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OPEN

Apple latent spherical virus(ALSV)‑induced gene silencing
in a medicinal plant, Lithospermum
erythrorhizon
Yuki Izuishi1, Natsumi Isaka1, Hao Li1, Kohei Nakanishi1, Joji Kageyama1, Kazuya Ishikawa2,
Tomoo Shimada2, Chikara Masuta3, Nobuyuki Yoshikawa4, Hiroaki Kusano1 &Kazufumi Yazaki1*
Lithospermum erythrorhizon is a medicinal plant that produces shikonin, a red lipophilic
naphthoquinone derivative that accumulates exclusively in roots. The biosynthetic steps required tocomplete the naphthalene ring of shikonin and its mechanism of secretion remain unclear. Multiple
omics studies identified several candidate genes involved in shikonin production. The functions ofthese genes can be evaluated using virus-induced gene silencing (VIGS) systems, which have beenshown advantageous in introducing iRNA genes into non-model plants. This study describes thedevelopment of a VIGS system using an apple latent spherical virus (ALSV) vector and a target gene,
phytoene desaturase (LePDS1). Virus particles packaged in Nicotiana benthamiana were inoculated
into L. erythrorhizon seedlings, yielding new leaves with albino phenotype but without disease
symptoms. The levels of LePDS1 mRNAs were significantly lower in the albino plants than in mockcontrol or escape plants. Virus-derived mRNA was detected in infected plants but not in escape andmock plants. Quantitative PCR and deep sequencing analysis indicated that transcription of anotherhypothetical PDS gene (LePDS2) also decreased in the defective leaves. Virus infection, however, had
no effect on shikonin production. These results suggest that virus-based genetic transformation andthe VIGS system silence target genes in soil-grown L. erythrorhizon.
Abbreviations
qRT-PCR	Quantitative reverse-transcriptase mediated polymerase chain reaction
VIGS	Virus-induced gene silencing
PDS	Phytoene desaturase
ALSV	Apple latent spherical virus
ACT​	Actin
UTR​	Untranslated region
CMV	Cucumber mosaic virus
Lithospermum erythrorhizon is a Boraginaceaeous medicinal plant that produces a unique red naphthoquinone,
shikonin, which accumulates exclusively in its roots. The dried roots have been used as a crude drug in Asian
countries, with shikonin derivatives being major active pharmaceutical components of these herbal medicines.
Shikonin and its stereo-isomer alkannin have been reported to have various biological activities, including
antibacterial, anti-inflammatory, anti-oxidant, antitumor, anti-angiogenic1, and anti-topoisomerase2 activities.
These compounds have also been found to enhance granulation and glucose u­ ptake3 and to reduce a­ diposity4.
These natural pigments are also used as dyes, especially for cloth, and to have other commercial uses. Although
1

Laboratory of Plant Gene Expression, Research Institute for Sustainable Humanosphere, Kyoto University,
Gokasho, Uji 611‑0011, Japan. 2Graduate School of Science, Kyoto University, Sakyo‑ku, Kyoto 606‑8502,
Japan. 3Research Faculty of Agriculture, Hokkaido University, Kita 9 Nishi 9, Kita‑ku, Sapporo 060‑8589,
Japan. 4Agri‑Innovation Center, Iwate University, Morioka 3‑18‑8, Iwate 020‑8550, Japan. *email: yazaki@
rish.kyoto‑u.ac.jp
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shikonin appears to be a simple compound, many steps are necessary for its chemical synthesis, making the supply of shikonin exclusively dependent on natural resources. L. erythrorhizon, however, is an endangered species,
preventing widespread continuous cultivation.
Biologic approaches are currently used to synthesize high value plant products in unicellular organisms,
such as yeasts. Production of a target compound, however, requires the determination of its entire biosynthetic
pathway and the identification of all genes and proteins involved in its biosynthesis. Shikonin is a meroterpene
synthesized via two independent biosynthetic pathways, a common phenylpropanoid pathway and an isopreniod­pathway5. Although several enzymes and genes involved in the initial steps of shikonin biosynthesis have been
identified to ­date6–10, genes and proteins involved in later steps have not yet been identified, especially those
involved in the crucial steps required to form the naphthalene ring.
To identify candidate genes involved in these later steps of shikonin biosynthesis, we performed large-scaletranscriptome and comparative proteome analyses, based on results showing that shikonin production can be
strictly regulated by many chemical and physical f­ actors9. For example, shikonin production is strongly inhibitedby ammonium ion and illumination, but is markedly enhanced in M9 medium. Shikonin production is also
tissue specific, being produced solely in the epidermis, but not in other root tissues, such as inside the central­cylinder6. These characteristics enabled the identification of more than 10 putative candidate genes involved in
shikonin ­biosynthesis9 and more than 16 putative candidate genes involved in shikonin secretion from ­cells11.
These genes of interest may be characterized using the hairy root transformation technique developed in
L. erythrorhizon. This method can be used to introduce an exogenous gene for overexpression or an RNAi to
suppress the expression of an endogenous g­ ene12. The recent development of a new protocol has dramatically
improved the transformation efficiency of this ­system13. However, stable transformation is time-consuming,with several months required to evaluate the physiological function of a gene. The present study utilized avirus-induced gene silencing (VIGS) system to establish a method for functional analyses of genes of interestin L. erythrorhizon plants.
One major advantage of VIGS is that special equipment is not required to introduce a foreign gene fragment
into a non-model plant. Within a short period of time after infection, the virus becomes wide-spread throughout
the entire plant body, allowing evaluation shortly after infection. One disadvantage of VIGS is that the hostvirus interaction is highly species-specific. Thus if a plant virus is unable to infect a plant species, the introduced
gene fragment cannot be evaluated. This study assessed the abilities of apple latent spherical virus (ALSV) andcucumber mosaic virus (CMV), both domestic plant viruses found in Japan, to infect L. erythrorhizon.

