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Article

The Silencing of the StPAM16-1 Gene Enhanced the Resistance of Potato Plants to the Phytotoxin Thaxtomin A

by
Lu Liu
1,2,†,
Shuangwei Song
1,2,†,
Ning Liu
3,4,
Zhiqin Wang
1,2,
Yonglong Zhao
1,2,
Naiqin Zhong
1,2,5,
Pan Zhao
1,2,5,* and
Haiyun Wang
1,*
1
State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
2
Engineering Laboratory for Advanced Microbial Technology of Agriculture, Chinese Academy of Sciences, Beijing 100101, China
3
Beijing Vegetable Research Center (BVRC), Beijing Academy of Agriculture and Forestry Science, Beijing 100097, China
4
National Engineering Research Center for Vegetables (NERCV), State Key Laboratory of Vegetable Biobreeding, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China
5
Key Laboratory of Potato Industry Integration and Development Enterprises in Inner Mongolia Autonomous Region, Hulunbuir 021000, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2025, 26(3), 1361; https://doi.org/10.3390/ijms26031361
Submission received: 6 January 2025 / Revised: 26 January 2025 / Accepted: 27 January 2025 / Published: 6 February 2025
(This article belongs to the Special Issue Genetic Regulation of Plant Growth and Protection)
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Abstract

:
Potato common scab (CS) caused by Streptomyces scabiei is a severe disease that threatens tuber quality and its market value. To date, little is known about the mechanism regulating the resistance of potato to CS. In this study, we identified a presequence translocase-associated motor 16 gene from potato (designated StPAM16-1) that is involved in the response to the phytotoxin thaxtomin A (TA) secreted by S. scabiei. The StPAM16-1 protein was localized in the mitochondria, and the expression of the gene was upregulated in potato leaves treated with TA. The suppression of StPAM16-1 in potato led to enhanced resistance to TA and S. scabiei. Protein interaction analyses revealed that StPAM16-1 interacted with the subunit 5b of the COP9 signalosome complex (StCSN5). Similar to that of StPAM16-1, the expression levels of StCSN5 significantly increased in potato leaves treated with TA. These results indicated that StPAM16-1 acted as a negative regulator and was functionally associated with StCSN5 in the immune response of potato plants against CS. Our study sheds light on the molecular mechanism by which PAM16 participates in the plant immune response. Furthermore, both StPAM16-1 and StCSN5 could be potential target genes in the molecular breeding of potato cultivars with increased resistance to CS.

1. Introduction

Potato common scab (CS), caused by pathogenic Streptomyces spp., is a severe soil-borne disease worldwide [1]. The symptoms of CS include scab-like superficial, raised and/or pitted lesions at infection sites, which results in a decrease in the quality and marketability of potato tubers [2]. Previous studies revealed that the phytotoxin thaxtomin, which is secreted by pathogenic Streptomyces spp., including S. scabiei, is the key pathogenic factor of CS [3]. As the predominant pathogenic component of the thaxtomin family, thaxtomin A (TA) affects plant growth and development in various ways, such as by inhibiting plant cellulose biosynthesis [4] and seedling growth [5], causing dramatic cell hypertrophy [6], altering Ca2+ and H+ flow [7,8] and triggering programmed cell death (PCD) [9]. Although several approaches such as crop rotation, chemical treatment and biological control have been shown to alleviate this disease, control efficiency is limited [10]. Given that disease resistance in potato cultivars is the foundation of the integrated method, studying the genetics of tolerance in breeding is regarded as one of the best options for controlling CS. To date, the strongest source of resistance has been a Phureja group clone, and a major dominant resistance gene is in the populations of hybrids between Solanum tuberosum dihaploids and Phureja group clones [11,12]. Research on the genome evolution and diversity of wild and cultivated potatoes, as well as the graph pangenome [13,14,15], can provide critical insights into the potato genome and guide breeders in potato breeding. At present, the identification and functional characterization of defense-related genes to generate CS-resistant cultivars via molecular breeding are some of the desirable strategies for controlling CS.
The presequence translocase-associated protein import motor (PAM) subunit PAM16 is required for protein precursors to be translocated into the mitochondrial matrix [16]. In Arabidopsis, AtPAM16 may play a negative role in plant immunity by importing a negative regulator of plant immunity to mitochondria to inhibit the overaccumulation of ROS, and a single Atpam16 mutant plant exhibited increased resistance to virulent pathogens [17]. Atpam16-1 and Atpam16-2 double-mutant plants showed enhanced SUPPRESSOR OF NPR1-1, CONSTITUTIVE 1 (SNC1)-mediated immunity [17]. The mechanism of non-target site resistance to the herbicide thaxtomin A conferred by the gene pam16 mutant in Marchantia polymorpha increases ROS levels or decreases thaxtomin A metabolism in weeds [18]. The homologous gene thaxtomin resistance 1 (TXR1) was first identified during the screening of a TA-resistant mutant of Arabidopsis [5]. The study revealed that the uptake of TA by txr1-transformed plants was lower than that by wild-type (WT) plants, but the metabolism of TA did not differ between the WT and txr1 mutant plants. These results implied that PAM16 (TXR1) likely plays an important role in plant responses to biotic stresses. In human cells, a protein homologous to yeast PAM16 named Magmas (mitochondria-associated granulocyte-macrophage colony-stimulating factor signaling molecule) was found to be an essential part of the mitochondrial precursor protein import machinery [19]. In addition, it was reported that Magmas can regulate cellular ROS levels by controlling ROS production and scavenging [20]. Therefore, it is of great interest to study how PAM16 regulates the plant immune system as a mitochondrial intima input protein and to analyze its physiological functions and mechanisms, for example, in potato responses to S. scabiei infection and TA treatment.
COP9 signalosome (CSN) is a conserved and nucleus-localized protein complex that is fractionated as a 450–550 kDa complex in gel filtration columns [21,22,23]. Studies have shown that CSN regulates the activity of CULLIN-RING E3 ubiquitin ligases (CRLs) by removing the ubiquitin-like protein RUB1 (related to ubiquitin 1 in plants)/NEDD8 (neural precursor cell-expressed developmentally downregulated-8 in animals), which interact with CRLs and thus affect the development and defense against pathogens and herbivorous insects [24]. CSN consists of eight subunits, among which CSN5 is a metalloprotease with a conserved JAMM (JAB1 MPN domain metalloprotease) motif and requires zinc ions as an activator [21,25]. CSN is widely present in eukaryotes. In plants, CSN plays an important role in regulating development and stress responses [26,27,28,29,30] and acts as an inhibitor of photomorphogenesis in Arabidopsis [31].
The identification and functional characterization of defense-related genes not only provide information for the interaction between potato and CS but also help in identifying candidate genes for molecular breeding to generate CS-resistant cultivars. In this study, we identified a phytotoxin TA-responsive gene, StPAM16-1, from tetraploid potato (Solanum tuberosum L.) cv. Shepody and found that the relative expression of StPAM16-1 was induced by TA treatment and that StPAM16-1 was localized in mitochondria. We characterized the function of StPAM16-1 in response to TA treatment by VIGS and RNAi. StPAM16-1 could interact with StCSN5 via yeast two-hybrid and split luciferase complementation assays. Our results demonstrated that StPAM16-1 acted as a negative regulator of the defense response to TA in potato. As the expression of StCSN5 was also induced by TA treatment, we speculated that the interaction between StPAM16-1 and StCSN5 may reflect a functional link between the two proteins in the immune response of potato to CS.

