Establishment of virus-induced gene silencing system and functional analysis of ScbHLH17 in Senecio cruentus

Journal Pre-proof Establishment of virus-induced gene silencing system and functional analysis of ScbHLH17 in Senecio cruentus Yajun Li, Yuting Liu, Fangting Qi, Chengyan Deng, Chenfei Lu, He Huang, Silan Dai PII:

S0981-9428(19)30540-6

DOI:

https://doi.org/10.1016/j.plaphy.2019.12.024

Reference:

PLAPHY 5981

To appear in:

Plant Physiology and Biochemistry

Received Date: 25 October 2019 Revised Date:

12 December 2019

Accepted Date: 19 December 2019

Please cite this article as: Y. Li, Y. Liu, F. Qi, C. Deng, C. Lu, H. Huang, S. Dai, Establishment of virus-induced gene silencing system and functional analysis of ScbHLH17 in Senecio cruentus, Plant Physiology et Biochemistry (2020), doi: https://doi.org/10.1016/j.plaphy.2019.12.024. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Masson SAS.

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Highlights A rapid transient gene knock-down technology was first established in Senecio cruentus. S. cruentus is a good material for anthocyanin research. VIGS system was obtained in capitulum of S. cruentus by silencing ScANS. ScbHLH17 was identified to positively regulate the anthocyanin biosynthesis.

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Establishment of virus-induced gene silencing system and functional analysis of ScbHLH17 in Senecio cruentus

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Abstract Virus-induced gene silencing (VIGS) is a technology for rapid gene functional analysis that depends on the degradation of viral RNA and is part of the natural defense mechanism in plants. Senecio cruentus is an important Compositae ornamental species that is plentiful and available in a variety of colors and has a typical blue variety that is rare in Compositae. These advantages make it a good material for studying the anthocyanin biosynthesis and blue flower formation mechanism. With the development of gene sequencing technology, the functions of many candidate genes that may be involved in anthocyanin biosynthesis in S. cruentus need to be identified. However, a stable and rapid genetic transformation system of S. cruentus is still lacking. Here, we screened two cultivars, ‘Venezia’ and ‘Jseter’, chosen ScPDS and ScANS as test genes, and investigate the effect of developmental periods, bacterial cell concentrations and infection methods on gene silencing efficiency. The results showed that the silencing efficiency of S. cruentus leaves was low (13%), and it was less affected by the parameters. However, the transcription factor gene ScbHLH17 was still silenced by VIGS, which resulted in the loss of anthocyanin accumulation in leaves, and the expression levels of anthocyanin biosynthesis pathway (ABP) structural genes, including ScCHI, ScDFR3 and ScANS, were decreased significantly. The result proved that ScbHLH17 was an important transcription factor that regulated flower color formation in S. cruentus. In addition, ScANS-silencing phenotypes were observed in S. cruentus capitulum by vacuum-infiltrating S1 stage buds for 10 min after scape injection. In general, the present study provided important technical support for the study of anthocyanin metabolism pathways in S. cruentus. Keywords: Senecio cruentus, VIGS, anthocyanin, bHLH transcription factor

74

1. Introduction

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Virus-induced gene silencing, referring to the insertion of a target gene fragment into a virus vector and infecting plants, which can inhibit endogenous genes and induce corresponding physiological and phenotypic changes, is a gene function analysis method involving molecular identification. With the rapid development of next-generation sequencing technology, a growing number of genes closely related to specific traits have been isolated. An efficient and rapid VIGS system has become a necessary technology for preliminary gene functional analysis and screening compared with traditional transgenic technology, which is labor-intensive and time-consuming (Dommes et al., 2018).

Yajun Lia,b,c,d,1, Yuting Liua,b,c,d,1, Fangting Qia,b,c,d, Chengyan Denga,b,c,d, Chenfei Lua,b,c,d, He Huanga,b,c,d,*1, Silan Daia,b,c,d,** a

Beijing Key Laboratory of Ornamental Plants Germplasm Innovation & Molecular Breeding, Beijing, 100083, China

b

National Engineering Research Center for Floriculture, Beijing, 100083, China

c

Beijing Laboratory of Urbanand Rural Ecological Environment, Beijing, 100083, China

d

School of Landscape Architecture, Beijing Forestry University, Beijing, 100083, China

*

Corresponding author. School of Landscape Architecture, Beijing Forestry University, Beijing, 100083, China. author. School of Landscape Architecture, Beijing Forestry University, Beijing, 100083, China. E-mail addresses: [email protected](H.Huang). 1 Contributed equally to this work. ∗∗Corresponding

