Development of transgenic watermelon resistant to Cucumber mosaic ...

Transgenic Res (2012) 21:983–993 DOI 10.1007/s11248-011-9585-8

ORIGINAL PAPER

Development of transgenic watermelon resistant to Cucumber mosaic virus and Watermelon mosaic virus by using a single chimeric transgene construct Ching-Yi Lin • Hsin-Mei Ku • Yi-Hua Chiang Hsiu-Yin Ho • Tsong-Ann Yu • Fuh-Jyh Jan

•

Received: 6 May 2011 / Accepted: 19 December 2011 / Published online: 28 December 2011 Ó Springer Science+Business Media B.V. 2011

H.-M. Ku Y.-H. Chiang H.-Y. Ho Department of Agronomy, National Chung Hsing University, Taichung 402, Taiwan

Single or multiple transgene copies randomly inserted into various locations in the genome were confirmed by Southern blot analysis. Transgenic watermelon R0 plants were individually challenged with CMV, CGMMV or WMV, or with a mixture of these three viruses for resistance evaluation. Two lines were identified to exhibit resistance to CMV, CGMMV, WMV individually, and a mixed inoculation of the three viruses. The R1 progeny of the two resistant R0 lines showed resistance to CMV and WMV, but not to CGMMV. Low level accumulation of transgene transcripts in resistant plants and small interfering (si) RNAs specific to CMV and WMV were readily detected in the resistant R1 plants by northern blot analysis, indicating that the resistance was established via RNA-mediated post-transcriptional gene silencing (PTGS). Loss of the CGMMV CP-transgene fragment in R1 progeny might be the reason for the failure to resistant CGMMV infection, as shown by the absence of a hybridization signal and no detectable siRNA specific to CGMMV in Southern and northern blot analyses. In summary, this study demonstrated that fusion of different viral CP gene fragments in transgenic watermelon contributed to multiple virus resistance via PTGS. The construct and resistant watermelon lines developed in this study could be used in a watermelon breeding program for resistance to multiple viruses.

T.-A. Yu Department of Molecular Biotechnology, DA-YEH University, Changhua 51591, Taiwan

Keywords Watermelon Transgenic plant Viral resistance

Abstract Watermelon, an important fruit crop worldwide, is prone to attack by several viruses that often results in destructive yield loss. To develop a transgenic watermelon resistant to multiple virus infection, a single chimeric transgene comprising a silencer DNA from the partial N gene of Watermelon silver mottle virus (WSMoV) fused to the partial coat protein (CP) gene sequences of Cucumber mosaic virus (CMV), Cucumber green mottle mosaic virus (CGMMV) and Watermelon mosaic virus (WMV) was constructed and transformed into watermelon (cv. Feeling) via Agrobacterium-mediated transformation.

Ching-Yi Lin and Hsin-Mei Ku equally contributed to this work.

Electronic supplementary material The online version of this article (doi:10.1007/s11248-011-9585-8) contains supplementary material, which is available to authorized users. C.-Y. Lin Y.-H. Chiang H.-Y. Ho F.-J. Jan (&) Department of Plant Pathology, National Chung Hsing University, Taichung 402, Taiwan e-mail: [email protected]

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Abbreviations CMV Cucumber mosaic virus CGMMV Cucumber green mottle mosaic virus WMV Watermelon mosaic virus siRNA Small interfering RNA

