Upload
others
View
0
Download
0
Embed Size (px)
Citation preview
1
Efficient production of saffron crocins and picrocrocin in 1
Nicotiana benthamiana using a virus-driven system 2
3
Maricarmen Martía,1, Gianfranco Direttob,1, Verónica Aragonésa, Sarah 4
Fruscianteb, Oussama Ahrazemc, Lourdes Gómez-Gómezc,*, José-Antonio Daròsa,* 5
6
aInstituto de Biología Molecular y Celular de Plantas (Consejo Superior de 7
Investigaciones Científicas-Universitat Politècnica de València), 46022 Valencia, Spain 8
bItalian National Agency for New Technologies, Energy, and Sustainable Development, 9
Casaccia Research Centre, 00123, Rome, Italy 10
cInstituto Botánico, Departamento de Ciencia y Tecnología Agroforestal y Genética, 11
Facultad de Farmacia, Universidad de Castilla-La Mancha, Campus Universitario s/n, 12
02071 Albacete, Spain 13
14
1M.M and G.D. contributed equally to this work. 15
16
*Corresponding authors e-mail addresses: [email protected] (L.Gómez-17
Gómez), [email protected] (J.A. Daròs). 18
19
20
ABSTRACT 21
22
Crocins and picrocrocin are glycosylated apocarotenoids responsible, respectively, 23
for the color and the unique taste of the saffron spice, known as red gold due to its 24
high price. Several studies have also shown the health-promoting properties of these 25
compounds. However, their high costs hamper the wide use of these metabolites in 26
the pharmaceutical sector. We have developed a virus-driven system to produce 27
remarkable amounts of crocins and picrocrocin in adult Nicotiana benthamiana 28
plants in only two weeks. The system consists of viral clones derived from tobacco 29
etch potyvirus that express specific carotenoid cleavage dioxygenase (CCD) enzymes 30
from Crocus sativus and Buddleja davidii. Metabolic analyses of infected tissues 31
demonstrated that the sole virus-driven expression of C. sativus CsCCD2L or B. 32
davidii BdCCD4.1 resulted in the production of crocins, picrocrocin and safranal. 33
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
2
Using the recombinant virus that expressed CsCCD2L, accumulations of 0.2% of 34
crocins and 0.8% of picrocrocin in leaf dry weight were reached in only two weeks. 35
In an attempt to improve apocarotenoid content in N. benthamiana, co-expression 36
of CsCCD2L with other carotenogenic enzymes, such as Pantoea ananatis phytoene 37
synthase (PaCrtB) and saffron -carotene hydroxylase 2 (BCH2), was performed 38
using the same viral system. This combinatorial approach led to an additional crocin 39
increase up to 0.35% in leaves in which CsCCD2L and PaCrtB were co-expressed. 40
Considering that saffron apocarotenoids are costly harvested from flower stigma 41
once a year, and that Buddleja spp. flowers accumulate lower amounts, this system 42
may be an attractive alternative for the sustainable production of these appreciated 43
metabolites. 44
45
Keywords: Apocarotenoids; crocins; picrocrocin; carotenoid cleavage dioxygenase; viral 46
vector; tobacco etch virus; potyvirus 47
48
1. Introduction 49
50
Plants possess an extremely rich secondary metabolism and, in addition to food, 51
feed, fibers and fuel, also provide highly valuable metabolites for food, pharma and 52
chemical industries. However, some of these metabolites are sometime produced in very 53
limiting amounts. Plant metabolic engineering can address some of these natural 54
limitations for nutritional improvement of foods or to create green factories that produce 55
valuable compounds (Martin and Li, 2017; Yuan and Grotewold, 2015). Yet, the complex 56
regulation of plant metabolic pathways and the particularly time-consuming approaches 57
for stable genetic transformation of plant tissues highly limit quick progress in plant 58
metabolic engineering. In this context, virus-derived vectors that fast and efficiently 59
deliver biosynthetic enzymes and regulatory factors into adult plants may significantly 60
contribute to solve some challenges (Sainsbury et al., 2012). 61
Carotenoids constitute an important group of natural products that humans cannot 62
biosynthesize and must uptake from the diet. These compounds are synthesized by higher 63
plants, algae, fungi and bacteria (Rodriguez-Concepcion et al., 2018). Yet, they play 64
multiple roles in human physiology (Fiedor and Burda, 2014). Carotenoids are widely 65
used as food colorants, nutraceuticals, animal feed, cosmetic additives and health 66
supplements (Fraser and Bramley, 2004). In all living organisms, carotenoids act as 67
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
3
substrates for the production of apocarotenoids (Ahrazem et al., 2016a). These are not 68
only the simple breakdown products of carotenoids, as they act as hormones and are 69
involved in signaling (Eroglu and Harrison, 2013; Jia et al., 2018; Walter et al., 2010). 70
Apocarotenoids are present as volatiles, water soluble and insoluble compounds, and 71
among those soluble, crocins are the most valuable pigments used in the food, and in less 72
extent, in the pharmaceutical industries (Ahrazem et al., 2015). Crocins are glycosylated 73
derivatives of the apocarotenoid crocetin. They are highly soluble in water and exhibit a 74
strong coloring capacity. In addition, crocins are powerful free radical quenchers, a 75
property associated to the broad range of health benefits they exhibit (Bukhari et al., 2018; 76
Christodoulou et al., 2015; Georgiadou et al., 2012; Nam et al., 2010). The interest in the 77
therapeutic properties of these compounds is increasing due to their analgesic and 78
sedative properties (Amin and Hosseinzadeh, 2012), and neurological protection and 79
anticancer activities (Finley and Gao, 2017; Skladnev and Johnstone, 2017). Further, 80
clinical trials indicate that crocins have a positive effect in the treatment of depression 81
and dementia (Lopresti and Drummond, 2014; Mazidi et al., 2016). Other apocarotenoids, 82
such as safranal (2,6,6-trimethyl-1,3-cyclohexadiene-1-carboxaldehyde) and its precursor 83
picrocrocin (β-D-glucopyranoside of hydroxyl-β-cyclocitral), have also been shown to 84
reduce the proliferation of different human carcinoma cells (Cheriyamundath et al., 2018; 85
Jabini et al., 2017; Kyriakoudi et al., 2015) and to exert anti-inflammatory effects (Zhang 86
et al., 2015). 87
Crocus sativus L. is the main natural source of crocins and picrocrocin, 88
respectively responsible for the color and flavor of the highly appreciated spice, saffron 89
(Tarantilis et al., 1995). Crocin pigments accumulate at huge levels in the flower stigma 90
of these plants conferring this organ with a distinctive dark red coloration (Moraga et al., 91
2009). Yet, these metabolites reach huge prices in the market, due to the labor intensive 92
activities associated to harvesting and processing the stigma collected from C. sativus 93
flowers (Ahrazem et al., 2015). Besides C. sativus, gardenia (Gardenia spp.) fruits are 94
also a commercial source of crocins, but at much lower scale. In addition, gardenia fruits 95
do not accumulate picrocrocin (Moras et al., 2018; Pfister et al., 1996). Some other plants, 96
such as Buddleja spp., also produce crocins, although they are not commercially exploited 97
due to low accumulation (Liao et al., 1999). All these plants share the common feature of 98
expressing carotenoid cleavage dioxygenase (CDD) activities, like the saffron CCD2L or 99
the CCD4 subfamily recently identified in Buddleja spp. (Ahrazem et al., 2017). 100
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
4
In C. sativus and Buddleja spp., zeaxanthin is the precursor of crocetin (Fig. 1). 101
Cleavage of the zeaxanthin molecule at the 7,8 and 7′,8′ double bonds render one 102
molecule of crocetin dialdehyde and two molecules of 4-hydroxy-2,6,6-trimethyl-1-103
cyclohexene-1-carboxaldehyde (HTCC) (Ahrazem et al., 2017, 2016c; Frusciante et al., 104
2014). Crocetin dialdehyde is further transformed to crocetin by the action of aldehyde 105
dehydrogenase (ALDH) enzymes (Demurtas et al., 2018; Gómez-Gómez et al., 2018, 106
2017). Next, crocetin serves as substrate of glucosyltransferase (UGT) activities that 107
catalyze the production of crocins through transfer of glucose to both ends of the molecule 108
(Côté et al., 2001; Demurtas et al., 2018; Moraga et al., 2004; Nagatoshi et al., 2012). The 109
HTCC molecule is also recognized by UGTs, resulting in production of picrocrocin (Fig. 110
1) (Diretto et al., 2019a). In C. sativus, the plastidic CsCCD2L enzyme catalyzes the 111
cleavage of zeaxanthin at 7,8;7′,8′ double bonds (Ahrazem et al., 2016c; Frusciante et al., 112
2014), which is closely related to the CCD1 subfamily (Ahrazem et al., 2016b, 2016a). 113
In Buddleja davidii, the CCD enzymes that catalyze the same reaction belong to a novel 114
group within the CCD4 subfamily of CCDs (Ahrazem et al., 2016a). These B. davidii 115
enzymes, BdCCD4.1 and BdCCD4.3, also localize in plastids and are expressed in the 116
flower tissue (Ahrazem et al., 2017). 117
Due to its high scientific and commercial interest, crocetin and crocins 118
biosynthesis has arisen great attention, and attempts to metabolically engineer their 119
pathway in microbial systems have been reported, although with limited success (Chai et 120
al., 2017; Diretto et al., 2019a; Tan et al., 2019; Wang et al., 2019). More in detail, the 121
use of the saffron CCD2L enzyme resulted in a maximum accumulation of 1.22 mg/l, 122
15.70 mg/l, and 4.42 mg/l of crocetin in, respectively, Saccharomyces cerevisiae (Chai et 123
al., 2017; Tan et al., 2019) and Escherichia coli (Wang et al., 2019). In a subsequent study 124
aimed to confirm, in planta, the role of a novel UGT in picrocrocin biosynthesis (Diretto 125
et al., 2019a), Nicotiana benthamiana leaves were transiently transformed, via 126
Agrobacterium tumefaciens, with CCD2L, alone or in combination with UGT709G1, 127
which led to the production of 30.5 g/g DW of crocins, with a glycosylation degree 128
ranging from 1 to 4 (crocin 1-4) (unpublished data). Although promising, these results 129
cannot be considered efficient and sustainable in the context of the development of an 130
industrial system for the production of the saffron high-value apocarotenoids. 131
Here we used a viral vector derived from Tobacco etch virus (TEV, genus 132
Potyvirus; family Potyviridae) (Bedoya et al., 2010) to transiently express a series of CCD 133
enzymes, alone or in combination with other carotenoid and non-carotenoid biosynthetic 134
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
5
enzymes, in N. benthamiana plants. Interestingly, tissues infected with viruses that 135
expressed CsCCD2L or BdCCD4.1 acquired yellow pigmentation visible to the naked 136
eye. Metabolite analyses of these tissues demonstrated the accumulation of crocins and 137
picrocrocin, reaching levels attractive enough for their commercial production, up to 138
0.35% and 0.8% of DW, respectively, in leaves in which CsCCD2L and Pantoea ananatis 139
phytoene synthase (PaCrtB) were simultaneously expressed. 140
141
2. Materials and Methods 142
143
2.1. Plasmid construction 144
145
Plasmids were constructed by standard molecular biology techniques, including 146
PCR amplifications of cDNAs with the high-fidelity Phusion DNA polymerase (Thermo 147
Scientific) and Gibson DNA assembly (Gibson et al., 2009), using the NEBuilder HiFi 148
DNA Assembly Master Mix (New England Biolabs). To generate a transformed N. 149
benthamiana line that stably expresses TEV NIb, we built the plasmid p235KNIbd. This 150
plasmid is a derivative of pCLEAN-G181 (Thole et al., 2007) and between the left and 151
right borders of A. tumefaciens transfer DNA (T-DNA) contains two in tandem cassettes 152
in which the neomycin phosphotransferase (NPTII; kanamycin resistance) and the TEV 153
NIb are expressed under the control of CaMV 35S promoter and terminator. The exact 154
nucleotide sequence of the p235KNIbd T-DNA is in Supplementary Fig. S1. Plasmids 155
pGTEVΔNIb-CsCCD2L, -BdCCD4.1, -BdCCD4.3, -CsCCD2L/PaCrtB, -156
CsCCD2L/CsBCH2 and -CsCCD2L/CsLPT1 were built on the basis of pGTEVa (Bedoya 157
et al., 2012) that contains a wild-type TEV cDNA (GenBank accession number 158
DQ986288 with the two silent and neutral mutations G273A and A1119G) flanked by the 159
CaMV 35S promoter and terminator in a binary vector that also derives from pCLEAN-160
G181. These plasmids were built, as explained above, by PCR amplification of CsCCD2L 161
(Ahrazem et al., 2016c), BdCCD4.1 and BdCCD4.3 (Ahrazem et al., 2017), PaCrtB 162
(Majer et al., 2017), CsBCH2 (Castillo et al., 2005) and CsLPT1 (Gómez-Gómez et al., 163
2010) cDNAs and assembly into a pGTEVa version in which the NIb cistron was deleted 164
(pGTEVaΔNIb) (Majer et al., 2015). The exact sequences of the TEV recombinant clones 165
(TEVΔNIb-CsCCD2L, -BdCCD4.1, -BdCCD4.3, -CsCCD2L/PaCrtB, -166
CsCCD2L/CsBCH2 and -CsCCD2L/CsLPT1) contained in the resulting plasmids are in 167
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
6
Supplementary Fig. S2. The plasmid expressing the recombinant TEVΔNIb-aGFP clone 168
(Supplementary Fig. S2) was previously described (Majer et al., 2015). 169
170
2.2. Plant transformation 171
172
The LBA4404 strain of A. tumefaciens was sequentially transformed with the 173
helper plasmid pSoup (Hellens et al., 2000) and p235KNIbd. Clones containing both 174
plasmids were selected in plates containing 50 µg/ml rifampicin, 50 µg/ml kanamycin 175
and 7.5 µg/ml tetracycline. A liquid culture grown from a selected colony was used to 176
transform N. benthamiana (Clemente, 2006). Briefly, N. benthamiana leaf explants were 177
incubated with the A. tumefaciens culture at an optical density (600 nm) of 0.5 for 30 min 178
and then incubated on solid media. Four days later, explants were transferred to plates 179
with organogenesis media containing 100 µg/ml of kanamycin. Explants were repeatedly 180
transferred to fresh organogenesis plates every three weeks until shoots emerged. These 181
shoots were first transferred to rooting media and, when roots appeared, cultivated in a 182
greenhouse with regular soil until seeds were harvested. Transformation with the NIb 183
cDNA was confirmed by PCR. Batches of seedlings from four independent transformed 184
lines were screened for efficient complementation of TEV clones lacking NIb by 185
mechanical inoculation of TEVΔNIb-Ros1 (Bedoya et al., 2010). 186
187
2.3. Plant inoculation 188
189
A. tumefaciens C58C1 competent cells, previously transformed with the helper 190
