Transgenic Res (2012) 21:1319–1327 DOI 10.1007/s11248-012-9611-5
ORIGINAL PAPER
Transient expression of artificial microRNAs targeting Grapevine fanleaf virus and evidence for RNA silencing in grapevine somatic embryos Noe´mie S. Jelly • Paul Schellenbaum Bernard Walter • Pascale Maillot
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Received: 11 October 2011 / Accepted: 7 March 2012 / Published online: 17 March 2012 Ó Springer Science+Business Media B.V. 2012
Abstract Grapevines are affected worldwide by viruses that compromise fruit yield and quality. Grapevine fanleaf virus (GFLV) causes fanleaf degeneration disease, a major threat to grapevine production. Transgenic approaches exploiting the RNA silencing machinery have proven suitable for engineering viral resistance in several crop species. However, the artificial microRNA (amiRNA)-based strategy has not yet been reported in grapevine. We developed two amiRNA precursors (pre-amiRNAs) targeting the coat protein (CP) gene of GFLV and characterised their functionality in grapevine somatic embryos. To create these pre-amiRNAs, natural pre-miR319a of Arabid-
Electronic supplementary material The online version of this article (doi:10.1007/s11248-012-9611-5) contains supplementary material, which is available to authorized users. N. S. Jelly P. Schellenbaum B. Walter P. Maillot (&) Laboratoire Vigne, Biotechnologies et Environnement, Universite´ de Haute-Alsace, 33 rue de Herrlisheim, 68008 Colmar, France e-mail:
[email protected] Present Address: N. S. Jelly Institut de Biologie Mole´culaire et Cellulaire, UPR9022 CNRS, Universite´ de Strasbourg, 15 rue Rene´ Descartes, 67084 Strasbourg, France
opsis thaliana was modified by overlapping PCR in order to replace miR319a with two amiRNAs targeting different regions of the CP gene: amiRCP-1 or amiRCP2. Transient expression of these two pre-amiRNA constructs was tested in grapevine somatic embryos after co-cultivation with Agrobacterium tumefaciens. Expression of amiRCP-1 and amiRCP-2 was detected in plant tissues by an endpoint stem-loop RT-PCR as early as 1 day after a 48-h co-cultivation, indicating active processing of pre-amiRNAs by the plant machinery. In parallel, GUS-sensor constructs (GCP1 and GCP-2) were obtained by fusing the target sequence of amiRCP-1 or amiRCP-2 to the 30 terminus of the GUS gene. Co-transformation assays with GUSsensors and the pre-amiRNA constructs provided evidence for in vivo recognition and cleavage of the 21-nt target sequence of GUS-sensors by the corresponding amiRNA. This is the first report of amiRNA ectopic expression in grapevine. The constructs we developed could be useful for engineering GFLVresistant grapes in the future. Keywords Vitis vinifera Fanleaf degeneration Agrobacterium tumefaciens Transient transformation Abbreviations amiRNA Artificial microRNA GFLV Grapevine fanleaf virus GUS b-Glucuronidase CP Coat protein
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Introduction Grapevine is an economically important crop that is grown worldwide for the production of fresh fruits, juices, raisins, wines and liquors. Several biotic factors, including viral diseases, affect this crop. Among them, fanleaf degeneration is a major threat. This disease decreases yield and reduces fruit quality. It is mainly caused by Grapevine fanleaf virus (GFLV), a nepovirus of the family Secoviridae (Sanfac¸on et al. 2009) that is specifically transmitted by the ectoparasitic nematode Xiphinema index (Brown et al. 1995). GFLV dissemination is mainly caused by the worldwide exchange of plant grafts and propagation materials (Martelli 1978). Despite rigorous sanitary controls, producers lack efficient technologies to control the infection since nematicide chemicals tend to be prohibited. Transgenic approaches to control this virus are therefore attractive