Department of Plant Pathology, Cornell University NYSAES, Geneva, NY 14456, USA1
Department of Plant Pathology, National Chung Hsing University, Taichung 402, Taiwan, ROC2
Author for correspondence: Dennis Gonsalves. Fax +1 315 787 2389. e-mail dg12{at}nysaes.cornell.edu
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Abstract |
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Introduction |
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Various models have been proposed to explain the mechanisms that trigger PTGS and produce virus resistance in transgenic plants that express a transgene that is homologous to the attacking virus. These include an RNA threshold model (Dougherty & Parks, 1995 ; Smith et al., 1994
), an ectopic pairing and aberrant RNA model (Baulcombe, 1996
; Baulcombe & English, 1996
; English et al., 1996
) and a dsRNA-induced PTGS model (Metzlaff et al., 1997
; Montgomery & Fire, 1998
; Waterhouse et al., 1998
). However, all of these models propose a common sequence-specific RNA-degradation process. Briefly, RNA-dependent RNA polymerase synthesizes short antisense RNA from the transgene mRNA and the antisense RNA binds to the complementary regions of the mRNA in the cytoplasm to form RNA duplexes, which are then degraded by dsRNA-specific nucleases (Dalmay et al., 2000a
, b
; Mourrain et al., 2000
). Viral RNA in the cytoplasm is also a target for degradation. Indeed, several recent papers report the identification of small RNA molecules, 2125 nt in length, that correspond to sense and antisense pieces of the dsRNA or transgene that is introduced into the cytoplasm (Bass, 2000
; Dalmay et al., 2000a
, b
; Hamilton & Baulcombe, 1999
).
Papaya ringspot virus (PRSV), from the genus Potyvirus, is the major limiting factor for economic papaya production throughout the tropics and subtropics, including the state of Hawaii (Gonsalves, 1998 ). Two transgenic cultivars, Rainbow and SunUp, that are resistant to PRSV in Hawaii were recently commercialized (Gonsalves, 1998
; Manshardt, 1999
). SunUp was derived from transgenic papaya line 55-1 (Fitch et al., 1992
) and is homozygous for a single insert of the CP gene of PRSV HA 5-1 (Tennant et al., 2001
), a mild mutant of PRSV HA (Yeh & Gonsalves, 1984
). Rainbow is a hybrid of SunUp and the non-transgenic cultivar Kapoho. It is therefore hemizygous for the CP gene (Manshardt, 1999
). Tennant et al. (1994
, 2001
) reported that Rainbow and hemizygous plants of line 55-1 are resistant to PRSV isolates from Hawaii that share at least 97% nt identity to the CP transgene but are susceptible to isolates from outside Hawaii that have 8994% identity to the transgene. In contrast, SunUp is resistant to a number of isolates from outside Hawaii.
We recently developed infectious transcripts of PRSV HA (Chiang & Yeh, 1997 ), which provide us with a unique opportunity to produce PRSV HA chimeras that are different from PRSV HA in their CP sequences. Such chimeras can be used to determine the relative importance of CP sequence similarity in breaking the resistance of Rainbow. We constructed a series of such CP recombinants by using whole or partial CP sequences of PRSV YK, a PRSV isolate with 90% nt identity to PRSV HA in the CP sequence (Wang & Yeh, 1997
). Recombinant viruses were able to overcome the resistance of Rainbow but the symptoms varied from very mild to severe, depending on the region of the CP gene that was substituted.
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Methods |
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Transgenic papaya lines.