Introduction Watermelon [Citrullus lanatus (Thunb.) Matsum & Nakai, Family Cucurbitaceae] is one of the major cucurbit crops in the world, especially in tropical and subtropical regions. Its cultivation is hampered by numerous viral diseases that often cause economic losses in most production areas (Gaba et al. 2004; Papayiannis et al. 2005). The cucurbit crops are susceptible to at least 35 viruses (Provvidenti 1996; Papayiannis et al. 2005), among which Cucumber mosaic virus (CMV), Cucumber green mottle mosaic virus (CGMMV), and Watermelon mosaic virus (WMV) are the most damaging viruses in watermelon. CMV is the type species of the genus Cucumovirus in the Family Bromoviridae and is a positive-sense RNA virus with a tripartite genome (Palukaitis et al. 1992). The CMV coat protein (CP) gene is located at the 30 end of RNA 3 and is expressed from subgenomic RNA 4 (Palukaitis et al. 1992). Commonly distributed worldwide, CMV has a very wide host range and attacks over 1,000 plant species (Lovisolo 1981; Roossinck 2002). CMV can be transmitted by mechanical inoculation either artificially or by aphids in a nonpersistent manner (Palukaitis et al. 1992). Symptoms in cucurbits caused by CMV infection are systemic mosaic, mottling, distorted and curled leaves, and plant stunting (Palukaitis et al. 1992). CGMMV, a single stranded positivesense RNA virus, belongs to the genus Tobamovirus in the family Virgoviridae (Ugaki et al. 1991; Wang and Stubbs 1994). The CP gene of CGMMV is located at the 30 end of the genomic RNA and is expressed via the subgenomic mRNA of open reading frame 4 (Ugaki et al. 1991). This virus is transmitted mechanically through seeds and soil contamination and can cause severe diseases in cucumber and watermelon with systemic green mottle mosaic on foliage and fruit deterioration (Buttner et al. 1995). WMV, a member of the genus Potyvirus in the family Potyviridae, is transmitted in a nonpersistent manner by aphids (Milne et al. 1969; Shukla et al. 1994) and contains a single

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Transgenic Res (2012) 21:983–993

stranded positive-sense RNA (Shukla et al. 1994). Its CP gene is localized at the 30 end of the open reading frame and is expressed by proteolytic cleavage by the virusencoded polyprotein (Shukla et al. 1994). WMV is distributed worldwide and causes severe yield loss in cucurbit crops, including watermelon (Purcifull et al. 1998). Typical symptoms induced by WMV on cucurbit plants include leaf mottling and mosaic, chlorosis, tip stunting or bunching, and reduced fruit yield and quality (Purcifull et al. 1984). To develop transgenic cucurbits with multiple virus resistance, CP-mediated protection based on the concept of pathogen-derived resistance (PDR) (Sanford and Johnston 1985) provides an effective strategy of plant protection against various viruses. For example, transgenic squash with multiple viral CP genes are resistant to Zucchini yellow mosaic virus (ZYMV), WMV (Clough and Hamm 1995; Fuchs and Gonsalves 1995; Klas et al. 2006), and CMV (Tricoli et al. 1995; Fuchs et al. 1998) and transgenic cantaloupe with multi-viral CP genes are resistant to ZYMV, WMV and CMV (Fuchs et al. 1997). However, multi-viral CP genes in these cases are under the control of independent promoters which might result in unequal expression of each transgene. To overcome this problem, a single chimeric gene consisting of linked viral segments was constructed to successfully develop resistance in transgenic Nicotiana benthamiana against Turnip mosaic virus (TuMV) and Tomato spotted wilt virus (TSWV) (Jan et al. 2000b). Recently, this strategy was successfully applied to produce transgenic oriental melon resistant to ZYMV and Papaya ringspot virus (PRSV W) by expression of viral CP gene segments (Wu et al. 2010). Although several transgenic cucurbits with multi-viral resistance have been reported, such as squash, cantaloupe and oriental melon, there were few reports of transgenic watermelon with virus resistance. Only Park et al. (2005) developed a single viral resistant transgenic watermelon rootstock by transforming the CP gene of CGMMV, and a double resistant transgenic watermelon was generated against the potyviruses ZYMV and PRSV W (Yu et al. 2011). In this study, a single chimeric gene consisting of viral CP gene segments of CMV, CGMMV and WMV, which represent distinct plant virus families, was constructed and transformed into watermelon by Agrobacterium tumefaciens-mediated transformation. Several transgenic R0 watermelon lines exhibited resistance to these three viruses. Molecular analyses


of transgenic watermelon lines demonstrated that the multiple resistance is induced by RNA-mediated posttranscriptional gene silencing (PTGS) (Hamilton and Baulcombe 1999; Hammond et al. 2000).