2. Results

2.1. Identification of StPAM16-1

A previous study reported that AtPAM16/AtTXR1 is a TA-responsive gene that plays a negative role in the defense process in Arabidopsis [5]. In the aim of improving the disease tolerance of potato plants through genetic manipulation, we selected PAM16 as the candidate gene and identified AtPAM16/AtTXR1 orthologs in the potato proteome via sequence alignment via the Potato Genomics Resource (http://solanaceae.plantbiology.msu.edu/; accessed on 15 November 2021) and NCBI databases (https://blast.ncbi.nlm.nih.gov/; accessed on 15 November 2021). A total of four proteins with high homology to AtPAM16/AtTXR1 were identified, among which the protein with the highest homology was named StPAM16-1 (PGSC0003DMP400044341). The other three homologous proteins were named StPAM16-2 (PGSC0003DMP400051461), StPAM16-3 (PGSC0003DMP400051472) and StPAM16-4 (PGSC0003DMP400026161), respectively. The sequence alignment and domain analysis of StPAM16-1, StPAM16-2, StPAM16-3, StPAM16-4, AtPAM16, AtPAM16L and ScPAM16 were carried out using DNAMAN. StPAM16-1 contains a highly conserved DnaJ domain (Figure 1a), which mediates chaperone binding [32]. Furthermore, the phylogenetic analysis of StPAM16-1 and its homologous proteins was carried out by MEGA 7.0. As shown in Figure 1b, PAM16 is present in various eukaryotes, and StPAM16-1 shares high homology with the PAM16 of S. lycopersicum.

2.2. StPAM16-1 Expression Is Induced by TA and Defense-Related Plant Hormones

To test whether StPAM16-1 participates in the response to TA in potato, StPAM16-1 transcript levels were determined by quantitative reverse transcription PCR (RT-qPCR). The results showed that the relative expression of StPAM16-1 was significantly upregulated at 6 h post-treatment and then gradually recovered to the background level (Figure 2a). Then, we examined the relative expression of StPAM16-1 after treatment with several defense-related phytohormones, including salicylic acid (SA), jasmonic acid (JA) and ethylene (ETH). The results showed that the expression of StPAM16-1 was upregulated after treatment with SA, JA and ETH (Figure 2b), with the highest enhancement occurring after SA treatment.
In addition, the relative expression of StPAM16-1 in different tissues was detected by RT-qPCR. As shown in Figure 2c, StPAM16-1 was expressed in all tissues but with relatively higher levels in the roots, tubers and stolons, which is closely related to its disease-related function.

2.3. StPAM16-1 Localizes to Mitochondria

To further understand the biological function of StPAM16-1, the subcellular distribution of the proteins was analyzed using a tobacco transient expression system. An StPAM16-1-GFP fusion gene driven by the 35S promoter was constructed in the vector and transformed into competent Agrobacterium cells. The transformants were then injected into tobacco leaf cells. As shown in Figure 3, StPAM16-1-GFP fusion proteins were colocalized with mitochondrial markers, indicating that StPAM16-1 is localized to this organelle.