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The efficiency of VIGS closely depends on the interaction between the plant and virus (Burch-Smith et al., 2010). First, establishment of a VIGS system in plants depends on the infectivity of virus vectors. Certain viruses can be used as VIGS vectors, such as TRV (Tobacco rattle virus), TMV (Tobacco rattle virus), CymMV (Cymbidium mosaic virus), BSMV (Barley stripe mosaic virus), ALSV (Apple latent spherical virus) and BMV (Brome mosaic virus) (Dommes et al., 2018). BSMV is the most widely used vector among monocots, including many agronomical important crop species such as Zea mays, Triticum aestivum, Oryza sativa and Brachypodium distachyon (Lange et al., 2013). The use of CymMV is relatively mature in gene function research for orchid. VIGS vectors developed from TRV (Liu et al., 2002; Ratcliff et al., 2001) have been used for gene function analysis in many plant species, including plants in Solanaceae, Ranunculaceae, Compositae and Rosaceae. The TRV vector can even infect meristem and silence genes in flower organs and pollen (Hsieh et al., 2013a; Hsieh et al., 2013b; Lange et al., 2013; Sui et al., 2018; Dommes et al., 2018). Second, a successful VIGS system depends on whether the silencing phenotype can appear systemically after virus infection. For some species, such as Solanum lycopersicum (Yan et al., 2012), Physalis floridana (Zhang et al., 2014), Catharanthus roseus (Sung et al., 2015) and Solanum nigrum (Meng et al., 2016), the silencing phenotypes can be visualized in flowers and fruits after infecting young leaves, allowing for the analysis of gene function throughout the whole plant development process. For species in which the silencing phenotype cannot be transferred systemically, assistive technology needs to be applied to silence genes in the inflorescence and fruit, utilizing methods including petal disc infection, grafted-accelerated VIGS, scape with scratch wounding infection and fruit injection (Dai et al., 2012; Yan et al., 2018; Deng et al., 2012; Bai et al., 2012). Furthermore, the length of the target gene, the infection method, the developmental period and the culture environmental parameters could all affect the silencing efficiency of VIGS (Li et al., 2018). Most researchers believe that a target gene fragment between 300 and 500 bp is more efficient for inducing silencing of endogenous genes (Burch-Smith et al., 2010). Syringe infiltration and vacuum infiltration are two commonly used Agrobacterium-mediated viral infection methods, in which vacuum infiltration is laborless and can achieve higher silencing efficiency when it is applied for seedlings, while needle-free syringe infiltration is usually applied to the leaves, and needle injection can be applied to meristematic tissues such as flowers and fruits (Bai et al., 2016). For the infection period and the culture environment, higher silencing efficiency can be achieved when the young plant tissue is infected and cultured in an environment of temperature 20 °C - 25 °C and humidity 50% - 70% post-infiltration (Fu et al., 2006; Jiang et al., 2011). VIGS technology has been widely used for characterizing gene function in horticultural plants, such as studies on inflorescence development, secondary metabolism and fruit maturation. It was proved that AP3/AGL homology determined the inflorescence development in Phalaenopsis by VIGS using the CymMV vector (Hsu et al., 2015). Silencing of RhAG by VIGS significantly increased the number of petals through an increased production of petaloid stamens, which mimicked the impact of low-temperature treatments in Rosa hybrida (Ma et al., 2015). For research on secondary metabolism, VIGS combined with LC-MS was used in C. roseus, and Payne et al proved that CrNPF2.9 could export strictosidine, the central intermediate to the antitumor MIA pathway, into the cytosol from the vacuole (Payne et al., 2017). S. cruentus, belonging to the Senecio genus of Compositae, is an important and ideal material

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for the research of flower color of ornamental plants, as it can accumulate three different kinds of anthocyanin, including pelargonidin, cyanidin and delphindin. We have isolated several candidate genes that may be involved in the ABP in S. cruentus, such as ScCHI, ScDFRs, ScF3'H, and ScF3'5'H for structural genes, and ScbHLH17, ScbHLH21, ScbHLH22 for regulatory genes (Jin et al., 2016). However, the establishment of a high-efficiency regeneration and transformation system for the functional analysis of these genes was difficult and time-consuming. In the present study, a high-efficiency TRV-based VIGS protocol was established, and efficient gene knock-down phenotypes were achieved in both leaves and capitulum, providing technical support for the rapid analysis of anthocyanin metabolism-related gene function in this species.

