Efficient production of transgenic citrus plants ... - Springer Link

Plant Cell Reports (2000) 19 : 427–433

Q Springer-Verlag 2000

A. Domínguez 7 J. Guerri 7 M. Cambra 7 L. Navarro P. Moreno 7 L. Penã

Efficient production of transgenic citrus plants expressing the coat protein gene of citrus tristeza virus

Received: 7 April 1999 / Revision received: 17 June 1999 7 Accepted: 24 June 1999

Abstract The coat protein gene of citrus tristeza virus (CTV) has been introduced into Mexican lime (Citrus aurantifolia Swing.) plants by using an improved Agrobacterium-mediated genetic transformation system. Internodal stem segments from greenhouse-grown seedlings were co-cultivated with A. tumefaciens strain EHA 105 carrying the binary plasmid pBI 121/CTV-CP in a medium rich in auxins that provided the explant cells with the proper treatment to shift them to a competent state for transformation. The transformation frequency was enhanced, and this allowed us to recover 42 transgenic plants from 1200 explants. Regenerated shoots were identified as transformants by performing b-glucuronidase (GUS) assays and subsequently by PCR amplifications of the CTV-CP transgene. Southern analyses revealed that at least one copy of the CTV-CP gene was integrated in all PCR positive plants. Interestingly, 70% of them had linked T-DNAs arranged at one locus. Copy number of the CTV-CP gene varied from one to six among the transgenic lines. Half of them showed truncated T-DNAs in which the left border was lost. Expression of the CTV-CP transgene was demonstrated in 38 out of 42 plants by western analysis and DASI-ELISA. No correlation was found between coat protein expression and transgene copy number or integration pattern. Key words Lime 7 Genetic transformation 7 Coat-protein-mediated resistance 7 Citrus tristeza virus 7 Agrobacterium tumefaciens

Communicated by G. Pelletier A. Domínguez 7 J. Guerri 7 M. Cambra 7 L. Navarro P. Moreno 7 L. Penã (Y) Dpto. Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias (IVIA), Apartado Oficial, E-46113-Moncada, Valencia, Spain e-mail: lpenya6ivia.es Tel: c34-96-1391000 Fax: c34-96-1390240

Introduction Citrus tristeza virus (CTV), an aphid-transmitted closterovirus, is the causal agent of one of the most economically important diseases of Citrus. In nature, this virus is restricted to citrus species and it causes two main diseases: (1) decline and death of most citrus species grafted on sour orange (Citrus aurantium L.), and (2) stem pitting, stunting and reduced fruit yield and quality of some citrus varieties regardless of the rootstock (Bar-Joseph et al. 1989). Damage caused by the first disease, which is caused by most isolates, can be avoided by using CTV resistant or tolerant rootstocks. Losses from the second syndrome are more difficult to avoid and are highly dependent on the citrus species and the virus isolates. Some species, like Mexican lime, are very sensitive and show symptoms with most CTV isolates (Roistacher 1991), while others like grapefruit (C. paradisi Macf.) or some varieties of sweet orange (C. sinensis L. Osb.) are affected only by some virulent isolates. In citrus areas where these virulent isolates are common, cross-protection with mild CTV isolates (Costa and Müller 1980) has been assayed to reduce yield losses, but this approach has been successful only in some areas, and protection afforded is sometimes temporary. Since the initial report of coat protein (CP)mediated resistance to tobacco mosaic virus in transgenic tobacco plants (Powell-Abel et al. 1986), this approach of engineering resistance has proved to be applicable to a range of more than 20 virus species in different transgenic hosts (Beachy 1997). In citrus, sensitivity of Mexican lime to CTV has made this species an excellent model system to investigate coat-protein-mediated resistance against this virus. Genetic transformation of lime with the CP gene of CTV has previously been reported by Gutiérrez et al. (1997). However, only nine b-glucuronidase (GUS) positive plants were recovered from almost 7000 explants. Furthermore, some of them were chimeras

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which failed to express the CTV CP in western analyses. Efficient production of transgenic lime plants has been shown by Pérez-Molphe-Bach and OchoaAlejo (1998) and by our group (Penã et al. 1997) using A. rhizogenes and A. tumefaciens, respectively, as transformation vectors. Here, we refine our transformation procedure and use it to generate more than 40 transgenic plants carrying the coat protein gene of CTV. Transgene integration patterns and expression are also investigated and discussed.

