Transgenic Res (2013) 22:1191–1205 DOI 10.1007/s11248-013-9721-8
ORIGINAL PAPER
Transgenic accumulation of a defective cucumber mosaic virus (CMV) replicase derived double stranded RNA modulates plant defence against CMV strains O and Y in potato Valentine Otang Ntui • Kong Kynet • Pejman Azadi • Raham Sher Khan • Dong Poh Chin • Ikuo Nakamura • Masahiro Mii Received: 8 February 2013 / Accepted: 31 May 2013 / Published online: 8 June 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract Cucumber mosaic virus is an important plant pathogen with a broad host range encompassing many plant species. This study demonstrates the production of transgenic potato lines exhibiting complete resistance to cucumber mosaic virus strain O and Y by post transcriptional gene silencing. Two constructs were used, one, pEKH2IN2CMVai, contains inverted repeat of 1,138 bp fragment of a defective CMV replicase gene derived from RNA2 of cucumber mosaic virus strain O (CMV-O), while the other, TRV-based VIGS vector (pTRV2CMVai), contains the same fragment of the replicase gene, but without inverted repeat. These constructs were used to produce transgenic potato
Electronic supplementary material The online version of this article (doi:10.1007/s11248-013-9721-8) contains supplementary material, which is available to authorized users. V. O. Ntui (&) K. Kynet R. S. Khan D. P. Chin I. Nakamura M. Mii Laboratory of Plant Cell Technology, Graduate School of Horticulture, Chiba University, 648 Matsudo, Matsudo, Chiba 271-8510, Japan e-mail:
[email protected] V. O. Ntui Department of Genetics/Biotechnology, Faculty of Science, University of Calabar, PMB 1115, Calabar, Nigeria P. Azadi Tissue Culture and Gene Transformation Department, Agricultural Biotechnology Research Institute of Iran (ABRII), P. O. Box 31535-1897, Karaj, Iran
lines of cultivar ‘Danshaku’, a susceptible genotype to CMV. Transgenic lines derived from pEKH2IN2CMVai accumulated small interfering RNA (siRNA) before and after virus challenge, whereas those derived from pTRV2CMVai showed siRNA expression after virus challenge. When transgenic lines were challenged with CMV-O or CMV-Y, four lines exhibited complete (100 %) resistance to both strains, whereas the other lines had high levels of resistance. Infectivity of CMV-O was lower than that of CMV-Y in the highly resistant plants. There were no significant differences with regard to resistance between plants derived from pEKH2IN2CMVai and those obtained from pTRV2CMVai. The presence of CMV-specific siRNA in the resistant phenotypes indicates that the resistance was acquired through RNA silencing. Keywords dsRNA PTGS Replicase gene siRNA Solanum tuberosum Virus induced gene silencing
Introduction RNAi-mediated silencing, generally referred to as post-transcriptional gene silencing in plants (Reviewed in Susi et al. 2004), is a coordinated series of sub-cellular events that ultimately lead to the posttranscriptional termination of gene expression. It is a mechanism in which RNAs are specifically degraded if they are similar in sequence to a transgene,
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especially if it is designed to produce double stranded (ds) RNA (Chuang and Meyerowitz 2000). The most important feature of RNA silencing is the cleavage of dsRNA into short dsRNA fragments known as ‘‘short interfering RNAs (siRNAs)’’ of 21–25 bp in length (Bonfim et al. 2007; Elbashir et al. 2001), by a dsspecific ribonuclease termed ‘Dicer’, which occurs in plants as a family of four enzymes with different functions (Schauer et al. 2002). The strand of siRNA complementary to the target RNA becomes incorporated into the RNA-induced silencing complex (RISC) leading to mRNA degradation and gene silencing (Bonfim et al. 2007). Since dsRNA is the potent activator, any RNA virus which replicates via dsRNA intermediate will induce PTGS in plants. Recently, virus induced gene silencing (VIGS) has been developed as one of the techniques of PTGS in plants. VIGS is a RNA-silencing technique that uses viral vectors carrying a fragment of a gene of interest to generate double-stranded RNA, which initiates the silencing of the target gene (Burch-Smith et al. 2006). This activates the anti-viral RNA silencing pathway, resulting in down-regulation of the host gene transcript. As compared to other PTGS-based methods requiring genetic transformation steps, a ‘‘functional knock-down’’ for a plant gene can be created using VIGS within a matter of weeks without having to go through the rigorous work of transformation and tissue culture (reviewed in Purkayastha and Dasgupta 2009). Several viral vectors have been developed for VIGS and they have been successfully used in reverse genetics studies of a variety of processes occurring in plants (Senthil-Kumar et al. 2007; Senthil-Kumar and Udayakumar 2006; Liu et al. 2002a; Ratcliff et al. 2001). Cucumber mosaic virus (CMV) is a tripartite, positive sense RNA virus, which infects and causes yield losses to many plant species. Few sources of natural resistance to the virus are known. The CMV genome consists of three genomic RNAs (1, 2 and 3), and all three genomic RNAs are necessary for systemic infections in plants (Palukaitis et al. 1992). RNAs 1 and 2 encode the 1a and 2a proteins, respectively, which are components of the CMV replicase. RNA 3 encodes two proteins, 3a movement protein and the viral coat protein, which is expressed from subgenomic RNA 4. The 3a protein facilitates the movement of CMV RNA from cell-to-cell, although this may be through stabilization of RNA,
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as accumulation of all viral RNA species is decreased in the presence of defective forms of the coat protein. The concept of pathogen-derived resistance has been used to engineer virus resistance against several virus groups, including CMV in many plant species. However, pathogen-derived resistance to CMV often show partial resistance or very narrow spectrum resistance to the virus. Various levels of resistance were achieved via replicase-mediated resistance (Anderson et al. 1992). The occurrence of CMVspecific short RNAs derived by expression of dsRNA in transgenic tobacco resulted in high levels of resistance to the virus (Kalantidis et al. 2002). Therefore, RNAi-mediated gene silencing especially the expression of dsRNA remains an important tool to obtaining complete resistance to CMV. Potato is the world’s fourth most agronomically important food crop after wheat, maize and rice, notably due to its high productivity and its high starch, vitamin and protein content. It is affected by many diseases, producing substantial economic losses worldwide. Viruses are very wide spread in this crop and cause severe yield loss (Missiou et al. 2004). It has been reported that potato is highly susceptible to CMV at high temperatures (exceeding 24 °C) and that CMV could pose serious threat to potato crops grown in warmer climates or in moderates climates in semitropical and high land tropical areas (Celebi-Toprak et al. 2003). Thus, producing potato cultivars which would be resistant to CMV even at high temperatures would be of great economic importance. Currently, there is no report on the production of transgenic potato resistant to CMV by PTGS. In this study, we generated two constructs, which were used to produce potato lines exhibiting complete resistance to cucumber mosaic virus. In one, we generated an RNAi construct containing inverted repeat of 1,138 bp fragment of a defective CMV replicase gene derived from RNA2 of cucumber mosaic virus strain O (CMV-O). In the other, we used TRV-based VIGS vectors containing the same fragment of the replicase gene. Through genetic transformation techniques, we produced transgenic potato lines of cultivar ‘Danshaku’ (a susceptible genotype to CMV) that express fragments of the replicase gene sequence of CMV in the form of intramolecular dsRNA. Of the transgenic lines obtained, 6 lines from each construct were tested for resistance to CMV-O or CMV-Y at 24 and 30 °C. Four of the transgenic lines
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tested exhibited complete resistance to the CMV strains at both temperatures. The presence of homologous CMV siRNA in the plants correlates with the resistant phenotype showing that the resistance was acquired through RNA silencing.