2.4. Suppression of StPAM16-1 Expression Improves Tolerance of Potato Plants to Thaxtomin A and S. scabiei

To investigate the function of StPAM16-1 in response to TA, we first assessed the disease tolerance of potato plants with decreased StPAM16-1 expression levels via virus-induced gene silencing (VIGS). To test the efficiency of the VIGS system, StPDS, which encodes a 15-cis-phytoene desaturase, was used as a marker. Approximately 40 days after injection, the albino phenotype appeared on StPDS-silenced potato leaves, and StPDS silencing was verified by RT-qPCR, which confirmed the efficiency of VIGS (Figure 4a,b). Then, we constructed the VIGS vector pgR107-StPAM16-1 and transformed it into Agrobacterium. About 40 days after injection, total RNA was extracted from the leaves to determine the relative expression of StPAM16-1 via RT-qPCR. The results showed that the relative expression of StPAM16-1 was significantly lower in StPAM16-1-silenced potato leaves than in the control (Figure 4c). Subsequently, StPAM16-1-silenced and control potato plants were treated with 10 μM TA to test their tolerance. As shown in Figure 4d, there were more black necrotic spots on the control potato leaves than on the StPAM16-1-silenced potato leaves. Then, we used trypan blue to stain potato leaves treated with TA or sterile water and found that the necrosis of control potato leaves was more severe than that of StPAM16-1-silenced leaves. These results indicated that silencing StPAM16-1 increased the tolerance of potato plants to TA.
To further explore the effects of StPAM16-1 in response to TA, the expression of StPAM16-1 was suppressed by the RNAi approach, and an in-depth analysis was carried out. The pANDA35K-StPAM16-1 RNAi vector was constructed and subsequently transformed into potato plants via Agrobacterium-mediated transformation. The relative expression of StPAM16-1 in RNAi potato plants was measured by RT-qPCR. Three RNAi plants with different expression levels of StPAM16-1 were selected and named RNAi1, RNAi2 and RNAi3. As shown in Figure 5a, the relative expression levels of StPAM16-1 in the three StPAM16-1-RNAi plants were significantly lower than those in the WT plant, while the expression of StPAM16-2, StPAM16-3 and StPAM16-4 did not change in the WT and StPAM16-1-RNAi potato plants. Compared with the WT plants, the StPAM16-1-RNAi plants also exhibited a dwarf phenotype (Figure 5b). This finding is consistent with a previous report on Arabidopsis [5]. Therefore, StPAM16-1-RNAi plants were selected for further studies.
We treated WT and three StPAM16-1-RNAi potato leaves with 10 μM TA. As shown in Figure 5c, the tolerance of StPAM16-1-RNAi potato leaves to TA was significantly enhanced as compared to the WT control. Further, WT and StPAM16-1-RNAi plants grown for 3–4 weeks were inoculated with S. scabiei. Potato tubers were harvested after 3 months, and the disease incidence of tubers was counted. The results showed that there were a large number of scab patches on the WT tubers, while there were fewer scab patches on the StPAM16-1-RNAi tubers (Figure 5d). Meanwhile, the incidence rate, disease index and relative control effect of the WT and StPAM16-1-RNAi tubers were also calculated. Compared with those of the WT tubers, the incidence rate (Figure 5e) and disease index (Figure 5f) of the StPAM16-1-RNAi tubers significantly decreased, while the relative control effect clearly increased (Figure 5g). These results indicated that the inhibition of StPAM16-1 improved the tolerance of potato plants to S. scabiei.

2.5. StPAM16-1 Interacts with StCSN5

To elucidate the functional mechanism of StPAM16-1, the cDNA of potato leaves treated with TA was used to construct a yeast two-hybrid library. The cDNA was cloned into the yeast expression vector pGADT7 for prey protein screening, and StPAM16-1 cDNA was inserted into the pGBKT7 vector as bait. Four candidate proteins that interact with StPAM16-1 were initially screened from the yeast two-hybrid library (Table 1), including COP9 signalosome complex subunit 5b-like (StCSN5), anthocyanin-3-O-glucosyltransferase 2-like and fructose-diphosphate aldolase-like protein and the uncharacterized protein LOC102596406.
Among these four candidate proteins, CSN was reported to play an important role in regulating development and stress response [33]. Hence, we selected StCSN5 to verify the interaction between StPAM16-1. Yeast two-hybrid assays revealed a strong interaction between StPAM16-1 and StCSN5 (Figure 6a). Moreover, the interaction between StPAM16-1 and StCSN5 in plant cells was verified by the split luciferase complementation assay. As shown in Figure 6b, when an Agrobacterium culture containing cLUC-StPAM16-1/nLUC-StCSN5 vectors was injected into tobacco leaves, strong fluorescence was produced on the leaves. However, no fluorescence was detected in tobacco leaves injected with Agrobacterium cultures containing nLUC/cLUC-StPAM16-1, nLUC-StCSN5/cLUC or nLUC/cLUC. The results confirmed that StPAM16-1 and StCSN5 could interact in plant cells.