Materials and methods Vector construction The partial CP genes of a CMV-Chia Yi isolate originated from Taiwan (unpublished), a CGMMV SH strain (Ugaki et al. 1991) and a WMV-Florida isolate (Quemada et al. 1990) were used as the templates for constructing a hybrid transgene with three viral CP gene segments. The cDNA segments corresponding to the partial CP coding sequences of CMV [1428–1877 nucleotide (nt) of RNA 3], CGMMV (5774–6223 nt) and WMV (9332–9781 nt) were amplified from total RNA extracted from N. benthamiana infected with the respective viruses by reverse transcription-polymerase chain reaction (RT-PCR) with their individual CP specific primers (Table S1 in electronic supplementary material). The amplified CP gene segments were cloned into TOPO-clone vector (PCRÒII-TOPOÒ, Invitrogen, Carlsbad, CA), and the resulting plasmids were named TOPO-C, TOPO-G and TOPO-W. To link the three viral cDNA segments of CP gene, the TOPO-C, TOPO-G and TOPO-W were individually digested with NcoI/BamHI, BamHI/PstI and PstI/XhoI; the released segments (named CGW fragment) from three TOPO plasmids were fused together by ligase and sub-cloned into a plant expression vector, pEPJ-m/2 N, which carrying a silencer DNA [the middle half N gene (m/2 N) of Watermelon silver mottle virus (WSMoV), 378 bp on 2864–3247 nt of S RNA, GenBank Accession No. NC_003843] (Shih 2002). The resulting product was digested with HpaI/SacI and the segments containing the expression cassette was introduced into the binary vector pGA482G (Jan 1998) to generate pGA482G-WoCGW (Fig. S1 in electronic supplementary material). Agrobacterium tumefaciens LBA4404 was transformed with pGA482G-WoCGW by electroporation (Gene pulserÒ II, Bio-Rad Laboratories Inc., Richmond, CA). Watermelon transformation The protocol for watermelon transformation was modified from a previous report by Li (2005).

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Watermelon seeds [C. lanatus (Thunb.) Matsum & Nakai cv. Feeling] (Known-You seed Co. Ltd., Taiwan) were manually peeled to remove the episperm before sterilization for 5 min in 1% Clorox (Clorox Company, USA) followed by 3 rinses with sterilized water. The sterilized seeds were deposited on SH medium (pH 5.7) [4.4 g MS salt including Gamborg B5 Vitamin per liter (Duchefa Biochemie, the Netherlands), 3% sucrose, 1,000 mg/l myo-inositol, 1 mg/l nicotinic acid and 0.8% agar] for germination in the dark at 27°C. Cotyledons from 3-day-old seedling were cut near the proximal end, excised into four segments and used as explants for transformation. The explants were soaked in 20 ml Murashige and Skoog liquid medium (pH 5.7) (4.4 g MS salt including Gamborg B5 Vitamin per liter and 3% sucrose), adding 200 ll overnight culture of A. tumefaciens and 20 ll acetosyringone (AS) (200 lM) for 10 min and then placed on the co-culture medium [SH medium containing 1.5 mg/l 6-benzylaminopurine (BA)] for 4 days under 16 h light/8 h dark cycles. Subsequently, the co-cultivated explants (4,800 explants) were transferred to the selection medium (co-culture medium containing 250 mg/l carbenicillin and 100 mg/l kanamycin) and incubated for 2–3 weeks. The explants that developed small shoots were transferred to elongation medium [SH medium containing 0.2 mg/l BA, 0.02 mg/l a-naphthaleacetic (NAA), 200 mg/l carbenicillin and 200 mg/l kanamycin] and incubated for 6 weeks under 16 h light/8 h dark cycles. The green shoots were propagated on elongation medium with 100 mg/l kanamycin by micropropagation. The propagated shoots were transferred to rooting medium [SH medium containing 1 mg/l indolebutyric acid (IBA), 200 mg/l carbenicillin] and maintained for 7 days in the dark and another 7 days in the light. Plantlets with developing roots were transplanted to soil and vermiculite mixture under greenhouse conditions for further analysis. Screening transgenic plant by polymerase chain reaction (PCR) Transgene insertion in transformed watermelon was confirmed by polymerase chain reaction (PCR) with specific primers for the CGW transgene (upstream primer of the CMV CP gene/downstream primer of the WMV CP gene) and plant selection marker gene

