Molecular Breeding 10: 1–10, 2002. © 2002 Kluwer Academic Publishers. Printed in the Netherlands.
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Pathogen-derived resistance to Citrus tristeza virus (CTV) in transgenic mexican lime (Citrus aurantifolia (Christ.) Swing.) plants expressing its p25 coat protein gene Antonio Domínguez, Alfonso Hermoso de Mendoza, José Guerri, Mariano Cambra, Luis Navarro, Pedro Moreno and Leandro Peña* Dpto. Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias (IVIA), Apartado Oficial, E-46113 Moncada, Valencia, Spain; *Author for correspondence (e-mail:
[email protected]; phone: +34-96-13910000; fax: +34-96-1390240) Received 22 February 2001; accepted in revised form 17 October 2001
Key words: Citrus, Closterovirus, Plant virus, Protection, Transgenic trees Abstract The p25 coat protein (CP) gene of Citrus tristeza virus (CTV) was incorporated to Mexican lime plants and forty-two transgenic lines were produced, 25 containing the p25 CP gene of the severe CTV strain T-305 and 17 with that of the mild strain T-317. When plants propagated from each transgenic line were graft-inoculated with CTV T-305 or aphidinoculated with T-300, two types of response to viral challenge were observed: some lines developed CTV symptoms similar to those of non-transgenic controls, whereas others exhibited protection against the virus. This protection consisted of a proportion of plants, ranging from 10 to 33%, that were resistant to CTV, and the rest of them that showed a significant delay in virus accumulation and symptom onset. Protection was efficient against non-homologous CTV strains and was generally accompanied by high accumulation of p25 CP in the protected lines, which suggest a CP-mediated protection mechanism in most cases. This is the first report demonstrating pathogen-derived resistance in transgenic plants against a Closterovirus member in its natural host. Introduction Citrus is a major fruit tree crop in the world, and its ⬇ 100 million tons annual production represents 25% of the world fruit production (FAO 2001). Citrus tristeza virus (CTV), a member of genus Closterovirus, is the causal agent of the most economically important viral disease of this crop. CTV produces two main field diseases depending on the isolates: common isolates cause decline and death of most scion varieties grafted on sour orange (Citrus aurantium L.) rootstock, whereas highly virulent isolates additionally cause stem pitting on different scion varieties regardless the rootstock, resulting in reduced vigor, yield and fruit quality. CTV is considered a most serious threat to the citrus industry world-wide (Bar-Joseph et al. 1989). CTV has filamentous virions 2000 × 10–12 nm in size, composed of one molecule of single stranded
RNA of positive polarity and two capsid proteins (CP) of 25 kDa (p25) and 27 kDa (p27) that coat 95% and 5% of the particle length, respectively, conferring to the virions a ⬙rattlesnake⬙ structure (Agranovsky et al. 1995; Febres et al. 1996). The CTV genomic RNA has 19.2 kb (Karasev et al. 1995) and is organized in 12 open reading frames (ORFs) and untranslated regions (UTRs) at the 5’ and 3’ termini. ORF1a encodes a 349 kDa polyprotein containing two papain-like protease, methyltransferase and helicase-like domains. Translation of the polyprotein is thought to occasionally continue through the polymerase-like domain (ORF1b) by a +1 frameshift mechanism (Karasev et al. 1995). The ten ORFs 3’ proximal are expressed via 3’ co-terminal subgenomic mRNAs that are promoted internally (Hilf et al. 1995), and they include genes encoding the minor and major CPs, and several other proteins of 33-, 6-, 65-, 61-, 18-, 13-,
2 20- and 23 kDa (Pappu et al. 1994; Karasev et al. 1995). CTV is readily transmitted with infected buds and locally spread by several aphid species in a semi-persistent mode. It can be also experimentally transmitted mechanically but with low efficiency. In nature, its host range is limited to citrus, and only occurs in phloem-associated cells (Bar-Joseph et al. 1989). Up to date, breeding programs in citrus species have shown poor results mainly because of their complex reproductive biology, with many cases of apomixis and cross- and self-incompatibility, their high heterozigosity, and their long juvenile periods. Also the mode of inheritance of most important agronomic characters is largely unknown. Regarding CTV resistance, only the tolerant rootstocks Troyer and