). Then, we calculated the relative expression levels of the target gene and the internal reference gene using the 2 −∆∆CT method [24]. The primers used in this study are listed in Supplementary Table S1.

Subcellular Localization of StNPR1 Protein
Using a pretest, we selected the StNPR1 gene-which is upregulated in response to biological stress-as the research object and obtained the full length of the StNPR1 gene using the cDNA extracted previously via PCR. StNPR1 was cloned into a pCambia1302::eGFP vector using the homologous recombination method to generate a STNPR1-eGFP fusion protein. The 35S::eGFP and 35S::StNPR1-eGFP were transferred into Agrobacterium tumefaciens LBA4404 using the liquid nitrogen freeze-thaw method. We determined the subcellular localization of StNPR1 via transient transfection into the leaf epidermal cells of 1-month-old Nicotiana benthamiana. Subcellular localization experiments were conducted according to Chen et al. [25]. The transformed tobacco leaf epidermal cells were observed via confocal microscopy.

Overexpression of StNPR1 in Potato
The 35S::STNPR1-GUS vector was constructed via homologous recombination and by connecting StNPR1 to the PBI121 vector. The T-DNA region of the vector contained a Kanamycin sulfate resistance gene, which could be used for screening transgenic plants.
The 35S::StNPR1-GUS vector was transformed into Agrobacterium tumefaciens LBA4404 via the freeze-thawing process. The micropotato genetic transformation system was used to cut the micropotato (0.5-1.0 cm in diameter) into slices approximately 2 mm thick. The potato tuber slices were infected with the above Agrobacterium suspension (OD 600 = 0.5) for 7 min. The infected potato tuber slices were co-cultured in MS medium (1 mg/L of 6-Benzylaminopurine + 1.5 mg/L of Indole-acetic acid + 50 µmol/L of Acetosyringone) at 27 • C in the dark for 2 days. The potato tuber slices were then transferred to MS differentiation medium (1.0 mg/L of 6-Benzylaminopurine + 1.0 mg/L of Indole-acetic acid + 2 mg/L of Zeatin + 50 mg/L of Kanamycin + 250 mg/L of Ceftiofur). When the regenerated seedlings grew to 1-2 cm, they were placed in MS medium (50 mg/L Chlormequat chloride + 75 mg/L Kanamycin + 250 mg/L Ceftiofur) for rooting selection in order to obtain transgenic plants. GUS gene-specific primers were used for PCR identification. The expression of the StNPR1 gene in transgenic potato was detected using RT-qPCR. The process of RNA extraction and cDNA synthesis is described above.

Analysis of Resistance of StNPR1 Transgenic Plants to R. solanacearum
The StNPR1-overexpressed potato and wild-type potato were placed in a greenhouse for 60 days. Potato leaves were cultured in vitro at 28 • C with a constant temperature, and the light cycle was 16/8 h. Isolated potato leaves were inoculated with 20 uL of the R. solanacearum bacterial suspension (OD 600 = 0.8) to spread the bacteria into the veins. The disease symptoms in the isolated leaves of the overexpressed and wild-type plants were compared between days 1 and 4 after inoculation with the pathogen [10]. The apoptotic cells affected by bacteria were observed using the trypan blue staining method, and samples were taken at the same time [26]. Then, the cells were quickly frozen in liquid nitrogen and stored in the refrigerator at −80 • C for use.

Statistical Analysis
The expression analysis data were statistically analyzed for three biological replicates and three technical replicates, and the data are shown as the mean ± standard deviation. Statistical analysis was performed using SPSS 20.0. The difference was statistically significant with a probability (p) value ≤ 0.05, as determined by t-test.

