Masamichi Nishiguchi

The complete nucleotide sequence of cucumber green mottle mosaic virus (SH strain) genomic RNA.

The complete nucleotide sequence of the genomic RNA of cucumber green mottle mosaic virus watermelon strain SH (CGMMV-SH) was determined using cloned cDNA. This sequence is 6421 nucleotides long containing at least four open reading frames, which correspond to 186K, 129K, 29K and 17.3K proteins. The 17.3K protein is the coat protein. Sequence analysis shows that CGMMV-SH is very closely related to another watermelon strain. CGMMV-W, although three amino acid substitutions in the 29K protein were found between these strains. The sequence was also compared to those of other tobamoviruses, tobacco mosaic virus (TMV) vulgare, TMV-L (a tomato strain) and tobacco mild green mosaic virus reported by other groups. It shows 55 to 56% identity with these viruses. The size and location of the open reading frames are very similar to those of TMV but the 129K and 186K proteins are composed of 1142 and 1646 amino acids, being larger than those of TMV by 27 and 31 amino acids, respectively. The deduced amino acid sequences of these proteins are highly homologous to those of TMV, especially in the readthrough downstream region of the 186K protein.

Single amino acid substitution in 30K protein of TMV defective in virus transport function

Involvement of the tobacco mosaic virus (TMV) coded 30K protein in a virus transport function within the infected plant has been suggested. Previously a temperature sensitive mutant, TMV Ls 1, that is defective in cell-to-cell movement at a restrictive temperature, was reported. To demonstrate a relationship between the 30K protein and the transport function, the nucleotide sequences of the 30K and coat protein cistrons of the mutant, TMV Ls 1, and the wild type, TMV L (tomato strain) were compared. A single base substitution which causes replacement of a proline codon in the L strain by a serine codon was found in the Ls 1 mutant. Results support the notion that the 30K protein is responsible for the virus transport function.

Behaviour of a Temperature Sensitive Strain of Tobacco Mosaic Virus in Tomato Leaves and Protoplasts

Summary A temperature-sensitive (ts) strain, Ls1, was isolated from a culture of L, a tomato strain of tobacco mosaic virus (TMV). Ls1 caused smaller necrotic local lesions than L in leaves of hypersensitive tobacco plants. A temperature shift treatment (22 °C for 3 days, 32 °C for 2 days and 22 °C for 1 day) allowed L to produce necrotic lesions surrounded by a collapsed area, whereas Ls1 caused necrotic lesions without a collapsed surrounding area, suggesting that Ls1 did not spread outside the lesions at 32 °C. In tomato leaf discs at 22 °C, infectivity of Ls1 increased in parallel with that of L, but at 32 °C the increase in infectivity of Ls1 was negligible and contrasted with the large increase of infectivity of L. Few mesophyll cells from Ls1-inoculated discs incubated at 32 °C were stained by fluorescent antibody but many of those incubated at 22 °C were stained. In protoplasts at 32 °C, however, Ls1 and L infected and multiplied similarly. When leaves inoculated with Ls1 were kept at 20 to 25 °C for 24 h, and discs were then prepared and cultured for 12 h at 32 or 22 °C, the proportion of mesophyll cells containing virus antigen did not increase at 32 °C but infectivity increased greatly. This suggests that Ls1 multiplied readily at 32 °C in already infected cells. At 22 °C the proportion of infected cells and the infectivity of leaf extracts increased rapidly. The growth curve of Ls1 in leaf discs at 32 °C resembled the so-called ‘one-step growth curve’ of Ls1 or L in protoplasts, and not the growth curve of L in discs, in which the virus could spread from cell to cell. These results suggest that Ls1 is a ts strain that multiplies normally at the non-permissive temperature but has a malfunction in cell-to-cell movement.

