Journal of Experimental Botany, Vol. 54, No. 388, pp. 1675-1683,
July 1, 2003
© 2003 Oxford University Press
Virus-induced silencing of sterol biosynthetic genes: identification of a Nicotiana tabacum L. obtusifoliol-14
-demethylase (CYP51) by genetic manipulation of the sterol biosynthetic pathway in Nicotiana benthamiana L.*
Received 5 December 2002; Accepted 2 April 2003
,
,
Institut de Biologie Moléculaire des Plantes du CNRS, Département Isoprénoïdes, Institut de Botanique, 28 rue Goethe, F-67083 Strasbourg, France
* The nucleotide sequences reported in this paper have been submitted to GenBank with the accession numbers AF116915 and AY065641. Present address: Department of Biochemistry and Genetics, University of Newcastle, UK. To whom correspondence should be addressed. Fax: +33 (0)3 90 24 18 84. E-mail: Hubert.Schaller{at}ibmp-ulp.u-strasbg.fr
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Abstract |
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Obtusifoliol-14
-demethylase (CYP51) is implicated in plant sterol biosynthesis. An Arabidopsis expressed sequence tag encoding a CYP51 was used as a probe to isolate Nicotiana tabacum L. cDNAs. Two types of cDNA clones were identified. Nt CYP51-1 and Nt CYP51-2 shared 97% identity together and around 75% with other plant CYP51s. The function of the encoded enzyme has been demonstrated in planta by manipulating the sterol biosynthetic pathway at the gene level. The endogenous CYP51 of Nicotiana benthamiana was silenced upon inoculation of the plantlets with POTATO VIRUS X::Nt CYP51-1 transcripts. This resulted in the accumulation of obtusifoliol, the substrate of CYP51, and other 14-methyl sterols, with a concomitant growth reduction phenotype. Virus-induced gene silencing was also applied to another steroidogenic enzyme, the 
7-sterol-C5(6)-desaturase, and this resulted in the accumulation of 7-sterols in infected plants instead of the pathway end-products 5-sterols.
Key words: CYP51, gene silencing, Nicotiana, obtusifoliol-14-demethylase, sterol biosynthesis.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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CYP51 is a unique orthologous P450 which catalyses 14
-demethylation of sterol biosynthetic intermediates in animal, fungi, plants, Mycobacterium tuberculosis, and Dictyostelium discoideum (Yoshida et al., 2000). Lanosterol-14-demethylase was first characterized in yeast (Yoshida and Aoyama, 1984; Kalb et al., 1986) then in mammals (Trzaskos et al., 1986; Aoyama et al., 1996), and eburicol-14-demethylase was isolated in fungi of agricultural interest (Délye et al., 1997a). Obtusifoliol-14-demethylase was cloned and characterized in Sorghum bicolor (L.) Moench (Bak et al., 1997) and in wheat (Cabello-Hurtado et al., 1999); antisense CYP51 Arabidopsis thaliana (L.) Heynh were recently generated and these plants showed a semi-dwarf phenotype and a longer life span than control plants (Kushiro et al., 2001). Regulated CYP51 expression has been mostly studied in animals, particularly in relation to the biological function of meiosis-activating steroids (Yamashita et al., 2001; Rozman et al., 2002).
The inhibition of sterol-14-demethylation by azole pesticides used in agriculture and medicine has driven a considerable research effort to understand the biochemical basis of CYP51 affinity for these chemicals (Podust et al., 2001; Lamb et al., 2001). Azole resistance has been attributed to amino acid substitution in CYP51 for a number of resistant fungi (Lamb et al., 1997; Délye et al., 1997b; Marichal et al., 1999; Favre et al., 1999), overexpression of CYP51 in fungi (Kontoyiannis et al., 1999) and in plants (Grausem et al., 1995), or active efflux of the azoles in fungi (Sanglard et al., 1995).