Identification, Phylogeny, and Characterization of Potato NPR1-like Genes
The conserved BTB/POZ (PF00651) and ankyrin repeat (PF00023) structural domains of the NPR1 family members were identified via Blast search and HMMER analysis in the potato genome database and then screened by using pfam and the NCBI-CDD website to remove the duplicate sequences and structural domain variants. A total of six NPR1 family members were identified in potato and named StNPR1-6 ( Table S2). The amino acid sequence encoded by StNPR4 was the shortest (461aa), and the amino acid sequence encoded by StNPR1 was the longest (627aa). The molecular weight of the NPR1 family ranged from 50,646.89 kDa to 69,593.25 kDa, while the theoretical isoelectric point ranged from 5.66 to 6.46, indicating that the proteins encoded by these six members were weakly acidic. The SPUD DB website was used to analyze the chromosome location of the potato NPR1 gene family members, and two genes were found on chromosome 7, namely StNPR1 and StNPR2. StNPR3 was distributed on chromosome 2, and the StNPR4 gene was distributed on chromosome 4. Meanwhile, StNPR5 and StNPR6 were distributed on chromosome 10 (Table S2).
We analyzed the evolutionary relationships of the NPR1 family members, and the results showed that the NPR1 family members could be classified into three subclasses (I, II, and III). Among them, subclass I's yellow area included StNPR1, Arabidopsis thaliana NPR1/2, Nicotiana tabacum NPR1, Ipomoea batatas NPR1, Solanum lycopersicum NPR1, and other proteins, indicating that they are evolutionarily close and may have a common origin. Additionally, AtNPR1 was shown to play an important role in improving plant disease resistance [36]. In subclass II's dark-red area, StNPR2 and StNPR3 were in the same branch with AtNPR3/4, Oryza sativa NPR3, Zea mays NPR1, and other proteins, suggesting that the StNPR2 and StNPR3 genes may be involved in the negative regulation of SAR [37]. In branch III's light-blue area, StNPR4, StNPR5, and StNPR6, three members of potato, were in the same branch as AtNPR5/6, NtBOP3/4, and OsNPR5. Additionally, it was speculated that these seven genes may be related to plant organ development [16] (Figure 1). Overall, the NPR1 gene was relatively conserved during evolution.
remove the duplicate sequences and structural domain variants. A total of six NPR1 family members were identified in potato and named StNPR1-6 ( Table S2). The amino acid sequence encoded by StNPR4 was the shortest (461aa), and the amino acid sequence encoded by StNPR1 was the longest (627aa). The molecular weight of the NPR1 family ranged from 50,646.89 kDa to 69,593.25 kDa, while the theoretical isoelectric point ranged from 5.66 to 6.46, indicating that the proteins encoded by these six members were weakly acidic. The SPUD DB website was used to analyze the chromosome location of the potato NPR1 gene family members, and two genes were found on chromosome 7, namely StNPR1 and StNPR2. StNPR3 was distributed on chromosome 2, and the StNPR4 gene was distributed on chromosome 4. Meanwhile, StNPR5 and StNPR6 were distributed on chromosome 10 (Table S2).
We analyzed the evolutionary relationships of the NPR1 family members, and the results showed that the NPR1 family members could be classified into three subclasses (I, II, and III). Among them, subclass I's yellow area included StNPR1, Arabidopsis thaliana NPR1/2, Nicotiana tabacum NPR1, Ipomoea batatas NPR1, Solanum lycopersicum NPR1, and other proteins, indicating that they are evolutionarily close and may have a common origin. Additionally, AtNPR1 was shown to play an important role in improving plant disease resistance [36]. In subclass Ⅱ's dark-red area, StNPR2 and StNPR3 were in the same branch with AtNPR3/4, Oryza sativa NPR3, Zea mays NPR1, and other proteins, suggesting that the StNPR2 and StNPR3 genes may be involved in the negative regulation of SAR [37]. In branch Ⅲ's light-blue area, StNPR4, StNPR5, and StNPR6, three members of potato, were in the same branch as AtNPR5/6, NtBOP3/4, and OsNPR5. Additionally, it was speculated that these seven genes may be related to plant organ development [16] (Figure 1). Overall, the NPR1 gene was relatively conserved during evolution.  (Table S3); and the three evolutionary branches are distinguished by different colors. The red triangle represents the position of the potato NPR1-related proteins in the three branches of the phylogenetic tree.  (Table S3); and the three evolutionary branches are distinguished by different colors. The red triangle represents the position of the potato NPR1-related proteins in the three branches of the phylogenetic tree.