for use in grapevine. Recently, the discovery of RNA silencing has made it possible to engineer virus-resistant crops. RNA silencing has roles in a great variety of processes, including transcriptional regulation. This mechanism is mediated by the binding of small non-coding RNAs of 19–30 nucleotides in length to their complementary target sequence. These small RNAs can be divided into two main categories: small interfering RNAs (siRNAs) and microRNAs (miRNAs). siRNAs originate from the cleavage of long exogenous or endogenous double stranded RNAs, leading to the production of several siRNAs corresponding to different portions of both strands of their precursors (Wassenegger and Krczal 2006). miRNAs arise from the processing of a long endogenous transcript called miRNA precursor (premiRNA) forming a localised hairpin structure (Bartel 2004); two cuts in this stem-loop structure lead to the production of a single species of miRNA, in contrast to siRNAs (Papp et al. 2003). Natural pre-miRNAs can be engineered to generate artificial miRNAs (amiRNAs) which can trigger the silencing of RNAs of interest in a sequence-specific manner (Schwab et al. 2006). This strategy produced highly efficient and specific gene silencing in Arabidopsis (Schwab et al. 2006), rice (Warthmann et al. 2008), Physcomitrella patens (Khraiwesh et al. 2008), tomato (Fernandez et al. 2009), and the green alga Chlamydomonas reinhardtii (Molnar et al. 2009). The use of amiRNAs allowed functional analyses of endogenous genes such as phenylalanine ammonia-lyase (PAL) genes
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in Populus trichocarpa (Shi et al. 2010), or genes involved in the circadian clock in Arabidopsis (Kim and Somers 2010; Salome´ et al. 2010). amiRNAs were also able to silence viral sequences, conferring efficient resistance against Cucumber mosaic virus to tobacco (Qu et al. 2007), Arabidopsis (Duan et al. 2008), and, more recently, tomato (Zhang et al. 2011). Transgenic Arabidopsis lines expressing amiRNAs against Turnip yellow mosaic virus and Turnip mosaic virus proved resistant against both viruses (Niu et al. 2006). In addition, tobacco lines resistant to both Potato viruses X and Y (Ai et al. 2011), as well as transgenic wheat resistant to Wheat streak mosaic virus (Fahim et al. 2011), were also obtained using this technology. In an effort to induce resistance against GFLV, numerous transgenic grapevine lines expressing GFLV-derived gene constructs have been obtained and characterised (Krastanova et al. 1995; Mauro et al. 1995; Xue et al. 1999; Vigne et al. 2004; Gambino et al. 2005; Maghuly et al. 2006; Valat et al. 2006; JardakJamoussi et al. 2009; Gambino et al. 2010). However, only a few reports address the efficiency of these constructs to induce viral resistance. In field trials, transgenic grapevines expressing the coat protein (CP) gene of GFLV displayed a delay in viral infection (Vigne et al. 2004). After electroporation with purified particles of GFLV, protoplasts of a transgenic rootstock expressing the viral CP gene showed a decrease in virus titer (Valat et al. 2006). Other studies showed viral resistance in transgenic Nicotiana benthamiana expressing GFLV sequences, whereas grapevines transformed with the same sequences remained susceptible to the virus (Go¨lles et al. 2000). So far, the use of amiRNAs has not been reported in grapevine. Based on results obtained in other crops, this approaches is promising for the development of transgenic grapevines resistant to GFLV. Here we report the design and cloning of two pre-amiRNAs directed against GFLV and their transient expression in grapevine (V. vinifera cv. ‘Chardonnay’), leading to the silencing of their target sequences. Materials and methods Plasmid construction A 2,161-bp fragment containing the GUS sequence was amplified by PCR using the two primers GUS-5