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neomycin phosphotransferase II (NPTII) (NOS promoter/terminator for NPTII), respectively (Table S1 in electronic supplementary material). Total DNA was extracted from the transformed plants as described by Fulton (1995) and 1 ll (0.5–1 lg) of the extracted DNA was used as a template in 50 ll PCR amplification. The PCR reaction was performed with an initial cycle at 93°C for 5 min; followed by 34 cycles at 93°C for 1 min, 55°C for 1 min, 72°C for 2 min and a final cycle at 72°C for 10 min. The PCR products were monitored by electrophoresis on 0.8% agarose gel. PCR-positive plants were evaluated for viral resistance.

generated by Dr. F.-J. Jan’s Lab, National Chung Hsing University, Taichung, Taiwan. The AP-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was used as the secondary antibody (5,000-fold dilution) for the detection of rabbit antibodies. The absorbance at 405 nm was recorded using the ELISA reader (Multiskan Ex, labsystems Inc., Franklin, MA) for 10–30 min after the addition of substrate (SigmaAldrich Fine Chemicals, Milwaukee, WI, USA). The reaction was considered positive when the A405 value was at least twofold higher than that of the negative control.

Resistance evaluation

Southern hybridization

The virus inocula were prepared from 1:50 (w/v) diluted leaf homogenates of zucchini squash plants infected with CMV or WMV or horned melon [Cucumis metuliferus (Naud) Mey line Acc. 2459] infected with CGMMV in 10 mM phosphate buffer (0.033 M KH2PO4, 0.067 M K2HPO4; pH 7.0). Test watermelons were mechanically inoculated by rubbing the top three youngest leaves that were dusted with carborundum (600 mesh). Non-transformed watermelon cv. Feeling which is susceptible to the three viruses was used as a control. Inoculated plants were monitored for symptom development for 5 weeks. The R0 lines with resistance were selffertilized for seed production. To verify the inheritance of virus resistance in transgenic progeny, seeds of R1 lines were germinated in soil in a greenhouse. After growing to the 5–6 leaves stage, these R1 plants were mechanically inoculated with CMV, CGMMV or WMV individually and with a mixture of all three viruses combined for the evaluation of their resistance to respective viral infection.

Total DNA was extracted from leaves of the transgenic and non-transgenic watermelon plants as described by Fulton (1995). Fifteen lg of total DNA were digested with HpaI and separated by electrophoresis on 1% agarose gel in 1X TAE buffer and transferred to a nylon membrane (GeneScreen PlusÒ membrane, Perkin Elmer Life Science, USA). Blots were hybridized with a-32P-dATP (Pharmacia, Feinberg and Vogelstein 1983) labeled probes prepared from m/2 N of WSMoV, which were digested with NcoI from the plasmid. After post-hybridization and wash, the membrane was autoradiographed by exposing to Kodak X-ray film with appropriate length of time at -80°C.

Indirect enzyme-linked immunosorbent assay (ELISA) Indirect ELISA (Yeh and Gonsalves 1984) was performed to detect viral infection in watermelon plants. The upper leaves of inoculated plants were collected 30 days post inoculation (dpi), and 1:20 (w/v) diluted homogenates of leaf tissues were assayed (Yeh and Gonsalves 1984) using 5,000-fold dilution of the antiserum to CMV or CGMMV and 2,000-fold dilution of the antiserum to WMV. All antisera were

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Transcript and siRNA detection in transgenic watermelon plants by northern blot hybridization Total RNA was extracted from leaves of transgenic plants by the method described by Napoli et al. (1990). To detect transgene expression level, 10 lg of total RNA was electrophoresed on a formaldehyde-containing agarose gel (Sambrook and Russell 2001). Total plant RNA was visualized by staining with ethidium bromide. The hybridization was performed at 60°C using a-32P dATP labeled probe corresponding to the CGW transgene and obtained by random primer labeling. The membranes were rinsed with 2X SSC, washed twice with 2X SSC, 1% SDS, 0.5X SSC, 1% SDS and 1X SSC at 60°C. Signals were visualized by autoradiography on X-ray film at -80°C. For siRNA detection, 50 lg of total RNA was separated by electrophoresis on a 15% polyacrylamide (19:1) gel


containing 7 M urea in 0.5X Tris–borate-EDTA (TBE) and transferred to a GeneScreen PlusÒ membrane. The hybridization was performed at 42°C using a-32P dATP labeled probe of the CGW transgene obtained by random primer labeling. The DNA probes corresponded to the CMV-, CGMMV- and WMV-CP fragments were digested from the plasmid. Membranes were washed twice with 2X SSC, 0.2% sodium dodecyl sulfate (SDS) at 42°C for 10 min modified from the protocol described by Lin et al. (2011). Signals were visualized by autoradiography on X-ray film at -80°C.