plasmid pCLEAN-S48 (Thole et al., 2007), were electroporated with different plasmids 191
that contained the TEV recombinant clones and selected in plates with 50 µg/ml 192
rifampicin, 50 µg/ml kanamycin and 7.5 µg/ml tetracycline. Liquid cultures of selected 193
colonies were brought to an optical density of 0.5 (600 nm) in agroinoculation solution 194
(10 mM MES-NaOH, pH 5.6, 10 mM MgCl2 and 150 μM acetosyringone) and incubated 195
for 2 h at 28ºC. Using a needleless syringe, these cultures were used to infiltrate one leaf 196
of five-week-old plants of the selected N. benthamiana transformed line that stably 197
expresses TEV NIb. After inoculation, plants were kept in a growth chamber at 25ºC 198
under a 12 h day-night photoperiod. Leaves were collected at different dpi as indicated; 199
in the case of the time-course experiments, leaf samples were collected at 8 time-points 200
(0, 4, 8, 11, 13, 15, 18 and 20 dpi). 201
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
7
202
2.4. Analysis of virus progeny 203
204
Viral progeny was analyzed by reverse transcription (RT)-PCR using RevertAid 205
reverse transcriptase (Thermo Scientific) and Phusion DNA polymerase followed by 206
electrophoretic separation of the amplification products in 1% agarose gels that were 207
stained with ethidium bromide. RNA from N. benthamiana plants was purified from 208
upper non-inoculated leaves at 15 dpi using silica-gel columns (Zymo Research). For 209
virus infection diagnosis, a cDNA corresponding to the TEV CP cistron (804 bp) was 210
amplified. Aliquots of the RNA preparations were subjected to RT using primer PI (5’-211
CTCGCACTACATAGGAGAATTAGAC-3’), followed by PCR amplification with 212
primers PII (5’-AGTGGCACTGTGGGTGCTGGTGTTG-3’) and PIII (5’-213
CTGGCGGACCCCTAATAG-3’). To analyze the potential deletion of the inserted CDD 214
cDNAs, RNA aliquots were reverse transcribed using primer PIV (5’- 215
GCTGTTTGTCACTCAATGACACATTAT-3’) and amplified by PCR with primers PV 216
(5’-AAAATAACAAATCTCAACACAACATATAC-3’) and PVI (5’- 217
CCGCGGTCTCCCCATTATGCACAAGTTGAGTGGTAGC-3’). 218
219
2.5. Metabolite extraction and analysis 220
221
Polar and apolar metabolites were extracted from 50 mg of lyophilized leaf tissue. 222
For polar metabolites analyses (crocins and picrocrocin), the tissues were extracted in 223
cold 50% methanol. The soluble fraction was analyzed by HPLC-DAD-HRMS and 224
HPLC-DAD (Ahrazem et al., 2018; Moraga et al., 2009). The liposoluble fractions 225
(crocetin, HTCC, -cyclocitral, carotenoids and chlorophylls) were extracted with 0.5:1 ml 226
cold extraction solvents (50:50 methanol and CHCl3), and analyzed by HPLC-DAD-227
HRMS and HPLC-DAD as previously described (Ahrazem et al., 2018; Castillo et al., 228
2005; D’Esposito et al., 2017; Fasano et al., 2016). Metabolites were identified on the 229
basis of absorption spectra and retention times relative to standard compounds. Pigments 230
were quantified by integrating peak areas that were converted to concentrations in 231
comparison with authentic standards. Chlorophyll a and b were determined by measuring 232
absorbance at 645 nm and 663 nm as previously described (Richardson et al., 2002). Mass 233
spectrometry carotenoid, chlorophyll derivative and apocarotenoid identification and 234
quantification was carried out as previously described (Ahrazem et al., 2018; Diretto et 235
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
8
al., 2019a; Rambla et al., 2016). Mass spectrometry analysis of other isoprenoids 236
(tocochromanols and quinones) were performed as reported before (Sulli et al., 2017). 237
Picrocrocin derivatives, as reported by (Moras et al., 2018) and (D’Archivio et al., 2016), 238
were tentatively identified according to their m/z accurate masses as included in the 239
PubChem database for monoisotopic masses or by using the Mass Spectrometry Adduct 240
Calculator from the Metabolomics Fiehn Lab for adduct ions; and were further validated 241
by isotopic pattern ratio and the comparison between theoretical and experimental m/z 242
fragmentation, by using the MassFrontier 7.0 software (Thermo Fisher Scientific). 243
244
2.6. Subcellular localization of crocins 245
246
To determine the subcellular localization of crocins in N. benthamiana tissues, LSCM 247
was performed with a Zeiss 7080 Axio Observer equipped with a water immersion 248
objective lens (C-Apochromat 40X/1.20 W). An argon ion laser at 458 nm was used for 249
excitation. Crocins and chloroplast autofluorescence were detected with 520 to 540 nm 250
and 660 to 750 nm band-pass emission filters, respectively. Images were processed using 251
the FIJI software (http://fiji.sc/Fiji). 252
253
2.7. Statistics and bioinformatics 254
255
Statistical validation of the data (ANOVA+ Tukey’s t-test) and heatmap 256
visualization were performed as previously described (Cappelli et al., 2018; Grosso et al., 257
2018). 258
259
3. Results 260
261
3.1. TEV recombinant clones that express CCD enzymes in N. benthamiana 262
263
The goal of this work was to develop a heterologous system to efficiently produce 264
highly appreciated and scarce apocarotenoids in plant tissues. For this purpose, we 265
planned to use an expression vector derived from TEV, more specifically TEVΔNIb 266
(Bedoya et al., 2010), recently employed to rewire the lycopene biosynthetic pathway 267
from the plastid to the cytosol of plant cells (Majer et al., 2017). Since CCD is the first 268
enzyme in the apocarotenoid biosynthetic pathway (Fig. 1), we started exploring the 269
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
9
virus-based expression of a series of CCD enzymes from well-known crocins producing 270
plants: namely C. sativus CsCCD2L (Ahrazem et al., 2016c), and B. davidii BdCCD4.1 271
and BdCCD4.3 (Ahrazem et al., 2017). 272
In TEVΔNIb, the approximately 1.6 kb corresponding to nuclear inclusion b 273
(NIb) cistron, coding for the viral RNA-dependent RNA polymerase, is deleted to 274
increase the space to insert foreign genetic information. Then, the viral recombinant 275
clones only infect plants in which NIb is supplied in trans (Bedoya et al., 2010). We chose 276
N. benthamiana as the biofactory plant for apocarotenoid production because of the 277
amenable genetic transformation, the high amounts of biomass achievable and the fast 278
growth. For this aim, as the first step in our work, we transformed N. benthamiana leaf 279
tissue using Agrobacterium tumefaciens and regenerated adult plants that constitutively 280
expressed TEV NIb under the control of Cauliflower mosaic virus (CaMV) 35S promoter 281
and terminator. To screen for transformed lines that efficiently complemented TEVΔNIb 282
deletion mutants, we inoculated the plants with TEVΔNIb-Ros1, a viral clone in which 283
the NIb cistron was replaced by a cDNA encoding for the MYB-type transcription factor 284
Rosea1 from Antirrhinum majus L., whose expression induces accumulation of colored 285
anthocyanins in infected tissues and facilitates visual tracking of infection (Bedoya et al., 286
2010, 2012). On the basis of anthocyanin accumulation, we selected a N. benthamiana 287
transformed line that efficiently complemented the replication and systemic movement 288
of TEVΔNIb-Ros1 (Supplementary Fig. S3). Then, by self-pollination and TEVΔNIb-289
Ros1 inoculation of the progeny, we selected a transformed homozygous line that was 290
used in all subsequent experiments. 291
Next, on the context of a TEV lacking NIb, we constructed three recombinant 292
clones (Fig. 2A) to express C. sativus CsCCD2L (TEVΔNIb-CsCCD2L), B. davidii 293
BdCCD4.1 and BdCCD4.3 (TEVΔNIb-BdCCD4.1 and TEVΔNIb-BdCCD4.3). These 294
CCDs are plastidic enzymes that are encoded in the nucleus and contain native amino-295
terminal transit peptides. To target these enzymes to plastids, we inserted their cDNAs in 296
a position corresponding to the amino terminus of the viral polyprotein in the virus 297
genome (Majer et al., 2015). CCD cDNAs also contained a sequence corresponding to an 298
artificial NIaPro cleavage site at the 3’ end to mediate the release of the heterologous 299
protein from the viral polyprotein. Based on previous observations, we used the -8/+3 site 300
that splits NIb and CP in TEV. The exact sequences of these clones are in Supplementary 301
Fig. S2. 302
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
10
Finally, transformed N. benthamiana plants that express NIb were 303
agroinoculated with the three TEV recombinant clones. As a control in this experiment, 304
some plants were agroinoculated with TEVΔNIb-aGFP (Fig. 2A and Supplementary Fig. 305
S2), which is a recombinant clone that expresses GFP from the amino-terminal position 306
in the viral polyprotein. Interestingly, 7 days post-inoculation (dpi), even before 307
symptoms were observed, we noticed a distinctive yellow pigmentation in systemic 308
tissues of the plants agroinoculated with TEVΔNIb-CsCCD2L. Symptoms of infection 309
were soon observed (approximately 8 dpi) in plants agroinoculated with TEVΔNIb-aGFP, 310
-CsCCD2L and -BdCCD4.1. Distinctive yellow pigmentation was also observed in 311
symptomatic tissues of plants agroinoculated with TEVΔNIb-BdCCD4.1, although with 312
some delay comparing to TEVΔNIb-CsCCD2L. Yellow pigmentation of symptomatic 313
tissues in plants inoculated with TEVΔNIb-CsCCD2L was more intense than that in 314
tissues of plants inoculated with TEVΔNIb-BdCCD4.1. Plants inoculated with 315
TEVΔNIb-BdCCD4.3 showed mild symptoms of infection at approximately 13 dpi and 316
yellow pigmentation was never observed. Fig. 2B shows pictures of representative leaves 317
of all these plants at 13 dpi. Reverse transcription (RT)-PCR analysis confirmed TEV 318
infection in all inoculated plants (Fig. 2C). Observation of symptomatic tissues with a 319
fluorescence stereomicroscope confirmed expression of GFP only in plants inoculated 320
with the TEVΔNIb-aGFP control (Supplementary Fig. S4). Analysis of viral progeny at 321
15 dpi by RT-PCR indicated that, while the inserts of TEVΔNIb-aGFP, -CsCCD2L are 322
perfectly stable in the recombinant virus, those of TEVΔNIb-BdCCD4.1 and TEVΔNIb-323
BdCCD4.3 are partially and completely lost, respectively, at this time post-inoculation 324
(Fig. 2D). Based on distinctive yellow color, these results suggested that the virus-driven 325
expression of CsCCD2L or BdCCD4.1 in N. benthamiana tissues may induce 326
apocarotenoid accumulation. 327
328
3.2. Analysis of apocarotenoids in infected tissues 329
330
Symptomatic leaf tissues from plants infected with TEVΔNIb-aGFP, TEVΔNIb-331
CsCCD2L and TEVΔNIb-BdCCD4.1 were subjected to extraction and analysis by high 332
performance liquid chromatography-diode array detector-high resolution mass 333
spectrometry (HPLC-DAD-HRMS) to determine their apocarotenoid and carotenoid 334
profiles. Tissues from mock-inoculated controls were also included in the analysis. The 335
tissues from the plants infected with TEVΔNIb-BdCCD4.3 were discarded due to the 336
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
11
absence of pigmentation and rapid deletion of BdCCD4.3 cDNA in the viral progeny. 337
Analysis of the polar fraction of tissues infected with TEVΔNIb-CsCCD2L and 338
TEVΔNIb-BdCCD4.1 showed a series of peaks with maximum absorbance from 433 to 339
439 nm. These peaks were not observed in extracts from tissues from mock-inoculated 340
plants or plants infected with the TEVΔNIb-aGFP control (Fig. 3). Mass spectrometry 341
chromatograms evidenced the presence, for all the fore mentioned peaks, of the crocetin 342
aglycon (m/z 329.1747 (M+H)), thus supporting the presence of crocins (Supplementary 343
Fig. S5). Further analyses of the sugar moieties conjugated with crocetin lead to the 344
identification of crocins with different degrees of glycosylation from one glucose 345
molecule to five in the infected tissues in which CCDs were expressed (Fig. 4A and 346
Supplementary Table S1). However, predominant crocins were those conjugated with 347
three and four glucose molecules. Interestingly, CCD-infected N. benthamiana leaves, 348
displayed a different pattern of crocin accumulation compared to that typical from saffron 349
stigma (Fig. 3A), with trans-crocin 4, followed by trans-crocin 3 and 2, being the most 350
abundant crocin species. Minor changes were also observed between TEVΔNIb-351
CsCCD2L and TEVΔNIb-BdCCD4.1 samples (Supplementary Table S2): in the former, 352
trans-crocin 3 was the most abundant (30.84%), followed by cis-crocin 4 (17.04%) and 353
trans-crocin 2 (16.84%); whereas similar amounts in cis-crocin 5, cis-crocin 4 and trans-354
crocin 3 were found in the latter (20.92, 20.62 and 18.31%, respectively). Picrocrocin, 355
safranal and several derivatives were also identified in the polar fraction of tissues 356
infected with TEVΔNIb-CsCCD2L and TEVΔNIb-BdCCD4.1 (Fig. 4B and 357
Supplementary Table S1). All these polar apocarotenoids were absent in the control 358
tissues non-infected and infected with TEVΔNIb-aGFP (Fig. 4B). Overall, and in 359
agreement with the most intense visual phenotype, TEVΔNIb-CsCCD2L-infected tissues 360
displayed a higher accumulation in all the linear and cyclic apocarotenoids than 361
TEVΔNIb-BdCCD4.1-infected tissues (Fig. 4, Table 1 and Supplementary Table S1). 362
In the polar and apolar fractions of TEVΔNIb-CsCCD2L and TEVΔNIb-363
BdCCD4.1-infected leaves, we also detected the presence of crocetin dialdehyde and 364
HTCC, which are the products of the enzymatic cleavage step, and crocetin and safranal, 365
which are produced by the activity of ALDH enzymes and the spontaneous 366
deglycosilation of picrocrocin, respectively (Fig. 4 and Supplementary Table S1). None 367
of these metabolites were found in the TEVΔNIb-aGFP-infected and mock-inoculated 368
control tissues. Subsequently, the levels of different carotenoids and chlorophylls were 369
also investigated in the apolar fractions (Fig. 5 and Supplementary Table S3). As 370
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
12
expected, virus infection negatively affected the isoprenoid pools, with most of 371
carotenoids and chlorophylls reduced in all infected tissues compared to those from 372
mock-inoculated plants. However, the comparison between tissues infected with 373
TEVΔNIb-aGFP and both CCD viruses highlighted a series of alterations at metabolite 374
level. The most striking differences were in the contents of phytoene and phytofluene, as 375