136

2. Materials and methods

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2.1 Plant materials Two S. cruentus cultivars, ‘Venezia’ and ‘Jester’, were used as plant materials, of which the seeds were purchased from Syngenta Company, USA. Leaves of ‘Venezia’ and ‘Jester’ could accumulate similar anthocyanin components with the ray florets of the capitulum, which make them good materials for ABP gene functional analysis. ‘Venezia’ seedlings at the 2~4-leave stage were used for VIGS system establishment, while the carmine and blue cultivars of ‘Jester’ were used for verification of the system. ‘Venezia’ plants with flower buds developing to S1 stage were used for the capitulum infection experiment. The capitulum developmental stages have been described previously, and S1 stage was defined as when the ray florets were 0-5 mm in length and not yet out of bract (Jin et al., 2016). The seedlings were grown in an illumination incubator (20 °C, 60% relative humidity, 12 h light /8 h dark cycle). 2.2 Construction of VIGS vectors ScPDS and ScANS were used as test genes to establish the VIGS system since the silencing of these genes yielded a photobleaching phenotype or loss of anthocyanin accumulation, respectively. ScbHLH17 was a candidate regulatory gene for the functional verification of ABP. According to our previous study, it was expressed highly in carmine and blue varieties but was not expressed in white variety (Jin et al., 2016). TRV vectors were constructed by ligation-independent cloning (LIC) as previously reported (Dong et al., 2007). Sequences of ScPDS, ScANS and ScbHLH17 were identified from the previous S. cruentus transcriptome database. Fragments 300-500 bp in size (ScPDS and ScANS in a conserved region, ScbHLH17 in a specific region) for each transcript were PCR amplified with gene-specific primers, adding to LIC1 and LIC2 (adapter sequence of LIC TRV vector) in the 5' terminal of upstream and downstream primers, respectively (Table S1). These fragments were subsequently cloned into TRV2-based pYY13 vector using T4 DNA polymerase after being digested with PstI (New England Biolabs, Ipswich, MA) in advance. The plasmid profile and vector construction process were shown in Fig.S1 and Fig.S2, respectively. Recombinant plasmids were transformed into E.coli DH5α, and colonies were PCR-screened to select positive clones for sequencing. The correct recombinant plasmids were transformed into Agrobacterium strain GV3101 (ZOMANBIO, Beijing, China; Koncz et al., 1986) by using a freeze-thaw method (Yan et al., 2012).

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2.3 Leaf infiltration and optimization of the VIGS system To determine whether S. cruentus could be infected by TRV, ScPDS was used as a test gene of the infection of abaxial leaf surfaces. Agrobacterium colonies carrying TRV-VIGS vectors (pTRV1, pTRV2 and pTRV2 derivates) were shaken at 28 °C in LB medium containing 50 mg/L rifampicin and 50 mg/L kanamycin. After 16 h, the cells were harvested and resuspended to OD600 =1.5 in infiltration buffer containing 10 mM MgCl2, 200 mM acetosyringone and 10 mM MES at pH 5.6 and incubated at room temperature for 3 h. Before infiltration, bacteria carrying pTRV1 and pTRV2 (or pTRV2 derivates) were mixed in a 1:1 volume ratio. The infiltration mixture was introduced on the abaxial leaf surface in 2- or 4-leaf stage seedlings with a 1-ml needle-free syringe until the water-stained area accounted for more than 2/3 of the leaf surface (Fig. 1A). In previous studies, the inoculated seedlings were usually placed at 8-16 °C for dark culture to improve the entry of Agrobacterium into plant cells (Tang et al., 2017; Kim et al., 2017). The inoculated S. cruentus seedlings suffered freezing damage in the dark culture at 8 °C by the pre-experiment. Therefore, the seedlings were grown at 10 °C for 2 d and at 15 °C for 1 d under dark and were then cultured at 20 °C under normal light in 60% relative humidity. After determining that S. cruentus could be infected by TRV vectors, VIGS system optimization was conducted through setting up a total of eight groups of experiments (Y1-Y8) with different conditions, including plant developmental periods, bacterial cell concentration and infection methods (Table 1); both ScPDS and ScANS were used in these system optimization experiments. For leaf and shoot apical meristem syringe injection (SI), the infiltration mixture was introduced into the surface as above and was also injected into the shoot apical meristem. For vacuum infiltration (VI), the seedlings were submerged into infiltration mixture and pulled by a vacuum pump until the pressure reached 0.8 kg/cm2, and the vacuum pressure was then maintained for 5 min, after which it was released rapidly to attain atmospheric pressure. This was repeated once, and the treated seedlings were then rinsed with distilled water, repotted in soil, and moved to an illumination incubator. After infection, we observed the seedlings every three days, and the survival rate and silencing efficiency were recorded at 30 days post-infiltration (dpi) (survival rate = numbers of living plants / numbers of infected plants, silence efficiency = numbers of exhibiting silencing phenotype plants / numbers of infected plants). To confirm if the VIGS system was suitable for other S. cruentus varieties, ScPDS-silenced and ScANS-silenced experiments were carried out in the carmine and blue varieties of ‘Jester’. The optimized VIGS system was also applied to study the gene function of ScbHLH17 in S. cruentus. All experiments were included a noninfiltrated control (WT, no treatment) and a negative control (NC, infecting pTRV2 empty vector and pTRV1). 2.4 Infection of capitulum in S. cruentus For silencing genes in capitulum, the infiltration mixture containing pTRV1 and pTRV2-ScANS was introduced into buds at S1 stage from the scapes using needle injection, and the whole capitulum was then submerged in infiltration mixture and subjected to vacuum. The vacuum pressure was 0.8 kg/cm2 for 10 min and was then released quickly to attain atmospheric pressure. This process was repeated once, after which the inoculated plants were grown in the same conditions described for the leaf infiltration. 2.5 Anthocyanin content determination and gene expression analysis Leaf photobleaching and the faded color of the abaxial surface could be observed in ScPDS-silenced and ScANS-silenced plants, respectively. Freeze dried tissue was used for the

