Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Kent Ridge, Singapore 117543
Correspondence
Sek-Man Wong
dbswsm{at}nus.edu.sg
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ABSTRACT |
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INTRODUCTION |
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METHODS |
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Isolation, electroporation of Dendrobium cultivar Sonia and N. benthamiana protoplasts and GFP imaging.
Protoplast suspensions (1x106) of Dendrobium cultivar Sonia and N. benthamiana were isolated and electroporated with 10 µg of in vitro transcripts of various ORSV and TMV mutant constructs, according to methods described previously (Hu et al., 1998). Protoplasts of Dendrobium cultivar Sonia, and leaves of C. quinoa and N. benthamiana were harvested and observed for green fluorescence emission under an inverted fluorescence microscope (ZEISS Axiovert 135) using a 366 nm filter at 36 h post-inoculation (p.i.) and at 3, 7, 20 and 30 days p.i.
Northern and Western blot analyses.
At 24 h p.i., protoplasts were pelleted by centrifugation, resuspended in 100 µl sterile water and homogenized in 100 µl RNA extraction buffer (200 mM Tris/HCl pH 8·5, 1 M NaCl, 1 % SDS, 2 mM EDTA). After three phenol/chloroform extractions, total RNA was precipitated with 3 vols ethanol and washed with 70 % ethanol. Plant total RNA was extracted using Trizol reagent (Life Technologies), according to the manufacturer's protocol. Equal amounts of total RNA (0·5 µg for positive-strand RNA, 3·0 µg for negative-strand RNA) were analysed on 1·2 % agarose-formaldehyde denaturing gels. After electrophoresis and blotting to positively charged nylon membranes (Roche), digoxigenin (DIG)-UTP-labelled RNA probes of ORSV CP-specific cRNA (nt 61975718 for positive-strand RNA detection; nt 57186197 for negative-strand RNA detection) and TMV-specific cRNA (nt 64015100) were used.
Total proteins from inoculated leaves, upper non-inoculated leaves with systemic mosaic symptoms and from protoplasts of Dendrobium cultivar Sonia were extracted after grinding samples in liquid nitrogen and resuspended in 500 µl lysis buffer (8 M urea, 10 % SDS, 1 % Triton X-100 and 10 mM Tris/HCl, pH 7·5). Protoplasts were vortexed for 3 min and tissue debris was removed by centrifugation. The supernatant was concentrated and subjected to Western blot analysis. Each sample contained total proteins from 1x106 protoplasts. Protein samples (10 µg) were probed with anti-ORSV CP antiserum and a secondary antibodyAP conjugate after 15 % SDS-PAGE.
RT-PCR and DNA sequencing.
To confirm the sequences from virus particles extracted from upper non-inoculated leaves after co-inoculation of N. benthamiana with tOT1+tORSV.GFP, tOT1+tORSV.GFPm or tOT1.GFP+tORSV (TTC), viral RNA was isolated from the purified virus using phenol/chloroform and precipitated with ethanol. Long-template RT was performed using 0·5 µg viral RNA with an enhanced AMV reverse transcriptase (Sigma) in a final volume of 20 µl using primer A (5'-TAAGCCAGCTTAGACGGG-3', corresponding to nt 57415758 of ORSV-S1). PCR was performed using 5 µl of the cDNA synthesized, 40 mM dNTPs, 32 pM each primer and 2·5 U AccuTaq polymerase (Sigma) in a final volume of 50 µl, using primers A and B (5'-CCACCGTTCTAGACGCCC-3', complementary to nt 182199 of ORSV-S1). For detecting the GFP gene in the inoculated and upper non-inoculated leaves, primers E (5'-GGCAATACAGTCATTATA-3', complementary to nt 44014418 of ORSV-S1) and F (5'-CAAGAATTGGGACAACTCC-3', corresponding to nt 2846 of the GFP gene) were used. The PCR was carried out with an initial denaturation at 98 °C for 30 s, then 2 cycles of 94 °C for 30 s, 48 °C for 1 min and 68 °C for 12 min and 30 cycles of 94 °C for 30 s, 58 °C for 1 min and 68 °C for 12 min.
To verify the sequence at nucleotide 211, total RNAs were extracted with Trizol reagent. Two RT-PCR products were obtained from total RNA isolated from the inoculated protoplasts or the upper non-inoculated leaves from plants inoculated with tOT1+tORSV.GFP, tOT1+tORSV.GFPm or tOT1+tORSV (TTC) using the Titan-One RT-PCR kit (Roche), using primers C (5'-ATGGCACACTTCCAACAAAC-3', corresponding to nt 6382 of ORSV-S1) and D (5'-CGTAATAGAACCAAATGG-3', corresponding to nt 359376 of ORSV-S1). RT-PCR products were cloned into PGEM-T Easy vector (Promega) and sequenced with primer D.