Results

ALSV has been shown to infect a broad spectrum of plant species, and has been used as a viral vector including
for gene ­silencing14. To determine if ALSV can infect L. erythrorhizon, we assessed its ability to knock down
expression of the phytoene desaturase (PDS) gene in this plant species. Successful infection and PDS genesilencing should result in the appearance of albino leaves, a representative phenotype of the knockdown of PDS­function15,16.

ALSV‑mediated VIGS phenotype of the PDS gene in L. erythrorhizon.

Transcriptome analysisof L. erythrorhizon identified a PDS gene, designated LePDS1 (accession no. LC512725). A 234 nucleotide fragment of this gene (Fig. 1A) was cloned into ALSV using a method involving rub-inoculation following Agribactrium-mediated inoculation of Nicotiana benthamiana (see the Methods section for more detail) (Fig. 1B)17. The
packaged virus was inoculated onto the leaves of young L. erythrorhizon seedlings during the stage of first leaf
expansion (Fig. 1C). After cultivation for about 3 weeks, newly developed leaves above the inoculated leaves ofseveral plants showed albino phenotypes. Although the albino phenotype was reproducibly observed, about 70%
of inoculated seedlings did not have albino leaves and were considered to have escaped infection (Fig. 1D). Viral
RNA was detected in infected plants that showed the albino phenotype, but not in plants that escaped infection
or in mock control plants, which were treated with water instead of ALSV (Fig. 1E).

Abundance of PDS transcript in infected L. erythrorhizon.

The expression of PDS mRNA wasassessed by qRT-PCR five weeks after viral inoculation of L. erythrorhizon leaves located at levels ranging fromL1 (older) to L10 (younger) toward the shoot tips (Fig. 2A). The albino phenotype was observed in leaves at
L5–L10, but not in leaves at L1–L4 (Fig. 2B). To compare the expression of PDS mRNA detected by qRT-PCR
in leaves and plants, its level was normalized to that of an internal standard, actin 7 (LeACT7) mRNA (Fig. 2C),
which is ubiquitously expressed in these ­plants10. PDS mRNA expression was lower in infected than in escape ormock plants, with the differences in PDS mRNA expression at L2–L8 being significantly lower in infected than inescape (p = 4.08 × 10–11) or mock infected (p = 6.89 × 10–15) plants, with no difference between escape and mock
infected plants (p = 0.53). A comparison of the relative expression of PDS mRNA in each leaf of each infected,
escape, and mock control plant showed that PDS expression was almost constitutive, with differences within
plants being generally statistically not significant, although PDS expression was significantly higher in L1 thanin other leaves of infected plants (p = 0.014). These results strongly suggest that PDS expression was significantly
inhibited by ALSV-mediated VIGS in infected plant leaves.