well as zeaxanthin and lutein. While the formers increased in the TEVΔNIb-CsCCD2L 376
and TEVΔNIb-BdCCD4.1-infected tissues, the levels of zeaxanthin and lutein were 377
strongly reduced (Fig. 5A and Table S3A). On the contrary, no significant alterations in 378
chlorophyll levels were observed in tissues infected with the two CCD viruses when 379
compared to the TEVΔNIb-aGFP-infected control (Fig. 5B and Supplementary Table 380
S3B). Other typical leaf isoprenoids, such as tocochromanols and quinones, displayed 381
only few changes in CCD versus control tissues: for instance, -/-tocopherol, α-382
tocopherol quinone and menaquinone-8 increased, whereas plastoquinone was reduced 383
in CsCCD2L and BdCCD4.1-infected tissues compared to mock-inoculated and GFP-384
infected leaves (Fig. 5C and Supplementary Table S3C). 385
386
3.3. Subcellular accumulation of apocarotenoids in tissues infected with CCD-expressing 387
viruses 388
389
On the basis of the differential autofluorescence of chlorophylls and carotenoids, 390
we analyzed the subcellular localization of crocins that accumulate in symptomatic 391
tissues of plants infected with TEVΔNIb-CsCCD2L and TEVΔNIb-BdCCD4.1. Laser 392
scanning confocal microscopy (LSCM) images showed an intense fluorescence signal 393
from crocins in the vacuoles, while tissues infected with TEVΔNIb only showed 394
chlorophyll fluorescence (Fig. 6). 395
396
3.4. Time course accumulation of crocins and picrocrocin in inoculated plants 397
398
Next, we researched the point with the maximum apocarotenoid accumulation in 399
inoculated plants. For this aim, we carried out a time-course analysis focusing only on the 400
virus recombinant clone TEVΔNIb-CsCCD2L that induces the highest apocarotenoid 401
accumulation in N. benthamiana tissues. After plant agroinoculation, upper systemic 402
tissues were collected at 0, 4, 8, 11, 13, 15, 18 and 20 dpi from three independent replicate 403
plants per time point. Pigments were extracted and analyzed by HPLC-DAD. In these 404
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
13
tissues, accumulation of total crocins increased from approximately 8 to 13 dpi, reaching 405
a plateau up to the end of the analysis (20 dpi) (Fig. 7A). Picrocrocin levels showed 406
identical behavior (Fig. 7B). Accumulation of lutein, -carotene and chlorophylls was 407
also evaluated in all these samples. In contrast to apocarotenoids, accumulation of lutein 408
and -carotene (Fig. 7C) as well as chlorophylls a and b (Fig. 7D) dropped as infection 409
progressed. These results indicate that the virus-driven system to produce apocarotenoids 410
in N. benthamiana developed here reaches the highest yield in only 13 days after 411
inoculating the plants and this yield is maintained during at least one week with no 412
apparent loss. This experiment also revealed the remarkable accumulation of 2.18±0.23 413
mg of crocins and 8.24±2.93 mg of picrocrocin per gram of N. benthamiana dry weight 414
leaf tissue with the sole virus-driven expression of C. sativus CsCCD2L. 415
416
3.5. Virus-based combined expression of CsCCD2L and other carotenogenic and non-417
carotenogenic enzymes 418
419
In order to further improve apocarotenoid accumulation in inoculated N. 420
benthamiana plants, we combined expression of CsCCD2L with two carotenoid enzymes 421
(Fig. 8A), acting early and late in the biosynthetic pathway, namely Pantoea ananatis 422
phytoene synthase (PaCrtB), and saffron -carotene hydroxylase2 (CsBCH2); or with a 423
non-carotenoid transporter, the saffron lipid transfer protein 1 (CsLTP1), involved in the 424
transport of secondary metabolites (Wang et al., 2016). Interestingly, virus-based co-425
expression of CsCCD2L and PaCrtB induced a stronger yellow phenotype (Fig. 8B), and 426
a higher content in crocins (3.493±0.325 mg/g DW; Fig. 8C and Table S4). Improved 427
crocins levels were also observed when CsCCD2L was co-expressed with CsBCH2, 428
although at a lesser extent in this case (2.198±0.338 mg/g DW). On the contrary, co-429
expression of LPT1 did not result in an increment of total apocarotenoid content (Fig. 8C 430
and Table S4). Interestingly, when compared to tissues in which CsCCD2L alone was 431
expressed, a preferential over-accumulation for higher glycosylated crocins was 432
observed, while lower glycosylated species were down-represented (Fig. 8D). 433
434
4. Discussion 435
436
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
14
The carotenoid biosynthetic pathway in higher plants has been manipulated by 437
genetic engineering, both at the nuclear and transplastomic levels, to increase the general 438
amounts of carotenoids or to produce specific carotenoids of interest, such as 439
ketocarotenoids (Apel and Bock, 2009; Diretto et al., 2019b, 2007; Farré et al., 2014; 440
Giorio et al., 2007; Hasunuma et al., 2008; Nogueira et al., 2019; Zhu et al., 2018). 441
However, these projects also showed that the stable genetic transformation of nuclear or 442
plastid genomes from higher plants is a work-intensive and time-consuming process, 443
which frequently drives to unpredicted results, due to the complex regulation of metabolic 444
pathways. Although no exempted of important limitations, chief among them is the 445
amount of exogenous genetic information plant virus-derived expression systems can 446
carry, they represent an attractive alternative for some plant metabolic engineering goals. 447
Here, we used a virus-driven system that allows the production of noteworthy amounts 448
of appreciated apocarotenoids, such as crocins and picrocrocin, in adult N. benthamiana 449
plants in as little as 13 days (Fig. 7). For this, we manipulated the genome of a 10-kb 450
potyvirus, a quick straight-forward process that can be easily scaled-up for the high-451
throughput analysis of many enzymes and regulatory factors and engineered versions 452
thereof. 453
In this virus-driven system, the expression of an appropriate CCD in N. 454
benthamiana is sufficient to trigger the apocarotenoid pathway in this plant. Recently, it 455
was observed the same situation in a transient expression system (Diretto et al., 2019a). 456
These observations indicate that the zeaxanthin cleavage is the limiting step of 457
apocarotenoid biosynthesis in this species, while aldehyde dehydrogenation and 458
glycosylation steps can be efficiently complemented by endogenous orthologue enzymes. 459
A similar situation was previously observed with other secondary metabolites. Indeed, a 460
large portion of hydroxyl- and carboxyl-containing terpenoid compounds heterologously 461
produced in plants are glycosylated by endogenous UGTs. For example, in transgenic 462
maize expressing a geraniol synthase gene from Lippia dulcis, geranoyl-6-O-malonyl-β-463
D-glucopyranoside was the most abundant geraniol-derived compound (Yang et al., 464
2011). In another study, N. benthamiana plants transiently expressing the Artemesia 465
annua genes of the artemisin biosynthetic pathway accumulated glycosylated versions of 466
intermediate metabolites (Ting et al., 2013). Likewise, in tomato plants expressing the 467
chrysanthemyl diphosphate synthase gene from Tanacetum cinerariifolium, the 62% of 468
trans-chrysanthemic acid was converted into malonyl glycosides (Xu et al., 2018). 469
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
15
Zeaxanthin, the crocins precursor, is not a major carotenoid in plant leaves grown 470
under regular light conditions, although its level increases during high light stress 471
(Demmig-Adams et al., 2012). However, the efficient virus-driven production of all the 472
intermediate and final products of the apocarotenoid biosynthetic pathway in N. 473
benthamiana (Fig. 4) suggests that CCD is able to intercept the leaf metabolic flux in 474
order to diverge it towards the production of apocarotenoids. Similarly, the observed 475
reduction in lutein (Fig. 5A) may result from the CCD activity on the -ring of this 476
molecule, which confirms, at least for the C. sativus enzyme, the previous in vitro data 477
showing that lutein can act as substrate of this cleavage activity (Frusciante et al., 2014). 478
Although N. benthamiana leaves infected with CsCCD2L and BdCCD4.1 recombinant 479
viruses are able to accumulate crocins, it has to be mentioned that, at the qualitative level, 480
their profiles are distinct with respect to the saffron stigma: while in the formers the major 481
crocin species are the trans-crocin 3, together with cis-crocin 4, trans-crocin 2, cis-crocin 482
3 and cis-crocin 5, saffron stigmas are characterized by the presence, in decreasing order 483
of accumulation, of trans-crocin 4, trans-crocin 3 and trans-crocin 2. These results suggest 484
not only the implication of promiscuous glucosyltransferase enzymes differing form those 485
in saffron, but also the different conditions in which crocins are produce in saffron and 486
N. benthamiana, which implies respectively the absence or presence of light during this 487
process, which can effectively influence the cis- or trans- configuration of crocetin 488
(Rubio-Moraga et al., 2010). A higher bioactivity of the trans- forms has been previously 489
demonstrated, at least for crocetin (Zhang et al., 2017), thus making the crocins pool 490
accumulated in the CsCCD2L and BdCCD4.1 leaves a good source of bioactive 491
molecules. The C. sativus and B. davidii enzymes also generated a different crocins 492
pattern: this finding can be explained by a distinct specificity and kinetics, which might 493
influence the subsequent glucosylation step. Another remarkable observation is the 494
dramatic increase of phytoene and phytofluene levels in N. benthamiana tissues as a 495
consequence of the virus-driven expression of CCDs (Fig. 5A). These are upstream 496
intermediates in the carotenoid biosynthetic pathway. Previous reports indicated that the 497
overexpression of downstream enzymes or the increased storage of end-metabolites in 498
the carotenoid pathway can drain the metabolic flux towards the synthesis and 499
accumulation of the end-products (Diretto et al., 2007; Lopez et al., 2008). In our virus-500
driven system, expression of CsCCD2L and BdCCD4.1 seem to induce the same effect, 501
promoting the activities of the phytoene synthase (PSY) and phytoene desaturase (PDS), 502
which results in a higher accumulation of their metabolic intermediate products phytoene 503
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
16
and phytofluene. In this context, the observed amounts of phytoene and phytofluene 504
might be a consequence of PSY and PDS catalyzing the rate-limiting steps of the pathway 505
in N. benthamiana, as previously described in other species (Maass et al., 2009; Xu et al., 506
2006). Levels of additional plastidic metabolites in the groups of tocochromanols and 507
quinones only showed minor fluctuations, which indicates that perturbations in 508
carotenoid and apocarotenoid biosynthesis do not necessarily affect other related 509
pathways that share common precursors, such as geranylgeranyl diphosphate (GGPP) 510
(Fig. 5C). Our results also indicate that the decrease in the chlorophyll levels do not 511
depend on CCD expression, but on viral infection, since it was also observed in the control 512
tissues infected with TEVΔNIb-aGFP (Fig. 5B). This finding agrees with the effect of 513
plant virus infection on photosynthetic machinery (Li et al., 2016). 514
In our virus-driven experiments, CsCCD2L was more efficient in the production 515
of crocetin dialdehyde and crocins than BdCCD4.1. This suggests that the C. sativus CCD 516
used here (CsCCD2L) is definitively a highly active enzyme, completely compatible with 517
the virus vector and the host plant. Indeed, C. sativus stigma is the main commercial 518
source for these apocarotenoids (Castillo et al., 2005; Rubio Moraga et al., 2013). 519
Apocarotenoids are also present, although at lower levels, in the flowers of Buddleja spp., 520
where they are restricted to the calix. 521
Virus-driven expression of CsCCD2L in adult N. benthamiana plants allowed the 522
accumulation of the notable amounts of 2.18±0.23 mg of crocins and 8.24±2.93 mg of 523
picrocrocin per gram (dry weight) of infected tissues in only 13 dpi (Fig. 7). These 524
amounts are much higher than those obtained with a classic A. tumefaciens-mediated 525
transient expression of the CCD2L enzyme in N. benthamiana leaves, alone or in 526
combination with UGT709G1 (30.5 g/g DW of crocins (unpublished data) and 4.13 g/g 527
DW of picrocrocin (Diretto et al., 2019a), and highlight the opportunity to exploit the 528
viral system for the production of valuable secondary metabolites. A huge range of values 529
has been reported for crocins and picrocrocin in saffron in different studies. Crocins 530
reported values in saffron samples from Spain ranged from 0.85 to 32.40 mg/g dry weight 531
(ALONSO et al., 2001). Other reports showed 24.87 mg/g from China (Tong et al., 2015), 532
26.60 mg/g from Greece (Koulakiotis et al., 2015), 29.00 mg/g from Morocco (Gonda et 533
al., 2012), 32.60 mg/g from Iran, 49.80 mg/g from Italy (Masi et al., 2016) or 89.00 mg/g 534
from Nepal (Li et al., 2018). In Gardenia fruits, crocins levels range from 2.60 to 8.37 535
mg/g (Ouyang et al., 2011; Wu et al., 2014). The picrocrocin content ranges from 7.90 to 536
129.40 mg/g in Spanish saffron. In Greek saffron, 6.70 mg/g were reported (Koulakiotis 537
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
17
et al., 2015), and 10.70-2.16 mg/g in Indian, 21.80-6.15 mg/g in Iranian and 42.20-280.00 538
mg/g in Moroccan saffron (Lage and Cantrell, 2009). The different values obtained in 539
those samples are also a consequence of the different procedures followed to obtain the 540
saffron spice. Most methods involve a dehydration step by heating, which causes the 541
conversion of picrocrocin to safranal. Although lower than those from natural sources, 542
the crocins and picrocrocin contents of N. benthamiana tissues achieved with our virus-543
driven system may still be attractive for commercial purposes because, in contrast to C. 544
sativus stigma or Gardenia spp. fruits, N. benthamiana infected tissues can be quickly 545
and easily obtained in practically unlimited amounts. Moreover, the virus-driven 546
production of crocins and picrocrocin in N. benthamiana may still be optimized by 547
increasing, for instance, the zeaxanthin precursor. PSY over-expression in plants was 548