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RESULTS |
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Phe50 plays an important role in infectivity of ORSV
Phe50 of the 126/183-kDa proteins is highly conserved among the 13 tobamoviruses compared (data not shown), except for SHMV, which contains Cys50. In order to ascertain whether a change to nucleotide 211 would affect ORSV RdRp activity, different nucleotide residues were introduced into the codon for Phe50 through site-directed mutagenesis. Four mutants tORSV.Ser (TCT), tORSV.Tyr (TAT), tORSV.Cys (TGT) and tORSV.Trp (TGG) did not infect the local-lesion host C. quinoa or systemic host N. benthamiana. Only mutant tORSV.Phe (TTC) could induce local lesions and systemic symptoms identical to those produced by tOT2 (Table 1). Although tORSV.Tyr (TAT) possesses similar charge and hydrophobicity as Phe, it did not infect the test plants. These results suggested that it was the amino acid rather than the nucleotide change that resulted in the loss of infectivity of OT1.
The ability of each mutant to replicate was also studied in protoplasts of the orchid Dendrobium cultivar Sonia. The accumulation of viral plus-strand RNA and CP was detected only for tORSV.Phe (TTC) and not for tORSV.Ser (TCT), tORSV.Tyr (TAT), tORSV.Cys (TGT) or tORSV.Trp (TGG) (Fig. 1b and c). These results were consistent with those observed in whole plants. No minus-strand RNA synthesis was detected at 36 h p.i., except for tOT2 and tORSV.Phe (TTC) (Fig. 1d
), demonstrating that mutants with substitution of Phe50 in the 126/183-kDa proteins could not replicate. Moreover, results from time-course experiments confirmed that there was no detectable minus-strand RNA synthesis in the protoplasts transfected with mutant tOT1 (Fig. 1d
). Taken together, it is concluded that the mutants tested with a substitution at Phe50 prevented minus-strand RNA synthesis.
Importance of Phe50 in replication of TMV
To investigate the importance of Phe50 in the replication of tobamoviruses, similar mutations were introduced into TMV. Inoculation with in vitro transcripts of three mutants pTMV.Ser (TCT), pTMV.Phe (TTC) and pTMV.Tyr (TAT) (Fig. 3a) produced typical local lesions on inoculated leaves 24 days p.i. for wild-type TMV and tTMV.Phe (TTC) on N. tabacum Xanthi nc. Test plants inoculated with tTMV.Ser (TCT) or tTMV.Tyr (TAT) were not infected (Fig. 3a
). In addition, after electroporation of N. benthamiana protoplasts with the transcripts from the mutants, the RNA blot showed that only wild-type tTMV.Phe (TTT) and mutant tTMV.Phe (TTC) were able to replicate, and not tTMV.Ser (TCT) or tTMV.Tyr (TAT) (Fig. 3b
) at 24 h p.i. These results showed that Phe50 is probably highly preferred in the replication of tobamoviruses.
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At 7 days p.i., on C. quinoa inoculated with tORSV.GFP and tOT1+tORSV.GFP on opposite halves of the same leaf, necrotic lesions of two different sizes appeared. Small lesions (SL) were found on both half-leaves, whereas large lesions (LL) were only found on the half-leaf inoculated with tOT1+tORSV.GFP, which resembled similar sized lesions from those infected with OT2 alone (Fig. 4i). Only tOT1 was detected from the LL in the Northern blot, probed with a CP gene of ORSV (Fig. 5f
). Western blot further confirmed the presence of ORSV CP from the LL but not from the SL (Fig. 5g
). This showed that replication of tOT1 was complemented by tORSV.GFP.
Complementation of tOT1.GFP+tORSV (TTC)
To confirm the complementation event further, N. benthamiana plants were co-inoculated with tOT1.GFP+tORSV (TTC). ORSV (TTC) was a mutant construct of OT2 (TTT), with its Phe50 encoded by a different codon. Green fluorescent foci on the inoculated leaves at 8 days p.i. (Fig. 4j) were found in 4 of 20 plants, and the codon (TCT) derived from tOT1 was also detected using RT-PCR and sequencing. Systemic symptoms appeared in 19 of the 20 co-inoculated plants at 12 days p.i. None of the four plants showing green fluorescence on the inoculated leaves showed any green fluorescence on the upper non-inoculated leaves (Table 2
). This result verified that tOT1 was complemented by a replication-competent mutant on the inoculated leaves and that long-distance movement of tOT1.GFP was not supported by the CP derived from tORSV (TTC).
