Using BLASTP analysis of the potato genome, 28 members of the RPP13 gene family were identified and designated as StRPP13-1 through StRPP13-28. These genes were distributed across 12 chromosomes ( Figure 1 ), with chromosomes 1, 2, 4, and 8 each harboring four RPP13 genes (e.g., StRPP13-1 to StRPP13-4 on chromosome 1). The remaining genes were distributed across other chromosomes, such as chromosomes 9 and 12, which also contained multiple RPP13 genes (e.g., StRPP13-18 to StRPP13-20 and StRPP13-25 to StRPP13-28). This distribution pattern suggested that the RPP13 gene family exhibited a widespread and evolutionarily conserved organization within the potato genome.

The physicochemical properties of the RPP13 proteins revealed amino acid lengths ranging from 781 to 1714, with molecular weights varying between 89,918.09 Da and 195,237.26 Da. The theoretical isoelectric points (pI) ranged from 5.42 to 9.00, indicating a broad spectrum of acid-base properties. Most RPP13 proteins had instability indices below 50, suggesting that they were relatively stable within the cellular environment. Moreover, the GRAVY (Grand Average of Hydropathicity) values for all RPP13 family proteins were negative, reflecting their hydrophilic nature. Subcellular localization predictions indicated that all RPP13 proteins were localized in the cytoplasm ( Supplementary Table S1 ).

A phylogenetic analysis of the RPP13 gene family members from potato and Arabidopsis thaliana classified the 28 RPP13 genes into four evolutionary clades ( Figure 2 ). Group I included genes such as StRPP13-1, StRPP13-4, and StRPP13-15. Group II contained StRPP13-18, StRPP13-9, along with two Arabidopsis homologs (AT3G50950.1 and AT3G14470.1), indicating strong evolutionary relationships. The members of Group III and Group IV demonstrated divergent evolutionary trajectories, with Group III genes such as StRPP13-3 and StRPP13-5 exhibiting unique conservation patterns. This classification revealed the evolutionary diversity of the RPP13 gene family in potato, providing a foundation for future functional studies.

Gene structure analysis revealed that the number of exons in the RPP13 genes ranged from 1 to 6. Most genes, such as StRPP13-1 and StRPP13-5, contained 2 to 3 exons, while StRPP13-26 exhibited a more complex gene structure with 6 exons, suggesting that this gene might possess intricate transcriptional and functional regulation mechanisms. Additionally, StRPP13-9 was the longest gene in the family; despite having only 3 exons, it featured significantly extended introns, indicating that it might have unique regulatory characteristics ( Figure 3A ).

In the conserved motif analysis, 20 distinct conserved motifs were identified in the RPP13 proteins ( Figure 3B ). Motifs 1 through 4 were widely distributed across all RPP13 proteins, underscoring their evolutionary conservation. Motifs 16, 19, and 20 were predominantly present in Group III, suggesting their possible involvement in specific disease resistance functions. Functional domain analysis further confirmed the functional conservation of the RPP13 family proteins. All proteins were found to contain disease resistance-related domains such as NB-ARC, RX-CC, and LRR, with StRPP13-25 and StRPP13-26 in Group III also containing additional domains, including PLN00113 and the COG3903 superfamily domains, indicating that these proteins might play specialized roles in pathogen defense ( Figure 3C ).

Collinearity analysis revealed the conservation and evolutionary relationships of the RPP13 gene family both within and across species. Intraspecific collinearity analysis showed significant collinearity relationships of the RPP13 genes on chromosomes 1, 2, 3, 8, 9, and 12. Particularly notable were the collinearity observed between chromosomes 2 and 3, and between chromosomes 8 and 9, suggesting the occurrence of tandem duplications or segmental duplications, which might have contributed to the expansion of this gene family. However, in the central chromosomal regions, collinearity between StRPP13-19 and StRPP13-20 was the only observed segment, indicating that these genes might have been subjected to specific selective pressures during evolution ( Figure 4A ).

Interspecific collinearity analysis revealed a degree of collinearity between the potato RPP13 gene family and those in other Solanaceae species, such as tomato and tobacco, particularly on chromosomes 1, 2, and 12. This suggested that these gene families might have originated from a common ancestor and undergone conserved evolution within Solanaceae species. Specifically, synteny analysis with tomato suggested that these genes were highly aligned with chloroplast-related disease defense pathways, further supporting their conserved functional characteristics. In contrast, collinearity with Arabidopsis thaliana was limited, with only a few collinear segments observed on chromosomes 2 and 3. The collinearity with tobacco was more complex, indicating that multiple chromosomal rearrangement events might have occurred during evolution ( Figure 4B ).

