Department of Plant Pathology, Cornell University, Ithaca, NY 14853, USA1
Author for correspondence: William Wintermantel. Present address: USDA-ARS, 1636 E. Alisal Street, Salinas, CA 93905, USA. Fax +1 831 755 2814. e-mail bwinter{at}pwa.ars.usda.gov
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Abstract |
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Introduction |
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In some cases, transgenic plants exhibit more than one resistance phenotype, which can complicate analysis of mechanisms. For example, Pang et al. (1994) found that coat protein-mediated resistance against tospoviruses was RNA-mediated when directed against closely related isolates, but protein-mediated when directed against more distantly related tospoviruses. Tenllado et al. (1995)
found that transgenic plants containing the 54 kDa gene of Pepper mild mottle virus exhibited two different resistance responses, one complete and the other delayed. Further studies have suggested protein involvement in this system as well (Tenllado et al., 1996
). In addition, although TMV replicase-mediated resistance was shown to be protein-mediated in protoplasts (Carr et al., 1992
), research by Marano & Baulcombe (1998)
found that transgenic tobacco plants containing a gene encoding the TMV 54 kDa protein exhibited transgene silencing. The silencing was shown to contribute to virus resistance.
Replicase-mediated resistance to Cucumber mosaic virus (CMV) is complex, and encompasses at least two separate mechanisms which contribute to the resistance, one reducing virus replication (Carr et al., 1994 ), the other restricting long-distance virus movement (Wintermantel et al., 1997
). A second type of movement restriction may exist at the cell-to-cell level (Nguyen et al., 1996
; Canto & Palukaitis, 1999
). CMV RNA2 is the target of both components of the resistance; however, the block on virus movement involves interactions in the central region of the RNA, while replication inhibition involves sequences throughout its entire length (Hellwald & Palukaitis, 1995
). Although the viral target of the resistance has been identified, it has not been determined whether the transgene mRNA, derived from CMV RNA2, or its protein product is the effector of replicase-mediated resistance. We address this question, beginning with a series of transgenic plants containing translatable and nontranslatable transgenes, designed to determine whether transgene translatability or protein expression is required for resistance. This work is coupled with an analysis of the relationship between resistance and transgene copy number, steady-state transgene mRNA levels and symptomatology. Together, the results indicate a complex resistance which is difficult, although not impossible, to achieve without a translatable transgene, and probably protein expression. While transgene translatability has sometimes been identified as a facilitator of homology-dependent gene silencing (Cassidy & Nelson, 1995
; Tanzer et al., 1997
), some aspects of the resistance described here clearly do not fit the typical gene-silencing model for transgene-mediated resistance.
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Methods |
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Plant transformation constructs and transformations.
All constructs used for plant transformation (Fig. 1A) were developed from pCMV N/B-23 (Anderson et al., 1992
). First, the CMV RNA2 transgene sequence in pCMV N/B-23 was excised by digestion with BamHI, which flanks the transgene, and subsequently cloned into the BamHI site of pBluescriptII SK(+) (Stratagene), to yield pBW40. The SphI site in pBW40 was removed by digestion with SphI, followed by treatment with T4 DNA polymerase (Sambrook et al., 1989
). Restriction enzyme analysis and DNA sequencing were used to verify removal of the start codon. This new construct, pBW42, was digested with BamHI, and the restriction fragment containing the CMV RNA 2 sequence was inserted into the binary plant transformation vector pROK2 (Sleat et al., 1988
) to form pCMV2-PP (Fig. 1A
). Restoration of the deletion in pCMV N/B-23 was accomplished by replacing a StyI fragment from pBW42, containing a deletion between nucleotides 18501951, with a StyI fragment from pFny-209, a full-length cDNA clone of Fny-CMV RNA2 (Fig. 1A
). Restriction analysis and DNA sequencing were used to verify complete restoration of the missing sequence. The new construct was digested with BamHI, and the fragment containing the CMV RNA 2 sequence was inserted into the plant transformation vector pROK2 to form pCMV2-FLP. To generate a nontranslatable transgene, pBW42 was digested with SalI, and the overhang was filled in using the Klenow fragment of DNA polymerase I (Sambrook et al., 1989
), creating a new PvuI site, which shifted translation out of frame, resulting in early termination (Fig. 1A
, B
). This new construct was cleaved with BamHI, and the fragment containing the CMV RNA 2 sequence was inserted into the plant transformation vector pROK2 to form pCMV2-NP (Fig. 1A
). Restriction analysis and DNA sequencing were used to verify the sequence changes and the orientation of the transgene in pCMV2-NP, pCMV2-PP and pCMV2-FLP. Translation of all constructs was tested in a wheat germ cell-free in vitro translation system (Roberts & Patterson, 1973
) prior to insertion of the transgene cassette into the plant transformation vector. All translatable constructs produced translation products of the expected size. It was not possible to clearly identify the small 3 kDa polypeptide of pCMV2-NP; however, no significant translation products were detected with this construct (data not shown).
