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Involvement of THH1, an Arabidopsis thaliana homologue of the TOM1 gene, in tobamovirus multiplication

¹ Plant–Microbe Interactions Research Unit, National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan
² Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan
³ Graduate School of Life Science, Hokkaido University, Sapporo 060-8589, Japan
⁴ CREST, Japan Science and Technology Corporation, Kawaguchi 322-0012, Japan

Correspondence
Masayuki Ishikawa
ishika32{at}affrc.go.jp

	ABSTRACT

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The TOM1 and TOM3 genes of Arabidopsis thaliana encode homologous proteins that are required for tobamovirus multiplication. Although the A. thaliana genome encodes another TOM1-like gene, THH1, the tobamovirus coat protein (CP) does not accumulate to a detectable level in the tom1 tom3 double mutant. Here, double and triple mutants of tom1, tom3 and thh1 were generated to investigate whether THH1 functions to support tobamovirus multiplication. In the tom1 thh1 double mutant, the tobamovirus CP accumulated to a level that was detectable, but lower than that in the tom1 single mutant. In tom1 tom3 double-mutant lines overexpressing THH1, the tobamovirus CP accumulated to a level similar to that observed in wild-type plants. These results suggest that THH1 supports tobamovirus multiplication, but to a lesser extent than TOM1 and TOM3. The expression level of THH1 is lower than that of TOM1 and TOM3, which might explain the smaller contribution of THH1 to tobamovirus multiplication.

	MAIN TEXT

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Virus multiplication depends on both viral and host factors (reviewed by Buck, 1999; Ahlquist et al., 2003; Noueiry & Ahlquist, 2003). The genomes of positive-strand RNA viruses encode one or more polypeptides that are required for the replication of the virus genomes. These proteins, called replication proteins, are synthesized by translation of the genomic RNA after invasion into host cells. The genomes of all known positive-strand RNA viruses replicate via negative-strand RNA in replication complexes, which contain replication proteins and template RNA and are formed in association with intracellular membranes.

The tobamoviruses are positive-strand RNA viruses and their genomes encode replication proteins of approximately 130 and 180 kDa. In addition to these viral factors, the proteins TOM1 and TOM2A are required for efficient multiplication of tobamoviruses in the host Arabidopsis thaliana. TOM1 is a seven-pass transmembrane protein that interacts with both the tobamovirus replication proteins and with TOM2A, a four-pass transmembrane protein that is also required for the efficient multiplication of tobamoviruses (Yamanaka et al., 2000; Tsujimoto et al., 2003). TOM1 and tobamovirus RNA-dependent RNA polymerase activity show similar fractionation patterns during membrane-flotation analysis of tobamovirus-infected protoplast lysates by iodixanol-gradient centrifugation. This observation, together with its ability to interact with tobamovirus replication proteins, suggests that TOM1 plays a direct role in the formation and/or maintenance of membrane-bound tobamovirus replication complexes (Hagiwara et al., 2003).

The A. thaliana genome encodes two TOM1 homologues, TOM3 and THH1. Amino acid identity is 56 % between TOM1 and TOM3, 58 % between TOM1 and THH1 and 88 % between TOM3 and THH1. Previous studies have demonstrated that the tom1 single mutation inhibits tobamovirus multiplication moderately, but not completely, and that simultaneous mutations in the TOM1 and TOM3 genes suppress tobamovirus coat protein (CP) accumulation to an undetectable level (Yamanaka et al., 2002). These results indicate that TOM1 and TOM3 support tobamovirus multiplication. However, because it was not clear whether THH1 also supports tobamovirus multiplication in A. thaliana, we investigated the issue in this study.

A mutant line of A. thaliana (GABI-Kat 046D04) (Rosso et al., 2003) that contains a T-DNA fragment inserted in the fifth exon of the THH1 gene (Fig. 1) was obtained from the Max Planck Institute for Plant Breeding Research. The mutation, named thh1-1, had no apparent effect on plant growth or development (data not shown). The CP of TMV-Cg, a tobamovirus that infects wild-type A. thaliana plants systemically (Ishikawa et al., 1991; Yamanaka et al., 1998), accumulated in the thh1-1 single-mutant plants to a level similar to that found in wild-type plants (Fig. 2a). Because the tom3-1 single mutation (Yamanaka et al., 2002) (Fig. 1) does not affect TMV-Cg CP accumulation, and the tom1-2 single mutation (Yamanaka et al., 2000) (Fig. 1) reduces the level of its accumulation (Fig. 2a), the contribution of TOM1 to TMV-Cg multiplication is larger than that of THH1 or TOM3.

