Primescript kit

Total RNA was extracted from collected sample after various stress treatments using Trizol (Invitrogen) method. Then the first strand cDNA was synthesized using of prime script TM kit (Takara) following the manufacturer’s protocols. The NanoDrop instrument (Thermo Scientific NanoDrop 2000C, USA) was used to measure the concentration of cDNA. Later on qRT-PCR was carried out using SYBR^® Premix Ex Taq^TM II (TaKaRa) in an iCycler iQ^TM Multicolor PCR Detection System (Bio-Rad, USA). The amplification cycling parameters of qRT-PCR were as follow: 95°C for 1 min and followed by 40 cycles at 95°C for 10 s, 56°C for 15 s, and 72°C for 15 s. All the primers used for qRT-PCR were shown in Supplementary Table S1. Relative expression of genes was calculated as described by Livak and Schmittgen (2001) (link) and CaUbi3 gene was used as the reference gene in this study (Wan et al., 2011 (link)).

Total RNA was extracted using TRIzol (Invitrogen) following the manufacturer’s specifications. cDNA was synthesized from the total RNA using PrimeScript^TM Kit (Takara, Dalian, China). Transcription levels were measured in duplicate by PowerUp^TM SYBR^TM Green Master Mix (Invitrogen). Relative expression levels of lncRNA and mRNA were normalized to GAPDH expression. The primer sequences were shown in Table S1.

Total RNA was extracted from the intestine, and the integrity was tested by RNA electropherogram. Afterwards, using the Prime ScriptTM kit (Takara, Japan) to reverse transcription of the extracted total RNA into cDNA. The qRT-PCR was performed by Mastercycler ep realplex (Eppendorf, Germany), and reaction volume was 10 μL (primers (0.4 μL), cDNA (0.5 μL), SYBR Green qPCR Mix (High ROX) (Aidlab Biotechnologies Co. Ltd., China) (5 μL), and sterile nonenzyme water (4.1 μL)). The qPCR program was set as 95°C for 2 min, followed by 40 cycles of 95°C for 15 s, Tm for 30 s, and 72°C for 30 s. Adopting the 2^−ΔΔCt method to calculate the genes expression [44 (link)]. According to the result of amplification efficiency, then selected 18s ribosome RNA as the internal reference gene of this study. The primer sequences are shown in Table 2.

Total RNA was extracted from the roots of Y3 plants which were inoculated with virulent strain of P. capsici and reverse transcribed into cDNA using a PrimeScript^TM Kit (Takara, Bio Inc, China) following the manufacturer’s protocols, and then the cDNA was used as a template to isolate the CaPTI1 gene according to the transcriptome database of compatible and incompatible interactions of pepper (Y3) with P. capsici. After that CaPTI1 gene was cloned into pMD19-T vector (Takara) and sequenced by Sangon Biotech (Shanghai) Co. Ltd.
The molecular weight (MW) and isoelectric point (pI) of CaPTI1 were predicted by ExPASy¹. Conserved domain of CaPTI1 protein was identified in Conserved Domain of NCBI². The full length of CaPTI1 and other ERF proteins were used to construct the neighbor joining phylogenetic tree by MEGA 6.05 with 1000 bootstrap replicates. Multiple sequence alignments of the AP2/ERF domain in CaPTI1 and other ERFs were performed by DNAMAN 5.0.