Transgenic Res (2011) 20:503512 DOI 10.

1007/s11248-010-9432-3

ORIGINAL PAPER

Genetic transformation with untranslatable coat protein gene of sugarcane yellow leaf virus reduces virus titers in sugarcane
Yun J. Zhu Heather McCafferty Greg Osterman Steven Lim Ricelle Agbayani Axel Lehrer Susan Schenck Ewald Komor

Received: 7 June 2010 / Accepted: 16 July 2010 / Published online: 27 July 2010 Springer Science+Business Media B.V. 2010

Abstract Sugarcane yellow leaf syndrome, characterized by a yellowing of the leaf midrib followed by leaf necrosis and growth suppression, is caused by sugarcane yellow leaf virus (SCYLV). We produced SCYLV-resistant transgenic sugarcane from a susceptible cultivar (H62-4671) and determined the amount of virus present following inoculation. The transgenic plants were produced through biolistic bombardment of cell cultures with an untranslatable coat protein gene. Presence of the transgene in regenerated plants was conrmed using PCR and Southern blot analysis. The transgenic lines were inoculated by viruliferous aphids and the level of SCYLV in the plants was determined. Six out of nine transgenic lines had at least 103-fold lower virus titer than the non-transformed, susceptible parent line. This resistance level, as
Y. J. Zhu (&) H. McCafferty G. Osterman S. Lim R. Agbayani A. Lehrer S. Schenck Hawaii Agriculture Research Center, 94-340 Kunia Road, Waipahu, HI 96797, USA e-mail: jzhu@harc-hspa.com G. Osterman R. Agbayani Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, HI 96822, USA A. Lehrer Currently with Hawaii Biotech Inc, 99-193 Aiea Heights Drive, Aiea, HI 96701, USA E. Komor University of Bayreuth, 95440 Bayreuth, Germany

measured by virus titer and symptom development, was similar to that of a resistant cultivar (H78-4153). The selected SCYLV-resistant transgenic sugarcane lines will be available for integration of the resistance gene into other commercial cultivars and for quantication of viral effects on yield. Keywords Transgenic Sugarcane Monocot Virus resistance Coat protein gene Abbreviations BA Benzyladenine IBA Indolebutyric acid MS Murashige and Skoog plant culture medium NAA Naphthaleneacetic acid, 2, 4-D: 2, 4-dichlorophenoxyacetic acid PCR Polymerase chain reaction RT-PCR Reverse transcription polymerase chain reaction SCYLV Sugarcane yellow leaf virus YLS Yellow leaf syndrome Ubi Ubiquitin nptII Neomycin phosphotransferase SCYLVcp Sugarcane yellow leaf virus coat protein TPI Triosephosphate isomerase

Introduction In the late 1980s, a leaf yellowing disease called yellow leaf syndrome (YLS) was reported worldwide

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on sugarcane (Borth et al. 1994; Comstock et al. 1994; Moutia and Saumtally 1999; Rassaby et al. 2003; Vega et al. 1997). In some cases, the appearance of YLS was reported to be associated with substantial yield losses (Rassaby et al. 2003; Vega et al. 1997). Viral particles found in the phloem of affected leaves were characterized as those of a luteovirus (Vega et al. 1997) which was named sugarcane yellow leaf virus (SCYLV). Analysis of viral RNA-sequences revealed that SCYLV belongs to a distinct subgroup that had probably originated from the recombination of two other luteovirus species (Moonan et al. 2000). Antibodies specic for the puried virus were produced to assay for the presence of the virus in sugarcane tissues (Scagliusi and Lockhart 2000). In subsequent surveys, using this antibody in a tissue blot immunoassay, many symptomatic and asymptomatic plants were found to contain the virus (Schenck et al. 1997; Schenck and Lehrer 2000). Preliminary tests in Hawaii and Louisiana have indicated that the infection is detrimental to plant performance even when the plants show no symptoms (Schenck and Lehrer 2000; Lehrer et al. 2009). In Hawaii, SCYLV is widespread in all susceptible cultivars and has been shown to be spread by certain common aphid species, although only Melanaphis sacchari is considered important for eld spread (Lehrer et al. 2007). The genetic complexity and low fertility of sugarcane hamper traditional breeding efforts and make sugarcane a prime candidate for improvement through genetic engineering (Ingelbrecht et al. 1999). Furthermore, results from crossing cultivars suggest that SCYLV-resistance is a dominant trait. Unfortunately, it is apparently accompanied by undesirable traits which means that the majority of commercial cultivars are susceptible. Therefore the transgenic approach with an untranslatable gene-segment is a promising concept to introduce SCYLV-resistance in high-yielding cultivars. Sugarcane is capable of producing regenerable calli and this means that direct manipulation of specic traits, such as virus resistance, is possible (Ingelbrecht et al. 1999). There have been numerous reports of genetically engineered virus resistance in plants. For a review of the different technologies involved see Baulcome (1996). Posttranscriptional gene silencing has already been demonstrated in sugarcane by transformation

