日木直病 報 60: 27-35 (1994)
Ann. Phytopath. Soc. Japan 60: 27-35 (1994)
Replication of Cauliflower Mosaic Virus ORF I Mutants in Turnip Protoplasts
Seiji TSUGE*,•õ, Kappei KOBAYASHI*,•õ•õ, Hitoshi NAKAYASHIKI*,
Tetsuro OKUNO* and Iwao FURUSAWA*
Abstract We succeeded in infecting turnip protoplasts with a cloned cauliflower mosaic virus (CaMV) DNA, pCa122, which contains 1.2 copy of CaMV genomic DNA and a plasmid expressing open reading frame (ORF) VI products (pEXP6) using polyethylene glycol. Fluorescent antibody staining showed that up to 50% of protoplasts were infected. It was difficult to detect the progeny DNA and the viral protein in protoplasts inoculated with pCa122 alone. Co-transfection with the plasmid pEXP6 produced larger fluorescing specks in each infected protoplasts and increased the accumulation of the progeny DNA and some other viral proteins to detectable levels. Using this protoplast system, three CaMV ORF I insertional mutants which were not infectious on turnip plants were tested for their infectivity on turnip protoplasts. Viral DNA and products accumulated in infected proto- plasts to the same extent of the wild type DNA-infected protoplasts. These results indicate that ORF I product is not required for multiplication of CaMV in protoplasts, but is indispensable for infection on whole plants, strongly supporting that ORF I product is involved in cell-to-cell movement of CaMV. (Received May 6, 1993)
Key words: cauliflower mosaic virus, protoplasts, movement protein.
INTRODUCTION
Cauliflower mosaic virus (CaMV) has an 8kb circular double stranded DNA genome which encodes six major open reading frames (ORFs) I to VI on the same DNA strand17). Functions have been assigned to these ORF products except for the product of ORF III. It is tempting to study the gene expression of CaMV in relation to the functions of genes in protoplasts8). However, no one has been successful in infecting protoplasts with cloned CaMV DNAs although infection of turnip protoplasts with virion DNA has been reported22). We found that co-inoculation with a plasmid which expresses the CaMV ORF VI product (P6) under the control of the CaMV 35S promoter by means of polyethylene glycol (PEG) led to efficient infection of turnip protoplasts with cloned CaMV DNAs. P6 has been shown to function as a trans-activator for other genes of CaMV2) and also to be a major component of viroplasm where viral replication occurs3,18). Therefore, gene expression and viral replication are thought to be activated by P6 in protoplasts inoculated with the cloned CaMV DNA. Using this protoplast system we analyzed three CaMV mutants having 27bp (in-frame) and 31bp (frame-shift) non-viral sequence inserted in ORF I which was thought to encode a cell-to-cell movement protein of CaMV21). We report here that CaMV ORF I mutant DNAs, which cannot infect systemically on turnip plants, replicated in protoplasts to the
* Laboratory of Plant Pathology , Faculty of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-01, Japan 京 都 大 学 農 学 部 † Present address: Laboratory of Plant Pathology , Faculty of Agriculture, Kyoto Prefectural University, Sakyo- ku, Kyoto 606, Japan 京 都 府 立 大 学 農 学 部 †† Present address: Department of Microbiology , Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602, Japan 京 都 府 立 医 科 大 学 28 日本植 物 病 理 学会 報 第60巻 第1号 平 成6年2月 same level of the wild type DNA. This indicates that the ORF I product (P1) is not essential for the replication of CaMV in single cells and supports the recent findings that CaMV ORF I encodes a cell-to-cell movement protein21).
