Laboratory of Bioresource Technology, Graduate School of Frontier Sciences, The University of Tokyo, 202 Bioscience Bldg, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan
Correspondence
Shigetou Namba
snamba{at}ims.u-tokyo.ac.jp
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
![]() |
MAIN TEXT |
---|
![]() ![]() ![]() ![]() |
---|
|
Infectivity assays of each ASGV cDNA clone were performed as follows. RNA transcribed in vitro was immediately used to inoculate C. quinoa seedlings at the five- to ten-leaf stage after adjusting the concentration of the transcribed RNA to 1 µg µl-1, as described by Ohira et al. (1995). Plants were tested for virus infection by RT-PCR with an AMV RNA PCR kit (Takara) using total RNA extracted by Isogen (Nippon Gene) and ASGV-specific primers. The amplified cDNA of infecting viruses was detected by agarose gel electrophoresis and sequenced with a DNA sequencer following the manufacturer's protocol (Applied Biosystems). RNA transcribed in vitro from pITCL (ASGV-wt) induced symptoms characteristic of ASGV. These consist of severe chlorotic and necrotic local lesions on inoculated C. quinoa leaves, seen about 1 week post-inoculation (p.i.) (Fig. 2
a) and chlorotic mottling and malformation on upper leaves (Fig. 2b
). In contrast, ASGV-RM21, which has a silent nucleotide substitution within ORF1, usually induced no symptoms (data not shown) or occasionally very mild symptoms, with only a few, small chlorotic spots on inoculated leaves (Fig. 2c
) and no symptoms on upper leaves (Fig. 2d
). Dwarfing was observed in the plants infected with ASGV-wt, but no dwarfing occurred in those infected with ASGV-RM21 (Fig. 2e
). RT-PCR analysis confirmed the presence of ASGV-RM21 progeny in the asymptomatic upper leaves (data not shown). The attenuated symptoms caused by ASGV-RM21 were observed also in additionally tested plants including Nicotiana glutinosa and Vigna sesquipedalis cv. Kurodane-Sanjaku. In N. glutinosa plants, ASGV-wt induced mosaic, chlorotic spots or faint ring spots with etched surface on the systemically infected leaves (Fig. 2f
), while ASGV-RM21 infected asymptomatically (Fig. 2g
). In V. sesquipedalis, ASGV-wt produced necrotic spots on the upper leaves (Fig. 2h
), but ASGV-RM21 showed no infectivity (Fig. 2i
). The stability of the ASGV-RM21 mutation was tested after four serial passages in C. quinoa plants using crude leaf extracts as an inoculum. Inoculated plants invariably produced attenuated symptoms identical to those induced by the original ASGV-RM21 (data not shown). The complete nucleotide sequences of the progeny viruses were determined using 26 internal primers. No nucleotide alteration during these passages was detected, indicating that the single nucleotide mutation of ASGV-RM21 is stable. In order to confirm that the single U to C substitution at nucleotide 4646 is responsible for the attenuated symptoms with ASGV-RM21, the C at nucleotide 4646 of pRM21 was mutagenized back to the wild-type T nucleotide using a QuikChange site-directed mutagenesis kit (Stratagene). As expected, transcripts of the resultant construct produced identical symptoms to ASGV-wt (data not shown). Similarly, when the T at nucleotide 4646 of pITCL was mutagenized into C, transcripts of the resultant construct produced no symptoms, as in the case of pRM21 (data not shown).
|
|
The relationship between viral genetic information and the symptoms induced by the virus in host plants has long attracted much attention. Reduced virus replication or reduced cell-to-cell or long-distance movement, or combinations of these factors, may cause attenuated symptoms. Amino acid alterations in viral replicase causing attenuated symptoms have been well documented (Nishiguchi et al., 1985; Holt et al., 1990
; Lewandowski & Dawson, 1993
; Hagiwara et al., 2002
). In addition, it has been reported that alteration of the amino acids in CP or MP modifies symptom development (Shintaku & Palukaitis, 1992
; Banerjee et al., 1995
; Rao & Grantham, 1995
; Fujita et al., 1996
; Moreno et al., 1997
; Andersen & Johansen, 1998
; Szilassy et al., 1999
; Takeshita et al., 2001
; de Assis Filho et al., 2002
). It has also been implied that symptom development is a complex process involving interactions between viral genes or their products and host plant factors. The viral RNA 3'- and 5'-noncoding sequences can also affect the expression of disease symptoms (Rodr
guez-Cerezo et al., 1991
; Zhang et al., 1994
; van der Vossen et al., 1996
). In this study, RM21 and the other two substitution mutations at nucleotide 4646 of the ASGV genome, which are responsible for the attenuated pathogenic phenotype, are translationally silent. To our knowledge, this is the first example of silent mutations in the coding region of a plant viral sequence affecting viral symptoms.