The commercial transgenic papaya SunUp and Rainbow used in this work were originally derived from transgenic line 55-1 (Manshardt, 1999 ). Line 55-1 was developed by transforming the Hawaiian papaya cultivar Sunset with the CP gene of PRSV HA 5-1 (Fitch et al., 1992
), which is a nitrous acid-induced mutant from the parent strain PRSV HA (Yeh & Gonsalves, 1984
). Comparison of the 3'-terminal 2235 nt of HA with its mild mutant HA 5-1 showed 99·4% identity (Wang & Yeh, 1992
). Their CP gene sequences differ by 2 nt but their 3' non-coding region (NCR) sequences are identical. The CP-homozygous SunUp was from the R3 generation and was obtained by crossing R0 transgenic line 55-1 with hermaphroditic Sunset and then self-crossing of progenies (Manshardt, 1999
). Rainbow is an F1 derived from a cross of SunUp and non-transgenic cultivar Kapoho. Rainbow and SunUp express the transgene from sequence positions 9257 to 10168 of PRSV HA 5-1, which corresponds to the entire CP gene (Quemada et al., 1990
) and 51 nt of the 3' NCR (Fig. 1B
). Additionally, the 5' end of PRSV CP contains an extra 48 nt that encode 16 amino acids of cucumber mosaic virus (CMV) CP, and an extra 22 nt of the CMV 3' NCR is fused to the end of the PRSV 3' NCR (Ling et al., 1991
).
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Clone p3'YKCP, which contains the 1·2 kb 3' region of PRSV YK, was constructed by using RTPCR with an upstream primer, 5' GGCAGGGCCCCATATGTGTCTG 3', that contains a created ApaI site (underlined) between positions 9053 and 9074 of YK and with an oligo(dT) oligonucleotide that has a 5'-terminal NotI site as a downstream primer. A full-length hybrid virus, designated pHA-3'YK, was obtained by replacing the ApaINotI fragment of pT3-HAG with the corresponding region of p3'YKCP (Fig. 1A, B
). Thus, clone HA-3'YK (Fig. 1B
) contains the entire sequence of PRSV HA except that the 3'-proximal 1·2 kb, consisting of 200 nt of the nuclear inclusion b (NIb) gene, the complete CP gene (861 nt) and 209 nt of the 3' NCR, is from PRSV YK.
We constructed six other recombinant clones that contained YK sequences in the 5' region, the central region or the 3' region of the CP gene (Fig. 1B). These six full-length chimeric CP constructs were obtained by replacing cDNA fragments with the common restriction enzyme sites (ApaI, SwaI, EcoRI and NotI) between pT3-HAG and pHA-3'YK (Fig. 1B
). Clones YK-AS, YK-SE, YK-EN, YK-AE and YK-SN were constructed by exchanging the ApaISwaI, SwaIEcoRI, EcoRINotI, ApaIEcoRI and SwaINotI restriction fragments of pT3-HAG with those from pHA-3'YK. YK-AS/EN was obtained by replacing the SwaIEcoRI fragment of pHA-3'YK with the corresponding fragment from pT3-HAG. The sequences of the recombinants were verified by digestion with enzymes NdeI, SpeI and SacII to identify replacement regions between HA and YK and by sequencing to confirm the replacement.
Inoculation of papaya.
RNA transcripts were synthesized in vitro by T3 RNA polymerase from NotI-linearized plasmids as described by Chiang & Yeh (1997) . Capped RNA transcripts were then applied mechanically onto non-transgenic plants of papaya (Carica papaya) with three true leaves. Initially, inocula (1 g leaves in 15 ml buffer) were from papaya infected with the in vitro transcript. Subsequently, non-transgenic papaya and another systemic host, Cucumis metuliferus (Naud.), were also inoculated and used as the source of recombinant virus for subsequent tests. However, only tissues from up to three inoculation transfers were used as inocula. After that, inocula were again obtained from the original papaya that was infected by the in vitro transcripts. Papaya plants were inoculated at a young stage, with 56 true leaves, or at an older stage, with 1012 true leaves. All inoculated plants were kept in a greenhouse at 2124 °C and observed for symptoms for 90 days.
Virus detection.
Total RNA was extracted from papaya leaves as described by Levy et al. (1994) . The 3' region of the viral genome was amplified by RTPCR with upstream and downstream primers respectively corresponding to PRSV HA positions 88688897 and 1008310117. The RTPCR-generated DNA fragments were sequenced with an ABI 373 automated sequencer (DNA Sequencing Services, Cornell University, Ithaca, NY, USA).