MATERIALS AND METHODS
Plasmids. The plasmid pCa122 has an 1.2 copy of CaMV CM1841 DNA which comprises a complete CaMV DNA and an additional PvuII-BstEII fragment (#6,321-#8,031/#0-#127: nucleotide number refers to Gardner et al. 10)). The plasmid was constructed by using pCaMV10 which has one copy of CM1841 DNA inserted in pBR322 at its unique SalI site (#4,834)10. The plasmid pCaMV10 was digested with PvuII and religated for removing the shorter PvuII fragment, creating pCa0.8. Large SalI-SphI (filled out) fragment of pCa0.8 was ligated with the shortest SalI-BstEII (filled in) fragment
(#4,834-#8,031/#0-#127) of pCaMV10, creating the pCa122 (Fig. 1). Plasmid pEXP6 which has CaMV ORF VI region 3•Œ to the CaMV 35S promoter region was constructed as follows; the PvuII-ScaI fragment (#6,321-#7,640) of CaMV DNA was inserted into pUC118 at HindIII site after filling in, and a new SaiI site was introduced to the 35S RNA cap site (#7,433) by site-directed mutagenesis using oligonucleotide (5•Œ-CATTTGGAGTCGACACGCTGAA-3•Œ)14, to obtain pMC. The 3•Œend sequence of nopaline synthase gene was excised from pBI221 (Clontech Laboratories), and inserted into SacI-EcoRI sites of pMC, to create pMCT. The EcoRV fragment of CaMV DNA
(#5,711-#7,343) was inserted into the respective site of pBluescript II SK-. A SalI site was introduced to the cap site of 19S RNA by site-directed mutagenesis using oligonucleotide (5•Œ-CTGAGAAAGTCGA- CCTCCAAGC-3•Œ), and the SalI-EcoRV fragment of the plasmid was inserted into pMCT which had been digested with SalI and Smal to create pEXP6. This plasmid was used for co-inoculation of turnip protoplasts with cloned CaMV DNA. Construction of ORF I mutants. First, we constructed a plasmid pUCK191 (a derivative of pUC19) which has kanamycin resistance (Kmr) gene (derived from Tn903) with KpnI and SmaI (XmaI)
Fig. 1. Schematic representation of pCa122. CaMV CM1841 DNA is shown by a thin line with ORFs indicated by arrows on it, and DNA derived from pBR322 is shown by an open box. Thin arrows inside the DNA represent RNA transcripts of CaMV. Zero are the start positions of a nucleotide number of CM1841 (refer to Gardner et al.10)). Black and shaded arrowheads indicate CIaI and XbaI sites, respectively. Ann. Phytopath. Soc. Japan 60 (1). February, 1994 29
Fig. 2. Restriction sites flanking on both sides of kanamycin resistance gene (Kmr) in pUCK191. Kmr is derived from Tn903, and the other region of this plasmid is derived from pUC19 in which DraI fragment, containing ampicillin resistance gene, is deleted. Thick and thin arrows indicate that each regions are located in the same orientation, respectively.
sites flanking on either side in the same orientation, and SalI (AccI), XbaI and Bam HI sites flanking on either outside of the KpnI and SmaI (XmaI) sites in the same orientation (Fig. 2). Dral fragment containing a part of ampicillin resistance gene was removed from the plasmid. CaMV mutants having an inserted sequence in ORF I region were constructed in a similar way as described by Dixon et al.4) The plasmid pCa122 was partially digested with ClaI in the presence of 80ƒÊg/ml ethidium bromide to obtain full length linear DNA molecules. The DNAs were then ligated with the Kmr gene excised from pUCK191 by digestion with AccI, and introduced into Escherichia coli. The transformants were selected on medium containing both ampicillin and kanamycin. The Kmr gene was removed from the recom- binant CaMV DNA by digestion with KpnI and relegation for generating insertional mutants containing a 27bp inserted sequence including KpnI, SmaI BamHI and XbaI sites at any one of ClaI sites of pCa122. Alternatively, the Kmr gene was removed by XmaI digestion followed by filling-in and relegation, resulting in mutants having a 31bp inserted sequence containing KpnI, BamHI, XbaI and Eco52I sites. The plasmids having a 27bp and a 31bp inserted sequence at the ClaI site in the ORF I of CaMV (#822) were identified by restriction enzyme analysis, and designated pCaMV822-1 and pCaMV822-2, respec- tively. Similarly, we constructed other insertional mutants which have a 31bp inserted sequence at the XbaI site in the ORF I (#839) and named pCaMV839-2. Nucleotide sequences in the ORF I region of mutant DNAs were determined using Sequenase kit (USB) according to the manufacturer's instruction. Inoculation of turnip protoplasts with cloned CaMV DNA. Turnip protoplasts were prepared as described by Furusawa and Okuno6). To the pellet of protoplasts (1•~106) added were 0.5ml of 10mM MES buffer (pH 5.8) containing 0.5M mannitol, 40mM CaCl2, 50ƒÊg of cloned CaMV DNA, 20