2.6. Expression of StCSN5 Is Significantly Induced by TA and Several Defense-Related Plant Hormones

Given that the expression of StPAM16-1 was induced by TA, we speculated that StCSN5 expression might also respond to TA treatment. To verify this hypothesis, RT-qPCR was used to analyze the relative expression of StCSN5 after TA treatment. Figure 6 shows that the transcription of StCSN5 was indeed induced by TA treatment and peaked at 6 hpi. This expression pattern of StCSN5 is similar to that of StPAM16-1. Hence, it can be speculated that the functions of these two genes are closely related to the responses of potato plants to TA and S. scabiei infection (Figure 7a).
Furthermore, we tested the expression of StCSN5 in potato plants after defense-related hormone treatment by RT-qPCR. The results showed that the expression of StCSN5 was induced by JA and ETH (Figure 7b) treatments.

3. Discussion

As a serious potato disease, CS has been one of the most prevalent concerns for farmers and industry [34]. In our previous study, we found that the phytotoxin TA secreted by pathogenic S. scabiei could trigger a series of host immune responses in potato [35], including changes in many metabolic pathways, enzymatic activities and ROS accumulation. Here, we identified a thaxtomin responsive gene from potato, StPAM16-1, that negatively regulates potato immune responses against TA and CS.
In this study, through a series of experiments, we showed that the relative expression of StPAM16-1 in potato leaves was induced by TA (Figure 2a) and that StPAM16-1-inhibited potato plants generated by VIGS and RNAi acquired increased resistance to TA and S. scabiei (Figure 4 and Figure 5). Plants are constantly confronted with the invasions of a variety of pathogens, including bacteria, fungi, viruses, nematodes and oomycetes [36]. To combat these attacks, plants have an evolved delicate and two-layered innate immune system, i.e., pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) [37]. Many plant proteins participate and play either positive or negative roles in these immune processes in different plant-pathogen interaction systems [38,39]. During the interaction between potato and pathogens, the expression of the StLysM-RLK05 gene was upregulated in tubers infected with S. scabiei [40], and the expression of StLysM-RLK05 was significantly upregulated in resistant tubers compared with susceptible tubers infected with S. scabiei, suggesting a positive regulatory role of the protein in the immune response. In another study, Tai et al. reported that the expression of two MYB and three bHLH genes was correlated with CS resistance, indicating that these genes may contribute to the defense response of potato against CS [41]. Similarly, in Arabidopsis thaliana, AtPAM16/AtTXR1 participated in the response to TA and the pathogens Pseudomonas syringae and Hyaloperonospora arabidopsidis Noco2 [5,17]. Above all, we believe that StPAM16-1, which is similar to AtPAM16, is a negative regulator of the immune response of potato plants to TA and S. scabiei infection.
Similar to the AtPAM16 protein [12], the subcellular distribution analysis revealed that StPAM16-1 is a mitochondria-localized protein (Figure 3). The translocation of mitochondrial preproteins was significantly decreased in the yeast pam16 mutant compared with the WT [16,42]. Similarly, a subunit of the Arabidopsis PAM complex, AtPAM16, was found to bind to the TIM23 complex and participate in translocating mitochondrial preproteins into the matrix [43,44]. Additionally, a previous study reported that TXR1 (PAM16) was a regulator of a transport system, and the increased resistance of the Arabidopsis mutant txr1 to TA was due to a decrease in the rate of toxin uptake [5]. In our study, we observed that the expression of StPAM16-1 was significantly induced by TA treatment, suggesting that upregulated StPAM16-1 might play the same role as AtTXR1 in facilitating the entry of TA toxins into potato cells. Based on this information, we speculated that StPAM16-1 plays a role in response to TA and S. scabiei possibly by translocating a preprotein into mitochondria. This preprotein could be TA or a mitochondrial preprotein related to TA. As TA is only a cyclic dipeptide molecule containing tryptophan and phenylalanine residues [45,46], it is likely that TA binds to mitochondrial preproteins and is co-translocated into the mitochondrial matrix. However, such a role of StPAM16-1 in the translocation of mitochondrial preproteins needs to be further assessed.
To investigate the functional mechanism of StPAM16-1, we screened four proteins that interact with StPAM16-1 from a yeast two-hybrid library and verified the interaction between StPAM16-1 and StCSN5 via yeast two-hybrid and split luciferase complementation assays (Figure 6). As the fifth subunit of CSN [47], CSN5 plays an important role in regulating plant growth, development and various abiotic or biotic stress tolerances. It interacts with CRLs to regulate protein ubiquitination and degradation by regulating the ubiquitin/26S proteasome system [33]. It also interacts with CRLs to regulate various plant hormone-related signal transduction pathways, such as auxin, jasmonic acid and salicylic acid signals [26,27,28,29]. In Arabidopsis, CSN5 interacts with the ascorbic acid (AsA) biosynthetic enzyme GDP-Man pyrophosphorylase (VTC1) to inhibit the synthesis of AsA, thus negatively regulating the salt stress tolerance of plants [30]. It also interacts with CRLs to assist in the plant response to cold stress through ubiquitination [48]. Cui et al. reported that the silencing of the CSN5 gene in susceptible grapevine enhanced resistance to powdery mildew [49]. The expression levels of the SA-associated marker genes PR1, PR3 and PAD4 were higher in the CSN5-RNAi plants and lower in the CSN5-overexpressing plants than in the WT plants.
Several studies have reported that CSN5 regulates plant resistance to pathogen and pest invasion by activating the JA pathway while inhibiting the SA pathway. Shang et al. [29] reported that tomato CSN4 and CSN5 play critical positive roles in resistance against root-knot nematodes (RKNs), which is positively related to JA content. The downregulation of tomato CSN5 resulted in reduced resistance to Botrytis cinerea and Manduca sexta larvae, which corresponded with reduced JA content [50]. Zhang et al. [51] reported that CSN5 negatively regulates the resistance of wheat to Puccinia triticina, as well as Puccinia striiformis f. sp. tritici, which is closely related to the SA pathway [28]. In our study, the expression of StCSN5 was significantly induced by SA, JA and ETH (Figure 7), suggesting that StCSN5 is also involved in the response to phytohormone signaling. The StPAM16-1 protein contains a highly conserved DnaJ domain (Figure 1a) that binds to the heat shock protein for its proper folding, thus playing key roles in reactive oxygen species (ROS) scavenging and signaling [52]. When StPAM16-1 interacts with StCSN5, we hypothesize that the binding of StPAM16 to StCSN5 may assure the correct folding of StCSN5 and enable it to participate in the regulation of resistance pathways such as phytohormone signaling response. Moreover, as StPAM16-1 interacted with StCSN5 and the expression levels of both genes were induced by TA, we speculated that after TA treatment, an increase in the amount of the StCSN5 protein might be needed to degrade the accumulated StPAM16-1 protein to assist in plant defense. However, it is also possible that StPAM16-1 and StCSN5 form a complex to synergistically regulate the host immune response.
Further investigations will focus on the correlation between StPAM16-1 and defensive pathways, including phytohormone, metabolism and R-related genes. Generating StCSN5-silenced plants and investigating the biochemical activity of StCSN5 may provide more insights into the function of StPAM16-1-StCSN5 in the host immune response.