with an untranslatable piece of the Sorghum mosaic virus (SrMV) (Ingelbrecht et al. 1999). They examined transgenic sugarcane plants over a two year period and found that when they were challenged with a strain of SrMV they fell into three groups, susceptible plants which showed symptoms, recovery plants which produced new healthy leaves after initially showing symptoms and immune plants which never showed symptoms. However, as reported by Lehrer and Komor (2008), symptoms are not a reliable measure for the presence of SCYLV, because some cultivars contain high virus titer with only marginal symptom expression. Recently, we developed a highly sensitive quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) assay and detected the virus in cultivars previously thought (based on TBIA) to be immune to virus infection (Zhu et al. 2010). We produced transgenic sugarcane with an untranslatable coat protein gene of SCYLV with the aim of improving resistance to this virus. We examined the plant resistance by presence of visible symptoms and also by measuring the virus titer within the plants.

Materials and methods DNA constructs pFM395 construct was kindly provided by Dr. E. Mirkov (Texas A&M Agriculture Experiment Station, Weslaco, TX). This construct contains the the untranslatable coat protein of SCYLV. It was obtained by introducing a stop codon immediately after the ATG start in a sense orientation in relation to the maize ubiquitin promoter (ubi). SCYLV construct was co-bombarded with pUbi9nptII (named pHA9), a selection plasmid that contains the neomycin phosphotransferase II gene (nptII) driven by a sugarcane ubiquitin promoter, SCubi9. Figure 1 shows the core features of the pFM395 and pHA9 plasmids used for transformation. Plant material and transformation A Hawaiian cultivar, H62-4671, a Saccharum spp. hybrid, was used for transformation. Particle gun bombardment of embryogenic calli of H62-4671 was

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SphI PstI PstI SalI

505

XhoI

EcoRI

pFM395
'UBIp(int1) YLS-S

EcoRI

and relative humidity ranging from 30 to 60%, under natural daylight. Genomic DNA extraction, PCR analysis The sugarcane callus from tissue culture plates or leaf tissue from greenhouse-grown plants was excised, immediately frozen in liquid nitrogen, and stored in a -80C freezer until genomic DNA was extracted. Genomic DNA for use in PCR was isolated by the CTAB method (Doyle and Doyle 1990). PCR amplication of the SCYLVcp gene was carried out using a set of primers; YLS462 50 -GTC TCC ATT CCC TTT GTA CAG C -30 and YLS111 50 -TCT CAC TTT CAC GGT TGA CG-30 to produce a 351 bp product. The primers used to amplify a 700 bp product from the NPT II gene were 50 -AGA GGC TAT TCG GCT ATG AC-30 and 50 -GTC AAG AAG GCG ATA GAA GG30 . The PCR reactions were carried out in 50 lL volume consisting of 1 lL template DNA, 1 lL of each primer (25 lm), 4 lL dNTPs (2.5 mM each), 5 lL 109 Taq buffer (Promega), 4 lL MgCl2, 1 unit of Taq polymerase (Promega), and 33 lL H2O. The PCR reaction conditions were: 30 cycles of 30 s denaturation at 94C, 50 s primer annealing at 55C, and 80 s primer extension at 72C. A nal 5 min incubation at 72C was performed to ensure fulllength products were obtained. The amplied PCR products were separated by electrophoresis in 0.8% agarose gel (Sambrook et al. 1989). Southern blotting Plants were initially tested by PCR and those that were PCR-positive for the SCYLVcp gene were then examined by Southern analysis (Southern 1975) to conrm integration of the transgene. DNA for Southern blotting was isolated using the protocol of Dellaporta et al. (1983). Ten lg of DNA were digested to completion by EcoRI and blotted to Hybond N? membranes (Amersham) by capillary alkaline transfer. EcoRI digestion of plasmid pFM395 yielded a 1.3 Kb fragment containing an intact copy of the untranslatable coat protein gene. The 1.3 Kb fragment was gel puried and labeled with 32P by the random priming method (Feinberg and Vogelstein 1983) and used as a probe. Hybridization and stringency washes were performed as previously described by Church and Gilbert (1984).