Carrizo citrange (C. sinensis (L.) Osb. x Poncirus trifoliata (L.) Raf.), obtained in 1909 in a cross made to incorporate cold tolerance to citrus fruits (Van Vuuren et al. 1993), have reached wide use. They have practically substituted sour orange rootstocks in certain citrus areas, as California and Spain, where citrange rootstocks perform well and CTV isolates are relatively mild. General resistance to CTV has been found in Poncirus trifoliata (L.) Raf., and strain-specific resistance in Citrus grandis (L.) Osb. and in some citrus relatives, as Fortunella crassifolia Swing. and Severinia buxifolia (Poir.) Tenore among others. A resistance gene from P. trifoliata has been characterized and mapped (Gmitter et al. 1996; Mestre et al. 1997a; Fang et al. 1998), however, because of the complex genetics of citrus, it is extremely difficult to introgress this resistance gene into citrus varieties. On the other hand, cloning of this gene is under way in several laboratories (Deng et al. 2000; Yang et al. 2001). Presently, the only possibility to protect susceptible commercial varieties from virulent CTV isolates is classical cross protection with mild CTV isolates (Costa and Müller 1980). Although it has been successfully used to protect Pera sweet orange (C. sinensis (L.) Osb.) in Brazil, and Marsh grapefruit (C. paradisi Macf.) in South Africa (Van Vuuren et al. 1993), protection afforded is sometimes temporal. Pathogen-derived resistance (PDR) could provide an efficient alternative to control CTV, by using transgenic citrus plants expressing viral genes or sequences able to disrupt the virus infection cycle (Sanford and Johnston 1985). The CP gene of tobacco mosaic virus (TMV) was used in the first demonstration of virus-derived resistance in transgenic plants
(Powell-Abel et al. 1986). Since then, this strategy has been demonstrated to be applicable to more than 35 viruses from taxonomically diverse families and in many host plants (Beachy 1997). To achieve PDR against CTV, we have produced more than 40 transgenic lines of Mexican lime (Citrus aurantifolia (Christ.) Swing.) carrying the p25 gene from a mild and a severe CTV strains. Mexican lime was chosen because it is very sensitive to this virus (Roistacher 1991). Transgenic plants were challenged with homologous and heterologous CTV strains by graft- or aphid-inoculation under greenhouse conditions. It is shown here that several transgenic plants were partially or totally protected against CTV infection.
Materials and methods Cloning and lime transformation The full-length p25 CP gene from the severe CTV strain T-305 and the mild strain T-317 (Moreno et al. 1993) were reverse transcribed and PCR-amplified (RT-PCR) with primers 5’-TTGGATCCATGGACGGAAAC-3’ and 5’-TTGGATCCTCAACGTGTGTTG-3’ carrying BamHI sites at both 5’ ends, and after digestion with BamHI were cloned into pMOG 180 (Mogen International). The resulting plasmids, pMOG T305 and pMOG T317, harbored the p25 CP genes under the control of the Cauliflower mosaic virus (CaMV) 35S promoter, with double enhancer and the Alfalfa mosaic virus (AMV) RNA 4 leader sequence, and the nopaline synthase gene (nos) terminator sequence. Correct cloning and insert orientation was confirmed by sequencing. The expression cassettes were subcloned into the plant transformation vector pBI 121 (Clontech) at the unique HindIII site, between the 35S/uidA/nos and nos/nptII/nos cassettes, generating pBI T305 and pBI T317. These plasmids were transferred to Agrobacterium tumefaciens strain EHA 105 by electroporation. Transgenic Mexican lime plants were obtained by Agrobacterium-mediated transformation of internodal stem segments from 6 to 12-month-old greenhousegrown plants, as described in Domínguez et al. (Deng et al. 2000). To analyze the integrity of the p25 CP expression cassette and to estimate the number of copies of the p25 CP gene inserted in the transgenic lime plants, Southern blot assays were performed. DNA was ex-