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(50 -TGGAGAGAACACGGGGGACT-30 ) and GUS-3 (50 -TCGCAAGACCGGCAACAGGA-30 ) with pCambia1301 as a template (Cambia, Canberra, Australia). Using the GATEWAYTM recombinational cloning system (Invitrogen, Carlsbad, CA), it was first cloned into the entry pCRÒ8 (Invitrogen), and then into the binary vector pK7WG2D (Karimi et al. 2002) to generate pNJ-GUS. This binary vector thus contains the GUS reporter gene under the control of the constitutive Cauliflower mosaic virus 35S promoter. The sequences of amiRNAs designed to target the CP gene encoded by RNA2 of GFLV-F13 (GenBank Accession No. X16907) were obtained using the Web MicroRNA Designer (WMD) platform (http://wmd3. weigelworld.org) (Fig. S1). The pre-amiRNAs were engineered by overlapping PCR according to Schwab et al. (2006) with primers obtained as a final output with the WMD platform (Fig. S2, Table S1 in supplementary material). The template used was the plasmid pRS300, corresponding to a 404-bp fragment containing the natural Arabidopsis pre-miR319a sequence cloned into pBluescript SK?. The resulting pre-amiRNAs were cloned into pK7WG2 (Karimi et al. 2002) to generate pNJ-amiRCP-1 and pNJamiRCP-2, respectively for amiRCP-1 and amiRCP-2 (Fig. S3) GUS-sensor constructs were obtained by fusing the target sequence of amiRCP-1 or amiRCP-2 to the GUS gene at its 30 terminal sequence (Fig. S4). Mutagenesis was performed by PCR, using pCambia1301 as a template, and the oligonucleotides GUS5 (see above) and GUSCP1-3 (50 -AAAAACGTGAGAAGGTCCGTCTCGCAAGACCGGCAACAGG A-30 ) or GUSCP2-3 (50 -CCATACGAAATAGT CT TCTCGCAAGACCGGCAACAGGA-30 ) containing the target sequences (underlined) of amiRCP-1 and amiRCP-2, respectively. The resulting GUS-sensors were cloned into pK7WG2D according to the procedure described above, giving pGCP-1 and pGCP-2.
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micro-salts and vitamins, 60 g.L-1 sucrose, 2.5 g.L-1 activated charcoal, 20 lM IAA (indole-3-acetic acid), 10 lM NOA (2-naphtoxyacetic acid) and 1 lM BAP (6-benzylaminopurine). A set of 16–20 embryos was placed in each Petri dish (60 mm of diameter). Cultures were incubated at 25 °C in the dark for 48 h before co-culture. The A. tumefaciens strain GV3101 (pMP90) was transformed with the expression vector of interest. It was cultured in YEB medium (Vervliet et al. 1975) supplemented with rifampicin at 50 mg/L for the selection of the chromosomal background of the bacterium, gentamycin at 20 mg/L for the selection of the helper plasmid pMP90, and spectinomycin at 75 mg/L for the selection of the pK7WG2(D)-derived plasmid. Before co-culture, the bacterium was precultured on a solid medium for 48 h at 28 °C. A single colony was transferred into 5 mL of liquid medium supplemented with 2 mM MgSO4 and grown for 8 h at 28 °C with agitation. Thirty lL of the pre-culture were used to inoculate 25 mL of the same medium. The culture was incubated at 28 °C for 14 h to reach a final OD600nm of 0.2–0.5. The cells were pelleted by a 5-min centrifugation at 5,000 rpm. Bacteria were resuspended in acidic (pH 5.7) YEB medium supplemented with acetosyringone (100 lM) for additional 2-h incubation at 28 °C with agitation. After pelleting, the cells were washed 3 times in a 10 mM MgSO4 solution and re-suspended in the same solution at a final OD600nm of 0.4–0.5. A droplet of 25–40 lL of bacteria suspension was put on each embryo. Cocultures were incubated for 48 h at 27 °C in the dark. Embryos were then transferred onto a fresh medium containing cefotaxime (200 mg/L) and carbenicilline (200 mg/L) to kill the agrobacteria. Cultures were incubated for 1 day at 27 °C in the dark, prior to GUS staining or RNA analysis. Embryos were frozen in liquid nitrogen and stored at -80 °C prior to RNA analysis.