Sequence and Structural Analysis of StNPR1-like Genes and Proteins
To further explore the potential functions of the StNPR1-like gene family, the structural features and nucleotide sequences of the six potato NPR genes were systematically analyzed. The number of introns in this family was 1-4, among which, StNPR1 was divided into five segments by four introns. Both StNPR2 and StNPR3 were divided into four fragments by three introns. StNPR4, StNPR5, and StNPR6 consisted of one intron and two exons (

Expression Analysis of StNPR1-like Genes in Different Tissues and Organs
In order to investigate the tissue-specific expression of the StNPR1-like gene members in potatoes, the expression patterns were analyzed via qRT-PCR in different potato tissues (roots, stems, leaves, flowers, and tubers) ( Figure 3). The results showed that the StNPR1-like genes were expressed in potato roots, above-ground stems, leaves, flowers, and tubers, with significant differences in expression in different organs. StNPR1 was expressed at moderate levels, with higher expression levels in the leaf and stolon tissues. StNPR2 had the greatest expression level in the leaves and tubers. StNPR3 was highly expressed in the leaves, followed by tubers. StNPR4 had the highest expression level in the flowers. The expression of StNPR5 in the tubers was significantly higher than that of other genes in this family. StNPR6 showed strong expression levels in the stems, leaves, and tubers, but minor expression in other organs. In order to further clarify the biological conservation of the potato StNPR protein motifs, domains, amino acid residue mutation sites, and protein functional residues, multiple sequence alignment was performed on the six StNPRs and Arabidopsis thaliana (AtNPR1 to AtNPR6), Oryza sativa (OsNPR1), and Nicotiana tabacum (NtNPR1) NPRs with defined function protein sequences ( Figure 2B). The amino acid sequence mutation sites npr1-1 (His334Tyr), npr1-2 (Cys150tyr), and nim1-2 (His300tyr) of the Arabidopsis AtNPR1 allele mutants were highly conserved among the six StNPR, OsNPR1, and NtNPR1 proteins. The three cysteine residues (Cys82, Cys156, and Cys216) associated with oligomer formation in the AtNPR1 protein and the cysteine residues Cys82, Cys156, and Cys216 in the six StNPRs proteins were highly conserved. In addition, the AtNPR3/4 proteins contained the EAR motif (VDLNETP), which is essential for their transcriptional repression [14]. This transcriptional repressor motif is also intact in the StNPR3 protein. In conclusion, Arabidopsis and potato NPR1-like family members that are clustered in the same evolutionary branch share similar gene structures, conserved structural domain types, and positional arrangements, and are also highly conserved at key amino acid sites; presumably, their homologous genes have similar biological functions in potato.

Expression Analysis of StNPR1-like Genes in Different Tissues and Organs
In order to investigate the tissue-specific expression of the StNPR1-like gene members in potatoes, the expression patterns were analyzed via qRT-PCR in different potato tissues (roots, stems, leaves, flowers, and tubers) (Figure 3). The results showed that the StNPR1like genes were expressed in potato roots, above-ground stems, leaves, flowers, and tubers, with significant differences in expression in different organs. StNPR1 was expressed at moderate levels, with higher expression levels in the leaf and stolon tissues. StNPR2 had the greatest expression level in the leaves and tubers. StNPR3 was highly expressed in the leaves, followed by tubers. StNPR4 had the highest expression level in the flowers. The expression of StNPR5 in the tubers was significantly higher than that of other genes in this family. StNPR6 showed strong expression levels in the stems, leaves, and tubers, but minor expression in other organs.