4. Materials and Methods

4.1. Plant Growth and Treatments

The tetraploid potato (Solanum tuberosum L.) cultivar Shepody was micropropagated on solid Murashige and Skoog (MS) medium and subcultured every four weeks at 23 °C with a 16 h light–8 h dark photoperiod. Three or four weeks later, potato plants with the same node segments and similar lengths were transplanted into pots containing nutrient soil and vermiculite (v/v = 3:1) and grown in a greenhouse under the same conditions as those used for tissue-cultured potato plants. Potato plants were watered with tap water once a week and Hogland nutrient solution once a month. N. benthamiana plants were grown in the greenhouse at 26 °C with the same photoperiod as that of the potato plants. For TA and hormone treatments, potato plants grown for three weeks were treated with 10 μM TA, 1 mM SA, 50 μM JA or 1 mM ETH and sampled at the indicated time points for further analysis. Leaves treated with sterile water were used as controls. A total of 3 pots (25 cm in diameter) with 5 seedlings per pot were used to replicate each treatment.

4.2. Pathogen Cultivation and Inoculation

The S. scabiei strain 4.1765, a typical pathogenic isolate from the China General Microbiological Culture Collection Center (CGMCC), was used in this study. S. scabiei was cultured on oatmeal agar medium (OMA) plates at 28 °C. Then, the spores were scraped off and dissolved in sterile water to make the spore suspension. The concentration of the spore suspension was adjusted to approximately 1 × 107 CFU/mL using the flat colony counting method. A diluted spore suspension was used to inoculate 3-week-old potato plants.
CS disease assessment was performed as previously described. Tubers > 2 g were selected and washed under running water, and then CS severity was evaluated. The disease index was calculated by the following equation: Disease index = [∑(n × 1 + n × 2 + n × 3 + n × 4 + n × 5)/(N × 5)] × 100. (n = number of tubers corresponding to the numerical grade. N = total number of potato tubers assessed. 5 = high score on the severity of scale). The percentage of tuber area covered was 0. No symptoms of scab; 1. 0–12.5%; 2. 12.6–25%; 3. 26–50%; 4. 51–75%; 5. 76–100%. Control efficacy = (disease index of control − disease index of treated)/disease index of control × 100%. Rate of disease = [number of infected tubers/total number of tubers] × 100× 100%.

4.3. Subcellular Localization of StPAM16-1

The coding region of StPAM16-1 was amplified by PCR and fused with GFP under the control of the CaMV 35S promoter in the plant expression vector pGWB6. Plasmids containing GFP or mitochondrial markers were used as controls. The plasmids were transformed into Agrobacterium tumefaciens strain GV3101 competent cells, which were subsequently infiltrated into the leaves of 4-week-old N. benthamiana plants. After 48 h of incubation, fluorescent signals were visualized using a confocal laser scanning microscope (Leica TCS SP8, Leica Microsystems, Wetzlar, Germany).

4.4. Virus-Induced Gene Silencing

A specific cDNA fragment of StPAM16-1 was amplified by PCR and inserted into the plant expression vector pgR107. pgR107 and pgR107-StPDS were used as negative and positive controls, respectively. Then, the plasmids were transformed into A. tumefaciens strain GV3101 competent cells (containing pJIC SA_Rep), which were subsequently infiltrated into 2-week-old potato leaves [53]. Potato plants were incubated in the dark for 24 h and then transferred to a greenhouse. Potato plants harboring pgR107-StPDS were used as a positive control.