3 tracted from leaves according to Dellaporta et al. (1983). About 20 g of DNA were digested with HindIII, that excise the p25 CP expression cassette, or with DraI, that cut once the T-DNA near the RB. After electrophoresis in 1% agarose gels, the DNA was blotted to nylon membranes and probed with a digoxigenin (Roche)-labeled fragment of the coding region of the p25 CP gene, prepared by PCR following the manufacturer’s protocol. Accumulation of the p25 CP in each transgenic line was analyzed by Western blot. Leaf tissue was ground with extraction buffer consisting of 0.1 M Tris-HCl pH 6.8 and 1 mM phenyl-methyl-sulfonylfluoride (PMSF). Protein extracts were electrophoresed in 15% SDS-polyacrilamide gels, and electroblotted to Immobilon-PVDF membranes (Millipore). Proteins were probed with the monoclonal antibodies 3DF1 + 3CA5 to the CTV p25 CP protein (Cambra et al. 1990; Vela et al. 1986) as the primary antibodies, and goat anti-mouse IgG conjugated with alkaline phosphatase as the secondary antibody. Inoculation of the transgenic plants Buds from both transgenic and non-transgenic Mexican limes were propagated by grafting onto Troyer citrange rootstocks and maintained in a greenhouse for transgenic plants at 24–26/15–16 °C day/night temperatures, with relative humidity between 60 and 80 %, and natural light. When new shoots were 30–40 cm long, homogeneous plants were used in virus challenge assays. The inoculum source for graft and aphid inoculations were Pineapple sweet orange plants infected with CTV strains T-305 or T-300, maintained in containers in an insect-proof screenhouse at the IVIA. Bark chips of 0.75 to 1 cm 2 in size from the CTV-infected source plants were grafted onto the citrange rootstock of each transgenic or control plant, 1 to 2 cm below the bud union. Between 1 and 2 months after inoculation, CTV RNA was detected in all scions and then the inoculum chips were removed. RNA detection was done by print-capture reverse transcription followed by nested PCR in a single closed tube (PC-RT-NESTED-PCR) (Olmos et al. 1999). For this purpose, fresh sections of leaf petioles or bark pieces were pressed onto Whatman 3 MM paper, and CTV targets were extracted from tissue prints with 120 l of 0.5% Triton X100 in a microfuge tube. After vortex and incubation for 2 min at room temperature, 100 l of Triton extracts were transferred to tubes coated with monoclonal anti-
bodies 3DF1 + 3CA5 in carbonate buffer, as described in (Olmos et al. 1999). RT and nested PCR in a single tube were performed using primers to the CTV 3’ UTR region, which showed over 95% sequence identity in all CTV isolates characterized (López et al. 1998). External primers were: 5’-TAAACAAACACACACTCTAAGG-3’ and 5’CATCTGATTGAAGTGGAC-3’, and internal primers were: 5’-GGTTCACGCATACGTTAAGCCTCACTT-3’ and 5’-TATCACTAGACAATAACCGGATGGGTA-3’ (Olmos et al. 1999). The amplification product (131 bp) was detected in a 0.8% agarose gel stained with ethidium bromide. For aphid inoculation, donor plants infected with T-300, a highly transmissible CTV isolate, were obtained by propagating infected Pineapple sweet orange buds on Troyer citrange. New shoots were tested by Immunoprinting-ELISA (Garnsey et al. 1993) to confirm CTV infection. Virus-free aphids (Aphis gossypii Glover) were fed for three days on donor plants and sets of 400 viruliferous aphids were transferred to each receptor plant, essentially as described in (Hermoso de Mendoza et al. 1984). Four days later, aphids were killed with an insecticide. Evaluation of resistance After inoculation, CTV symptom development and virus accumulation in leaves of new shoots was monitored in three consecutive flushes, which spanned over a one-year period. Symptom intensity was rated on a 0–3 scale, in which 0 indicated a complete absence of symptoms and 3 indicated severe symptoms, including strong vein clearing and leaf distortion of young leaves, and vein corking of old leaves. Virus accumulation was estimated by a semiquantitative double-antibody sandwich ELISA (DASELISA) as described in Cambra et al. (1993), in the three flushes evaluated in each plant. Six-seven new leaves collected from different parts of the plants and the monoclonal antibodies 3DF1 + 3CA5 (Cambra et al. 1990; Vela et al. 1986) were used for these assays. A plant was considered infected when the absorbance values at 405 nm were at least twice those of noninoculated control plants.
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Figure 1. Western blot analysis of transgenic Mexican lime plants. Crude protein extracts were extracted from 10 plants transformed with the p25 CP gene from CTV T-305 (their name starts with an S), from 6 plants transformed with the p25 CP gene from CTV T-317 (their name starts with an M), from a control non-transformed lime plant (C), and from a CTV-infected non-transgenic lime plant (T-305). Total proteins were probed with the monoclonal antibodies 3DF1 + 3CA5 against the CTV p25 CP protein.