As for the mechanism of the reduced accumulation of the viruses with a mutation in nucleotide 4646, one can argue that the nucleotide is located in the promoter region of the subgenomic RNA for MP, since the mutation is located upstream from the coding region (ORF2) of the putative MP. Although the promoter for MP subgenomic RNA has not yet identified, a homologous sequence, 5'-GCTNGAN(2)TTNGAAAN(2)TTNGGN(2)AN(14)AATG-3', is located upstream from both the MP and CP genes, and might be part of the subgenomic promoter of both genes (Magome et al., 1997). Nucleotide 4646 is 104 and 141 bases upstream from the homologous sequence and the initiation codon of MP, respectively. If the MP subgenomic promoter is somehow affected by the mutation, then the transcription of MP subgenomic RNA should be affected. Therefore, we quantified the amount of genomic, MP subgenomic, and CP subgenomic RNA species using the data shown in Fig. 3(b)
(RNA signal intensity in inoculated leaves). The relative abundance (genomic RNA=100) of genomic RNA:MP subgenomic RNA:CP subgenomic RNA was 100 : 12 : 18 for ASGV-wt and 100 : 13 : 20 for ASGV-RM21, meaning that the relative abundance of MP subgenomic RNA compared to that of genomic RNA is not specifically reduced in ASGV-RM21, although the absolute abundance of all three RNA species is reduced equally. This suggests that the mutation at nucleotide 4646 does not affect MP subgenomic RNA transcription.
Another possible explanation of the reduced accumulation of RM21 is that nucleotide 4646 might be involved in the expression or activity of viral replicase, thereby affecting virus replication. A stable stemloop structure was predicted near nucleotide 4646 of the ASGV-wt genomic RNA using the program MFOLD (Zuker & Jacobson, 1998), and altered stemloop structures were predicted for genomes of ASGV mutants having any of three substitutions at nucleotide 4646 (data not shown). The mechanism of symptom attenuation caused by a single nucleotide substitution which is translationally silent might be related to the alteration of the secondary structure of the genome.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|
Banerjee, N., Wang, J.-Y. & Zaitlin, M. (1995). A single nucleotide change in the coat protein gene of tobacco mosaic virus is involved in the induction of severe chlorosis. Virology 207, 234239.[CrossRef][Medline]
de Assis Filho, F. M., Paguio, O. R., Sherwood, J. L. & Deom, C. M. (2002). Symptom induction by Cowpea chlorotic mottle virus on Vigna unguiculata is determined by amino acid residue 151 in the coat protein. J Gen Virol 83, 879883.
Fujita, Y., Mise, K., Okuno, T., Ahlquist, P. & Furusawa, I. (1996). A single codon change in a conserved motif of a Bromovirus movement protein gene confers compatibility with a new host. Virology 223, 283291.[CrossRef][Medline]
Greener, A., Callahan, M. & Jerpseth, B. (1997). An efficient random mutagenesis technique using an E. coli mutator strain. Mol Biotechnol 7, 189195.[Medline]
Hagiwara, K., Ichiki, T. U., Ogawa, Y., Omura, T. & Tsuda, S. (2002). A single amino acid substitution in 126-kDa protein of Pepper mild mottle virus associates with symptom attenuation in pepper; the complete nucleotide sequence of an attenuated strain, C-1421. Arch Virol 147, 833340.[CrossRef][Medline]
Holt, C. A., Hodgson, R. A. J., Coker, F. A., Beachy, R. N. & Nelson, R. S. (1990). Characterization of the masked strain of tobacco mosaic virus: identification of the region responsible for symptom attenuation by analysis of an infectious cDNA clone. Mol Plant Microbe Interact 3, 417423.[Medline]
Lewandowski, D. J. & Dawson, W. O. (1993). A single amino acid change in tobacco mosaic virus replicase prevents symptom production. Mol Plant Microbe Interact 6, 157160.
Lu, X., Hirata, H., Yamaji, Y., Ugaki, M. & Namaba, S. (2001). Random mutagenesis in a plant viral genome using a DNA repair-deficient mutator Escherichia coli strain. J Virol Methods 94, 3743.[CrossRef][Medline]
Magome, H., Terauchi, H., Yoshikawa, N. & Takahashi, T. (1997). Analysis of double-stranded RNA in tissues infected with apple stem grooving capillovirus. Ann Phytopathol Soc Jpn 63, 450454.
