Rnaiso plus regent

Total RNA samples of B. cinerea were isolated using the RNAiso Plus regent (Takara) according to the manufacturer’s instructions and stored at minus 80 degrees for further study. The RNase-free Recombinant DNase I (Takara) treatment was performed to eliminate residual genomic DNA, and the M-MLV Reverse Transcriptase (Promega, Madison, WI, USA was used to generate the first-strand cDNA.
Quantitative real-time reverse transcriptase PCR (qRT-PCR) was performed by the using of CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA with iTaq universal SYBR Green supermix (Bio-Rad), according to the manufacturer’s instructions. The B. cinerea Actin gene (Bcin16g02020) was used as reference gene for normalizing the RNA samples [26 (link),60 (link)]. Primer were designed across or flanking an intron (Table S1). For each gene detecting, qRT-PCR assays were repeated at least twice and each with three independent biological replicates.

Total RNA was extracted from HDFs with an RNAiso Plus regent (9109, TaKaRa, Otsu, Japan), followed by reverse transcription to cDNA using Evo M-MLV RT Premix (AG11706, Accurate Biotechnology, Changsha, China). A quantitative PCR analysis was conducted using a SYBR Green Premix Pro tag HS qPCR Kit (AG11701, Accurate Biotechnology) on a real-time PCR system (CFX Connect, Bio-Rad, CA, USA). GAPDH served as the internal control for normalizing the levels of target genes. The relative quantification of gene expression was calculated using the 2^−ΔΔCT method. Primer sequences are shown in Table 2.

Total RNA was extracted from frozen S. rolfsii samples using RNAiso Plus regent (TaKaRa, Kusatsu, Japan) and treated with DNase I. One microgram of RNA was used to synthesize first-strand cDNA using PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa, Japan). The RT-qPCR reaction was carried out using PowerUp SYBR Green PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) on the Applied Biosystems QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific). Each 20 μL reaction system contained 10 μL of 2 × PowerUp SYBR Green Master Mix, 1 μL of each forward and reverse primer (10 μM), 1 μL of cDNA, and 7 μL of ddH₂O. Cycling system was performed at 95 °C for 30 s, followed by 40 cycles of 95 °C for 10 s and 60 °C for 10 s. The specificity of RT-qPCR products was verified by a melting curve analysis from 65 °C to 95 °C. Three biological replicates with three technical replicates were set up for each treatment in RT-qPCR analysis. A series of diluted cDNAs (1×, 10×, 100×, 1000×, 10,000×) were used to construct a standard curve to calculate its correlation coefficient and slope value. The amplification efficiency was calculated with the equation [10^(1/−slope) − 1] × 100%.

The total RNA was isolated using RNAiso Plus regent (Takara, Dalian, China) per manufacturer’s instruction. To measure vRNA level in tissues or the transcriptional expression of cytokines in spleens, reverse transcription quantitative PCR (RT-qPCR) assays were performed using 2×Taq SYBRGreen qPCR Premix (Innovagene, Changsha, China) in a CFX Connect Real-Time PCR Detect System (Bio-rad) following the manufacturer’s protocols. Primers used in present study are presented in Table S1.

Genomic DNA was isolated from mycelium by using the CTAB method [59 (link)]. Total RNA of fungi and plants were isolated with RNAiso Plus regent (Takara, Dalian, China) according to the manufacturer’s protocols. To detect the expression pattern of cutinase gene in infected tissues, the hyphae of S. sclerotiorum wild-type strain were inoculated on Arabidopsis thaliana plant leaves. The hyphae were collected at 0, 1, 3, 6, 9, 12 and 24 h after inoculation. After RNA extraction, the first-strand cDNA was synthesized by the Easy Script One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). Additionally, the gene expression was detected by qPCR analysis [60 (link)]. The β-tubulin gene in S. sclerotiorum, the UBQ5 gene in A. thaliana, and the elongation factor EF1α gene in N. benthamiana were used as internal controls, respectively. The real-time RT-qPCR analysis was repeated at least three times, with three biological replicates for each repeat.

In the asexual stage, the RNA samples were isolated from 24-h YEPD cultures (mycelium) and 24-h CMC cultures (sporulation). During sexual development, the RNA samples were collected from 7-day carrot plat hyphae (0 day post self-crossing, 0 dps) and 3-, 5-, 7-day perithecia (3, 5, and 7 days post self-crossing). Total RNA was isolated with the RNAiso Plus regent (Takara, Shiga, Japan) according to the manufacturer’s protocols. Potential DNA contamination was removed, and first-strand cDNA was synthesized by the EasyScript One-step gDNA Removal and cDNA Synthesis SuperMix (Transgen Biotech, Beijing, China) according to the manufacturer’s instructions.
The gene transcript levels were assessed by quantitative real-time reverse transcriptase PCR (qRT-PCR). qRT-PCR was performed on a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, United States) with the iTaq Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, United States USA). The F. graminearum actin gene was used as the internal control. The gene expression was calculated with the 2^-ΔΔCt method, and the mean and standard deviation were calculated from three biological replicates (Livak and Schmittgen, 2001 (link)). The gene-specific primer pairs for qRT-PCR are listed in Supplementary Table S1.