The plant enzyme obtusifoliol-14-demethylase (Taton and Rahier, 1991) has been shown both in vitro and in planta to be the target site of triazoles (Taton et al., 1988; Maillot-Vernier et al., 1990). The herbicidal effect of the triazoles LAB170250F and 
-ketotriazole was previously used as a positive screen for the isolation of resistant lines in the model species Nicotiana tabacum L. in the framework of a somatic genetic approach of sterol biosynthesis (Maillot-Vernier et al., 1990; Schaller et al., 1992).The isolation of cDNAs encoding tobacco CYP51s which is a prerequisite to the characterization of the triazole resistant mutant lines is described in the present article. The functional identification of a tobacco CYP51 cDNA was achieved by manipulating the sterol profile of Nicotiana benthamiana L. using a gene silencing (Baulcombe, 1999) approach applied for the first time to the study of plant sterol metabolism: virus-induced gene silencing (VIGS) of CYP51 and also of a 7-sterol-C5(6)-desaturase gene resulted in a strong inhibition of sterol biosynthesis with a concomitant growth reduction phenotype in the case of the CYP51 silenced plants.
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Materials and methods |
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Isolation of Nt CYP51 cDNAs
cDNAs were isolated from Nicotiana tabacum L. var. xanthi libraries. Two libraries were previously prepared in
-Zap (Stratagene) from a wild-type (cv. xanthi line SH6) and from a mutant (line sterov; Maillot-Vernier et al., 1991) genotypes using leaf protoplast-derived callus cultures (Maillot-Vernier et al., 1990). The libraries were referred to as A and B, respectively. 300 000 pfu from library B were screened with 32P-random labelled cDNA probes prepared according to a standard procedure from a SalI–NotI 1.6 kb fragment purified from the amplified Arabidopsis thaliana (L.) Heynh EST clone 224D4T7 (Arabidopsis Biological Ressources Center, Ohio) after confirming the sequence of this EST. Hybridization of the probes to the pfu was carried out at 55 °C according to standard conditions (Sambrook et al., 1986). The size of the recovered clones was estimated by performing a standard PCR assay on diluted phages using T3 and T7 primers. The clones producing amplicons of more than 1.7 kb were further purified and sequenced. One of these clones named A613 was then used to generate the 32P-random labelled cDNA probes used to screen 300 000 pfu from library A in the same hybridization conditions as those for the previous screening.
Nucleotide sequence determination
cDNAs were isolated as pBluescript SK (Stratagene) derivatives which were sequenced on both strands with an automatic sequencer Perkin Elmer model 373 using T3, T7 and specific internal primers. The sequencing reaction was based on the incorporation of fluorescent dNTPs. Nucleotidic and deduced peptidic sequences were aligned with the software tools from the Genetics Computer Group package version 8.1 run with default parameters.
DNA gel blot hybridization
Tobacco wild-type (var. xanthi line SH6) leaf DNA was purified from a CsCl gradient after it was extracted as described by Dellaporta et al. (1983). Electrophoresis of DNA fragments and gel blot analysis was performed as described by Sambrook et al. (1986). The restricted tobacco genome was then probed with 32P-random labelled cDNA probes synthesized from Nt CYP51 (clone A613).
Plasmid constructs for VIGS
A 1008 bp fragment of Nt CYP51-1 (clone A613) was PCR-amplified using Taq DNA polymerase (Gibco BRL) and the primers CB1 (5'-cggattatcgattcaaatcctggcc-3') and CB2 (5'-cgatgagtcgacccttaggaatatc-3'). The resulting fragment was cloned in the sense orientation into the ClaI and SalI sites of potato virus X vector pP2C2S (Chapman et al., 1992; Baulcombe et al., 1995) to yield the construct PVX::CYP51. A 693 bp fragment of the cDNA Nt 5-DES1-1 encoding a 7-sterol-C5(6)-desaturase (Husselstein et al., 1999) was PCR-amplified using the primers CB7 (5'-CGATGAAT CGATTCACTTGAAGCGC-3') and CB8 (5'-ATATATGTCG ACCACTGGTGGCCTA-3'). This fragment was cloned into the ClaI and SalI sites of potato virus X vector pP2C2S to yield PVX::5-DES. The identity of the inserts was verified by sequencing.