Materials and methods RNAi vector construction To construct a binary vector containing sense and antisense orientation, plasmid Spuc IG221 was cut with SacI and SalI, and T4 DNA polymerase enzyme (New England Biolabs) was used to produce blunt end. Then, a fragment of reading frame cassette containing ccdB and chloramphenicol resistance gene (Invitrogen, New Zealand) was inserted. The Ligated plasmid was then transferred to E. coli strain DB3 by heat shock method and selected on LB agar medium containing 50 mg L-1 chloramphenical and spectinomycin. After confirming the insertion of ccdB fragment by restriction analysis, the plasmid was cut with XbaI. The ccdB fragment was inserted again in the antisense orientation to the blunt end fragment obtained previously and transferred to E. coli DB3. The orientation of two ccdB inserts was investigated by restriction analysis using EcoRI enzyme. The plasmid with sense and antisense-ccdB was cut and inserted to HindIII site of the binary vector, pEKH2 (Nakamura et al., unpublished) which contains selectable marker gene, kanamycin and hygromycin resistant genes yielding the RNAi plasmid vector pEKH2IN2. A 1,138 bp fragment spanning nucleotides 391–1,528 of a defective CMV replicase gene from RNA2 (CMV2-GDD) of cucumber mosaic virus strain O (CMV-O) was cloned by RT-PCR technique. A pair of primers, CMVO2ai-5P and CMVO2ai-3P (Supplementary Table 1), which anneal the vector pBluescript SK (-) containing the CMV were used. The resulting PCR product with attB1 and attB2 was cloned into Gateway entry vector, pCRÒ8/GW/TOPOÒ (Invitrogen, New Zealand) according to the instruction manual. The pCRÒ8/GW/TOPO contains attL1 and attL2 recombination sites. The transformants were sequenced and analyzed. The correct transformant containing the replicase gene was subcloned in the sense and antisense orientation to the RNAi destination vector, pEKH2IN2, described above, by eLR
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clonaseTM (Invitrogen, New Zealand) recombination reaction. The product was transformed into TOP10 chemical competent cells (Invitrogen, New Zealand) and selected on kanamycin-containing LB plates. Clones were verified by digestion with EcoRI. The clone with the correct insert (1,138 bp) was mobilized to Agrobacteriun strain EHA105 by triparental mating. A 278 bp fragment of catalase 1 1st intron derived from caster bean separates the 1,138 bp replicase gene sequences in the inverted repeat to ensure stability of the construct. In this construct, the CMV gene is driven by cauliflower mosaic virus (CaMV) 35S promoter. Cloning of CMV replicase gene into TRV-based VIGS vector pTRV1 (accession name YL192) and pTRV2 (accession name YL 156) VIGS, were obtained from Arabidopsis Biological Resource Centre (ABRC), Ohio State University, Columbus, USA. These vectors have been described elsewhere (Liu et al. 2002a). The pTRV2 destination vector contains the attR1 and attR2 recombination sites. The 1,138 bp fragment of the replicase gene of CMV after amplification and subsequent transfer to the Gateway entry vector was incubated with the pTRV2 destination vector and LR clonase enzyme was added. The mixture was transformed to TOP10 chemical competent cells and selected on LB plates containing kanamycin. Clones were verified by digestion with EcoRV or a mixture of HindIII and XhoI. The colony with the correct insert was transferred to Agrobacterium strain EHA105 by triparental mating. In this construct, the 1,138 bp fragment of the CMV gene was positioned between the coat protein (CP) gene of tobacco rattle virus (TRV) and nopaline synthase terminator (nosT) in a T-DNA, under the control of duplicate CaMV 35S promoter. Also, plasmid pTRV1 without the replicase gene sequence of CMV was used in this study. The pTRV1was mobilized to Agrobacterium strain EHA 105 by freeze and thaw. Plant material, transformation, and PCR analysis Potato tubers of variety ‘Danshaku’ were used for transformation. A. tumefaciens strain EHA105 containing pEKH2IN2CMVai, pTRV1 or pTRV2CMVai were grown overnight, re-suspended in inoculation medium and left at room temperature for 30 min before
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inoculation. Tuber discs, 2 mm thick and 5 9 5 mm were inoculated with A. tumefaciens containing pEKH2IN2CMVai, or a mixture of pTRV1 and pTRV2CMVai for 10 min. After inoculation and cocultivation, the explants were cultured and transformed potato plants were regenerated as previously described (Khan et al. 2006). To detect the presence of foreign genes, one well rooted shoot from each explant was selected as an independent clone and screened by PCR using primers specific for CMV (CMVO2ai-5P and CMVO2ai-3P) and NPTII (NPTII-F and NPTII-R) (Supplementary Table 1). Of the PCR positive plants, 6 lines from each constructs were multiplied and grown in vitro and in the greenhouse for further analysis. Southern hybridization Genomic DNA was extracted from fresh leaves of transgenic and control plants. Fifteen lg of the DNA was digested overnight with HindIII, separated on a 0.8 % agarose gel, transferred, and probed with CMV PCR-DIG labelled probe. Prehybridization (3 h) and hybridization (overnight) were carried out at 38 and 64 °C. Hybridization signals were detected by exposing the membrane to a detection film (Lumi-Film Chemiluminescent Detection Film; Roche Diagnostics, Mannheim, Germany) for 20 min. Northern blot analysis of total RNA Total RNA was isolated from potato leaves using Guanidine thiocyanate, 15 lg of the total RNA was denatured and separated on 1.5 % agarose gel as previously described (Ntui et al. 2010). Separated RNAs were transferred to nylon membrane (Immobilon-Ny ? Transfer Membrane; Millipore Co, Billerica, MA, USA) by capillary blotting and hybridized with CMV PCR-DIG labelled DNA probe at 50 °C in high SDS hybridization buffer. Posthybridization washes were done twice with 29 SSC/0.1 %SDS and twice with 0.19 SSC/0.1 %SDS at 50 °C. Hybridized probes were visualized using CDP-star reagent (Roche Applied Science). Analysis of plants for siRNA before and after virus challenge Transgenic potato lines together with the control plants were analyzed for siRNA accumulation by