Expression Analysis of StNPR1-like Genes under Biotic stress
To investigate the potential functions of the StNPR1-like genes in response to biological stress, the pathogenic bacterium R. solanacearum was used to treat potatoes, and the expression of the StNPR1-like genes was detected via qRT-PCR after pathogen stress ( Figure 4). The expression of the StNPR1 gene increased first and then decreased after treatment with R. solanacearum, and significantly different expression levels were observed between 3 and 12 h. The StNPR2 gene was significantly expressed at 6 h. The StNPR3 gene was only differentially expressed at 24 h. The expression of the StNPR4 gene first decreased and then increased, and the expression was only significantly different at 6 h. The expression of StNPR5 and StNPR6 genes showed a downward trend, and significant differences in the StNPR5 gene expression were observed between 3 h

Expression Analysis of StNPR1-like Genes under Biotic Stress
To investigate the potential functions of the StNPR1-like genes in response to biological stress, the pathogenic bacterium R. solanacearum was used to treat potatoes, and the expression of the StNPR1-like genes was detected via qRT-PCR after pathogen stress (Figure 4). The expression of the StNPR1 gene increased first and then decreased after treatment with R. solanacearum, and significantly different expression levels were observed between 3 and 12 h. The StNPR2 gene was significantly expressed at 6 h. The StNPR3 gene was only differentially expressed at 24 h. The expression of the StNPR4 gene first decreased and then increased, and the expression was only significantly different at 6 h. The expression of StNPR5 and StNPR6 genes showed a downward trend, and significant differences in the StNPR5 gene expression were observed between 3 h and 24 h. At 12 h, no statistically significant differences were observed in the expression levels of four genes, namely StNPR2, StNPR 3, StNPR4, and StNPR 6, while StNPR1 was significantly upregulated. Taken together, the StNPR1 gene, upregulated in response to pathogenic bacterial stress, was selected as the next research object to verify its biological function.  The R. solanacearum bacterial suspension was used to treat potato seedlings at the 8-9-leaf stage and water was used as the control (CK). * indicates significant differences in t-tests (* p < 0.05, ** p < 0.01).

Subcellular Localization of StNPR1 Gene in Tobacco
Sequence analysis showed that StNPR1 contains a nuclear localization signal (NLS) at the C-terminal of the StNPR1 amino acid sequence, and that the STNPR1 protein may be localized in the nucleus. We performed the subcellular localization of the StNPR1 protein using Nicotiana benthamiana leaves as a model and then observed it under a fluorescent microscope. The StNPR1-EGFP fusion protein was localized in the nucleus, while the EGFP, as the control protein, showed fluorescence throughout the cells. These results indicate that the StNPR1 protein is localized in the nucleus ( Figure 5). Real-time quantitative PCR (qRT PCR) to validate the relative expression of NPR1 homologous genes in potatoes infected with R. solanacearum. The R. solanacearum bacterial suspension was used to treat potato seedlings at the 8-9-leaf stage and water was used as the control (CK). * indicates significant differences in t-tests (* p < 0.05, ** p < 0.01).

Subcellular Localization of StNPR1 Gene in Tobacco
Sequence analysis showed that StNPR1 contains a nuclear localization signal (NLS) at the C-terminal of the StNPR1 amino acid sequence, and that the STNPR1 protein may be localized in the nucleus. We performed the subcellular localization of the StNPR1 protein using Nicotiana benthamiana leaves as a model and then observed it under a fluorescent microscope. The StNPR1-EGFP fusion protein was localized in the nucleus, while the EGFP, as the control protein, showed fluorescence throughout the cells. These results indicate that the StNPR1 protein is localized in the nucleus ( Figure 5).

Overexpression of the StNPR1 Gene Enhances Potato Resistance to R. solanacearum
To further investigate the function of the StNPR1 gene in potato disease resistan two StNPR1 transgenic plants were obtained using the Agrobacterium tumefaciens-infect potatoes micropotato transformation method. Further analysis was conducted on ove expressed 1 (OE-1) and overexpressed 3 (OE-3) plants ( Figure 6B). The isolated leaves transgenic plants were inoculated with the pathogen R. solanacearum to observe an measure the disease spots. The results showed that the lesions on OE-1 and OE-3 leav were significantly smaller than those on the wild type ( Figure 6C). Trypan blue dye w used to stain the leaves of the overexpressed potato, and the wild-type potato was ino ulated with R. solanacearum to observe the cell death of different plants. The number cell deaths in the leaves of both the OE-1 and OE-3 overexpression lines was significant lower than that of WT at different times after R. solanacearum treatment, which was co sistent with the spot area results ( Figure 6D,E). The differences in the disease symptom between the leaves of the transgenic and wild-type plants were further confirmed.

