Potato and pathogen
The virus-free potato ‘Atlantic’ was obtained through tissue culture and maintained at the Research Center of Potato Breeding in Inner Mongolia Agricultural University, Hohhot, China. This cultivar is known to be highly susceptible to R. solani (strain PR11, AG3). R. solani AG-3 strain PR11 was isolated from an infected potato tuber in Wuchuan county of Inner Mongolia, which was confirmed to be highly pathogenic on stems and tubers of the ‘Atlantic’ potato. The culture was stored on potato sucrose agar [PSA (PSA, containing potato 200 g, sucrose 20 g, agar 15 g, distilled water 1000 mL)] at 4°C in the Department of Plant Pathology, Inner Mongolia Agricultural University.
Potato planting and inoculation
The potato was initially grown on Murashige and Skoog (MS) medium under conditions of a photo flux density of 3000 to 4000 Lx, 50 to 60% relative humidity, and a 16 h day and 8 h night cycle at 25°C for 30 d. Subsequently, the plants were transferred to pots (18 cm × 20 cm) and grown under natural environmental conditions at the farm of Inner Mongolia Agricultural University for additional 30 d.
R. solani was cultivated on PSA at 25℃ for 5 d. Disks from 5-day-old R. solani cultures were then transferred to the center of a new PSA plate and incubated for 7 d at 25 ± 1℃. The resulting mycelium was scraped from the plates use sterile toothpicks, and the mycelium was put into sterile tube, and then rapidly frozen in liquid nitrogen to grind. The mycelium suspension was diluted to 1 × 107 mycelium / mL for inoculation.
The potato subterraneous stems were inoculated with 3 mL of mycelial suspension (1 × 107 mycelium/mL) and covered with soil. For sodium silicate treatments (Group SS), 500 mL of MS nutrient solution containing NaSiO3 · 9H2O (concentration 3.02 kg / L) was applied once a week. As for the control group (Group CK), mock potato plants were treated with MS nutrient solution following the same procedure.
To assess the effects of the treatments, potato subterraneous stems were taken at 4, 8, and 12 days post application (dpa), with three replicates per treatment. The sample were rapidly frozen in liquid nitrogen and stored at -80℃ for further analysis. There were three replicates in each treatment.
Library construction and RNA-seq
For RNA extraction, an equal amount of collected tissues from each replicate at each time point (4, 8 and 12 dpa) was used. Total RNA was extracted according to the manufacturer’s instructions (Takara Biomedical Technology, Beijing, China). RNA integrity, purity, and concentrations were assessed using 1% agarose gels, and a NanoPhotometer® spectrophotometer (IMPLEN, CA, USA), Qubit 2.0 Fluorometer (Life Technologies, CA, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA).
Messenger RNA (mRNA) was subjected to purification, and subsequent construction of complementary DNA (cDNA) libraries. This construction process included cDNA end repair, adapter ligation, and cDNA amplification following the methodologies for preparing Illumina RNA-seq libraries of Novogene Bioinformatics Technology Co., Ltd. (Beijing, China). To quantify the final libraries, their concentrations were determined. The libraries were then sequenced on an Illumina HiSeqTM 4000 platform, generating paired-end reads of 125/ 150 bp. This sequencing method allowed for the generation of high-quality and comprehensive data for further analysis.
Sequence alignment
To ensure the quality of the reads obtained from sequencing, initial quality control analysis was conducted using FastQC (v0.19.7). Low-quality sequences (Qphred ≤ 20), as well as sequences containing adapter and poly-N contamination were identified and filtered out. Only high-quality reads were retained for subsequent analyses. This step ensured that only high-quality reads were retained for further analyses.
The reference genome was indexed using Hisat2 (v2.0.5). The paired-end reads were then aligned to the reference genome using Hisat2 (v2.0.5) to accurately map the reads. Hisat2 was utilized for counting the number of reads mapped to each gene.
Subsequently, the mapped reads from each sample were assembled by StringTie (v1.3.3b) in a reference-based approach. FeatureCounts (v1.5.0-p3) was used to count the reads mapped to each gene. FPKM (fragments per kilobase of transcript per million mapped reads) values were calculated for each gene based on its length and the corresponding read count. Read alignment and expression quantification were performed separately for each sample. Genes meeting the criteria of having an FPKM value > 4 and low variation across three biological replicates (coefficient of variation < 30%) were considered reliable and included in subsequent analyses. This stringent selection ensured the inclusion of only robust and consistent genes for further analysis.
Identification and functional enrichment analysis of differentially expressed genes (DEGs)
The identification of DEGs was performed using the DESeq2 R package (1.16.1), which provides statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting p-values were adjusted using the Benjamini and Hochberg’s approach to control the false discovery rate (FDR). Genes with an adjusted p-value < 0.05, as determined by DESeq2, were considered differentially expressed. This threshold ensured a reasonable balance between the sensitivity and specificity of identifying significant changes in gene expression levels.
GO and KEGG pathway
To gain insights into the functional significance of the differentially expressed genes (DEGs), gene ontology (GO) enrichment analysis was performed using the clusterProfler R package, which corrects for gene length bias. GO terms with corrected p-values below 0.05 were deemed significantly enriched among the DEGs.
KEGG pathway analysis was conducted to understand the high-level functions and utilities of the biological system. The clusterProfiler R package was utilized to test the statistical enrichment of differentially expressed genes in KEGG pathways. This analysis provides a broader understanding of the biological pathways that may be influenced by the observed gene expression changes, thereby revealing potential functional implications. Both GO enrichment analysis and KEGG pathway analysis enable the interpretation of the biological significance and underlying mechanisms associated with the DEGs.
Real-time qPCR analysis
To validate the results obtained from RNA-seq, reverse-transcriptase quantitative PCR (RT-qPCR) was performed using the same biological replicates. RNA extraction and cDNA synthesis were conducted according to the manufacturer’s instructions (TaKaRa, Beijing, China). Quantitative real-time PCR was carried out using Applied Biosystems equipment (Thermo Fisher, Massachusetts, USA). A total of 16 genes were selected for qPCR validation of the RNA-seq results, with the potato EF-1α gene serving as an internal control. The primer sequences used for quantitative real-time PCR were shown in Table S14. These specific primer sequences enabled the amplification and quantification of the target genes, providing valuable information for confirming the RNA-seq results.
The PCR amplification were performed in a 20 µL reaction volume, containing 10 µL of 2 × SYBR Green I Master (Roche, Switzerland), 0.4 µL of 10 µM each primer, 1 µL of 10-fold diluted cDNA and 8.2 µL of PCR grade water. A negative control without target cDNA was included in each PCR run. The thermal cycling procedure involved an initial step at 95℃ for 5 min, followed by 45 cycles of 95℃ for 10 s, 58℃ for 10 s, and 72℃ for 10 s. This was followed by a melting curve analysis with a procedure of 95℃ for 5 s, annealing temperature for 1 min, and 97℃ continuous monitoring to determine the specificity of PCR amplification. Relative mRNA expression levels were calculated following modified 2−∆∆CT method (Livak, 2001) and expressed as mean ± standard deviation (S.D.). Amplification efficiency of all genes was determined using quantitative real-time PCR with 10-fold serial diluted cDNA as template. Statistical analysis was conducted using SPSS v. 20.0 software, allowing for the evaluation of the significance of the observed differences.
Ethics approval and consent to participate (Not applicable)
The plant collection and use was in accordance with all the relevant guidelines. The virus-free potato ‘Atlantic’ was obtained through tissue culture and maintained at the Research Center of Potato Breeding in Inner Mongolia Agricultural University, Hohhot, China. This cultivar is known to be highly susceptible to R. solani (strain PR11, AG3). R. solani AG-3 strain PR11 was isolated from an infected potato tuber in Wuchuan county of Inner Mongolia, which was confirmed to be highly pathogenic on stems and tubers of the ‘Atlantic’ potato. Above work were did by prof. xiaoyu zhang. Our lab have permission to collect Potato plant.