shown to increase the flux of the whole biosynthetic carotenoid pathway (Nisar et al., 549
2015), as well as the zeaxanthin pool (Lagarde et al., 2000; Römer et al., 2002). In 550
addition, the virus-based cytosolic expression of a bacterial PSY (Pantoea ananatis crtB) 551
also induced general carotenoid accumulation, and particularly increased phytoene levels 552
in tobacco infected tissues (Majer et al., 2017). On the other side, BCH overexpression 553
resulted in increased zeaxanthin and xanthophyll contents, both in microbes and 554
plants (Arango et al., 2014; Du et al., 2010; Lagarde et al., 2000). In this context, we 555
selected PaCrtB and CsBCH2, together with CsLTP1, involved in carotenoid transport, 556
as additional targets to improve apocarotenoid contents in N. benthamiana leaves, which 557
allowed a further increase of 1.9 and 1.6 fold in CsCCD2L/PaCrtB and CsCCD2L/ 558
CsBCH2, respectively, compared to the sole expression of CsCCD2L (Fig. 8). Other 559
targets to improve apocarotenoid accumulation could include the knocking out of 560
enzymatic steps opposing zeaxanthin accumulation, as zeaxanthin epoxydase (ZEP), 561
which uses zeaxanthin as substrate to yield violaxanthin, or lycopene -cyclase (LCY-) 562
that converts lycopene in -carotene, thus diverging the metabolic flux towards the 563
synthesis of (lutein), rather than xanthophylls (as zeaxanthin), which will be 564
object of future attempts. 565
In summary, here we describe a new technology that allows production of 566
remarkable amounts of highly appreciated crocins and picrocrocin in adult N. 567
benthamiana plants in as little as 13 dpi. This achievement is the consequence of N. 568
benthamiana, a species that accumulate negligible amounts of apocarotenoids, only 569
requiring the expression of an appropriate CCD to trigger the apocarotenoid biosynthetic 570
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
18
pathway, and can be further improved through the combination with the overexpression 571
of early or late carotenoid structural genes. 572
573
Acknowledgements 574
575
We thank K. Schreiber and C. Mares (IBMCP, CSIC-UPV, Valencia, Spain) for 576
technical assistance during plant transformation. We thank M. Gascón and M.D. Gómez-577
Jiménez (IBMCP, CSIC-UPV, Valencia, Spain) for helpful assistance with LSCM 578
analyses. We thank D. Dubbala (IBMCP, CSIC-UPV, Valencia, Spain) for English 579
revision. This work was supported by grants BIO2016-77000-R and BIO2017-83184-R 580
from the Spanish Ministerio de Ciencia, Innovación y Universidades (co-financed 581
European Union FEDER funds). M.M. was the recipient of a predoctoral fellowship from 582
the Spanish Ministerio de Educación, Cultura y Deporte (FPU16/05294). G.D. and 583
L.G.G. are participants of the European COST action CA15136 (EUROCAROTEN). 584
L.G.G. is a participant of the CARNET network (BIO2015-71703-REDT and BIO2017‐585
90877‐RED). 586
587
Conflict of interest 588
589
The authors declare no conflict of interest. 590
591
References 592
593
Ahrazem, O., Argandoña, J., Fiore, A., Aguado, C., Luján, R., Rubio-Moraga, Á., 594
Marro, M., Araujo-Andrade, C., Loza-Alvarez, P., Diretto, G., Gómez-Gómez, L., 595
2018. Transcriptome analysis in tissue sectors with contrasting crocins 596
accumulation provides novel insights into apocarotenoid biosynthesis and 597
regulation during chromoplast biogenesis. Sci. Rep. 8. 598
https://doi.org/10.1038/s41598-018-21225-z 599
Ahrazem, O., Diretto, G., Argandoña, J., Rubio-Moraga, A., Julve, J.M., Orzáez, D., 600
Granell, A., Gómez-Gómez, L., 2017. Evolutionarily distinct carotenoid cleavage 601
dioxygenases are responsible for crocetin production in Buddleja davidii. J. Exp. 602
Bot. 68, 4663–4677. https://doi.org/10.1093/jxb/erx277 603
Ahrazem, O., Gómez-Gómez, L., Rodrigo, M.J., Avalos, J., Limón, M.C., 2016a. 604
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
19
Carotenoid cleavage oxygenases from microbes and photosynthetic organisms: 605
Features and functions. Int. J. Mol. Sci. https://doi.org/10.3390/ijms17111781 606
Ahrazem, O., Rubio-Moraga, A., Argandoña-Picazo, J., Castillo, R., Gómez-Gómez, L., 607
2016b. Intron retention and rhythmic diel pattern regulation of carotenoid cleavage 608
dioxygenase 2 during crocetin biosynthesis in saffron. Plant Mol. Biol. 91, 355–609
374. https://doi.org/10.1007/s11103-016-0473-8 610
Ahrazem, O., Rubio-Moraga, A., Berman, J., Capell, T., Christou, P., Zhu, C., Gómez-611
Gómez, L., 2016c. The carotenoid cleavage dioxygenase CCD2 catalysing the 612
synthesis of crocetin in spring crocuses and saffron is a plastidial enzyme. New 613
Phytol. 209, 650–663. https://doi.org/10.1111/nph.13609 614
Ahrazem, O., Rubio-Moraga, A., Nebauer, S.G., Molina, R.V., Gómez-Gómez, L., 615
2015. Saffron: Its Phytochemistry, Developmental Processes, and Biotechnological 616
Prospects. J. Agric. Food Chem. 63, 8751–8764. 617
https://doi.org/10.1021/acs.jafc.5b03194 618
ALONSO, G.L., SALINAS, M.R., GARIJO, J., SÁNCHEZ-FERNÁNDEZ, M.A., 619
2001. COMPOSITION OF CROCINS AND PICROCROCIN FROM SPANISH 620
SAFFRON (CROCUS SATIVUS L.). J. Food Qual. 24, 219–233. 621
https://doi.org/10.1111/j.1745-4557.2001.tb00604.x 622
Amin, B., Hosseinzadeh, H., 2012. Evaluation of aqueous and ethanolic extracts of 623
saffron, Crocus sativus L., and its constituents, safranal and crocin in allodynia and 624
hyperalgesia induced by chronic constriction injury model of neuropathic pain in 625
rats. Fitoterapia 83, 888–895. https://doi.org/10.1016/j.fitote.2012.03.022 626
Apel, W., Bock, R., 2009. Enhancement of carotenoid biosynthesis in transplastomic 627
tomatoes by induced lycopene-to-provitamin A conversion. Plant Physiol. 151, 59–628
66. https://doi.org/10.1104/pp.109.140533 629
Arango, J., Jourdan, M., Geoffriau, E., Beyer, P., Welsch, R., 2014. Carotene 630
hydroxylase activity determines the levels of both α-carotene and total carotenoids 631
in orange carrots. Plant Cell 26, 2223–2233. 632
https://doi.org/10.1105/tpc.113.122127 633
Bedoya, L., Martínez, F., Rubio, L., Daròs, J.A., 2010. Simultaneous equimolar 634
expression of multiple proteins in plants from a disarmed potyvirus vector. J. 635
Biotechnol. 150, 268–275. https://doi.org/10.1016/j.jbiotec.2010.08.006 636
Bedoya, L.C., Martínez, F., Orzáez, D., Daròs, J.A., 2012. Visual tracking of plant virus 637
infection and movement using a reporter MYB transcription factor that activates 638
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
20
anthocyanin biosynthesis. Plant Physiol. 158, 1130–1138. 639
https://doi.org/10.1104/pp.111.192922 640
Bukhari, S.I., Manzoor, M., Dhar, M.K., 2018. A comprehensive review of the 641
pharmacological potential of Crocus sativus and its bioactive apocarotenoids. 642
Biomed. Pharmacother. 98, 733–745. https://doi.org/10.1016/j.biopha.2017.12.090 643
Cappelli, G., Giovannini, D., Basso, A.L., Demurtas, O.C., Diretto, G., Santi, C., 644
Girelli, G., Bacchetta, L., Mariani, F., 2018. A Corylus avellana L. extract 645
enhances human macrophage bactericidal response against Staphylococcus aureus 646
by increasing the expression of anti-inflammatory and iron metabolism genes. J. 647
Funct. Foods 45, 499–511. https://doi.org/10.1016/j.jff.2018.04.007 648
Castillo, R., Fernández, J.A., Gómez-Gómez, L., 2005. Implications of carotenoid 649
biosynthetic genes in apocarotenoid formation during the stigma development of 650
Crocus sativus and its closer relatives. Plant Physiol. 139, 674–689. 651
https://doi.org/10.1104/pp.105.067827 652
Chai, F., Wang, Y., Mei, X., Yao, M., Chen, Y., Liu, H., Xiao, W., Yuan, Y., 2017. 653
Heterologous biosynthesis and manipulation of crocetin in Saccharomyces 654
cerevisiae. Microb. Cell Fact. 16, 54. https://doi.org/10.1186/s12934-017-0665-1 655
Cheriyamundath, S., Choudhary, S., Lopus, M., 2018. Safranal Inhibits HeLa Cell 656
Viability by Perturbing the Reassembly Potential of Microtubules. Phytother. Res. 657
32, 170–173. https://doi.org/10.1002/ptr.5938 658
Christodoulou, E., Kadoglou, N.P., Kostomitsopoulos, N., Valsami, G., 2015. Saffron: 659
A natural product with potential pharmaceutical applications. J. Pharm. Pharmacol. 660
67, 1634–1649. https://doi.org/10.1111/jphp.12456 661
Clemente, T., 2006. Nicotiana (Nicotiana tobaccum, Nicotiana benthamiana). Methods 662
Mol. Biol. 343, 143–154. https://doi.org/10.1385/1-59745-130-4:143 663
Côté, F., Cormier, F., Dufresne, C., Willemot, C., 2001. A highly specific 664
glucosyltransferase is involved in the synthesis of crocetin glucosylesters in Crocus 665
sativus cultured cells. J. Plant Physiol. 158, 553–560. https://doi.org/10.1078/0176-666
1617-00305 667
D’Archivio, A.A., Giannitto, A., Maggi, M.A., Ruggieri, F., 2016. Geographical 668
classification of Italian saffron (Crocus sativus L.) based on chemical constituents 669
determined by high-performance liquid-chromatography and by using linear 670
discriminant analysis. Food Chem. 212, 110–116. 671
https://doi.org/10.1016/j.foodchem.2016.05.149 672
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
21
D’Esposito, D., Ferriello, F., Molin, A.D., Diretto, G., Sacco, A., Minio, A., Barone, A., 673
Di Monaco, R., Cavella, S., Tardella, L., Giuliano, G., Delledonne, M., Frusciante, 674
L., Ercolano, M.R., 2017. Unraveling the complexity of transcriptomic, 675
metabolomic and quality environmental response of tomato fruit. BMC Plant Biol. 676
17, 66. https://doi.org/10.1186/s12870-017-1008-4 677
Demmig-Adams, B., Cohu, C.M., Muller, O., Adams, W.W., 2012. Modulation of 678
photosynthetic energy conversion efficiency in nature: From seconds to seasons, 679
in: Photosynthesis Research. pp. 75–88. https://doi.org/10.1007/s11120-012-9761-680
6 681
Demurtas, O.C., Frusciante, S., Ferrante, P., Diretto, G., Azad, N.H., Pietrella, M., 682
Aprea, G., Taddei, A.R., Romano, E., Mi, J., Al-Babili, S., Frigerio, L., Giuliano, 683
G., 2018. Candidate Enzymes for Saffron Crocin Biosynthesis Are Localized in 684
Multiple Cellular Compartments. Plant Physiol. 177, 990–1006. 685
https://doi.org/10.1104/pp.17.01815 686
Diretto, G., Ahrazem, O., Rubio-Moraga, Á., Fiore, A., Sevi, F., Argandoña, J., Gómez-687
Gómez, L., 2019a. UGT709G1: a novel uridine diphosphate glycosyltransferase 688
involved in the biosynthesis of picrocrocin, the precursor of safranal in saffron 689
(Crocus sativus). New Phytol. 224, 725–740. https://doi.org/10.1111/nph.16079 690
Diretto, G., Al-Babili, S., Tavazza, R., Papacchioli, V., Beyer, P., Giuliano, G., 2007. 691
Metabolic engineering of potato carotenoid content through tuber-specific 692
overexpression of a bacterial mini-pathway. PLoS One 2, e350. 693
https://doi.org/10.1371/journal.pone.0000350 694
Diretto, G., Frusciante, S., Fabbri, C., Schauer, N., Busta, L., Wang, Z., Matas, A.J., 695
Fiore, A., Rose, J.K.C., Fernie, A.R., Jetter, R., Mattei, B., Giovannoni, J., 696
Giuliano, G., 2019b. Manipulation of β‐carotene levels in tomato fruits results in 697
increased ABA content and extended shelf‐life. Plant Biotechnol. J. pbi.13283. 698
https://doi.org/10.1111/pbi.13283 699
Du, H., Wang, N., Cui, F., Li, X., Xiao, J., Xiong, L., 2010. Characterization of the β-700
carotene hydroxylase gene DSM2 conferring drought and oxidative stress 701
resistance by increasing xanthophylls and abscisic acid synthesis in rice. Plant 702
Physiol. 154, 1304–1318. https://doi.org/10.1104/pp.110.163741 703
Eroglu, A., Harrison, E.H., 2013. Carotenoid metabolism in mammals, including man: 704
Formation, occurrence, and function of apocarotenoids. J. Lipid Res. 705
https://doi.org/10.1194/jlr.R039537 706
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
22
Farré, G., Blancquaert, D., Capell, T., Van Der Straeten, D., Christou, P., Zhu, C., 2014. 707
Engineering complex metabolic pathways in plants. Annu. Rev. Plant Biol. 65, 708
187–223. https://doi.org/10.1146/annurev-arplant-050213-035825 709
Fasano, C., Diretto, G., Aversano, R., D’Agostino, N., Di Matteo, A., Frusciante, L., 710
Giuliano, G., Carputo, D., 2016. Transcriptome and metabolome of synthetic 711
Solanum autotetraploids reveal key genomic stress events following 712
polyploidization. New Phytol. 210, 1382–94. https://doi.org/10.1111/nph.13878 713
Fiedor, J., Burda, K., 2014. Potential role of carotenoids as antioxidants in human health 714
and disease. Nutrients 6, 466–488. https://doi.org/10.3390/nu6020466 715
Finley, J.W., Gao, S., 2017. A Perspective on Crocus sativus L. (Saffron) Constituent 716
Crocin: A Potent Water-Soluble Antioxidant and Potential Therapy for 717
Alzheimer’s Disease. J. Agric. Food Chem. 65, 1005–1020. 718
https://doi.org/10.1021/acs.jafc.6b04398 719
Fraser, P.D., Bramley, P.M., 2004. The biosynthesis and nutritional uses of carotenoids. 720
Prog. Lipid Res. 43, 228–265. https://doi.org/10.1016/j.plipres.2003.10.002 721
Frusciante, S., Diretto, G., Bruno, M., Ferrante, P., Pietrella, M., Prado-Cabrero, A., 722
Rubio-Moraga, A., Beyer, P., Gomez-Gomez, L., Al-Babili, S., Giuliano, G., 2014. 723
Novel carotenoid cleavage dioxygenase catalyzes the first dedicated step in saffron 724
crocin biosynthesis. Proc. Natl. Acad. Sci. U. S. A. 111, 12246–12251. 725
https://doi.org/10.1073/pnas.1404629111 726
Georgiadou, G., Tarantilis, P.A., Pitsikas, N., 2012. Effects of the active constituents of 727
Crocus Sativus L., crocins, in an animal model of obsessive-compulsive disorder. 728
Neurosci. Lett. 528, 27–30. https://doi.org/10.1016/j.neulet.2012.08.081 729
Gibson, D.G., Young, L., Chuang, R.Y., Venter, J.C., Hutchison 3rd, C.A., Smith, 730
H.O., 2009. Enzymatic assembly of DNA molecules up to several hundred 731
kilobases. Nat. Methods 6, 343–345. https://doi.org/10.1038/nmeth.1318 732
Giorio, G., Stigliani, A.L., D’Ambrosio, C., 2007. Agronomic performance and 733
transcriptional analysis of carotenoid biosynthesis in fruits of transgenic HighCaro 734
and control tomato lines under field conditions. Transgenic Res. 16, 15–28. 735
https://doi.org/10.1007/s11248-006-9025-3 736
Gómez-Gómez, L., Feo-Brito, F., Rubio-Moraga, A., Galindo, P.A., Prieto, A., 737
Ahrazem, O., 2010. Involvement of lipid transfer proteins in saffron 738
hypersensitivity: Molecular cloning of the potential allergens. J. Investig. Allergol. 739
Clin. Immunol. 20, 407–412. 740
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
23
Gómez-Gómez, L., Pacios, L.F., Diaz-Perales, A., Garrido-Arandia, M., Argandoña, J., 741
Rubio-Moraga, Á., Ahrazem, O., 2018. Expression and interaction analysis among 742
