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Analysis of tobamovirus multiplication in Arabidopsis thaliana mutants defective in TOM2A homologues

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

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

	ABSTRACT

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The TOM2A gene of Arabidopsis thaliana encodes a four-pass transmembrane protein that is required for efficient multiplication of a tobamovirus, TMV-Cg. In this study, the involvement of three TOM2A homologues in tobamovirus multiplication in A. thaliana was examined. T-DNA insertion mutations in the three homologues, separately or in combination, did not affect TMV-Cg multiplication, whereas, in the tom2a genetic background, some combinations reduced it. This result suggests that the TOM2A homologues are functional in enhancing TMV-Cg multiplication, but their contribution is much less than TOM2A. Interestingly, the multiplication of another tobamovirus, Tomato mosaic virus, was not drastically affected by any combinations of the mutations in TOM2A and its homologues as far as we examined.

	MAIN TEXT

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The genus Tobamovirus includes Tobacco mosaic virus, Tomato mosaic virus (ToMV) and Youcai mosaic virus (this virus is identical to TMV-Cg and, in this report, referred to as TMV-Cg for consistency with our previous publications), as well as many other related viruses (Gibbs, 1999). TMV-Cg is closely related to other crucifer-infecting tobamoviruses and multiplies to higher levels in Arabidopsis thaliana than ToMV (Li et al., 1983; Lartey et al., 1996; Yamanaka et al., 1998). The genome of a tobamovirus is a non-segmented positive-strand RNA and encodes 130 and 180 kDa proteins that participate in RNA replication and other functions, a 30 kDa protein that is required for virus cell-to-cell movement and the coat protein (CP) (reviewed by Ishikawa & Okada, 2004).

Virus multiplication depends not only on virus-coded factors, but also on host-derived factors (reviewed by Buck, 1999; Ahlquist et al., 2003). Quantitative and qualitative changes in these host factors cause a wide range of effects on virus multiplication. Previously, a mutant of A. thaliana, YS241 (Ohshima et al., 1998), was isolated and plant lines B1-113 and B1-234 were obtained by back-crossing YS241 plants to the wild-type plants (accession Col-0). The B1-113 line carries defects in both TOM2A, which encodes a four-pass transmembrane protein (Fig. 1a), and TOM2B, which encodes a small basic protein. In B1-113 plants and protoplasts, multiplication of both TMV-Cg and ToMV was significantly reduced (Tsujimoto et al., 2003). It was further demonstrated that (i) the TOM2A protein is co-purified with the 130 and 180 kDa replication proteins from solubilized membranes from ToMV-infected protoplasts (Nishikiori et al., 2006); (ii) TOM2A localizes mainly on vacuolar membranes, with which the 130 and 180 kDa replication proteins and the activity to synthesize tobamovirus-related RNAs are partly associated (Hagiwara et al., 2003); and (iii) TOM2A interacts in yeast with TOM1 (Tsujimoto et al., 2003), another transmembrane protein required for tobamovirus multiplication that interacts with the helicase-like domain polypeptide of the 130 and 180 kDa replication proteins (Yamanaka et al., 2000). Based on these observations, it was proposed that, acting with TOM1, TOM2A plays important roles in the formation and/or maintenance of TMV-Cg replication complexes on membranes. The function of TOM2B has not yet been determined.