In vitro transcription and inoculation of plants
Infectious recombinant PVX RNA molecules were produced by in vitro transcription of the PVX derivatives. The constructs were first linearized with SpeI restriction, then transcripts were produced with the RibomaxTM Large Scale RNA Production System T7 kit (Promega). The infectious RNA were resuspended in 50 mM phosphate buffer containing macaloid (0.05%) and rubbed onto the top leaves of 4–5-week-old Nicotiana benthamiana L. plantlets in the presence of a small amount of celite powder (Baulcombe et al., 1995). Plants were grown in a greenhouse with 16/8 h light/dark and 23/19 °C cycles for an additional time of 2–8 weeks. Lighting at 200 µmol radiation m–2 s–1 on average was obtained by complementing daylight, if necessary, with Phillips 600 W white bulbs.
Sterol analysis
Leaves of Nicotiana benthamiana were harvested at different days post inoculation (dpi) and lyophilized. The dry material was saponified with 6% (w/v) KOH in methanol at 80 °C for 2–3 h. Sterols were then extracted with 3 vols of n-hexane and an acetylation reaction was performed on the dried residue for 1 h at 70 °C in toluene with a mixture of pyridine/acetic anhydride (0.5:1, v:v). Steryl acetates were resolved by TLC on Merck precoated silica plates with one run of dichloromethane as a single band (Rf=0.5). Purified steryl acetates were separated and identified by gas chromatography (GC) using a Varian 8300 gas chromatograph with a flame ionization detection, a glass capillary column (DB 1) and H2 as a carrier gas (2 ml min–1). The temperature program included a steep ramp from 60–220 °C (30 °C min–1) followed by a 2 °C min–1 ramp from 220–280 °C. Data from the detector were monitored with the Varian Star computer program. Amounts of steryl acetates were quantified using cholesterol as an internal standard. Sterols structures were confirmed by GC-mass spectrometry using an Agilent 6890 GC system coupled to a mass detector, according to published data (Schmitt and Benveniste, 1979).
Measurements of RNA levels
Total RNA from leaf material of Nicotiana benthamiana were isolated with TRIzol® reagent (Invitrogen Life Technologies). A reverse transcription reaction was performed on 1 µg of total RNA with 200 units of M-MLV reverse transcriptase (Promega), 200 ng of random hexamers (Boeringher Mannheim) and 500 µM dNTPs in a final volume of 20 µl. Real-time PCR assays were performed with SYBR Green PCR master mix (Applied Biosystems) in a final volume of 25 µl including appropriate primer pairs designed with the software Primer Express (Applied Biosystems). Assays were run in duplicates on a GeneAmp 5700 sequence detection system (Applied Biosystems). The amplification program consisted of 40 cycles of a first step at 95 °C for 15 s then a second step at 60 °C for 1 min. The relative quantification of gene expression was performed using the comparative CT (threshold cycle) method in which the amount of target, normalized to an endogenous reference and relative to a calibrator, is given by the formula 2–CT (http://docs.appliedbiosystems.com). The accumulation of the viral CYP51 was measured with primers CB3 (5'-TGTCGGCCCATTTCTTTAAGG-3') and CB4 (5'-GGCCAAAAGTAGGCACATTGA-3').The dosage of the endogenous CYP51 RNA was done with primers CB5 (5'-CCTGA AATCGACTGGAATGCA-3') and CB6 (5'-GCTTTCGGCGCT TGTACTTC-3'). A Nicotiana tabacum ACTIN gene (tob 103 GeneBank accession number U60495
[GenBank]
) was used as an endogenous reference. The amplification reaction on that reference target was done with primer pairs CB9 (5'-TGCTGATCGTATGAGCA AGGAA-3') and CB10 (5'-GGTGGAGCAACAACCTTAAT CTTC-3') designed with the software Primer Express.