saffron ALDHs and crocetin dialdehyde. Int. J. Mol. Sci. 19. 743
https://doi.org/10.3390/ijms19051409 744
Gómez-Gómez, L., Parra-Vega, V., Rivas-Sendra, A., Seguí-Simarro, J.M., Molina, 745
R.V., Pallotti, C., Rubio-Moraga, Á., Diretto, G., Prieto, A., Ahrazem, O., 2017. 746
Unraveling massive crocins transport and accumulation through proteome and 747
microscopy tools during the development of saffron stigma. Int. J. Mol. Sci. 18. 748
https://doi.org/10.3390/ijms18010076 749
Gonda, S., Parizsa, P., Surányi, G., Gyémánt, G., Vasas, G., 2012. Quantification of 750
main bioactive metabolites from saffron (Crocus sativus) stigmas by a micellar 751
electrokinetic chromatographic (MEKC) method. J. Pharm. Biomed. Anal. 66, 68–752
74. https://doi.org/10.1016/j.jpba.2012.03.002 753
Grosso, V., Farina, A., Giorgi, D., Nardi, L., Diretto, G., Lucretti, S., 2018. A high-754
throughput flow cytometry system for early screening of in vitro made polyploids 755
in Dendrobium hybrids. Plant Cell. Tissue Organ Cult. 132, 57–70. 756
https://doi.org/10.1007/s11240-017-1310-8 757
Hasunuma, T., Miyazawa, S., Yoshimura, S., Shinzaki, Y., Tomizawa, K., Shindo, K., 758
Choi, S.K., Misawa, N., Miyake, C., 2008. Biosynthesis of astaxanthin in tobacco 759
leaves by transplastomic engineering. Plant J. 55, 857–868. 760
https://doi.org/10.1111/j.1365-313X.2008.03559.x 761
Hellens, R.P., Edwards, E.A., Leyland, N.R., Bean, S., Mullineaux, P.M., 2000. pGreen: 762
a versatile and flexible binary Ti vector for Agrobacterium -mediated plant 763
transformation. Plant Mol.Biol. 42, 819–832. 764
Jabini, R., Ehtesham-Gharaee, M., Dalirsani, Z., Mosaffa, F., Delavarian, Z., Behravan, 765
J., 2017. Evaluation of the Cytotoxic Activity of Crocin and Safranal, Constituents 766
of Saffron, in Oral Squamous Cell Carcinoma (KB Cell Line). Nutr. Cancer 69, 767
911–919. https://doi.org/10.1080/01635581.2017.1339816 768
Jia, K.P., Baz, L., Al-Babili, S., 2018. From carotenoids to strigolactones. J. Exp. Bot. 769
https://doi.org/10.1093/jxb/erx476 770
Koulakiotis, N.S., Gikas, E., Iatrou, G., Lamari, F.N., Tsarbopoulos, A., 2015. 771
Quantitation of crocins and picrocrocin in saffron by hplc: Application to quality 772
control and phytochemical differentiation from other crocus taxa. Planta Med. 81, 773
606–612. https://doi.org/10.1055/s-0035-1545873 774
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
24
Kyriakoudi, A., O’Callaghan, Y.C., Galvin, K., Tsimidou, M.Z., O’Brien, N.M., 2015. 775
Cellular Transport and Bioactivity of a Major Saffron Apocarotenoid, Picrocrocin 776
(4-(beta-D-Glucopyranosyloxy)-2,6,6-trimethyl-1-cyclohexene-1-carboxaldehyde). 777
J. Agric. Food Chem. 63, 8662–8668. https://doi.org/10.1021/acs.jafc.5b03363 778
Lagarde, D., Beuf, L., Vermaas, W., 2000. Increased production of zeaxanthin and other 779
pigments by application of genetic engineering techniques to Synechocystis sp. 780
strain PCC 6803. Appl. Environ. Microbiol. 66, 64–72. 781
https://doi.org/10.1128/AEM.66.1.64-72.2000 782
Lage, M., Cantrell, C.L., 2009. Quantification of saffron (Crocus sativus L.) metabolites 783
crocins, picrocrocin and safranal for quality determination of the spice grown 784
under different environmental Moroccan conditions. Sci. Hortic. (Amsterdam). 785
121, 366–373. https://doi.org/10.1016/j.scienta.2009.02.017 786
Li, S., Shao, Q., Lu, Z., Duan, C., Yi, H., Su, L., 2018. Rapid determination of crocins 787
in saffron by near-infrared spectroscopy combined with chemometric techniques. 788
Spectrochim. Acta - Part A Mol. Biomol. Spectrosc. 789
https://doi.org/10.1016/j.saa.2017.09.030 790
Li, Y., Cui, H., Cui, X., Wang, A., 2016. The altered photosynthetic machinery during 791
compatible virus infection. Curr. Opin. Virol. 17, 19–24. 792
https://doi.org/10.1016/j.coviro.2015.11.002 793
Liao, Y.H., Houghton, P.J., Hoult, J.R.S., 1999. Novel and known constituents from 794
Buddleja species and their activity against leukocyte eicosanoid generation. J. Nat. 795
Prod. 62, 1241–1245. https://doi.org/10.1021/np990092+ 796
Lopez, A.B., Van Eck, J., Conlin, B.J., Paolillo, D.J., O’Neill, J., Li, L., 2008. Effect of 797
the cauliflower or transgene on carotenoid accumulation and chromoplast 798
formation in transgenic potato tubers. J. Exp. Bot. 59, 213–223. 799
https://doi.org/10.1093/jxb/erm299 800
Lopresti, A.L., Drummond, P.D., 2014. Saffron (Crocus sativus) for depression: A 801
systematic review of clinical studies and examination of underlying antidepressant 802
mechanisms of action. Hum. Psychopharmacol. https://doi.org/10.1002/hup.2434 803
Maass, D., Arango, J., Wüst, F., Beyer, P., Welsch, R., 2009. Carotenoid crystal 804
formation in arabidopsis and carrot roots caused by increased phytoene synthase 805
protein levels. PLoS One 4, e6373. https://doi.org/10.1371/journal.pone.0006373 806
Majer, E., Llorente, B., Rodríguez-Concepción, M., Daròs, J.A., 2017. Rewiring 807
carotenoid biosynthesis in plants using a viral vector. Sci. Rep. 7. 808
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
25
https://doi.org/10.1038/srep41645 809
Majer, E., Navarro, J.A., Daròs, J.A., 2015. A potyvirus vector efficiently targets 810
recombinant proteins to chloroplasts, mitochondria and nuclei in plant cells when 811
expressed at the amino terminus of the polyprotein. Biotechnol. J. 10, 1792–1802. 812
https://doi.org/10.1002/biot.201500042 813
Martin, C., Li, J., 2017. Medicine is not health care, food is health care: plant metabolic 814
engineering, diet and human health. New Phytol. 216, 699–719. 815
https://doi.org/10.1111/nph.14730 816
Masi, E., Taiti, C., Heimler, D., Vignolini, P., Romani, A., Mancuso, S., 2016. PTR-817
TOF-MS and HPLC analysis in the characterization of saffron (Crocus sativus L.) 818
from Italy and Iran. Food Chem. 192, 75–81. 819
https://doi.org/10.1016/j.foodchem.2015.06.090 820
Mazidi, M., Shemshian, M., Mousavi, S.H., Norouzy, A., Kermani, T., Moghiman, T., 821
Sadeghi, A., Mokhber, N., Ghayour-Mobarhan, M., Ferns, G.A.A., 2016. A 822
double-blind, randomized and placebo-controlled trial of Saffron (Crocus sativus 823
L.) in the treatment of anxiety and depression. J. Complement. Integr. Med. 13, 824
195–199. https://doi.org/10.1515/jcim-2015-0043 825
Moraga, A.R., Nohales, P.F., Pérez, J.A., Gómez-Gómez, L., 2004. Glucosylation of the 826
saffron apocarotenoid crocetin by a glucosyltransferase isolated from Crocus 827
sativus stigmas. Planta 219, 955–966. https://doi.org/10.1007/s00425-004-1299-1 828
Moraga, Á.R., Rambla, J.L., Ahrazem, O., Granell, A., Gómez-Gómez, L., 2009. 829
Metabolite and target transcript analyses during Crocus sativus stigma 830
development. Phytochemistry 70, 1009–1016. 831
https://doi.org/10.1016/j.phytochem.2009.04.022 832
Moras, B., Loffredo, L., Rey, S., 2018. Quality assessment of saffron (Crocus sativus 833
L.) extracts via UHPLC-DAD-MS analysis and detection of adulteration using 834
gardenia fruit extract (Gardenia jasminoides Ellis). Food Chem. 257, 325–332. 835
https://doi.org/10.1016/j.foodchem.2018.03.025 836
Nagatoshi, M., Terasaka, K., Owaki, M., Sota, M., Inukai, T., Nagatsu, A., Mizukami, 837
H., 2012. UGT75L6 and UGT94E5 mediate sequential glucosylation of crocetin to 838
crocin in Gardenia jasminoides. FEBS Lett. 586, 1055–1061. 839
https://doi.org/10.1016/j.febslet.2012.03.003 840
Nam, K.N., Park, Y.M., Jung, H.J., Lee, J.Y., Min, B.D., Park, S.U., Jung, W.S., Cho, 841
K.H., Park, J.H., Kang, I., Hong, J.W., Lee, E.H., 2010. Anti-inflammatory effects 842
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
26
of crocin and crocetin in rat brain microglial cells. Eur. J. Pharmacol. 648, 110–843
116. https://doi.org/10.1016/j.ejphar.2010.09.003 844
Nisar, N., Li, L., Lu, S., Khin, N.C., Pogson, B.J., 2015. Carotenoid metabolism in 845
plants. Mol. Plant 8, 68–82. https://doi.org/10.1016/j.molp.2014.12.007 846
Nogueira, M., Enfissi, E.M.A., Welsch, R., Beyer, P., Zurbriggen, M.D., Fraser, P.D., 847
2019. Construction of a fusion enzyme for astaxanthin formation and its 848
characterisation in microbial and plant hosts: A new tool for engineering 849
ketocarotenoids. Metab. Eng. 52, 243–252. 850
https://doi.org/10.1016/j.ymben.2018.12.006 851
Ouyang, E., Zhang, C., Li, X., 2011. Simultaneous determination of geniposide, 852
chlorogenic acid, crocin1, and rutin in crude and processed fructus gardeniae 853
extracts by high performance liquid chromatography. Pharmacogn. Mag. 7, 267–854
270. https://doi.org/10.4103/0973-1296.90391 855
Pfister, S., Meyer, P., Steck, A., Pfander, H., 1996. Isolation and Structure Elucidation 856
of Carotenoid-Glycosyl Esters in Gardenia Fruits (Gardenia jasminoides Ellis) and 857
Saffron (Crocus sativus Linne). J. Agric. Food Chem. 44, 2612–2615. 858
https://doi.org/10.1021/jf950713e 859
Rambla, J.L., Trapero-Mozos, A., Diretto, G., Moraga, A.R., Granell, A., Gómez, L.G., 860
Ahrazem, O., 2016. Gene-metabolite networks of volatile metabolism in Airen and 861
Tempranillo grape cultivars revealed a distinct mechanism of aroma bouquet 862
production. Front. Plant Sci. 7. https://doi.org/10.3389/fpls.2016.01619 863
Richardson, A.D., Duigan, S.P., Berlyn, G.P., 2002. An evaluation of noninvasive 864
methods to estimate foliar chlorophyll content. New Phytol. 153, 185–194. 865
https://doi.org/10.1046/j.0028-646X.2001.00289.x 866
Rodriguez-Concepcion, M., Avalos, J., Bonet, M.L., Boronat, A., Gomez-Gomez, L., 867
Hornero-Mendez, D., Limon, M.C., Meléndez-Martínez, A.J., Olmedilla-Alonso, 868
B., Palou, A., Ribot, J., Rodrigo, M.J., Zacarias, L., Zhu, C., 2018. A global 869
perspective on carotenoids: Metabolism, biotechnology, and benefits for nutrition 870
and health. Prog. Lipid Res. 70, 62–93. 871
https://doi.org/10.1016/j.plipres.2018.04.004 872
Römer, S., Lübeck, J., Kauder, F., Steiger, S., Adomat, C., Sandmann, G., 2002. 873
Genetic engineering of a zeaxanthin-rich potato by antisense inactivation and co-874
suppression of carotenoid epoxidation. Metab. Eng. 4, 263–272. 875
https://doi.org/10.1006/mben.2002.0234 876
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
27
Rubio-Moraga, A., Trapero, A., Ahrazem, O., Gómez-Gómez, L., 2010. Crocins 877
transport in Crocus sativus: The long road from a senescent stigma to a newborn 878
corm. Phytochemistry 71, 1506–1513. 879
https://doi.org/10.1016/j.phytochem.2010.05.026 880
Rubio Moraga, A., Ahrazem, O., Rambla, J.L., Granell, A., Gómez Gómez, L., 2013. 881
Crocins with High Levels of Sugar Conjugation Contribute to the Yellow Colours 882
of Early-Spring Flowering Crocus Tepals. PLoS One 8. 883
https://doi.org/10.1371/journal.pone.0071946 884
Sainsbury, F., Saxena, P., Geisler, K., Osbourn, A., Lomonossoff, G.P., 2012. Using a 885
virus-derived system to manipulate plant natural product biosynthetic pathways. 886
Methods Enzym. 517, 185–202. https://doi.org/10.1016/B978-0-12-404634-887
4.00009-7 888
Skladnev, N. V., Johnstone, D.M., 2017. Neuroprotective properties of dietary saffron: 889
More than just a chemical scavenger? Neural Regen. Res. 890
https://doi.org/10.4103/1673-5374.198976 891
Sulli, M., Mandolino, G., Sturaro, M., Onofri, C., Diretto, G., Parisi, B., Giuliano, G., 892
2017. Molecular and biochemical characterization of a potato collection with 893
contrasting tuber carotenoid content. PLoS One 12. 894
https://doi.org/10.1371/journal.pone.0184143 895
Tan, H., Chen, X., Liang, N., Chen, R., Chen, J., Hu, C., Li, Qi, Li, Qing, Pei, W., Xiao, 896
W., Yuan, Y., Chen, W., Zhang, L., 2019. Transcriptome analysis reveals novel 897
enzymes for apo-carotenoid biosynthesis in saffron and allows construction of a 898
pathway for crocetin synthesis in yeast. J. Exp. Bot. 70, 4819–4834. 899
https://doi.org/10.1093/jxb/erz211 900
Tarantilis, P.A., Tsoupras, G., Polissiou, M., 1995. Determination of saffron (Crocus 901
sativus L.) components in crude plant extract using high-performance liquid 902
chromatography-UV-visible photodiode-array detection-mass spectrometry. J. 903
Chromatogr. A 699, 107–118. https://doi.org/10.1016/0021-9673(95)00044-N 904
Thole, V., Worland, B., Snape, J.W., Vain, P., 2007. The pCLEAN dual binary vector 905
system for Agrobacterium-mediated plant transformation. Plant Physiol. 145, 906
1211–1219. 907
Ting, H.M., Wang, B., Rydén, A.M., Woittiez, L., Van Herpen, T., Verstappen, F.W.A., 908
Ruyter-Spira, C., Beekwilder, J., Bouwmeester, H.J., Van der Krol, A., 2013. The 909
metabolite chemotype of Nicotiana benthamiana transiently expressing artemisinin 910
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
28
biosynthetic pathway genes is a function of CYP71AV1 type and relative gene 911
dosage. New Phytol. 199, 352–366. https://doi.org/10.1111/nph.12274 912
Walter, M.H., Floss, D.S., Strack, D., 2010. Apocarotenoids: Hormones, mycorrhizal 913
metabolites and aroma volatiles. Planta. https://doi.org/10.1007/s00425-010-1156-914
3 915
Wang, B., Kashkooli, A.B., Sallets, A., Ting, H.M., de Ruijter, N.C.A., Olofsson, L., 916
Brodelius, P., Pottier, M., Boutry, M., Bouwmeester, H., van der Krol, A.R., 2016. 917
Transient production of artemisinin in Nicotiana benthamiana is boosted by a 918
specific lipid transfer protein from A. annua. Metab. Eng. 38, 159–169. 919
https://doi.org/10.1016/j.ymben.2016.07.004 920
Wang, W., He, P., Zhao, D., Ye, L., Dai, L., Zhang, X., Sun, Y., Zheng, J., Bi, C., 2019. 921
Construction of Escherichia coli cell factories for crocin biosynthesis. Microb. Cell 922
Fact. 18. https://doi.org/10.1186/s12934-019-1166-1 923
Wu, X., Zhou, Y., Yin, F., Mao, C., Li, L., Cai, B., Lu, T., 2014. Quality control and 924
producing areas differentiation of Gardeniae Fructus for eight bioactive 925
constituents by HPLC-DAD-ESI/MS. Phytomedicine 21, 551–559. 926
https://doi.org/10.1016/j.phymed.2013.10.002 927
Xu, C.J., Fraser, P.D., Wang, W.J., Bramley, P.M., 2006. Differences in the carotenoid 928
content of ordinary citrus and lycopene-accumulating mutants. J. Agric. Food 929
Chem. 54, 5474–5481. https://doi.org/10.1021/jf060702t 930
Xu, H., Lybrand, D., Bennewitz, S., Tissier, A., Last, R.L., Pichersky, E., 2018. 931
Production of trans-chrysanthemic acid, the monoterpene acid moiety of natural 932
pyrethrin insecticides, in tomato fruit. Metab. Eng. 47, 271–278. 933
https://doi.org/10.1016/j.ymben.2018.04.004 934
Yang, T., Stoopen, G., Yalpani, N., Vervoort, J., de Vos, R., Voster, A., Verstappen, 935
F.W.A., Bouwmeester, H.J., Jongsma, M.A., 2011. Metabolic engineering of 936
geranic acid in maize to achieve fungal resistance is compromised by novel 937