Effect of virus infection on shikonin biosynthesis in roots. Because VIGS in L. erythrorhzon wasdeveloped to suppress genes of interest involved in shikonin production, virus infection itself should not affectthe production of shikonin. To assess whether ALSV infection altered shikonin biosynthesis, roots were harvested from virus- and mock-infected plants and shikonin derivatives were quantitatively analyzed (Fig. 3).
Assessment of ALSV abundance in RNA samples from roots showed that, similar to leaves, the virus was present

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Figure 1.  Generation of albino leaves in L. erythrorhizon plants by infection with ALSV containing a VIGS
trigger against LePDS1. (A) Schematic representation of the LePDS1 transcript. The VIGS trigger fragment
and PCR primers were set on the region encoding LePDS1 protein. (B) Construction of an ALSV vector. The
trigger fragment was inserted in the reading frame of the ALSV polyprotein without any stop codons or frame
shifts. The PCR primers to detect ALSV included the trigger fragment within the amplicon, with the productexpected to be 0.5-kb long. The bipartite ALSV RNAs were encoded downstream of the CaMV 2 × 35S promoter
in the pBICP35-based plasmids (pBICAL2 and pBICAL1). Abbreviations: LB and RB, T-DNA borders; 42 KP,
42 K movement protein; VP25, VP20 and VP24, capsid proteins; PRO-C, protease cofactor; HEL, NTPbinding helicase; C-PRO, cysteine protease; POL, RNA polymerase; NOS, terminator. (C) Young seedlings of
L. erythrorhizon just before inoculation. Scale bar = 1 cm. (D) L. erythrorhizon plant grown for five weeks after
inoculation. Left, an "infected" plant with albino leaves; center, an "escape" plant without albino leaves despitevirus inoculation; right, a “mock” infected plant. Scale bar = 3 cm. (E) Detection of ALSV in L. erythrorhizon
by RT-PCR. Abbreviations: infected, defective plant with albino leaves; escape, healthy plant despite virus
inoculation; mock, plant treated with inoculation solution without virus; PC, positive control of PCR. The arrow
indicates the band size corresponding to the PCR amplified fragment.
only in the roots of infected plants, not in the roots of escape and mock infected plants (Fig. 3A). HPLC analysisof the amounts and compositions of shikonin derivatives in the harvested roots showed no significant differences among the three plant types, indicating that ALSV infection of L. erythrorhizon plants did not influence
their production of shikonin (Fig. 3B). The major shikonin derivatives in all three plant types were acetylshikonin, isobutyrylshikonin, and α-methylbutylshikonin, accompanied by minor derivatives, including shikonin,
β-hydroxyisovalerylshikonin, and β,β-dimethylacrylshikonin (Fig. 3C). These results indicate that the shikoninbiosynthesis pathway was not disturbed by ALSV infection of and proliferation in plant tissues, including intheir roots, or by the silencing of a gene unrelated to shikonin production.