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Results |
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Isolation and sequence analysis of tobacco CYP51
An Arabidopsis thaliana 5'-truncated EST was initially found on the basis of its high identity with the monocot CYP51s (Bak et al., 1997; Cabello-Hurtado et al., 1999) and was used as a probe to isolate tobacco CYP51 from callus cDNA libraries. In a first screening of library B, 14 clones (among 26) presented an insert size of at least 1.7 kb and were analysed further. Complete sequencing of these showed that they were of two types which were assigned to Nt CYP51-1 and Nt CYP51-2. Ten of these 14 clones were represented by A613 (1913 bp) which is the Nt CYP51-1 described here (GenBank accession number AF116915 [GenBank] ) and the four remaining clones were represented by G321 (1725 bp) which is the Nt CYP51-2 reported here (GenBank accession number AY065641 [GenBank] ). The two cDNAs shared 95% identity at the nucleotide level. In order to enrich this P450 family with other members, library A was screened using Nt CYP51-1 to synthesize 32P-labelled random probes. Twenty-four clones were recovered of which 13 contained an insert of more than 1.7 kb. Sequencing of these led to the identification of three full length Nt CYP51-1 clones and two full length Nt CYP51-2 clones. In order to check the representativeness of CYP51 in the tobacco genome, a PCR synthesized-ORF contained in the cDNA Nt CYP51-1 was then used as a probe in a DNA gel blot experiment which is shown in Fig. 1. The autoradiography of the hybridized nylon membrane showed 2–3 bands when the genome was digested with restriction enzymes having no sites within the cDNAs or four bands when the genome was digested with HindIII which is a conserved unique site. Taken together, these results indicate that CYP51 in tobacco might be represented by two genes at least.
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Both cDNAs encode polypeptides of 487 amino acids which share 97% identity and present typical features of cytochrome P450s including an N-terminal hydrophobic anchor, a proline-rich domain and a conserved haem binding region (Werck-Reichhart and Feyereisen, 2000). The residues which differ between the two proteins are mostly conservative changes (data not shown). Tobacco CYP51s exhibit a high identity with other plant CYP51: 75% with the Sorghum bicolor (Bak et al., 1997) and Triticum aestivum (Cabello-Hurtado et al., 1999) polypeptides, and 80% and 70% with the Arabidopsis thaliana CYP51A1 and CYP51A2, respectively (Yoshida et al., 2000; http://www.biobase.dk/P450/).
In planta functional identification of Nt CYP51s
A central 1 kb fragment of the ORF of Nt CYP51-1 was cloned into the viral PVX-based pP2C2S vector (Baulcombe et al., 1995). Additionally, a 0.7 kb fragment of the ORF of Nt 5-DES-1 was cloned into the PVX vector. Nt 5-DES-1 is a 7-sterol-C5(6)-desaturase which has been previously reported (Husselstein et al., 1999). Because both tabacum and benthamiana of the Nicotiana genus share an almost identical codon usage (http://www.kazusa.or.jp/codon/), an efficient post-transcriptional silencing of the endogenous CYP51 and 5-DES messages is expected upon inoculation of N. benthamiana with the viral transcripts of PVX::CYP51 and PVX::5-DES, respectively, resulting in reduced amounts of CYP51 and 5-DES enzymes. Subsequently, this genetic inhibition of the sterol pathway should result in the accumulation of the substrates of the enzymes CYP51 and 5-DES.
Leaves of greenhouse-grown N. benthamiana were inoculated with buffer, PVX, PVX::CYP51 or PVX::5-DES transcripts. The sterol profiles of young growing leaves or stems from the upper third of the inoculated plants were determined at various dpi. The biochemical phenotype of PVX::CYP51 and PVX::5-DES1 plants at 24 dpi is given in Table 1. Leaf and stem samples of PVX::CYP51-inoculated plants accumulated over 50% of obtusifoliol, the substrate of CYP51, and its metabolites. Obtusifoliol may indeed undergo C241-methylation, C4-demethylation and/or 24-isomerization/reduction as indicated in Fig. 2. This pool of 14-methyl-8-sterols accumulated at the expense of the pathway end-product; 5-sterols campesterol, sitosterol and stigmasterol essentially. The opposite ratios of obtusifoliol to 14-methyl-24(241)-dihydrofecosterol and 14-methyl fecosterol in leaves and stems of PVX::CYP51 (Table 1) plants would probably reflect the tissue-specificity of some genes and enzymes (i.e. C4-demethylase, 24-isomerase/reductase) of the sterol pathway in N. benthamiana.