Overexpression of the StNPR1 Gene Enhances Potato Resistance to R. solanacearum
To further investigate the function of the StNPR1 gene in potato disease resistance, two StNPR1 transgenic plants were obtained using the Agrobacterium tumefaciens-infected potatoes micropotato transformation method. Further analysis was conducted on overexpressed 1 (OE-1) and overexpressed 3 (OE-3) plants ( Figure 6B). The isolated leaves of transgenic plants were inoculated with the pathogen R. solanacearum to observe and measure the disease spots. The results showed that the lesions on OE-1 and OE-3 leaves were significantly smaller than those on the wild type ( Figure 6C). Trypan blue dye was used to stain the leaves of the overexpressed potato, and the wild-type potato was inoculated with R. solanacearum to observe the cell death of different plants. The number of cell deaths in the leaves of both the OE-1 and OE-3 overexpression lines was significantly lower than that of WT at different times after R. solanacearum treatment, which was consistent with the spot area results (Figure 6D,E). The differences in the disease symptoms between the leaves of the transgenic and wild-type plants were further confirmed.

Effect of StNPR1 Overexpression on Defensive Enzyme Activities
To further analyze the enhanced resistance of the StNPR1-overexpression potato to R. solanacearum, the activities of the defense enzymes CHI, GLU, PPO, and PAL in transgenic and wild-type plants were analyzed 0, 1, 2, 3, and 4 dpi. The results showed that the CHI activity of the OE-1 and OE-3 overexpression lines was significantly higher than that of the WT at 2, 3, and 4 dpi (Figure 7). The GLU activity of OE1 and OE-3 was

Effect of StNPR1 Overexpression on Defensive Enzyme Activities
To further analyze the enhanced resistance of the StNPR1-overexpression potato to R. solanacearum, the activities of the defense enzymes CHI, GLU, PPO, and PAL in transgenic and wild-type plants were analyzed 0, 1, 2, 3, and 4 dpi. The results showed that the CHI activity of the OE-1 and OE-3 overexpression lines was significantly higher than that of the WT at 2, 3, and 4 dpi (Figure 7). The GLU activity of OE1 and OE-3 was significantly higher than that of the WT, and the GLU activity of OE-1 and OE-3 reached a peak at 3 dpi. The PAL activity of the overexpression lines was also significantly higher than that of WT, and their PAL activity showed an ascending-descending-ascending-descending trend. The PPO enzyme activity in the overexpression lines increased rapidly to a peak at 2 dpi and then gradually decreased. Additionally, the difference in PPO activity between the overexpression lines and wild-type plants was significant. In conclusion, the overexpression of the StNPR1 gene enhanced the activities of CHI, GLU, PPO, and PAL, which may be related to potato's resistance to R. solanacearum.
Genes 2023, 14, x FOR PEER REVIEW 12 of 18 than that of WT, and their PAL activity showed an ascending-descending-ascendingdescending trend. The PPO enzyme activity in the overexpression lines increased rapidly to a peak at 2 dpi and then gradually decreased. Additionally, the difference in PPO activity between the overexpression lines and wild-type plants was significant. In conclusion, the overexpression of the StNPR1 gene enhanced the activities of CHI, GLU, PPO, and PAL, which may be related to potato's resistance to R. solanacearum.

Effect of Overexpression of StNPR1 on ROS Content and Antioxidant Enzyme Activity
In order to explore the effect of StNPR1 overexpression on the content of reactive oxygen species and the activities of antioxidant enzymes in potato plants, the H2O2 content and the activities of SOD, POD, and CAT in leaves inoculated with R. solanacearum were measured. As shown, the H2O2 accumulation in transgenic plants was significantly lower compared to wild-type plants after inoculation with the pathogen (Figure 8). The CAT and SOD activities of both the overexpression and wild-type lines showed a trend of increasing and then decreasing, with a peak at 2 dpi, and the CAT and SOD activities of the transgenic lines were significantly higher than those of the wild-type plants. Compared with the wild-type plants, the POD enzyme activity of the transgenic plants was

