Byoung Eun Min

Plant virus cDNA chip hybridization for detection and differentiation of four cucurbit-infecting Tobamoviruses

A plant virus cDNA chip was developed by using viral cDNA clones and microarray technology. The cDNA chip was designed for detection and differentiation of the four species of selected cucurbit-infecting tobamoviruses [target viruses: Cucumber green mottle mosaic virus (CGMMV); Cucumber fruit mottle mosaic virus (CFMMV); Kyuri green mottle mosaic virus (KGMMV); and Zucchini green mottle mosaic virus (ZGMMV)]. The chip consisted of cDNA clones of the four cucurbit-infecting tobamoviruses, two target-related tobamoviruses, and another three unrelated plant viruses. Polymerase chain reaction products were amplified from the selected cDNA clones and arrayed onto slide glass. The cDNA chip, which was called cucurbit-virus chip, detected successfully specific target viruses. When applied to probes made from ZGMMV-infected samples, ZGMMV reacted strongly with its homologous cDNA and moderately reacted with KGMMV and CFMMV, while it did not react with CGMMV on the same chip. CGMMV probe gave strong signal intensity to its homologous cDNA spot and weakly reacted with ZGMMV, KGMMV, and CFMMV. The signal intensity of all combinations of probe and target was correlated significantly with nucleotide sequence identities between the probes and target viruses based on scatter diagrams. The signals could be made as image files for specific virus detection, and this could be useful for virus identification and differentiation. This is the first report of plant virus detection by using cDNA chip technology.

Evidence for novel viruses by analysis of nucleic acids in virus-like particle fractions from Ambrosia psilostachya

To test the hypothesis that many viruses remain to be discovered in plants, a procedure was developed to sequence nucleic acids cloned randomly from virus-like particle fractions of plant homogenates. As a test of the efficiency of the procedure we targeted Ambrosia psilostachya, western ragweed, plants growing at the Tallgrass Prairie Preserve of northeastern Oklahoma. Amplifiable nucleic acid was found in the fractions from six of twelve specimens and sequences were characterized from four of them. Evidence was obtained for the presence of viruses belonging to two families (Caulimoviridae, Flexiviridae). Multiple viral species were found in two of the four specimens and their level within the isolated nucleic acid population varied from less than 1-37%. None of the sequences were derived from reported sequences of known viruses. Thus, the analysis of nucleic acid from virus-like particles is a useful tool to expand our knowledge of the universe of viruses to non-cultivated species.

Zucchini green mottle mosaic virus is a new tobamovirus; comparison of its coat protein gene with that of kyuri green mottle mosaic virus

Summary. A novel virus we call zucchini green mottle mosaic virus (ZGMMV) was isolated from zucchini squash and its properties were determined. The size and shape of its virions, and other properties suggest that the virus is a tobamovirus. The coat protein (CP) genes of ZGMMV and kyuri green mottle mosaic virus (KGMMV), which also infects zucchini squash plants, were cloned and their nucleotides sequences were determined. The CP genes of ZGMMV and KGMMV are composed of 161 amino acid residues, and they share 77.6% amino acid identity. Western blot analysis showed that the two viruses are serologically related but not identical. Comparison of the sequences with those of sixteen other tobamoviruses revealed that the two viruses had much higher identity to cucumber green mottle mosaic virus (CGMMV), another tobamovirus infectious to cucurbit plants, than other tobamoviruses. The nucleotide and amino acid sequences of ZGMMV were from 29.5 to 78.4% and from 29.3 to 77.6% identical, respectively, to those of other tobamoviruses. The predicted virion assembly origins of the two tobamoviruses were located in the CP region of the genomic RNAs, and the predicted secondary structures were more similar to that of CGMMV than those of other tobamoviruses. The seventeen tobamo-viruses could be classified into three main subgroups based on the cphylogenetic tree analysis on the CP gene, and ZGMMV and KGMMV formed a third subgroup together with CGMMV and sunn-hemp mosaic virus (SHMV). These results show that ZGMMV is a previously unknown member of the Tobamovirus genus.