Results Vector construction and transformation To evaluate the resistance efficacy of a chimeric gene construct, segments of the partial CP genes of CMV, CGMMV and WMV, were fused to the Cauliflower mosaic virus (CaMV) 35S promoter and m/2 N of WSMoV and cloned in the plant transformation binary vector pGA482G (Fig. S1 in electronic supplementary material). The pGA482G-WoCGW was introduced into A. tumefaciens LBA4404 for watermelon transformation. For generating transgenic watermelon, a total of 600 seeds of watermelon cultivar Feeling without episperm was sterilized and used for germination (Fig. S2a, b in electronic supplementary material). Cotyledons from 3-day-old seedlings were split into four pieces (Fig. S2c in electronic supplementary material) and co-cultured with A. tumefaciens LBA4404 containing pGA482G-WoCGW. After 4 days, the explants co-cultured with Agrobacteria swelled and turned green, then the explants were transferred to selection medium (Fig. S2d in electronic supplementary material). Two to three weeks later, small shoots developed (Fig. S2e, f in electronic supplementary material) and were elongated to 2 cm explants on elongation medium (Fig. S2g in electronic supplementary material). The propagated shoots were transferred to rooting medium (Fig. S2h in electronic supplementary material) and the plantlets with developing roots were transplanted to soil containing a vermiculite mixture (Fig. S2i in electronic supplementary material) for further analysis. In summary, a 0.23% transformation rate (11 PCR positive lines/ 4,800 explants, 600 seeds) was obtained in this study.

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Transgene confirmation in putative watermelon plants Transgene presence in putative transgenic watermelon was detected by PCR using primers specific to fragment GCW and NPTII-specific primers (Table S1 in electronic supplementary material). Among 60 putative R0 lines obtained, 11 lines had an expected 1.35 kb product of the transgene (Fig. 1a) and an expected 1.1 kb product of the NPTII gene (Fig. 1b). Southern blot was performed to confirm the integration and the copy number of transgene in transgenic watermelon lines. Random insertion of the transgene and one to two copies of the transgene in each line were detected (Fig. 1c). Evaluation of virus resistance in transgenic watermelon lines All generated R0 lines showed normal morphology and three to ten plants in each R0 line were evaluated for their resistance against virus infection by mechanical inoculation with CMV, CGMMV and WMV, alone or together. The non-transformed susceptible plants showed symptoms at 4–7 days post inoculation

Fig. 1 Characterization of transgenic watermelon lines by PCR and Southern blot hybridization. PCR amplification of (a) a 1.35 kb fragment amplified with primers specific to fragment CGW that spans from the 50 CMV CP gene fragment to the 30 MWV fragment in pGA482G-WoCGW, b a 1.1. kb fragment amplified using primers specific to NOS promoter and terminator of NPTII gene from pGA482G-WoCGW. M marker, P plasmid, N non-transformed susceptible plant. c Southern blot analysis of pGA482G-WoCGW transgenic watermelon plants. The a-32P labeled WSMoV m/2 N gene segment was used as the probe

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Table 1 Evaluation of transgenic watermelon R0 plants mechanically inoculated with Cucumber mosaic virus, Cucumber green mottle mosaic virus, Watermelon mosaic virus or mixture of three viruses pGA482G-WoCGW

