Mirna first strand cdna synthesis

We extracted total RNA using the miRcute Plant miRNA Isolation Kit (Tiangen, Beijing, China). Expression of miRNA was detected by stem–loop qRT-PCR as described [62 (link)]. First-strand cDNA was synthesised by miRNA First Strand cDNA Synthesis (Vazyme, Nanjing, China) with the stem-loop RT primer. The small nuclear RNA U6 (in fungi) and 5.8 rRNA (in plant) was used as control. Transcript levels of genes were analysed by qRT-PCR, which was performed using RealStar Green Power Mixture (GenStar, Beijing, China). MdEF1α of M. domestica and G6PDH of V. mali were used as the reference genes, respectively. Relative expression of genes was calculated using the 2^-ΔΔCt method [63 (link)]. All primers used in this study are listed in Table S5 (see online supplementary material).

Samples of different treatments were collected at 0, 24, 36, 48, 60 and 72 h post inoculation (hpi). Total RNA was extracted using the RNeasy^® Plant Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. Expression of Tr-milRNA1 was determined by stem-loop qRT–PCR as previously described [25 (link)]. First strand cDNA was synthesized by miRNA First Strand cDNA Synthesis (Vazyme Biotech, Nanjing, China) with the stem-loop RT primer based on the manufacturer’s instructions. PCR detection was performed by a Tr-milRNA1-specific forward primer and a universal reverse primer. The NJAU 4742 18S rRNA biogenesis gene (18S) was used as a control. qRT–PCR was performed using the miRNA Universal SYBR qPCR Master Mix (Vazyme Biotech, Nanjing, China) according to the manufacturer’s instructions. For the determination of transcript levels of the corresponding target genes, cDNA synthesis was completed by the PrimeScript RT Reagent Kit (RR036A, Takara, Dalian, China) according to the manufacturer’s instructions. qRT-PCR was performed using SYBR Premix Ex Taq II (RR820A, Takara, Dalian, China) and the CFX connect^TM Real-Time system (Bio–Rad, Hercules, CA, USA). Transcription levels of the target genes were normalized by the 2^−ΔCt method, and translation elongation Factor 1 alpha (Tef) was used as the housekeeping gene. All primers used in this study are listed in Table S1.