glycosylation patterns. Metab. Eng. 13, 414–425. 938
https://doi.org/10.1016/j.ymben.2011.01.011 939
Yuan, L., Grotewold, E., 2015. Metabolic engineering to enhance the value of plants as 940
green factories. Metab. Eng. 27, 83–91. 941
https://doi.org/10.1016/j.ymben.2014.11.005 942
Zhang, C., Ma, J., Fan, L., Zou, Y., Dang, X., Wang, K., Song, J., 2015. 943
Neuroprotective effects of safranal in a rat model of traumatic injury to the spinal 944
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
29
cord by anti-apoptotic, anti-inflammatory and edema-attenuating. Tissue Cell 47, 945
291–300. https://doi.org/10.1016/j.tice.2015.03.007 946
Zhang, Y., Fei, F., Zhen, L., Zhu, X., Wang, J., Li, S., Geng, J., Sun, R., Yu, X., Chen, 947
T., Feng, S., Wang, P., Yang, N., Zhu, Y., Huang, J., Zhao, Y., Aa, J., Wang, G., 948
2017. Sensitive analysis and simultaneous assessment of pharmacokinetic 949
properties of crocin and crocetin after oral administration in rats. J. Chromatogr. B. 950
Analyt. Technol. Biomed. Life Sci. 1044–1045, 1–7. 951
https://doi.org/10.1016/j.jchromb.2016.12.003 952
Zhu, Q., Zeng, D., Yu, S., Cui, C., Li, J., Li, H., Chen, J., Zhang, R., Zhao, X., Chen, L., 953
Liu, Y.G., 2018. From Golden Rice to aSTARice: Bioengineering Astaxanthin 954
Biosynthesis in Rice Endosperm. Mol. Plant 11, 1440–1448. 955
https://doi.org/10.1016/j.molp.2018.09.007 956
957
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
30
Table 1. Ratios of (A) crocins- and (B) picrocrocin-related apocarotenoid levels in tissues 958
infected with TEVΔNIb-CsCCD2L and TEVΔNIb-BdCCD4.1. 959
960
A CsCCD2L/BdCCD4.1
crocetin dialdehyde 3.206
cis-crocetin 1.139
trans-crocetin 1.113
cis-crocin 1 3.318
trans-crocin 1 1.755
trans-crocin 2 3.605
trans-crocin 2' 2.420
cis-crocin 3 2.137
trans-crocin 3 4.915
trans-crocin 4 5.580
cis-crocin 5 2.448
trans-crocin 5 -
B CsCCD2L/BdCCD4.1
HTCC 1.573
picrocrocin isomer1 4.237
picrocrocin isomer2 1.405
picrocrocin-derivivative4 8.577
picrocrocin-derivivative7 6.474
safranal 3.805
961
962
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
31
Figures 963
964
965
Fig. 1. Schematic overview of the crocins biosynthesis pathway in C. sativus and B. 966
davidii. CrtB, phytoene synthase; PDS, phytoene desaturase; BCH2, carotene 967
hydroxylase; CsCCD2L, C. sativus carotenoid cleavage dioxygenase 2L; BdCCD4.1 and 968
4.3, B. davidii carotenoid cleavage dioxygenase 4.1 and 4.3; ALDH, aldehyde 969
dehydrogenase; UGT74AD1, UDP-glucosyltransferase 74AD1. 970
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
32
971
972
Fig. 2. Inoculation of N. benthamiana plants that stably express NIb with TEVΔNIb 973
recombinant clones that express different CCDs. (A) Schematic representation of 974
TEVΔNIb genome indicating the position where the GFP (green box) and the different 975
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
33
CCDs (CsCCD2L, orange box; BdCCD4.1, yellow box; and BdCCD4.3, gray box) were 976
inserted. The sequence of the artificial NIaPro cleavage site to mediate the release of the 977
recombinant proteins from the viral polyprotein is also indicated. The black triangle 978
indicates the exact cleavage site. Lines represent TEV 5’ and 3’ UTR and boxes represent 979
P1, HC-Pro, P3, P3N-PIPO, 6K1, CI, 6K2, VPg, NIaPro and CP cistrons, as indicated. 980
Scale bar corresponds to 1000 nt. (B) Pictures of representative leaves from plants mock-981
inoculated and agroinoculated with TEVΔNIb-aGFP, TEVΔNIb-CsCCD2L, TEVΔNIb-982
BdCCD4.1 and TEVΔNIb-BdCCD4.3, as indicated, taken at 13 dpi. Scale bars 983
correspond to 5 mm. (C) Virus diagnosis and (D) analysis of the progeny of recombinant 984
TEV in N. benthamiana plants mock-inoculated and agroinoculated with TEVΔNIb-985
aGFP, TEVΔNIb-CsCCD2L, -BdCCD4.1 and -BdCCD4.3. RNA was extracted at 15 dpi 986
and subjected to RT-PCR amplification. PCR products were separated in a 1% agarose 987
gel that was stained with ethidium bromide. Representative samples of triplicate analyses 988
are shown. (C and D) Lanes 0, DNA marker ladder with sizes (in bp) on the left; lanes 1, 989
RT-PCR controls with no RNA added; lanes 2, mock-inoculated plants; lanes 3 to 6, 990
plants inoculated with TEVΔNIb-aGFP (lanes 3), TEVΔNIb-CsCCD2L (lanes 4), 991
TEVΔNIb -BdCCD4.1 (lanes 4) and TEVΔNIb -BdCCD4.3 (lanes 5). The arrows point 992
to the bands corresponding to (C) the TEV CP, and (D) the full-length viral progenies 993
cDNAs. 994
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
34
995
996
Fig. 3. Heterologous production of crocins in N. benthamiana tissues infected with 997
recombinant viruses expressing CCD enzymes. (A) Chromatographic profile run on an 998
HPLC-PDA-HRMS and detected at 440 nm of the polar fraction of tissues infected with 999
TEVΔNIb-aGFP, TEVΔNIb-CsCCD2L and TEVΔNIb-BdCCD4.1. The profile of 1000
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
35
saffron stigma is also included as a control. Peaks abbreviations correspond to: c-cr, cis-1001
crocetin; t-cr, trans-crocetin; c-cr1, cis-crocin 1; t-cr1, trans-crocin 1; t-cr2, trans-crocin 1002
2; t-cr2ʹ, trans-crocin 2ʹ; c-cr3, cis-crocin 3, t-cr3, trans-crocin 3; c-cr4, cis-crocin 4; t-1003
cr4, trans-crocin 4; c-cr5, cis-crocin 5; t-cr5, trans-crocetin 5. (B) Absorbance spectra of 1004
the major crocins detected in the polar extracts of N. benthamiana tissues infected with 1005
TEVΔNIb-CsCCD2L and TEVΔNIb-BdCCD4.1. Analyses were performed at 13 dpi. 1006
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
36
1007
Fig. 4. Apocarotenoid content and composition of N. benthamiana tissues from mock-1008
inoculated plants and plants infected with TEVΔNIb-aGFP, TEVΔNIb-CsCCD2L and 1009
TEVΔNIb-BdCCD4.1. (A) Crocetin dialdehyde, crocetin and crocins accumulation. (B) 1010
Levels of safranal, picrocrocin and its derivatives, and HTCC. Data are averages ± sd of 1011
three biological replicates and expressed as % of fold internal standard (IS) levels. 1012
Analyses were performed at 13 dpi. 1013
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
37
1014
Fig. 5. Relative quantities of (A) carotenoids (B) chlorophylls and (C) quinones and 1015
tocochromanols detected by HPLC-DAD-HRMS in tissues of mock-inoculated and 1016
infected (TEVΔNIb-aGFP, TEVΔNIb-CsCCD2L and TEVΔNIb-BdCCD4.1) N. 1017
benthamiana plants. Data are averages ± sd of three biological replicates and expressed 1018
as fold internal standard (IS). Analyses were performed at 13 dpi. 1019
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
38
1020
1021
Fig. 6. LSCM images of crocins and chlorophyll autofluorescence in symptomatic leaves 1022
of N. benthamiana plants infected with TEVΔNIb, TEVΔNIb-CsCCD2L and TEVΔNIb-1023
BdCCD4.1, as indicated, at 13 dpi. Merged images of both fluorescent signals and images 1024
under white light are also shown. Scale bars indicate 10 μm. 1025
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
39
1026
1027
Fig. 7. Time-course accumulation of (A) crocins, (B) picrocrocin, (C) β-carotene and 1028
lutein, and (D) chlorophyll a and b, as indicated, in systemic tissues of N. benthamiana 1029
plants agroinoculated with TEVΔNIb-CsCCD2L. Data are average ± sd of at least three 1030
biological replicates. 1031
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
40
1032
Fig. 8. Apocarotenoid accumulation in N. benthamiana tissues inoculated with viral 1033
vectors carrying CsCCD2L, alone or in combination with PaCrtB, CsBCH2 and CsLTP1. 1034
(A) Schematic representation of TEVΔNIb-CsCCD2L/PaCrtB, - CsCCD2L/CsBCH2 and 1035
CsCCD2L/CsLPT1. The cDNAs corresponding to CsCCD2L, PaCrtB, CsBCH2 and 1036
CsLPT1 are indicated by orange, yellow, red and purple boxes respective. Other details 1037
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
41
are the same as in Fig. 2A. (B) Representative leaves from plants mock-inoculated and 1038
agroinoculated with TEVΔNIb-aGFP, TEVΔNIb-CsCCD2L, TEVΔNIb-1039
CsCCD2L/PaCrtB and TEVΔNIb-CsCCD2L/CsBCH2, at 13 dpi. (C) Total crocins, 1040
picrocrocin and safranal contents, expressed as mg/g of DW. Asterisks indicate 1041
significant differences according a t-test (pValue: *<0.05; **<0.01; ns, not significant) 1042
carried out in the comparisons CsCCD2L vs CsCCD2L+PaCrtB, CsCCD2L+CsBCH2 or 1043
CsCCD2L+CsLTP1. (D) Relative accumulation of single crocins, picrocrocin and 1044
safranal, normalized on the CsCCD2L-alone sample and visualized as heatmap of log2 1045
values. Data are average ± sd of three biological replicates. Analyses were performed at 1046
13 dpi. 1047
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
42
SUPPLEMENTARY FIGURES 1048
1049 Fig. S1. Nucleotide sequence of the cDNA transferred to the Nicotiana benthamiana 1050 transformed line using Agrobacterium tumefaciens to prepare the virus-drive expression 1051
system. 1052 1053 TGGCAGGATATATTGTGGTGTAAACGTTCCTGCGGCGGTCGAGATGGATCTTGGCAGGATATATTGTGGT1054 GTAAACGTTCCTGCGGCCGCAAGGGCTATTGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCG1055 GATTCCATTGCCCAGCTATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATG1056 CCATCATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCC1057 CCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTG1058 ATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCTTCCTCTATATAAGGA1059 AGTTCATTTCATTTGGAGAGGACACGCTGAAATCACCAGTCTCTCTCTACAAATCTATCTCTCTCGAGCT1060 TTCGCAGATCTGTCGATCGACCATGGGGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGG1061 GTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGC1062 TGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTCCAGGA1063 CGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACT1064 GAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTC1065 CTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCC1066 ATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAG1067 GATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGC1068 CCGACGGCGAGGATCTCGTCGTGACACATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCG1069 CTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACC1070 CGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTC1071 CCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGGA1072 TCGATCCTCTAGCTAGAGTCGATCGACAAGCTCGAGTTTCTCCATAATAATGTGTGAGTAGTTCCCAGAT1073 AAGGGAATTAGGGTTCCTATAGGGTTTCGCTCATGTGTTGAGCATATAAGAAACCCTTAGTATGTATTTG1074 TATTTGTAAAATACTTCTATCAATAAAATTTCTAATTCCTAAAACCAAAATCCAGTACTAAAATCCAGAT1075 CCCCCGAATTAATTCGGCGTTAATTCAGTACATTAGCGGCCGCGATTCCATTGCCCAGCTATCTGTCACT1076 TTATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCCAT1077 CGTTGAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAA1078 GAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATAGATTCCATTGCCCAGCTATCTGTC1079 ACTTTATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGC1080 CATCGTTGAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAA1081 AAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATG1082 ACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGTA1083 TTAAAATCTTAATAGGTTTTGATAAAAGCGAACGTGGGGAAACCCGAACCAAACCTTCTTCTAAACTCTC1084 TCTCATCTCTCTTAAAGCAAACTTCTCTCTTGTCTTTCTTGCGTGAGCGATCTTCAACGTTGTCAGATCG1085 TGCTTCGGCACCAGTACAACGTTTTCTTTCACTGAAGCGAAATCAAAGATCTCTTTGTGGACACGTAGTG1086 CGGCGCCATTAAATAACGTGTACTTGTCCTATTCTTGTCGGTGTGGTCTTGGGAAAAGAAAGCTTGCTGG1087 AGGCTGCTGTTCAGCCCCATACATTACTTGTTACGATTCTGCTGACTTTCGGCGGGTGCAATATCTCTAC1088 TTCTGCTTGACGAGGTATTGTTGCCTGTACTTCTTTCTTCTTCTTCTTGCTGATTGGTTCTATAAGAAAT1089 CTAGTATTTTCTTTGAAACAGAGTTTTCCCGTGGTTTTCGAACTTGGAGAAAGATTGTTAAGCTTCTGTA1090 TATTCTGCCCAAATTTGAAATGGGGGAGAAGAGGAAATGGGTCGTGGAAGCACTGTCAGGGAACTTGAGG1091 CCAGTGGCTGAGTGTCCCAGTCAGTTAGTCACAAAGCATGTGGTTAAAGGAAAGTGTCCCCTCTTTGAGC1092 TCTACTTGCAGTTGAATCCAGAAAAGGAAGCATATTTTAAACCGATGATGGGAGCATATAAGCCAAGTCG1093 ACTTAATAGAGAGGCGTTCCTCAAGGACATTCTAAAATATGCTAGTGAAATTGAGATTGGGAATGTGGAT1094 TGTGACTTGCTGGAGCTTGCAATAAGCATGCTCATCACAAAGCTCAAGGCGTTAGGATTCCCAACTGTGA1095 ACTACATCACTGACCCAGAGGAAATTTTTAGTGCATTGAATATGAAAGCAGCTATGGGAGCACTATACAA1096 AGGCAAGAAGAAAGAAGCTCTCAGCGAGCTCACACTAGATGAGCAGGAGGCAATGCTCAAAGCAAGTTGC1097 CTGCGACTGTATACGGGAAAGCTGGGAATTTGGAATGGCTCATTGAAAGCAGAGTTGCGTCCAATTGAGA1098 AGGTTGAAAACAACAAAACGCGAACTTTCACAGCAGCACCAATAGACACTCTTCTTGCTGGTAAAGTTTG1099 CGTGGATGATTTCAACAATCAATTTTATGATCTCAACATAAAGGCACCATGGACAGTTGGTATGACTAAG1100 TTTTATCAGGGGTGGAATGAATTGATGGAGGCTTTACCAAGTGGGTGGGTGTATTGTGACGCTGATGGTT1101 CGCAATTCGACAGTTCCTTGACTCCATTCCTCATTAATGCTGTATTGAAAGTGCGACTTGCCTTCATGGA1102 GGAATGGGATATTGGTGAGCAAATGCTGCGAAATTTGTACACTGAGATAGTGTATACACCAATCCTCACA1103 CCGGATGGTACTATCATTAAGAAGCATAAAGGCAACAATAGCGGGCAACCTTCAACAGTGGTGGACAACA1104 CACTCATGGTCATTATTGCAATGTTATACACATGTGAGAAGTGTGGAATCAACAAGGAAGAGATTGTGTA1105 TTACGTCAATGGCGATGACCTATTGATTGCCATTCACCCAGATAAAGCTGAGAGGTTGAGTGGATTCAAA1106 GAATCTTTCGGAGAGTTGGGCCTGAAATATGAATTTGACTGCACCACCAGGGACAAGACACAGTTGTGGT1107
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
43
TCATGTCACACAGGGCTTTGGAGAGGGATGGCATGTATATACCAAAGCTAGAAGAAGAAAGGATTGTTTC1108 TATTTTGGAATGGGACAGATCCAAAGAGCCGTCACATAGGCTTGAAGCCATCTGTGCATCAATGATCGAA1109 GCATGGGGTTATGACAAGCTGGTTGAAGAAATCCGCAATTTCTATGCATGGGTTTTGGAACAAGCGCCGT1110 ATTCACAGCTTGCAGAAGAAGGAAAGGCGCCATATCTGGCTGAGACTGCGCTTAAGTTTTTGTACACATC1111 TCAGCACGGAACAAACTCTGAGATAGAAGAGTATTTAAAAGTGTTGTATGATTACGATATTCCAACGACT1112 GAGAATCTTTATTTTCAGTAACTCTGGTTTCATTAAATTTTCTTTAGTTTGAATTTACTGTTATTCGGTG1113 TGCATTTCTATGTTTGGTGAGCGGTTTTCTGTGCTCAGAGTGTGTTTATTTTATGTAATTTAATTTCTTT1114 GTGAGCTCCTGTTTAGCAGGTCGTCCCTTCAGCAAGGACACAAAAAGATTTTAATTTTATTCGCTGAAAT1115 CACCAGTCTCTCTCTACAAATCTATCTCTCTCTATTTTCTCCATAAATAATGTGTGAGTAGTTTCCCGAT1116 AAGGGAAATTAGGGTTCTTATAGGGTTTCGCTCATGTGTTGAGCATATAAGAAACCCTTAGTATGTATTT1117 GTATTTGTAAAATACTTCTATCAATAAAATTTCTAATTCCTAAAACCAAAATCCAGGGGCCCTCGACGTT1118 CCTTGACAGGATATATTGGCGGGTAAACTAAGTCGCTGTATGTGTTTGTTTG 1119 1120
The cDNAs of the Neomycin phosphotransferase II (NPTII) and Tobacco etch virus 1121 (TEV) NIb are in blue and black, respectively. Initiation Met and stop codons are 1122
underlined. Note that an ATG has been added to the 5’ end of NIb cDNA that naturally 1123 lacks this codon because it is produced by proteolytic processing. A. tumefaciens T-DNA 1124
right border (RB) with overdrive (underlined) is in blue on yellow background and 1125
double left border (LB) is in blue on red background. Cauliflower mosaic virus 1126 (CaMV) 35S promoter and terminator are in red and fuchsia, respectively. Note that 1127 NIb is expressed from a partially duplicated version of CaMV 35S promoter. NIb open 1128 reading frame (ORF) is flanked by a modified version of the 5’ and 3’ untranslated 1129
regions (UTRs) of Cowpea mosaic virus (CPMV) RNA-2. 1130 1131
1132
