Real time system

All samples were homogenized in Trizol reagent following the manufacturer’s protocol. The extracted RNA was reversely transcribed into cDNA using PrimeScript™ One Step RT-PCR Kit (Takara, Kyoto, Japan). qRT-PCR was performed to examine the gene expression levels in the collected samples, with 18s rRNA being used as the internal control. Fungal load abundance of RNAi groups was evaluated by analyzing the transcript abundance of B. bassiana 18s rRNA as a fungal-specific primer [55 (link)]. All these primers used in the present study were listed in Table S2. qPCR was performed using BIO-RAD Real-Time System (Bio-Rad, Hercules, CA, USA) with the following process: initial denaturation at 95 °C for 30 s, then 40 cycles of denaturation at 95 °C for 5 s, annealing, and extension at 60 °C for 34 s. The melting curves for each PCR reaction were checked to verify the formation of primer dimers or sample contamination.
The expression levels of these genes were quantified using the 2^ΔΔCt method. These data were presented as the mean ± standard deviation (SD) with at least three replicates. One-way ANOVA analysis and Tukey’s test were used to compare the differential gene expression levels among different time points. The student t test was used to compare the mean of two groups.

First-strand cDNA was synthesized from total RNA using the PrimeScript RT reagent kit with gDNA Eraser (TaKaRa, Japan) according to the manufacturer’s instructions. qRT-PCR was performed using SYBR Green PCR master mix (TransGen, China) on a Bio-Rad Real-Time system (Bio-Rad, Hercules, USA). FvACTIN was used as an internal control [83 (link)]. Primers designed from the conserved region of each cDNA were used for qRT-PCR analyses (Additional file 9: Table S9). Relative expression levels were calculated using the 2 ^−ΔΔCT method.

To validate RNA-seq results, the expression patterns of 21 randomly selected genes from various functional categories and regulation patterns were analysed by qRT-PCR at five different time points post infection (Additional file 1: Table S1). Three biological replicates were collected independently and immediately frozen in liquid nitrogen. Total RNA isolation was performed as described above, and 1 μg of total RNA was reverse-transcribed using PrimerScript First-Strand cDNA Synthesis Kit (Takara, Dalian, China). qRT-PCR was performed in a 25 μl reaction mixture containing 12.5 μl 2 × SYBR Master Premix ExTaq (Takara), 1 μl cDNA template (1:5 dilution), and 1 ul of each forward and reverse primer for the selected gene of interest. All primers were designed based on cDNAs using Primer Premier 5.0 (Additional file 1: Table S1). Three biological and three technical relipcates per sample were analysed using Bio-Rad Real-Time System (BioRad, Hercules, CA, USA) based on the 2^-ΔΔCT method [32 (link)]. qRT-PCR conditions were 95 °C for 2 min followed 40 cycles at 95 °C for 5 s, 60 °C for 30 s and 65 °C for 5 s. Actin11 (GeneBank: 209698678) was used as a reference gene.