Materials and methods Plasmid vector construct The coding sequence of the CP gene of CTV severe strain T-305 (Moreno et al. 1993) was obtained from ds-RNA purified from CTV-infected tissue (Moreno et al . 1990) by reverse transcription and PCR amplification in a single step (RT-PCR). RT-PCR was performed at 42 7C for 45 min, and PCR included 30 cycles of 30 s at 94 7C, 30 s at 45 7C and 40 s at 72 7C, plus a final segment of 2 min at 72 7C. Primers used were 5b-TTGGATCCATGGACGGAAAC-3b and 5b-TTGGATCCTCAACGTGTGTTG-3b, which carry BamHI restriction sites at both 5b ends. RT-PCR amplification product digested with BamHI gave rise to a fragment of 673 nucleotides that was cloned into the vector pMOG180 (Mogen International, Leiden, The Netherlands) at its unique BamHI site, which is located between the cauliflower mosaic virus (CaMV) 2!35S promoter plus the alfalfa mosaic virus (AlMV) RNA 4 leader sequence and the nopaline synthase gene (nos) terminator sequence. The resulting plasmid carrying the CTV-CP expression cassette flanked by unique HindIII and EcoRI restriction sites was digested with EcoRI and the protruding end formed was filled in with the klenow fragment of the E. coli DNA polymerase I. Subsequently, a HindIII adaptor was linked and the plasmid was digested with HindIII to release the CTV-CP expression cassette. This was subcloned into the plant transformation binary vector pBI-121 (Jefferson et al. 1987) at the unique HindIII site. All manipulations were performed according to Sambrook et al. (1989). The resulting plasmid is referred to as pBI121/CTV-CP. In this vector, the CTV-CP expression cassette is flanked by the nptII expression cassette, used as a selectable marker gene, and the uidA expression cassette, used as a reporter and selectable marker gene. Transformation and regeneration pBI 121/CTV-CP was introduced in Agrobacterium tumefaciens EHA 105 (Hood et al. 1993) by electroporation (Singh et al. 1993). Greenhouse-grown Mexican lime seedlings, 6–12 months old, were used as source of tissue for transformation. Internodal stem segments were inoculated with Agrobacterium at 10 7 cell/ml in MS salts (Murashige and Skoog 1962), 0.2 mg/l thiamine hydrochloride, 1 mg/l pyridoxine hydrochloride, 1 mg/l nicotinic acid and 3% (wt/vol) sucrose for 15–30 min, blotted dry with sterile filter paper and transferred to co-cultivation medium for 3 days. To assess factors affecting the transformation frequency, different co-cultivation treatments were performed and compared. Agrobacterium-inoculated explants were co-cultivated on: (1) tomato cell suspension medium (TCSM), composed of MS salts, 1 mg/l thiamine hydrochloride, 1 mg/l pyridoxine hydrochloride, 1 mg/l nicotinic acid, 3% wt/vol sucrose, 2 mg/l indole-3acetic acid, 1 mg/l 2-isopentenyl-adenine, 2 mg/l 2,4-dichlorophenoxyacetic acid, 0.8% (wt/vol) agar; (2) TCSM with a filter paper

on the top (TCSMcP); (3) TCSM plus 2 ml of 6- to 7-day-old tomato cell suspensions in liquid TCSM on the top (TCSMcC); and (4) TCSM with a filter paper and tomato cell suspensions on the top (feeder plates) (TCSMcPcC). After co-cultivation, transgenic plant regeneration was carried out as described in Penã et al. (1997). Briefly, explants were blotted dry with sterile filter paper and transferred to shoot regeneration medium (SRM) consisting of MS salts, 0.2 mg/l thiamine hydrochloride, 1 mg/l pyridoxine hydrochloride, 1 mg/l nicotinic acid, 3% (wt/vol) sucrose, 1 mg/l benzylaminopurine, 10 g/l agar, 100 mg/l kanamycin for selection of transgenic shoots and 250 mg/l vancomycin and 500 mg /l cefotaxime to control bacterial growth. In all media pH was set at 5.7. The cultures were maintained in the dark for 15 days and then transferred to a 16-h photoperiod. Regenerating shoots were excised and divided into two pieces (Penã et al. 1995). Basal portions were tested for histochemical GUS activity and apical portions were shoot tip grafted in vitro on Troyer citrange seedlings. A new grafting of the in vitro-growing plants on vigorous rootstocks allowed rapid acclimatisation and development of plants under greenhouse conditions. Absence of Agrobacterium contamination in the regenerated shoots was demonstrated by PCR, according to Cubero et al. (1999). The transformation frequency was evaluated after 6 weeks on SRM as the total number of blue spots (GUS-expressing cell clusters at the cut ends of the explants, visible under a stereo microscope at 50!magnification) per Agrobacterium-inoculated explant!100.

PCR and Southern analyses GUS-positive plantlets were screened for the presence of the CTV-CP transgene by PCR using the same CTV-CP-specific primers described above. DNA was extracted from leaf pieces according to McGarvey and Kaper (1991). A standard PCR technique was used to detect the transgene. Reactions were performed in a thermal cycler under the following conditions: 30 cycles of 94 7C for 30 s, 55 7C for 30 s, 72 7C for 40 s plus a final segment of 72 C for 2 min. For Southern analyses, DNA was isolated from leaves according to Dellaporta et al. (1983). Twenty micrograms of HindIII- or DraI-digested DNA samples were separated on 1% (wt/vol) agarose gels and blotted to nylon membranes (HybondN c, Amersham). Filters were probed with a digoxigenin (DIG) (Boehringer-Mannheim) labelled fragment of the coding region of the CTV-CP gene prepared by PCR following the suppliers’ instructions. Subsequently, filters were stripped (Reed and Mann 1985) and reprobed with a DIG-labelled fragment of the uidA transgene, and again stripped and reprobed with a DIG-labelled fragment of the nptII transgene. The two last probes were also prepared by PCR. The uidA primers were 5b-ACGTCCTGTAGAAACCCCAACC-3b and 5b-TCCCTGCTGCGGTTTTTCAC-3b, and the nptII primers were 5b-GACGAGGCAGCGCGGCTAT-3b and 5b-AAGAAGGCGATAGAAGGCGA-3b. Reactions were performed as for CTV-CP except that DNA synthesis of uidA probe was at 72 7C for 1 min.

Western and ELISA analyses Leaf tissue from transgenic plants was used to obtain crude protein extracts for western analyses. The extraction buffer was 0.1 m Tris-HCl pH 6.8, 1 mm PMSF. Protein extracts were fractionated by electrophoresis on SDS-PAGE (14% polyacrylamide) and electroblotted to Immobilon-PVDF membranes (Millipore) using a semidry system (Biorad) following the suppliers’ instructions. Immunodetection was performed using the monoclonal antibody MCA-13 (kindly gifted by Dr. S. Garnsey, Horticultural Research Laboratory, Orlando, Fla., USA) to the CTV CP (Permar et al. 1990) as the primary anti-

429 body, and goat anti-mouse IgGs (Boehringer-Mannheim) conjugated with alkaline phosphatase as the secondary antibody. A double-antibody sandwich indirect ELISA (DASI-ELISA) was carried out as described in Cambra et al. (1993), again using the monoclonal antibody MCA-13 to the CTV coat protein.

Results and discussion Improvement of the Mexican lime transformation procedure We have previously reported the positive influence of using tomato feeder plates as transformation enhancers during co-cultivation of lime explants with Agrobacterium (Penã et al. 1997). Feeder plates seemed to stimulate the number of competent cells for transformation in the cut ends of the explants. To investigate the influence of the components of the feeder plates (the tomato cell suspensions, the filter paper layer and the culture medium rich in auxins) in the genetic transformation of lime explants, we have evaluated them separately and in combination. As shown in Fig. 1a, the TCSM basal medium was responsible for the increased lime transformation frequency previously attributed to the feeder plates as a whole. The role of the tomato cell suspensions and the filter paper was even detrimental, as they drastically decreased transformation frequency in comparison with co-cultivation of explants in TCSM. Not only was the transformation frequency much higher in explants co-cultivated in TCSM, but so were the percentage of explants with blue spots (Fig. 1b) and the maximum number of blue spots per explant (Fig. 1c). The liquid layer provided by the cell suspension affected the explants negatively, favouring excessive formation of abnormal callus. In contrast, the filter paper layer between the basal medium and the explants precluded

Fig. 1a–c Influence of co-cultivation treatments on transformation of Mexican lime explants. Explants were co-cultivated on TCSM, TCSMcP, TCSMcC or TCSMcPcC (feeder plates). a Transformation frequency expressed as the total number of transformation events (blue spots) per explant!100. b Number of explants with blue spots per total number of explants,!100. c Maximum number of blue spots per explant. The minimum number of blue spots was 0 in all treatments. Vertical bars indicate SE from three experiments with 50 explants for each treatment

callus formation at their cut ends. Therefore, TCSM was used for co-cultivation in further experiments. As we have previously shown for Carrizo citrange (Cervera et al. 1998), phytohormones (mainly auxins) in TCSM seem to play a crucial role in inducing dedifferentiated cells competent for transformation in new callus tissue from the cambial ring (Ghorbel et al. 1999). These results also suggest that auxin supply could be excessive when the explants are soaked in liquid TCSM in the feeder plates, and insufficient when a filter paper is placed between the explants and TCSM. The transformation frequency using feeder plates could not be compared with that reported by us before (Penã et al. 1997), since different binary plasmids with different T-DNA cassettes and T-DNA sizes were used in both cases. The CTV-CP transgene was integrated in Mexican lime plants at variable copy number and insertion sites Forty-two transgenic lines were recovered from four transformation experiments comprising approximately 300 explants each, giving a transformation efficiency (transgenic regenerants per inoculated explant!100) of 3.5%. Histochemical GUS activity allowed us to identify transgenic shoots regenerated, and PCR assays indicated that these regenerants indeed carried the CTV-CP transgene (results not shown). Southern analyses were performed to confirm the integration of the CTV-CP gene and to study the TDNA organisation in all 42 lime transgenic plants. Plant DNA was digested with HindIII or DraI and sequentially hybridised with CTV-CP, uidA and nptII probes. Results of the Southern analysis from a group of 20 transgenic limes (CP.1 to CP.20) are shown in Fig. 2. Integration of at least one intact copy of the CTVCP transgene was demonstrated in all the plants, since HindIII-digested DNA from all samples released an approximately 1.8 kb fragment, corresponding to the CTV-CP expression cassette, which hybridised to the CTV-CP probe (Fig. 2a). Lines CP.2 to CP.4, CP.6, CP.9, and CP.12 to CP.17 showed additional bands of higher and/or lower molecular weights, suggesting that rearrangements had occurred affecting the CTV-CP cassette (Fig. 2a).

430 Fig. 2a–f Southern analyses of 20 transgenic limes (CP.1 to CP.20) carrying the CTV-CP gene. a–c Southern blots from DNA digested with HindIII. The same filter was hybridised to CTV-CP (a) probe, and successively, after stripping, was hybridised to uidA (b) and nptII (c) probes. Bands marked with p showed a signal with both the uidA and the nptII probes. d–e Southern blots from DNA digested with DraI. The same filter was hybridised to CTVCP (d) probe, and after stripping to uidA (e) probe. Control DNA extracted from a non-transformed Mexican lime plant. Molecular weight is indicated in kilobases. f Schematic representation of pBI121/CTV-CP T-DNA, indicating restriction sites and probes used. Sizes are indicated in base pairs

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When HindIII filters were sequentially stripped and reprobed with uidA and nptII, bands of the same sizes, which became superimposed in autoradiographs, were detected for both uidA and nptII probes in plants CP.1 to CP.9, CP.11, CP.14 to CP.17 and CP.19 (Fig. 2b,c). This indicated the presence in transgenic lime plants of linked T-DNAs, arranged as tandem or multicopy repeats at high frequency. Multicopy transformation events at the same locus have been widely reported in the genetic transformation of plants. However, little is known about how multimers are formed. De Neve et al. (1997) demonstrated that T-DNAs from two Agrobacterium strains carrying two different marker genes integrated as tandem repeats at the same locus, suggesting that ligation of single T-DNAs had occurred in plant cells prior to integration. More recently, experiments with rice transformation by particle bombardment (Kohli et al. 1998) suggested ligation of foreign DNA preintegration, and a second phase in the integration mechanism in which the original site of integration of transgenic DNA would act as a hot spot, facilitating subsequent integration of successive copies at the same locus. Chen et al. (1998) have also shown that co-bombardment of 14 plasmids on rice embryogenic tissues resulted in co-integration at the same locus of transgenes from different plasmids at high frequency. Here we report that 70% (29 out of 42) of the transgenic lime plants had linked T-DNAs arranged at one locus. Occurrence of this feature at such a high frequency in individual transgenic plants supports the hypothesis that ligation of foreign DNA molecules, preintegration and/or during integration, does not occur randomly but must be somehow favoured. Plant DNA was also digested with DraI, for which T-DNA has a unique restriction site, generating hybridising fragments which were composed of both T-DNA and plant DNA flanking the integration site. The length of these fragments varied depending on the location of the nearest DraI site in the flanking plant DNA and the integrity of the inserted T-DNA. Filters were first hybridised with the CTV-CP probe and later sequentially stripped and hybridised with the uidA and nptII probes (Fig. 2d,e, results not shown). The number of fragments indicated the minimum number of TDNAs integrated. The copy number in the different transgenic lines ranged from one to six (Fig. 2d,e). A high copy number of T-DNA inserts at the same or different loci has been associated with silencing of transgenes [reviewed in Vaucheret et al. (1998); Matzke and Matzke (1998)]. However, we did not observe this effect and all transgenic lines showed consistent histochemical GUS activity irrespective of their uidA transgene copy number. This may be explained by our selection scheme of transformants, since only regenerated shoots showing GUS activity were recovered, and those with silenced uidA expression would have been eliminated (see Materials and methods). HindIII and DraI filters also allowed us to partially characterise tandem T-DNAs. Thus, no evidence

suggesting the existence of T-DNA inverted repeats was observed in any of the transgenic lines. Inverted repeats centred on the nptII gene would generate approximately 5 kb Hind III fragments for the nptII probe, and inverted repeats centred on the uidA gene would generate HindIII fragments of approximately 7.2 kb for the uidA probe. Some of the transformants showed bands of these sizes but their remaining band pattern showed that those bands did not correspond to inverted repeats. For instance, CP.10 and CP.14 showed a 7.2 kb HindIII fragment for the uidA probe (Fig. 2b), but CP.10 integrated just one T-DNA copy, as shown in HindIII and DraI filters, and the 7.2 kb HindIII fragment of CP.14 also hybridised to the nptII probe, suggesting that it was a tandem direct repeat (Fig. 2). Selection of transformants based on GUS activity probably precluded recovery of transgenic plants carrying T-DNAs arranged as inverted repeats, since this type of configuration has been consistently related to silencing of transgenes (Stam et al. 1997, 1998). Half of the transgenic lime plants (20 out of 42) showed variable transgene copy number for the three probes when DNA was digested with DraI, indicating truncation of T-DNAs. For instance, the CP.2 line showed three hybridising bands with the CTV-CP probe and just two bands with the uidA probe (Fig. 2d,e), suggesting that one of the three T-DNAs integrated into the CP.2 line lacked the uidA gene, which is situated at the left border (LB) of the T-DNA (Fig. 2f). Similar patterns were observed in CP.4 to CP.6, CP.12, CP.14 to CP.15 and CP.18 to CP.19 lines (9 out of 20 in Fig. 2d,e). No difference in copy number was noticed between nptII and CTV-CP transgenes (results not shown). All these results indicated that many integration events corresponded to incomplete insertions of T-DNA in transgenic lime plants, in which the right border (RB; nptII cassette) remained conserved in all cases, but the LB (uidA cassette) was frequently lost. This agrees with the model of Tinland et al. (1995) of integration of the T-DNA into the plant genome, which suggests that Agrobacterium VirD2 protein has an important role in preserving the RB of the T-strand during integration. In two transformation experiments, half of the lime explants were co-cultivated with Agrobacterium carrying pBI 121/CTV-CP and the other half were cocultivated with Agrobacterium carrying pBI 121, as a control. The transformation frequency was two to three times higher with pBI 121 than with pBI 121/CTV-CP (results not shown). Since a similar transformation frequency should be expected for both T-DNAs, these differences probably mean that pBI 121/CTV-CP tended to lose the LB during integration much more frequently than pBI 121. It is conceivable that the size of the T-DNA could be directly related to the frequency of LB truncations of the T-DNA. Recent data from our laboratory (Cervera et al. submitted) support this possibility: in transgenic citranges carrying

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T-DNAs with two or with three expression cassettes, transformation efficiencies were 78% for a T-DNA with two cassettes and 32% for a T-DNA with three cassettes, and plants for the second group had frequently integrated T-DNAs without the LB. Controversial results in transformation frequencies reported by us previously (Penã et al. 1997; a T-DNA with two cassettes was used) and in this report could be also explained in the same way. Therefore, GUS expression may not be a reliable indicator of the actual transformation frequency (and probably not of transformation efficiency either), at least when the uidA expression cassette is placed at the LB and a long TDNA must be integrated into the plant genome. Transgenic lime plants showed different levels of CTV-CP expression, not correlated with transgene copy number and integration patterns Expression of the CTV-CP gene was investigated by western analyses and DASI-ELISA. Western analyses demonstrated that in most samples a protein product that co-migrated with purified CTV CP was immunoFig. 3a–b Protein expression analyses of 20 transgenic limes (CP.1 to CP.20) carrying the CTV-CP gene. a Western blot assay immunoreacted with MCA-13 monoclonal antibody against CTV CP. CTV CP Purified CTV CP from non-transformed Mexican lime plant infected with CTV strain T-305 (50 ng); CTV crude protein extract from a non-transformed Mexican lime plant infected with CTV strain T-305; Control crude protein extract from a non-transformed Mexican lime plant. Arrow indicates migration of CTV CP. b DASI-ELISA reacted with MCA-13 monoclonal antibody against CTV CP. Absorbance was measured at 490 nm. Control Extract from a non-transformed Mexican lime plant. Vertical bars indicate SE from two measures

reactive with the monoclonal antibody MCA-13. A filter with a group of 20 transgenic lines (CP.1 to CP.20) is shown in Fig. 3a. CTV-CP gene expression was detected in 38 out of 42 transgenic plants. CP.2, CP.4, CP.18 and CP.19 showed no detectable reaction (Fig. 3a). Levels of expression ranging from very high in CP.10, CP.13, CP.14 and CP.16 to negligible in CP.5, CP.15 and CP.20 were observed in the coat-proteinexpressing lines. DASI-ELISA confirmed expression of the CTV CP in the transgenic plants, and expression levels corresponded with those obtained in the western analyses (Fig. 3b). As for the uidA transgene, no correlation was found between expression of CTV-CP gene and copy number or T-DNA integration pattern (Figs. 2, 3). Variable levels of CTV-CP expression among the transgenic lines will allow study of the effect of CP accumulation in protection against CTV. Furthermore, lines without detectable protein expression could be of interest if they transcribe CTV-CP mRNA, since RNAmediated resistance is a highly efficient mechanism against viral infections (Baulcombe 1996). It has been widely reported that engineered resistance to viruses requires the production of numerous independent transformants to be able to select at least one with the appropriate level of gene expression to achieve efficient resistance (Fuchs and Gonsalves 1995; Scorza et al. 1994; Tricoli et al. 1995). We have developed a reliable genetic transformation system for Mexican lime that allowed us to incorporate the CP gene of CTV in more than 40 transgenic plants. This is the first demonstration of CTV-CP gene expression in transgenic lime plants. Considering the wide range of integration and expression patterns, it is expected that some line(s) will provide a level of resistance sufficient to efficiently control CTV.

433 Acknowledgements The authors wish to thank J.A. Pina and M.T. Gorris for their technical assistance. This research was supported by grant no. SC97-102 from the Instituto Nacional de Investigaciones Agrarias.

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