Co-cultivation of somatic embryos with A. tumefaciens
GUS expression assay
Somatic embryos were obtained from embryogenic cultures of V. vinifera cv. ‘Chardonnay’ according to the protocol described in Maillot et al. (2009). Embryos at the cotyledonary-stage, approximately 4–8 mm in length, were collected and individually transferred onto a solid medium at pH 5.7 containing Murashige and Skoog (1962) half strength major salts,
Embryos were dipped into 0.3 mL of a GUS staining solution (Jefferson 1987) and vacuum-infiltrated (500 mmHg) in a desiccator for 10 min. Samples were incubated at 37 °C overnight. GUS activity was evaluated according to a scale ranging from 0 to 5, as shown in Fig. 2a. Marks were given as follows: 0 = no coloration; 1 = sparse, localised blue spots;
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2 = cotyledons exhibiting localised spots with pale and small blue zones; 3 = blue coloration covering up to approximately 50 % of the surface of cotyledons; 4 = blue coloration covering almost 100 % of the surface of cotyledons, with limited intensity; 5 = full and intense coloration of both cotyledons. A two-sided Wilcoxon rank sum test with continuity correction was performed to analyse the data, using the R statistical software, version 2.13.0 (RDC Team 2011). This nonparametric test allows the comparison of two populations by ranking each observation, the null hypothesis being that both populations are equal. The continuity correction factor was considered because of binomial distribution of the observations.
RNA analysis Total RNA isolation was performed from 100 mg of embryo tissues using ConcertTM Plant RNA Reagent (Invitrogen, Carlsbad, CA), according to the standard protocol. RT-PCR analyses were carried out using SuperscriptTM II Reverse Transcriptase (Invitrogen) following the manufacturer’s indications. Primers used for amplification of VvActin (GenBank Accession No. AF369525) were VvAct-5 (50 -TGCTATC CTTCGTCTTGACCTTG-30 ) and VvAct-3 (50 -GGA CTTCTGGACAACGGAATCTC-30 ) according to Schellenbaum et al. (2008). Mature amiRNAs were amplified and detected using stem-loop RT PCR adapted from VarkonyiGasic et al. (2007). Starting from 1 lg of total RNA, cDNA was synthesized with M-MuLV reverse transcriptase (Euromedex, Mundolsheim, France) in the presence of a stem-loop primer containing 6 nt complementary to the 30 sequence of the miRNA of interest. An endpoint PCR was performed using 4 lL of cDNA as a template with the following conditions: 95 °C for 1 min; 45 cycles of 95 °C for 30 s, 56 °C for 20 s, 72 °C for 30 s; 72 °C for 3 min. The forward primer contained a 15-nt sequence corresponding to the 50 sequence of the miRNA and 6 random nucleotides. The reverse primer was complementary to the stem-loop sequence. PCR products were resolved on a 3 % agarose gel and stained with GelRedTM (Biotium Inc., Hayward, CA) in 19 Tris–acetate-EDTA buffer. As a control for miRNA expression, the endogenous miR166c was simultaneously detected by stem-loop RT-PCR. Table S2 in supplementary material shows
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the primers used for miR166c, amiRCP-1 and amiRCP-2 detection.