Northern blot analysis was used to estimate viral RNA accumulation. Ten µg total RNA, extracted 45 days post-inoculation (p.i.) from transgenic Rainbow and non-transgenic papaya, was electrophoresed in a denaturing formaldehyde1·2% agarose gel and blotted onto a Gene Screen Plus nylon membrane as described by the manufacturers manual (DuPont). A 32P-labelled, random-primed, ApaI/NotI-digested, 1·2 kb DNA fragment from pT3-HAG, which contained 200 bp of NIb, the complete CP gene (861 nt), 209 bp of the NCR and a 36 residue poly(A) sequence from pT3-HAG, was used as a probe. Bark extracts from stems of plants that did not have symptoms on leaves but had water-soak lesions on the stems 4 months after inoculation were assayed by double-antibody sandwich ELISA (Clark & Adams, 1977 ) with antiserum to intact PRSV HA virus (Ling et al., 1991
).
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Results |
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Seven full-length PRSV HA recombinant constructs were generated by replacing segments of the HA genome with corresponding segments from YK (Fig. 1). Since a suitable restriction enzyme site at the 5' end of the CP gene in PRSV HA was not available, an ApaI site in the NIb gene 200 bp upstream from the CP gene was chosen to perform the DNA replacements between HA and YK. Consequently, recombinant viruses HA-3'YK, YK-AS, YK-AE and YK-AS/EN also contained 200 bp of NIb from YK. A NotI site was created downstream of the poly(A) tail to make constructs HA-3'YK, YK-EN, YK-SN and YK-AS/EN. The NotI restriction site was also used to linearize the plasmids prior to in vitro transcription (Chiang & Yeh, 1997
). Thus, recombinants HA-3'YK, YK-EN, YK-SN and YK-AS/EN contained an extra 158 nt of the 3' NCR sequence from YK compared with the transgene (Fig. 1
).
Comparisons of the YK segments of the recombinant viruses to corresponding regions of the transgene are shown in Fig. 1. The replacement segments of recombinant clone HA-3'YK showed 76 nt differences out of 861 in the PRSV HA 5-1 CP sequence and 8 nt differences out of 51 in the 3' NCR region. The YK replacement segment of the recombinant YK-AS had the lowest nucleotide sequence identity to the CP transgene (87·5%; 33 of 263 nt different); this region corresponded to the variable N terminus and part of the core region of the CP (Shukla et al., 1988
). The YK segment of recombinant YK-SE had an identity of 92·3% (32 of 415 nt different); the YK segment originated from the core region of the CP (Shukla et al., 1988
). The recombinant YK-EN contained a YK segment that corresponded to the conserved C-terminal and core regions of the CP (94·0% identity; 11 of 183 nt different) and the first 51 nt of the 3' NCR (84·3% identity; 8 of 51 nt different). Recombinants YK-AE, YK-SN and YK-AS/EN contained combinations of two YK segment replacements, as shown in Fig. 1(B)
.
The biological activity of the recombinants was tested on non-transgenic papaya. Papaya mechanically inoculated with in vitro transcripts corresponding to the recombinants showed symptoms similar to those induced by PRSV HA. Symptoms developed 811 days p.i. and consisted of severe mosaic, leaf distortion and stunting of the plants. RTPCR and sequencing from the inoculated non-transgenic plants verified that the infection was from the proper recombinant viruses (data not shown). Furthermore, these recombinants appeared stable in that they induced similar symptoms in non-transgenic papaya following serial passages for over a year.
The relative titres of several recombinants (HA-3'YK, HA-AE and HA-AS/EN) in non-transgenic papaya were also compared with those of HA and YK. Two or three selected non-inoculated leaves (top expanded leaf at 8, 12 and 14 or 16 days p.i.) were monitored for virus by ELISA at about 2 day intervals up to 20 days p.i. Additionally, comparisons of local lesion production on Chenopodium quinoa were done with HA-3'YK, HA and YK. ELISA readings of HA-AE and HA-AS/EN were similar to HA and YK over time (Fig. 2AC
). Virus was first detected by ELISA in the top expanded leaf (Fig. 2A
) at 1214 days p.i. and detection by ELISA coincided with the appearance of symptoms on the sampled leaves. The virus titre was maximal in all leaves starting 1618 days p.i. The recombinant HA-3'YK also showed virus titres similar to those of HA and YK (data not shown). Furthermore, leaf extracts of papaya sampled 15 days after inoculation with HA, YK or HA-3'YK induced similar numbers of local lesions (Table 1
). In similar tests, ELISA analysis of Cucumis metuliferus inoculated with the isolates showed that these plants also developed similar titres (data not shown). Taken together, these results show that the recombinants HA-3'YK, HA-AE and HA-AS/EN replicate and move in a similar way to HA and YK in non-transgenic papaya and Cucumis metuliferus.