CGMMV

CMV

WMV

n

R

D

S

n

R

D

6

10

10

–

–

10

10

–

13

10

8

–

2

10

14

10

10

–

–

10

8 other lines total

24

–

–

24

Control

10

–

6

46

–

14

58

–

Control

12

–

S

Mix

n

R

D

S

n

R

D

S

–

R0 line 10

10

–

–

10

10

–

–

10

10

10

–

–

10

–

–

10

10

–

–

10

10

–

10

10

–

–

9

–

–

9

9

–

–

9

9

–

–

9

10

10

–

–

10

10

–

–

10

10

–

–

10

3

43

39

19

1

19

39

14

6

19

ND

1

57

34

19

3

12

35

17

5

13

ND

–

12

6

–

–

6

6

–

–

6

R1 line

Phenotypes of transgenic plants after inoculation were compared with the same stage of virus infected, nontransformed watermelon plants. n, number of plants inoculated; R, plants with no symptoms on systemic leaves 30 days post inoculation (dpi); D, plants with delayed symptom development for more than 7 days when compared with infected-nontransformed watermelon plants; S, plants with symptoms developed at the same time as those on non-transformed susceptible controls; ND, data not detected. The non-transformed plants showed symptom in 4–7 dpi with CMV, in 7–10 dpi with WMV and in 7–14 dpi with CGMMV, respectively

(dpi) with CMV, 7–10 dpi with WMV and 7–14 dpi with CGMMV. Based on symptom development, 51 individual plants derived from 8 transgenic lines exhibited typical disease symptoms 4–14 days after inoculation with CMV, CGMMV and WMV, alone or together (Table 1). For transgenic line 13, 8 out of 10 individuals showed resistance to CGMMV and 10 out of 10 individuals plants showed resistance to WMV but none of them displayed resistance against CMV and mix infection (Table 1). For transgenic lines 6 and 14, 10 out of 10 plants of each line showed resistance to all three viruses individually as well as to all three collectively. No symptom was visible on these two transgenic lines during the observation period of 5 weeks (Table 1, Fig. 2a–d). The absence of virus in transgenic watermelon plants was shown by indirect ELISA using polyclonal antibodies to CMV-CP, CGMMV-CP and WMV-CP in 15 and 30 days after inoculation. Viruses were detected in symptomatic nontransformed plants by indirect ELISA. To verify the inheritance of virus resistance in transgenic progeny, seeds of transgenic lines 6 and 14 that showed resistance to the three viruses were tested. For line 6, 14 out of 39 plants were resistant to WMV (Fig. 2e) and 19 out of 39 were resistant to CMV (Fig. 2f) at 30 dpi. Resistant plants remained asymptomatic for the duration of the experiment. Unexpectedly, only 3 out of 46 plants expressed a delay in

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symptom expression following CGMMV inoculation compared to CGMMV-infected controls and the remaining 43 plants were susceptible (Table 1). Similarly, for line 14, 19 out of 34 plants and 17 out of 35 plants were resistant to CMV and WMV, respectively, and remained symptomless for the duration of the experiment but 57 out of 58 were symptomatic following inoculation with GCMMV (Table 1). Transcript and siRNA detection in transgenic watermelon plants The expression of transgene in transgenic R1 lines was monitored by northern blot hybridization. High level transgene CGW transcripts was detected from the susceptible transgenic line 26, while a lower level of transgene transcripts was detected from the resistant lines 6 and 14. Non-transformed susceptible plants did not show any signal (Fig. 3a). Accumulation of siRNA was analyzed by using probes specific to the transgene of CMV-CP, WMVCP and CGMMV-CP, respectively. A detectable accumulation of siRNA specific to the CMV-CP (Fig. 3b) and WMV-CP (Fig. 3c) was observed in resistant R1 plants from lines 6 and 14, respectively, but not in the susceptible line 54 (Fig. 3b, c). However, no siRNA specific to the CGMMV-CP


Fig. 2 Evaluation of viral resistance in transgenic watermelon lines. a Transgenic watermelon R0 lines inoculated with CMV 20 days post inoculation (dpi). Non-transformed plant showed severe symptoms (left), a resistant line showed symptomless (right). b Transgenic watermelon R0 lines inoculated with CGMMV 20 dpi. Non-transformed plant showed severe symptoms (left), a resistant line showed no symptoms (right). c Transgenic watermelon R0 lines inoculated with WMV 15 dpi. Non-transformed plant showed severe symptoms (left), a resistant line showed no symptoms (right). d Transgenic watermelon R0 lines mix-inoculated with three viruses 20 dpi. Non-transformed plant showed severe symptoms (left), a resistant line showed no symptoms (right). e and f, the progeny (R1) plants of resistant lines inoculated with WMV and CMV, respectively (30 dpi). S, a non-transformed R1 plants inoculated with WMV or CMV; R, resistant R1 plants inoculated with WMV or CMV; CK, non-transformed controls inoculated with buffer

fragment was observed in R1 plants of lines 6 and 14 (Fig. 3d).