NOS3

pHA9
Scubi pro NPTII NOS3

Fig. 1 Diagrams showing the core features of the pFM395 and pHA9 constructs used for transformation of sugarcane

performed using the method of Ma et al. (2000) with some modications. Embryogenic calli were initiated from leaf whorls, containing the shoot apical meristems, in 19 Murashige & Skoog with macro and micronutrients (Murashige and Skoog 1962) (Caisson Labs) with 3 mg/L 2,4-D (MS3) and maintained on MS1 medium (MS medium with 1 mg/L 2,4-D). Four to seven days prior to bombardment, calli were cut into 34 mm pieces and transferred to fresh MS1 medium. They were transferred to MS1 supplemented with sorbitol and mannitol (MS1 with 0.2 M sorbitol and 0.2 M mannitol) 4 h prior to bombardment. Approximately 5 lg of DNA (pFM395: pHA9, 1:1) per shot was used for bombardment. Each plate was bombarded twice. One day after bombardment, calli were transferred to MS1 medium for recovery. After 5 days, the calli were placed on selection medium, MS1 with 100 mg/L of geneticin (G418). The cultures were maintained under a controlled temperature and light regime (approximately 22.8C with 14 h light). They were maintained on selection medium for 1216 weeks, with transfers every 3 weeks. During transfer, if a piece of callus fragmented into several pieces each daughter callus was assigned the same line number to minimize the duplication of lines. Plantlets were regenerated from selected calli on MS0 (MS medium, no hormones) with 100 mg/L of G418. Regenerated plantlets were rst rooted on MS medium with 4 mg/L naphthaleneacetic acid (NAA) and 2.5 g/L phytagel (Sigma Aldrich) (MPII) with no selection and then in liquid MPII medium before potting. Plants were also regenerated from non-transformed callus as a negative tissue culture control. All plants were maintained in the greenhouse in 10 cm pots with Sunshine #4 professional growing mix (Sungro Horticulture, Canada). Greenhouse conditions were 25 4C

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Semi-quantitative and real-time quantitative RT-PCR analysis RNA was isolated from 1 g (fresh weight) of sugarcane plants infected with SCYLV using an RNeasy kit, (Qiagen) according to manufacturers instructions. For semi-quantitative RT-PCR, 50 lg of total RNA was treated with DNase (RQ1, Promega) and puried using phenol:chloroform according to RQ1 product instructions. Reverse transcription was performed using 5 lg of total RNA which was incubated for 5 min at 65C with 500 ng polydT primer and 12.5 mM of each dNTP in a nal volume of 12 lL. After centrifugation (30 s; 10,0009g), the mixture was incubated for 2 min at 42C with 4 lL of 59 rst strand buffer (Invitrogen) and 1 lL of 0.1 M DTT, 1 lL of SuperScript II RNase H- reverse transcriptase (200 U/uL, Invitrogen) was added and the mixture was incubated for 50 min at 42C. The reaction was terminated by heating the sample for 15 min at 70C. 2.5 lL of cDNA was used in a standard PCR reaction. PCR conditions were as follows: 95C for 30 s; 35 amplication cycles of 95C for 30 s, 55C for 30 s, 72C for 45 s; 72C for 45 s. PCR products were visualized on a 2% agarose gel stained with ethidium bromide. For each PCR reaction, the cycle number was optimized in order to remain in the exponential phase of the PCR reaction. Conserved primers were designed to the ORF0 sequences to cover all known SCYLV strains available in the GenBank data library (accession numbers AM072750.1, AM072751.1, AF157029 AY236971.1, AM072752.1, AM072753.1, AJ249447.1, AM072754.1, AM072755.1 and AM072756.1). SCYLV primers, 50 -AGA TCT ATG CTT TTC AAC GAA TTC-30 and 50 -GTC GAC CCA GTT GTA AAC GGG AGT G -30 were used (201 bp product). TPI (Triosephosphate isomerase) primers 50 -CAA TGA CTG GAG CAA CGT AG -30 and 50 - GTA ACA GAG CCT CCG TAG AT-30 were used as an internal control based on the gene sequence from sugarcane (200 bp product). The mRNA level of SCYLV was determined in parallel by real-time quantitative RT-PCR. For realtime quantitative RT-PCR, 1 lg of total RNA was treated with DNase (RQ1, Promega) and puried using phenol:chloroform according to RQ1 product instructions. First strand synthesis was carried out using random hexamer primers, as previously

