*4.7. Gene Transcription Level Analysis by qRT-PCR*

After a 6 days of pre-cultivation of the CMCs, the *A. flavus* mycelium elicitor was added at 25 mg/L. Then, the CMCs (experimental group, EG) and the sterile water-treated CMCs (the check group, CK) were cultured for another 48 h.

The cultures were frozen in liquid nitrogen and ground into a powder with a mortar and pestle, and RNA extraction was performed as described by Liu et al. [34]. The HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, Jiangsu, China) was used to treat 1 μg of total RNA with DNase to remove genomic DNA, after which cDNA was synthesized according to manufacturer instructions.

Transcript levels of the genes encoding the 40S ribosomal protein S9 (*RPS9*, the housekeeping gene), *D4H*, *G10H*, *GES*, *IRS*, *LAMT*, *SGD*, *STR*, *TDC*, and the transcription factor *ORCA3* were monitored in CMC cultures. Primer sequences for *RPS9*, *IRS*, *LAMT* [49], *D4H*, *G10H*, *GES*, *STR*, *TDC*, *ORCA3* [50], *SGD* [27], and *GES* [25] are shown in Table S6.

The qRT-PCR mixture was prepared using ChamQ SYBR qPCR Master Mix (Q311-02) (Vazyme, Nanjing, Jiangsu, China), and reactions were performed using a LightCycler® 480 II System (Roche, Basel, Switzerland). Amplification included a holding stage of 30 s at 95 ◦C and 40 cycles, each consisting of 10 s at 95 ◦C followed by 30 s at 60 ◦C. Melt curve analysis at 95–60–95 ◦C was then used to verify the specificity of the amplicons. The expression stability of the qRT-PCR results was assayed using the LightCycler® 480 II Software (Roche, Basel, Switzerland). All samples were measured in triplicate.

**Supplementary Materials:** The following are available online. Figure S1: HPLC-MS/MS spectra of the alkaloids in six-day-old suspensions of *C. roseus* CMCs. (a) UV chromatogram at 280 nm; (1) ajmalicine; (2) catharanthine; (3) vindoline. (b) Total ion current (TIC) chromatogram. (c) MS spectra of the identified alkaloids. Figure S2: Gene Ontology (GO) functional classification of assembled unigenes. A total of 61,829 unigenes were assigned to at least one GO term and grouped into three main GO categories and 56 groups (26 groups in the "biological process" domain, 20 in the "cellular component" domain, and 10 in the "molecular function" domain). The y-axis indicates the number of genes in a sub-category. Figure S3: Functional classification and pathway assignment of assembled unigenes by the Kyoto Encyclopedia of Genes and Genomes (KEGG). A total of 34,367 unigenes were classified to the five main KEGG metabolic pathways: cellular processes (A), environmental information processing (B), genetic information processing (C), metabolism (D), and organismal systems (E). The y-axis represents the name of the KEGG metabolic pathway. The x-axis indicates the number of unigenes annotated to the KEGG metabolic pathway and the ratio of their number to the total number of annotated unigenes. Figure S4: Correlation coefficient between the callus vs. CK (a) and EG vs. CK (b) samples. The x-axis indicates the log10 (FPKM + 1) of Sample 1 and the y-axis the log10 (FPKM + 1) of Sample 2, and R<sup>2</sup> was the square of the Pearson correlation coefficient. Figure S5: Correlation coefficient between the callus vs. CK (a) and EG vs. CK (b) samples. The x-axis indicates the log10 (FPKM + 1) of Sample 1 and the y-axis the log10 (FPKM + 1) of Sample 2, and R2 was the square of the Pearson correlation coefficient. Figure S6: Venn diagram of DEGs from callus vs. CK and EG vs. CK samples. Figure S7: GO enrichment analysis of DEGs in callus vs. CK and EG vs. CK samples. The x-axis indicates the comparative combination and the y-axis the number of DEGs. Figure S8: Partial results of KEGG pathway analysis of DEGs in callus vs. CK and EG vs. CK samples. The x-axis indicates the KEGG pathway and the y-axis the number of DEGs. Table S1: Summary of sequencing reads after filtering. Table S2: Summary of the sequence assembly results. Table S3: Summary of functional annotation for assembled unigenes. Table S4: Summary of KEGG pathways involved in the *Catharanthus roseus* transcriptome. Table S5: Gene transcription factor analysis of unigenes. Table S6: Sequences of primers for *Catharanthus roseus* genes used in the qRT-PCR assay.

**Author Contributions:** Methodology, J.Z. (Jianhua Zhu), J.Z. (Jiachen Zi) and C.L.; investigation, C.L., P.Z., L.X. and C.C..; formal analysis, J.Z. (Jianhua Zhu), J.L., C.L. and C.C.; resources, J.Z. (Jianhua Zhu), J.Z. (Jiachen Zi) and R.Y.; writing—original draft preparation, C.L., J.L., P.Z, L.X. and C.C.; writing—review and editing, J.Z. (Jianhua Zhu) and R.Y.; supervision, J.Z. (Jianhua Zhu) and R.Y.; project administration, J.Z. (Jianhua Zhu); funding acquisition, J.Z. (Jianhua Zhu) and R.Y.

**Funding:** This study was supported by the National Natural Science Foundation of China (Nos. 81573568 and 81673571), the Guangdong Province Natural Science Fund for Distinguished Young Scholars (No. 2016A030306009), and the Key Laboratory of Chemical Biology (Ministry of Education) Open Projects Fund (Project No. CB-201707).

**Conflicts of Interest:** The authors declare no conflict of interest.