Results Expression of the 35S:GUS construct One day after a 48-h co-cultivation with GV3101 (pMP90) carrying the pNJ-GUS plasmid, embryos showed necrosis on the cotyledon margins, characteristic of A. tumefaciens infection. After GUS expression assay by histochemical staining, intense and homogeneous blue coloration was detected, showing expression of the 35S:GUS cassette of pNJ-GUS at a high level. GUS expression was essentially localised in the two differentiated cotyledons and rarely visible in the rootlet. Expression of amiRNAs The developed transient expression system was used to test whether amiRNAs derived from modified athpre-miR319a could be expressed in grapevine. Embryos were co-cultivated for 48 h with GV3101 (pMP90) carrying pNJ-amiRCP-1 or pNJ-amiRCP-2, and harvested 1 day after co-cultivation for amiRNA detection. Two independent transient assays were carried out, using a set of 10–12 embryos (100 mg) for RNA isolation. An end-point stem-loop RT-PCR was performed to detect amiRNAs expression in cocultivated embryos. miR166c was chosen as an internal control, since its constitutive expression was previously detected in several grapevine tissues, such as leaves, roots, stems, inflorescences, berries, and in vitro callus cultures (Mica et al. 2010). As shown in Fig. 1, expression of miR166c and Vvactin was detected in all samples tested, assessing the integrity of total RNA. Expression of amiRCP-1 and amiRCP-2 was detected in embryos co-cultivated with GV3101 (pMP90) carrying the plasmids pNJamiRCP-1 and pNJ-amiRCP-2, demonstrating the functionality of the two constructions in transformed plant tissues. These two amiRNAs were not expressed in embryos co-cultivated with the Agrobacterium carrying the plasmid pK7WG2, used as a negative control. In parallel, reactions performed without reverse transcriptase gave no amplification product (data not shown).
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experiments 1 and 2) (Fig. 2c). These results indicate that amiRCP-1 and amiRCP-2 induce silencing of their corresponding target in GUS-sensors, when transiently expressed in grapevine somatic embryos.
Discussion Fig. 1 Detection of amiRNAs expression in ‘Chardonnay’ somatic embryos by stem-loop RT-PCR. amiRNAs were amplified in embryos co-cultivated with A. tumefaciens GV3101 (pMP90) carrying pNJ-amiRCP constructs. miR166c (stem-loop RT-PCR) and VvActin (RT-PCR) used as internal controls were expressed in all samples. Experiments 1 and 2 were performed independently
GUS-sensor assay To confirm the biological functionality of amiRCP-1 and amiRCP-2, we performed a GUS-sensor assay. Two independent transient assays were carried out for both amiRCP-1 and amiRCP-2. GUS-sensor constructs consisted of the GUS encoding sequence fused to the amiRNA target sequence (21 nt) in the 30 UTR. To investigate amiRNA-induced gene silencing, co-transformation assays were conducted with a mix of two Agrobacterium carrying a GUS-sensor construct (pGCP-1 or pGCP-2) and the corresponding pNJamiRCP construct (pNJ-amiRCP-1 or pNJ-amiRCP-2), respectively (ratio 1:3). Co-expression of the two constructs should result in the silencing of the GUS gene by targeting the added 21 nt (Fig. S4). As a control, pNJ-amiRCP constructs were replaced with the vector pK7WG2 by keeping the same ratio. After histochemical detection of the GUS protein activity, embryos showed different levels of coloration. GUS expression was evaluated following the 0–5 scale shown in Fig. 2a, according to the surface and intensity of cotyledons staining. For pGCP-1 (Fig. 2b), embryos co-transformed with pK7WG2 showed significantly higher levels of GUS expression compared to embryos co-transformed with pNJ-amiRCP-1, according to the two-sided Wilcoxon rank sum test with continuity correction (p = 5.5E-08 and p = 6.7E-05, respectively for experiments 1 and 2). Similarly, after a co-transformation with pNJ-amiRCP-2, a significant decrease of GUS-sensor expression was observed, compared to co-transformation with pK7WG2 (p = 1.1E-08 and p = 3.0E-05, respectively for