Transgenic cucumbers harboring the 54-kDa putative gene of Cucumber fruit mottle mosaic tobamovirus are highly resistant to viral infection and protect non-transgenic scions from soil infection

Cucumber fruit mottle mosaic tobamovirus (CFMMV) causes severe mosaic symptoms and yellow mottling on leaves and fruits and, occasionally, severe wilting of cucumber (Cucumis sativus L.) plants. No genetic source of resistance against this virus has been identified in cucumber. The gene coding for the putative 54-kDa replicase gene of CFMMV was cloned into an Agrobacterium tumefaciens binary vector, and transformation was performed on cotyledon explants of a parthenocarpic cucumber cultivar. R1 seedlings were screened for resistance to CFMMV by symptom expression, back inoculation on an alternative host and ELISA. From a total of 14 replicase-containing R1 lines, eight resistant lines were identified. Line I44 – homozygous for the putative 54-kDa replicase gene – was immune to CFMMV infection by mechanical and graft inoculation, and to root infection following planting in CFMMV-infested soil. A substantial delay of symptom appearance was observed following infection by three additional cucurbit-infecting tobamoviruses. When used as a rootstock, line I44 protected susceptible cucumber scions from soil infection by CFMMV. This paper is the first report on protection of a susceptible cultivar against a soil-borne viral pathogen, by grafting onto a transgenic rootstock.

Determination of complete nucleotide sequence of Hibiscus latent Singapore virus: Evidence for the presence of an internal poly(A) tract

Summary.We have sequenced the complete genome of a hibiscus-infecting tobamovirus, Hibiscus latent Singapore virus (HLSV). The experimental host range of HLSV is similar to that of another distinct species of hibiscus infecting tobamovirus, Hibiscus latent Fort Pierce virus (HLFPV). The genomic structure of HLSV is similar to other tobamoviruses in general. It consists of a 5′ untranslated region (UTR), followed by ORFs encoding for a 128 kDa protein and a 186 kDa readthrough protein, a 30 kDa movement protein (MP), 18 kDa coat protein (CP) and a 3′ UTR. The unique feature of HLSV is the presence of a poly(A) tract within its 3′ UTR. In our previous work, we have reported MP and CP sequences of HLSV and its phylogenetic analysis. Here we report the complete nucleotide sequence of HLSV, phylogenetic analysis of the nucleotide and amino acid sequences of 128/186 kDa ORFs and the presence of a uniquely located poly(A) tract within the 3′ UTR.

Completion of nucleotide sequence and generation of highly infectious transcripts to cucurbits from full-length cDNA clone of Kyuri green mottle mosaic virus

Summary. The nucleotide sequence of the genome of the type strain of Kyuri green mottle mosaic virus (KGMMV-C1) has been completely determined. The genome structure and sequence of the virus were compared to those of Yodo strain of KGMMV (KGMMV-Y). The genome of KGMMV-C1 is 6,514 nucleotides long consisting of 5’ and 3’ nontranslated regions (NTRs) and four open reading frames coding for 131 kDa and 189 kDa viral replicases, 28 kDa movement protein and 17 kDa coat protein. The nucleotide and amino acid sequences identities of the four encoded proteins and two NTRs between KGMMV-C1 and KGMMV-Y were 85.6% to 93.9% and 87.6% to 95.5%, respectively. Full-length cDNA of KGMMV-C1 was directly amplified by reverse-transcription polymerase chain reaction (RT-PCR) with a set of 5’-end primer anchoring T7 RNA promoter sequence and 3’-end primer. This full-length RT-PCR product allowed RNA to be transcribed in vitro. The T7 promoter-anchored RT-PCR product was cloned and used as templates for transcription for plant inoculation test. Capped transcript RNAs transcribed from the full-length cDNA clone as well as capped transcript RNAs from the uncloned RT-PCR products were infectious and caused symptoms characteristic of KGMMV when mechanically inoculated to systemic host plants such as zucchini squash, cucumber and Nicotiana benthamiana. Transcript-derived progeny virus was indistinguishable from the wild-type virus with the same biological and biochemical properties. To our know-ledge, this is the first report of the generation of a biologically active KGMMV clone, driven by the T7 promoter, that is highly infectious to cucurbitaceous plants.