Effect of Overexpression of StNPR1 on ROS Content and Antioxidant Enzyme Activity
In order to explore the effect of StNPR1 overexpression on the content of reactive oxygen species and the activities of antioxidant enzymes in potato plants, the H 2 O 2 content and the activities of SOD, POD, and CAT in leaves inoculated with R. solanacearum were measured. As shown, the H 2 O 2 accumulation in transgenic plants was significantly lower compared to wild-type plants after inoculation with the pathogen (Figure 8). The CAT and SOD activities of both the overexpression and wild-type lines showed a trend of increasing and then decreasing, with a peak at 2 dpi, and the CAT and SOD activities of the transgenic lines were significantly higher than those of the wild-type plants. Compared with the wild-type plants, the POD enzyme activity of the transgenic plants was higher, and the difference was significant. In summary, the results showed that the transgenic plants had lower levels of reactive oxygen species accumulation and higher SOD, CAT, and POD enzyme activities than the wild-type plants under the same pathogen infection conditions.

Effect of Transgenic Potato Overexpression of StNPR1 on SA/JA-Signaling-Related Genes
To investigate the StNPR1-gene-mediated defense response, a transcriptional analysis of the SA and JA signaling-pathway-related genes was performed on transgenic lines and wild-type plants after pathogen infection. The results showed that the transcription levels of StPR1, the NPR1 downstream transcription factor StWRKY70, and the phenylalanine ammonia-lyase gene StPAL were significantly increased in the OE-1 and OE-3 transgenic plants compared with the WT plants. In contrast, the expression levels of StICS1 and StPAD4 were not significantly different in the transgenic lines and WT plants. The JA biosynthetic gene AOS and the JA response gene VSP1 showed similar expression patterns to BnPDF1.2. These results suggest that the overexpression of the StNPR1 gene activates the expression of SA defense genes and suppresses the JA defense response after R. solanacearum infection (Figure 9).

Discussion
The NPR1 gene plays a central regulatory role in plant disease resistance. It can improve the ability of plants to resist a variety of diseases, and it is a cross-site between a variety of disease resistance pathways in plants [6]. Members of the NPR1 family have been isolated and identified in an increasing number of plants, with six members identified in Arabidopsis thaliana, four in rice, nine in poplar [38], and five in Persea americana [39]. In this study, we identified six NPR1-like genes in the potato genome and con-