View larger version (49K): [in this window] [in a new window]   Fig. 1. TOM2A and its homologues in A. thaliana and the mutants carrying T-DNA insertions in these genes. (a) Alignment of the deduced amino acid sequences of the TOM2A family members. The deduced amino acid sequences of the TOM2A, TOM2AH1, TOM2AH2 and TOM2AH3 gene products were obtained from The Arabidopsis Information Resource (http://www.arabidopsis.org/) and aligned using the program CLUSTAL W (Thompson et al., 1994). Identical amino acid residues among all four TOM2A family member proteins and those among two or three of the proteins are shown with black and grey backgrounds, respectively. Transmembrane regions (numbered I–IV) predicted by the program SOSUI (Hirokawa et al., 1998) are indicated by boxes. (b) The positions of T-DNA insertions in the mutants used in this study. The coding and non-coding regions of the exons are indicated by filled and open boxes, respectively. Introns are shown by lines. The positions of T-DNA insertions in TOM2A (tom2a-1; GABI-Kat 505G01), TOM2AH1 (tom2ah1-1; SALK_034692), TOM2AH2 (tom2ah2-1; SALK_022303) and TOM2AH3 (tom2ah3-1; GABI-Kat 343E12) are also indicated. In this study, the tom2a-1, tom2ah1-1, tom2ah2-1 and tom2ah3-1 mutations were designated 2a, h1, h2 and h3, respectively. The presence and absence of the T-DNA insertions in the TOM2A, TOM2AH1, TOM2AH2 and TOM2AH3 loci were checked by PCR using three primers: two specific for the TOM2A, TOM2AH1, TOM2AH2 and TOM2AH3 genomic sequences (5'-TTACATTCGATTTGACTAATGGAG-3' and 5'-ATCATCAACCCAGAAACAAGAATC-3' for TOM2A; 5'-CGGGAGGACATGGACTCATAGC-3' and 5'-TTTGCAAATGGGTCGGAATTG-3' for TOM2AH1; 5'-AAACACATGCAGCCAAAAACA-3' and 5'-TCCAGTTGGATCATACGGAAG-3' for TOM2AH2; and 5'-ACACAAGAAAGAATTCAGGAAGGA-3' and 5'-GAGAAGACATCTGGAGCAACAGGA-3' for TOM2AH3) and one specific for the left border of the T-DNA (5'-CCCATTTGGACGTGAATGTAGACAC-3' for TOM2A and TOM2AH3; and 5'-CGTGGACCGCTTGCTGCAACT-3' for TOM2AH1 and TOM2AH2). Plants homozygously carrying the T-DNA insertions were selected and used for the experiments. (c) mRNA levels of TOM2A and its homologues in the 2a, h1, h2 and h3 single mutant plants and wild-type Col-0 plants (wt). Semi-quantitative RT-PCR was performed by using primer sets: 5'-ATGGCTTGTAGAGGTTGTTTGGA-3' and 5'-TCACATGATGGTGCATCGGCCCT-3' for TOM2A (25 cycles); 5'-ATGCGACACAATTGTTGCCATGTC-3' and 5'-TTAAGCTGATGGAGTGTGGCTCTG-3' for TOM2AH1 (25 cycles); 5'-ATGCGGCGTAATTGCTGCCATGTT-3' and 5'-TCAACCTTTCGGGTTCACCGGTG-3' for TOM2AH2 (25 cycles); 5'-ATGGTGAGGATCGTGAGAAGTTG-3' and 5'-TCACCTCTCACCGGTTTGCCCTTTC-3' for TOM2AH3 (25 cycles); and 5'-CATCAGGAAGGACTTGTACGG-3' and 5'-GATGGACCTGACTCGTCATAC-3' for Actin1 (25 cycles). (d) Effects of triple and quadruple mutations on plant growth. Plants were grown at 23 °C with 16 h illumination per day (Fujisaki et al., 2004) and photographed 5 weeks after sowing. Note that all the single and double mutants grew normally.

View larger version (47K): [in this window] [in a new window]   Fig. 2. Effects of the single and multiple mutations in TOM2A family members on virus multiplication. (a) Accumulation of TMV-Cg (upper panel) or ToMV (lower panel) CPs in plants carrying single mutations in either the TOM2A, TOM2AH1, TOM2AH2 or TOM2AH3 genes. Virus inoculation was performed as described previously (Fujisaki et al., 2006). Inoculated leaves were harvested 14 days after infection and CPs were detected by SDS-PAGE and Coomassie brilliant blue staining. As a control, B1-234 mutant and wild-type (wt) plants were inoculated and analysed in parallel. A sample from mock-inoculated leaves of wt plants was also run in the left-most lanes (M). Asterisks show plant-derived protein bands that serve as loading controls. (b) Accumulation of TMV-Cg, ToMV or CMV CPs in wt, the 2a single, and 2a/h1/h3, 2a/h2/h3 and h1/h2/h3 triple mutant plants. Inoculated leaves were harvested 14 days after infection (TMV-Cg and ToMV) or 4 days after infection (CMV) and the accumulation of the CPs was examined as described for (a). Asterisks show plant-derived protein bands that serve as loading controls.