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Tobacco genomic DNA isolation and analysis.
Genomic DNA was isolated from several small (1·01·5 cm long) tobacco leaves according to the procedure of Fulton et al. (1995) with the following modifications: leaves were pulverized in liquid nitrogen prior to grinding, and extracted twice to eliminate proteinaceous contaminants. DNA was resuspended in sterile, nuclease-free water and either used immediately or stored frozen at -20 °C. Ten µg of tobacco genomic DNA was digested with either EcoRI or BamHI, and electrophoresed at 40 V overnight in 1% agarose1x TBE. Southern (alkali) blotting and hybridization of tobacco genomic DNA were performed on Amersham Hybond-N+ nylon membrane at 65 °C, according to the manufacturers recommendations. Blots were hybridized to a denatured PCR-amplified, gel-purified DNA fragment corresponding to RNA2 of Fny-CMV, labelled with [32P]dATP, with detection by autoradiography.
Total plant RNA isolation and analysis.
Total plant RNA was prepared by grinding 15 to 20 small (34 cm long) tobacco leaves in liquid nitrogen, followed by suspension, vortexing and extraction three times in a mixture of 5 ml RNA extraction buffer (50 mM TrisHCl, pH 8·0; 10 mM EDTA, 2% SDS) and 5 ml phenolchloroform, followed by ethanol precipitation of the aqueous phase. Total RNA concentration and quality were determined by spectrophotometry and gel electrophoresis. Ribonuclease protection assays were performed using the RPAII kit (Ambion) and [32P]UTP-labelled antisense transcript probes made from a cDNA clone containing a 417 nucleotide XbaIXhoI fragment from Fny-CMV RNA2 (Fig. 1A).
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Results |
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None of the plants transformed with vector sequence alone (23 lines) showed delays or any level of resistance as primary transformants or progeny (data not shown). Only one of 61 lines transformed with the nontranslatable frameshift construct, pCMV2-NP (hereafter referred to as NP lines), produced resistance (line NP7), and one other NP line exhibited a slight delay in the appearance of symptoms. All other NP lines became infected at the same rate as nontransformed control plants (Table 1, Fig. 2
). Because line NP7 produced a partial resistance phenotype, unlike all other lines containing nontranslatable transgenes, the transgene from this line was amplified by PCR and sequenced to determine if the reading frame had been restored. Results verified that the transgene in line NP7 remained nontranslatable (data not shown). When R2 progeny of resistant line NP7 were tested for resistance, approximately 50% of the plants tested exhibited delayed infection or resistance (data not shown). In contrast, transformation with the two constructs which allowed for translation of considerable portions of the transgene (pCMV2-PP and pCMV2-FLP) resulted in many transgenic lines exhibiting delayed infection compared with controls (Table 1
, Fig. 2
), as well as more lines showing substantial levels of resistance (Table 1
). This implied that transgene translatability or the CMV 2a protein encoded by the transgene may have a role in replicase-mediated resistance to CMV.