Discussion The production of virus-resistant transgenic watermelon remains challenging. It is hindered by a very low efficiency of transformation. In this study, the watermelon cv. Feeling, a commercial variety for fresh fruit production, was used for transformation

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experiments. A 0.23% transformation rate was obtained (11 PCR positive plants/4,800 explants, 600 seeds) which was similar to that of Park et al. (2005). However, these authors used a translatable full length of CP gene of CGMMV to generate transgenic watermelon resistant to CGMMV. Yu et al. (2011) evaluated the transformation efficiency of three commercial watermelon cultivars by A. tumefaciensmediated transformation and showed the highest transformation efficiency with cultivar Feeling (12 PCR positive plants/130 seeds). A higher bacterial population in our co-cultivation procedure may have caused a decrease in transformation efficiency compared to that of Yu et al. (2011) who have used only 1/4 in volume of the bacterial culture that was employed in this study. Other factors such as the selection pressure of antibiotics, condition of explants, and composition of culture medium might also have influenced a lower efficiency. RNA-mediated resistance is an effective strategy to protect plants against RNA viruses and has been demonstrated in transgenic squash with the CP gene of Squash mosaic virus (SqMV) (Jan et al. 2000c; Pang et al. 2000), transgenic cantaloupe with the CP gene of PRSV W (Krubphachaya et al. 2007) and transgenic oriental melon with the CP genes of ZYMV and PRSV W (Wu et al. 2009, 2010). The first report of transgenic virus resistance in watermelon was with the commercial cultivar Gongdae [C. lanatus (Twinser)], which was used as a rootstock (Park et al. 2005). The translatable CGMMV CP was detected in one of the resistant plants while it was not detected in the other five resistant plants. This result indicated that there was no correlation between virus resistance and the levels of transgene transcripts or translatable products (Park et al. 2005). Previously, a transgenic watermelon resistant to two potyviruses was generated by transforming an untranslatable chimeric construct consisting of the CP gene fragments of ZYMV and PRSV W (Yu et al. 2011). Several transgenic lines resistant to ZYMV and PRSV W were obtained through the RNAmediated PTGS mechanism, as shown by the accumulation of siRNA (Yu et al. 2011). In this study, transformation of a chimeric gene construct derived from three distinct viruses including CMV, CGMMV and WMV was designed and transgenic R0 watermelon lines 6 and 14 were resistant to CMV, CGMMV and WMV alone or in mix-infection. The R1 progeny derived from lines 6 and 14 exhibited resistance to

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Fig. 3 Northern hybridization to detect the expression of transgene transcripts and the accumulation of siRNA in the transgenic R1 watermelon plants. a Northern blot analysis of CGW transgene transcript levels in the resistance R1 transgenic watermelon of line 6 and line 14. b Accumulation of CMV CP specific siRNA to the CMV CP transgene in the resistant R1 plants of line 6, line 14 and a susceptible (S) line. c Accumulation

of specific siRNA to the WMV CP transgene in the resistant R1 plants of line 6, line 14 and a susceptible line. d Accumulation of specific siRNA to the CGMMV CP transgene in the resistant R1 plants of line 6, line 14 and a susceptible line. S, susceptible line 26; N, non-transformed susceptible watermelon; EtBr, samples of RNA were used as loading control. The sizes of the markers are indicated on the left

both CMV and WMV but not to CGMMV (Table 1). The transcription of the transgene (three viral partial CP, CGW fragment) was detected at low level in the R1 plants with resistance to CMV and WMV while at high level in susceptible plants using northern blot analysis (Fig. 3) and the accumulation of siRNAs specific to CMV- and WMV-CP genes was observed in resistant R1 plants, which provided evidence for the occurrence of siRNA-induced PTGS (Vaucheret et al. 1998; Hamilton and Baulcombe 1999). However, loss of CGMMV-resistance in the progeny derived from resistant R0 watermelon lines was observed. When R1 progeny was analyzed by Southern hybridization, no signal was detected by the probe corresponding to the sequence of the CGMMV CP fragment alone but a detectable signal was revealed clearly when using a probe to the CGW fragment alone, which consisted of the three viral partial CP gene sequences as the probe (Fig. S3 in electronic supplementary material).