Complete nucleotide sequence and genome organization of sweet potato feathery mottle virus (S strain) genomic RNA:the large coding region of the P1 gene

SummaryThe complete nucleotide sequence of a sweet potato feathery mottle virus severe strain (SPFMV-S) genomic RNA was determined from overlapping cDNA clones and by directly sequencing viral RNA. The viral RNA genome is 10 820 nucleotides long, excluding the poly(A) tail and contains one open reading frame (ORF) starting at nucleotide 118 and ending at 10 599, potentially encoding a polyprotein of 3 493 amino acids (Mr 393 800). The ORF was followed by a 3′ untranslated region of 221 nucleotides. The deduced polyprotein includes P1 (74K), HC-Pro (52K), P3 (46K), 6K1, CI (72K), 6K2, NIa-VPg (22K), NIa-Pro (28K), NIb (60K) and coat (35K) proteins, after an analysis of protein cleavage sites analogous to other potyvirus polyproteins. The polyprotein had a high level of amino acid identity with those of other potyviruses, except in the regions of P1 and P3. The P1 of SPFMV-S RNA has 664 amino acid residues, and is the largest and least similar to those of other potyviruses. HC-Pro and CI show high identity with those of other potyviruses. P3 has relatively low identity, however, the length of P3 was within the range of variability among other potyviruses. The 6K1 protein between P3 and C1 is also highly similar to those of other potyviruses. This is the first report on the complete nucleotide sequence of the sweet potato-infecting virus.

Tobamovirus-resistant tobacco generated by RNA interference directed against host genes

Two homologous Nicotiana tabacum genes NtTOM1 and NtTOM3 have been identified. These genes encode polypeptides with amino acid sequence similarity to Arabidopsis thaliana TOM1 and TOM3, which function in parallel to support tobamovirus multiplication. Simultaneous RNA interference against NtTOM1 and NtTOM3 in N. tabacum resulted in nearly complete inhibition of the multiplication of Tomato mosaic virus and other tobamoviruses, but did not affect plant growth or the ability of Cucumber mosaic virus to multiply. As TOM1 and TOM3 homologues are present in a variety of plant species, their inhibition via RNA interference should constitute a useful method for generating tobamovirus‐resistant plants.

The genome structure of kyuri green mottle mosaic tobamovirus and its comparison with that of cucumber green mottle mosaic tobamovirus

Summary. The genome of the Y strain of kyuri green mottle mosaic virus (KGMMV-Y) has been completely sequenced. Its genomic structure and sequence show it to be a typical tobamovirus, that is closest to, but distinct from, that of cucumber green mottle mosaic tobamovirus (CGMMV). The genomic sequence of KGMMV-Y was compared in detail with that of the SH strain of CGMMV. The sequences of their 5′- and 3′-untranslated regions were 74% and 63% identical. The amino acid sequences of the shorter and longer (read through) RNA replicase components, movement protein (MP) and coat protein (CP) were 58, 58. 60 and 46% identical, respectively. The KGMMV-Y genome sequence was also compared partly to that of another strain of KGMMV, KGMMV-C. The CP sequences of KGMMV-Y and KGMMV-C differed by 20 amino acid residues, suggesting that their relationship is more distant than the relationship between CGMMV-SH and CGMMV-W whose CP sequences are identical. The MPs of KGMMV-Y and KGMMV-C, however, differ only by one amino acid residue, although three amino acid substitutions are present in the MPs between CGMMV-SH and CGMMV-W. Two long stretches, one in the RNA replicase and the other in the MP, were highly conserved in KGMMV and CGMMV.

Further investigation of a temperature-sensitive strain of tobacco mosaic virus: its behaviour in tomato leaf epidermis.

Summary A temperature-sensitive (ts) strain of tobacco mosaic virus (TMV), Ls1, was further investigated with regard to its behaviour at permissive (22 °C) and nonpermissive (32 °C) temperatures at the primary infection sites in tomato leaf epidermis. Tomato leaflets were inoculated with Ls1 or a wild-type strain, L, cultured at 22 or 32 °C and the epidermis isolated; virus spread was followed by staining with fluorescent antibody after enzymic digestion of the upper cell wall. Profiles of epidermis stained with fluorescent antibody indicated that Ls1 multiplies in primary-infected cells but that it cannot move to the neighbouring cells at the nonpermissive temperature.

Overexpression of a host factor TOM1 inhibits tomato mosaic virus propagation and suppression of RNA silencing.