Results Production of transgenic lime plants and analysis of expression of the CTVp25 CP transgene Transgenic plants of Mexican lime were produced to express the full-length p25 CP gene of CTV. Based on the presence of the p25 CP gene, 42 transgenic lines were selected, 25 carrying the p25 CP gene from the severe CTV strain T-305, and 17 transformed with the p25 CP gene from the mild strain T-317. Lines harboring the p25 CP gene from T-305 were referred to as S(from severe)CP.1 to SCP.17, SCP.19, SCP.25 to SCP.29, SCP.41 and SCP.42, and those with the p25 CP gene from T-317 were called as M(from mild)CP.18, MCP.20 to MCP.24 and MCP.30 to MCP.40. Transgene integration patterns were usually complex with almost half of the plants showing T-DNA truncations (Domínguez et al. (2000); results not shown). Copy number of the p25 CP transgene was also variable in the transgenic lines, ranging from one to six (Table 1). Accumulation of the p25 CP protein was detected in all transgenic lines, except SCP.2. SCP.4, SCP.18 and SCP.19 (Figure 1; results not shown), and p25 CP accumulation was estimated to range between 1 and 4, by measuring band intensity with the MacBas Image program (Table 1). No correlation was found between p25 CP transgene copy number and p25 protein accumulation. Protection of transgenic lime plants against CTV inoculated by grafting CTV T-305 causes severe symptoms in Mexican lime, as vein clearing, leaf distortion, stem pitting, vein corking and marked stunting, whereas CTV T-317 causes only mild vein clearing (Moreno et al. 1993).
To investigate PDR against the homologous and heterologous strains, CTV T-305 was graft-inoculated onto both transgenic lines carrying T-305- and T-317derived p25 CP transgenes. In the initial challenge assays, two plants from each transgenic line were inoculated and observed for CTV symptoms in three successive flushes after inoculation. Those lines showing symptoms similar to those of non-transformed controls were discarded, and only lines exhibiting any level of protection were selected for further evaluation. Sixteen independent transgenic lines were used in a new experiment, SCP.1, SCP.6, SCP.10, SCP.11, SCP.13 to SCP.16, SCP.19, SCP.25, MCP.18, and MCP.20 to MCP.24. Eight to ten propagated plants from each transgenic line and the same number of non-transformed lime controls were inoculated with CTV T-305 and evaluated for resistance. All plants were individually tested for the presence of CTV in the scion by PC-RT-NESTED-PCR, and upon virus detection, the inoculum bark chip was removed to limit the inoculum dose (Figure 2). Symptoms started to appear in the first flush after inoculation in all the control plants, whereas transgenic lines showed symptoms at different times and could be separated in two groups: those that showed typical CTV T-305 symptoms developing basically in the same way as non-transformed controls (SCP.1, SCP.6, SCP.14, MCP.18, SCP.19, MCP.20 to MCP.23 and SCP.25), and those exhibiting different levels of protection against the virus (SCP.10, SCP.11, SCP.13, SCP.15, SCP.16 and MCP.24) (Table 1). Lines in the first group showed the same infection rate as the control plants, and their symptom intensity and virus accumulation were also comparable throughout the three flushes investigated (Table 1). Lines included in the second group showed some pro-
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Figure 2. PC-RT-NESTED-PCR analysis to detect the presence of CTV in transgenic and control scions before removing the bark chips used as virus inoculum. CTV RNA detection in extracts from two bark pieces (two lanes) from the transgenic plants SCP.15.1 to SCP.15.8. C corresponds to a non-transgenic control plant, and T-305 corresponds to a plant infected with CTV T-305. M is a 100 bp ladder marker.