ƒÊg of pEXP6 and 50ƒÊg of salmon sperm DNA (as a carrier), followed by mixing with 0.9ml of 0.5M mannitol containing 40% PEG, MW. 4000 (SIGMA) and 40mM CaCl2. Protoplasts were incubated for 30min on ice with gentle shaking, diluted with 10ml of ice cold 0.5M mannitol containing 40mM CaCl2, and incubated for additional 30min on ice. After washing three times with 0.5M mannitol containing 50mM glycine and 50mM CaCl2, pH 8.513), protoplasts were cultured as described previously7). Infection of protoplasts was determined by fluorescent antibody staining7). Inoculation of turnip plants with cloned CaMV DNA. Turnip plants (Brassica raga L. cv. Marubakomatsuna) were grown under natural daylight in a greenhouse at controlled temperature of 22•}3•Ž. Ten microlitter of pCa122 or its derivatives (1ƒÊg/ƒÊl) was mixed with an equal volume of sterilized water containing carborundum (50%
RESULTS
Infection of turnip protoplasts with cloned CaMV DNA The plasmid pC122 was found to be infectious when inoculated on turnip leaves without removing the bacterial plasmid region. Therefore, we used pCa122 for infection tests of turnip protoplasts with cloned CaMV DNA. Infection of turnip protoplasts was assessed by fluorescent antibody staining using antibody to CaMV virions after 72hr of incubation (Fig. 3). Up to 50% of protoplasts were infected with cloned CaMV DNA either in the presence or in the absence of pEXP6 which expresses P6. However, fluorescing specks were small in protoplasts inoculated in the absence of pEXP6 compared to those observed in protoplasts co-inoculated with pEXP6 (data not shown). Accumulation of viral products in co-transfected protoplasts was analyzed by western blotting methods. ORF II (P2) and III products (P3) were accumulated enough to be detected in protoplasts co-inoculated with pCa122 and pEXP6, although we failed to detect these viral proteins in protoplasts inoculated with pCa122 alone (Fig. 4). Accumula- tion of viral antigen and probably viral replication in each protoplast seemed to be increased.by pEXP6 used for transfection. Therefore, protoplasts were inoculated with cloned CaMV DNA together with pEXP6 in all following experiments.
Fig. 3. Immunofluorescent staining of protoplasts inoculated with pCa122 using antibody to CaMV virion. An arrow indicates fluorescing specks. Ann. Phytopath. Soc. Japan 60 (1). February, 1994 31
A
B
Fig. 4. Analysis of accumulation of P2 (A) and P3 (B) products in protoplasts inoculated with pCa122 alone or co-inoculated with pCa122 and pEXP6. Total proteins extracted from protoplasts 72hr of incuba- tion were fractionated by SDS-PAGE and detected by western blotting method using antiserum against P2 (A) or P3 (B) product. Lane 1, pCa122; 2, pCa122+pEXP6; 3, pEXP6; 4, mock-inoculation. Arrows indicate P2 (A) and P3 (B) products, respectively.
Fig. 5. Nucleotide sequences inserted in ORF I region of each mutants. Open boxes indicate ORFs whereas lines represent the non-coding regions. The expanded nucleotide sequences shows the inserted sequences (hatched boxes) in ORF I. Non-viral inserted sequences and their joint viral sequences are indicated by capital letters and small letters, respectively. The termination codons are indicated by under line. 32 日本植 物 病 理 学会 報 第60巻 第1号 平 成6年2月
Fig. 6. An electron micrograph of a section of turnip protoplasts inoculated with pCaMV822-2 and cultured for 72hr. The arrow indicates the viral particle aggregates represented by inclusion-body-like matrix.
(A) (B) Fig. 7. Analysis of progeny virion DNAs in protoplasts inoculated with CaMV ORF I mutants. DNAs were extracted from protoplasts inoculated with a wild type or mutants DNA and cultured for 72hr. Extracted DNA were digested with Bam HI and analyzed by Southern blotting method using nick-translated pCa122-specific probe. (B) is exposed longer than (A). Lane 1, pCa122+pEXP6; 2, pCaMV822-1+pEXP6; 3, pCaMV822-2+pEXP6; 4, pEXP6. The length of CaMV DNAs are indicated on the right.