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Reversion rather than recombination occurred in systemic leaves of N. benthamiana
To study whether recombination had occurred and, if so, whether it had led tOT1 to restore its replication function, a construct designated pORSV.GFPm was made (Fig. 2c). As shown in Table 2
, at 3 days p.i., both tOT1 and tORSV.GFPm were detected in the co-inoculated leaves of all 20 plants, whereas no tOT1 was detected in leaves inoculated by tOT1 alone at 3 days p.i., as determined by RT-PCR and sequencing. The number of plants showing green fluorescent foci on inoculated leaves was recorded. At 18 days p.i., mosaic symptoms appeared on three infected plants. Long-template RT-PCR and sequencing using viral RNA extracted from virus particles purified from the upper non-inoculated leaves revealed that silent marker sequences from tORSV.GFPm (AAtTTTagt) were not detected, whereas the wild-type TTT sequence was found. The sequences of tOT1 (TCT) and tORSV.GFPm were not detected in the total RNA extracted from the upper non-inoculated leaves up to 20 days p.i. In addition, results from the complementation experiment of tOT1.GFP+tORSV (TTC) showed that no recombination but rather reversion occurred in two of the 20 co-inoculated plants.
To investigate further whether reversion could occur rapidly in the newly infected plant cells, protoplasts of N. benthamiana were co-inoculated with tOT1+tORSV.GFPm. Only tOT1 or tORSV.GFPm sequences were detected from 50 cDNA clones of RT-PCR products amplified from the region flanking nt 182376 of ORSV. No wild-type virus TTT sequence was detected. Therefore, reversion was not detected in the co-inoculated protoplasts by 48 h p.i.
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DISCUSSION |
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The 110 aa region downstream of the core MT in the 126/183-kDa proteins of ToMV was shown to bind to its 3' UTR (Osman & Buck, 2003). A similar binding domain might also be present in ORSV. Aromatic amino acids, including Phe and Tyr, are known to cross-link with nucleic acids (Golden et al., 1999
). However, alteration of Ser50 to Tyr did not restore the replication. Therefore, neither Tyr50 nor Phe50 may participate in direct binding to RNA. In the Ras superfamily, a group of small GTPases that function as molecular switches or timers to regulate cell fate, Phe156 is conserved and mutation of Phe results in disruption of secondary structure (Quilliam et al., 1995
). Similarly, it is possible that substitution of Phe50 may result in a conformational change that renders it non-functional. Similar results obtained from the TMV mutants further suggest that Phe50 may play an important role in replication among all tobamoviruses. The exact effects of altering Phe50 could be studied if the complete 126/183-kDa proteins could be expressed and crystallized. However, it is still a challenge to express and purify large membrane-bound RdRp proteins of TMV from E. coli (Osman & Buck, 2003
) and only limited success has been achieved with partially purified RdRp of Bamboo mosaic virus (Li et al., 1998
). Since OT1 and OT2 share the same known individual domains with only one amino acid change outside of those domains, studies using individual domains are unable to distinguish the differences between OT1 and OT2.
Trans-complementation is a common phenomenon among animal and plant viruses (Atabekov et al., 1999; Garcia-Arenal et al., 2001
; Heldsinger & Sharmeen, 2001
; Lough et al., 2001
; Osbourn et al., 1990
; Saphire et al., 2002
; van Kuppeveld et al., 2002
). A full-length replication-defective mutant of the coxsackie B3 virus 2B protein can be complemented by its wild-type virus (van Kuppeveld et al., 2002
). Replicase components produced by a replication-competent mutant of TMV are able to complement replication-defective mutants with various deletions in trans (Knapp et al., 2001
; Lewandowski & Dawson, 1998
; Ogawa et al., 1991
, 1992
). However, efficient trans-complementation of a full-length replication-deficient genome is uncommon among tobamoviruses (Ogawa et al., 1991
). Full-length defective RNA encoding only the 126-kDa protein could not be complemented in trans by a construct encoding the functional 126/183-kDa proteins (Lewandowski & Dawson, 1998
; Ogawa et al., 1992
), as it contains sequences that are inhibitory to replication (Lewandowski & Dawson, 1998
). Efficient replication requires the 183-kDa protein to form a heterodimer with the 126-kDa protein which has already bound to the target RNA (Lewandowski & Dawson, 2000
). Recent evidence shows that the helicase domain interacts with itself to form hexamer-like oligomers (Goregaoker et al., 2001
). Our results showed that full-length tOT1 was trans-complemented by various replication-competent mutants in both protoplasts and host plants (Fig. 5
and Table 2
). The results thus confirmed that a full-length replication-defective mutant could be complemented by a replication-competent ORSV, indicating that there is no inhibitory RNA sequence in ORSV.