tection. A percentage of plants ranging from 25 to 33%, depending on the transgenic line, did not develop symptoms in any of the three flushes and did not contain virus as determined by DAS-ELISA, whereas all the control plants became infected and were symptomatic by the second flush after inoculation (Figures 3 and 4, Table 1). These transgenic plants were considered resistant to CTV infection and the rest of the transgenic plants from the same lines showed symptom attenuation (Figures 3 and 4) and lower virus accumulation compared to control plants (Table 1). None of these plants showed vein corking in old leaves, a symptom shown by many control plants as well as plants from the susceptible transgenic lines (results not shown). Significant differences were also observed in virus accumulation and symptom intensity between transgenic lines showing a susceptible phenotype and those showing protection (Figure 4, Table 1). This protection was more evident in the two first flushes post-inoculation, but was markedly reduced by the third flush (Figure 4, Table 1). To confirm these results, transgenic lines SCP.10, SCP.11, SCP.13, SCP.15, SCP.16 and MCP.24, were subjected to a new challenge experiment. Furthermore, the two SCP.15 plants that escaped virus infection in the previous challenge were also included in this experiment as independent lines, referred to as SCP.15-5 and SCP.15-7. Eight to ten graft-propagated plants from each transgenic line and the same number of control plants were graft-inoculated with T-305
and virus resistance evaluated as before. Results with lines SCP.10, SCP.11, SCP.13, SCP.15, SCP.16 and MCP.24 were similar to those obtained previously (not shown), indicating that the protection pattern against CTV exhibited by these transgenic lines was consistent. Regarding plants propagated from SCP.15-5 and SCP.15-7, 33% (2 out of 9) of the SCP.15-5 plants and 20% (2 out of 10) of the SCP.15-7 plants were resistant to CTV, a protection pattern similar to that observed in the original SCP.15 line (25%; or 2 out of 8 plants resistant to CTV). Protection of transgenic lime plants against CTV aphid-inoculation Selected transgenic lines included in the last graft-inoculation experiment were challenged with CTV T-300 by aphid inoculation, the natural via of CTV dispersal under field conditions. Ten graft-propagated plants from lines SCP.10, SCP.11, SCP.13, SCP.15, SCP.16, MCP.24 and the corresponding non-transgenic controls were inoculated by aphid feeding and evaluated for resistance. Virus transmission was efficient since all non-transgenic controls became infected and CTV was serologically detected in the first flush post-inoculation (Figure 4c). As observed in graft-inoculation tests, a significant ratio, ranging from 10 to 30%, of the transgenic plants from the six lines escaped virus infection (Figure 4; results not shown). In the rest of the transgenic plants, symptom development and virus accumulation were delayed,
1 4 6 2 4 2 3 2 2 1 2 1 1 3 1 2 –
number
2 2 4 0 0 1 1 2 1 2 4 2 4 1 3 3 0
accumulation
CP transgene copy CP transgene a
7/8(2.0) 5/8(2.1) 3/8(1.3) 5/9(2.2) 6/9(2.1) 5/8(2.3) 7/10(2.5) 5/8(2.2) 7/8(2.1) 6/9(2.4) 4/10(1.8) 6/8(1.1) 5/8(0.9) 4/8(2.0) 4/9(1.7) 4/8(1.4) 8/10(2.5)
(ELISA OD )
c
ELISA+ / Total b
1 st flush
e
7/8(1.1) 4/8(1.6) 3/8(0.6) 5/9 (1.9) 6/9(1.1) 3/8(2.0) 6/10(1.6) 4/8(1.5) 5/8(1.1) 4/9(1.7) 4/10(0.8) 4/8(0.7) 5/8(0.9) 0/8(0.7) 3/9(1.1) 4/8(1.5) 8/10(1.9)
Total (Sint. Int. )
d
V.Clear.-L.Dist./
7/8(2.2) 6/8(2.2) 4/8(2.0) 6/9(2.2) 8/9(2.1) 7/8(2.4) 9/10(2.5) 7/8(2.4) 8/8(2.3) 8/9(2.6) 6/10(2.1) 7/8(1.9) 5/8(1.0) 5/8(2.3) 5/9(1.9) 6/8(1.5) 10/10(2.6)
(ELISA OD )
c
ELISA+ / Total b
2 nd flush
e
7/8(1.3) 6/8(2.2) 4/8(1.5) 6/9(1.9) 8/9(2.3) 7/8(2.5) 8/10(2.0) 6/8(1.8) 7/8(1.7) 7/9(1.9) 5/10(1.0) 6/8(1.0) 5/8(1.5) 3/8(0.9) 5/9(1.3) 6/8(2.2) 10/10(2.2)
Total (Sint. Int. )
d
V.Clear.-L.Dist./
8/8(2.4) 8/8(2.5) 8/8(2.2) 9/9(2.2) 9/9(2.4) 8/8(2.3) 10/10(2.7) 8/8(2.6) 8/8(2.4) 9/9(2.6) 7/10(2.1) 7/8(2.0) 6/8(2.1) 6/8(2.3) 6/9(2.2) 7/8(2.1) 10/10(2.5)
(ELISA OD )
c
ELISA+ / Total b
3 rd flush
8/8(2.7) 8/8(2.6) 8/8(2.4) 9/9(1.9) 9/9(2.3) 8/8(2.4) 10/10(2.2) 8/8(2.3) 8/8(2.4) 9/9(2.1) 7/10(1.6) 7/8(2.5) 6/8(2.0) 6/8(1.8) 6/9(1.7) 7/8(2.0) 10/10(2.3)
Total d (Sint. Int. e)
V.Clear.-L.Dist./
a Accumulation rate of CTV-CP transgenic protein in different lines estimated by Western and MacBas Image program. 0: not detectable, 1: low, 2: medium; 3: high, 4: very high. b Number of ELISA positive plants 1 total inoculated plants. c Average optical density in ELISA positive plants. d Number of plants showing vein-clearing and/or leaf-distorsion symptoms / total inoculated plants. e Average intensity of vein-clearing and/or leaf-distorsion symptoms in symptomatic plants. 0: asymptomatic; 1: mild; 2: medium, 3: severe.
