Sybr green premix kit

Total RNA was extracted with the Polysaccharide Polyphenol Plant RNA Extraction Kit (Chengdu Fuji Biotechnology, China). According to the instruction manual, ~1 µg of total RNA was transcribed into cDNA using HiScript Q RT SuperMix for qPCR (+gDNA wiper) (Vazyme, China). The primer sequences for the semiquantitative RT-PCR and qRT-PCR assays were designed using Primer 5 (in the program EMBOSS Explorer) and were listed in Table S3. The method and program of Shi et al.⁶³ were employed for semiquantitative RT-PCR. qRT-PCR was performed using a SYBR® Green Premix kit (TaKaRa Biotechnology, China). The program of Jin et al.^{64 (link)} was used for qRT-PCR. Each cDNA was analyzed in triplicate, after which the average threshold cycle (Ct) was calculated for each sample. Relative expression levels were calculated with the 2^−ΔΔCt method^{65 (link)}. Tubulin was analyzed in parallel as a reference control for P. bretschneideri, and AtActin was used for Arabidopsis.

Total RNA was extracted and reversely transcribed using the PrimeScript RT reagent Kit (TaKaRa, Japan) according to the protocols recommended by the manufacturer. The cDNA was subjected to qRT-PCR detection using a SYBR Green Premix Kit (TaKaRa, Japan). The relative expression was calculated using the 2^-ΔΔCT method for the following genes: TNFα, IL1β, IL6, IL10, CCL18, MRC1, CD80, HLA-DRα, PAI1, and THBS1.

The phenotypes strains (WT, Δppci1, and Δppci1-Com) were inoculated in PDA (Potato dextrose agar) and YES (Yeast extract supplement) medium. For the purpose of colony morphology and mycelial growth, all the strains were cultured at 37 °C in the dark. After 5 d colony diameters were measured. For conidia analysis, all strains were inoculated and cultured on YES medium at 37 °C in the dark. After 2 d, the hyphae were cut and observed under a microscope [44 (link)]. The qRT-PCR was used with the Real-Time PCR system (Thermo Scientific, Finland) and SYBR Green Premix kit (Takara, Dalian, China). The 2^−ΔΔCT method was used to evaluate the expression level of the target gene [30 (link)]. For sclerotial analysis, all strains were inoculated and cultured on WKM (Wickerham) agar medium at 37 °C in the dark. After 7 d, conidia on the surface of the medium were washed away by 75% ethanol, and the sclerotia was examined under the microscope [54 (link)].

The qRT-PCR assay was performed following the former method described by Zhang (2016) [55 ]. To remove possible residual G-DNA, the total RNA (5 µg) was firstly treated with DNase I (Thermo Fisher Scientific, Waltham, MA, USA). Then, the G-DNA free RNA (1 mg) was reverse-transcribed into cDNA by using the Revert Aid First-strand cDNA Synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA). Finally, the qRT-PCR was performed with the SYBR Green Premix kit (Takara, Dalian, China), and the instrument used is M×3000p thermocycler (Agilent Technologies). The method of 2´∆∆Ct was applied to evaluate the expression levels of corresponding target genes. The primers of the qRT-PCR assay were listed in Table S1.

YTHDF1, TRIM44, LGR4, SGTA, DDX20, and FZD8 expression in cells were measured by qRT-PCR. Briefly, total RNA was extracted by the Trizol method, cDNA was reverse-transcribed (Takara, Japan) into cDNA, and the cDNA products were used for qPCR using the SYBR Green Premix kit (Takara, Japan). GAPDH was used as the reference gene and relative gene expression was calculated by the $2^{-\mathrm{\Delta }\mathrm{\Delta }{\mathrm{C}}_{\mathrm{T}}}$  method. The primers used in this study are shown in Table 1.
Table 1

The primers used in this study

Primer ID	5′–3′
YTHDF1-F	CAGCACCGATCCCGACATAG
YTHDF1-R	CTGGCTTCCTGAAGACGATGA
TRIM44-F	GCCAGGAAGATAGGCAGCTCAT
TRIM44-R	CTTCAGTCCACCTGAGTCTTTGC
LGR4-F	GGAGCATTTGATGGTAATCCACTC
LGR4-R	CCATGCTTGCACCACGAATGAC
SGTA-F	CGGTAGAAGACAGTGACCTTGC
SGTA-R	TCTGCTGAGTCCTCCTCGGAAG
DDX20-F	AATCAGCGTCTTGATGCTATGGC
DDX20-R	ACAACCAGATTCACCTTCTCAGC
FZD8-F	GCTCTACAACCGCGTCAAGACA
FZD8-R	AAGGTGGACACGAAGCAGAGCA
GAPDH-F	GAGTCAACGGATTTGGTCGT
GAPDH-R	GACAAGCTTCCCGTTCTCAG