Serological Studies on Tomato Golden Mosaic Virus, a Geminivirus

Abstract

SUMMARYParticles of tomato golden mosaic virus (TGMV), purified by an improved procedure, were used to prepare an antiserum which in gel double-diffusion tests with homologous virus gave a single precipitin line and had a titre of 1/256. TGMV was shown to be serologically related to another geminivirus, cassava latent virus, and distantly related to a leafhopper-transmitted geminivirus, beet curly top virus. No relationship could be detected to four other leaf hopper-transmitted geminiviruses.Golden mosaic disease of the tomato Lycopersicon esculentum Mill is a serious problem in Brazilian horticulture (Costa, 1976). The causal agent, tomato golden mosaic virus (TGMV), is transmitted in nature by the whitefly Bemisia tabaci Genn., but it can also be transmitted by mechanical inoculation to a number of Nicotiana species which serve as convenient hosts for virus propagation. TGMV has been placed in the geminivirus group (Matthews, 1982) because its particles have geminate morphology, its capsid polypeptide species has a mol. wt. of 28 000 and its genome is covalently closed circular single-stranded DNA (Matyis et al., 1975;Hamilton et al., 1981). The genome of TGMV, like that of two other geminiviruses, bean golden mosaic virus (Haber et al., 1981) and cassava latent virus (CLV) (Stanley & Gay, 1983), consists of two distinct DNA molecules of similar size (approx. 2600 nucleotides) Bisaro et al., 1982).In the present communication we report an improved purification procedure for TGMV and studies of its serological relationships with other geminiviruses.TGMV (Brazilian isolate kindly supplied by Dr A. S. Costa) was propagated in Nicotiana benthamiana by inoculation of the expanding upper leaves of plants grown to the six to eight leaf stage which were then kept for 14 days in a glasshouse maintained at a temperature of 25 to 28 °C with a relative humidity of 70 to 80~ and 15 klux light intensity supplied by a mixture of mercury and incandescent bulbs with an 18 h photoperiod. Inocula consisted of resuspended virus pellets (see below) which were either used immediately or stored at -20 °C for up to 4 months without loss of infectivity.For virus isolation 250 g of leaf material was homogenized in 500 ml buffer (0.1 M-trisodium citrate, 0.759/0 sodium sulphite, 5 mM-disodium EDTA, 1 ~ 2-mercaptoethanol, 0.325~o Lascorbic acid, adjusted to pH 7 with NaOH). The homogenate was made 2-5 9/0 (by vol.) in Triton X-100, stirred for 16 h at 4 °C and then filtered through muslin. The filtrate was centrifuged at I0000 g for 15 min and the supernatant was collected and centrifuged at 40000 rev/min in a Beckman Ti45 rotor for 2 h. The virus pellets were resuspended in 12 ml CEM buffer (0-01 Mtrisodium citrate, 1 mM-disodium EDTA, 0.05~ 2-mercaptoethanol, adjusted to pH 7 with NaOH) and the suspension was divided into three aliquots, each of which was overlaid onto a 1 ml cushion of 20~ (w/v) sucrose in CEM buffer and centrifuged for 3 h at 40000 rev/min in a Beckman SW50.1 rotor. The re-pelleted virus was resuspended in a total ...

Serological Relationships and Genome Homologies among Geminiviruses

“…The relationship between ACMV and EuMV has also been detected by geldiffusion precipitin tests (R. W. Fulton, personal communication). The apparent relationships previously reported of BCTV with ACMV (Sequeira & Harrison, 1982) and TomGMV (Stein et al, 1983) were not confirmed and were possibly caused by anti-host antibodies in the serum. No relationship was detected in the test reported here between BCTV and any of the whiteflytransmitted viruses.…”

“…However, Sequeira & Harrison (1982) detected a serological relationship between ACMV and bean golden mosaic virus (BGMV), and Cohen et al (1983) found that ACMV is serologically related to squash leaf curl virus (SLCV). Similarly, tomato golden mosaic virus (TomGMV) was found to be serologically related to ACMV and BGMV (Stein et al, 1983) and, in addition, weak relationships were reported between beet curly top virus (BCTV) and both ACMV and TomGMV (Sequeira & Harrison, 1982;Stein etal., 1983). Of these viruses, ACMV, BGMV, SLCV and TomGMV are transmitted by the whitefly Bemisia tabaci, whereas the others have leafhopper vectors.…”

DNA sequences essential for replication of the B genome component of tomato golden mosaic virus.

“…Twenty-one days post-inoculation, DNA was extracted from all plants as described previously (Stein, Coutts, and Buck, 1983). The presence or absence of replicated A and B DNA was detected by DNA gel blot analysis using 32P-labeled A-specific or 6-specific riboprobes prepared from TGMV subclones in pGEM plasmids (Promega, Madison, WI).…”

Identification of the Coat Protein Gene of Tomato Golden Mosaic Virus

“…TGMV capsid polypeptide was subjected to SDS-PAGE in three lanes of a 15 ~ polyacrylamide gel alongside marker proteins and then immobilized on nitrocellulose by Western blotting (Towbin et al, 1979). The nitrocellulose sheet from the blot was cut into three portions: one portion was stained with amido black, the second was probed with an antiserum prepared to TGMV virions (Stein et al, 1983) and the third was probed with the AR1 fusion protein antiserum. Detection of bound antibodies with a Protein A-peroxidase conjugate by probing with the antisera, followed by incubation with 4-chloro-l-naphthol and hydrogen peroxide, were carried out as described by Sherwood (1987).…”

“…None of the protein products of these ORFs has been directly identified. We now present evidence which proves that ORF AR1 codes for the coat protein.TGMV was isolated from systemically infected Nicotiana benthamiana plants and purified by sucrose density gradient centrifugation as described by Stein et al (1983). To ensure that the preparation was completely free from host proteins, virus was further purified by centrifugation for 20 h at 115 000 g in a caesium sulphate density gradient in a Beckman SW50.1 rotor.…”