To determine the contribution of the TOM2A homologues to tobamovirus multiplication, A. thaliana mutants carrying T-DNA insertions in the TOM2A homologues [tom2ah1-1 (hereafter, ‘h1’; SALK_034692), tom2ah2-1 (‘h2’; SALK_022303) and tom2ah3-1 (‘h3’; GABI-Kat 343E12); Fig. 1b] were utilized. Plant lines that are homozygous for each mutation were established and the specific reduction in accumulation of corresponding mRNAs to undetectable levels was confirmed (Fig. 1c). Plant lines carrying multiple mutations in 2a, h1, h2 and/or h3 were also generated. Among all possible combinations of the mutations, the 2a/h1/h2 triple and the quadruple (2a/h1/h2/h3) mutations led to growth defects, whereas the other single, double and triple mutations had no apparent effect on plant growth (Fig. 1d; data not shown). Seeds of the 2a/h1/h2 triple mutant germinated normally, but the plants started to show growth defects around 3–5 weeks after sowing. The quadruple mutant started to show similar, but more severe, growth defects around 2–3 weeks after sowing (Fig. 1d) and did not produce seeds (data not shown) under the growth conditions described by Fujisaki et al. (2004). These observations suggest that TOM2A and its three homologues play a parallel and essential role in the plant life cycle and that the contribution of TOM2AH3 to plant growth is the least among the TOM2A family members.

To investigate the contribution of the three TOM2A homologues to tobamovirus multiplication, plants carrying h1, h2 and/or h3 mutations were established. In these plants, including the h1/h2/h3 triple mutant, TMV-Cg CP accumulated to wild-type levels (Fig. 2a, b; data not shown). To more sensitively detect the contribution of the TOM2A homologues to TMV-Cg multiplication, TMV-Cg CP accumulation in the normally growing triple mutants 2a/h1/h3 and 2a/h2/h3 was compared with that in the 2a single mutant (Fig. 2b). Quantification of the CP bands in Fig. 2(b) by the Image J program (http://rsb.info.nih.gov/ij/) suggested that accumulation levels of TMV-Cg CP in the 2a/h1/h3 and 2a/h2/h3 triple mutant leaves were 60 and 40 %, respectively, of that in the 2a single mutant leaves 14 days after infection [the difference between 2a/h1/h3 or 2a/h2/h3 and 2a was significant according to the Student's t-test (P<0.05)].

It has been demonstrated that the tom2a mutation inhibits TMV-Cg multiplication in protoplasts (Tsujimoto et al., 2003). TMV-Cg multiplication in 2a/h1/h3, 2a/h2/h3 and h1/h2/h3 triple and 2a single mutant protoplasts was then examined. Consistent with our previous report (Tsujimoto et al., 2003), the 2a single mutation led to low level accumulation of TMV-Cg CP and additional h1/h3 and h2/h3 mutations in the 2a background further reduced CP accumulation levels (Fig. 3a). Quantification of the CP bands in Fig. 3(a) showed that the accumulation levels of TMV-Cg CP in 2a/h1/h3 and 2a/h2/h3 triple mutant protoplasts were 6 and 15 %, respectively, of that in 2a single mutant protoplasts [difference between 2a/h1/h3 or 2a/h2/h3 and 2a was significant according to the Student's t-test (P<0.05)]. The reduced accumulation of TMV-Cg CP in the triple mutant protoplasts was associated with parallel decreases in the accumulation of genomic and CP subgenomic RNAs of TMV-Cg (Fig. 3b). In h1/h2/h3 triple mutant protoplasts, TMV-Cg-related molecules accumulated as in wild-type protoplasts. These results indicate that the three TOM2A homologues contribute to TMV-Cg multiplication, but their contribution is much less than that of TOM2A.