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All six recombinant viruses with various segments of YK CP (Fig. 1) induced severe symptoms on Cucumis metuliferus and non-transgenic papaya (Table 3
), but variable reactions appeared on Rainbow and SunUp plants. None of the recombinants with only partial CP sequences of YK were able to infect SunUp with the exception of recombinant YK-SN, which infected only 16% of the inoculated plants and caused very mild symptoms, consisting of a few small yellow spots (Table 3
). However, Rainbow plants that were challenged with these recombinant viruses developed a range of symptoms, which were milder than those caused by HA-3'YK (Fig. 3D
F
; Table 3
). The symptoms were grouped into three types. Type I symptoms (Fig. 3D
) were characterized by extensive vein clearing on leaves early in the test (45 days p.i.) and leaf distortion at a later stage (90 days p.i.). Type II symptoms (Fig. 3E
) were less severe than type I and consisted of many vein flecks in newly developed leaves (45 days p.i.), with variable symptom expression later on (90 days p.i.). Type III symptoms (Fig. 3F
) consisted of a few vein flecks at 45 days p.i., and the new leaves were symptomless at 90 days p.i.
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Northern blot analysis of total RNA from Rainbow plants infected with different recombinants revealed that symptom severity was correlated with viral RNA accumulation (Fig. 4). Rainbow plants infected with YK-SE, YK-EN or YK-SN, which caused type I symptoms, and YK-AE or YK-AS/EN, which caused type II symptoms, had relatively high levels of RNA accumulation (Fig. 4
). In contrast, very little or no viral RNA was detected in Rainbow plants infected with YK-AS (type III symptoms). As expected, a large amount of viral RNA was detected in non-transgenic plants infected by PRSV HA, while no viral RNA was detected in Rainbow plants inoculated with HA. The weaker signals in the YK- and HA-3'YK-infected plants (Fig. 4
) were probably due to the relatively low similarity of the probe to the YK segment (described in Methods). The probe was derived from PRSV HA, which has 88·8% sequence identity to YK in the corresponding region (Wang & Yeh, 1997
).
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Some Rainbow plants that initially showed type II symptoms recovered at a later stage, with leaves being symptomless, although the stems still showed water-soak lesions. Virus was apparently still present in the stem tissue, since ELISA analysis of six recovered Rainbow plants gave 2-to 5-fold higher readings than mock-inoculated Rainbow plants (A405 of 1·81·0 compared with 0·4).
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Discussion |
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Our conclusion that the virulence of our recombinants on Rainbow is affected more by the position rather than the degree of sequence similarity is based on several observations. The YK-AS recombinant, which has a 263 nt YK segment with 87·5% sequence identity (33 mismatched nt) to the corresponding region of the transgene, induced very mild type III symptoms on Rainbow, in contrast to the more prominent type I symptoms induced by YK-SE and YK-EN recombinants, which respectively have 92·3 and 91·9% nt identity to the transgene. Furthermore, the length of the YK replacement segment does not account for the different symptoms induced, since the YK segments in the YK-EN and YK-AS recombinants are similar in length (234 and 263 nt; Fig. 1). Also, the different symptoms that the recombinants produced on Rainbow are apparently not due to their inherent capacity to replicate, as all recombinants produced severe symptoms on non-transgenic plants.
A plausible explanation for the differential virulence of the recombinants on Rainbow is that the PTGS mechanism is preferentially targeted to the middle and 3' regions of the virus transgene. Thus, recombinant YK-AS, which has 99·9% identity to the middle and 3' end of the virus transgene, would be degraded more effectively (and thus produce very mild type III symptoms) than YK-SN, which has only 92·2% identity to the middle and 3' end of the transgene. This would also explain the type I symptoms produced by the YK-SE and YK-EN recombinants. Other investigations on homology-dependent virus resistance have shown that the PTGS mechanism is preferentially directed against the 3' end region of the transgene (e.g. English et al., 1996 ; Metzlaff et al., 1997
; Sijen et al., 1996
; Sonoda et al., 1999
). On the other hand, reports have shown (i) that the target sites of some transgenic lines are scattered throughout the transgene (Jacobs et al., 1999
; Sonoda et al., 1999
) and (ii) that the 5'- and 3'-terminal coding regions of the mRNA may be relatively inefficient targets for the silencing machinery (Jacobs et al., 1999
).