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
44
Fig. S2. Nucleotide sequences of TEVΔNIb (sequence variant DQ986288 that include 1133
the two silent mutations G273A and A1119G, in red, and lacks the whole NIb cistron) 1134 and the derived recombinant viruses TEVΔNIb-aGFP, -CsCCD2L, -BdCCD4.1, -1135 BdCCD4.3, -CsCCD2L/PaCrtB, -CsCCD2L/CsBCH2 and -CsCCD2L/CsLPT1. The 1136
boundaries of viral cistrons are indicated on blue background. Initiation Met and stop 1137 codons are underlined. 1138 1139 >TEVΔNIb (8004 bp) 1140 AAAATAACAAATCTCAACACAACATATACAAAACAAACGAATCTCAAGCAATCAAGCATTCTACTTCTAT1141 TGCAGCAATTTAAATCATTTCTTTTAAAGCAAAAGCAATTTTCTGAAAATTTTCACCATTTACGAACGAT1142 AGCCATGGCACTCATCTTTGGCACAGTCAACGCTAACATCCTGAAGGAAGTGTTCGGTGGAGCTCGTATG1143 GCTTGCGTTACCAGCGCACATATGGCTGGAGCGAATGGAAGCATTTTGAAGAAGGCAGAAGAAACCTCTC1144 GTGCAATCATGCACAAACCAGTGATCTTCGGAGAAGACTACATTACCGAGGCAGACTTGCCTTACACACC1145 ACTCCATTTAGAGGTCGATGCTGAAATGGAGCGGATGTATTATCTTGGTCGTCGCGCGCTCACCCATGGC1146 AAGAGACGCAAAGTTTCTGTGAATAACAAGAGGAACAGGAGAAGGAAAGTGGCCAAAACGTACGTGGGGC1147 GTGATTCCATTGTTGAGAAGATTGTAGTGCCCCACACCGAGAGAAAGGTTGATACCACAGCAGCAGTGGA1148 AGACATTTGCAATGAAGCTACCACTCAACTTGTGCATAATAGTATGCCAAAGCGTAAGAAGCAGAAAAAC1149 TTCTTGCCCGCCACTTCACTAAGTAACGTGTATGCCCAAACTTGGAGCATAGTGCGCAAACGCCATATGC1150 AGGTGGAGATCATTAGCAAGAAGAGCGTCCGAGCGAGGGTCAAGAGATTTGAGGGCTCGGTGCAATTGTT1151 CGCAAGTGTGCGTCACATGTATGGCGAGAGGAAAAGGGTGGACTTACGTATTGACAACTGGCAGCAAGAG1152 ACACTTCTAGACCTTGCTAAAAGATTTAAGAATGAGAGAGTGGATCAATCGAAGCTCACTTTTGGTTCAA1153 GTGGCCTAGTTTTGAGGCAAGGCTCGTACGGACCTGCGCATTGGTATCGACATGGTATGTTCATTGTACG1154 CGGTCGGTCGGATGGGATGTTGGTGGATGCTCGTGCGAAGGTAACGTTCGCTGTTTGTCACTCAATGACA1155 CATTATAGCGACAAATCAATCTCTGAGGCATTCTTCATACCATACTCTAAGAAATTCTTGGAGTTGAGGC1156 CAGATGGAATCTCCCATGAGTGTACAAGAGGAGTATCAGTTGAGCGGTGCGGTGAGGTGGCTGCAATCCT1157 GACACAAGCACTTTCACCGTGTGGTAAGATCACATGCAAACGTTGCATGGTTGAAACACCTGACATTGTT1158 GAGGGTGAGTCGGGAGACAGTGTCACCAACCAAGGTAAGCTCCTAGCAATGCTGAAAGAACAGTATCCAG1159 ATTTCCCAATGGCCGAGAAACTACTCACAAGGTTTTTGCAACAGAAATCACTAGTAAATACAAATTTGAC1160 AGCCTGCGTGAGCGTCAAACAACTCATTGGTGACCGCAAACAAGCTCCATTCACACACGTACTGGCTGTC1161 AGCGAAATTCTGTTTAAAGGCAATAAACTAACAGGGGCCGATCTCGAAGAGGCAAGCACACATATGCTTG1162 AAATAGCAAGGTTCTTGAACAATCGCACTGAAAATATGCGCATTGGCCACCTTGGTTCTTTCAGAAATAA1163 AATCTCATCGAAGGCCCATGTGAATAACGCACTCATGTGTGATAATCAACTTGATCAGAATGGGAATTTT1164 ATTTGGGGACTAAGGGGTGCACACGCAAAGAGGTTTCTTAAAGGATTTTTCACTGAGATTGACCCAAATG1165 AAGGATACGATAAGTATGTTATCAGGAAACATATCAGGGGTAGCAGAAAGCTAGCAATTGGCAATTTGAT1166 AATGTCAACTGACTTCCAGACGCTCAGGCAACAAATTCAAGGCGAAACTATTGAGCGTAAAGAAATTGGG1167 AATCACTGCATTTCAATGCGGAATGGTAATTACGTGTACCCATGTTGTTGTGTTACTCTTGAAGATGGTA1168 AGGCTCAATATTCGGATCTAAAGCATCCAACGAAGAGACATCTGGTCATTGGCAACTCTGGCGATTCAAA1169 GTACCTAGACCTTCCAGTTCTCAATGAAGAGAAAATGTATATAGCTAATGAAGGTTATTGCTACATGAAC1170 ATTTTCTTTGCTCTACTAGTGAATGTCAAGGAAGAGGATGCAAAGGACTTCACCAAGTTTATAAGGGACA1171 CAATTGTTCCAAAGCTTGGAGCGTGGCCAACAATGCAAGATGTTGCAACTGCATGCTACTTACTTTCCAT1172 TCTTTACCCAGATGTCCTGAGTGCTGAATTACCCAGAATTTTGGTTGATCATGACAACAAAACAATGCAT1173 GTTTTGGATTCGTATGGGTCTAGAACGACAGGATACCACATGTTGAAAATGAACACAACATCCCAGCTAA1174 TTGAATTCGTTCATTCAGGTTTGGAATCCGAAATGAAAACTTACAATGTTGGAGGGATGAACCGAGATAT1175 GGTCACACAAGGTGCAATTGAGATGTTGATCAAGTCCATATACAAACCACATCTCATGAAGCAGTTACTT1176 GAGGAGGAGCCATACATAATTGTCCTGGCAATAGTCTCCCCTTCAATTTTAATTGCCATGTACAACTCTG1177 GAACTTTTGAGCAGGCGTTACAAATGTGGTTGCCAAATACAATGAGGTTAGCTAACCTCGCTGCCATCTT1178 GTCAGCCTTGGCGCAAAAGTTAACTTTGGCAGACTTGTTCGTCCAGCAGCGTAATTTGATTAATGAGTAT1179 GCGCAGGTAATTTTGGACAATCTGATTGACGGTGTCAGGGTTAACCATTCGCTATCCCTAGCAATGGAAA1180 TTGTTACTATTAAGCTGGCCACCCAAGAGATGGACATGGCGTTGAGGGAAGGTGGCTATGCTGTGACCTC1181 TGAAAAGGTGCATGAAATGTTGGAAAAAAACTATGTAAAGGCTTTGAAGGATGCATGGGACGAATTAACT1182 TGGTTGGAAAAATTCTCCGCAATCAGGCATTCAAGAAAGCTCTTGAAATTTGGGCGAAAGCCTTTAATCA1183 TGAAAAACACCGTAGATTGCGGCGGACATATAGACTTGTCTGTGAAATCGCTTTTCAAGTTCCACTTGGA1184 ACTCCTGAAGGGAACCATCTCAAGAGCCGTAAATGGTGGTGCAAGAAAGGTAAGAGTAGCGAAGAATGCC1185 ATGACAAAAGGGGTTTTTCTCAAAATCTACAGCATGCTTCCTGACGTCTACAAGTTTATCACAGTCTCGA1186 GTGTCCTTTCCTTGTTGTTGACATTCTTATTTCAAATTGACTGCATGATAAGGGCACACCGAGAGGCGAA1187 GGTTGCTGCACAGTTGCAGAAAGAGAGCGAGTGGGACAATATCATCAATAGAACTTTCCAGTATTCTAAG1188 CTTGAAAATCCTATTGGCTATCGCTCTACAGCGGAGGAAAGACTCCAATCAGAACACCCCGAGGCTTTCG1189 AGTACTACAAGTTTTGCATTGGAAAGGAAGACCTCGTTGAACAGGCAAAACAACCGGAGATAGCATACTT1190 TGAAAAGATTATAGCTTTCATCACACTTGTATTAATGGCTTTTGACGCTGAGCGGAGTGATGGAGTGTTC1191 AAGATACTCAATAAGTTCAAAGGAATACTGAGCTCAACGGAGAGGGAGATCATCTACACGCAGAGTTTGG1192
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
45
ATGATTACGTTACAACCTTTGATGACAATATGACAATCAACCTCGAGTTGAATATGGATGAACTCCACAA1193 GACGAGCCTTCCTGGAGTCACTTTTAAGCAATGGTGGAACAACCAAATCAGCCGAGGCAACGTGAAGCCA1194 CATTATAGAACTGAGGGGCACTTCATGGAGTTTACCAGAGATACTGCGGCATCGGTTGCCAGCGAGATAT1195 CACACTCACCCGCAAGAGATTTTCTTGTGAGAGGTGCTGTTGGATCTGGAAAATCCACAGGACTTCCATA1196 CCATTTATCAAAGAGAGGGAGAGTGTTAATGCTTGAGCCTACCAGACCACTCACAGATAACGTGCACAAG1197 CAACTGAGAAGTGAACCATTTAACTGCTTCCCAACTTTGAGGATGAGAGGGAAGTCAACTTTTGGGTCAT1198 CACCGATTACAGTCATGACTAGTGGATTCGCTTTACACCATTTTGCACGAAACATAGCTGAGGTAAAAAC1199 ATACGATTTTGTCATAATTGATGAATGTCATGTGAATGATGCTTCTGCTATAGCGTTTAGGAATCTACTG1200 TTTGAACATGAATTTGAAGGAAAAGTCCTCAAAGTGTCAGCCACACCACCAGGTAGAGAAGTTGAATTCA1201 CAACTCAGTTTCCCGTGAAACTCAAGATAGAAGAGGCTCTTAGCTTTCAGGAATTTGTAAGTTTACAAGG1202 GACAGGTGCCAACGCCGATGTGATTAGTTGTGGCGACAACATACTAGTATATGTTGCTAGCTACAATGAT1203 GTTGATAGTCTTGGCAAGCTCCTTGTGCAAAAGGGATACAAAGTGTCGAAGATTGATGGAAGAACAATGA1204 AGAGTGGAGGAACTGAAATAATCACTGAAGGTACTTCAGTGAAAAAGCATTTCATAGTCGCAACTAATAT1205 TATTGAGAATGGTGTAACCATTGACATTGATGTAGTTGTGGATTTTGGGACTAAGGTTGTACCAGTTTTG1206 GATGTGGACAATAGAGCGGTGCAGTACAACAAAACTGTGGTGAGTTATGGGGAGCGCATCCAAAGACTCG1207 GTAGAGTTGGGCGACACAAGGAAGGAGTAGCACTTCGAATTGGCCAAACAAATAAAACACTGGTTGAAAT1208 TCCAGAAATGGTTGCCACTGAAGCTGCCTTTCTATGCTTCATGTACAATTTGCCAGTGACAACACAGAGT1209 GTTTCAACCACACTGCTGGAAAATGCCACATTATTACAAGCTAGAACTATGGCACAGTTTGAGCTATCAT1210 ATTTTTACACAATTAATTTTGTGCGATTTGATGGTAGTATGCATCCAGTCATACATGACAAGCTGAAGCG1211 CTTTAAGCTACACACTTGTGAGACATTCCTCAATAAGTTGGCGATCCCAAATAAAGGCTTATCCTCTTGG1212 CTTACGAGTGGAGAGTATAAGCGACTTGGTTACATAGCAGAGGATGCTGGCATAAGAATCCCATTCGTGT1213 GCAAAGAAATTCCAGACTCCTTGCATGAGGAAATTTGGCACATTGTAGTCGCCCATAAAGGTGACTCGGG1214 TATTGGGAGGCTCACTAGCGTACAGGCAGCAAAGGTTGTTTATACTCTGCAAACGGATGTGCACTCAATT1215 GCGAGGACTCTAGCATGCATCAATAGACTCATAGCACATGAACAAATGAAGCAGAGTCATTTTGAAGCCG1216 CAACTGGGAGAGCATTTTCCTTCACAAATTACTCAATACAAAGCATATTTGACACGCTGAAAGCAAATTA1217 TGCTACAAAGCATACGAAAGAAAATATTGCAGTGCTTCAGCAGGCAAAAGATCAATTGCTAGAGTTTTCG1218 AACCTAGCAAAGGATCAAGATGTCACGGGTATCATCCAAGACTTCAATCACCTGGAAACTATCTATCTCC1219 AATCAGATAGCGAAGTGGCTAAGCATCTGAAGCTTAAAAGTCACTGGAATAAAAGCCAAATCACTAGGGA1220 CATCATAATAGCTTTGTCTGTGTTAATTGGTGGTGGATGGATGCTTGCAACGTACTTCAAGGACAAGTTC1221 AATGAACCAGTCTATTTCCAAGGGAAGAAGAATCAGAAGCACAAGCTTAAGATGAGAGAGGCGCGTGGGG1222 CTAGAGGGCAATATGAGGTTGCAGCGGAGCCAGAGGCGCTAGAACATTACTTTGGAAGCGCATATAATAA1223 CAAAGGAAAGCGCAAGGGCACCACGAGAGGAATGGGTGCAAAGTCTCGGAAATTCATAAACATGTATGGG1224 TTTGATCCAACTGATTTTTCATACATTAGGTTTGTGGATCCATTGACAGGTCACACTATTGATGAGTCCA1225 CAAACGCACCTATTGATTTAGTGCAGCATGAGTTTGGAAAGGTTAGAACACGCATGTTAATTGACGATGA1226 GATAGAGCCTCAAAGTCTTAGCACCCACACCACAATCCATGCTTATTTGGTGAATAGTGGCACGAAGAAA1227 GTTCTTAAGGTTGATTTAACACCACACTCGTCGCTACGTGCGAGTGAGAAATCAACAGCAATAATGGGAT1228 TTCCTGAAAGGGAGAATGAATTGCGTCAAACCGGCATGGCAGTGCCAGTGGCTTATGATCAATTGCCACC1229 AAAGAGTGAGGACTTGACGTTTGAAGGAGAAAGCTTGTTTAAGGGACCACGTGATTACAACCCGATATCG1230 AGCACCATTTGTCACTTGACGAATGAATCTGATGGGCACACAACATCGTTGTATGGTATTGGATTTGGTC1231 CCTTCATCATTACAAACAAGCACTTGTTTAGAAGAAATAATGGAACACTGTTGGTCCAATCACTACATGG1232 TGTATTCAAGGTCAAGAACACCACGACTTTGCAACAACACCTCATTGATGGGAGGGACATGATAATTATT1233 CGCATGCCTAAGGATTTCCCACCATTTCCTCAAAAGCTGAAATTTAGAGAGCCACAAAGGGAAGAGCGCA1234 TATGTCTTGTGACAACCAACTTCCAAACTAAGAGCATGTCTAGCATGGTGTCAGACACTAGTTGCACATT1235 CCCTTCATCTGATGGCATATTCTGGAAGCATTGGATTCAAACCAAGGATGGGCAGTGTGGCAGTCCATTA1236 GTATCAACTAGAGATGGGTTCATTGTTGGTATACACTCAGCATCGAATTTCACCAACACAAACAATTATT1237 TCACAAGCGTGCCGAAAAACTTCATGGAATTGTTGACAAATCAGGAGGCGCAGCAGTGGGTTAGTGGTTG1238 GCGATTAAATGCTGACTCAGTATTGTGGGGGGGCCATAAAGTTTTCATGAGCAAACCTGAAGAGCCTTTT1239 CAGCCAGTTAAGGAAGCGACTCAACTCATGAGTGAATTGGTGTACTCGCAAAGTGGCACTGTGGGTGCTG1240 GTGTTGACGCTGGTAAGAAGAAAGATCAAAAGGATGATAAAGTCGCTGAGCAGGCTTCAAAGGATAGGGA1241 TGTTAATGCTGGAACTTCAGGAACATTCTCAGTTCCACGAATAAATGCTATGGCCACAAAACTTCAATAT1242 CCAAGGATGAGGGGAGAGGTGGTTGTAAACTTGAATCACCTTTTAGGATACAAGCCACAGCAAATTGATT1243 TGTCAAATGCTCGAGCCACACATGAGCAGTTTGCCGCGTGGCATCAGGCAGTGATGACAGCCTATGGAGT1244 GAATGAAGAGCAAATGAAAATATTGCTAAATGGATTTATGGTGTGGTGCATAGAAAATGGGACTTCCCCA1245 AATTTGAACGGAACTTGGGTTATGATGGATGGTGAGGAGCAAGTTTCATACCCGCTGAAACCAATGGTTG1246 AAAACGCGCAGCCAACACTGAGGCAAATTATGACACACTTCAGTGACCTGGCTGAAGCGTATATTGAGAT1247 GAGGAATAGGGAGCGACCATACATGCCTAGGTATGGTCTACAGAGAAACATTACAGACATGAGTTTGTCA1248 CGCTATGCGTTCGACTTCTATGAGCTAACTTCAAAAACACCTGTTAGAGCGAGGGAGGCGCATATGCAAA1249 TGAAAGCTGCTGCAGTACGAAACAGTGGAACTAGGTTATTTGGTCTTGATGGCAACGTGGGTACTGCAGA1250 GGAAGACACTGAACGGCACACAGCGCACGATGTGAACCGTAACATGCACACACTATTAGGGGTCCGCCAG1251 TGATAGTTTCTGCGTGTCTTTGCTTTCCGCTTTTAAGCTTATTGTAATATATATGAATAGCTATTCACAG1252 TGGGACTTGGTCTTGTGTTGAATGGTATCTTATATGTTTTAATATGTCTTATTAGTCTCATTACTTAGGC1253
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
46
GAACGACAAAGTGAGGTCACCTCGGTCTAATTCTCCTATGTAGTGCGAGAAAAAAAAAAAAAAAAAAAAA1254 AAAAAAAAAAAAAAAAAAAAAAAA 1255 1256 >TEVΔNIb-aGFP (insert between possitions 144 and 145 of TEVΔNIb; 1257 artificial NIaPro cleavage site is on gray background) 1258 ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAA1259 ACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTT1260 CATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAG1261 TGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACG1262 TCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGG1263 CGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCAC1264 AAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGG1265 TGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACAC1266 CCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAA1267 GACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCA1268 TGGACGAGCTGTACAAGACGACTGAGAATCTTTATTTTCAGGGGGAGAAG 1269 1270 >TEVΔNIb-CsCCD2L (insert between possitions 144 and 145 of TEVΔNIb; 1271 artificial NIaPro cleavage site is on gray background) 1272 ATGGAATCTCCTGCTACTAAATTACCTGCACCTCTGCTGATGTTATCTTCTTCTCCATTCCTTCTCCCTT1273 CTCCTAATAAGAGCTCCTCCATCTTCCTTCCACGTAAATTAGGGCCGCTACCTCCAAAATATTATTATTA1274 CAATTGCTGCCATCCTAAGAGTAGATCAATCTCAGTAGTATCAATGGCAAATAAGGAGGAGGCAGAGACC1275 AGTAAGAAGAAGCCCAAACCATTAAAAGTACTAATTACCAAAGTGGATCCGAAGCCGAGGAAGGGCATGG1276 CATCCGTCGCAGTGGACTTACTCGAGAAGGCCTTTGTATACCTATTGTCCGGAAATTCTGCAGCTGATCG1277 TAGTAGTAGTAGTGGTCGTCGTCGTCGTAAAGAGCATTACTACCTCTCCGGCAATTATGCGCCCGTCGGA1278 CACGAAACCCCGCCCTCCGACCACCTCCCCATTCATGGATCCCTTCCTGAATGCTTGAATGGAGTGTTTC1279 TGAGAGTTGGTCCTAACCCCAAGTTTGCTCCCGTAGCCGGATACAATTGGGTCGATGGAGATGGAATGAT1280 TCATGGATTGCGTATTAAAGATGGAAAAGCAACTTATCTATCTCGATATATTAAAACGTCACGGTTTAAA1281 CAAGAAGAATATTTTGGAAGAGCAAAATTTATGAAGATTGGAGATCTAAGGGGATTGCTTGGGTTCTTTA1282 CGATCTTAATACTAGTACTTCGAACAACATTGAAAGTAATAGACATTTCATATGGAAGAGGGACGGGTAA1283 TACAGCTCTTGTGTATCATAATGGCTTACTATTGGCTCTATCAGAAGAAGATAAACCTTATGTTGTTAAA1284 GTTTTAGAAGATGGAGACTTGCAAACTCTTGGGATATTGGATTATGACAAGAAATTGTCACATCCATTCA1285 CCGCTCATCCAAAGATCGATCCGTTAACTGATGAGATGTTTACCTTTGGATATTCCATCTCGCCTCCGTA1286 TCTTACTTATCGAGTCATTTCCAAGGATGGAGTGATGCAAGATCCAGTGCAAATCTCAATTACATCCCCT1287 ACCATAATGCATGATTTTGCTATTACTGAAAATTATGCCATCTTCATGGACCTGCCCTTGTATTTCCAAC1288 CAGAGGAAATGGTAAAGGGGAAATTTGTCTCTTCATTTCACCCTACAAAAAGAGCTCGTATCGGTGTGCT1289 TCCACGATATGCAAAAGACGAGCATCCAATTCGATGGTTCGATCTTCCAAGTTGCTTCATGACTCATAAT1290 GCAAATGCTTGGGAAGAGAATGATGAAGTTGTGCTATTCACATGTCGCCTTGAGAGTCCTGATCTTGACA1291 TGCTTAGTGGACCTGCGGAAGAAGAGATTGGGAATTCAAAAAGTGAGCTTTACGAAATGAGGTTCAATTT1292 GAAAACTGGAATTACTTCACAAAAGCAACTATCTGTACCTAGTGTTGATTTTCCTCGGATCAACCAAAGT1293 TATACTGGCAGGAAACAGCAATATGTTTATTGTACTCTTGGCAACACCAAGATTAAGGGCATTGTGAAGT1294 TTGATCTGCAAATTGAACCAGAAGCCGGAAAGACAATGCTTGAAGTTGGAGGAAATGTACAAGGCATCTT1295 TGAGTTGGGACCTAGAAGATATGGTTCAGAGGCAATATTTGTGCCATGCCAACCTGGCATCAAATCTGAT1296 GAGGATGACGGTTACTTGATATTCTTTGTACACGACGAAAACAATGGGAAATCTGAGGTCAATGTCATCG1297 ATGCAAAGACAATGTCTGCAGAACCTGTGGCTGTTGTGGAACTTCCAAGCAGGGTTCCATATGGATTCCA1298 TGCCTTGTTTCTGAATGAGGAAGAACTTCAGAAGCACCAAGCAGAGACAACAACCGAAAATTTATATTTC1299 CAGTCTGGAACA 1300 1301 >TEVΔNIb-BdCCD4.1 (insert between possitions 144 and 145 of TEVΔNIb; 1302 artificial NIaPro cleavage site is on gray background) 1303 ATGAACTCCCTTTATTCTTCCTCTTTCCTTTCCAAATTTCCAATGAATGTTTCATCACTTGGAATAATTG1304 ACAAGAACCCTCAAACAAAGATTAATCCCATTTATTCCGTACCAACGTTCCATGCCCGTCTAACCGAAAA1305 ACCCTTTCGCGGAAAGCCTTTATATGAGAGGAGAAGAAATGATGAATCTTTGTATGCAAAAATGTTGAAA1306 TCATTAGATGATTTCATATGCAAGTTCATGAGTGAAGTACCCCTGGACCCCTCCAACGATCCAAAACACA1307 TACTGGTGGGCAACTTTGCACCGGTGGACGAGCTTCCTCCGACTCCATGTGAGGTGGTGGAAGGCTCCCT1308 TCCGATATGTGTGGAGGGTGCATACATCCGCAACGGTCCCAACCCTCCGATCACCCCTCACGGTCCGTAC1309 CATCTCTTTGATGGAGATGGAATGCTTCACTCCATCACAATCTCAAAAGGCAAAGCCACCTTTTGTAGTC1310 GTTATGTTAAGACTTATAAATATTTGGTGGAACAAAACATAGGCTATCCGGTCTTCCCAAGTCCTTTCTC1311 CTCCTTTAATAATGGCCTCTCGGCTTCTATAGCGCGTTTAGTCGTATTAACGGTAGCACGAGTGCTGGTT1312 ACTAGGTGTTTGGATCCTAGACCTAATGGGTTTGGAACGGGGAATACTAGTTTAGGTTTATTTGGCGGGA1313 AACTCTTTGCGCTATGTGAGTCTGATCTTCCTTATGCTATAAAAGTGACATCGGATGGGGATATAATAAC1314
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
47