Here we outline a system for Agrobacterium tumefaciens-mediated transient expression of exogenous genes in grapevine embryos. Transient assays have been increasingly employed in plants and provide a rapid and efficient method for genetic and functional studies of plants. Moreover, the level of gene expression is generally higher than in stable transformants, allowing easier detection of mRNAs or proteins (Wroblewski et al. 2005). Particularly, in species recalcitrant to regeneration such as grapevine, transient assays represent an alternative to stable transformation which remains unpredictable and timeconsuming. Recently, two methods for leaf agroinfiltration in different species have been successfully adapted for use in several grapevine varieties (SantosRosa et al. 2008; Zottini et al. 2008). Either vacuum infiltration or injection with a needleless syringe was used to infiltrate leaf tissues with an A. tumefaciens suspension. The efficiency of both methods was shown to largely depend on the physiological status of the plant material. In fact, seemingly trivial characteristics such as the initial leaf position appear to be significant for efficient transformation. Interestingly, leaves from in vitro-grown plants were more susceptible to transformation than greenhouse-grown plants, thereby suggesting that tissue structure is critical for infection. In our system, co-cultivated embryos displayed a high sensitivity to A. tumefaciens as evidenced by intense blue staining of cotyledons. Somatic embryos consist of actively proliferating cells, and this characteristic potentially makes them more susceptible to transformation. This would also be consistent with the observation that tissues undergoing rapid cell proliferation, such as parenchyma of young leaves, exhibit higher levels of transient expression than do older tissues (Wroblewski et al. 2005). As a result, the amount of transformed cells in co-cultivated embryos was sufficient to perform RNA analysis and detect the expression of amiRNAs. We also demonstrated that co-expression of two different constructs is possible,
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Fig. 2 Level of GUS-sensor expression in ‘Chardonnay’ somatic embryos. a Scale used for assessment of GUS expression level, ranging from 0 (no coloration) to 5 (full and intense blue coloration of cotyledons). b, c Percentage of embryos in each class according to the defined 0–5 scale for
pGCP-1 (b) and pGCP-2 (c), in the presence and absence of amiRCP-1 or amiRCP-2, respectively. Experiments 1 and 2 correspond to two independent transient assays. n number of embryos
which enables in vivo studies of molecular interactions. Compared to leaf agro-infiltration, the co-cultivation of embryos with A. tumefaciens does not require specific equipment nor delicate manipulation of tissues. This method has the added benefit that it avoids potential injury to leaf tissues caused by injection via syringe or by vacuum pressure which is sometimes observed during leaf agro-infiltration (Zottini et al. 2008). Our system uses homogeneous cultures of somatic embryos obtained by recurrent somatic embryogenesis. We succeeded with the
cultivar ‘Chardonnay’, but this transient expression system could be adapted to other grapevine varieties, as supported by the successful transformation of young embryos of a few other Vitis vinifera cultivars, and one grapevine hybrid (Li et al. 2008). In addition, this system could be useful for transient transformation of other plant species. We used this transient assay to assess the functionality of amiRNAs targeting GFLV. The amiRNA technology is highly efficient and specific to knock down genes of interest. For example, the expression of amiRNAs targeting GUN4 and LEAFY genes led to
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phenotypes similar to those of null mutants in Arabidopsis, illustrating the specificity and effectiveness of these amiRNAs (Schwab et al. 2006). Here, we designed and cloned two amiRNAs targeting GFLV by modifying an Arabidopsis premiRNA, ath-pre-miR319a. amiRNA-mediated virus resistance has been shown in several species, mainly by targeting viral silencing suppressors (e.g. Niu et al. 2006; Ai et al. 2011) or other genes such as the CP gene (Jiang et al. 2011). In nepoviruses, no silencing suppressor has been characterised to date (Jovel et al. 2007; Dupuis 2010). Furthermore, grapevines have been historically transformed with the CP gene (Krastanova et al. 1995; Mauro et al. 1995; Xue et al. 1999; Vigne et al. 2004; Gambino et al. 2005; Maghuly et al. 2006; Valat et al. 2006; JardakJamoussi et al. 2009; Gambino et al. 2010), therefore we decided to design amiRNAs against the CP gene of GFLV. The target sequence was developed according to the sequence of the GFLV-F13 reference strain (Serghini et al. 1990; Ritzenthaler et al. 1991). amiRCP-1 and amiRCP-2, designed using WMD2 software, were predicted to efficiently target the CP gene. In support of the amiRNAs specificity, no offtargets were found within published sequences of Vitis species. Constructs containing ath-pre-miR319a modified with the sequences of amiRCP-1 and amiRCP-2 were transiently expressed in ‘Chardonnay’ somatic embryos. The resulting amiRNAs were detected in plant tissues by stem-loop RT-PCR, in which the specific amplification of miRNAs occurred by the use of a long stem-loop primer to initiate reverse transcription. This technique has been widely used for the detection and quantification of miRNAs (Chen et al. 2005; Varkonyi-Gasic et al. 2007) as well as amiRNAs (Tang et al. 2010). Its high sensitivity and rapidity makes it an attractive alternative to northern blot analyses which require several micrograms of RNA and preferably radioactive probes. Successful amplification of amiRNAs by stem-loop PCR is evidence that the transgene is expressed at sufficient levels to be easily detected in co-cultivated somatic embryos. The absence of amiRNA amplification in embryos cocultivated with A. tumefaciens carrying the vector pK7WG2 highlights the absence of endogenous miRNAs with sequences similar to those of amiRCP1 and amiRCP-2 in grapevine embryo tissues. Altogether, our results indicate that the modified ath-pre-
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miR319a was effectively processed to release amiRNAs targeting the CP gene of GFLV in the plant tissues. Expression of amiRNAs using this precursor was previously reported in other species such as Populus trichocarpa (Shi et al. 2010), tomato (Fernandez et al. 2009), tobacco (Jiang et al. 2011) and the moss Physcomitrella patens (Khraiwesh et al. 2008). Our results confirm the suitability of this precursor for designing plant amiRNAs. In order to demonstrate the efficiency of our constructs for silencing GFLV sequences, we used transient expression assays based on the use of GUSsensors. We performed co-transformations with a pGCP and the corresponding pNJ-amiRCP constructs. In the presence of amiRCP-1 or amiRCP-2, GUS expression was altered compared to the control. Differences in the level of GUS expression were readily observable after histochemical staining, and were ranked based on a 0–5 scale. We showed that GUS-sensors were significantly inhibited when coexpressed with amiRNAs, strongly suggesting that the decrease in GUS expression was a result of amiRNAmediated gene silencing. These GUS-sensor assays, with constructs easily obtained by a one-step PCRdirected mutagenesis, thereby allowed us to assess the complete functionality of the two developed amiRNAs. As suggested by the results obtained in experiment 1 (Fig. 2b, c), the amiRCP-2 could be more efficient than the amiRCP-1 for reducing the expression of the GUS-sensor. This could be explained by a better recognition of its target, possibly due to a better RISC-accessibility of this target (Kertesz et al. 2007), or by a higher level of amiRNA expression (Qu et al. 2007; Ai et al. 2011; Jiang et al. 2011). The real higher efficiency of amiRCP-2 should consequently be verified in tissues challenged with GFLV. Finally, our results are consistent with GUS-sensor assays performed for silencing sequences of Cucumber mosaic virus in N. benthamiana leaves (Duan et al. 2008). This infers that, if targets are RISC-accessible within the viral genome, amiRCP-1 and amiRCP-2 could potentially induce cleavage of viral RNA in GFLV-infected cells. Acknowledgments This study was funded by the French Ministry for Research. The authors thank Sascha Laubinger (University of Tuebingen, Germany) for his input concerning stem-loop RT-PCR, Marc Lollier (Universite´ de Haute-Alsace, France) for statistical analysis, Leon Otten (Institut de Biologie Mole´culaire des Plantes, France) for providing A. tumefaciens
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