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northern blot hybridization before and after challenge with CMV-O or Y. Total RNA was isolated as described above, small RNAs were enriched from the total RNA by polyethylene glycol (PEG; MW8000) as described by Smith and Eamens (2012). Thirty micrograms of small RNAs were electrophoresed on a 17 % polyacrylamide (acrylamide:bisacrylamide 19:1) gel containing 7 M urea and 10x TBE buffer (0.9 M Tris–HCl, 0.9 M Boric acid, 20 mM EDTA). The gel was stained with 0.5 x TBE containing ethidium bromide (0.5 lg/ml) and photographed. Nucleic acids from gel were transferred to Immobilon-NY ? membrane (Millipore Corporation, Billerica, MA, USA) in a semi-dry cell (Semi-dry blotting apparatus NA-1512, Nippon Edido, Tokyo, Japan) for 1 h at 10 V/400 mA. The RNAs were UV fixed and hybridized with CMV RNA probe obtained by in vitro transcription of CMV-replicase gene in the antisense and sense orientation using T7 RNA polymerase (Roche Applied Science, Indianapolis, IN, USA). In order to improve the signal, the probe was hydrolyzed with carbonate buffer (60 mM sodium carbonate and 40 mM sodium bicarbonate), and incubating at 60 °C for 3 h, according to the formula reported by Hamilton and Baulcombe (1999). Prehybridization (30 min) and hybridization (overnight) were performed at 37 °C. Posthybridization membrane was washed with 29 SSC for 2 9 5 min at room temperature, followed by 0.19 SSC/0.2 %SDS for 2 9 15 min at 50 °C. Signals for siRNA were detected using CDP-star (Roche Applied Science), as described in the DIG system and DIG application manual. Evaluation of transgenic potato lines for resistance to CMV-O or Y- in vitro and ex vivo bioassays For in vitro bioassay, 4 week-old in vitro transgenic and wild-type plants were sap inoculated with CMV-O or CMV-Y inoculums obtained from CMV-O and CMV-Y-infected tobacco plants. Sap extract was prepared in 50 mM sodium phosphate buffer containing 0.4 % sodium sulphite, pH 7.5. The sap collected after a brief centrifugation (3,500 rpm for 5 min) was used directly by gently rubbing onto two completely opened young leaves with the help of carborundum as an abrasive agent (Mbanzibwa et al. 2009; Azadi et al. 2011). The inoculated plants were maintained in a growth room at 25 ± 2 °C, 16 h photoperiod, 30–40 lmol m-2 s-1 cool white fluorescent light
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and 70 % relative humidity. The plants were observed daily for disease development; pictures were taken at 30 days post inoculation (dpi). Five plants per line were inoculated with each virus strain. To evaluate transgenic potato plants for resistance to CMV ex vivo, rooted plantlets were hardened and grown in the greenhouse. After 6 weeks, 5 plants per line per construct were inoculated with CMV-O or CMV-Y as described above and maintained in a walkin growth chamber at 24 or 30 °C. In all, 20 plants per line were used for each assay. The inoculated plants were monitored for symptom development every alternate day for a period of 6 weeks. The number of plants showing mosaic symptoms was recorded. Also, the number of leaves per plant showing mosaic symptoms was counted and recorded at 15 and 30 days post-inoculation (dpi). Disease symptom severity, the amount of leaf area affected by the disease was expressed on a scale of 0–4 grades (0, no symptom; 1, faint mosaic, \25 % of leaf area; 2, yellow mosaic malformation, 26–50 % of leaf area; 3, severe mosaic up to 75 % leaf area and 4, severe mosaic, [76 % of leaf area), and disease index (D.I) calculated as described by Krishnamoorthy et al. (2004) and Ntui et al. (2010). P b n D:I ¼ ðN 1ÞT where n: number of leaves in each grade; b: grade; N: number of grades used in the scale; T: total number of leaves scored. Plants with a disease index of 0.0 % were considered as not susceptible (completely resistant), those with a disease index\25 % as having high resistance, those with a disease index of 25.1–50 % as having moderate resistance, those with a disease index of 50.1–75 % as susceptible, and those with a disease index [75 % as highly susceptible. RT-PCR analysis of CMV-O challenged transgenic plants Total RNA was extracted from transgenic and wildtype plants after challenge with CMV-O using Guanidine thiocyanate as described above. The RNAs were extracted 4 weeks after infection from new leaves that emerged from infected plants. After DNase treatment, the RNAs were subjected to RT-PCR using
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the Superscript III RNase H reverse transcriptase (RT) kit (Invitrogen). The cDNAs were then used as templates for the amplification of CMVO-CP using primers CMV-O CP-F and CMV-O CP-R (Supplementary Table 1). Also, PCR analysis of rice actin gene (RAc1; X16280) was performed as a control to check the quality of cDNA synthesized in the RT-PCRs using specific primers (Supplementary Table 1). Dot-immunobinding assay (DIBA) New leaves produced from infected transgenic and wild-type plants were collected for DIBA. Ten milligram of each leaf sample were ground in a centrifuge tube containing 100 ll TBS buffer (20 mM Tris/HCl, 500 mM NaCl, pH 7.5, and 0.5 % Tween 20) to release the sap. The mixture was centrifuged at 10,000 rpm for 10 min, 25 °C. Then, 10 ll each of two concentrations; 1 and 10-1 (10 times dilution with TBS buffer) were gently dropped on separate positions on the surface of a PVDF membrane (Bio-Rad), which has been partitioned according to the number of samples and pre-soaked in TBS buffer. The membrane was air-dried and submerged in Western blot blocking solution for 30 min, followed by soaking in primary antibody raised against CP of CMV for 1 h and then washed three times (10 min x 3) in TBS buffer. To detect the presence of CMV, the membrane was submerged in ECL anti-rabbit lgG immunoglobulin (Boehringer) as the secondary antibody and stained with VEC SK-4800 kit (vector NovaRED substrate kit for peroxidase, Burlingame). All incubations were done at room temperature. Statistical analysis The experiments on the effect of temperature were subjected to analysis of variance (ANOVA) test using a completely randomised design (CRD). Means were separated by least significant difference (LSD) test at the 5 % probability level (p B 0.05). All computations were performed using SPSS 17.0 statistical package for Windows (SPSS, Inc., Chicago, IL, USA). All percent data were checked for normality distribution and subjected to arc sine (Hx) transformation before statistical analysis.