TCTTGATCGCCATGATTTTTACACCGGTGATCCGTTACTCAGAATGACTGCACACCCAAAGATTGATTTA1315 GAGACTGGACAAGTCTTCGCTTTCAGATGTGACATAACATCTCCATTCTTGACATTTTTCCGAATCGATT1316 CATCTGGAAGAAAAGGCCCCGATTTTCCAATCTTCTCCTTGCACGGACCGTCACTCATTCATGATTTTGC1317 TCTTACCAAACATTATGTCATATTCCAGGATACACAGGTTGAACTTAATCCAATGGAGATAACTAGAGGA1318 AGATCACCAATTGTAGTTGACCATACCAAAGTGCCCCGGCTAGGCATTTTACCACGCAACGCCATGAATG1319 AAACTGAACTGTCGTGGATTGATGTACCAGGATTAAACATGTCACACTTTATTAATGCTTGGGAAGAAGA1320 TGGCGGAAACAAAATAGTAATTGTTGCTTTTAATGTATTATCAGGGTTGGATTCCCTATATTTGTTCAAT1321 TCCTCAATGGAAAAAATTACAATTGACCTCAAGGCAAAGAAGATTGAAAGACATCCATTGTCCACTAAAA1322 ATCTCGAGTTAGGAGTTATTAATCCTGCTTACGCCGGAAAGAAAAACAAGTATGTATATGCGGCAATTAT1323 AAGTCAAACACCAAAGGCAGCAGGGGTGATTAAGCTTGACATATCACAGCTCCCTAATGCTGATAGTAGT1324 AATTGCATGGTGGCTAGCCGACTTTATGGTTCGAGCTGTTATGGTGGTGAACCTTACTTTGTCGCAAGGG1325 AGCCTGATAATCCAGAGGCTGAAGAGGATGATGGTTATCTAGTCACATACATGCATGATGAGAATAGCGA1326 GGAATCGTGGTTCTTGGTTATGAACGCCAAATCCCCAACTCTTGACATTGTTGCTAGTGTTAGATTACCT1327 GGTAGAGTGCCTTATGGCTTCCACGGACTTTTTGTCAGGGAAGGTGACCTCAACAAGCTGACAACCGAAA1328 ATTTATATTTCCAGTCTGGAACA 1329 1330 >TEVΔNIb-BdCCD4.3 (insert between possitions 144 and 145 of TEVΔNIb; 1331 artificial NIaPro cleavage site is on gray background) 1332 ATGGACACCCTTTCTTCCTTTTTCCTTCAAAAATATGTTCCAAGTCATTCACTCACACCATCCCCGCCAT1333 TACTCCTTAAATTCAATATTTCATCTCTTAAAATTGATAAAAAATGCGATAGATTAAATCATCAAAAATC1334 TATGATCACAACATCAATTTTGCATCAAAAAAAGCTTAAAACGTTAAACACTCCGAAAAACCCTTCAACC1335 TCAGAAATTACAAAAATAAAAAAACAATCTTTTCTTACTATTTTCTTCAACTCATTAGATAATTTCATAT1336 GCAATTTCATAGACCCCCCACTCCGCCCCTCCATCGACCCTAACCACGTCCTCTCCGGCAACTTCGCCCC1337 CGTCGGCGAGCTCCCCCCCACCGCGTGCGACGTCGTCGAGGGCTCCCTTCCGCCGTCGTTAAACGGCGCG1338 TACATTCGCAACGGCCCAAACCCTCAGTTCATCCCCCGTGGGCCCTACCACCTATTCGACGGCGATGGCA1339 TGCTCCACGCCGTCACCATCTCCGGCGGCAAAGCCACATTCTGCAGCCGCTTCGTCAAGACTTATAAGTA1340 CACGCTGGAGCGAGAGATCGGCTCGCCGGTTATTCTGAACGTGTTCTCCGCCTTCAACGGCTTGCTGGCT1341 TCGCTGGCGCGCTGCGCCGTATCATCAGGCCGCGTCCTCACCGGACAATACGACCCCCGCCGCGGCGCCG1342 GCCGCGCCAACACAAGCTTAGCGTTGATTAGCGGAAAGTTATTCGCTCACGAAGAATGTGACCTTCCCTA1343 CGGAATAAAATTAACATGTGATGGCGATATAATCACTTTAGGCCGCCATGAATTTCTCGGTGAGCAGTTT1344 ACGGCGATGACGGCGCACCCGAAAATCGACATGGAAACCGGCGAGACCTTTGCTTTCAGATACAGTTTCC1345 TGCCGCCGTTCTTAACATATTTCAGGATCAACGGGGATGGAATTATGCAACACGAAGTGCCGATTTTCTC1346 CTTGAAGCAATCGTCATTCATCCACGACTTCGCAATATCCAAAAACTACGTCGTTTTCCCAGATACGCAG1347 ATTGTGATAAAACCTAGAGATATGTTGAGAGGGAGGGTGCCATTAAGAGTTGATCTTGAAAAAGTGCCGA1348 GGCTTGGGATTATGCCGAGATACGCGGTGGATGAGAGGGAAATGTGGTGGGTTGATGTGTCGGGGTTTAA1349 TATAATACACGCGGTGAATGCGTGGGAGGAGGAAGACGACGGCGGCGACACCATTGTGATGGTGGCGCCG1350 AATGCTTTGGGGGTGGAGCATGTGTTGGAGCGTATGGATTTGGTTCACTCATCCTTGGAAAGAATTCAAA1351 TAAATTTGAAGGAGAAGAGTGTGACGAGACGCCCGGTGTCCACAGCAAATCTCGAGTTTGCGGTCATCAA1352 TCCGGCTTACGTTGGGAAGAAGAACAGGTATGTGTATGCAGCAGTAGGAGATCCAATGCCAAAGATAGTA1353 GGTGTTGTGAAGGTGGACTTATCACTTTCCACGGCCAACTCCGGCGACTGCACGGTGGCTAGACGCCTAT1354 ACGGGCCCGGCTGCTACGGCGGTGAACCATTTTTTGTGGCAAGGGAGCCGGATAATCCGGCGGCGGAGGA1355 GGATGATGGCTATCTAATAACTTATATGCATAATGAAAATACTGAAGAATCAAAGTTTTTAGTAATGGAT1356 GCAAAATCCCCTACTCTTCAAATTCTTGCTGCTGTTAAGCTACCCCAACGAGTTCCCTATGGCTTCCATG1357 GAATCTTTGTGCCGGAATCTCACTTGCGTAAACTAACAACCGAAAATTTATATTTCCAGTCTGGAACA 1358
1359 >TEVΔNIb-CsCCD2L/PaCrtB 1360 (insert between possitions 144 and 145 of TEVΔNIb; artificial NIaPro 1361 cleavage site is on gray background) 1362 ATGGAATCTCCTGCTACTAAATTACCTGCACCTCTGCTGATGTTATCTTCTTCTCCATTCCTTCTCCCTT1363 CTCCTAATAAGAGCTCCTCCATCTTCCTTCCACGTAAATTAGGGCCGCTACCTCCAAAATATTATTATTA1364 CAATTGCTGCCATCCTAAGAGTAGATCAATCTCAGTAGTATCAATGGCAAATAAGGAGGAGGCAGAGACC1365 AGTAAGAAGAAGCCCAAACCATTAAAAGTACTAATTACCAAAGTGGATCCGAAGCCGAGGAAGGGCATGG1366 CATCCGTCGCAGTGGACTTACTCGAGAAGGCCTTTGTATACCTATTGTCCGGAAATTCTGCAGCTGATCG1367 TAGTAGTAGTAGTGGTCGTCGTCGTCGTAAAGAGCATTACTACCTCTCCGGCAATTATGCGCCCGTCGGA1368 CACGAAACCCCGCCCTCCGACCACCTCCCCATTCATGGATCCCTTCCTGAATGCTTGAATGGAGTGTTTC1369 TGAGAGTTGGTCCTAACCCCAAGTTTGCTCCCGTAGCCGGATACAATTGGGTCGATGGAGATGGAATGAT1370 TCATGGATTGCGTATTAAAGATGGAAAAGCAACTTATCTATCTCGATATATTAAAACGTCACGGTTTAAA1371 CAAGAAGAATATTTTGGAAGAGCAAAATTTATGAAGATTGGAGATCTAAGGGGATTGCTTGGGTTCTTTA1372 CGATCTTAATACTAGTACTTCGAACAACATTGAAAGTAATAGACATTTCATATGGAAGAGGGACGGGTAA1373 TACAGCTCTTGTGTATCATAATGGCTTACTATTGGCTCTATCAGAAGAAGATAAACCTTATGTTGTTAAA1374 GTTTTAGAAGATGGAGACTTGCAAACTCTTGGGATATTGGATTATGACAAGAAATTGTCACATCCATTCA1375
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
48
CCGCTCATCCAAAGATCGATCCGTTAACTGATGAGATGTTTACCTTTGGATATTCCATCTCGCCTCCGTA1376 TCTTACTTATCGAGTCATTTCCAAGGATGGAGTGATGCAAGATCCAGTGCAAATCTCAATTACATCCCCT1377 ACCATAATGCATGATTTTGCTATTACTGAAAATTATGCCATCTTCATGGACCTGCCCTTGTATTTCCAAC1378 CAGAGGAAATGGTAAAGGGGAAATTTGTCTCTTCATTTCACCCTACAAAAAGAGCTCGTATCGGTGTGCT1379 TCCACGATATGCAAAAGACGAGCATCCAATTCGATGGTTCGATCTTCCAAGTTGCTTCATGACTCATAAT1380 GCAAATGCTTGGGAAGAGAATGATGAAGTTGTGCTATTCACATGTCGCCTTGAGAGTCCTGATCTTGACA1381 TGCTTAGTGGACCTGCGGAAGAAGAGATTGGGAATTCAAAAAGTGAGCTTTACGAAATGAGGTTCAATTT1382 GAAAACTGGAATTACTTCACAAAAGCAACTATCTGTACCTAGTGTTGATTTTCCTCGGATCAACCAAAGT1383 TATACTGGCAGGAAACAGCAATATGTTTATTGTACTCTTGGCAACACCAAGATTAAGGGCATTGTGAAGT1384 TTGATCTGCAAATTGAACCAGAAGCCGGAAAGACAATGCTTGAAGTTGGAGGAAATGTACAAGGCATCTT1385 TGAGTTGGGACCTAGAAGATATGGTTCAGAGGCAATATTTGTGCCATGCCAACCTGGCATCAAATCTGAT1386 GAGGATGACGGTTACTTGATATTCTTTGTACACGACGAAAACAATGGGAAATCTGAGGTCAATGTCATCG1387 ATGCAAAGACAATGTCTGCAGAACCTGTGGCTGTTGTGGAACTTCCAAGCAGGGTTCCATATGGATTCCA1388 TGCCTTGTTTCTGAATGAGGAAGAACTTCAGAAGCACCAAGCAGAGACAACAACCGAAAATTTATATTTC1389 CAGTCTGGAACA 1390 (insert between possitions 6981 and 6982 of TEVΔNIb; sequences to 1391 complete native NIaPro cleavage sites are on yellow background) 1392 GGGGAGAAGATGAATAATCCGTCGTTACTCAATCATGCGGTCGAAACGATGGCAGTTGGCTCGAAAAGTT1393 TTGCGACAGCCTCAAAGTTATTTGATGCAAAAACCCGGCGCAGCGTACTGATGCTCTACGCCTGGTGCCG1394 CCATTGTGACGATGTTATTGACGATCAGACGCTGGGCTTTCAGGCCCGGCAGCCTGCCTTACAAACGCCC1395 GAACAACGTCTGATGCAACTTGAGATGAAAACGCGCCAGGCCTATGCAGGATCGCAGATGCACGAACCGG1396 CGTTTGCGGCTTTTCAGGAAGTGGCTATGGCTCATGATATCGCCCCGGCTTACGCGTTTGATCATCTGGA1397 AGGCTTCGCCATGGATGTACGCGAAGCGCAATACAGCCAACTGGATGATACGCTGCGCTATTGCTATCAC1398 GTTGCAGGCGTTGTCGGCTTGATGATGGCGCAAATCATGGGCGTGCGGGATAACGCCACGCTGGACCGCG1399 CCTGTGACCTTGGGCTGGCATTTCAGTTGACCAATATTGCTCGCGATATTGTGGACGATGCGCATGCGGG1400 CCGCTGTTATCTGCCGGCAAGCTGGCTGGAGCATGAAGGTCTGAACAAAGAGAATTATGCGGCACCTGAA1401 AACCGTCAGGCGCTGAGCCGTATCGCCCGTCGTTTGGTGCAGGAAGCAGAACCTTACTATTTGTCTGCCA1402 CAGCCGGCCTGGCAGGGTTGCCCCTGCGTTCCGCCTGGGCAATCGCTACGGCGAAGCAGGTTTACCGGAA1403 AATAGGTGTCAAAGTTGAACAGGCCGGTCAGCAAGCCTGGGATCAGCGGCAGTCAACGACCACGCCCGAA1404 AAATTAACGCTGCTGCTGGCCGCCTCTGGTCAGGCCCTTACTTCCCGGATGCGGGCTCATCCTCCCCGCC1405 CTGCGCATCTCTGGCAGCGCCCGCTCACGACTGAGAATCTTTATTTTCAG 1406 1407 >TEVΔNIb-CsCCD2L/CsBCH2 1408 (insert between possitions 144 and 145 of TEVΔNIb; artificial NIaPro 1409 cleavage site is on gray background) 1410 ATGGAATCTCCTGCTACTAAATTACCTGCACCTCTGCTGATGTTATCTTCTTCTCCATTCCTTCTCCCTT1411 CTCCTAATAAGAGCTCCTCCATCTTCCTTCCACGTAAATTAGGGCCGCTACCTCCAAAATATTATTATTA1412 CAATTGCTGCCATCCTAAGAGTAGATCAATCTCAGTAGTATCAATGGCAAATAAGGAGGAGGCAGAGACC1413 AGTAAGAAGAAGCCCAAACCATTAAAAGTACTAATTACCAAAGTGGATCCGAAGCCGAGGAAGGGCATGG1414 CATCCGTCGCAGTGGACTTACTCGAGAAGGCCTTTGTATACCTATTGTCCGGAAATTCTGCAGCTGATCG1415 TAGTAGTAGTAGTGGTCGTCGTCGTCGTAAAGAGCATTACTACCTCTCCGGCAATTATGCGCCCGTCGGA1416 CACGAAACCCCGCCCTCCGACCACCTCCCCATTCATGGATCCCTTCCTGAATGCTTGAATGGAGTGTTTC1417 TGAGAGTTGGTCCTAACCCCAAGTTTGCTCCCGTAGCCGGATACAATTGGGTCGATGGAGATGGAATGAT1418 TCATGGATTGCGTATTAAAGATGGAAAAGCAACTTATCTATCTCGATATATTAAAACGTCACGGTTTAAA1419 CAAGAAGAATATTTTGGAAGAGCAAAATTTATGAAGATTGGAGATCTAAGGGGATTGCTTGGGTTCTTTA1420 CGATCTTAATACTAGTACTTCGAACAACATTGAAAGTAATAGACATTTCATATGGAAGAGGGACGGGTAA1421 TACAGCTCTTGTGTATCATAATGGCTTACTATTGGCTCTATCAGAAGAAGATAAACCTTATGTTGTTAAA1422 GTTTTAGAAGATGGAGACTTGCAAACTCTTGGGATATTGGATTATGACAAGAAATTGTCACATCCATTCA1423 CCGCTCATCCAAAGATCGATCCGTTAACTGATGAGATGTTTACCTTTGGATATTCCATCTCGCCTCCGTA1424 TCTTACTTATCGAGTCATTTCCAAGGATGGAGTGATGCAAGATCCAGTGCAAATCTCAATTACATCCCCT1425 ACCATAATGCATGATTTTGCTATTACTGAAAATTATGCCATCTTCATGGACCTGCCCTTGTATTTCCAAC1426 CAGAGGAAATGGTAAAGGGGAAATTTGTCTCTTCATTTCACCCTACAAAAAGAGCTCGTATCGGTGTGCT1427 TCCACGATATGCAAAAGACGAGCATCCAATTCGATGGTTCGATCTTCCAAGTTGCTTCATGACTCATAAT1428 GCAAATGCTTGGGAAGAGAATGATGAAGTTGTGCTATTCACATGTCGCCTTGAGAGTCCTGATCTTGACA1429 TGCTTAGTGGACCTGCGGAAGAAGAGATTGGGAATTCAAAAAGTGAGCTTTACGAAATGAGGTTCAATTT1430 GAAAACTGGAATTACTTCACAAAAGCAACTATCTGTACCTAGTGTTGATTTTCCTCGGATCAACCAAAGT1431 TATACTGGCAGGAAACAGCAATATGTTTATTGTACTCTTGGCAACACCAAGATTAAGGGCATTGTGAAGT1432 TTGATCTGCAAATTGAACCAGAAGCCGGAAAGACAATGCTTGAAGTTGGAGGAAATGTACAAGGCATCTT1433 TGAGTTGGGACCTAGAAGATATGGTTCAGAGGCAATATTTGTGCCATGCCAACCTGGCATCAAATCTGAT1434 GAGGATGACGGTTACTTGATATTCTTTGTACACGACGAAAACAATGGGAAATCTGAGGTCAATGTCATCG1435 ATGCAAAGACAATGTCTGCAGAACCTGTGGCTGTTGTGGAACTTCCAAGCAGGGTTCCATATGGATTCCA1436
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
49
TGCCTTGTTTCTGAATGAGGAAGAACTTCAGAAGCACCAAGCAGAGACAACAACCGAAAATTTATATTTC1437 CAGTCTGGAACA 1438 (insert between possitions 6981 and 6982 of TEVΔNIb; sequences to 1439 complete native NIaPro cleavage sites are on yellow background) 1440 GGGGAGAAGATGTCGGCCAGAATCTCCCCCTCCGCCACCACCCTCGCCGCCTCCTTCCGCCGCCCTCCGT1441 CTGGCGCACGCATCCTCCTCCTCCCTTCGCTCCCTGTCCGCCGCCCCGTCGAACTCCGGCCGCCGTTGCT1442 TCATCGTCGGCGTCGGACGGCGACAGTGTTTTTCGTTCTCGCCGAAGAAAAAACAACTCCTTTTCTTGAC1443 GACGTGGAGGAAGAGAAGAGTATTGCGCCGTCAAATCGGGCGGCTGAGAGGTCGGCGCGGAAGCGGTCGG1444 AGCGGACCACGTACCTCATAGCCGCGGTTATGTCGAGCTTGGGCATCACATCCATGGCCGCCGCAGCCGT1445 CTACTACCGCTTCGCTTGGCAAATGGAGGGAGGGGATGTGCCAGTGACGGAGATGGCGGGAACGTTCGCT1446 CTCTCGGTCGGGGCGGCGGTGGGGATGGAGTTCTGGGCCAGGTGGGCCCACCGGGCCCTCTGGCACGCGT1447 CGCTCTGGCACATGCACGAGTCCCACCACCGGCCGAGGGAGGGCGCTTTCGAACTCAACGACGTCTTCGC1448 CATAATCAACGCGGTCCCCGCCATAGCCCTGCTCAACTTCGGCTTCTTCCACAGAGGTCTTCTCCCCGGC1449 CTCTGTTTCGGCGCCGGGCTGGGGATCACGCTGTTTGGTATTGCGTACATGTTCGTCCACGACGGGCTAG1450 TCCACCGGCGGTTCCCTGTGGGGCCCATAGCCGACGTGCCCTACTTCCAGCGCGTCGCCGCTGCTCACCA1451 GATCCACCACTCGGAGAAGTTCGAAGGGGTGCCCTATGGACTGTTCATGGGGCCCAAGGTCGGTGCGGTT1452 TCTTTTTGTTGTTTTCCTCTCTTAACGACTGAGAATCTTTATTTTCAG 1453 1454 >TEVΔNIb-CsCCD2L/CsLPT1 1455 (insert between possitions 144 and 145 of TEVΔNIb; artificial NIaPro 1456 cleavage site is on gray background) 1457 ATGGAATCTCCTGCTACTAAATTACCTGCACCTCTGCTGATGTTATCTTCTTCTCCATTCCTTCTCCCTT1458 CTCCTAATAAGAGCTCCTCCATCTTCCTTCCACGTAAATTAGGGCCGCTACCTCCAAAATATTATTATTA1459 CAATTGCTGCCATCCTAAGAGTAGATCAATCTCAGTAGTATCAATGGCAAATAAGGAGGAGGCAGAGACC1460 AGTAAGAAGAAGCCCAAACCATTAAAAGTACTAATTACCAAAGTGGATCCGAAGCCGAGGAAGGGCATGG1461 CATCCGTCGCAGTGGACTTACTCGAGAAGGCCTTTGTATACCTATTGTCCGGAAATTCTGCAGCTGATCG1462 TAGTAGTAGTAGTGGTCGTCGTCGTCGTAAAGAGCATTACTACCTCTCCGGCAATTATGCGCCCGTCGGA1463 CACGAAACCCCGCCCTCCGACCACCTCCCCATTCATGGATCCCTTCCTGAATGCTTGAATGGAGTGTTTC1464 TGAGAGTTGGTCCTAACCCCAAGTTTGCTCCCGTAGCCGGATACAATTGGGTCGATGGAGATGGAATGAT1465 TCATGGATTGCGTATTAAAGATGGAAAAGCAACTTATCTATCTCGATATATTAAAACGTCACGGTTTAAA1466 CAAGAAGAATATTTTGGAAGAGCAAAATTTATGAAGATTGGAGATCTAAGGGGATTGCTTGGGTTCTTTA1467 CGATCTTAATACTAGTACTTCGAACAACATTGAAAGTAATAGACATTTCATATGGAAGAGGGACGGGTAA1468 TACAGCTCTTGTGTATCATAATGGCTTACTATTGGCTCTATCAGAAGAAGATAAACCTTATGTTGTTAAA1469 GTTTTAGAAGATGGAGACTTGCAAACTCTTGGGATATTGGATTATGACAAGAAATTGTCACATCCATTCA1470 CCGCTCATCCAAAGATCGATCCGTTAACTGATGAGATGTTTACCTTTGGATATTCCATCTCGCCTCCGTA1471 TCTTACTTATCGAGTCATTTCCAAGGATGGAGTGATGCAAGATCCAGTGCAAATCTCAATTACATCCCCT1472 ACCATAATGCATGATTTTGCTATTACTGAAAATTATGCCATCTTCATGGACCTGCCCTTGTATTTCCAAC1473 CAGAGGAAATGGTAAAGGGGAAATTTGTCTCTTCATTTCACCCTACAAAAAGAGCTCGTATCGGTGTGCT1474 TCCACGATATGCAAAAGACGAGCATCCAATTCGATGGTTCGATCTTCCAAGTTGCTTCATGACTCATAAT1475 GCAAATGCTTGGGAAGAGAATGATGAAGTTGTGCTATTCACATGTCGCCTTGAGAGTCCTGATCTTGACA1476 TGCTTAGTGGACCTGCGGAAGAAGAGATTGGGAATTCAAAAAGTGAGCTTTACGAAATGAGGTTCAATTT1477 GAAAACTGGAATTACTTCACAAAAGCAACTATCTGTACCTAGTGTTGATTTTCCTCGGATCAACCAAAGT1478 TATACTGGCAGGAAACAGCAATATGTTTATTGTACTCTTGGCAACACCAAGATTAAGGGCATTGTGAAGT1479 TTGATCTGCAAATTGAACCAGAAGCCGGAAAGACAATGCTTGAAGTTGGAGGAAATGTACAAGGCATCTT1480 TGAGTTGGGACCTAGAAGATATGGTTCAGAGGCAATATTTGTGCCATGCCAACCTGGCATCAAATCTGAT1481 GAGGATGACGGTTACTTGATATTCTTTGTACACGACGAAAACAATGGGAAATCTGAGGTCAATGTCATCG1482 ATGCAAAGACAATGTCTGCAGAACCTGTGGCTGTTGTGGAACTTCCAAGCAGGGTTCCATATGGATTCCA1483 TGCCTTGTTTCTGAATGAGGAAGAACTTCAGAAGCACCAAGCAGAGACAACAACCGAAAATTTATATTTC1484 CAGTCTGGAACA 1485 (insert between possitions 6981 and 6982 of TEVΔNIb; sequences to 1486 complete native NIaPro cleavage sites are on yellow background) 1487 GGGGAGAAGATGGCTCGCCAAGTTGCTCTGTGCTCTGTCCTGGTGATCGCTGCCTTCCTCTTCATCTCCT1488 CCCCTCGTGCCGAGAGCGCAGTCACCTGCAGCACTGTGGCCTCTGCGGTATCACCGTGCATCGGTTATGT1489 TCGCGGCCAGGGCGCACTGACTGGCGCGTGCTGCAGCGGCGTGAAGGGCCTTGCTGCAGCGGCAACCACC1490 CCCCCTGACCGTCGGACTGCATGCAGCTGCCTCAAGTCAATGGCTGGCAAAATCAGCGGCCTGAACCCTA1491 GTCTCGCAAAAGGCCTCCCCGGCAAGTGCGGTGCCAGCGTCCCCTATGTCATCAGCACATCCACTGACTG1492 CACTAAGGTGGCAACGACTGAGAATCTTTATTTTCAG 1493
1494
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
50
1495 1496 Fig. S3. Complementation of a TEV mutant that lacks NIb in the transformed N. 1497 benthamiana line that stably expresses TEV NIb. (A) Wild-type and (B) NIb-expressing 1498
N. benthamiana plants were mechanically inoculated with TEVΔNIb-Ros1, a 1499 recombinant virus in which the NIb cistron is replaced by the Rosea1 visual marker that 1500 induces anthocyanin accumulation. Pictures were taken 11 days post-inoculation (dpi). 1501
1502
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
51
1503
1504 Fig. S4. Fluorescence stereomicroscope analysis of tissues from plants mock-inoculated 1505 and agroinoculated with TEVΔNIb-aGFP, TEVΔNIb-CsCCD2L, -BdCCD4.1 and -1506 BdCCD4.3. Pictures at 15 dpi were taken with a Leica MZ 16 F stereomicroscope under 1507 white light and under UV illumination with the GFP2 (Leica) fluorescence filter. Scale 1508 bars correspond to 5 mm. 1509
1510
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
52
1511 1512 Fig. S5. Crocetin ion extraction (m/z 329.1747 (M+H+)) in control and CsCCD2L and 1513 BdCCD4.1 polar fraction chromatograms. Metabolites were extracted from infected 1514
leaves and run on an HPLC-PDA-HRMS system. The apocarotenoids have an accurate 1515 mass and a chromatographic mobility identical to those of the authentic standards, when 1516 available. 1517
1518
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint
53
SUPPLEMENTARY TABLES 1519
1520
Table S1. LC-HRMS of crocins- (A) and picrocrocin-related (B) apocarotenoid levels in 1521
infected leaves. Data are avg ± sd of, at least, three biological replicates; nd (not detected). 1522
Values are reported as fold level between the signal intensities of the apocarotenoid 1523
metabolite on the signal intensity of the internal standard (formononetin). 1524
1525
Table S2. Relative abundance, as % on the total, of apocarotenoid levels in CsCCD2L 1526
and BdCCD4.1 leaves. (A) crocins-related metabolites and (B) picrocrocin-related 1527
metabolites. 1528
1529
Table S3. LC-HRMS of carotenoid- (A), chlorophyll-related (B), and tocochromanols 1530
and quinones (C) levels in infected leaves. Data are avg ± sd of at least three biological 1531
replicates; nd (not detected). Values are reported as fold level between the signal 1532
intensities of the apocarotenoid metabolite on the signal intensity of the internal standard 1533
(a-tocopherol acetate). Asterisks indicate significant differences according a t-test 1534
(pValue: *<0.05; **<0.01) carried out in the comparisons Not infected vs GFP, CCD2L 1535
vs GFP and CCD4.1 vs GFP. 1536
1537
Table S4. LC-HRMS of crocins- (A) and picrocrocin and safranal (B) apocarotenoid 1538
levels in infected leaves. Data are avg ± sd of at least three biological replicates; nd (not 1539
detected). Values are reported as absolute amounts (in mg/g DW) by using authentical 1540
standards and external calibration curves. Asterisks indicate significant differences 1541
according a t-test (pValue: *<0.05; **<0.01) carried out in the comparisons CsCCD2L 1542
vs CsCCD2L+PaCrtB, CsCCD2L+CsBCH2 or CsCCD2L+CsLTP1. 1543
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint