Trizol a reagent

To detect gene expression after CCN infection, two-leaf stage seedlings were cultured in sterilized soil (four seedlings each pot), and inoculated with nematodes (300 J2s/pot). The root tissues were respectively collected at 0, 30 h and 3, 9 days. All these samples were stored at -80°C for RNA extraction.
Total RNA was extracted using the TRIzol-A⁺ reagent (Tiangen, Beijing, China) according to the manufacturer’s instructions. The cDNA synthesis was carried out using the ReverTra Ace qPCR RT Kit (TOYOBO). QPCR was conducted as described (Wang et al., 2013 (link)). Gene-specific primers were designed to amplify PCR products about 100–200 bp, and listed in Supplementary Table 1. Elongation factor1-α (EF1α) mRNA was employed as an internal control for normalization (Livak and Schmittgen, 2001 (link)). Each sample or treatment was tested in three biological repeats and experiment was performed for three times. The differences were analyzed by t-test and data were presented by software Origin 8.6.

The viral loads in the tissues were determined by qPCR with absolute quantification. Based on the methods performed by Ryncarz AJ et al. (24 (link)), the binding site designed by the primer was located in the HSV-1 gG gene region, and the gG gene was constructed on the p-GMT plasmid as a standard DNA sample. The primers were F: TCCTSGTTCCTMACKGCCTCCC and R: GCAGICAYACGTAACGCACGCT. Viral genomic DNA was extracted from tissues using an AxyPrep™ Body Fluid Viral DNA/RNA Miniprep kit (Central Avenue Union City, CA, USA). The TaqMan probe (Sangon Biotech, Shanghai, China) had the sequence 5’-6FAMCGTCTGGACCAACCGCCACACAGGTTAMRA. The reactions were performed using Premix Ex Taq™ (Probe qPCR; TaKaRa, Dalian, China) on a CFX96 Connect Real-Time System (Bio-Rad, Hercules, CA, USA).
For the relative expression of cytokines in tissues and cells, total RNA was extracted with TRIzol-A+ Reagent (Cat# DP421, Tiangen) according to the manufacturer’s protocol. Gene expression was expressed as the fold-change (2^−ΔΔCt) relative to the levels in samples from PBS-injected mice or virus-uninfected cells used for calibration. The reactions were performed using a One-Step SYBR Prime Script™ PLUS RT-PCR kit (TaKaRa, Dalian, China). The specific primers used are listed in Table S1.

The roots, shoots, and leaves of Q9 and 65 potato plants were collected separately. Each leaf sample was obtained by sampling the completely expanded fourth leaf. Moreover, we selected the stem tissue between the fourth leaf and the fifth leaf. In addition, we washed and dried the water after sampling the root tissue of the plants. Then, 36 samples (two cultivars, N0 and N1 treatment, three tissues) with three biological replicates were prepared for RNA extraction (TRIzol-A+ reagent, TIANGEN BIOTECH, Beijing) followed by treatment with RNase-free DNase I (TaKaRa). RNA quantity was measured with a Nanodrop and Qubit 2.0 Fluorometer (Life Technologies, CA, USA). RNA quality was evaluated with an Agilent Bioanalyzer Model 2100 (Agilent Technologies, Palo Alto, CA). Samples with an RNA integrity number (RIN) value greater than 6.6 were deemed acceptable according to the Illumina transcriptome sequencing protocol of the Beijing Allwegene Technology Company (Beijing, China) [24 (link)].

The genomic DNA was extracted from the paired tissues using the TIANamp Genomic DNA Kit (TianGen Biotech, Beijing, China) according to the manufacturer’s instruction. Total RNA extraction from the tissue samples was performed with TRIZOL-A reagent (TianGen Biotech, Beijing, China) according to the manufacturer’s instruction. The quality and quantity of all DNA and RNA samples were assessed on Nanodrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA).

Total RNA was extracted using TRIzol-A+ reagent (TIANGEN BIOTECH, Beijing, China) and then treated with RNase-free DNase I (TaKaRa, Dalian, China), according to the manufacturer’s protocol. RNA concentration was measured using a Nanodrop Qubit 2.0 Fluorometer (Life Technologies, CA, United States). RNA quality was evaluated using Agilent Bioanalyzer Model 2,100 (Agilent Technologies, Palo Alto, Canada). According to the Illumina transcriptome sequencing protocol, samples with an RNA integrity number (RIN) value >7.5 were deemed acceptable. All sequencing reads were submitted to the Sequence Read Archive (SRA) and National Center for Biotechnology Information (NCBI).

According to the manufacturer’s protocol, TRIzol-A+ reagent (TianGen, Beijing, China) was used to extract total RNA from the tissues of mice or dendritic cells collected at different time points after infection, and the One Step TB Green™ Prime Script™ PLUS RT-PCR Kit (TaKaRa Bio, Dalian, China) was used for amplification. Mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was the normalization control gene. The relative expression levels of inflammatory cytokines in mouse tissues were normalized to their levels in the blank control group by using the comparative Ct (ΔΔCt) method. The specific primer sets used are listed in Supplementary Table S1.

Total RNA samples were extracted and reverse transcribed into cDNA with TRIzol-A⁺ reagent (Tiangen Biotech, Beijing, China) and a FastKing RT kit (with gDNase) (Tiangen Biotech, Beijing, China). The 96 RT-PCR system (Thermo Fisher Scientific, USA) was used for quantitative real-time PCR (qRT-PCR) analysis with 2 × M5 HiPer SYBR Premix EsTaq (with Tli RNaseH) (Mei5 Biotechnology, Beijing, China) and the primers listed in Table S1. Fungal 18S rRNA was considered an endogenous standard, and the relative transcript levels of target genes were computed with the threshold cycle (2^-ΔΔCt) method (50 (link)).