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Results Generation of the RNAi constructs and potato transformation To engineer potato resistant against different strains of CMV, we generated two constructs. In one, we produced a plasmid containing inverted repeat of 1,138 bp fragment spanning between nucleotides 391–1,528 of a defective CMV replicase gene derived from RNA2 of cucumber mosaic virus strain O (CMVO). We selected this fragment because the sequence analysis from database shows that majority of the strains show similarity of over 95 % in this region. To ensure stability of the inverted repeat in Escherichia coli, the two cDNA fragments were separated by a 278 bp unrelated spacer sequence. Although the spacer is short, shorter spacers have been found to be relatively stable and effective (Reviewed in Hirai and Kodama 2008). The whole cassette was cloned in the plant transformation vector, pEKH2IN2 under the control of CaMV 35S promoter, yielding pEKH2IN2CMVai (Supplementary Fig. 1A). In the other, we used TRV-based VIGS vector containing the same fragment of the replicase gene. The cDNA fragment was inserted in the attR1 and attR2 recombination sites of the binary vector pTRV2 under the control of duplicated CaMV 35S promoter, yielding pTRV2CMVai (Supplementary Fig. 1B). This vector does not contain inverted repeat of the CMV gene, however, when the vector is introduced into plants, the transgene synthesizes another RNA strand during the course of viral infection, producing double stranded RNA (dsRNA). Also, pTRV1, without the CMV fragment (Supplementary Fig. 1C) was mobilized into A. tumefaciens and used in mixture with pTRV2CMVai. Potato tuber discs of variety ‘‘Danshaku’’ were transformed with the binary vectors described above via A. tumefaciens-mediated transformation. A total of 40 independent plant lines were regenerated. Screening of the transgenic lines by PCR resulted in the amplification of the 1,138 bp fragment of the CMV gene and 800 bp fragment of the nptII gene in 31 out of the 40 plantlets (data not shown). Of these lines, 15 belonged to pEKH2IN2CMVai and 16 belonged to pTRV2CMVai. Since all the transgenic lines were phenotypically normal and no stunting was observed, 6 lines were selected at random from each construct (pEKH2IN2CMVai and pTRV2CMVai) and subjected to Southern and northern blot analyses to
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determine the copy number and expression of the transgene. Southern blot analysis revealed 5 plants with a single locus and 7 with 2 or more loci (Supplementary Fig. 2A). Northern hybridization revealed the accumulation of transgene transcript in transgenic plants before (Supplementary Fig. 2B) and after challenge with CMV (Supplementary Fig. 2C). Enhanced resistance of transgenic potato plants to CMV strains O and Y in vitro The 12 lines used for Southern and northern blot analyses (6 from each construct) and the wild-type plants were challenged with either CMV strain O or Y in vitro. Upon inoculation with CMV-O, the wild-type plants developed systemic symptoms in leaves in a few days, whereas the transgenic lines showed no sign of disease development (data not shown). Similar observations were made upon inoculation with CMVY. At 30 days post inoculation (dpi), more than 80 % of the leaves of the wild-type plants showed characteristic severe mosaic symptoms. The effect was more severe in the wild-type plants inoculated with CMV-Y (Fig. 1B, a, b) than in those inoculated with CMV-O (Fig. 1A, a, b). None of the transgenic plants inoculated with CMV-O, irrespective of the construct from which they were derived, showed sign of disease development under the same period. In contrast, at 30 dpi, 2 transgenic lines (T6 and T9) showed moderate symptoms after inoculation with CMV-Y (Fig. 1B, c, d, only line T6 is shown here). Challenging transgenic potato plants with CMV-O or Y ex vivo To further screen transgenic plants for resistance to CMV, 6 week-old hardened transgenic and wild-type plants were challenged with CMV-O or Y, and grown in a walk-in growth chamber. For each CMV strain, 5 plants per line per construct were analyzed. Altogether, 20 plants per line were used for each assay. Typical mosaic symptoms appeared on new emerging young leaves of the wild-type plants 2 days post inoculation (dpi). At 15–30 dpi, all the wild-type plants showed characteristics severe mosaic symptoms, which included mild to severe mosaic bleaching of leaves (Fig. 1C, D), leaf deformation and stunted growth. Among the transgenic plants tested, 4 lines, E1, E5, E9 and T1, were asymptomic, under the period
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Fig. 1 Resistance of Solanum tuberosum lines to (cucumber mosaic virus) CMV infection. A, B In vitro evaluation of Solanum tuberosum lines infected with CMV-O (A) or CMV-Y (B). a, b Wild-type plants, arrows indicate mosaic symptoms. c, d Transgenic line T6 derived from pTRV2CMVai showing 100 % protection after infection with CMV-O and mild symptoms (arrow) after infection with CMV-Y. e, f Transgenic line E1 derived from pEKH2IN2CMVai showing 100 % resistance. The Upper row represents start of inoculation. The
lower row represents 30 days post inoculation (dpi). C, D Ex vivo evaluation of Solanum tuberosum lines infected with CMV-O (C) or CMV-Y (D). WT, wild type plant, full symptoms, and plants reduced to vines. T6, transgenic line 6 derived from pTRV2CMVai. This line showed mild symptoms after infection with CMV strain Y. E1, transgenic line 1 derived from pEKH2INCMVai, resistant phenotype. Photos were taken at 30 days post inoculation (dpi)