4.5. Vector Construction and Potato Transformation

The specific cDNA fragment of StPAM16-1 was amplified by PCR and inserted into the plant expression vector pANDA35K under the control of the CaMV 35S promoter. Then, the plasmid was transformed into GV3101 competent cells. For potato transformation, the stems of 3-week-old sterile potato plants were cut into 1 cm long pieces and immersed in A. tumefaciens resuspension solution for 10 min. After removing the excess solution, the stems were cultured on MS1 (MS + 4 mg/mL zeatin + 1 mg/mL indole-3-acetic acid) medium in the dark for 2 d. Then, the stems were transferred to MS2 (MS1 + 100 mg/L kanamycin + 200 mg/L cephalosporins) medium for 3–4 weeks to produce callus. The seedlings grown from callus were transplanted into MS3 (MS1 + 100 mg/L kanamycin) medium, and transgenic identification was carried out after the plants developed roots.

4.6. RNA Extraction and RT-qPCR Analysis

Total RNA was extracted from various potato tissues using a Plant Total RNA Extraction Kit (Tiangen, Beijing, China) according to the manufacturer’s instructions. The same tissues from 5 plants in one pot were pooled together as one sample for RNA extraction. Three replicates were performed for each tissue sample. Then, cDNA was synthesized from 2 μg of RNA using TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix (Transgene, Beijing, China). RT-qPCR was performed using SYBR® Green Real-time PCR Master Mix (Toyobo, Osaka, Japan). All RT-qPCR experiments were performed using a CFX96 Touch instrument (Bio-Rad, Hercules, CA, USA) with three technical replicates and biological replicates. The relative expression of genes was calculated by the 2−∆∆Ct method. StActin (PGSC0003DMG400027746) was used as the reference gene. The primers used for RT-qPCR are shown in Table S1.

4.7. Y2H Assay

The Y2H assay was performed according to the Matchmaker Gold Yeast Two-Hybrid System’s instructions (Clontech, Mountain View, CA, USA). The ORFs of StPAM16-1 and StCSN5 were inserted into pGBKT7 and pGADT7, respectively. The plasmids BD-53/AD-T, BD-StPAM16-1/AD, BD/AD-StCSN5, BD/AD and BD-StPAM16-1/AD-StCSN5 were co-transformed into yeast strain AH109 competent cells, which were subsequently cultured on DDO medium (SD medium/-Leu/-Trp) at 28–30 °C for 3–5 days. The yeast cells were subsequently resuspended and cultured in QOD medium (SD medium/-Leu/-Trp/-His/-Ade) supplemented with 40 μg/mL X-α-Gal at 30 °C. Then, the growth of colonies was observed and photographed.

4.8. Split Luciferase Complementation Assays

For the firefly luciferase complementation imaging assay [47], the ORF regions of StPAM16-1 and StCSN5 were inserted into the plant expression vectors pCAMBIA1300-CLuc and pCAMBIA1300-NLuc, respectively, and subsequently transformed into Agrobacterium cell strain GV3101. The Agrobacterium cultures were mixed and co-injected into the leaves of 4-week-old N. benthamiana plants. The plants were cultured in the dark for 24 h and then transferred to a greenhouse for 24 h. The leaves were sprayed with 0.5 mM D-luciferin potassium salt solution, and the Luc signals were captured using a plant in vivo imaging system (NightSHADE LB 985, Berthold Technologies, Baden, Germany).

4.9. Trypan Blue Staining

Trypan blue staining was performed as described previously [35]. Briefly, potato leaves were placed in a small beaker and washed with distilled water three times. The trypan blue staining solution (0.02% m/v trypan blue; ethanol-phenol-water-83% lactic acid = 2:1:1:1) was added to the beaker and boiled for 5–10 min. Then, 2.5 g/mL chloral hydrate solution was added to the beaker for decolorization, and the leaves were photographed.

4.10. Phylogenetic Analysis

PAM16-1 and relative homologous proteins sequences were downloaded from Potato Genomics Resource (http://solanaceae.plantbiology.msu.edu/, accessed on 15 November 2021) and NCBI (http://blast.ncbi.nlm.nih.gov/, accessed on 15 November 2021) databases. Multiple sequence alignment and phylogenetic analysis were performed using DNAMAN and MEGA7.0 software. Phylogenetic tree was constructed using Neighbor-joining method.

4.11. Statistical Analysis

Statistical analysis was performed using GraphPad Prism 9.3.0 (GraphPad Software, Boston, MA, USA). Three independent replications were performed for each test, and all values are reported as the mean with standard deviations (SDs). Student’s t test (unpaired, two-tailed) was used to test for statistical significance for two groups compared at the 95% significance level (p < 0.05) or 99% significance level (p < 0.01).