described. For the PCR part, primers specic for SCYLV and TPI were used with Platinum SYBR Green qPCR Supermix UDG (Invitrogen) as per manufacturers instructions. Aphid vector inoculation Approximately 56-month-old transgenic plants along with non-transformed control plants of the same age were infected by placing ten viruliferous aphids (Melanaphis sacchari) on the top visible dewlap leaf lamina and allowing them to feed for 1 week. Viruliferous aphids were collected from a plant of H73-6110 which was known to be infected with SCYLV and upon which the aphids had been allowed to feed freely. Newly emerged leaves were harvested, screened for presence of the virus by tissue blots (approximately one month post inoculation) and used for RT-PCR and qRT-PCR. The experiments were repeated three times. Tissue blot immunoassay Regenerated plants were screened for SCYLV infection with a tissue blot immunoassay (TBIA) modied from the method of Schenck et al. (1997). Tissue blots were made from leaf midribs that were transversely cut with a razor blade and immediately pressed onto a nitrocellulose TransBlot membrane (Biorad Laboratories, Hercules, CA, USA). The protein impregnated membranes were immersed for 15 min in chloroform to remove chlorophyll that might otherwise interfere with the analysis of the colorimetric assay (Fitch et al. 2001). The membranes impregnated with plant and viral proteins were then removed from the chloroform and dried at room temperature. The dried membranes were immersed in TBS buffer (0.05 M Trizma base, 0.05 M NaCl, pH 7.5) and blocked in 2% nonfat dry milk powder (dissolved in TBS buffer) for 1 h at room temperature. The membranes were then rinsed in a PBSTween solution containing 0.137 M NaCl, 1.5 mM KH2PO4, 8.1 mM Na2HPO47H2O and 0.05% Tween 20 (pH 7.4). The rinsed membranes were placed into a SCYLV polyclonal antibody IgG solution from rabbits (Dr B.E.L. Lockhart, University of Minnesota, St Paul, USA) containing 1 lg per 3 mL IgG in TBS plus 1% dry milk and incubated overnight at 4C. After three 10 min rinses at room temperature in

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PBS-Tween on a shaking platform, the membranes treated with primary antibody were incubated in an alkaline phosphatase-conjugated goat anti-rabbit IgG solution (1 lg per 2 mL in TBS plus 1% dry milk, BioRad) for 3 h at room temperature. The primary antibody was raised in rabbits to detect SCYLV while the secondary antibody was obtained from goat to detect rabbit protein. The proteins treated with secondary antibody were given three 10 min rinses in PBS-Tween and then incubated for 1030 min in the dark in a substrate solution (one tablet of 5bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium [BCIP/NBT, SigmaAldrich] per 10 mL distilled water). After rinsing in distilled water and drying on a paper towel, the membranes were inspected under a binocular microscope for purple color development in the phloem tissue. A plant was scored as positive if in any of the three experiments a positive reaction to the antibody was observed.