Cis-acting element analysis of the promoter regions (2000 bp upstream) of 28 potato RPP13 genes revealed the presence of multiple elements associated with environmental stress responses, plant growth and development, hormone responses, and light responses ( Figure 5 ). Among these, stress-responsive elements such as MYC, STRE, and ARE were particularly prominent, with the highest abundance observed in StRPP13-5, StRPP13-20, and StRPP13-26. Additionally, StRPP13-26 contained the largest number of WUN-motif, suggesting its involvement in wound response and pathogen defense mechanisms.

Plant growth and development-related elements, such as AAGAA-motif, O2-site, and GCN4-motif, were widely distributed among the genes, particularly in StRPP13-22 and StRPP13-15 in Group I and Group IV, indicating that these genes might play crucial roles in cell division and tissue differentiation.

Hormone response elements, such as ERE and ABRE, were highly enriched in StRPP13-14, StRPP13-21, and StRPP13-3, suggesting their involvement in hormone-regulated processes. Light response elements, including Box 4, G-box, and GT1-motif, were significantly increased in StRPP13-18, StRPP13-20, and StRPP13-21, indicating that these genes might function in light-regulated growth processes.

The cis-acting elements identified in the promoters of the RPP13 gene family highlighted their critical roles in potato’s disease resistance, environmental stress response, hormone regulation, and light-responsive processes. Furthermore, the analysis suggested that these genes not only participated in disease defense mechanisms but might also influence the plant’s light response by regulating photosynthesis-related pathways and chloroplast metabolism, providing a more comprehensive view of their function in both defense and broader physiological processes.

To further investigate the specific subcellular localization of the key disease resistance gene StRPP13-11, a transient expression system using Arabidopsis protoplasts was employed. The Arabidopsis protoplast system, known for its efficient transient expression, allowed for the rapid evaluation of gene subcellular localization. The subcellular localization of the StRPP13-11 protein was determined by observing the GFP signal from the fusion protein under a laser confocal microscope.

The results showed that the GFP control (35S-GFP) was evenly distributed in the cytoplasm, whereas the GFP signal of the StRPP13-11-GFP fusion protein was mainly concentrated in the chloroplast region ( Figure 7 ), with a high overlap with the red autofluorescence of chlorophyll. This localization result indicated that the StRPP13-11 protein is localized in the chloroplast. Chloroplasts are essential organelles in plant cells, responsible for photosynthesis and serving as hubs for numerous metabolic and signaling pathways. The chloroplast localization of StRPP13-11 suggested that it might exert direct influence on pathogen infection by regulating photosynthesis or chloroplast function as part of its defense mechanisms. This finding provided an important clue for further investigating its specific functions within the chloroplast.

The potato genome data and its gff annotation file (DM 1-3 516 R44) were downloaded from the Spud DB online database (http://spuddb.uga.edu/). The amino acid sequences of Arabidopsis thaliana RPP13 genes were obtained from the TAIR database (https://www.arabidopsis.org/) and used as references for BLASTP analysis. The specific locus names of the Arabidopsis RPP13 genes used in this study are AT1G59218.1, AT1G58390.1, AT3G46530.1, AT3G46710.1, AT3G46730.1, AT1G50180.1, AT3G07040.1, AT3G50950.1, and AT3G14470.1. A local BLASTP analysis was conducted using the Diamond software (E-value set to 10^{^-3}). The presence of characteristic domains of the RPP13 proteins, such as RX-CC, NB-ARC, and LRR, was confirmed using the Conserved Domain Database (CDD) of the National Center for Biotechnology Information (NCBI).

The physicochemical properties of the potato RPP13 proteins, including amino acid sequence length, theoretical isoelectric point (pI), molecular weight (Mw), instability index, aliphatic index, and grand average of hydropathicity (GRAVY) index, were calculated using the ProtParam tool (https://web.expasy.org/protparam/). Additionally, the subcellular localization of the RPP13 proteins was predicted using the CELLO online tool (http://cello.life.nctu.edu.tw/). Finally, the chromosomal locations of the RPP13 genes were extracted from the genome annotation file and visualized using the Gene Location Visualize from GTF/GFF module of the Tbtools software (v2.097).

The amino acid sequences of RPP13 proteins from potato and Arabidopsis thaliana were aligned using the Clustal algorithm. A phylogenetic tree was constructed using the Neighbor-Joining (NJ) method in MEGA11 software with 1000 bootstrap replicates to assess branch confidence. The constructed phylogenetic tree was visualized and enhanced using the iTOL tool (https://itol.embl.de/). Bootstrap values are displayed at the corresponding nodes to indicate the confidence level of each branch.