View larger version (29K): [in this window] [in a new window]   Fig. 1. Organization of TOM1, TOM3 and THH1. (a) Intron–exon organization. Exons are shown in boxes. Open and filled boxes indicate non-coding and coding regions, respectively. The positions of the tom1-1, tom1-2, tom1-3 and tom3-1 mutations (Yamanaka et al., 2000, 2002) and the T-DNA insertion in the thh1-1 mutant (GABI-Kat 046D04) are shown. The mutant alleles used in this study are indicated by bold type. (b) Alignment of amino acid sequences of TOM1, TOM3 and THH1. The deduced amino acid sequences of TOM1, TOM3 and THH1 were aligned by using the CLUSTAL W program (Thompson et al.,1994). Identical residues are indicated by asterisks. The amino acid residues corresponding to the mutation sites (tom1-1, a point mutation at the splicing-acceptor site immediately upstream of the I157 codon; tom1-2, W68 to stop; tom1-3, one-base insertion atV148; tom3-1, W45 to stop; thh1-1, T-DNA insertion at the N151 codon) are shown on a black background.

View larger version (18K): [in this window] [in a new window]   Fig. 2. Accumulation of the TMV-Cg, CMV and TCV CPs in the tom1-2, tom3-1 and thh1-1 single, double and triple mutants. Rosette leaves of 3- to 4-week-old plants were inoculated with TMV-Cg (a), CMV (b) or TCV (c) virion RNAs (0.1 µg µl–1 each) and inoculated leaves were harvested at 2 and 5 d.p.i. Total proteins were extracted and subjected to SDS-PAGE. Protein samples from 0.25, 0.025 and 0.05 mg (fresh weight) leaf tissue inoculated with TMV-Cg, CMV and TCV, respectively, were loaded per lane. CPs were detected by using specific rabbit antisera (Yoshii et al., 2004) and alkaline phosphatase-conjugated goat anti-rabbit IgG secondary antibody, followed by a colour reaction with 5-bromo-4-chloro-3-indolyl phosphate in combination with nitro blue tetrazolium. The levels of accumulation of the CPs were quantified densitometrically by using the Image J program (http://rsb.info.nih.gov/ij/). The graphs show means+SD of relative CP accumulation in four to 12 plants (mean CP accumulation in wild-type Col-0 at 5 d.p.i.=100 % for each virus).

The multiplication of TMV-Cg in the double and triple mutants was examined. At 2 days post-inoculation (d.p.i.), no infection of any plants with the tom1-2 mutation was detected, whereas the tom3-1 and thh1-1 single mutations allowed TMV-Cg to multiply to wild-type levels. In tom3-1 thh1-1 double-mutant plants, TMV-Cg CP accumulation was detectable, but lower than that in tom3-1 or thh1-1 single-mutant or wild-type plants (Fig. 2a; P<0.01, Student's t-test). Even at 5 d.p.i., TMV-Cg CP accumulation was not detected at all in the tom1-2 tom3-1 double mutant or the triple mutant (Fig. 2a). By contrast, the tom1-2 thh1-1 double mutation allowed TMV-Cg CP to accumulate to a detectable, but lower, level than that in tom1-2 single-mutant plants at 5 d.p.i. (Fig. 2a; P<0.01, Student's t-test). These results suggest that THH1 functions to support TMV-Cg multiplication in A. thaliana, but with an activity lower than that of the TOM3 gene. In the tom1-2 tom3-1 double mutant, in which THH1 is the only gene of the three to be functional, non-specific degradation of tobamovirus RNA and/or host defences would overcome the replication, resulting in no detectable amplification of tobamoviruses. On the other hand, in the wild-type genetic background, loss of TOM3 or THH1 alone does not affect tobamovirus multiplication, probably because another factor, but not the TOM1 plus THH1 or TOM3 function, limits overall efficiency of tobamovirus multiplication.

To confirm the involvement of THH1 in TMV-Cg multiplication more directly, the tom1-2 tom3-1 double mutant was transformed by using a gene cassette in which the THH1 coding sequence was placed under the control of the cauliflower mosaic virus 35S RNA promoter. Semi-quantitative RT-PCR showed that the THH1 mRNA was overexpressed in T2 plants that carried the T-DNA insert, whilst the expression level of the THH1 mRNA in tom1 tom3 double-mutant plants that did not carry the transgene was similar to that in wild-type Col-0 plants (Fig. 3). When plants were inoculated with TMV-Cg, the level of CP accumulation in the tom1 tom3 double-mutant plants that overexpressed THH1 mRNA was similar to that in wild-type Col-0 plants (Fig. 3). Consistent with earlier results, TMV-Cg CP accumulation was not detected in the tom1 tom3 double-mutant plants that did not carry the transgene (Fig. 3). This result indicates that THH1 functions to support TMV-Cg multiplication in a manner parallel to the actions of TOM1 and TOM3. Because TOM1 and TOM3 are implicated in tobamovirus RNA replication, reduction of TMV-Cg multiplication by the tom1, tom3 and/or thh1 mutations observed in inoculated plant leaves is most likely to be caused by the inhibition of replication, rather than other processes of infection.