of study, 6 weeks (Fig. 1C, D, data are shown only for line E1 and T6). The other 7 transgenic lines showed mild to moderate symptoms at 30 dpi. Of the two CMV strains used, CMV-Y caused more severe effect (Fig. 1D) than CMV-O (Fig. 1C), irrespective of the transgene construct used, suggesting that the transgenic plants are more resistant to CMV-O, the strain from which the transgene sequence was derived than from a closely related strain Y. We also examined the effect of temperature on resistance of transgenic potato plants to CMV, ex vivo. One group of transgenic plants was challenged with CMV-O or Y, and grown at 24 °C, while another group was grown at 30 °C after challenge with either
strain. First, we checked the infectivity of CMV-O and Y by recording the number of plants per line showing mosaic symptoms, over a period of 15 days (Fig. 2). Only four selected transgenic lines and the wild-type plant are presented in Fig. 2. In the plants inoculated with CMV-O, disease symptoms appeared in the wild type plants 3 dpi at both temperatures. In the transgenic lines, some of the lines remained without symptoms, under the period of study, while others showed delayed symptoms, which started on 7 and 5 dpi, at 24 and 30 °C, respectively (Fig. 2A, B). By 12 dpi, all wild-type plants were infected, whereas less than 35 % of the transgenic lines showed disease symptoms. Upon inoculation with CMV-Y, symptoms
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Fig. 2 Symptom development in wild-type (WT) and representative T0 transgenic potato lines after inoculation with CMV-O A, B or CMV-Y C, D and grown at 24 °C A, C or 30 °C B, D. E3 and E4, transgenic lines derived from pEKH2IN2CMVai; T6 and T10, transgenic lines derived from pTRV2CMVai. For each CMV strain, 5 plants per line per construct were analyzed. Altogether, 20 plants per line were used for each temperature assay
were visible in the wild-type plants 2 dpi, while transgenic plants showed mosaic symptoms 4 and 3 dpi, at 24 and 30 °C, respectively (Fig. 2C, D). The pattern of disease development is summarized in Fig. 2. Second, we measured the degree of virulence by evaluating the number of leaves per plant showing mosaic symptom every alternate day, but the final classification was recorded at 15 and 30 dpi. Symptom severity was calculated according to a standard scale of 0 (asymptomic) to 4 (severe mosaic,[76 % of leaf area). Based on the above scale, infection indices were calculated according to the formula shown in experimental procedures. For plants inoculated with CMV-O, 4 lines, E1, E5, E9 and T1 were symptom free at 15 and 30 dpi, irrespective of the growth conditions (Table 1). These plants were classified as not susceptible (NS, infection index = 0 %) to the CMV strain, at the temperatures under study. In the remaining transgenic lines, varying degree of disease indices, which were generally less than 15 %, were observed (Table 1). Therefore, these transgenic lines were classified as having high resistance (HR), according to the disease rating. Compared to the transgenic lines, the wild-type plants had higher disease indices, which were greater than 75 %, hence they were classified as being highly susceptible
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(Tables 1). Disease indices recorded for 30 °C were higher than those recorded for 24 °C (Table 1). When CMV-Y was inoculated, the pattern of resistance was similar to the one observed for CMVO. The four transgenic lines, E1, E5, E9 and T1 remained symptom free even at 30 °C, 30 dpi (Table 1). Disease indices for the other transgenic lines were lower than 25 %, therefore, they were considered as having high resistance. The wild-type plants, irrespective of the culture conditions, had very high infection index and showed high susceptibility (Table 1). Comparing the two temperatures, plants grown at 24 °C had lower indices than those grown at 30 °C; CMV strain and days post inoculation notwithstanding. A comparison of disease severity caused by the CMV strains showed that plants inoculated with CMV-O had lower disease indices than those inoculated with CMV-Y both at 15 dpi (Supplementary Fig. 3A) and at 30 dpi (Supplementary Fig. 3B). Analysis of small interfering RNA (siRNA) expression Prior to challenging the plants with CMV, northern blot analysis was carried out to detect CMV- specific siRNAs in transgenic and wild-type plants. Six weekold hardened green house plants were used for the
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Table 1 Disease indices at 15 and 30 days post inoculation (dpi) of wild-type (WT) and T0 transgenic potato plants inoculated with CMV-O and Y, and grown at 24 °C or 30 °C Line
Disease indexa (%) 15 dpi 24 °C
Mean disease indexb (%)
30 dpi 30 °C
24 °C
Disease ratingc
30 °C
CMV-O WT
63.6 ± 0.98f
80.1 ± 1.16e
84.2 ± 0.17e
98.3 ± 0.23 g
81.5 ± 0.64c
HS
E1
0 ± 0a
0 ± 0a
0 ± 0a
0 ± 0a
0 ± 0a
NS
E3
3.4 ± 0.18b
5.9 ± 0.24b
8.3 ± 0.23bc
8.8 ± 0.23b
6.6 ± 0.22b
HR
E4
2.9 ± 0.41b
5.6 ± 0.64b
8.8 ± 0.13c
10.3 ± 0.22 cd
6.9 ± 0.35b
HR
E5 E9
0 ± 0a 0 ± 0a
0 ± 0a 0 ± 0a
0 ± 0a 0 ± 0a
0 ± 0a 0 ± 0a
0 ± 0a 0 ± 0a
NS NS
E11
6.6 ± 0.22c
10 ± 0.38 cd
9.1 ± 0.53c
11 ± 0.18de
9.2 ± 0.33b
HR
T1
0 ± 0a
0 ± 0a
0 ± 0a
0 ± 0a
0 ± 0a
NS
T3
8.3 ± 0.4 de
8.6 ± 0.45c
7.1 ± 0.32b
9.7 ± 0.57bcd
8.42 ± 0.44b
HR
T6
8.9 ± 0.54e
9.4 ± 0.18 cd
10.8 ± 0.09d
11.7 ± 0.14e
10.2 ± 0.24b
HR
T9
7 ± 0.57 cd
10.5 ± 0.39d
7.1 ± 0.56b
11.7 ± 0.79e
9.1 ± 0.58b
HR
T10
7.8 ± 0.5cde
9.7 ± 0.43 cd
11.8 ± 0.28d
13.9 ± 0.12f
10.8 ± 0.33b
HR
T13
7.8 ± 0.75cde
9 ± 0.58 cd
11.4 ± 0.25d
9.4 ± 0.11b
9.4 ± 0.42b
HR
LSD
1.32
1.57
1.33
1.35
6.11
CMV-Y WT
64 ± 0.86 g
89 ± 1.0 g
88.2 ± 0.11 h
106.6 ± 0.19 h
86.9 ± 0.54c
HS
E1
0 ± 0a
0 ± 0a
0 ± 0a
0 ± 0a
0 ± 0a
NS
E3
5 ± 0.18b
8.8 ± 0.12b
14.7 ± 0.23 cd
15.9 ± 0.2b
11.1 ± 0.16ab
HR
E4
6.3 ± 0.3c
12.5 ± 0.15d
13.9 ± 0.13b
22.4 ± 0.23c
13.77 ± 0.2b
HR
E5
0 ± 0a
0 ± 0a
0 ± 0a
0 ± 0a
0 ± 0a
NS
E9 E11
0 ± 0a 8.3 ± 0.22d
0 ± 0a 10.3 ± 0.22c