5. Conclusions

Potato CS is a soil-borne disease that seriously threatens tuber quality and its market value. To date, an effective approach to controlling CS is lacking. In this study, we identified a gene designated StPAM16-1 that responds to the phytotoxin TA and demonstrated that StPAM16-1 acted as a negative regulator of CS defense in potato. Importantly, we found that StPAM16-1 localized to the mitochondria and interacted with the COP9 signalosome subunit StCSN5, and the expression of StCSN5 significantly increased in potato leaves after they were treated with TA. These results provide important clues for understanding the functional mechanism of StPAM16-1. The identification of important regulators involved in the interaction between potato and S. scabiei could provide candidate genes for the genetic manipulation of plants with increased disease resistance. Based on our results, we propose that StPAM16-1 can be used as a target gene for gene editing to develop new varieties of potato resistant to CS. Moreover, after the function of StCSN5 was verified, StCSN5 could also be used as a target gene.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/ijms26031361/s1.

Author Contributions

L.L., S.S., N.L. and H.W. conceived and designed this study; L.L., S.S., Z.W. and Y.Z. performed the experiments and data analyses; L.L. drafted the original manuscript; L.L., H.W., P.Z. and N.Z. reviewed and edited the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Hebei Science and Technology Major Project, grant number 22287501Z; the Key Technologies R & D Program of Inner Mongolia, grant number 2021GG0300; and the Priority Research Program of Chinese Academy of Science, grant number XDA24020104.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data generated or analyzed during this study are included in the published article and Supplementary Materials.