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anthocyanin analysis. Samples of the ground freeze-dried petal tissue (0.1 g) were initially extracted in 5 ml of hydrochloric acid, methanol (1: 99, v: v) for 24 hours at 4 °C in the dark. The optical density at the absorption maximum peak of the extract was measured with an absorption spectrum (500- 600 nm) using a spectrophotometerand, and the anthocyanin content was calculated according to the molar extinction coefficient 93.2 (Karl, 1978) Reverse transcription (RT)-PCR was performed to compare the expression between the silenced tissue and the control group. The conditions for RT-PCR were as follows: one cycle for denaturation (94 °C, 5 min), followed by 35 cycles (94 °C for 50 s, 56 °C for 40 s, 72 °C for 30 s) and holding at 10 °C forever. Quantitative real-time PCR was carried out to compare the expression levels of ABP genes, including three regulatory genes (ScbHLH17, ScbHLH21, and ScbHLH22) and five structural genes (ScCHS2, ScCHI, ScF3H1, ScDFR3 and ScANS) between the ScbHLH17-silenced leaves, the NC and the WT. The conditions for qRT-PCR were as follows: 1 initial cycle of denaturation (95 °C for 4 min), followed by 39 cycles of amplification (at 94 °C for 50 s, 60 °C for 40 s) and at 72 °C for 30 s. The sequences of these primers are shown in Table S1.

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3. Results

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3.1 S. cruentus ‘Venezia’ leaves could be infiltrated with TRV A 436-bp fragment of the ScPDS gene was amplified and cloned into the pTRV2 vector to form pTRV2-ScPDS. Then, the infiltration mixture containing pTRV1 and pTRV2-ScPDS was introduced into ‘Venezia’. Forty seedlings at the 2-leaf stage were infiltrated through syringe-infiltration on the abaxial leaf surface. Two of 40 inoculated seedlings exhibited photobleaching symptoms in newly developed leaf at 18 dpi (leaves became white, see red arrow in Fig. 1B). RT-PCR showed that the presence of pTRV1 was detected in both NC and ScPDS-silenced leaves, while pTRV2-ScPDS was only detected in ScPDS-silenced leaves and was absent in WT (Fig. 1C). These results confirmed that TRV vectors could infiltrate S. cruentus and that pTRV2-ScPDS successfully induced transient gene knock-down to exhibit photobleaching phenotypes. Nevertheless, not all plants showed the silencing phenotype, and the silencing phenotypes could not transfer systemically throughout the whole plants, as the newly developed leaves returned to green (black arrow in Fig. 1B). These findings indicated that the VIGS system could be applied on S. cruentus leaves, but further exploration was needed to improve the efficiency of gene silencing and to enable systematic virus transfer.

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Fig. 1. TRV reorganization of vector-infiltrated S. cruentus leaves. (A) Seedlings infiltrated with TRV on the abaxial leaf surface. (B) ScPDS- infiltrated leaf exhibiting the photobleaching phenotype (red arrow), WT: blank control without treatment, NC: negative control, infected with the pTRV2 empty vector. TRV2-ScPDS: infected with pTRV2-ScPDS. (C) Gene expression in inoculated seedlings: no presence of pTRV1 and pTRV2-ScPDS in CK, and the presence of only pTRV1 in NC, while both were present in pTRV1 and pTRV2-ScPDS in silencing phenotype plants (SP).