Simultaneous ALSV silencing of PDS genes in L. erythrorhizon. Because our findings suggested
that the albino phenotype was partial in L. erythrorhizon, we suspected that the silencing efficiency was not sufficient or that these plants also expressed an isoform of PDS. Careful search of our transcriptome data identified
another PDS paralogue, LePDS2 (accession no. LC512726), with a nucleotide sequence highly similar to that of
LePDS1, including 91% identity in the coding region. LePDS2 was found to encode a polypeptide of the samelength as LePDS1 (90% identity). The nucleotide sequences of the 5′UTR and 3′UTR regions of these two genes,
however, differed markedly (Fig. 4A). Assembly of the genomic DNA sequence of L. erythrorhizon from theavailable Genbank dataset (SRP108575) showed that the LePDS2 gene had the same exon–intron structure as
14 coding exons of LePDS1, but their lengths and intron sequences differed (Fig. 4B). A phylogenetic analysis of
their amino acid sequences showed that LePDS1 and LePDS2 differed at 53 of 581 amino acid residues, but thatthese members of the phytoene desaturase family differed markedly from those of the zeta-carotene desaturase
family (Fig. 4C). These findings suggested that LePDS1 and LePDS2 evolved in a recent ancestor of L. erythrorhizon after splitting from a single, broadly conserved PDS gene.
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Figure 2.  Abundance of LePDS mRNA in ALSV infected plants. (A) Schematic representation of an L.
erythrorhizon plant with expansion of 12 true leaves. One of the first four leaves of the seedlings, including thecotyledon, was inoculated with virus. L1–L10 represent true leaves 3–12, respectively. (B) Leaves from infected
and escape plants. Scale bar = 3 cm. (C) Relative LePDS mRNA expression measured by qRT-PCR normalized
relative to actin 7 (LeACT7) mRNA expression in the same samples. Error bars represent standard errors (n = 3).
The box indicates comparisons of leaves L2–L8 in infected, escape, and mock infected plants (n = 21).
Compared with LePDS1, LePDS2 differs at six nucleotides within the VIGS trigger (Fig. 4A). To evaluate thesilencing efficiency of these two close paralogues by VIGS, the ratio of their transcript levels was evaluated byamplicon sequencing (Fig. 4D,E) using identical sequences of LePDS1 and LePDS2 (Fig. 4A). The read numberof LePDS2 was slightly but significantly smaller than that of LePDS1 in escape (n = 7, p = 1.50 × 10–9) and mockinfected (p = 2.10 × 10–6) plant leaves, suggesting that the levels of expression of LePDS1 and LePDS2 are almost
the same (6:4) in a normal environment (Fig. 4D). In infected plants, however, the read number of LePDS1 wasmuch smaller than that of LePDS2 (n = 7, p = 8.19 × 10–12) (Fig. 4D) and the LePDS1:LePDS2 ratio in infected(n = 7) plant leaves differed significantly from the ratios in escape (n = 14, p = 7.17 × 10–11) and mock infected(n = 14, p = 5.19 × 10–9) plant leaves (Fig. 4D). These results suggested that the designed sequence in this VIGS
experiment preferentially knocked down LePDS1 rather than LePDS2, although the expression of LePDS2 was
negatively regulated to a lesser extent. qRT-PCR measurements of individual and combined expression of LePDS1and LePDS2 and of the LePDS1: LePDS2 ratio in albino L5–L8 leaves expected by amplicon sequencing (Fig. 4E)
showed that LePDS1 expression was significantly lower in leaves of infected than of escape (n = 4, p = 1.17 × 10–6)
and mock infected (n = 4, p = 7.00 × 10–5) plants, Similarly, LePDS2 expression, was significantly lower in leavesof infected than of escape (n = 4, p = 1.22 × 10–6) and mock treated (n = 4, p = 1.68 × 10–3) plants. These results
suggest that the VIGS simultaneously knocked down the expression of both the LePDS1 and LePDS2 genes.
However, the silencing efficiency for both LePDS genes did not reach 100%, resulting in a pale green phenotype.

Other plant virus vectors for VIGS in L. erythrorhizon.

In Japan, the number of wild L. erythrorhizon
plants has markedly decreased in the last several decades, with one of the major reasons being the susceptibilityof these plants to plant viruses, such as cucumber mosaic virus (CMV). We therefore assessed the use of othervirus vectors, such as CMV and tobacco rattle virus (TRV), for VIGS in L. erythrorhizon. Infection of theseplants with domestic CMV yielded a dwarf phenotype (Fig. S1A,B), whereas infection with TRV did not inducea marked phenotypic change (Fig. S2). The use of TRV is strictly limited in Japan, as is the transfer of infectious
materials from other countries. This limits further investigations with TRV, as this pathogenic virus originatedin the United States.
A CMV vector for V­ IGS18 was used to silence the L. erythrorhizon dark-inducible gene-2 (LeDI-2), which is
involved in shikonin p­ roduction19, in cultured shoots, with a GFP fragment of nearly the same length used as acontrol (Fig. S1C,D). Similar to findings with ALSV, CMV silenced the LeDI-2 gene in cultured shoots, parallel tothe reduction in shikonin production (Fig. S1), suggesting that CMV can be utilized for VIGS in L. erythrorhizon.

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Figure 3.  Biosynthesis of shikonin derivatives in ALSV infected L. erythrorhizon roots. (A) Relative abundanceof ALSV RNA in roots, as measured by qRT-PCR and normalized relative to actin 7 (LeACT7) mRNA
expression. Also shown is a gel image of the PCR products. Error bars represent standard errors (n = 3). ND, no
amplicon detected by qPCR. (B) Spectrophotometric measurements of amounts of shikonin derivatives in root
extracts. Error bars represent standard errors (infected, n = 9; escape, n = 13; mock, n = 6). (C) Representative
photographs of roots and HPLC chromatograms of the extracts. The compounds were identified by comparison
with standards. a, shikonin; b, α-hydroxyisovalerylshikonin; c, acetylshikonin; d, isobutyrylshikonin; e, β,βdimethylacrylshikonin; f, β-methylbutylshikonin.