0 ± 0a 14.5 ± 0.16c
0 ± 0a 15.8 ± 0.33b
0 ± 0a 12.2 ± 0.22ab
NS HR
T1
0 ± 0a
0 ± 0a
0 ± 0a
0 ± 0a
0 ± 0a
NS
T3
9.4 ± 0.22ef
15.4 ± 0.08e
14.5 ± 0.19c
23.4 ± 0.34d
15.67 ± 0.21b
HR
T6
10 ± 0.15f
17.3 ± 0.2f
19.7 ± 0.04 g
29.7 ± 0.48 g
19.17 ± 0.21b
HR
T9
8.3 ± 0.2d
15.6 ± 0.9e
15.8 ± 0.33e
25 ± 0.15e
16.2 ± 0.40b
HR
T10
9.7 ± 0.2f
17.2 ± 0.24f
16.7 ± 0.04f
26.4 ± 0.27f
17.5 ± 0.19b
HR HR
T13
8.8 ± 0.13de
16.7 ± 0.22f
15 ± 0.32d
25 ± 0.54e
16.4 ± 0.30b
LSD
0.821
1.17
0.48
0.80
12.63
a
Disease indices were calculated using the equation shown in experimental procedures after severity scores as indicated in Fig. S6
b
Mean disease index is the average of the pool of disease indices calculated at 15 and 30 days post inoculation
c
Disease rating is based on the mean disease index. Lines with a disease index of 0 % were considered not susceptible (NS, 100 % resistance), those with a disease index \25 % as having high resistance (HR, [75 % resistance), those with a disease index of 25.1–50 % as being moderately resistant (MR, 50–74 % resistance), those with a disease index of 50.1–75 % as being susceptible (S, 25–49 % resistance), and those with a disease index [75 % as highly susceptible (HS, \25 % resistance), under the period of study. E1 to E11, transgenic lines derived from pEKH2IN2CMVai; T1 to T13, transgenic lines derived from pTRV2CMVai. Mean values followed by different lower case letters along a vertical array for a particular CMV strain denote a statistically significant difference at p B 0.0 5, as determined by Least Significant difference (LSD) test
analysis. To exclude a potential latent infection with CMV, which might result in virus-derived siRNA that are not of transgene origin, the plants were subjected
to RT-PCR and DIBA. As expected, no CMV signal was detected in any of the lines studied (data not shown). All transgenic plant lines (lines E1-E11),
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which were derived from pEKH2IN2CMVai were found to accumulate siRNAs. Conversely, no siRNAs were detected in the wild-type plants and transgenic lines (T1-T13) derived from pTRV2CMVai (Fig. 3A). After challenge with either CMV strain O or Y, at day 15, the wild-type plant and three transgenic lines selected from each construct were analyzed for the presence of siRNA. The analysis showed detectable levels of siRNAs in all the transgenic lines tested even in the lines derived from pTRV2CMVai (Fig. 3B). This suggests that in pTRV2CMVai, a VIGS vector, synthesizes dsRNA, which is cleaved to siRNAs only during the course of viral infection. siRNA signal was also observed in the inoculated susceptible wild-type plants. We also tested the effect of temperature on the accumulation of siRNA. Transgenic and wild-type plants were inoculated with either strain of CMV and grown at 24 or 30 °C. At 15 dpi, northern blot analysis was performed to detect the expression of siRNA. The result showed accumulation of siRNA signals in all transgenic and wild-type plants tested (Supplementary Fig. 4). High levels of siRNA expression were noted in transgenic plants cultivated at 30 °C than in those grown at 24 °C. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of CMV-O challenged potato plants At 15 dpi, total RNA was extracted from leaves of transgenic and wild-type plants challenged with CMV-O and subjected to RT-PCR to detect CMV-O coat protein (CP) using primers CMVO-CP-F and CMVO-CP-R (Supplementary Table 1) designed to amplify 600 bp CMV-O CP. Amplification of the viral RNA was not detected in all 6 transgenic lines (E1E11) derived from pEKH2IN2CMVai (Fig. 4A) and 4 of the transgenic lines (T1, T3, T9 and T13) derived from pTRV2CMVai (Fig. 4A). In lines T6 and T10, very low signal of the CP messages were seen (Fig. 4A). Clear amplification of viral RNA was detected in the wild-type plants (Fig. 4A). Dot-immunobinding assay analysis of CMV-O and Y challenged plants To confirm the result of RT-PCR we did Dotimmunobinding assay to examine the presence of the
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CMV in new leaves of the challenged plants. Two control (tobacco and potato) and 6 transgenic lines were used for the assay. The analysis indicate that the leaves of tobacco and potato infected control plants showed high intensity of CP signal of CMV-O and Y even at low concentration (10-1, 10 times dilution) compared to the transgenic plants (Fig. 4B). No signals of the virus was detected in lines E1, E3, E4 and T1 at both concentration of plant sap, whereas in lines T6 and T10, very low intensity of CMV-Y CP signal was detected. This result is in agreement with the RT-PCR of CP mRNA reported earlier. Correlation of copy number of the transgene and disease index of transgenic plants In view of the variable response of transgenic plants to CMV we decided to check whether there is a correlation between copy number of the transgene and disease index. Disease index obtained at 30 dpi were used for the analysis. The results show insignificant positive relationship (R = 0.2980 to 0.3369; p [ 0.05) between these two variables both at 24 and 30 °C, irrespective of the CMV strain used (Supplementary Fig. 5), suggesting that the disease index or the levels of resistance observed in the transgenic lines is independent of the transgene copy number. For example, among the plants that showed complete resistance to both CMV strains, one line (E9) had one transgene locus; two lines (T1 and E1) had two transgene loci; and one line (E5) had 3 transgene loci. Furthermore, 4 of the transgenic lines having one transgene locus each, exhibited high resistance to both strains of CMV, while lines T6 and T10 having 7 and 6 transgene copies, respectively, also showed high resistance similar to the one observed in the lines having one copy of the transgene.