3.2 Silencing of the ScPDS and ScANS genes in S. cruentus ‘Venezia’ leaves After determining that S. cruentus could be infiltrated with pTRV vectors, we optimized several parameters, including developmental periods (2-leaf and 4-leaf), bacterial cell concentration (OD600 =1.5 and OD600 =2.0), and infection methods (SI and VI) that may improve the silencing efficiency using both ScPDS and ScANS as test genes. The results are shown in Table 1, Table 2 and Fig. 2. The photobleaching phenotype was observed in the newly developed leaves at 12 dpi obtained from seedlings inoculated with pTRV2-ScPDS vectors, but not in those obtained from WT, NC (Fig. 2A and B). Meanwhile, the faded abaxial leaf phenotypes of the carmine cultivar were detected in the newly developed leaves collected from the seedlings inoculated with pTRV2-ScANS at 18 dpi (Fig. 2C). After approximately 30 dpi, no newly silencing seedlings were observed. The silencing phenotype could not transfer systemically in most seedlings, except for one seedling in which the photobleaching phenotype was exhibited on six newly developed leaves – the third and fourth leaves even turned completely white (black arrow in Fig. 2B; Y8 treatment). Determination of anthocyanin content revealed a significant decline in ScANS-silenced leaves (Fig. 2D). RT-PCR was performed on all treatment groups (Fig. 2E and F). The presence of both pTRV1 and pTRV2-ScPDS or pTRV2-ScANS was detected in all of the seedlings exhibiting the silencing phenotype (Y1, Y4, and Y8 treatments for TRV2-ScPDS; Y1, Y2, Y3, Y4, Y5, Y6, and Y8 treatments for TRV2-ScANS). The highest silencing efficiencies of TRV2-ScPDS and TRV2-ScANS at the 2-leaf stage both appeared in treatment Y1, which were 12.50% and 13.21%, respectively. The highest silencing efficiencies at the 4-leaf stage were 8.33% and 13.3%, respectively (Table 1 and 2). But generally, all treatments could lead to the gene silencing, and the silencing efficiency was around 10%, with no significant difference. Therefore, it was believed that VIGS could be applied in S. cruentus, regardless of whether the development period was in the 2-leaf stage or the 4-leaf stage, the

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bacterial solution concentration was OD600 = 1.5 or OD600 = 2.0, and the infection method was vacuum infiltration or injection infiltration. Table 1 S. cruentus leaves were infiltrated with pTRV1-ScPDS OD600

Y1

Developmental period 2L

Surviving plants 40

Survival rate 100%

Silenced plants 5

Silencing efficiency 13%

Y2

2L

1.5

SI

Y3

2L

2

VI

30

20

67%

0

0

45

22

50%

0

Y4

2L

2

0

SI

30

30

100%

3

10%

Y5

4L

1.5

VI

Y6

45

33

73%

0

0

4L

1.5

SI

24

17

71%

0

0

Y7

4L

2

VI

39

21

54%

1

3%

Y8

4L

2

SI

24

10

42%

2

8%

NO.

276 277 278 279 280

Infiltrated plants 40

2L: seedlings at the 2-leaf stage; 4L: seedlings at the 4-leaf stage; VI: vacuum infiltration; SI: leaf and shoot apical meristem syringe injection.

Table 2 S. cruentus leaves were infiltrated with pTRV1-ScANS OD600

Y1

Developmental period 2L

Y2

2L

1.5

SI

Y3

2L

2

VI

Y4

2L

2

SI

Y5

4L

1.5

Y6

4L

Y7 Y8

NO.

281 282

1.5

Infiltration methods VI

Surviving plants 39

Survival rate 75%

Silenced plants 7

Silencing efficiency 13%

30

30

100%

2

7%

43

40

93%

2

5%

30

30

100%

3

10%

VI

40

28

70%

2

5%

1.5

SI

24

18

75%

1

4%

4L

2

VI

39

8

21%

0

0

4L

2

SI

45

45

100%

6

13%

1.5

Infiltration methods VI

Infiltrated plants 53

2L: seedlings at the 2-leaf stage; 4L: seedlings at the 4-leaf stage; VI: vacuum infiltration; SI: leaf and shoot apical meristem syringe injection.

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Fig. 2. S. cruentus leaves were infiltrated with pTRV1-ScPDS and pTRV2-ScANS by different methods. (A)

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3.3 Verification of the VIGS system in cultivars of S. cruentus ‘Jester’ To verify whether the ‘Venezia’ leaf VIGS system was suitable for other S. cruentus cultivars, carmine and blue cultivars of S. cruentus ‘Jester’, which are commonly used in flower markets, were selected to repeat the VIGS experiment using the Y1 treatment. The infiltration mixture containing pTRV1 and pTRV2 or its derivatives (OD600=1.5) was introduced into leaves at the 2-leaf stage by vacuum infiltration. The results showed that the ‘Jester’ seedlings infiltrated with pTRV2-ScPDS showed a photobleaching phenotype at approximately 15 dpi (Fig. 3A and B), and the presence of both pTRV1 and pTRV2-ScPDS was detected in the photobleached leaves (Fig. 3C). The seedlings infiltrated with pTRV2-ScANS developed faded carmine abaxial leaf phenotypes at approximately 20 dpi (Fig. 3D). The anthocyanin contents and gene expression levels of the faded leaf parts were analyzed, and the results showed that the anthocyanin accumulation decreased significantly and that both pTRV1 and pTRV2-ScANS were detected (Fig. 3F). The inoculation on ‘Jester’ exhibited 1.33-fold higher ScPDS silencing efficiency than ‘Venezia’(16.12% in ‘Jester’ and 12.50% in ‘Venezia’) and 1.14-fold higher ScANS silencing efficiency (15.10% in ‘Jester’ and 13.21% in ‘Venezia’) (Table 3). These observations indicated that the VIGS system in ‘Venezia’ could be