Discussion

The roots of L. erythrorhizon have been used in crude drugs and as natural dyes for many centuries. Because ofits marked decrease in the wild, due both to overharvesting and the worldwide spread of plant viruses, L. erythrorhizon is considered an endangered species. Using a multiple omics approach, we have identified several genesthat may be involved in shikonin biosynthesis and secretion. To analyze the function of each candidate gene andits possible involvement in shikonin biosynthesis and secretion, we established a VIGS system in L. erythrorhizon. Compared with the RNAi approach to analyzing gene function, in which hairy roots are generated using
Rhizobium rhizogenes13, the VIGS approach is more rapid. Specifically, the RNAi-induced hairy root formation
approach requires at least 6 weeks from infection to see the phenotype associated with suppression of the geneof interest. In contrast, the VIGS-mediated approach takes only about 3 weeks to observe these phenotypes.
ALSV is distributed thorough the body of various plant species. For example, ALSV was shown to be present in root tissues of soybean and pea plants, and ALSV-mediated VIGS was reported effective in these plant­tissues20,21. This virus is ubiquitously distributed in most cell types, including the epidermis, of various plant
species, making this virus useful for the knockdown of genes preferentially expressed in epidermal ­cells22,23. ALSV
was able to infect L. erythrorhizon without inducing any obvious phenotypic differences. PDS was selected as amodel gene for silencing of expression by VIGS. Knockdown of PDS in soil grown plants resulted in an albino
phenotype without any growth defect. The albino appearance of newly developed leaves is a representative phenotype of the PDS gene in plant ­biology15. The spread of ALSV was also confirmed in the infected plant body, as
the virus was also detected in root tissues in L. erythrorhizon. This finding is very important, as this study aimed
to evaluate gene function in roots, the sole source of shikonin derivatives.
Shikonin production was not significantly affected by ALSV infection. Specifically, the total shikonin content
and the composition of shikonin derivatives in the roots of infected plant roots were the same as those in the rootsof both escape and mock infected plants. As shikonin derivatives specifically accumulate in the root epidermisof these plants, the wide distribution of ALSV, including in the epidermis, is also advantageous. Taken together,
these findings suggest that ALSV-mediated VIGS may provide a powerful tool for analysis of the functions ofgenes involved in shikonin biosynthesis in L. erythrorhizon (Fig. S3).
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Figure 4.  Simultaneous knockdown of PDS family genes by VIGS. (A) Schematic representation of LePDS1and LePDS2 transcripts. The green color represents nucleotides identical in LePDS2 and LePDS1; the black color
represents mismatched nucleotides in LePDS2 relative to LePDS1; and the white color represents regions in
LePDS2 non-homologous to those in LePDS1. The arrow indicates the mismatched region of the VIGS trigger
relative to LePDS2. NGS analysis indicates the amplicon for deep sequencing. Scale bar = 0.3 kb. (B) Schematic
representation of the genomic structures of LePDS1 and LePDS2. Numbers represent coding exons, with coding
regions indicated in green. Scale bar = 1.0 kb. (C) Neighbor-joining tree of the phytoene desaturase (green)and zeta-carotene desaturase (blue) protein families. The organism and the Genbank accession numbers forsequences are indicated. L. erythrorhizon proteins are labeled in red. Numbers represents bootstrap valuesof 1,000 replicates. Scale bar = substitution of 0.1 amino acid. (D) Read count ratio of LePDS1 to LePDS2
transcripts in leaves measured by deep sequencing. Filled bars represent LePDS1/total and white bars represent
LePDS2/total. Error bars represent standard errors (n = 7). (E) Relative LePDS1 and LePDS2 expression
calculated from the read count ratio and total LePDS expression measured by qRT-PCR normalized to actin 7
gene (LeACT7) expression in the same samples (n = 4).
Analysis of deep sequencing data from L. erythrorhizon identified three PDS-like genes, with two classified
as being in the PDS gene family. These two genes may have diverged from a common PDS gene in a near ancestor of L. erythrorhizon, as we could not find two conserved PDS subfamilies among eudicots. Infection of plantleaves with virus containing the LePDS1 construct resulted in simultaneous knockdown of both PDS genes,
with LePDS1 being more strongly suppressed. The VIGS trigger sequence contains two regions, 49 and 156
nucleotides in length, that match LePDS2, with these two regions separated by a 29 nucleotide-long sequence
containing six nucleotide mismatches. In contrast, the full 234 nucleotide sequence of the VIGS trigger matches
that of LePDS1. These findings suggest that this trigger sequence was effective in knocking down gene expression,
but that knockdown of these two potentially functional LePDS genes showed statistically and physiologicallysignificant differences.
A CMV-based vector also showed potential in gene silencing. CMV was found to infect L. erythrorhizon, inducing growth arrest insertion of a DNA fragment into the CMV vector usually decreased the disease