View larger version (57K): [in this window] [in a new window]   Fig. 3. Multiplication of TMV-Cg in transgenic plants overexpressing the THH1 mRNA. A cDNA fragment covering the THH1 coding region was amplified by PCR from an A. thaliana cDNA pool synthesized by using a SMART PCR cDNA synthesis kit (Clontech) with the primers 5'-TGCGAATTCGTGGAGATGAGAAGCGGCGGC-3' and 5'-CGAGGATCCATGTTCATTGGATTTGATGGTACTGTGT-3'. Amplified THH1 cDNA was digested with EcoRI and BamHI and cloned between the EcoRI and BamHI sites of pGEM7Zf(+) (Promega) to obtain plasmid #850. Plasmid #850 was digested with XbaI and SacI and the resulting fragment containing the THH1 cDNA was inserted between the XbaI and SacI sites of pBI121 to create the T-DNA clone pBI-THH1, in which the THH1 cDNA was placed under the control of the cauliflower mosaic virus 35S RNA promoter. The tom1 tom3 double mutant was transformed with pBI-THH1 by using the Agrobacterium-mediated floral-dip method (Clough & Bent, 1998). Five T2 plants derived from asingle T1 plant (a kanamycin-resistant progeny of Agrobacterium-treated plants) were inoculated with TMV-Cg virion RNA (0.1 µg µl–1) and inoculated leaves were harvested at 5 d.p.i. CP accumulation was detected as described in Fig. 2. Two TMV-Cg-inoculated Col-0 plants, two TMV-Cg-inoculated tom1 tom3 double-mutant plants and one mock-inoculated Col-0 (M) plant were analysed alongside the five T2 plants. The T-DNA was confirmed to be present in plants by using PCR with a primer set specific for the 35S RNA promoter (5'-CAAAGCAAGTGGATTGATGTGATATC-3') and the NOS terminator (5'-CATGTTAATTATTACATGCTTAACGTAATTC-3'). Semi-quantitative RT-PCR was also performed by using a THH1 mRNA-specific primer set (5'-TTCCGTCATTGCTGTGATTC-3' and 5'-ATACAGCACGTGCCTGG-3') or an Actin1 mRNA-specific primer set (5'-CATCAGGAAGGACTTGTACGG-3' and 5'-GATGGACCTGACTCGTCATAC-3'). The resulting PCR products were separated by agarose-gel electrophoresis and visualized by ethidium bromide staining (three lower panels). Similar results were obtained for three other tom1 tom3 double-mutant lines transformed with pBI-THH1.

Cucumber mosaic virus (CMV) and Turnip crinkle virus (TCV) belong to taxonomic groups that are distinct from the genus Tobamovirus. The tom1 mutation did not influence the multiplication of either CMV or TCV (Ishikawa et al., 1991). The CMV and TCV CPs accumulated to wild-type levels in all of the single, double and triple mutants [difference not significant (P>0.01, Student's t-test); Fig. 2b, c], suggesting that the effect of these mutations is specific to the TMV-Cg multiplication. We previously found that co-infection of CMV with TMV-Cg affects TMV-Cg multiplication in tom1 protoplasts, but not in wild-type protoplasts, and proposed that CMV competes with TMV-Cg for the utilization of TOM1 homologues (Ishikawa et al., 1993). However, our present results do not suggest that CMV utilizes TOM3 or THH1 as host factors for multiplication.

In this study, we found that the tom1-2 tom3-1 thh1-1 triple mutant grows normally under our growth conditions (Fujisaki et al., 2004), even though each mutation is likely to inhibit the synthesis of functional protein product severely. Because TOM1 homologues are widely present not only in dicot species, but also in the monocot species rice (Asano et al., 2005), TOM1 and related genes must play an important role in some aspect of plant life cycles. The functioning of these genes might be necessary for plants to survive under more extreme wild conditions.

	ACKNOWLEDGEMENTS

 
We thank the Max Planck Institute for Plant Breeding Research for materials, Tetsuo Meshi and Yuko Ohashi for helpful discussions and Rena Satoh, Soko Kobayashi and Kumi Fujiwara for technical and general assistance. This work was supported in part by a Grant-in-Aid from the Ministry of Agriculture, Forestry and Fisheries of Japan and by the Core Research for Evolutional Science and Technology of the Japan Science and Technology Agency. K. F. is supported by the Japan Society for the Promotion of Science. G. B. R. is supported by a Japanese Government Scholarship.