Cactus mild mottle virus is a new cactus-infecting tobamovirus

Summary.A new cactus-infecting tobamovirus, Cactus mild mottle virus (CMMoV), was isolated from diseased grafted cactus, Gymnocalycium mihanovichii and its molecular properties were characterized. CMMoV is distantly related to known species of the genus Tobamovirus on the basis of serological and sequence analyses. Western blot analysis showed that CMMoV is serologically unrelated to Sammon’s Opuntia virus, which is the only known species of the genus Tobamovirus found in cactus plants. The 3′-terminal 2,910 nucleotides of CMMoV have been sequenced. The coat protein (CP) and movement protein (MP) genes encode 161 and 306 amino acids residues, respectively, and the 3′ untranslated region (UTR) consists of 229 nucleotides long. The nucleotide and amino acid sequences of the CP of CMMoV were 39.6% to 49.2% and 25.8% to 40.3% identical to other seventeen tobamoviruses, respectively. The MP shared 34.9% to 40.6% and 16.3% to 27.0% and 44.6% to 63.4% identities, respectively, at the amino acid and nucleotide levels with other members of the genus. Percentage identities of nucleotides of the 3′ UTR ranged from 42.5% to 63.4%. Phylogenetic tree analyses of the CP and MP suggest the existence of the fifth cactus-infecting subgroup in the genus Tobamovirus. Sequence analyses of these two viral proteins revealed that the highest amino acid sequence identity between the virus and seventeen other tobamoviruses was 40.6%, supporting the view that CMMoV is a new definite species of the genus Tobamovirus.

Molecular evidence supporting the confirmation of Maracuja mosaic virus as a species of the genus Tobamovirus and production of an infectious cDNA transcript

Summary.The complete genome sequence of maracuja mosaic virus (MarMV) was determined and analyzed. The full MarMV genome consisted of 6794 nucleotides, and this is the largest genome size among known tobamoviruses. The MarMV genome RNA contained four open reading frames (ORFs) coding for proteins of M(r) 126, 181, 34 and 18 kDa from the 5′ to 3′ end, respectively. The lengths of the 5′ nontranslated region (NTR) and the 3′ NTR were 54 and 177 nucleotides, respectively. Phylogenetic tree analysis revealed that these MarMV-encoded proteins are related to members of the Malvaceae- and Cucurbitaceae-infecting tobamoviruses. MarMV is different from other tobamoviruses and forms a new Passifloraceae-infecting subgroup. Western blot analysis showed that MarMV cross-reacted strongly with antibodies against Kyuri green mottle mosaic virus and Hibiscus latent Singapore virus. Synthesized capped transcripts from full-length cDNA of MarMV were infectious. These data clearly indicate that MarMV belongs to a separate species of the genus Tobamovirus.

Genome structure and complete sequence of genomic RNA of Daphne virus S

Summary.The complete genomic nucleotide sequence and structure of Daphne virus S (DVS), a daphne-infecting member of the genus Carlavirus, were determined. The genome of DVS was 8,739 nucleotides long, excluding the poly (A) tails. The genome of DVS contained six open reading frames coding for proteins of Mr 227 kDa (viral replicase), 25 kDa, 11 kDa and 7 kDa (triple gene block TGB) proteins 1, 2 and 3), 35 kDa (coat protein; CP), and 12 kDa from the 5′ to 3′ ends; respectively. This is the typical genome structure of members of the genus Carlavirus. Overall amino acid sequence similarities for the six ORFs of DVS were from 58.5% to 13.2% to those of the other carlaviruses. The 227 kDa replicase of DVS shared 45.5–39.2% amino acid similarities to that of 8 other known carlaviruses. Results from phylogenetic analyses of viral replicases and CPs demonstrated that DVS is a close relative of Helenium virus S and Chrysanthemum virus B. A total of 13 isolates of DVS shared 100–95.9% identities for the amino acid level and 99.5–81.0% identities for the nucleotide level. This is the first report of the complete genome sequence and structure of DVS and supports the conclusion that DVS is a typical species of the genus Carlavirus.

Complete sequence and genome structure of cactus mild mottle virus.

We have completed the genomic sequence of a tobamovirus, cactus mild mottle virus (CMMoV), and compared it to those of other known tobamoviruses. The complete genome sequence of CMMoV consists of 6,449 nucleotides. The genome RNA of the virus contains four open reading frames, encoding, from the 5′ to the 3′ end, the 120-kDa viral replicase, the 186-kDa viral polymerase, the 33-kDa movement protein and the 18-kDa coat protein. Overall amino acid similarities for the four viral proteins of CMMoV ranged from 16.3 to 44.4% compared to those of 20 other tobamoviruses. Phylogenetic analysis of the viral replicases and MP revealed that CMMoV is closely related to cucurbit-infecting tobamoviruses, while the CMMoV CP is more closely related to brassica- and solanaceous-infecting tobamoviruses.