Southern Blot Analysis

M T1 T2 T3 T4 T5 T6 T7 NT P1 P5 P10

1.3 Kb

Results Production of transgenic sugarcane From 20 plates of H62-4671 embryogenic calli that were bombarded, 12 transgenic plants, morphologically similar to the non-transformed H62-4671 plant, were produced. This is an average of 0.6 G418 resistant (G418R) plant lines/plate. Calli or plants which had a bleached appearance were discarded. Also, some calli failed to regenerate and these were also discarded. Transgenic lines were conrmed by PCR using primers specic for the nptII gene (gure not shown) and SCYLVcp gene (gure not shown). PCR-amplied NPT II and SCYLVcp genes were detected in all tested lines, while no bands were detected in the non-transformed tissue culture control (NT). One transformed line, T4 showed two bands in NPT II-specic PCR amplication (gure not shown), a weak one at the expected size of 700 bp and a stronger one at a lower size, indicating that T4 consisted of at least two copies of NPT II gene, with one being degraded. The same line had an intact copy of the SCYLVcp gene (gure not shown). Fragmentation of a plasmid introduced by biolistic bombardment is commonly observed in transgenic plants transformed using this method so it is not surprising to obtain pieces of the NPTII gene here. Southern blot

Fig. 2 Southern blot analysis of EcoRI-digested genomic DNA extracted from sugarcane leaves and probed with 1.3 Kb SCYLVcp probe. M: molecular size marker (u174 DNA HaeIII digest, New England Biolabs); NT: non-transformed H62-4671 control; T1-7: transgenic H62-4671 sugarcane lines transformed with the SCYLVcp construct. Molecular weight of the hybridizing product is indicated on the left. P: pFM395 plasmid DNA; P1, P5 and P10 are dilutions of plasmid DNA (pFM395) and represent 1, 5 and 10 copies, respectively

analysis conrmed the transgene integration. The PCR positive plants contained between 1 and 5 copies of the SCYLVcp transgene based on the intensity of the bands (Fig. 2). Test of transgenic sugarcane lines for resistance to SCYLV Seven randomly selected transgenic sugarcane lines (T1, T2, T3, T4, T5, T7, T9) along with controls (H62-4671, non-transformed parent line; pHM2-10, transgenic H62-4671 transformed with nptII selectable marker only; H65-7052, another non-transformed Hawaiian cultivar; and H73-6110, a virus source plant), were tested for resistance to SCYLV infection. The transgenic lines (T1-T9) and controls (H62-4671, pHM2-10, H65-7052) were tested by TBIA and RT-PCR for SCYLV prior to the infection experiment and proven to be virus-free (data not shown). H73-6110 was used as the virus source plant and was germinated from infected seed pieces of eld-grown plants. H73-6110 consistently tested

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positive for SCYLV. Sugarcane aphids were transferred onto the H73-6110 plants and allowed to feed for at least 1 month before being transferred onto other plants in order to transfer the virus. To check that the aphids were in fact carrying the virus and able to infect plants, some aphids were transferred onto virus-free plants of H62-4671 which had been obtained through tissue culture (Fitch et al. 2001). All of the tested H624671 plants became infected, conrming that the aphids were viruliferous and the tissue culture plants were still highly susceptible to the virus. The test was carried out by TBIA in which chlorophyll was removed from the membrane before adding the blocking reagent. This modication allowed for the unambiguous scoring of even faint viral infections in small, freshly-emerged leaves (Fig. 3a, b). The transgenic plants which were infected by viruliferous aphids were scored using tissue blot immunoassay for the presence of virus and compared to the controls (Table 1). Plants of the parent line H62-4671 were negative for virus before the inoculation and became positive after inoculation in all experiments, showing that the parent line is highly susceptible to the virus and that the aphids are viruliferous. Plants of cultivar H73-6110 which was the virus source plant on which aphids originally fed, gave positive results in all experiments. Control transgenic plants, pHM2-10, of cultivar H62-4671,