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Silencing of the endogenous Nicotiana benthamiana CYP51 was demonstrated using a real time PCR assay. The primer pair CB3/CB4 was used to amplify a 95 bp sequence present in the viral transcript PVX::CYP51 and the primer pair CB5/CB6 was designed to amplify a 73 bp sequence from the Nt CYP51-1 cDNA which was not included in the PVX::CYP51 construct. CB5/CB6 were therefore used to measure the endogenous level of CYP51 transcripts in N. benthamiana. In fact, preliminary assays have shown that these primer pairs designed from a tobacco sequence were able to amplify amplicons of the expected sizes from reverse-transcribed total RNAs from N. benthamiana leaves. Sequencing of the PCR product obtained with CB3/CB4 gave a 91 bp sequence displaying 97.81% identity with Nt CYP51-1 (data not shown). the level of CYP51 viral and endogenous transcripts was measured at 22 dpi, using as a reference the level of expression of the N. benthamiana orthologue of the tobacco ACTIN gene Tob103 with the primer pair CB9/CB10 previously shown to produce optimized PCR amplification curves (L Wentzinger, IBMP, personal communication). The results shown in Table 2 indicate an over 400-fold increase of the viral truncated CYP51 message in plants inoculated with PVX::CYP51, compared to controls. Secondly, the level of endogenous N. benthamiana CYP51 transcription in PVX::CYP51 plants is decreased to 16% of the wild-type level, demonstrating thus, together with the first observation, efficient silencing of CYP51. The results show also a consistent reduction of the level of CYP51 transcripts in PVX-inoculated controls, compared with the wild type, which could be tentatively explained by a modified metabolism of PVX-inoculated plants. Additionally, these results demonstrate that primer pairs CB3/CB4 and CB5/CB6 are equivalent for the purpose of endogenous CYP51 expression measurements in N. benthamiana.
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Leaf samples of PVX::5-DES1-inoculated plants contained up to 44% of
7-sterols compared with a maximum of 9% for the various controls (Table 1). The presence of 6% of 7-campesterol and 21% of 7-sitosterol at the expense of 5-sterols show that enzymes downstream to 7-sterol-C(5)6-desaturase are metabolizing the accumulated substrates of the latter as described in the Arabidopsis thaliana mutants ste1 (Gachotte et al., 1995), dwarf7 (Choe et al., 1999) and bul1 (Catterou et al., 2001) in which genetic blocks at the level of the 7-sterol-C5(6)-desaturase had been demonstrated. The growth of PVX::CYP51 and PVX::5-DES1 plants were monitored until 32 dpi. The first observation is that PVX-inoculated plants were reduced in size and altered in their morphology compared to control mock-inoculated plants (Fig. 3). A reasonable explanation for this effect is that PVX induces viral symptoms on N. benthamiana. A second and important observation is the severe morphological phenotype of PVX::CYP51 plants. Principally, these plants display a reduced stature due to the reduction of stem length between internodes when compared with controls (Table 3; Fig. 3). In addition, after 15 dpi the stems of PVX::CYP51 plants developed a few blackish dry elongated areas which were absent from PVX plants (data not shown). The last observation is that PVX::5-DES1 plants did not show any morphological phenotype compared to PVX control plants (data not shown).
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Discussion |
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The present paper reports the isolation and in planta functional identification of tobacco cDNAs encoding the sterol biosynthetic enzyme obtusifoliol-14
-demethylase. A cDNA library screening with Arabidopsis and then tobacco CYP51 probes led to the isolation of two cDNAs whose encoded products shared 97% identity. These may be orthologues of the Arabidopsis thaliana CYP51A1 or CYP51A2 (Yoshida et al., 2000; http://www.biobase.dk/P450/). In fact, Nt CYP51-1 and Nt CYP51-2 share 80% identity with At CYP51A1 and 70% identity with At CYP51A2. Because the Arabidopsis thaliana EST which was used as a probe in the screening procedure was in fact an EST of CYP51A2 (Kushiro et al., 2001), it is not excluded that one or more orthologues of the other putative obtusifoliol-14-demethylase from Arabidopsis thaliana might exist in the amphihaploid genome of tobacco.