The suggested preferential PTGS targeting to the middle and 3' end of the transgene does not account fully for the intermediate type II symptoms induced on Rainbow by the recombinants YK-AE and YK-AS/EN, which contain YK segments of the 5' end plus the middle or 3' end of the CP gene (Table 3; Fig. 1
). We would expect these recombinants to produce type I symptoms, since other recombinants with YK segments from the middle or 3' regions of the CP gene produced type I symptoms. Furthermore, these type II symptom-producing recombinants infected an average of 56% (23/41) of the inoculated Rainbow plants compared with 89% (54/61) of the plants inoculated with type I symptom-producing recombinants (Table 2
). Taken together, it seems that the presence of the YK 5' CP segment reduced the virulence of recombinants that would otherwise produce type I symptoms on Rainbow. Furthermore, these recombinants were as virulent as YK or HA on non-transgenic papaya, which rules out the possibility that the differences in symptoms were due the recombinants being inherently less virulent. At present, we have no explanation for this observation. It should be noted, however, that plants with type II symptoms at 45 days p.i. showed variable symptoms (type I or II or recovery; see Fig. 1
) at 90 days p.i. In contrast, plants with type I symptoms at 45 days p.i. still had the same symptoms at 90 days p.i. (Fig. 1
).
Additionally, the differential virulence of the HA-3'YK recombinant and YK on Rainbow and SunUp is difficult to explain solely by the concept of homology-dependent resistance. Since the genomes of HA-3'YK and YK have identical CP and 3' NCR sequences, they should have equal virulence on Rainbow and SunUp. Instead, HA-3'YK produced only mild symptoms on older Rainbow and on young SunUp plants, and did not infect older SunUp plants, whereas YK caused severe symptoms on Rainbow and SunUp plants that were inoculated at all stages (see Table 1). The observed differences are not due to HA-3'YK being inherently less virulent than YK, as our results show that HA-3'YK replicates and moves as well as YK and HA in non-transgenic papaya (Table 1
). Thus, our results suggest that virus sequences or genes that do not correspond to the transgene may affect the phenotypic reaction of the transgenic plant. Several reports (Anandalakshmi et al., 1998
; Brigneti et al., 1998
; Kasschau & Carrington, 1998
; Voinnet et al., 1999
) have shown that HC-Pro of potyviruses can act as a suppressor of PTGS. Thus, if the HC-Pro of YK is more effective than the HC-Pro of HA in suppressing PTGS of infected plants, YK would be expected to replicate better than HA-3'YK in Rainbow and SunUp plants. The HC-Pro of HA and YK share 86·5 and 95·6% nucleotide and amino acid sequence identity (Wang & Yeh, 1997
). The availability of infectious clones of HA (Chiang & Yeh, 1997
) and the stability of recombinants that contain segments of HA and YK (this work) will allow us to test experimentally whether HC-Pro contributes to the above observation on Rainbow and SunUp.
We also show here that zygosity and development stage affect the resistance of transgenic plants to recombinants. Our results confirm and extend those of Tennant et al. (2001) , who tested Rainbow and SunUp with different PRSV isolates but did not use recombinants. Others have shown similar effects of zygosity and development stage on the resistance of other virustransgenic plant systems (Goodwin et al., 1996
; Jan et al., 2000
; Pang et al., 1996
), but the experiments were not done with recombinant viruses.
Resistance-breaking strains could conceivably emerge through recombination of PRSV strains from Hawaii with transgenic papaya that express the CP or other genes of PRSV strains that overcome the resistance of Rainbow or SunUp. Thus, the RainbowPRSV system is a good model for investigating critically the risk of viruses arising through recombination of PRSV with heterologous PRSV transgenes in papaya.
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Acknowledgments |
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Received 22 November 2000;
accepted 16 July 2001.
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