ScPDS-silenced seedlings exhibited the photobleaching phenotype. (B) Photobleaching phenotype plants, red arrow indicates the earliest exhibiting the photobleaching phenotype in silencing seedlings, black arrow indicates that pTRV1-ScPDS was transferred systemically in silencing seedlings. (C) Faded carmine abaxial leaf phenotypes in ScANS-silenced seedlings. (D) Relative content of anthocyanin in ScANS-silenced leaves. (E) The presence of pTRV1 and pTRV2-ScPDS in treatments Y1-Y8. (F) The presence of pTRV1 and pTRV2-ScANS in treatments Y1-Y8. (G) Silencing efficiency in treatments Y1-Y8.

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applied to ‘Jester’ carmine and blue varieties and obtained higher silencing efficiency, which provided an important tool for identifying the gene functions of both ABP genes.

Fig. 3. VIGS system for carmine and blue varieties in S. cruentus ‘Jester’. (A) ScPDS-silenced leaves exhibited the photobleaching phenotype in the ‘Jester’ carmine variety. (B) ScPDS-silenced leaves exhibited the photobleaching phenotype in the ‘Jester’ blue variety. (C) Gene expression detection in ScPDS-silenced leaves. SP1 indicates the photobleaching phenotype seedlings in the carmine variety, SP2 indicates the photobleaching phenotype seedlings in the blue variety. (D) ScANS-silenced leaves faded. (E) Relative contents of anthocyanins in ScANS-silenced leaves. (F) Gene expression detection in ScANS-silenced leaves. SP1 represents faded leaves in the carmine variety, SP2 represents faded leaves in the blue variety. Table 3 Silencing efficiency of the VIGS system in the carmine and blue varieties in S. cruentus ‘Jester’ Vector

Infiltrated plants/strain

Surviving plants/strain

Survival rate

Silenced plants/strain

Silencing efficiency

pTRV2-ScPDS

62

25

40%

10

16 %

pTRV2-ScANS

60

53

93%

8

15%

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3.4 Silencing of the ScbHLH17 gene in S. cruentus ‘Jester’ leaves A 397-bp fragment of the ScbHLH17 gene was amplified and cloned into the pTRV2 vector to form pTRV2-ScbHLH17, which was introduced to ‘Jester’ seedlings at the 2-leaf stage. Faded carmine abaxial leaf phenotypes were observed at 30 dpi, and RT-PCR showed the presence of both pTRV1 and pTRV2-ScANS in ScbHLH17-silenced seedlings; the plasmids were absent in WT, NC (Fig. 4A and B). These findings indicated that the TRV vectors were successfully inoculated into the seedlings and led to the silencing of endogenous ScbHLH17. qRT-PCR was carried out to detect the effect of ScbHLH17 silencing on anthocyanin structural genes by examining the transcription of these genes in the leaves of WT, NC and ScbHLH17-silenced seedlings. The expression of ScbHLH17 was 6.05-fold lower in ScbHLH17-silenced leaves than in WT and NC. The transcription levels of five structural genes, including ScCHS2, ScCHI, ScF3H1, ScDFR3 and ScANS, were repressed to varying degrees in ScbHLH17-silenced leaves (Fig. 4C). These results demonstrated that ScbHLH17 is necessary for conferring anthocyanin biosynthesis in the abaxial leaf of ‘Jester’. To determine the specificity of VIGS silencing, two additional bHLH homologs (ScbHLH21 and ScbHLH22) grouped together with ScbHLH17 in the IIIf bHLH subgroup were

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included for the qRT-PCR analyses. Subsequent measurement showed that their expression was nearly unaffected in the VIGS suppressed leaves (Fig. S5). The results suggest there was only a low probability of incorrect targeting caused by VIGS suppression and that the observed silencing phenotypes most likely resulted from downregulation by expression of individual VIGS constructs.

Fig. 4. Silencing of ScbHLH17 in the leaves of S. cruentus ‘Jester’. (A) The black arrows indicated ScbHLH17-silenced tissue. (B) RT-PCR detected gene expression in silenced seedlings. (C) qRT-PCR detected the expression of ABP genes in ScbHLH17-silenced leaves.