Moreover, the absence of siRNA specific to CGMMV in R1 transgenic watermelon (Fig. 3d) with resistance to CMV and WMV but not CGMMV (Table 1) was observed. These results provided evidence that the CGMMC-CP fragment in R1 progeny might have been lost in the transmission process from R0 transgenic lines. The complexity of genome structure in the F1 watermelon hybrid cv. Feeling and other factors might lead to the loss of CGMMC-CP fragment and its function. The abnormal inheritance of transgene have been observed in several crops and different effects such as deletion, duplication, rearrangement, repeated sequence recombination as well as the interactions among genes may possibly occur (Yin et al. 2004). The genetic background of the recipient genome is one of the factors that influence the stability of the introduced transgenes (Yin et al. 2004). The reasons for the loss of the CGMMV-CP tansgene are unclear and warrant further investigations. To address the

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unstable segregation, resistant R0 transgenic watermelon lines developed in this study could be backcrossed to the parental watermelon (cv. Feeling). Alternatively, the construct developed in this study could be transformed to other breeding watermelon lines. Previous studies indicated that RNA-mediated viral resistance is often associated with two or more copies of the transgene (Sijen et al. 1996; Souza et al. 2005; Kung et al. 2009). In this study, resistant lines 6 and 14 and susceptible lines 54 and 5 all showed two copynumber insertions in Southern blot analysis (Fig. 1c), suggesting no association between the transgene copy number and viral resistance. These results were consistent with those of Hu et al. (2011). In our construct, a partial N gene of WSMoV was used as a silencer DNA (Shih 2002). Previous studies indicated that segments of the TSWV N gene of 110 bp or more could induce PTGS and confer resistance when linked to a transcribed DNA (designated as a silencer DNA) such as a green fluorescent protein (GFP) gene (Pang et al. 1997; Jan et al. 2000a). It was further demonstrated that a viral sequence can be a silencer DNA and confer resistance via PTGS as well as resistance to other viruses when it is linked to segments of other viral sequence fragments (Jan et al. 2000b; Lin et al. 2011). Base on these observations, it is possible that the transgenic lines developed in this study could also provide resistance against WSMoV. Due to the fact that WSMoV is a major limiting factor of cultivation of cucurbitaceae crops (Yeh et al. 1992; Yeh and Chang 1995), developing transgenic plants with resistance to WSMoV would be beneficial for management of WSMoV. However, it is very difficult to mechanically inoculate watermelon with the WSMoV isolate (wax gourd isolate) used in this study, thus we could not evaluate the effectiveness of WSMoV-resistance in our transgenic watermelon. In conclusion, we have successfully produced transgenic watermelon lines with multiple virus resistance by transforming a chimeric gene with linkage of CMV-, CGMMV-, and WMV-CP gene fragments via A. tumefaciens-mediated transformation and provided evidence that PTGS was responsible for the resistance in this study. Our work has great potential for the control of watermelon virus diseases in Taiwan and other production areas. Furthermore, our construct can be transformed into other cultivars to generate resistant transgenic lines which could be crossed to other

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transgenic watermelon lines that are resistant to other viruses for pyramiding multiple virus resistances. Acknowledgments We are grateful to Dr. Chung-Jan Chang, Professor, University of Georgia and Dr. Wen-Hsiung Ko, Professor Emeritus, University of Hawaii at Manoa for their critical review of this manuscript. This study was partially supported by grants (91AS-3.1.2-BQ-B1, 92AS-4.2.2-BQ-B1, 93AS-4.1.2-BQ-B1 and 94AS-5.1.4-BQ-B1) from the Bureau of Animal and Plant Health Inspection and Quarantine, Council of Agriculture, Executive Yuan, Taiwan, ROC.

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