**1. Introduction**

*Catharanthus roseus* (L.) Don is a perennial medicinal plant of the family Apocynaceae. At present, over 130 alkaloids have been isolated from *C. roseus*, most of which are terpenoid indole alkaloids (TIAs) [1]. Some TIAs such as vinblastine, vincristine, and ajmaline exhibit strong pharmacological activities, and some are widely used in the treatment of various diseases [2–5]. Ajmalicine is a potent antihypertensive reagent [5]. Vinblastine and vincristine, two bisindole alkaloids derived from coupling vindoline and catharanthine, are natural anticancer drugs and are still among the most valuable agents used to treat cancer [3,4].

These secondary metabolites are produced from the TIA biosynthetic pathway in *C. roseus*, which is complex and highly regulated (Figure 1) [2,6,7]. The TIA biosynthetic pathway consists of TIA feeder pathways and the downstream of the TIA biosynthetic pathway. The TIA feeder pathways are the monoterpenoid pathway and indole pathway [7]. The downstream of the TIA biosynthetic pathway in *C. roseus* starts with the formation of strictosidine from tryptamine and

secologanin, which is catalyzed by strictosidine synthase (STR) [8]. Then, strictosidine is deglucosylated by strictosidine β-D-glucosidase (SGD) to form strictosidine aglycone. Further enzymatic steps result in the formation of numerous TIAs, and the TIA biosynthetic pathway is classified into several specific branches. One branch of the TIA biosynthetic pathway produces ajmalicine and serpentine, a second branch catharanthine, a third vindoline, and a fourth lochnericine and horhammericine [7]. Genes encoding enzymes catalyzing the production of vindoline from tabersonine, such as deacetoxyvindoline 4-hydroxylase (D4H) and deacetylvindoline O-acyltransferase (DAT), have now been identified [9–14]. The formation of α-3 ,4 -anhydrovinblastine from vindoline and catharanthine is catalyzed by a major class III peroxidase (PRX1) [15]. Then, vinblastine and vincristine are formed through multiple enzymatic steps from α-3 ,4 -anhydrovinblastine (Figure 1) [7]. The TIA biosynthetic pathway in *C. roseus* is highly regulated by enzymes and transcription factors (TFs). In 2000, Fits and Memelink discovered octadecanoid-derivative responsive *Catharanthus* A P2-domain protein 3 (ORCA3), a jasmonate-responsive AP2/ERF transcription factor, in *C. roseus* using the T-DNA activation tagging technology [16]. Since overexpression of *ORCA3* increases the expression of key genes in the TIA biosynthetic pathway, it is considered to be the core transcription factor in the TIA biosynthetic pathway in *C. roseus* [6]. In addition, *ORCA3* expression could be induced by jasmonates [17].

**Figure 1.** Terpenoid indole alkaloid (TIA) biosynthetic pathways of *C. roseus*. Enzyme abbreviations: GES: geranial synthase; G10H: geraniol 10-hydroxylase; IRS: iridoid synthase; LAMT: loganic acid methyltransferase; SLS: secologanin synthase; AS: anthranilate synthase; TDC: tryptophan decarboxylase; STR: strictosidine synthase; SGD: strictosidine β-D-glucosidase; D4H: deacetoxyvindoline 4-hydroxylase; DAT: deacetylvindoline 4-*O*-acetyltransferase; T6,7E: tabersonine 6,7-epoxidase; T19H: tabersonine/lochnericine 19-hydroxylase; PRX1: vacuolar class III peroxidase. Single arrows denote single steps, and dotted arrows denote multiple or unidentified steps.

However, the content of the pharmacological secondary metabolites in *C. roseus* is very low [18], and commercial production has used a semi-synthetic route to couple catharanthine and vindoline [3]. Therefore, developing methods to increase the yields of TIAs in *C. roseus* has become a focus for domestic and foreign scholars [19]. In recent years, an increasing number of studies has shown that fungal elicitors play an important role in regulating biological secondary metabolic pathways and intracellular information transmission [20,21]. The fungal elicitor is an active substance derived from fungus, which can rapidly and specifically induce the expression of specific plant genes, thereby activating specific secondary metabolic pathways and increasing the accumulation of interesting secondary metabolites [20]. The treatment of plant cell culture systems with fungal elicitors has become an effective method for rapidly increasing the yield of target product in plant cell culture [20,21]. Although fungal elicitors are known to regulate the production of secondary metabolites, the inducting effect varies depending on factors such as fungal species, elicitor components, dose, time of addition, and type of plant cell culture [22–24]. In 2016, Tonk reported that a low-dose *A. flavus* fungal elicitor effectively increased the growth rate of callus, embryo biomass, germination rate, and alkaloid content in the embryos of *C. roseus*, as well as increasing the shoot and root lengths of germinated somatic embryos. Further, antioxidant enzyme activity assays showed that a low-dose *A. flavus* elicitor caused an allergic reaction in the mature and germinating somatic embryos of *C. roseus*, resulting in an increase in alkaloid content [24]. Therefore, *A. flavus* may be a good elicitor for promoting the TIA production of *C. roseus* cell cultures.

More interestingly, our previous research showed that *C. roseus* cambial meristematic cells (CMCs) exhibit good characteristics when compared to *C. roseus* dedifferentiated plant cells (DDCs), such as faster growth, higher yields, diverse bioactive TIAs, lower variability, and high expression of TIA biosynthesis genes. In addition, after two years of being cultured, *C. roseus* CMCs remained stable in both genetic traits and alkaloid content; the production of vindoline, catharanthine, and ajmaline in *C. roseus* CMCs could be enhanced by β-cyclodextrin and methyl jasmonate (MeJA) [25]. Therefore, *C. roseus* CMCs may be a good system for the investigation of the biosynthesis and regulation of TIAs.

However, there is no report on the effect of the *A. flavus* fungal elicitor on the TIA production of *C. roseus* CMCs so far. In this research, we investigated the inducing effect of the *A. flavus* fungal elicitor on the biosynthesis of TIAs in *C. roseus* CMCs, and the inducing mechanism was explored by transcriptome analysis and determination of the expression of TIA biosynthesis-related genes via the quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) technique.

#### **2. Results**