Infectivity of ORF I mutants on turnip plants and protoplasts Nucleotide sequences inserted in the ORF I region of each mutant are shown in Fig. 5. An in-frame mutant pCaMV822-1 had no termination codon in the inserted sequence, thus P1 of this mutant was predicted to have additional nine amino acids compared to that of the wild type. In the case of frame-shift mutants, pCaMV822-2 and pCaMV839-2 had a termination codon in the inserted sequence and just the downstream of the inserted sequence, respectively. Therefore, the P1 of these mutants was supposed to be approximately half size of the wild type's. Ann. Phytopath. Soc. Japan 60 (1). February, 1994 33
A
B
Fig. 8. Analysis of accumulation of P2 and P3 in protoplasts inoculated with CaMV ORF I mutants. Protoplasts inoculated and cultured for 72hr were suspended in sample buffer, fractionated by SDS-PAGE and detected by western blotting method using antiserum against P2 (A) or P3 (B). Lane 1, pCa122+pEXP6; 2, pCaMV822-1+pEXP6; 3, pCaMV822-2+pEXP6; 4, pCaMV839-2+pEXP6; 5, pEXP6. The arrows on the right indicate P2 and P3, respectively.
Turnip plants, when inoculated with any of ORF I mutants DNAs, did not develop systemic symptoms and no viral DNAs were detected in the upper leaves by dot-blot hybridization 6 weeks after inoculation (data not shown). These ORF I mutant DNAs were tested for their ability to replicate and to produce progenies in turnip protoplasts by fluorescent antibody staining using antibody to CaMV virions. Accumulation of viral antigen was indistinguishable between pCa122 and ORF I mutants including the in-frame mutant pCaMV822-1 and frame-shift mutants pCaMV822-2 and 839-2 (data not shown). Ultra-thin sections of protoplasts inoculated with the mutants were observed by electron microscopy. As shown in Fig 6, progeny virions were formed in mutant-infected protoplasts to the same extent as in wild type-infected protoplasts. To determine DNA sequences in mutated regions of progeny virus, virion DNAs were extracted from protoplasts infected with the mutants. The DNA was digested with BamHI, fractionated on agarose gel and hybridized with a 32P-labeled pCa122 probe. The wild type DNA was digested into two DNA fragments 7,809 and 222 by while ORF I mutant DNAs were separated into approximately 6,700, 1,100 and 222bp because of a new Bam HI site created in the inserted sequences (Fig. 7). These results indicated that ORF I mutants multiplicated in turnip protoplasts with mutated regions preserved. Expression of downstream ORF II and III in ORF I mutant-infected protoplasts was investigated by western blotting method using antiserum of P2 and P3. Little difference was observed between the wild type CaMV- and mutants-infected protoplasts in accumulation of either P2 or P3, indicating that mutations in the ORF I did not influence the expression of ORF II and III (Fig. 8).
DISCUSSION
Protoplasts are useful for the study of plant viruses20). In the case of CaMV, however, no one has succeeded in infecting protoplasts with cloned mutated viral DNA. In this paper we showed that turnip 34 日本植 物病 理学 会 報 第60巻 第1号 平 成6年2月
protoplasts were infected with CaMV DNAs by means of PEG using co-transfection with the plasmid
pEXP6 which expresses P6 under the control of CaMV 35S promoter. The results showed pEXP6 was not necessary for CaMV DNA infection, but might be involved in increasing rate of viral replication in
each infected protoplast. The protein P6 has been shown to function as a trans-activator for other genes
of CaMV2) and also to be a major component of viroplasm where viral replication occurs3,18). P6 will be
produced in pEXP6-co-transfected protoplasts to a higher level than in protoplasts inoculated with
pCa122 alone, resulting in more active viral replication in pEXP6-co-transfected protoplasts. Generally, plant viruses encode a protein involved in cell-to-cell movement of the virus. In CaMV,
P1 has been predicted to be involved in cell-to-cell movement based on several indirect evidences; partial
amino acid homology with the 30K protein of TMV12), accumulation in the cell-wall-enriched fraction1),
and association with the cell wall matrix close to the modified plasmodesmata16). Recently, using
•g agroinfection•h technique, an ORF I deletion mutant which is not infectious systemically on host plants
has been found to be infectious in initially infected cells21).