SCP.1 SCP.6 SCP.14 MCP.18 SCP.19 MCP.20 MCP.21 MCP.22 MCP.23 SCP.25 SCP.10 SCP.11 SCP.13 SCP.15 SCP.16 MCP.24 Control
Transgenic Line
Table 1. Symptom expression and virus accumulation in transgenic Mexican lime plants carrying the CTVp25 CP gene after graft-inoculation with CTV T-305.
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Figure 3. Transgenic and non-transgenic control plants of Mexican lime showing CTV symptoms of different intensity in young leaves from the second flush after graft-inoculation with CTV T-305. Severe leaf distortion and stunting symptoms caused by CTV T-305 in a nontransgenic control plant (left), delay in virus infection and symptom attenuation in a transgenic plant (centre), and resistance against CTV T-305 in other transgenic plant of the same line (right).
Figure 4. Evaluation of resistance in non-transgenic control Mexican limes and in transgenic lines expressing the CTV p25 CP gene. Comparison of the response to CTV T-305 graft-inoculation in the susceptible lines MCP.21 and SCP.25, and in the protected lines SCP.10 and SCP.16 (a, b). Protection against CTV T-300 in transgenic lines SCP.13, SCP.15 and MCP.24 inoculated by aphid feeding (c, d). Symptom intensity estimated in a 0 to 3 scale of vein clearing/leaf distortion in symptomatic plants. Vertical bars indicate SE.
and symptom severity was lower than in control plants. Again, differences were higher in the two first flushes post-inoculation (Figure 4).
Discussion Up to date, PDR has been demonstrated to be efficient to control virus diseases in many virus-plant systems (Baulcombe 1996; Beachy 1997). Most of these studies were done in herbaceous plants, and there are very few reports related to PDR in woody fruit trees (Ravelonandro et al. 2000). Here we have
8 attempted to develop PDR against CTV in transgenic citrus plants. Forty-two transgenic lines carrying either the p25 CP gene from the mild CTV strain T-317 or the highly virulent strain T-305 were produced. Some of the transgenic lines showed a susceptible phenotype similar to non-transformed controls, whereas others showed protection against CTV. This protection was characterized by a significant delay in CTV establishment and accumulation. In addition, a proportion of plants, ranging from 10-33 %, from six transgenic lines (SCP.10, SCP.11, SCP.13, SCP.15, SCP.16, and MCP.24) were resistant to CTV. These lines consistently showed protection in different challenge experiments by both graft- and aphid-inoculation. Inoculation failure was discarded since: (1) 100% of the non-transgenic control plants became infected in all evaluation experiments, indicating that the inoculation systems were highly efficient and reliable; (2) only these six lines exhibited the protection phenotype consistently; and (3) CTV was detected by PC-RT-NESTED-PCR in transgenic and control scions from all plants, before removing bark chips in graft-inoculation experiments. Mestre et al. (1997b) reported that CTV can be passively transported through the phloem tubes even in CTV-resistant citrus genotypes. Similarly, in our experiments viral RNA could be detected by RT-PCR in virus-resistant transgenic plants before removing the source of inoculum. A fraction of transgenic plants from the CTV-protected lines were fully resistant and did not show any sign of virus infection. When these resistant plants were propagated and re-inoculated with CTV by grafting, the resistance phenotype was again reproduced in a similar proportion of plants, while the rest of them showed a delay in virus accumulation and symptom attenuation. These findings could be explained by: (1) the dose of virus delivered to individual plants may be variable and in some cases it was sufficient to overcome resistance; (2) sequence variants of CTV are sometimes unevenly distributed in infected plants (D’Urso et al. 2000) and therefore, individual plants may have received inocula with different viral populations; or (3) the level of expression of the transgenes could vary during evaluation experiments, and this may affect the efficiency of the protection mechanism. Variation of the expression level of the transgenes during the course of evaluation tests was shown for the transgenic line SCP.15. Indeed, GUS activity changed after CTV inoculation and progressively increased in SCP.15 plants (results not