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phenotype, depending on insert length, with a longer insert associated with a weaker phenotype. We found that
the CMV vector containing a fragment of the LeDI-2 gene knocked down the expression of endogenous LeDI-2
in the shoots of L. erythrorhizon, with the production of shikonin derivatives being slightly but not significantly
reduced in these shoots. We previously reported that LeDI-2 antisense RNA knocked down LeDI-2 expression andthe production of shikonin derivatives in hairy root c­ ultures19. The results presented in this study were similar,
suggesting that CMV may act to suppress targeted genes in L. erythrorhizon.
Shikonin derivatives are secreted by cells and accumulate in the apoplastic space. The molecular mechanism
underlying shikonin secretion is of great importance. Shikonin is a very lipophilic metabolite and rapidly crystallizes under aqueous conditions. Plants contain many such hydrophobic secondary metabolites, including terpenoids, furanocoumarins, prenylated flavonoids and some a­ lkaloids5,24–27. Although the mechanisms underlying
their secretion remain undetermined, it is difficult to determine these mechanisms because only a few types ofcells secrete lipids in this manner. For example, secretory cells in glandular trichomes and epithelial cells in oil
cavities and resin ducts have been shown to secrete these compounds. Cultured L. erythrorhizon cells provide
a model system suitable for lipid secretion, because ca. 10% of shikonin is produced as ester derivatives, which
are secreted by cells, and plant masses can be easily enlarged in liquid suspension c­ ultures6. Our multiple omics
study identified 10 genes as being strongly involved in shikonin biosynthesis and 16 as being strongly involved
in shikonin s­ ecretion9. The VIGS methodology described in this study will likely be utilized for functional
analysis of these genes to determine the entire shikonin biosynthetic pathway and to understand mechanisms
underlying its secretion.

Materials and methods

Plant growth conditions. Lithospermum erythrorhizon and tobacco plants were grown in soil pots in aroom maintained at a temperature of 25 °C and 12 h/12 h day/night conditions. The cultured shoots of L. erythrorhizon were grown in a growth chamber set at 25 °C and exposed continuously to light using fluorescent lamps(85 µmol/m2 sec on average).
Vector construction and virus preparation. The trigger fragment of LePDS1 transcript was amplified

from a pool of L. erythrorhizon cDNA by RT-PCR, using the primers 5′-GCC​CTC​GAG​CTC​CTA​AGT​GTG​TAT​
G-3′ and 5′-CCG​GGA​TCC​CAC​GGA​CCT​CGG​AGT​C-3′. The amplified fragment was digested with BamHI and
XhoI and inserted into the BamHI and XhoI recognition sites of the plasmid p­ BICAL217. The resulting plasmidand pBICAL1 were introduced into Agrobacterium strain GV3101. Colonies on plates of the two strains weremixed in extraction buffer (10 mM MES-KOH, pH 5.7, 10 mM ­MgCl2, 0.15 mM acetosyringone). One of thetrue leaves of a tobacco plant (Nicotiana benthamiana) was covered after sprinkling with carborandom 600 mesh(nacalai tesque, Japan) during the stage of expansion of four leaves. A solution of Agrobacterium was placed on
the leaf, which was rubbed gently by a hand covered with a rubber glove (rub-inoculation), and the leaf waswashed with water to remove the carborandom. After three weeks of growth, the leaves were harvested from thetobacco plants and ground with a mortal and pestle in 1 mL of 100 mM phosphate buffer, pH 7.0, containing
0.3 g of diethyldithiocarbamic acid trihydrate. The lysate was cleared with a 0.45 µm syringe filter, and used toinoculate L. erythrorhizon.