View larger version (48K): [in this window] [in a new window]   Fig. 3. Effects of mutations in TOM2A family members on virus multiplication in protoplasts. Arabidopsis protoplasts (0.5–1.0x106) of the indicated genotypes were prepared from fully expanded leaves and inoculated with TMV-Cg, ToMV or CMV virion RNAs (3 µg each), according to the protocol obtained from J. Sheen's laboratory (http://genetics.mgh.harvard.edu/sheenweb/main_page.html). Consistent results were obtained in three independent experiments and a typical dataset is indicated here. (a) CP accumulation. Inoculated protoplasts were harvested at 20 h post-inoculation (h p.i.). Viral CPs were detected by the immunoblotting method (upper panels). Control samples derived from mock-inoculated wt protoplasts were run in the left-most lanes (M). Bands of ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (RuBisCOL) detected by Coomassie brilliant blue staining are shown as loading controls (lower panels). (b) Accumulation of virus-related RNAs. Protoplasts were harvested at 2 and 20 h p.i. and the accumulation of viral RNAs was examined by Northern blotting and hybridization (upper panels). ToMV RNAs were detected as described by Kubota et al. (2003) except that the NcoI–MluI fragment of ToMV cDNA was used as a probe. TMV-Cg and CMV RNAs were detected as described by Yoshii et al. (1998). Positions of tobamovirus genomic RNA (G) and CP subgenomic (sg) RNA are indicated on the left and those of CMV genomic RNAs 1, 2 and 3, and sg RNA4 are indicated on the right. rRNA bands detected by ethidium bromide staining are shown as loading controls (lower panels).

Cucumber mosaic virus (CMV), which belongs to a different taxonomic group than tobamoviruses and which is known to be insensitive to the tom2a mutation (Fig. 2b; Ohshima et al., 1998), also multiplied to wild-type levels in 2a/h1/h3, 2a/h2/h3 and h1/h2/h3 triple mutant plants and protoplasts (Figs 2b, 3a, b), indicating that the observed restriction of TMV-Cg multiplication is due to a specific effect.

In this study, it has been shown that at least some of the TOM2A homologues function to support TMV-Cg multiplication, but their contribution is much less than that of TOM2A. With regard to the respective contribution of each TOM2A homologue, a slight decrease in TMV-Cg CP accumulation was observed at an early time point of infection (4 days after inoculation) in 2a/h1 and 2a/h2 double mutants, but not in the 2a/h3 mutant, compared with that in 2a single mutant plants (data not shown). Therefore, the contribution to TMV-Cg multiplication is likely to be TOM2A>>TOM2AH1, TOM2AH2>TOM2AH3. This conclusion highlights the strong dependence of TMV-Cg multiplication on TOM2A.

For ToMV multiplication, 2a/h1/h3, 2a/h2/h3 and h1/h2/h3 triple mutations did not show any drastic effect. However, because the expression of TOM2A in the tom2a tom2b double mutant increases ToMV multiplication (Tsujimoto et al., 2003), TOM2A has some function to support ToMV multiplication at least in the tom2b genetic background. It is possible that TOM2A and its homologues (at least TOM2AH1 and TOM2AH2) play a parallel role in ToMV multiplication and the presence of one of these genes is enough for ToMV to accumulate to wild-type levels.

It is also possible that, in the wild-type TOM2B background, none of the TOM2A family members participates in ToMV multiplication. Even if this is the case, TOM2A should be positioned in close proximity to ToMV replication proteins, because TOM1 (and its homologues), which is absolutely required for the multiplication of several tobamovirus species including ToMV (Yamanaka et al., 2002; Asano et al., 2005; Fujisaki et al., 2006), interacts with both TOM2A and the replication proteins of tobamoviruses (Yamanaka et al., 2002; Tsujimoto et al., 2003). If crucifer-infecting tobamoviruses have evolved from a ToMV-like prototype virus, it seems plausible that the tobamovirus prototype gained the ability to specifically utilize TOM2A during this adaptation to cruciferous plants and achieved highly efficient multiplication in them.

	ACKNOWLEDGEMENTS

 
We thank SALK Institute Genomic Analysis Laboratory, Max Planck Institute for Plant Breeding Research and Arabidopsis Biological Resource Center for A. thaliana seeds and Tetsuo Meshi, Yuko Ohashi and the members of our laboratory for helpful discussions. This work was supported in part by the Core Research for Evolutional Science and Technology of the Japan Science and Technology Agency and by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN). K. F. is supported by the Japan Society for the Promotion of Science.

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Received 18 January 2008; accepted 20 February 2008.

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