shown). This is an indirect indication that p25 CP expression could also vary since both transgenes, uidA and p25 CP, were located at the same T-DNA and therefore they were inserted at the same site in the plant genome, and also expression of both transgenes was under the control of the CaMV 35S promoter. Influence of developmental stimuli in transgene expression and consequently in the efficiency of PDR has been previously reported (Pang et al. 1996). On the other hand, activation of expression of silenced marker transgenes after virus inoculation has been found associated to the expression of viral suppressors of gene silencing (Voinnet 2001). Interestingly, in our experiments fluctuation of GUS expression only occurred in transgenic plants from the SCP.15 line, suggesting that it was related with an specific integration pattern and/or integration site. Since GUS expression was stable in all the plants from the rest of the lines, variable dose of inoculum and/or inoculation with different viral populations better explained the characteristics of the protection phenotype for these transgenic plants. In all the 42 transgenic lines evaluated at least one intact copy of the p25 CP expression cassette was detected, and p25 CP accumulation was confirmed in 38 of these lines. Transgenic lines that showed protection against virus infection generally accumulated p25 CP at high or moderate level. Susceptibility was found in lines that expressed low level or no p25 CP, except for line SCP.14 that was highly susceptible and showed high level accumulation of p25 CP. These results suggest that in most cases protection was conferred by the accumulation of the p25 CP in the transgenic plants. Transgenic line SCP.15 was an exception, since it showed low p25 CP accumulation and high level of protection. Therefore, we can not discard a different protection mechanism, as RNAmediated resistance, for the SCP.15 line. The protection observed was not affected by the CTV strain used to prepare the p25 CP transgene nor by the inoculation system. Transgenic lines MCP.24, that carried the p25 CP gene from CTV T-317, and SCP.10, SCP.11, SCP.13, SCP.15 and SCP.16, carrying the p25 CP gene from CTV T-305, showed similar protection against CTV T-305 inoculated by grafting, or CTV T-300 inoculated by aphid feeding, although the ratio of transgenic lines that showed protection against T-305 was higher in those carrying the p25 CP gene from T-305 (5 out of 25) than in those with the p25 CP gene from T-317 (1 out of 17). Nucleotide identity between the p25 CP gene of T-305
9 and T-317 is 91.4% whereas amino acid identity is 94.6%, which indicates that homologous protection seemed to be more efficient in the transgenic plants. In citrus growing areas where highly virulent CTV isolates are common, cross protection with mild isolates has been the only approach to reduce yield losses in some genotypes (Costa and Müller 1980). Although only a moderate protection level was achieved in transgenic lime plants expressing the p25 CP gene in greenhouse experiments under aggressive challenge conditions, performance of these plants in field trials has to be determined. However, it should be stated that even if a 3-year delay is gained, this would be insufficient to protect the crop against CTV for the whole productive life of the tree. In summary, we report here protection against CTV in transgenic citrus plants expressing its p25 CP gene. This study demonstrates for the first time that PDR can be extended to a member of the Closteroviridae family of plant viruses and to its natural host. Furthermore, in the future it may provide a possible alternative to cross protection for the efficient control of the devastating tristeza disease.
Acknowledgements The authors wish to thank Dr Antonio Olmos, Carlos Marroquín, José Antonio Pina and Maria Teresa Gorris for their technical assistance. This research was supported by grants from the Instituto Nacional de Investigaciones Agrarias (SC97-102, SC97-098 and RTA01-120), from CICYT-European Union (1FD970822) and a fellowship provided by Generalitat Valenciana to the first author.
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