3.5 Reduced S. cruentus ray flower coloration as a result of ScANS VIGS in capitulum The infiltration mixture containing pTRV1 and pTRV2-ScANS (OD600=1.5) was introduced to capitulum by scape injection and vacuum infiltration (Fig. 5A). Two of seven inoculated plants showed silencing symptoms in which carmine ray florets faded to white at 15 dpi, with a silencing efficiency of 17.39% (Fig. 5B). The anthocyanin content in silenced inflorescences was significantly lower than that of the WT, with a slight decline observed in NC (Fig. 5C). Both pTRV1 and pTRV2-ScANS were detected in ScNAS-silenced capitulum, while pTRV1 was only present in NC, and neither pTRV1 nor pTRV2-ScANS were present in WT (Fig. 5D). The results indicated that pTRV2-ScANS could infiltrate the S. cruentus capitulum and resulted in the silencing of the endogenous ScANS gene, leading to the inhibition of anthocyanin biosynthesis and accumulation.

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Fig. 5. Silencing of ScANS in S. cruentus capitulum. (A) Infecting capitulum by scape injection and vacuum infiltration. (B)

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4. Discussion

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VIGS is a fast and highly effective transient gene knock-down technology that has been widely used for gene functional analysis. In Compositae, the VIGS system has been successfully established in Gerbera hybrid, Tagetes erecta, Senecio vulgaris and Matricaria inodora to study the flower development-related MADS-box genes ,the ABP regulatory MYB gene and the auxin transport genes (Deng et al., 2012; Zoulias, 2014; Tang et al., 2017). In the present study, the VIGS system of S. cruentus was established by using ScPDS and ScANS as test genes. The gene function of ScbHLH17 was investigated by this VIGS system, and the results indicated that ScbHLH17 could positively regulate the anthocyanin biosynthesis in S. cruentus. At the same time, we established the VIGS system in floral tissues using ScANS as a test gene. 4.1 Establishment of the VIGS system based on TRV The TRV vector is the most widely used vector in VIGS systems (Dommes et al., 2018). The silencing efficiency of TRV-VIGS can be affected by many factors, such as cultivar selection, developmental periods, bacterial cell concentration, infection methods and culture environment parameters, such as temperature and humidity. Although TRV has a wide range of hosts, there is a significant difference in sensitivity to TRV between species and cultivars (Senthilkumar and Mysore, 2014; Bennypaul et al., 2012; Deng et al., 2012). For instance, TRV sensitivity testing was carried out on 21 gerbera cultivars, and only 5 cultivars showed photobleached PDS-silencing symptoms in newly developed leaves (Deng et al., 2012). We also constructed the recombinant vector pTRV2-CmPDS to infect 15 Chrysanthemum morifolium cultivars and 2 wild species, C. vestitum and C. lavandulifolium, and all of these plants failed to be infected (data was not shown). For S. cruentus, both ‘Venezia’ and ‘Jester’ could be successfully infected by TRVs. However, the silencing efficiency was low (13%), even though the different parameters had been tested. In Solanaceae, such as P. floridana, the silencing efficiency could reach to 77% with syringe infiltration on the leaf (Zhang et al., 2014). And in Rosaceae, such as Malus carabapple, the silencing efficiency reached to 77% with vacuum infiltration (Zhang et al., 2016). In Compositae,

ScANS-silenced phenotype in capitulum. (C) Gene expression detection in silenced plants; SP1-4 show four capitulum with fading flowers. (D) Relative contents of anthocyanin in ScANS-silenced ray florets.