Discussion Post transcriptional gene silencing has been applied successfully in different plant species to protect them from RNA viruses, and has led to the development of virus resistant crops (Patil et al. 2011; Yadav et al. 2011, Krubphachaya et al. 2007; Missiou et al. 2004; Zhang et al. 2005; Kalantidis et al. 2002; Liu et al. 2002b). The aim of the present work was to engineer strong CMV resistance in potato by PTGS. To achieve
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Fig. 3 Northern blot analysis of defective CMV replicase specific siRNA accumulation in potato plants before (A) and after (B) inoculation with CMV-O or CMV-Y. The inoculated plants were analyzed for siRNA accumulation at 15 days post inoculation (dpi) by hybridization with CMV RNA probe
obtained by the in vitro transcription of CMV-replicase gene in the antisense and sense orientation using T7 RNA polymerase. WT, wild type; T1 to T13, transgenic lines derived from the pTRV2CMVai; E1-E11, transgenic plants derived from pEKH2IN2CMVai
Fig. 4 Screening of transgenic plants for detection of CMV virus after infection. A RT-PCR analysis for the detection of coat protein (CP) of cucumber mosaic virus in transgenic lines infected with CMV-O. Fifteen days after infection, total RNA was extracted from the plants and tested for the presence of the CP using CP labelled probe. Rice actin (RAc1) gene was used as an internal control for RNA input. WT, wild type; T1 to T13, transgenic lines derived from the pTRV2CMVai; E1-E11,
transgenic plants derived from pEKH2IN2CMVai. B Dotimmunobinding assay for detection CP of cucumber mosaic virus in potato plants infected with CMV-O (upper row) or CMV-Y (lower row). TIC, Tobacco infected control plant. PIC, Potato infected control plant. E1-E4, transgenic lines derived from pEKH2IN2CMVai. T1, T6 and T10, transgenic lines derived from pTRV2CMVai. 1 and 0.1 (10 times dilution) represent the concentration of plant sap used
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this, we produced two constructs. One, pEKH2IN2CMVai, contains inverted repeat of a defective CMV-O replicase gene. The other, TRVbased VIGS vector (pTRV2CMVai) contains the same fragment of the replicase gene, but without inverted repeat. In both vectors, dsRNA is produced when the transgene is expressed and/or when the virus is replicated as in the case of pTRV2CMVai. The TRV VIGS vector has often been used for gene silencing and the technique usually involves agroinfiltration or biolistic of plants (Burch-Smith et al. 2006; Liu et al. 2002a, b; Ratcliff et al. 2001). Here, we explored the possibility of using TRV VIGS in plant transformation and regeneration. The use of TRV VIGS vector in plant transformation did not affect gene silencing since transgenic plants were resistant to CMV. This suggests that TRV VIGS could be used effectively in plant transformation to produce transgenic plants. This is considered important because the phenotypes will be stable, the foreign insert will be maintained and inherited from generation to generation, and silencing will occur throughout the plant. It will also have advantage in institutions where agroinfiltration is not allowed, and the biolistic apparatus is not available. The plants transformed with either of the vectors were phenotypically normal and no stunting was observed, suggesting that the high level expression of the CMV transcript did not induce any growth abnormality in the transgenic plants. A unifying feature of RNA silencing is the production of siRNAs that act as specific determinants that downregulate gene expression (Hammond et al. 2000). These siRNA are not only indicative of PTGS but also play a key role in PTGS mechanism (Waterhouse et al. 2001). In this work, all transgenic plants derived from the construct containing inverted repeat (pEKH2IN2CMVai) produced significant levels of siRNA before and after inoculation with CMV-O or Y, whereas transgenic plants derived from the TRVVIGS produced high levels of siRNA only after infection with the CMV strains. This shows that during the course of viral infection, dsRNA was synthesized in the TRV VIGS derived plants. This could have occurred in two ways; it is possible that after CMV infection, pTRV1 co-transformed with pTRV2 supported active replication of TRV2 leading to formation of dsRNAs at late stages of plant development, or CMV stimulated TRV replication leading to the accumulation of siRNA derived from TRV2 RNA.
