**1. Introduction**

*Lycoris* belongs to the Amaryllidaceae family and consists of around 20 distinct species worldwide. They are native to Eastern Asia and distributed in moist warm temperature areas, especially in China, Japan and Korea [1]. Most species are valued for their stunningly vibrant and distinct coloration and striking blooms. As an important class of bulbous flowers, they also have low environmental requirements and strong adaptability, making them a good choice for landscaping [2]. In addition, the bulbs contain a large number of alkaloid compounds, which have anti-malarial and anti-tumor effects and are a treatment for senile dementia [3,4], so the plants also have important medicinal value. Due to the great ornamental and medicinal value of *Lycoris*, more and more transcriptomic data have been provided, and functional genes related to excellent breeding traits have been identified [5–8]. However, due to the lack of efficient genetic transformation, there has been no report on the gene function of *Lycoris* thus far. Moreover, *Lycoris* is a perennial bulbous flower; it takes 3–5 years to grow from a small bulb to a flowering plant [9]. In the study of the functional genes of flowering traits, a long period is always needed to observe the phenotype after conventional genetic transformation. Therefore, it is necessary to establish an efficient, rapid and appropriate transformation system to promote research on the molecular regulation mechanism of this genus.

**Citation:** Cheng, G.; Shu, X.; Wang, Z.; Wang, N.; Zhang, F. Establishing a Virus-Induced Gene Silencing System in *Lycoris chinensis*. *Plants* **2023**, *12*, 2458. https://doi.org/10.3390/ plants12132458

Academic Editors: Qian-Hao Zhu, Aiping Song and Yu Chen

Received: 1 June 2023 Revised: 21 June 2023 Accepted: 25 June 2023 Published: 27 June 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Virus-induced gene silencing (VIGS) is an excellent alternative to obtaining information about gene function by transiently knocking out the gene of interest [10]. It is a post-transcriptional gene silencing (PTGS)-based technology that utilizes natural defense mechanisms employed by plants to protect against invading viruses [11]. Virus-infected plants induce double-stranded RNA-mediated PTGS, which degrades viral RNA [12]. Recombinant viruses carrying partial sequences of host target genes are used to infect and spread throughout the plant [13]. Viral gene transcripts and plant target genes are recognized and degraded by endogenous PTGS, resulting in reduced target gene expression [14]. Compared with other transgenic technologies, VIGS technology can avoid plant transformation and has the advantages of short cycle, low cost and simple operation. At present, it has been applied to a variety of plants for gene function verification, such as Arabidopsis [15], tomato [11], pepper [16], *Lilium* [17], etc. VIGS has great advantages, especially in perennial woody plants and perennial herbaceous plants. In *Vernicia fordii*, VIGS can shorten the time to phenotype observation and identify phenotypes after loss-offunction of a gene of interest within a single generation [14]. For herbaceous plants, the transformation operation of VIGS is simple and fast and can function in different genetic backgrounds [12].

Many viruses have been used to develop VIGS vectors, such as tobacco mosaic virus (TMV) [18], potato virus X (PVX) [19], tomato golden mosaic virus (TGMV) [20], tobacco rattle virus (TRV) [21] and apple latent spherical virus (ALSV) [22]. Among these viral vectors, TRV has been widely used to construct VIGS vectors for silencing target genes in various bulbous perennial flowers; for example, VIGS experiments using TRV vectors in lily petals showed that anthocyanin accumulation was reduced when *LvMYB5* was silenced [23]. After silencing *NtPDS* with TRV vector in Chinese narcissus, extensive chlorosis of leaves was found [24]. However, it is unclear whether TRV vector-based VIGS can be used to reveal gene function in *Lycoris*, and there is no suitable VIGS system established by now.

*Cloroplastos Alterados 1* (*CLA1*) and *Phytoene Desaturase* (*PDS*) are the most commonly used indicator genes for VIGS system establishment. The *CLA1* gene is involved in chloroplast development and has shown a highly pronounced albino phenotype, so the silenced *CLA1* serves as a useful marker to determine silencing efficiency [25]. *CLA1* has mainly been used in cotton as a positive control for VIGS [26]. When the albino phenotype is observed, it indicates that the gene has been silenced. To determine whether *HyPRP1* is required for cotton resistance to Verticillium wilt, the marker gene *CLA1*-VIGS plants are used to determine the silencing efficiency of *HyPRP1* [27]. In upland cotton, using TRV-*CLA1* as a control, it was found that silencing of *GhCLCg-1* resulted in impaired salt tolerance [28]. It has also been successfully used in other species, such as *Arabidopsis* [14] and *Vernicia fordii* [29]. The gene *PDS* also produces an albino phenotype and is widely used as an indicator gene for the VIGS system [30]. The TRV-*PDS* system has been successfully applied in many ornamental plants. For example, in tree peonies, a typical albino phenotype was found in the newly sprouted top leaves of TRV-*PoPDS*-infected triennial tree peony seedlings using leaf syringe infiltration and seedling vacuum infiltration [31]. This shows that TRV-based VIGS technology can be applied to the high-throughput functional characterization of tree peony genes. In *Lilium* × *formolongi* [17], using the inoculation method of rubbing plus injection, albino was observed in newly developing leaves of TRV-*LhPDS*-infected lily seedlings 56 days after infiltration. In *Solanum pseudocapsicum* L. [32], the TRV-*SpPDS* system was used to infect leaves, and obvious albino was found. In *Catharanthus roseus* [33], the TRV-*CrPDS* system was used to infect roots, stems, leaves, and flowers, and it was found that all tissues showed obvious albino, and the phenotypes of leaves and flowers were more obvious. Nishii et al. used a TRV vector with a wide host range to silence the *SrPDS* gene in *Streptocarpus rexii* through *Agrobacterium* inoculation, and finally obtained silenced plants with albino phenotypes [34]. These examples demonstrate the wide application of the TRV-PDS system. However, the *PDS* gene is not only involved in chlorophyll content but also in carotenoid biosynthesis [35]. For example, in highbush blueberry, which is rich

in polyphenols and anthocyanins, a phenotype could occur without chlorophyll, but with red coloration after knockout of the *PDS* gene [36]. It suggests that the chlorophyll-lacking phenotype is varied in different species.

The leaves of *Lycoris* plants are linear and have the characteristics of longitudinal and orderly growth of vascular tissue. The surface of the leaves is covered with a thin waxy layer, and thus it is not easy to infiltrate the bacterial solution with the traditional infiltration method. In this study, we developed a leaf tip injection method, constructed TRV vectors using *LcCLA1* and *LcPDS* as reporter genes, and tested the feasibility of the TRV-VIGS system in spring-leafed *L. chinensis*, in which the young leaves emerge from the bulb in early spring when they are suitable for injection. Two weeks after infection, the leaves of *L. chinensis* injected with *LcCLA1* and *LcPDS* showed chlorosis, but the phenotype of leaves injected with *LcPDS* was not as obvious as that injected with *LcCLA1*. The expression level of *LcCLA1* in the chlorotic leaves was significantly lower than that in non-injected leaves. In terms of gene expression, the expression levels of *LcCLA1* and *LcPDS* in chlorotic leaves were significantly lower than those in uninfected leaves, while the gene expression of *LcCLA1* was lower, which indicated that *LcCLA1* was more suitable as an indicator gene in gene function studies. Therefore, TRV-*LcCLA1* can silence the genes in *L. chinensis* more effectively than the TRV-*LcPDS* VIGS system, which lays a good foundation for the gene function verification of *L. chinensis* in the future.