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Deng et al (2012) achieved a silencing efficiency of 67% in the gerbera cultivar ‘President’. However, the VIGS research in S. vulgaris and M. inodora only showed a silencing efficiency of 5% and 3.33%, respectively, with syringe infiltration (Zoulias, 2014). It indicated that the silencing efficiency of VIGS technology may varied among different species. In addition, the factors affecting silencing efficiency were not similar in different species. Infection during the cotyledon stage showed higher silencing efficiency than the 2- and 4-leaf stages in gerbera (Deng et al., 2012) but was lower than infection during the 1-leaf stage in S. nigrum (Meng et al., 2016); the seedling age had no effect on the silencing efficiency in C. roseus (Sung et al., 2014). The bacterial cell concentration showed no obvious influence on silencing efficiency in gerbera; however, the optimum concentrations were OD600=0.5 for leaf infection and OD600=1.0 for fruit infection in pepper (Deng et al., 2012; Zhang et al., 2015; Tian et al., 2014). For infiltration methods, vacuum infiltration is likely to be used for improving the silencing efficiency for species that are difficult to infect with syringe infiltration, such as gerbera ‘Terraregina’, which exhibited more intensive silencing symptoms with vacuum infiltration than that with syringe infiltration (Deng et al., 2012). However, syringe infiltration showed approximately 50% higher silencing efficiency compared with vacuum infiltration in Solanum pseudocapsicum (Xu et al., 2018). In our study, seedling development stage, bacterial cell concentration and infection methods showed no significant influence on the silencing efficiency. These findings show that VIGS systems are not similar in various species and need to be adjusted according to the development stage and tissues infected, even within the same species. 4.2 Application of VIGS for unsystemically injected flowers and fruits One of the aims in developing VIGS system for plants was to identify genes involved in flower and fruit development (Orzaezet al., 2009; Hiseh et al., 2012; Li et al., 2019). In our study, a silenced phenotype appearing in young leaves but could not transfer systemically to flower; therefore, the VIGS system in capitulum was established by scape injection and vacuum infiltration. There are several species in which silenced phenotypes cannot transfer systemically, such as T. erecta, Lilium and some woody plants. Bai et al (2016) injected pTRV2-CCD4 into white peach fruit before the commercial pick-up period and observed in the silencing phenotypes that fruits could accumulate carotenoids, which showed that CCD4 could promote the degradation of carotenoid. In addition to silencing genes involved in pigment synthesis, VIGS can also be applied to silence genes associated with flower shape and fragrance, such as RhAG and RhNUDX1 in rose (Yan et al., 2018) and GhGLO1 in gerbera (Deng et al., 2012). In this study, the VIGS system in S. cruentus capitulum was successfully established to study the genes related to flower color, but whether it can be applied to flower shape genes remains to be further tested. 4.3 Application of VIGS for gene-related pigment synthesis in leaves For gene functional analysis associated with the pigment synthesis pathway, stable genetic transformation is costly and extremely time-consuming. VIGS allows for the characterization of gene function in plants with colored leaves, effectively reducing the time and labor cost. In our study, ScbHLH17 was silenced by VIGS in leaves, and the expression levels of structural genes on ABP genes, including ScCHI, ScDFR3 and ScANS, decreased significantly. We also overexpressed ScbHLH17 in tobacco, and obtained a line that the corolla of flowers of transgenic tobacco plants were much darker pink than that of wide types (data not shown). These indicated that ScbHLH17 could positively regulate anthocyanin biosynthesis. Research of pigment-related genes has also been carried out in other plants with colorful leaves by VIGS. Infiltration with TRV2-McMYB10

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into the Malus cultivar ‘Red Begonia’ resulted in characteristic faded red leaves and low levels of anthocyanin biosynthetic gene transcripts (Zhang et al., 2016). Adhikary et al (2019) silenced AtriCYP76AD1 by VIGS in Amaranthus red plants and observed that AtriCYP76AD1 and the related genes AtriCYP76AD6 and AtriCYP76AD5 had diminished transcript abundance levels, leading to a strong decline in betacyanin accumulation and an increase in L-DOPA accumulation. VIGS was applied to silence CaMYB in the red-purple leaves of chili pepper to study the interactions between MYB and bHLH and WD40, in which MYC expression decreased while that of WD40 increased after CaMYB silencing. These findings indicated that MYB can induce MYC expression to coregulate the accumulation of anthocyanins and WD40 acted as a scaffold and participated in the interaction between MYC and MYB proteins (Zhang et al., 2015). We proved that ScbHLH17 is a transcription factor involved in regulating anthocyanin biosynthesis, but the MYB transcription factor, which can interact with ScbHLH17, remains to be investigated.

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5. Conclusions

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This paper describes the establishment of a TRV-based VIGS system in S. cruentus and demonstrates that VIGS is an effective tool to characterize gene function. Although the silencing efficiency is low (13%), we still successfully silenced ScbHLH17 by VIGS, and gene expression analysis in silenced leaves demonstrated that ScbHLH17 could positively regulate anthocyanin biosynthesis. ScANS silencing phenotypes were also exhibited in capitulum through scape injection and vacuum infiltration on S1 stage buds. This study provided a significant technological support for the research on anthocyanin biosynthesis pathway in S. cruentus and inflorescence development in Compositae plants.

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Author contributions

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HH and SLD conceived and designed this study. YJL, YTL, FTQ and HH performed the experiments. CYD and CFL carried out the data analysis. YJL and HH wrote this manuscript. All authors read and approved the final manuscript.

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Acknowledgments

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We are appreciated for Dr. Yule Liu, Tsinghua University, China, for his kindly providing the TRV vector plasmids. This work was supported by the National Natural Science Foundation of China (grant no. 31870693) and the Fundamental Research Funds for the Central Universities (No. 2015ZCQ-YL-03).

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Highlights A rapid transient gene knock-down technology was first established in Senecio cruentus. S. cruentus is a good material for anthocyanin research. VIGS system was obtained in capitulum of S. cruentus by silencing ScANS. ScbHLH17 was identified to positively regulate the anthocyanin biosynthesis.

Author contributions HH and SLD conceived and designed this study. YJL, YTL, FTQ and HH performed the experiments. CYD and CFL carried out the data analysis. YJL and HH wrote this manuscript. All authors read and approved the final manuscript.

Declaration of Interest Statement We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of the manuscript entitled.