Sybr green 2

Total RNA was extracted from 1 × 10⁷ tachyzoites of the Nc-1 wild-type strain and three ΔNcROP5 clones with TRIzol reagent (Invitrogen, USA). cDNA was synthesized using the EasyScript First-Strand cDNA Synthesis SuperMix kit (TransGen, China). Specific primers were designed using Primer Premier 5.0 (Hui et al., 2014 (link)), including primers for rhoptry necks (RON2 and RON4), rhoptrys (ROP4, ROP5, ROP7, ROP16, and ROP17), dense granules (GRA2, GRA6, and GRA7) and the endogenous reference gene NcActin (Supplementary Table S1). The specificity of these primers was evaluated using conventional quantitative real-time PCR (qRT-PCR). The qRT-PCR was conducted using the ABI Prism 7500 System (Biosystems Inc., USA) with SYBR Green II (Takara Biotechnology, Dalian, Co., Ltd) following manufacturer’s instructions. The resulting RNA concentrations were normalized using Ncactin (Wang et al., 2017 (link)), and the relative expression levels of the target genes were analyzed using the ABI Prism 7500 software v2.0.5 (Biosystems Inc., USA). The RT-PCR conditions were as follows: 94°C for 5 s, 40 cycles of 94°C for 5 s and 60°C for 30 s. The relative expression of genes was calculated using the 2^-ΔΔCt method and standard deviation was calculated from three replicates (Cárdenas-Mondragón et al., 2016 (link); Gagnaire et al., 2016 (link)).

The gel shift assay of cssDNA was carried out in agarose gel electrophoresis with TAE buffer. cssDNA of pUC119 (100 ng) in HKD buffer including 1 mM MgCl₂ was incubated at 4 °C for 10 min. E .coli SSB was purchased from Bio Academia. The agarose gels were stained by SYBRGreen II (Takara Bio Inc.). Images of gels were captured by using LuminoGraph (ATTO) and band intensities were analyzed by using ImageJ 1.48v.

Among the 25 plants assigned to cold or control conditions, we chose 15 plants of uniform growth for sampling. Three main spikes of BS366 or J411 in the control or cold conditions from the meiosis to the vacuolated stage were pooled together with two replicates. All samples were immediately frozen in liquid nitrogen and stored at − 80 °C for RNA extraction. Total RNA from spikes of both lines under cold and control conditions was extracted using TRIzol Reagent (Invitrogen Corp., Carlsbad, CA). The concentration and quality of total RNA were determined with a Nanodrop spectrophotometer and 1% agarose gel electrophoresis and subjected to transcriptome sequencing. For real-time qRT-PCR, cDNA was synthesized using the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara). Differentially expressed genes were validated with a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) using SYBR Green II (Takara). The expression levels of genes in samples were normalized using the endogenous wheat 18S gene with primer sequences 5′-TGCTGGAATCGGAATAGTTGAG-3′ and 5′-ACTACGCAGGCTCATCAAACAG-3′. The relative expression levels were calculated using the 2^−ΔΔCt method. Primer sequences were designed using Primer3 input version 4.0.0 (http://primer3.ut.ee/) and are listed in Additional file 2: Table S9.

Quantitative real-time PCR (qRT-PCR) was applied to study the expression alterations of VLR2 isoforms in E. sinensis after challenge assays. Reactions were carried out on an ABI PRISM 7500 Sequence Detection System (Applied Biosystems) using SYBR green II (TaKaRa) as the fluorescent dye. The β-actin was chosen as a reference gene for internal standardization. The PCR reaction was carried out in a total volume of 20 μL, containing 10 μL of TB Green Premix DimerEraser (TaKaRa), 0.4 μL of 50 ROX Reference Dye II (TaKaRa), 2 μL of the diluted cDNA, 0.6 μL of each primer (10 μM), and 6.4 μL of sterile distilled water. The PCR program was 95°C for 5 min, followed by 40 cycles of 95°C for 5 s and 55°C for 31 s. Dissociation curve analysis of the amplification products was performed at the end of each PCR reaction to confirm that only one PCR product was amplified. All samples were repeated in triplicates in the qRT-PCR analysis. Fold change for the gene expression relative to controls was determined by the 2^-△△Ct method (22 (link)).

The gene expression was examined by performing an RT-PCR analysis using previously described protocols^{55 (link)}. Total RNA was extracted using TRIZOL reagent and reversely transcribed using a first-strand cDNA synthesis kit (Takara, China) according to the manufacturer’s protocol. Real-time RT-PCR was performed using SYBR Green II (Takara, China) and inventoried assay on CFX96 Real-Time PCR platform (Bio-Rad, USA). The housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control for normalization. The gene-specific primers were synthesized by Sangon Biotech (China), and their sequences are listed in Supplementary File S4.

Total RNA was isolated using Trizol Reagent (Sangon Biotech, Shanghai, China) and cDNA synthesis was performed with the SYBR^® PrimeScript™miRNA RT-PCR Kit and PrimeScript™ RT Master Mix (Takara Biotech, Otsu, Japan) following the manufacturer's instructions for each reagent or kit. MiRNA and mRNA analyses were performed by qRT-PCR using SYBR Green II (Takara Biotech) according to manufacturer's protocol, with a CFX96^TM Real-time System (Bio-Rad, California, USA). Relative quantification of miR-486-5p was obtained by normalization to U6 expression levels, and relative quantification of CDK4 was obtained by normalized to 18S rRNA expression levels. The expression levels of mRNAs and miRNAs were determined by the 2^-ΔΔCt method for relative quantification of gene expression. ΔCt and ΔΔCt were calculated using the following formulae: ΔCt = Ct_miR-486-5p- Ct_U6 or Ct_CDK4 - Ct_18S and ΔΔCt = ΔCt_case - ΔCt_control.

Following the manufacturers' instructions, total RNA was isolated using Trizol Reagent (Sangon Biotech, Shanghai, China) and cDNA synthesis was performed with the PrimeScript™ 1st Strand cDNA Synthesis Kit or SYBR^® PrimeScript™miRNA RT-PCR Kit (TaKaRa). Quantitative RT-PCR analysis was performed using SYBR Green II (TaKaRa a) on a CFX96™ Real-time System (Bio-Rad, Shanghai, China) according to the manufacturer's protocol. The expression levels of miRNAs were normalized to the U6 expression level. The expression levels of mRNAs were normalized to the 18S expression level.