In this study, we confirmed that ORSV.GFPm could not move long distances and was not assembled, as shown by RT-PCR using appropriate primers, whereas OT1 was assembled in N. benthamiana protoplasts (Fig. 5c). Another result showed that OT1.GFP, which has CP substituted with GFP, was not detected in systemic leaves (Table 2
). It indicates that CP is required for efficient long-distance movement. Our observation of two sizes of necrotic lesions on C. quinoa may indicate differences in efficiency of cell-to-cell movement among the mutants tOT1+tORSV.GFP, tORSV.GFP and tOT2.
Since tOT1 alone is unable to replicate and tORSV.GFP is unable to move long-distances without CP, neither virus mutant could move into the upper non-inoculated leaves independently. Only virus mutants that acquire a functional RdRp gene and CP gene either through recombination or reversion will be able to move systemically and induce symptoms. Therefore, it is reasonable to speculate that reversion occurs in the inoculated leaves, as recombination has not been detected. Revertants were not detected in the inoculated leaves; this was probably due to the lack of detection sensitivity at early stages of infection.
If recombination between OT1 and ORSV.GFPm occurred during trans-complementation to restore the replication of OT1, the marker nucleotides in the silent mutations of ORSV.GFPm (AAtTTTagt) would appear in the recombinant viruses present in the upper non-inoculated leaves. Only the revertant sequence TTT was detected in the extracted virions. None of the silent marker sequences from ORSV.GFPm were detected in the total RNA or virions extracted from the upper non-inoculated leaves (Table 2), indicating that the tOT1 TCT sequence reverted without recombination. The reversion of a mutant Y1867F back to wild-type sequence after undergoing systemic spread has been reported in Tobacco vein mottling virus (Murphy et al., 1996
). Another example comes from mutant BW5.123 of Beet western yellow virus which reverted to its original wild-type sequence without complementation and/or recombination (Brault et al., 2000
).
Reversion of mutants could occur in vivo. In general, restoration of function by second or other site mutations have been reported. For example, in ToMV, restoration of the MP function occurred, resulting from a Leu to Ile mutation, rather than reversion (Kawakami et al., 2003). Another example is a mutation at the amino acid level in the HEL region of the TMV RdRp which reverted to the wild-type in nucleotide sequence (Goregaoker et al., 2001
). We observed exact reversion, but not second-site mutation, that resulted in restoration of replication in OT1. A mutant containing GUA in the 3' non-coding region of RNA3 of Brome mosaic virus reverted back to the conserved AUA wild-type sequence without any other mutation occurring (Rao & Hall, 1993
). A similar report showed that an infectious Rubella virus variant with a motif substitution of GDD with ADD in the RdRp region reverted back during infection (Wang & Gillam, 2001
). In these cases, it seems that reversion is likely to be the only way to restore function. In another example, a single nucleotide substitution in the amber codon of 130/180-kDa proteins of ToMV resulted in a mutant without expression of the 130-kDa protein. This mutant was unstable and the mutated codon rapidly reverted back to an amber codon, resulting in the expression of both 130-kDa and 180-kDa proteins (Ishikawa et al., 1986
). Here reversion occurred and resulted in the restoration of replication for OT1.
Reversion to Phe50 restored the replication function of OT1 (TCT). Since there are only two possible codons for Phe (TTT or TTC), mutation from TCT to TTC can also restore the replication function. However, this would require two mutation events. Reversion from TCT to TTT is more straightforward, as it involves a change in only one nucleotide to regain its function. Therefore, this mutation is more likely to occur, resulting in an exact reversion. During the replication of tOT1 complemented by the functional RdRp of tORSV.GFP, the host might also be involved in the selection of the revertant for replication in vivo. In Potato virus X, restoration of a stemloop structure required for accumulation indicated host selection (Miller et al., 1999).
Two lines of evidence have shown exact reversion of Ser50 to Phe50 in vivo [Table 2; tOT1+tORSV.GFP and tOT1.GFP+tORSV (TTC)], this may have resulted from virus mutation, virus adaptation, host selection (Domingo et al., 2002
; Rozanov et al., 1992
) or any of the above combinations. Failure to detect the reversion in protoplasts could be due to insufficient cycles, small population size, lack of host selection or that reversion was such a rare event that it escaped detection. The mechanism of exact reversion is still unclear. However, the error-prone RdRp and mutation may contribute to exact reversion during virus replication. Further understanding and investigation into the biochemistry of each component of the functional RdRp complex may provide new insights into the mechanism of exact reversion.
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ACKNOWLEDGEMENTS |
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Received 28 February 2004;
accepted 7 April 2004.