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The results of the plant transformation experiments suggested that transgene protein or mRNA translatability contributes to CMV replicase-mediated resistance. The occurrence of limited resistance in one nontranslatable line, however, implies that the transgene mRNA can also engender resistance, even if nontranslatable, suggesting that gene silencing or another RNA-based mechanism could sometimes be involved in CMV replicase-mediated resistance. In order to characterize the relationship between transgene mRNA and resistance, Northern blots were performed on total mRNA extracts from the same well characterized, R1 transgenic-resistant tobacco lines examined by Southern analysis (NB23 lines). Although the blots were capable of detecting as little as 10 pg of control RNA, transgene mRNA levels were too low for comparison (data not shown). Consequently, ribonuclease protection assays were used to examine steady-state transgene message levels in these plants, and their relationship with resistance. Ribonuclease protection assays were performed with 100 µg of total plant mRNA from each of the transgenic lines. Total RNA was hybridized to antisense CMV RNA2 transcript probe (Fig. 4A). Lines 1-2 and 1-8, which have the strongest resistance based on testing at very high inoculum doses (Anderson et al., 1992
) and which also exhibit suppression of virus replication (Carr et al., 1994
), also contained the highest steady-state levels of transgene mRNA. In contrast, line 1-1, which was chosen to represent a transgenic line with an extremely low level of resistance, contained the lowest levels of transgene mRNA among the lines examined. Lines 2-3 and 2-7, showing intermediate levels of resistance, contained low to intermediate levels of transgene mRNA compared with the other lines studied (Fig. 4A
). When sibling plants of lines representing each of the three levels of resistance were examined for steady-state transgene mRNA by ribonuclease protection assay, identical patterns were observed among siblings (Fig. 4B
). These results indicated a direct correlation between higher relative amounts of steady-state transgene message and resistance. Ribonuclease protection assays were also done on R1 plants of several FLP Lines. These plants also exhibited a range of resistance levels between lines (Fig. 4C
). In these experiments, the level of resistance was based on the percentage of plants infected in resistance tests at inoculum concentrations of either 5 or 50 µg/ml (data not shown). As with the partial protein constructs, those lines exhibiting the highest levels of resistance contained the most steady-state transgene mRNA, while those exhibiting progressively lower levels of resistance contained lower steady-state levels of transgene mRNA, although none of the mRNA levels were very high with any of the constructs in any plants, resistant or susceptible (Fig. 4C
). These results indicated the same mechanism was responsible for resistance with both constructs.
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Transgenic plants containing fully or partially translatable transgenes were far more effective in generating resistance or delayed symptom development than plants containing nontranslatable transgenes. To determine if stronger resistance was directly correlated with higher transgene protein levels in lines containing translatable transgenes, Western blots were performed on total protein extracts from transgenic plants exhibiting different levels of resistance. Although it was possible to detect the transgene protein by Western blotting (Carr et al., 1994 ), levels were very low, and it was not possible during the course of our studies to detect the transgene protein consistently or to compare protein levels between lines differing in resistance. In fact, the only lines in which the protein could be detected at all were those exhibiting the highest levels of resistance (data not shown). Gal-On et al. (1998)
confirmed the low level of the 75 kDa transgene protein in tomato plants transformed with pCMV N/B-23.
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Discussion |
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Translatability of the transgene is important for replicase-mediated resistance to CMV
The analysis of transgenic plants containing translatable and nontranslatable transgenes, and examination of steady-state mRNA levels suggest that, like some examples of RNA-mediated resistance, translatability of the transgene plays an important role in replicase-mediated resistance to CMV. Resistance can be obtained with either a full-length, fully translatable CMV RNA2 construct, or a CMV RNA2 construct with a small internal deletion, but which translates only the N-terminal approximately two-thirds of the protein (Anderson et al., 1992 ; Fig. 1
, Fig. 2
, Table 1
). In contrast, as shown here, a nearly identical construct that only differs by a small, 4 nucleotide insertion which shifts translation out of frame shortly after initiation, was unable to produce resistance in tests of 60 of 61 independent transgenic lines (Table 1
).
Local lesions formed on transgenic plants capable of producing full-length or nearly full-length transgene proteins, but not on those transgenic plants containing the nontranslatable transgene construct. These lesions are a visible representation of the extent of virus movement in the inoculated leaf (Wintermantel et al., 1997 ). CMV is unable to enter the vascular system within these lesions, and is confined to localized areas on the inoculated leaf. Some plants that exhibit lesions on the inoculated leaf do eventually become infected, although infection is delayed compared to infection of either nontransformed controls or lines containing the nontranslatable construct (Fig. 2
). In highly resistant lines, however, further cell-to-cell movement beyond the lesion and entry into the vascular system were precluded (Wintermantel et al., 1997
). The localized symptoms may be indicative of a role for the transgene protein in restricting virus cell-to-cell and/or long-distance movement through as yet unidentified cellular processes.