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In the four lines that were immune to CMV infections, it could be that both RNA transcript and genomic RNA were targeted by the silencing machinery (Bonfim et al. 2007). In the other highly resistant lines, it could be that the complete PTGS process is not yet fully activated, at least not to levels to fully suppress the systemic movement of the virus. High levels CMV siRNA was also detected in CMV inoculated wildtype plants. It is possible that CMV was actively replicating despite PTGS, whereas in the transgenic plants, the dsRNA transgene provides an additional defence mechanism leading to plant recovery. In the wild-type plant, there was a seemingly fragile balance between the plant and the virus: the plant suppressing the virus via PTGS (and possibly other mechanisms) and the virus responds by rapid replication and suppression of the host’s silencing mechanism (Kasschau and Carrington 1998; Voinnet et al. 1999). It has been reported that as a counterdefense, certain plant viruses encode proteins that can suppress the RNA silencing in order to overcome the defense mechanism. Cucumber mosaic virus (CMV)-encoded 2b protein was among the first suppressors identified that could inhibit post-transcriptional gene silencing (PTGS). The 2b protein is a protein that contains a nuclear localization signal required for long distance viral movement in some hosts and it suppresses silencing only in newly emerged tissues that develop after infection (Brigneti et al. 1998). In the case of the wild-type plant expressing high levels of siRNA, the 2b protein inhibited gene silencing possibly by sequestrating siRNAs and preventing them from entering the RISC. Because of the similar levels of expression of siRNA in the transgenic plants, we checked whether the variable response to CMV in the plants was due to the copy number of the transgene. Analysis showed insignificant positive correlation. It may be that the number of copies of the transgene and their association with the siRNA expression did not influence the resistance of transgenic plants to CMV. Kalantidis et al. (2002) reported strong positive correlation between copy number and resistance levels in tobacco plants containing dsRNA of coat protein gene of CMV. They indicated that lines with two or more transgene loci were more likely to become resistant to CMV than those with one locus. This assertion differs from our findings probably because of the target gene used. It is possible that both replicase and coat protein
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sequences operate by different mechanisms, hence the discrepancy in the relationship between copy number and resistance. To test transgenic plants for resistance to CMV, the plants were inoculated with CMV-O and Y in vitro and ex vivo. Although the CMV strains used for disease assay belong to the same subgroup and show high degree of similarity in their nucleotide sequence (Hayakawa et al. 1989), infectivity of CMV-O was slightly lower than that of CMV-Y in both assay. It has been reported that different CMV strains caused varying degrees of symptom severity in the same test species. For example, Roossinck and Palukaitis (1990) indicated that in some susceptible squash cultivars, the Fny-CMV strain showed severe systemic symptoms 1–3 days post inoculation, while the Sny-CMV strain showed mild systemic symptoms 5–7 days post inoculation. Similarly, Valkone and Watanabe (1995) reported CMV-specific symptoms in potato plants that were graft inoculated. It is very likely that multiple virus infections occur simultaneously which could results in synergistic effects, i.e. stronger disease symptoms under field conditions. Although we did not study the synergistic effect of CMV-O and CMV-Y in our transgenic plants the possibility that they may play a role in the suppression of RNA silencing in the transgenic plants cannot be ruled out in this study. However, sequence analysis show that the CMV-O nucleotide sequence used for hairpin in the transgenic plants and the strains used for infection (CMV-O and CMV-Y) in our experiments share 99 % identity. Furthermore, the nucleotide sequence of the majority of CMV strains found in database is highly conserved with greater than 95 % identity within the part of the CMV sequence used in this work. Therefore, taking into account the high levels of resistance obtained here, it is most likely that the resistance gained would last for a long period and would be effective under field conditions. It is also possible that the transgenic plants may display high levels of resistance against a broad range of CMV strains and other viruses. In order to determine that the transgenic lines were free of virus after challenge with the pathogens, such plants were screened by RT-PCR and DIBA. Exception of two lines, resistance was found to have the form of immunity, as no viral particle could be detected in the lines as shown by RT-PCR and DIBA. Since there is no virus load in these plants, it is important because the plants would not be a potential source of virus
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acquisition by aphids (whiteflies) when tried under field conditions. Virus symptoms severity in most plant species is influenced by environmental conditions, especially temperature. Szittya et al. (2003) indicated that RNA silencing-mediated plant defense is temperature dependent. At low temperature, both virus and transgene silencing was inhibited leading to susceptibility. Recently, Celebi-Toprak et al. (2003) suggested that resistance to CMV observed in many crop species could be overcome when inoculated plants were grown at elevated temperature. They showed that potato cultivars that were resistant to CMV at 24 °C became infected when grown at 30 °C. Kalantidis and his associates (2002) found that elevated temperature favoured the formation of CMV derived siRNA in tobacco, resulting to increased resistance. Bonfim and his associates (2007) reported reduced levels of AC1 siRNA when common bean plants were cultivated at low temperature. In our work, when inoculated potato lines were cultivated at elevated temperature (30 °C), siRNA signals were slightly more intense. In the highly resistant plants, despite the intense siRNA signals, disease index was slightly higher in the plants grown at 30 °C than at 24 °C, although the difference was not significant. It could be that at the elevated temperature, CMV replicated and spread a short distance/or faster before activation of PTGS machinery. Overall, it is possible that the transgenic potato lines developed here may display high levels of resistance against CMV infection when cultivated under field conditions. In the present study, we demonstrate that dsRNA could successfully be expressed in transgenic plants using a defective replicase gene. The mechanism associated with defective replicated mediated resistance to CMV is complex and involves separate elements contributing to the resistance: one impeding virus replication, the other restricting virus movement (Canto and Palukaitis 2001). We were interested in determining whether expression of a defective CMV replicase derived dsRNA in potato could result to increased levels of resistance to CMV more than-or-a type akin to coat protein derived dsRNA. The results presented here indicate that potato plants expressing the defective CMV replicase derived dsRNA showed detectable levels of siRNA, indicating that the resistance was acquired through gene silencing. In fact, four lines showed complete resistance to both strains
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of CMV as no symptoms were spotted even at the inoculated portions, suggesting that the replicase constructs used here are good candidates for PTGS. Therefore, the potential application of these constructs may not only be limited to resistance to CMV strains of subgroup I. These constructs maybe used to achieve broad spectrum resistance to other plant viruses in a wide range of important crop plants by adding more viral sequences to the existing transgene. As far as we are aware, this work represents the first example of replicase-mediated resistance resulting from the expression of dsRNA in transgenic plants, and the use of TRV VIGS vector in plant transformation and regeneration to achieve resistance by PTGS in potato. Extensive study of field resistance of transgenic lines to CMV-O and Y, other strains of CMV, and other viruses to further determine the efficacy of these constructs will be important future research. Acknowledgments The authors gratefully thank Professor Takahashi Hideki for kindly donating the CMV-O and Y strains. We thank Professor Hiroaki Kodama for assisting with the northern blot hybridization protocol for siRNA. This work was supported by Japan Society for the Promotion of Science (JSPS). V. O. Ntui is a recipient of JSPS fellowship.
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