*2.3. qRT-PCR Analysis of LcCLA1 and LcPDS Silencing Level*

After detection of viral infection in *L. chinensis* leaves, we next analyzed gene expression after infection using qRT-PCR. The gene expression of *LcCLA1* and *LcPDS* in untreated wild-type, empty-vector and virus-infected plants was compared to assess gene-silencing efficiency. For both genes, gene expression was significantly reduced in infected *Lycoris* leaves compared to wild-type and empty TRV vector plants (Figure 4). The efficiency of gene silencing was analyzed by monitoring the expression levels of *LcCLA1* in plants showing leaf bleaching phenotype. According to the results, it was found that the expression level of *LcCLA1* in pTRV1- and pTRV2-injected leaves was not significantly changed compared with CK. However, the expression level of *LcCLA1* in leaves injected with pTRV2-*LcCLA1* was about 75% lower than that in CK (Figure 4a). The etiolation phenotype of the leaves was consistent with the expression level of *LcCLA1* after silencing. The expression level of *LcPDS* was also analyzed in leaves injected with pTRV2-*LcPDS* but with less etiolation phenotype. From the results, it was found that the expression level of *LcPDS* in CK leaves was approximately the same as that in leaves injected with pTRV1 and pTRV2. However, the expression level of *LcPDS* in leaves injected with pTRV2-*LcPDS* was only about 50% of that in CK (Figure 4b). Compared with the silencing efficiency of *LcCLA1*, the silencing efficiency of *LcPDS* is much lower.

**Figure 4.** Relative expression levels of *LcCLA1* and *LcPDS* in not injected (CK), TRV vector-injected (pTRV1+pTRV2), pTRV2-*LcCLA1*- and pTRV2-*LcPDS*-injected leaves of *L. chinensis*. (**a**) Relative expression level of *LcCLA1*. (**b**) Relative expression level of *LcPDS*. Error bars represent standard errors, and different lowercase letters indicate significant differences at *p* ≤ 0.05.

#### **3. Discussion**

In this study, we demonstrated that the endogenous genes in *L. chinensis* can be effectively down-regulated using the TRV-VIGS system. Since the current genetic transformation system of *Lycoris* is very immature, it is difficult to rapidly verify the gene function of this genus. Breaking through this barrier would not only take a lot of time and effort but would also need to overcome a lot of technical challenges. Therefore, effective and low-cost technologies need to be developed to temporarily replace genetic transformation systems. We developed the TRV-VIGS system to contribute to the gene regulation research of *Lycoris*, especially in its flowering traits.

*Agrobacterium* infection is the most effective method of virus-based vector infestation of plants [37]. The silencing efficiency varies greatly with different injection methods for the same plant [32]. The silencing efficiency is related to the fluid volume of *Agrobacterium* entering the plant. Since the leaf surface of *Lycoris* has a waxy layer, it is difficult to get the *Agrobacterium* solution into the leaf using the normal method of injection infiltration. We inserted the solution by injection with the needle of a syringe from the tip of the leaf to fully infiltrate the leaf, thus greatly improving the injection efficiency. To visualize whether genes were being knocked down, we used two different reporter genes, *CLA1* and *PDS*, which are commonly used in VIGS and which produce a visible phenotype after successful silencing.

The *CLA1* gene is involved in chloroplast development and is a potent marker in the TRV-VIGS system [38]. The *CLA1* gene was selected as a reporter, and it was tested out to determine if the TRV-VIGS system can be well applied to the study of gene function in *Solanum melongena* [39]. In *Arabidopsis*, the bleaching phenotype after silencing *CLA1* was used as a visible indicator of the silencing efficiency of the TRV-VIGS system [40]. Up to now, *CLA1* has been used as a reporter gene for gene silencing many times in cotton studies. For example, when weather cold-induced changes in non-coding gene expression had a significant effect on the cold tolerance of cotton seedlings, TRV2-*CLA1* was used as a positive control to determine that silencing of lincRNA *XH123* resulted in increased sensitivity to cold injury [41]. When studying the function of the *CLE* gene family in cotton, *CLA* was again used as a positive control, and it was found that the silencing of *GhCLE5* in cotton resulted in dwarf seedlings [42]. Inhibition of *GauGRAS1* by VIGS resulted in glandless stems and petioles of *Gossypium australe* compared with positive control *TRV-CLA* and negative control TRV empty vector [43]. We isolated a homologue of *CLA1* from *L. chinensis* leaves, named *LcCLA1*. Plants were infiltrated with *Agrobacterium tumefaciens* expressing partial sequences of *LcCLA1*, and etiolation phenotype was observed on the injected leaves approximately two weeks after infiltration. qRT-PCR analysis revealed low levels of *LcCLA1* transcript in *L. chinensis* leaves inoculated with the TRV-VIGS system, which confirmed the silencing of this gene. This suggests that the expression of *LcCLA1* is significantly down-regulated in *L. chinensis* through the TRV-VIGS system, resulting in etiolation phenotype of the leaves.

