Total RNA was isolated from the stem phloem samples using an EASYspin Plus Complex Plant RNA Kit (Aidlab, Beijing, China), from the inoculated N. benthamiana leaves using a TransZol Plant RNA Kit (TransGen Biotech, Beijing, China), and from L. theobromae mycelia using TRIzol reagent (Invitrogen). We used 2 µg of each total RNA sample to synthesize cDNA using a Superscript III First-Strand Synthesis SuperMix Kit (Invitrogen). The qRT-PCRs were run on a 7500 Real-Time System (Applied Biosystems) following the manufacturer’s protocols. Relative gene expression levels were calculated using the comparative 2−ΔΔCT method (Livak and Schmittgen, 2001 (link)). The primers used are listed in Supplementary Table S1 .
Defense suppression tests were performed as previously reported (Chen et al., 2015 (link)). Briefly, 10-day-old seedlings of N. benthamiana were treated with 1 µM flg22 (Sangon Biotech, China). The expression of the PTI-associated genes NbACRE31, NbGRAS2, and NbPTI5 were determined by qRT-PCR. In addition, qRT-PCR was also used to determine the expression levels of the defense-related genes in the SA and jasmonic acid (JA) signaling pathways, including PATHOGENESIS RELATED PROTEIN 1 (NbPR1) and LINOLEATE 9S-LIPOXYGENASE 5 (NbLOX), which are specifically induced by SA signaling. The NbEF1α and NbTUBULIN6 genes were used as internal controls.
Defense suppression tests were performed as previously reported (Chen et al., 2015 (link)). Briefly, 10-day-old seedlings of N. benthamiana were treated with 1 µM flg22 (Sangon Biotech, China). The expression of the PTI-associated genes NbACRE31, NbGRAS2, and NbPTI5 were determined by qRT-PCR. In addition, qRT-PCR was also used to determine the expression levels of the defense-related genes in the SA and jasmonic acid (JA) signaling pathways, including PATHOGENESIS RELATED PROTEIN 1 (NbPR1) and LINOLEATE 9S-LIPOXYGENASE 5 (NbLOX), which are specifically induced by SA signaling. The NbEF1α and NbTUBULIN6 genes were used as internal controls.
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