Discussion
The NPR1 gene plays a central regulatory role in plant disease resistance. It can improve the ability of plants to resist a variety of diseases, and it is a cross-site between a variety of disease resistance pathways in plants [6]. Members of the NPR1 family have been isolated and identified in an increasing number of plants, with six members identified in Arabidopsis thaliana, four in rice, nine in poplar [38], and five in Persea americana [39]. In this study, we identified six NPR1-like genes in the potato genome and constructed a phylogenetic tree of NPR domains in potato, poplar, Arabidopsis, and rice. The NPR genes of potato were distributed in the three branches and were clearly divided into three subclasses with the six NPR1 family members of Arabidopsis thaliana, indicating that the potato gene family members may have a clear division of function.
The expression patterns of NPR1-like genes in different tissues have been reported in Arabidopsis [16] and wheat [40], but their tissue expression characteristics in potato have been rarely studied. In this study, the expression pattern of the NPR1-like gene was analyzed, and the results showed that the expression of the NPR1-like gene was tissue-specific. The expression of the NPR1-like gene was specific in StNPR3 in tubers and StNPR4 in flowers. These results indicate that they may play a special role in the growth and development of some organs, which is consistent with the tissue expression specificity of Arabidopsis thaliana NPR1-like genes [41]. The expression levels of StNPR4 and StNPR6 were similar in stems, leaves, and tubers, but minor in other organs. The results imply that StNPR4 and StNPR6 might have relevance to AtBop1/2 in plant growth and development [16,42].
Bacterial wilt induced by R. solanacearum is one of the main diseases of potato and is the most serious bacterial disease that seriously threatens the yield and quality of potato, but no effective control measures have been found yet [43]. In this study, the expression profile of the StNPR1-like gene was analyzed under treatment with R. solanacearum, and the expression of StNPR1 was significantly elevated after R. solanacearum treatment, while StNPR2/3 was not significantly changed under the disease stress. The downregulation of StNPR4/5/6 expression may be negatively regulated during infection, indicating that the StNPR1 gene may play an important role in R. solanacearum's defense. It has been reported that the constitutive expression of the AtNPR1 gene can induce disease resistance in rice, and the overexpression of the NPR1 gene in Arabidopsis thaliana and rice can improve their resistance to bacteria and fungi, indicating that NPR1 plays an extremely important role in the plant defense system. In this study, the StNPR1 gene was overexpressed in potato, and the transgenic plants showed a delayed onset of disease, a smaller lesion area, and a reduced number of dead cells. These results indicated that the overexpression of StNPR1 could improve the resistance of potato to R. solanacearum.
In the process of pathogen infection, the burst of ROS, mainly hydrogen peroxide and the superoxide radical anion, is related to the HR hypersensitivity reaction, which affects the acquisition of SAR. SOD, CAT, and POD play important roles in removing excessive ROS and oxidizing substances in cells. In this study, the H 2 O 2 concentration and the activities of CAT, SOD, and POD were determined in NPR1-overexpressed plants and WT plants. The content of H 2 O 2 in overexpressed plants was lower than that in wild-type plants, but the activities of SOD, CAT, and POD in overexpressed plants were higher than those in wild-type plants. CAT is an H 2 O 2 scavenger and plays an important role in H 2 O 2 scavenging [44], which resulted in a lower H 2 O 2 content in the overexpression plants compared to the WT plants. The excess of ROS and oxidized substances is unfavorable to plant growth. The increased activities of SOD, CAT, and POD help eliminate the excess production of ROS and oxidized substances, reduce oxidative stress during R. solanacearum infection, and maintain a dynamic balance that can not only produce ROS in order to resist exogenous infection, but also remove the excess ROS that will cause damage.
Salicylic acid and jasmonic acid mediate the basic signaling pathways of plant disease resistance and defense response. The SA signal transduction pathway is closely related to the JA signal transduction pathway, and the two pathways cooperate with each other and cross each other. Meanwhile, NPR1 is an important regulatory element at the intersection of multiple defense pathways. It can function as a receptor of SA and a positive regulator of SAR, which plays an important role in SA/JA signaling. Previous studies have shown that the SA signal transduction pathway has antagonistic effects with the JA and ET-mediated signal transduction pathways. The accumulation of SA can inhibit the expression of JA-regulated genes, and the occurrence of this antagonism also requires the participation of NPR1 [45]. The results of this study showed that the expression of disease-resistant genes related to the SA signal transduction pathway was enhanced in StNPR1-OE plants under R. solanacearum infection, and that the overexpression of StNPR1 led to the suppression of the expression of genes related to the JA signal pathway. StNPR1 positively regulated the SA defense response in the interaction between potato and R. solanacearum. In addition, NPR1 has been shown to inhibit JA-dependent defense responses in Arabidopsis thaliana.

Conclusions
In summary, this is the first time that six NPR1-like genes have been identified in potato and StNPR1-like genes have been analyzed in depth, including their molecular characteristics, chromosomal distribution, phylogenetic classification, gene structure, and amino acid residues. The expression of StNPR1-like genes was tissue-expression-specific. StNPR1 was significantly induced by R. solanacearum biological stress conditions. The StNPR1 protein subcellularly localized to the cytoplasm and depolymerized into monomers in the nucleus after exposed to pathogenic bacteria or SA treatment. Transgenic potatoes overexpressing StNPR1 showed a significantly enhanced resistance to R. solanacearum, possibly related to ROS accumulation and the SA-mediated defense response. Therefore, it is possible that StNPR1 could be an important candidate gene for genetic engineering in order to improve disease resistance.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/genes14061170/s1, Table S1: Primers used in this research; Table S2: Characteristics of potato NPR1-like gene family members; Table S3: Associated NPR1-like genes in various plants.