	REFERENCES

TOP ABSTRACT MAIN TEXT REFERENCES

 
Ahlquist, P., Noueiry, A. O., Lee, W.-M., Kushner, D. B. & Dye, B. T. (2003). Host factors in positive-strand RNA virus genome replication. J Virol 77, 8181–8186.[Free Full Text]

Asano, M., Satoh, R., Mochizuki, A. & 7 other authors (2005). Tobamovirus-resistant tobacco generated by RNA interference directed against host genes. FEBS Lett 579, 4479–4484.[CrossRef][Medline]

Buck, K. W. (1999). Replication of tobacco mosaic virus RNA. Philos Trans R Soc Lond B Biol Sci 354, 613–627.[Abstract/Free Full Text]

Clough, S. J. & Bent, A. F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16, 735–743.[CrossRef][Medline]

Fujisaki, K., Hagihara, F., Azukawa, Y., Kaido, M., Okuno, T. & Mise, K. (2004). Identification and characterization of the SSB1 locus involved in symptom development by Spring beauty latent virus infection in Arabidopsis thaliana. Mol Plant Microbe Interact 17, 967–975.[Medline]

Hagiwara, Y., Komoda, K., Yamanaka, T., Tamai, A., Meshi, T., Funada, R., Tsuchiya, T., Naito, S. & Ishikawa, M. (2003). Subcellular localization of host and viral proteins associated with tobamovirus RNA replication. EMBO J 22, 344–353.[CrossRef][Medline]

Ishikawa, M., Obata, F., Kumagai, T. & Ohno, T. (1991). Isolation of mutants of Arabidopsis thaliana in which accumulation of tobacco mosaic virus coat protein is reduced to low levels. Mol Gen Genet 230, 33–38.[CrossRef][Medline]

Ishikawa, M., Naito, S. & Ohno, T. (1993). Effects of the tom1 mutation of Arabidopsis thaliana on the multiplication of tobacco mosaic virus RNA in protoplasts. J Virol 67, 5328–5338.[Abstract/Free Full Text]

Neff, M. M., Neff, J. D., Chory, J. & Pepper, A. E. (1998). dCAPS, a simple technique for the genetic analysis of single nucleotide polymorphisms: experimental applications in Arabidopsis thaliana genetics. Plant J 14, 387–392.[CrossRef][Medline]

Noueiry, A. O. & Ahlquist, P. (2003). Brome mosaic virus RNA replication: revealing the role of the host in RNA virus replication. Annu Rev Phytopathol 41, 77–98.[CrossRef][Medline]

Rosso, M. G., Li, Y., Strizhov, N., Reiss, B., Dekker, K. & Weisshaar, B. (2003). An Arabidopsis thaliana T-DNA mutagenized population (GABI-Kat) for flanking sequence tag-based reverse genetics. Plant Mol Biol 53, 247–259.[CrossRef][Medline]

Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.[Abstract/Free Full Text]

Tsujimoto, Y., Numaga, T., Ohshima, K., Yano, M.-A., Ohsawa, R., Goto, D. B., Naito, S. & Ishikawa, M. (2003). Arabidopsis TOBAMOVIRUS MULTIPLICATION (TOM) 2 locus encodes a transmembrane protein that interacts with TOM1. EMBO J 22, 335–343.[CrossRef][Medline]

Yamanaka, T., Komatani, H., Meshi, T., Naito, S., Ishikawa, M. & Ohno, T. (1998). Complete nucleotide sequence of the genomic RNA of tobacco mosaic virus strain Cg. Virus Genes 16, 173–176.[CrossRef][Medline]

Yamanaka, T., Ohta, T., Takahashi, M., Meshi, T., Schmidt, R., Dean, C., Naito, S. & Ishikawa, M. (2000). TOM1, an Arabidopsis gene required for efficient multiplication of a tobamovirus, encodes a putative transmembrane protein. Proc Natl Acad Sci U S A 97, 10107–10112.[Abstract/Free Full Text]

Yamanaka, T., Imai, T., Satoh, R. & 7 other authors (2002). Complete inhibition of tobamovirus multiplication by simultaneous mutations in two homologous host genes. J Virol 76, 2491–2497.[Abstract/Free Full Text]

Yoshii, M., Nishikiori, M., Tomita, K., Yoshioka, N., Kozuka, R., Naito, S. & Ishikawa, M. (2004). The Arabidopsis cucumovirus multiplication 1 and 2 loci encode translation initiation factors 4E and 4G. J Virol 78, 6102–6111.[Abstract/Free Full Text]

Received 13 February 2006; accepted 19 April 2006.

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