The ORF I insertional mutants constructed here are predicted to produce either ORF I protein
having additional nine amino acids compared to that of the wild type or a truncated small ORF I protein.
Either type of the ORF I mutants were not infectious on turnip plants, but they were as infectious as the
wild type CaMV DNA on protoplasts and produced progeny virions containing DNA with the inserted
sequence preserved (Fig. 6, 7). The results indicate that ORF I product was dispensable for the
multiplication of CaMV in protoplasts and support the finding that the ORF I product is the cell-to-cell
movement protein of CaMV21).
Since ORF I, II and III are located very closely on the genome map of CaMV DNA, their products
are proposed to be translated by a •grelay race•h mechanism from the polycistronic mRNA5). If so,
translation efficiency of ORF II and III might be decreased in frame-shift mutants pCaMV822-2 and
pCaMV839-2 because of a long untranslated region, approximately 500bp, between the termination colon of a truncated ORF I and the initiation colon of ORF II (Fig. 4). Alternatively, even if ribosomes
which have finished translating a truncated ORF I pass through 62 (pCaMV822-2) or 47 bases
(pCaMV839-2) without being disengaged mRNA and re-initiate translation of remaining part of ORF I,
ORF II and III from the initiation colon in the same frame, the translation level is supposed to be
decreased. However, accumulation level of both P2 and P3 products in infected protoplasts was not
different between ORF I mutants and the wild type (Fig. 7). Therefore, it seems difficult to explain the
efficient translation of ORF II and III in these mutants by the •grelay race•h mechanism. Some other
translation system may be present.
Literature cited 1. Albrecht, H., Geldreich, A., Menissier De Murcia, J., Kirchherr, D., Mesnard, J.M. and Lebeurier, G. (1988). Cauliflower mosaic virus gene I product detected in a cell-wall-enriched fraction. Virology 163: 503-508. 2. Bonneville, J.M., Sanfacon, H., Ftitterer, J. and Hohn, T. (1989). Posttranscriptional traps-activation in cauliflower mosaic virus. Cell 59: 1135-1143. 3. Covey, S.N. and Hull, R. (1981). Transcription of cauliflower mosaic virus DNA. Detection of transcripts, properties and location of the gene encoding the virus inclusion body protein. Virology 111: 463-474. 4. Dixon, L.K., Koenig, I. and Hohn, T. (1983). Mutagenesis of cauliflower mosaic virus. Gene 25: 189-199. 5. Dixon, L.K. and Hohn, T. (1983). Initiation of translation of the cauliflower mosaic virus genome from a polycistronic mRNA: evidence from deletion mutagenesis. EMBO J. 3: 2741-2736. 6. Furusawa, I. and Okuno, T. (1978). Infection with BMV of mesophyll protoplasts isolated from five plant species. J. Gen. Virol. 40: 489-491. 7. Furusawa, I., Yamaoka, N., Okuno, T., Yamamoto, M., Kohno, M. and Kunoh, H. (1980). Infection of turnip protoplasts with cauliflower mosaic virus. J. Gen. Virol. 48: 431-435. 8. Fiitterer, J., Gordon, K., Sanfacon, H., Bonneville, J. and Hohn, T. (1990). Positive and negative control of translation by the leader sequence of cauliflower mosaic virus pregenomic 35S RNA. EMBO J. 9: 1697-1707. 9. Gardner, R.C. and Shepherd, R.J. (1980). A procedure for rapid isolation and analysis of cauliflower mosaic virus DNA. Virology 196: 159-161. 10. Gardner, R.C., Howarth, A.J., Hahn, P., Brown-Luedi, M., Shepherd, R.J. and Messing, J. (1981). The Ann. Phytopath. Soc. Japan 60 (1). February, 1994 35