Virus infection of L. erythrorhizon by rub‑inoculation. The inoculation solution described above wasplaced onto the true leaves of 21-day-old L. erythrorhizon seedlings and rubbed by hand as above. Mock control
leaves were inoculated with extraction buffer alone. The inoculated plants were washed with water and grown for
5 weeks to observe the albino phenotype. Total RNA was extracted using Qiagen RNeasy plant mini kits (Qiagen,Germany) and reverse transcribed with ReverTraAce (Toyobo, Osaka, Japan). PCR primers for detecting ALSV
containing an integrated trigger fragment were 5′-ACT​TCT​GAT​GGT​GTC​CTC​A-3′ and 5′-TAA​CTC​TTG​CAA​
GGT​GGT​CG-3′.
Quantification of transcript abundance. Total RNA was extracted and reverse transcribed as above.

The LePDS1 and LePDS2 transcripts were quantified by BioRad real-time PCR system (Bio-Rad, CA, USA) using
the primer pairs 5′-GCC​CTC​GAG​CCT​GAT​GAA​CTTTC-3′ and 5′-CCG​GGA​TCC​CGA​ACT​TCA​CCACC3′ and LeACT7 transcripts were quantified using the primers 5′-TTT​TGA​CTG​AGG​CAC​CCC​-3′ and 5′-TGA​
CAG​GAA​CTC​CCA​CTA​GCT-3′. LePDS1 and LePDS2 mRNAs were normalized relative to the levels of LeACT7mRNA in the same s­amples10. For amplicon sequencing, LePDS1 and LePDS2 cDNAs were PCR amplified
using the primers 5′-GTG​TAC​CAG​ATC​GAG​TTA​C-3′ and 5′-TGA​CAG​GAA​CTC​CCA​CTA​GCT-3′, attached
to adaptors and with four nucleotide tag sequences at their 5′ ends. Deep sequencing was performed by Macrogen Japan Corp. (Kyoto Japan) with a MiSeq system. The number of reads identical in LePDS1 and LePDS2
sequences were counted in silico with a Perl script.

Phylogenetic analysis. Amino acid sequences were aligned using a muscle algorithm. A phylogenetic tree
was determined using the neighbor-joining method with 1,000 bootstrap replicates and drawn using MEGA7­software28. The accession numbers are indicated in Fig. 4C.
Analysis of shikonin derivatives. Shikonin derivatives in L. erythrorhizon were extracted and analyzed quantitatively as ­described10. The extract was analyzed by HPLC as previously reported, with several­modifications29. Briefly, HPLC was performed on a Prominence system (Shimadzu), which was used with a TSK
gel column ODS-80 Tm (4.6 mm × 250 mm, TOSOH); a solvent system consisting of a 7:3 mixture of acetonitrile
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and water containing 1% acetic acid and 1% triethylamine; a flow rate of 1 ml/min; and an oven temperature of
40 °C; with detection at 520 nm.

Received: 24 February 2020; Accepted: 27 July 2020

Reference

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Acknowledgements

The authors thank Dr. Masanori Kaido (Kyoto University) for providing binary vectors of ALSV; Amato Pharmaceutical Products, Ltd. for providing L. erythrorhizon seeds; Dr. Tsuyoshi Nakagawa (Shimane University)
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www.nature.com/scientificreports/
for providing the pGWB505 ­vector30; Dr. Noboru Onishi (KIRIN Ltd.) and Dr. Koichiro Shimomura (ToyoUniversity) for providing cultured L. erythrorhizon shoots; and Dr. Takuji Ichino (Kyoto University) and Dr.
Kanade Tatsumi (Kyoto University) for cloning the LeACT7 and LeDI2 genes, respectively. This project was
supported in part by KAKENHI Grant No. JP19H05638 (to K.Y.). Additional support was provided by Mission
Research of RISH Kyoto University.

Author contributions

Y.I. performed all experiments and wrote the manuscript. H.L. and H.K. performed all informatics experiments
and wrote the manuscript. K.N. performed HPLC analysis of shikonin derivatives. N.I., J.K. and C.M. performed
all experiments involving CMV. K.I. and T.S. performed all experiments involving TRV. N.Y. designed the ALSV
vector system. K.Y. supervised the entire study and wrote the manuscript.

Competing interests

The authors declare no competing interests.

Additional information

Supplementary information is available for this paper at https​://doi.org/10.1038/s4159​8-020-70469​-1.
Correspondence and requests for materials should be addressed to K.Y.
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