The functional identification of the cloned CYP51 was done by virus-induced gene silencing. Because gene silencing is based on nucleotide sequence specificity (Baulcombe, 1999), one (Nt CYP51-1) of the two cDNAs was used in this experiment. The accumulation of the viral transcript of Nt CYP51 in N. benthamiana upon inoculation with PVX::CYP51 led to a dramatic decrease of the endogenous message and, subsequently, to a lack of the corresponding protein. The detection of obtusifoliol, substrate of CYP51, and of other 14-methyl-8-sterols in the silenced N. benthamiana proves that the cDNAs reported in this article are coding for obtusifoliol-14-demethylases. This result is in full accordance with the increased amount of obtusifoliol measured by Kushiro et al. (2001) in transgenic Arabidopsis expressing an antisense At CYP51A2 construct (Kushiro et al., 2001).
The biochemical phenotype of the CYP51-silenced plants is identical to the sterol profiles of tobacco cell cultures, seedlings or plants treated with the triazole experimental herbicides -ketotriazole and LAB170250F (Maillot-Vernier et al., 1990, 1991; Schaller et al., 1992) and therefore it was concluded that both genetic and chemical inhibition of CYP51 produce exact phenocopies in terms of biochemical composition.
The severe morphological phenotype of the CYP51-silenced plants indicates that a sterol profile consisting of obtusifoliol and 14-methyl-dihydrofecosterol as major compounds cannot sustain normal growth and development of plant tissues because of probable adverse effects on cell membranes as has been discussed in the case of the herbicidal properties of triazole inhibitors (Grausem et al., 1995). The severe growth phenotype of CYP51-silenced N. benthamiana also shares features of the CYP51A2 antisense Arabidopsis thaliana which were semi-dwarf plants (Kushiro et al., 2001).
The efficiency of VIGS applied to sterol biosynthesis genes has been further demonstrated using PVX::5-DES1 transcripts. The inhibition of the sterol pathway at the level of the 7-sterol-C5(6)-desaturase led indeed to the accumulation of 7-sterols at the expense of 5-sterols in the plant cells. Genetic defects of the 7-sterol-C5(6)-desaturase Arabidopsis thaliana gene have been reported and the sterol profiles of the corresponding genotypes displayed a predominant 7-sterol pathway (Catterou et al., 2001). Knock-out alleles of the gene were dwarf plants (Choe et al., 1999) but the leaky mutants ste1 (Gachotte et al., 1995) which accumulated 50–70% of 7-sterols had a morphology and growth similar to that of the wild type. The absence of a growth defect of PVX::5-DES1-inoculated N. benthamiana, which accumulated up to 50% of 7-sterols compared to the controls, is consequently in accordance with the previous observation.
VIGS in Nicotiana benthamiana has been successfully developed to characterize tobacco genes on the basis of biochemical or biological phenotypes. Different aspects of plant biology have been considered including cell wall biogenesis (Burton et al., 2000), chlorophyll biosynthesis (Hiriart et al., 2002) and cytokinesis (Nishihama et al., 2002). Silencing of a cellulose synthase gene or of the ChlH gene induced chimeric phenotypes with very large spherical cells ballooning from the undersurface of leaves in the first case (consistent with a reduced cellulose content) or with green and yellowish leaves in the second case. A chimeric phenotype of leaves comprising bleached areas was also observed in the case of the silencing of the gene encoding phytoene desaturase (Ruiz et al., 1998). The chimeric nature of all these phenotypes is most probably due to the fact that a gene silencing signal moves not only between cells through plasmodesmata, but also through vascular tissues close to which the most severe instances of a phenotype are described (Burton et al., 2000). Although it is clear from the phenotype of PVX::CYP51 plants that their altered sterol profile is not compatible with normal growth, it might be that a heterogenous (chimeric) sterol composition of a given plant tissue would, to some extent, prevent an accurate characterization of a morphological phenotype otherwise found (if not lethal) in the case of a defective mutation.
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Acknowledgements |
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We thank Professor David Baulcombe and colleagues from the Sainsbury Laboratory in Norwich for the pP2C2S PVX-based vector. We thank also Patrice Dunoyer and Sébastien Pfeffer from the IBMP in Strasbourg for helpful discussions about viral transcript inoculation to plants. Dr Dawn Little is warmly acknowledged for assistance in improving the English grammar.
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