containing the NPT II selectable marker only, were positive in all but one test, indicating that the NPT II transgene itself does not improve plant resistance to the virus. Results for the seven H62-4671 transgenic lines varied. For lines T1 and T3 no virus was detected in any blot, lines T2 and T7 showed two positive results, and lines T4, T5 and T9 gave 34 positive results which was similar to the result of the susceptible cultivar H65-7052 (Table 1). Most of the plants which gave positive results for SCYLV did not have visible symptoms. However, when the plants were moved into conditions of cold stress, typical leaf yellowing symptoms were expressed in the infected parent line, but not in the transgenic line T1 (Fig. 3c). T1 was the only transgenic line subjected to cold stress in the growth chamber. Determination of virus titer by RT-PCR and real-time qRT-PCR There was a strong visual co-variation between the results for RT-PCR (Fig. 4), as shown by agarose gel electrophoresis, and the real-time qRT-PCR. Tissue culture plants of H62-4671 served as a negative control, with no virus being detected by either RT-PCR or real-time RT-PCR. A resistant cultivar H78-4153 (based on previous TBIA) and susceptible

Fig. 3 Analysis of SCYLV-infected sugarcane leaves. a Representative tissue blot immunoassay of sugarcane leaf midribs using antibody against SCYLV coat protein from an uninfected leaf of parent line H62-4671, b representative tissue blot from an infected leaf of parent line H62-4671, c Phenotypes of

SCYLV-infected sugarcane leaves from plants under a cold stress (18C after 1 month); left susceptible leaf with typical yellowing symptoms (non-transformed control, H62-4671), right resistant leaf with no visible symptoms (transgenic line, T1)

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Table 1 Detection of SCYLV by TBIA in transgenic and control sugarcane H62-4671 plants infected with viruliferous aphids Lines Positives Exp #1 3/3 0/3 2/3 0/3 1/3 0/3 1/3 1/3 2/3 0/3 3/3 Positives Exp #2 3/3 0/3 0/3 0/3 1/3 1/3 0/3 1/3 3/3 2/3 3/3 Positives Exp #3 3/3 0/3 0/3 0/3 2/3 2/3 1/3 1/3 3/3 2/3 3/3 Positive plants/total plants tested 9/9 0/9 2/9 0/9 4/9 3/9 2/9 3/9 8/9 4/9 9/9

H62-4671 (non-transformed) T1 (transgenic H62-4671) T2 (transgenic H62-4671) T3 (transgenic H62-4671) T4 (transgenic H62-4671) T5 (transgenic line H62-4671) T7 (transgenic H62-4671) T9 (transgenic H62-4671) pHM2-10 (transgenic H62-4671 with npt II) H65-7052 (susceptible virus-free control) H73-6110 (virus source plant)

Plants for this experiment (except virus source plants, H73-6110) were derived from tissue culture through embryogenic calli and were initially free of virus. Five to six-month-old plants of uniform size were each inoculated in the greenhouse with 10 viruliferous aphids which had previously fed on virus source plants H73-6110. The aphids were allowed to feed for one week. One month later, tissue blots were taken from emerged leaves. For each plant, single leaves were blotted three times. The plant was scored as positive if any of three blots showed a positive reaction for the presence of bound antibody. There were three replicates in each experiment and the experiment was repeated three times throughout the year

Fig. 4 Relative quantication of virus titer using RT-PCR. a agarose gel electrophoresis of RT-PCR products. Primers used were for SCYLV and for TPI (endogenous internal control). b results from real-time quantitative RT-PCR using the same primers as for a. Tissue culture plants (nontransformed H62-4671), which do not contain any virus, were used as a negative control. The susceptible cultivar and the transgenic lines were H62-4671, while the resistant cultivar was H78-4153. The transgenic lines had been inoculated with viruliferous aphids 1 month prior

(a)
SCYLV TPI

(b)
SCYLV concentration

5 4.5 4

(Log scale)

3.5 3 2.5 2 1.5 1 0.5 0 T1 T2 T3 T4 T5 T6 T7 T8 T9

Transgenic H62-4671 lines

cultivar, non-transformed H62-4671, were included for comparison purposes. With RT-PCR and qRT-PCR, the resistant cultivar H78-4153 was detected to have SCYLV, however, with 103- to 104-fold lower titer than the susceptible cultivar H62-4671. It was found that, with the exception of T6

and T8, the virus titer for all transgenic lines was at least 103-fold lower than that of the susceptible parent line, H62-4671 (Fig. 4a). However, there was a low amount of virus in the inoculated plants of the transgenic lines T1, T2, T3, T5, T7 and T9, at a similar amount as in so-called resistant cultivar

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H78-4153 (Fig. 4b), denitely above the value for the virus-free line obtained by tissue culture. The amount of virus detected in line T4 was found to be lower than that for lines T6 and T8 but it was higher than the other transformed lines. Relationship between copy number and virus titer for transgenic sugarcane lines of H62-4671 An estimate of copy number was made from the results of Southern blotting. Transgenic lines T1, T2, T3, T4, T5, T6, T7 were included in this analysis. The virus titer for these lines was determined by real-time quantitative RT-PCR. A graph of copy number with respect to virus titer was made (gure not shown). Copy numbers of the transgene ranged from 1 to 5 copies while virus titer ranged from roughly 0.5 to 4.5 on a log scale. Lines T1 and T4, for example, both contained 1 copy of the transgene but had virus titers of 0.5 and 2.1, respectively. Also lines T2, T3, T5 and T7 all had virus titers below 1.0 but had copy numbers of 2, 3, 5 and 5 respectively. These results show that there was no direct correlation between copy number and virus titer.

Discussion Available sequences in Genbank indicate that the ORF of the coat protein is the most conserved of the ORFs of SCYLV (Zhu et al. in press). Consequently, an untranslatable fragment from the ORF of the coat protein is a good choice for producing transgenic lines with resistance to as many SCYLV strains as possible. Southern blots conrmed incorporation of the transgene into the chromosome, apparently with different copy numbers, estimated to be between 1 and 5 copies. Virus titer, as measured by real-time quantitative RT-PCR, was compared to the transgene copy number. There was no direct correlation found for the seven lines examined. Transformation efciency was low with 0.6 lines per plate. It is impossible to directly compare results here with previously published results as researchers have used different tissues and also different methods. Two examples of published transformation efciency are Bower et al. (1996) who reported 19.8 3.7 independent transgenic plants per bombardment from an optimized G418 selection regimen,

and Arencibia et al. (1998) who used Agrobacterium tumefaciens and reported transformation frequencies between 9.4 9 10-3 and 1.15 9 10-2. The virus titer after controlled inoculation with viruliferous aphids was taken as a measure of resistance to SCYLV, i. e. how well the virus is able to infect the sugarcane and replicate within the plant. In this study both TBIA and RT-PCR methods have been used to detect presence of the virus in sugarcane plants. However, TBIA was used here as it is a simple and fast method which is routinely used for screening multiple samples. The reaction to virus infection varied from almost complete resistance with hardly any virus being detected (lines T1 and T3) to only partial resistance for line T4, and no resistance (lines T6 and T8) in which the amount of virus detected was comparable to that of the non-transformed H62-4671. A similar variation in sugarcane resistance was reported previously by Ingelbrecht et al. (1999) using SrMV and in barley by Wang et al. (2000). In sugarcane resistant to SrMV, the recovery phenotype was characterized by a gradual reduction in mosaic symptoms in successive leaves until leaves emerged that were completely symptomless and virus free. The transmission of SCYLV by Melanaphis sacchari is a routinely practiced and successful method to produce virus-infected sugarcane (Schenck and Lehrer 2000). The non-transformed parent control, H62-4671, was 100% positive for SCYLV. Even the most resistant transgenic plants have a low virus titer, similar to that of naturally-resistant cultivars, but about 3 times higher than the virus-free line from tissue culture. SCYLV-multiplication is thus not completely suppressed. The SCYLV-resistance of the transgenics was also seen as an absence of symptom expression after a shift to cool temperature. However, symptoms are a less reliable indicator of virus-resistance compared with qRT-PCR, because there are differences in symptom expression among the Hawaiian cultivars. In one test location, leaf yellowing appeared on H657052 at 4 months after planting, whereas cultivar H73-6110 did not show yellowing until 10 months after planting. Generally, H73-6110 showed symptoms at a later stage of development than did H65-7052, and only when water stressed. RT-PCR and real-time qRT-PCR are 103- to 4 10 -fold more sensitive than the TBIA as shown previously (Zhu et al. 2010). In this earlier study,