Moreno, I. M., Bernal, J. J., de Blas, B. G., Rodriguez-Cerezo, E. & Graca-Arenal, F. (1997). The expression level of the 3a movement protein determines differences in severity of symptoms between two strains of tomato aspermy cucumovirus. Mol Plant Microbe Interact 10, 171179.[Medline]
Nishiguchi, M., Kikuchi, S., Kiho, Y., Ohno, T., Meshi, T. & Okada, Y. (1985). Molecular basis of plant viral virulence; the complete nucleotide sequence of an attenuated strain of tobacco mosaic virus. Nucleic Acids Res 13, 55855590.[Abstract]
Nishio, T., Kawai, A., Takahashi, T., Namba, S. & Yamashita, S. (1989). Purification and properties of citrus tatter leaf virus. Ann Phytopathol Soc Jpn 55, 254258.
Ohira, K., Ito, T., Kawai, A., Namba, S., Kusumi, T. & Tsuchizaki, T. (1994). Nucleotide sequence of the 3'-terminal region of citrus tatter leaf virus RNA. Virus Genes 8, 169172.[Medline]
Ohira, K., Namba, S., Rozanov, M., Kusumi, T. & Tsuchizaki, T. (1995). Complete sequence of an infectious full-length cDNA clone of citrus tatter leaf capillovirus: comparative sequence analysis of capillovirus genomes. J Gen Virol 76, 23052309.[Abstract]
Rao, A. L. N. & Grantham, G. L. (1995). A spontaneous mutation in the movement protein gene of brome mosaic virus modulates symptom phenotype in Nicotiana benthamiana. J Gen Virol 69, 26892691.
Rodrguez-Cerezo, E., Klein, P. G. & Shaw, J. G. (1991). A determinant of disease symptom severity is located in the 3'-terminal noncoding region of the RNA of a plant virus. Proc Natl Acad Sci U S A 88, 98639867.[Abstract]
Sambrook, J. & Russell, D. W. (2001a). Molecular Cloning: a Laboratory Manual, 3rd edn, vol. 3, pp. A8.40A8.55. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sambrook, J. & Russell, D. W. (2001b). Molecular Cloning: a Laboratory Manual, 3rd edn, vol. 1, pp. 7.317.50. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Shintaku, M. H. & Palukaitis, P. (1992). A single amino acid substitution in the coat protein of cucumber mosaic virus induces chlorosis in tobacco. Plant Cell 4, 751757.
Szilassy, D., Salánki, K. & Balázs, E. (1999). Stunting induced by cucumber mosaic cucumovirus-infected Nicotiana glutinosa is determined by a single amino acid residue in the coat protein. Mol Plant Microbe Interact 12, 11051113.[Medline]
Takeshita, M., Suzuki, M. & Takanami, Y. (2001). Combination of amino acids in the 3a protein and the coat protein of Cucumber mosaic virus determines symptom expression and viral spread in bottle gourd. Arch Virol 146, 697711.[CrossRef][Medline]
van der Vossen, E. A. G., Neeleman, L. & Bol, J. F. (1996). The 5' terminal sequence of alfalfa mosaic virus RNA3 is dispensable for replication and contains a determinant for symptom formation. Virology 221, 271280.[CrossRef][Medline]
Verwoerd, T. C., Dekker, B. M. & Hoekema, A. (1989). A small-scale procedure for the rapid isolation of plant RNAs. Nucleic Acids Res 17, 2362.[Medline]
Yoshikawa, N. & Takahashi, T. (1992). Evidence for translation of apple stem grooving capillovirus genomic RNA. J Gen Virol 73, 13131315.[Abstract]
Yoshikawa, N., Sasaki, E., Kato, M. & Takahashi, T. (1992). The nucleotide sequence of apple stem grooving capillovirus genome. Virology 191, 98105.[Medline]
Yoshikawa, N., Imaizumi, M., Takahashi, T. & Inouye, N. (1993). Striking similarities between the nucleotide sequence and genome organization of citrus tatter leaf and apple stem grooving capilloviruses. J Gen Virol 74, 27432747.[Abstract]
Zhang, L., Hanada, K. & Palukaitis, P. (1994). Mapping local and systemic symptom determinants of cucumber mosaic cucumovirus in tobacco. J Gen Virol 75, 31853191.[Abstract]
Zuker, M. & Jacobson, A. B. (1998). Using reliability information to annotate RNA secondary structures. RNA 4, 669679.
Received 21 February 2003;
accepted 29 April 2003.