The conserved motifs of the RPP13 genes were analyzed using the MEME suite (http://meme-suite.org/) with the maximum number of motifs set to 20, the minimum width to 6, and the maximum width to 50. Gene structure analysis was performed by extracting the coding sequences (CDS) and untranslated regions (UTR) of the potato RPP13 genes, and the results were visualized using TBtools v2.101.

The BLASTP program was employed to identify homologous RPP13 genes in potato (E-value threshold < e^-5). The collinearity relationships among potato RPP13 genes were analyzed using the MCScanX program, and the collinear blocks and duplicated gene pairs were visualized using TBtools.

Additionally, genome sequences and annotation files of Arabidopsis thaliana, tobacco, and tomato were downloaded from the Phytozome v13 (https://phytozome-next.jgi.doe.gov/), NCBI (https://www.ncbi.nlm.nih.gov/), and Sol Genomics Network (https://solgenomics.net/organism/solanum_lycopersicum/genome) websites, respectively. Collinearity between potato and Arabidopsis thaliana, tobacco, and tomato was analyzed using MCScanX, and the results were visualized with TBtools.

The 2000 bp sequences upstream of the start codon of each potato RPP13 gene were extracted and considered as the promoter regions. The promoter sequences were extracted using TBtools and analyzed using the PlantCARE database (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/) to predict cis-regulatory elements. The cis-regulatory elements associated with stress response, plant growth and development, hormone response, and light responsiveness were summarized and visualized using the Basic Biosequence View and HeatMap modules of TBtools.

The expression patterns of 28 RPP13 genes in 14 different tissues, including stolon, young tuber, flower, leaf, shoot apex, petiole, stem, mature tuber, root, tuber peel, tuber cortex, tuber pith, tuber sprout, and whole in vitro plant, were analyzed using the gene expression tool from the EnsemblPlants database (https://plants.ensembl.org/Solanum_tuberosum/Info/Index), and the results were visualized to show the expression differences across tissues.

Based on previous transcriptome sequencing data from our team, the expression changes of RPP13 genes were analyzed in the resistant cultivar Chunshu 10 and the susceptible cultivar Chunshu 11 after inoculation with scab pathogen Streptomyces scabies at 0 days and 10 days post-inoculation (De Torres Zabala et al., 2015). The two cultivars were grown in pots, and a spore suspension of 2×10^{^7} spores/mL was applied during the sprouting phase (Wanner, 2007). Samples were collected at 0 days (pre-inoculation) and 10 days post-inoculation, rapidly frozen in liquid nitrogen, and stored at -80°C for further analysis.

To further validate the expression patterns of RPP13 genes under scab infection, nine genes from the 28 RPP13 family members were randomly selected for qRT-PCR analysis. Primer design was performed using Primer 3 (https://primer3.ut.ee/), and all primer sequences used in this study are provided in Supplementary Table S2 . Total RNA was extracted from the samples using the TaKaRa MiniBEST Plant RNA Extraction Kit, and cDNA was synthesized using PrimeScript™ RT Master Mix (TaKaRa) (Ye et al., 2018). qRT-PCR was performed on the Applied Biosystems QuantStudio 6 system, with StActin97 used as the reference gene. Each gene’s expression level was determined using three biological replicates, and relative expression levels were calculated using the 2^−ΔΔCT method (Livak and Schmittgen, 2001).

The subcellular localization of the StRPP13-11 gene was analyzed using the Arabidopsis protoplast transient expression system. The StRPP13-11 gene was cloned into the 16318-hGFP vector, and seamless cloning was used to transfer the gene into Escherichia coli DH5α for cloning and plasmid extraction. The extracted plasmid was then introduced into Arabidopsis protoplasts via PEG-mediated transformation, followed by washing and purification with W5 solution. The transformed protoplasts were incubated overnight (Kobayashi et al., 2006).

After transformation, the subcellular localization of the StRPP13-11 protein was observed using a laser confocal microscope at excitation wavelengths of 405 nm and 488 nm to detect GFP fluorescence (Zhu et al., 2023).

Statistical analyses were performed using Microsoft Excel 2013 and SPSS 19.0. Gene expression data from qRT-PCR experiments and transcriptomic analyses were included in the analysis. Group comparisons were carried out using Duncan’s multiple range tests to evaluate differences among experimental groups. To ensure the validity of ANOVA results, assumptions of normality and homogeneity of variances were verified using the Shapiro-Wilk test and Levene’s test, respectively. Only data that met these assumptions were subjected. For repeated experiments, the mean values were calculated from three biological replicates, and data variability was expressed as the standard deviation (SD). Significant differences among groups were indicated with different letters. A p-value of less than 0.05 was considered statistically significant. Data visualization, including graphical representations of gene expression levels, was conducted using GraphPad Prism 9.