Line NP7, the one nontranslatable line exhibiting some level of resistance differed from other resistant transgenic lines in that it did not produce the local response on inoculated leaves that occurs when resistant lines expressing translatable transgene constructs are inoculated with high concentrations of CMV virions. The inability of CMV to produce lesions on NP7 inoculated leaves suggests that this line is fundamentally different from resistant lines containing translatable constructs. A possible explanation is that NP7 may not exhibit the restricted cell-to-cell or long-distance movement of CMV that is observed in resistant lines containing a translatable transgene (Nguyen et al., 1996 ; Wintermantel et al., 1997
). Alternatively, NP7 may simply represent a transformation anomaly in which transgene insertion has somehow interfered with normal processes necessary for virus infection.
Do our results mean RNA is not involved in replicase-mediated resistance to CMV?
This study demonstrates a role for transgene translatability in CMV replicase-mediated resistance, and suggests a role for the transgene protein in resistance as well. Others have also identified transgene translatability as contributing to RNA mediated resistance (Cassidy & Nelson, 1995 ; Tanzer et al., 1997
). Transgenic plants containing a nontranslatable coat protein transgene of Peanut stripe virus, for example, exhibited either resistance or delayed symptom development following inoculation, and like the plants in our study, failed to show recovery once infected. Plants containing translatable coat protein constructs produced either complete resistance, or delayed infection followed by recovery (Cassidy & Nelson, 1995
). Unlike the Cassidy & Nelson study, however, none of our nontranslatable lines exhibited a complete resistance phenotype, and recovery was not observed for any line containing translatable or nontranslatable CMV RNA 2 constructs. Furthermore, nearly all nontranslatable lines in our study became infected at the same rate as nontransformed controls.
The partial resistance to CMV exhibited by line NP7, the lone resistant line containing the nontranslatable construct, however, indicates that in some cases RNA may also facilitate resistance in the absence of protein. In addition, steady-state transgene mRNA levels were fairly low in all plants tested. This alone could be suggestive of transgene silencing (reviewed by Baulcombe, 1996 ), which is characterized by low steady-state levels of transgene mRNA correlated with high levels of resistance. Such inverse correlations have been found in transgenic resistant plants expressing translatable and nontranslatable coat protein genes (Lindbo et al., 1993
; Smith et al., 1994
), replicase genes (Mueller et al., 1995
) and other foreign genes (Flavell, 1994
). If replicase-mediated resistance to CMV were the result of a gene silencing effect, one might expect to find lower levels of steady-state transgene mRNA in highly resistant plants than in susceptible transgenic controls. Interestingly, our CMV resistant transgenic plants contain higher relative levels of transgene mRNA than more susceptible lines (Fig. 4
).
We do not rule out the possibility that translatability enhances RNA effects in this resistance, but there are a number of points suggesting that the protein itself contributes to CMV-replicase mediated resistance. (1) Only one of 60 transgenic NP lines producing a nontranslatable transgene mRNA with only minor sequence alterations produced resistance, and systemic symptoms appeared in susceptible NP lines at the same rate as in nontransformed plants (Fig. 2). In contrast, effective resistance, as well as many delayed infections, were obtained with plants containing any of the translatable constructs. (2) The lack of symptom production on inoculated leaves (Fig. 3
) of NP plants suggests the inability to produce a transgene protein changes the nature of the interaction between the virus and the transgenic host. (3) Lines containing translatable constructs with stronger resistance contain higher steady-state transgene mRNA levels than lines exhibiting less resistance. Although this does not mean RNA-mediated resistance is not operating in these plants, the pattern is not what would typically be expected for this type of resistance. (4) There is never recovery from infection as often occurs in RNA-mediated resistance.
Arguably, the most interesting aspect of CMV replicase-mediated resistance is the existence of at least two separate elements contributing to the resistance: one resulting in a reduced level of virus replication, the other restricting virus movement (Carr et al., 1994 ; Hellwald & Palukaitis, 1995
; Nguyen et al., 1996
; Wintermantel et al., 1997
; Canto & Palukaitis, 1999
). We do not contend that RNA-mediated resistance does not occur in CMV replicase-mediated resistance, rather that this resistance is likely the result of a complex mechanism in which both transgene mRNA and protein can contribute separately, or in a cumulative manner, to engender resistance to CMV.
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Acknowledgments |
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References |
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Received 22 September 1999;
accepted 24 November 1999.