A plant integral membrane protein TOM1 is involved in the multiplication of Tomato mosaic virus (ToMV). TOM1 interacts with ToMV replication proteins and has been suggested to tether the replication proteins to the membranes where the viral RNA synthesis takes place. We have previously demonstrated that inactivation of TOM1 results in reduced ToMV multiplication. In the present study, we show that overexpression of TOM1 in tobacco also inhibits ToMV propagation. TOM1 overexpression led to a decreased accumulation of the soluble form of the replication proteins and interfered with the ability of the replication protein to suppress RNA silencing. The reduced accumulation of the soluble replication proteins was also observed in a silencing suppressor-defective ToMV mutant. Based on these results, we propose that RNA silencing suppression is executed by the soluble form of the replication proteins and that efficient ToMV multiplication requires balanced accumulation of the soluble and membrane-bound replication proteins.

Overexpression of a rice heme activator protein gene (OsHAP2E) confers resistance to pathogens, salinity and drought, and increases photosynthesis and tiller number

Heme activator protein (HAP), also known as nuclear factor Y or CCAAT binding factor (HAP/NF-Y/CBF), has important functions in regulating plant growth, development and stress responses. The expression of rice HAP gene (OsHAP2E) was induced by probenazole (PBZ), a chemical inducer of disease resistance. To characterize the gene, the chimeric gene (OsHAP2E::GUS) engineered to carry the structural gene encoding β-glucuronidase (GUS) driven by the promoter from OsHAP2E was introduced into rice. The transgenic lines of OsHAP2Ein::GUS with the intron showed high GUS activity in the wounds and surrounding tissues. When treated by salicylic acid (SA), isonicotinic acid (INA), abscisic acid (ABA) and hydrogen peroxide (H2 O2 ), the lines showed GUS activity exclusively in vascular tissues and mesophyll cells. This activity was enhanced after inoculation with Magnaporthe oryzae or Xanthomonas oryzae pv. oryzae. The OsHAP2E expression level was also induced after inoculation of rice with M. oryzae and X. oryzae pv. oryzae and after treatment with SA, INA, ABA and H2 O2, respectively. We further produced transgenic rice overexpressing OsHAP2E. These lines conferred resistance to M. oryzae or X. oryzae pv. oryzae and to salinity and drought. Furthermore, they showed a higher photosynthetic rate and an increased number of tillers. Microarray analysis showed up-regulation of defence-related genes. These results suggest that this gene could contribute to conferring biotic and abiotic resistances and increasing photosynthesis and tiller numbers.

The cysteine-rich proteins of beet necrotic yellow vein virus and tobacco rattle virus contribute to efficient suppression of silencing in roots

Many plant viruses encode proteins that suppress RNA silencing, but little is known about the activity of silencing suppressors in roots. This study examined differences in the silencing suppression activity of different viruses in leaves and roots of Nicotiana benthamiana plants. Infection by tobacco mosaic virus, potato virus Y and cucumber mosaic virus but not potato virus X (PVX) resulted in strong silencing suppression activity of a transgene in both leaves and roots, whereas infection by beet necrotic yellow vein virus (BNYVV) and tobacco rattle virus (TRV) showed transgene silencing suppression in roots but not in leaves. For most viruses tested, viral negative-strand RNA accumulated at a very low level in roots, compared with considerable levels of positive-strand genomic RNA. Co-inoculation of leaves with PVX and either BNYVV or TRV produced an increase in PVX negative-strand RNA and subgenomic RNA (sgRNA) accumulation in roots. The cysteine-rich proteins (CRPs) BNYVV p14 and TRV 16K showed weak silencing suppression activity in leaves. However, when either of these CRPs was expressed from a PVX vector, there was an enhancement of PVX negative-strand RNA and sgRNA accumulation in roots compared with PVX alone. Such enhancement of PVX sgRNAs was also observed by expression of CRPs of other viruses and the well-known suppressors HC-Pro and p19 but not of the potato mop-top virus p8 CRP. These results indicate that BNYVV- and TRV-encoded CRPs suppress RNA silencing more efficiently in roots than in leaves.