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positive RT-PCR and qRT-PCR results were obtained for cultivars previously thought, based on the TBIA, to be immune to virus infection. The results for the transgenics and the parent line are generally in agreement with that observation (Table 1; Fig. 4). The parent line H62-4671 produced 9/9 positive results for the tissue blot and had the highest SCYLV level in qRT-PCR. Lines T1 and T3 which had 0/9 positives in the tissue blot had among the lowest level in qRT-PCR. However, lines T2 and T7 had a similarly low virus titer but reacted in 2/9 cases in TBIA, suggesting that the sensitivity of TBIA may be as low as of qRT-PCR in that particular case. The explanation is that the TBIA test is marked as positive if one of the vascular bundles shows a positive reaction, whereas PCR is performed with RNA-extracts from all vascular bundles so that a localized, high virus titer is diluted in the extraction process, in contrast to the TBIA. SCYLV-resistant cultivars are available and preliminary research indicates that resistance is inherited (S. Schenck, unpublished results). Production of SCYLV-resistant cultivars through breeding may therefore appear useful, however, the cultivars with highest yield are all susceptible to SCYLV and transgenic sugarcane plants of a Hawaiian cultivar with resistance to SCYLV may be the only promising way to reduce the incidence of SCYLV in the eld. Production of virus-free high-yielding lines by meristem culture would be another promising option, but the maintenance of virus-free seedcane elds may be problematic because virus-free susceptible sugarcane can quickly become infected. In one instance, nearly half of SCYLV-free H87-4094 stools growing near infected sugarcane which was infested with aphids became infected within 18 weeks, although plants in the plots located far from any other sugarcane could still remain uninfected 12 months after planting (Lehrer et al. 2007). In the eld, preventing virus spread by controlling aphids is not practical, since insecticides are not used on sugarcane plantations in Hawaii (Schenck and Lehrer 2000). Recently, Gilbert et al. (2009) reported the transformation of two Canal Point clones with a construct harbouring a fragment of the SCYLV coat protein in an antisense orientation and a modied antibacterial Cecropin B gene. In a eld trial, they compared the two transgenic lines with a transformation control (NPT II only), the non-transformed

parent and a tissue culture control. They found by using TBIA that compared to 98% infection in the non-transformed plants, one transgenic line had only 5% and the other was virus free. However, as TBIA has been shown to be relatively insensitive, it is reasonable to assume that both transformed lines contained a low virus titer. Regrettably, the yield of the two transformed lines was lower than that of the non-transformed plants (6.58.7 tons less sucrose/ha/ year), a result which highlights the importance of eld testing transgenic plants. Gilbert et al. (2009) found a reduction in virus detected even in the transformation control plants (the ones containing only NPT II). In our results there was no difference between plants containing NPT II and the nontransformed H62-4671. Yield tests of SCYLV-free lines in the eld obtained by meristem culture showed that the absence of SCYLV in a commercial cultivar increases yield, at least in a one year crop cycle (Lehrer et al. 2009), and a limited eld trial comparing plants of cv. H65-7052 carrying low and high SCYLV-titer showed that the eld plots with plants of higher virus titer developed YLS and yielded only 5460% of cane and sugar tonnage compared to plots with plants of low virus titer (Zhu et al. 2010). Therefore the transgenic approach to produce high yielding sugarcane cultivars with resistance to SCYLV seems to be a valuable option for Hawaiian sugarcane growers.
Acknowledgments We thank Dr. E. Mirkov, Texas A&M Agriculture Experiment Station, Weslaco, TX for providing pFM395 construct; Dr. Henrik Albert at USDA, ARS for providing the pHA9 construct containing selection marker; Dr. M. Fitch, USDA, ARS, for advice on sugarcane transformation and clonal propagation and Dr. M. Irey, US Sugar, for providing primer sequences for SCYLV. This work was partially supported by a cooperative agreement (No. CA 58-5320-3-460) between the U.S. Department of Agriculture, Agricultural Research Service, and the Hawaii Agriculture Research Center.

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