complete nucleotide sequence of an infectious clone of cauliflower mosaic virus by M13mp7 shotgun sequencing. Nucl. Acids Res. 9: 2871-2888. 11. Horikoshi, M., Mise, K., Furusawa, I. and Shishiyama, J. (1988). Immunological analysis of brome mosaic virus replicase. J. Gen. Virol. 69: 3081-3087. 12. Hull, R., Sadler, J. and Longstaff, M. (1986). The sequence of carnation etched ring virus DNA: compari- son with cauliflower mosaic virus and retroviruses. EMBO J. 5: 3083-3090. 13. Keller, W.A. and Melchers, G. (1973). The effect of high pH and calcium on tobacco leaf protoplast fusion. Z. Naturf orsch. 28C: 737-741. 14. Kunkel, T.A. (1985). Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc. Natl. Acad. Sci. USA 82: 488-498. 15. Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 227: 680-685. 16. Linstead, P.J., Hills, G.J., Plaskitt, K.A., Wilson, I.G., Harker, C.L. and Maule, A.J. (1988). The subcellular location of the gene 1 product of cauliflower mosaic virus is consistent with a function associated with virus spread. J. Gen. Virol. 69: 1809-1818. 17. Mason, W.S., Taylor, J.M. and Hull, R. (1987). Retroid virus genome replication. Adv. Virus Res. 321: 35- 96. 18. Mazzolini, L., Bonneville, J.M., Volovitch, M., Magazin, M. and Yot, P. (1985). Strand-specific viral DNA synthesis in purified viroplasms isolated from turnip leaves infected with cauliflower mosaic virus. Virology 145: 293-303. 19. Nakayashiki, H., Kobayashi, K., Tsuge, S., Okuno, T. and Furusawa, I. (1991). Characterization of in vitro recombinant cauliflower mosaic virus in aphid transmissibility. Ann. Phytopath. Soc. Japan 57: 634-640. 20. Takebe, I. (1977). Protoplasts in the study of plant virus replication. In Comprehensive Virology, vol. XI (H. Fraenkel-Conrat and R.R. Wagner eds.). Plenum Press, New York. pp. 237-283. 21. Thomas, C.L., Perbal, C. and Maule, J. (1993). A mutation of cauliflower mosaic virus gene I interferes with virus movement but not virus replication. Virology 192: 415-421. 22. Yamaoka, N., Furusawa, I. and Yamamoto, M. (1982). Infection of turnip protoplasts with cauliflower mosaic virus DNA. Virology 122: 503-505.
和 文 摘 要
津 下 誠 治 ・小林括 平 ・中屋敷 均 ・奥野哲 郎 ・古澤 巌:カ リフ ラ ワー モ ザ イ ク ウイ ル ス の オー プ ン リー デ ィ ング フ レ ー ムI変 異体 の コマツ ナ プ ロ トプ ラス トにお け る増殖
ク ロ ーニ ング され た カ リフ ラ ワー モ ザ イ クウ イル ス(CaMV)DNAを,ト ラ ン ス ア クテ ィベ ータ ー と して機 能 す る こ とが知 られ て い るCaMVの オ ー プ ン リー デ ィ ング フ レー ム(ORF)VIの 産物 を発 現 す るプ ラ ス ミ ドと とも に,ポ リエ チ レ ング リコール 法 を用 い て コマ ツナ プ ロ トプラス トに接種 す る こ とに よ り,最 高 約50%の 感 染率 が得 られた。また, 感 染 プ ロ トプラ ス トから は,ウ イ ル ス タ ンパ ク質,お よび子 孫 ウイ ル スDNAも 検 出 可能 とな った。 この系 を用 い,植 物 体 で感 染 性 を失 っ た3種 のCaMVのORFI挿 入 変 異体 の,コ マ ツ ナ プ ロ トプラ ス トへの感 染 性 を調べ た とこ ろ,ウ イ ル スDNA,タ ンパ ク質 とも に,野 生 株 と同程度 に蓄 積 してい る こ とが確認 さ れ た。 この結 果 は,CaMVのORF I 産 物 が,ウ イ ル スの細 胞 間移 行 性 に関 与 す るタ ンパ ク質で あ り,CaMVの 一 細 胞 で の複製,増 殖 に は関与 しな い とい う こ とを強 く支持 す る もの で あ る。