The gene *PDS* encodes an enzyme in carotenoid biosynthesis whose silencing leads to the albino phenotype of plant tissues [18], so it is often used as a marker in VIGS experiments. Up to now, VIGS has been reported to silence *PDS* in many plants, including *Solanum melongena* [39], *Capsicum annuum* [16], tomato [11], *Nicotiana benthamiana* [44], *Sorghum bicolor* [45], soybean [46], etc. In this study, the endogenous *PDS* gene also was selected as the reporter gene in *L. chinensis*. Injected leaves were significantly altered compared to controls, exhibiting a pronounced chlorotic phenotype, especially in the upper and middle sections of leaves. It indicated that the *PDS* gene could be selected as the indicator gene for VIGS system in *L. chinensis*. However, the chlorotic phenotype of *LcCLA1* was more obvious than that of *LcPDS*. qRT-PCR validation also showed that the expression of *LcCLA1* was lower than that of *LcPDS*. The reason may be that the *PDS* gene is not only involved in chlorophyll content but also carotenoid biosynthesis [18,35]. For example, in highbush blueberry that knock out the *PDS* gene, the leaves exhibited a phenotype without chlorophyll, but with red coloration [36]. Thus, our study suggested that the *PDS* gene may also be involved in carotenoid biosynthesis in *Lycoris*, leading to less albino phenotype in leaves.

There are many factors that affect silencing efficiency, such as the growth temperature after inoculation, the growth stage of the inoculated plants, the type of *Agrobacterium* strain, the inoculation method and the inoculum concentration. Different plants require different temperatures to produce a good silencing phenotype after VIGS. In the tomato, the optimal silencing phenotype was obtained when the growth temperature was 22 ◦C after inoculation [47]. In *S. pseudocapsicum*, silencing efficiency decreased when inoculated seedlings were grown at 18 ◦C or 30 ◦C [32]. We need to improve the growth temperature of the VIGS system between 20 ◦C and 25 ◦C. The growth stage of the target plant affects silencing efficiency. Gerbera seedlings at the early stages of vegetative development were much more sensitive to TRV VIGS than those at the middle and late stages [48]. We can try to inoculate between one and two weeks after leaf emergence. The optimal *Agrobacterium* strain for VIGS varies from plant to plant and has a strong impact on gene silencing efficiency. Studies have shown that both *Agrobacterium* strains GV2260 and GV3101 can be used in *N. benthamiana*, with GV2260 working best [18]. LBA4404 and GV2260 can be used on the tomato, but the silencing efficiency is very low, and GV3101 has the best silencing effect [49]. We will select LBA4404 and GV2260 to compare with GV3101 and select the strain with the best silencing efficiency. Due to the different inoculation methods

and characteristics of different infected plants, the concentration of the infection solution strongly affects the gene silencing efficiency of VIGS experiments. In the TRV-VIGS experiment of *Arabidopsis*, the most suitable concentration of *Agrobacterium* infection solution was OD600 = 1.5, and the silencing efficiency was almost 100% [15]. In addition, naked cotton seeds soaked in *Agrobacterium* inoculum with OD600 of 1.5 for 90 min showed the best silencing efficiency [50]. For vacuum infiltration, some studies have found that the optimal conditions for VIGS are vacuum treatment with *Agrobacterium* liquid with OD600 of 0.3 for 30–60 s, and co-cultivation with the same concentration of *Agrobacterium* for 15 h [51]. The above experiments show that different species require different concentrations of *Agrobacterium* inoculation; different inoculation methods also require different concentrations of *Agrobacterium*. In this study, the silencing of *LcCLA1* and *LcPDS* were successfully achieved by *Agrobacterium* infection at OD600 = 2.0 and cultured at 18–26 ◦C for 2 weeks. In addition, *L. chinensis* has an obvious chlorosis phenotype. Therefore, this can be used as the recommended concentration for the VIGS system of *Lycoris* plants.