Acknowledgments

We thank Guixian Xia at Institute of Microbiology, Chinese Academy of Sciences, for critically reading the manuscript and Lei Su for technical support with confocal microscopes. We also thank David Baulcombe (Sainsbury Laboratory, Norwich, UK) for kindly providing us with the PVX vector pGR107 and the helper plasmid pJIC SA_Rep.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Homology analysis of StPAM16-1. (a,b) show multiple sequence alignment and phylogenetic analysis of StPAM16-1 and its homologous proteins, respectively. DnaJ domain is underlined by solid lines. Different colors of background represent similarity of amino acid sequences (black: 100%; pink: 75%; blue: 50%). Scale bar stands for evolutionary distance.
Figure 1. Homology analysis of StPAM16-1. (a,b) show multiple sequence alignment and phylogenetic analysis of StPAM16-1 and its homologous proteins, respectively. DnaJ domain is underlined by solid lines. Different colors of background represent similarity of amino acid sequences (black: 100%; pink: 75%; blue: 50%). Scale bar stands for evolutionary distance.
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Figure 2. The expression pattern of StPAM16-1 in potato plants. (a,b) The relative expression of StPAM16-1 in potato leaves after TA (a), SA, JA and ETH (b) treatments. The asterisks denote statistically significant differences according to Student’s t test; * p < 0.05, ** p < 0.01. (c) The relative expression of StPAM16-1 in different organs of potato. Different letters indicate statistically significant differences as determined by Student’s t test. StActin was used as a reference gene. The values represent the means ± standard deviation (SD). Three biological replicates were performed.
Figure 2. The expression pattern of StPAM16-1 in potato plants. (a,b) The relative expression of StPAM16-1 in potato leaves after TA (a), SA, JA and ETH (b) treatments. The asterisks denote statistically significant differences according to Student’s t test; * p < 0.05, ** p < 0.01. (c) The relative expression of StPAM16-1 in different organs of potato. Different letters indicate statistically significant differences as determined by Student’s t test. StActin was used as a reference gene. The values represent the means ± standard deviation (SD). Three biological replicates were performed.
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Figure 3. Subcellular localization of StPAM16-1 in Nicotiana benthamiana leaf cells. Bar = 25 μm.
Figure 3. Subcellular localization of StPAM16-1 in Nicotiana benthamiana leaf cells. Bar = 25 μm.
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Figure 4. Increased tolerance of StPAM16-1-silenced potato plants to TA treatment. (a) Albino phenotype of control (PVX:00) and StPDS-silenced potato plants (PVX:StPDS) approximately 40 days after injection. (b) Relative expression of StPDS in PVX:00 and PVX:StPDS. (c) Relative expression of StPAM16-1 in PVX:00 and StPAM16-1-silenced potato plants (PVX:StPAM16-1). StActin was used as reference gene. Values represent means ± SD. Asterisks denote statistically significant differences as determined by Student’s t test, ** p < 0.01. Three biological replicates were performed. (d) Phenotypes of PVX:00 and PVX:StPAM16-1 after TA treatment, which were stained with trypan blue. Mock represents PVX:00 treated with sterile water.
Figure 4. Increased tolerance of StPAM16-1-silenced potato plants to TA treatment. (a) Albino phenotype of control (PVX:00) and StPDS-silenced potato plants (PVX:StPDS) approximately 40 days after injection. (b) Relative expression of StPDS in PVX:00 and PVX:StPDS. (c) Relative expression of StPAM16-1 in PVX:00 and StPAM16-1-silenced potato plants (PVX:StPAM16-1). StActin was used as reference gene. Values represent means ± SD. Asterisks denote statistically significant differences as determined by Student’s t test, ** p < 0.01. Three biological replicates were performed. (d) Phenotypes of PVX:00 and PVX:StPAM16-1 after TA treatment, which were stained with trypan blue. Mock represents PVX:00 treated with sterile water.
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Figure 5. Repression of StPAM16-1 expression enhanced tolerance of potato plants to TA and S. scabiei infection. (a) Relative expression levels of StPAM16-1 and its homologous genes in WT and StPAM16-1-RNAi potato plants. (b) Phenotypes of WT and StPAM16-1-RNAi potato plants. (c) Repression of StPAM16-1 expression increased tolerance of potato plants to TA treatment. (d) Phenotypes of WT and StPAM16-1-RNAi potato tubers after S. scabiei infection. Mock represents WT potato tubers treated with sterile water. (e) Rate of diseased tubers of WT and StPAM16-1-RNAi potato plants. (f) Disease index of WT and StPAM16-1-RNAi potato tubers. (g) Relative disease control effect of StPAM16-1-RNAi in potato tubers. Values represent means ± SD. Asterisks denote statistically significant differences according to Student’s t test; * p < 0.05, ** p < 0.01. Three biological replicates were performed.
Figure 5. Repression of StPAM16-1 expression enhanced tolerance of potato plants to TA and S. scabiei infection. (a) Relative expression levels of StPAM16-1 and its homologous genes in WT and StPAM16-1-RNAi potato plants. (b) Phenotypes of WT and StPAM16-1-RNAi potato plants. (c) Repression of StPAM16-1 expression increased tolerance of potato plants to TA treatment. (d) Phenotypes of WT and StPAM16-1-RNAi potato tubers after S. scabiei infection. Mock represents WT potato tubers treated with sterile water. (e) Rate of diseased tubers of WT and StPAM16-1-RNAi potato plants. (f) Disease index of WT and StPAM16-1-RNAi potato tubers. (g) Relative disease control effect of StPAM16-1-RNAi in potato tubers. Values represent means ± SD. Asterisks denote statistically significant differences according to Student’s t test; * p < 0.05, ** p < 0.01. Three biological replicates were performed.
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Figure 6. Interaction between StPAM16-1 and StCSN5. (a) Interaction between StPAM16-1 and StCSN5 was verified by yeast two-hybrid assay. +, positive control (BD-L53/AD-T); –, negative control (BD-Lam/AD-T). (b) Split luciferase complementation assay of interaction between StPAM16-1 and StCSN5 in plant cells.
Figure 6. Interaction between StPAM16-1 and StCSN5. (a) Interaction between StPAM16-1 and StCSN5 was verified by yeast two-hybrid assay. +, positive control (BD-L53/AD-T); –, negative control (BD-Lam/AD-T). (b) Split luciferase complementation assay of interaction between StPAM16-1 and StCSN5 in plant cells.
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Figure 7. Relative expression of StCSN5 in potato leaves after TA (a), SA, JA and ETH (b) treatments. StActin was used as reference gene. Values represent means ± SD. Asterisks denote statistically significant differences according to Student’s t test; * p < 0.05, ** p < 0.01. Three biological replicates were performed.
Figure 7. Relative expression of StCSN5 in potato leaves after TA (a), SA, JA and ETH (b) treatments. StActin was used as reference gene. Values represent means ± SD. Asterisks denote statistically significant differences according to Student’s t test; * p < 0.05, ** p < 0.01. Three biological replicates were performed.
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Table 1. StPAM16-1-interacting proteins.
Table 1. StPAM16-1-interacting proteins.
NumberProtein DescriptionNCBI Database Accession No.
1PREDICTED: COP9 signalosome complex subunit 5b-like proteinXP_006351153.1
2PREDICTED: anthocyanidin 3-O-glucosyltransferase 2-like proteinXP_006353729.1
3fructose-bisphosphate aldolase-like proteinNP_001275379.1
4PREDICTED: uncharacterized protein LOC102596406XP_006344642.1
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Liu, L.; Song, S.; Liu, N.; Wang, Z.; Zhao, Y.; Zhong, N.; Zhao, P.; Wang, H. The Silencing of the StPAM16-1 Gene Enhanced the Resistance of Potato Plants to the Phytotoxin Thaxtomin A. Int. J. Mol. Sci. 2025, 26, 1361. https://doi.org/10.3390/ijms26031361

AMA Style

Liu L, Song S, Liu N, Wang Z, Zhao Y, Zhong N, Zhao P, Wang H. The Silencing of the StPAM16-1 Gene Enhanced the Resistance of Potato Plants to the Phytotoxin Thaxtomin A. International Journal of Molecular Sciences. 2025; 26(3):1361. https://doi.org/10.3390/ijms26031361

Chicago/Turabian Style

Liu, Lu, Shuangwei Song, Ning Liu, Zhiqin Wang, Yonglong Zhao, Naiqin Zhong, Pan Zhao, and Haiyun Wang. 2025. "The Silencing of the StPAM16-1 Gene Enhanced the Resistance of Potato Plants to the Phytotoxin Thaxtomin A" International Journal of Molecular Sciences 26, no. 3: 1361. https://doi.org/10.3390/ijms26031361

APA Style

Liu, L., Song, S., Liu, N., Wang, Z., Zhao, Y., Zhong, N., Zhao, P., & Wang, H. (2025). The Silencing of the StPAM16-1 Gene Enhanced the Resistance of Potato Plants to the Phytotoxin Thaxtomin A. International Journal of Molecular Sciences, 26(3), 1361. https://doi.org/10.3390/ijms26031361

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