Mosaic Virus Coat Protein Gene Research Articles

Occurrence and molecular characterization of begomoviruses associated with cassava across agro-ecological zones in Cross River State, Nigeria

Cassava (Manihot esculenta Crantz) is a major root crop in sub-Saharan Africa, including Cross River State, Nigeria, where the crop is cultivated as a staple food in many communities. Cassava mosaic begomoviruses (CMBs) have limited cassava production across Africa. Thus, between March and August 2021, forty-five samples of symptomatic cassava leaves across the state were collected and evaluated for CMBs using molecular techniques. Partial sequencing of the African cassava mosaic virus (ACMV) and East African cassava mosaic virus (EACMV) coat protein genes produced sequence lengths of 620 bp for representative isolates from Okpoma (MZ507578), Ababene (MZ507579) and Idundu (MZ507580), and 554 bp for an Ovonum isolate, respectively. BLASTn results for MZ507578 and MZ507580 showed a percentage similarity of 99.35 and 99.03%, respectively with the nucleotide sequence of ACMV (MH251339) from Nigeria for MZ507579, percentage similarity was 99.84% with ACMV (MG250088) from Ghana. Conversely, the Ovonum isolate showed a 100% percentage similarity with EACMV (MW826364) from Nigeria. Phylogenetic analysis clustered the Calabar isolates (MZ507578, MZ507579 and MZ507580) with MH251339 from Nigeria, MG250088 from Ghana and EU155147 from Togo while the Ovonum isolate was grouped with MW826364 and MT571462, both from Nigeria. The two main CMBs in Nigeria, ACMV and EACMV, were identified and characterized from Cross River state in this study, indicating that disease monitoring should be continuous and more virus-resistant cultivars should be introduced for proper virus management.

Assessment of Yam mild mosaic virus coat protein gene sequence diversity reveals the prevalence of cosmopolitan and African group of isolates in Ghana and Nigeria

This study analyzed the genetic diversity of 18 Yam mild mosaic virus (YMMV, genus Potyvirus) isolates collected from field surveys in Ghana (N = 8) and Nigeria (N = 10) in 2012−13. The full coat protein (CP) encoding region of the virus genome was sequenced and used for comparison and phylogenetic analysis of the YMMV isolates available in the NCBI nucleotide database. The mean nucleotide (nt) diversity was 13.4% among the 18 isolates (17 from D. alata and one from D. rotundata), 11.4% within the isolates of Ghana and 7.4% within the isolates of Nigeria. The phylogenetic clustering of the 18 YMMV isolates did not show correlation with the country of origin, and they aligned with the reference sequences of four of the 11 YMMV monophyletic groups representing the cosmopolitan group and the African group of YMMV isolates. High sequence homology of 99% between the YMMV sequence from Nigeria (CP12-DaN6-1) and a previously reported sequence from Togo (GenBank Accession Number AF548514) suggests a prevalence of seed-borne virus spread within the region. Understanding YMMV sequence diversity in West Africa aid in the improvement of diagnostic assays necessary for virus indexing and seed certification.

Identification and phylogenetic analysis of a tobamovirus causing hibiscus (Hibiscus rosa-sinensis L.) mosaic disease in Iran

Hibiscus (Hibiscus rosa-sinensis L.) is a tropical perennial shrub frequently cultivated as an ornamental plant in southwest Iran. Virus-like symptoms including leaf mosaic, mottling and malformation have been observed on hibiscus plants during 2015–2017. Infectivity of the causal agent of the symptoms was proved through mechanical transmission to indicator plants. The cotton-melon aphid (Aphis gossypii G.) was shown to transmit the causal agent with a very low efficiency. A tobamoviral infection was detected in symptomatic leaves by ELISA. Tomato mosaic virus (ToMV) coat protein (CP) gene was amplified in infected samples and sequenced completely. Nucleotide comparison of the sequences showed 96–98% identity to Iranian isolates of ToMV. Phylogenetic analysis of nucleotide and amino acid sequence of ToMV isolates originating from various sources and diverse geographical locations was performed and ToMV isolates were classified into two clades, Clade I and Clade II. ToMV isolates obtained in this study were placed in Clade II and formed a separate subclade. Multiple alignment of amino acid sequences demonstrated several substitutions in CPs of hibiscus-infecting ToMV isolates which might contribute to the variations in symptoms induced by them. The results showed the first record of ToMV infecting an ornamental plant in Iran.

Inheritance and effectiveness of two transgenes determining PVY resistance in progeny from crossing independently transformed tobacco lines

Genetic transformation of plants allows us to obtain improved genotypes enriched with the desired traits. However, if transgenic lines were to be used in breeding programs the stability of inserted transgenes is essential. In the present study, we followed the inheritance of transgenes in hybrids originated from crossing two transgenic tobacco lines resistant to Potato virus Y (PVY): MN 944 LMV with the transgene containing Lettuce mosaic virus coat protein gene (LMV CP) and AC Gayed ROKY2 with PVY replicase gene (ROKY2). Progeny populations generated by successive self-pollination were analyzed with respect to the transgene segregation ratio and resistance to Potato virus Y in tests carried out under greenhouse conditions. The presence of the virus in inoculated plants was detected by DAS-ELISA method. The results demonstrated the Mendelian fashion of inheritance of transgenes which were segregated independently and stably. As a result, we obtained T4 generation of hybrid with both transgenes stacked and which was highly resistant to PVY.

Development and validation of protocols for product stewardship in transgenic white clover (Trifolium repens L.): detection of the AMV CP and npt2 transgenes in pollen, honey and honey bees

There are no current commercial releases of genetically modified white clover, but several research groups are working on traits such as virus resistance, stress tolerance and bloat safety that are likely to provide large economic benefits for livestock farmers. However, white clover pollen is a common constituent of honey produced by bees foraging white clover flowers. Therefore, there is a need to develop tools to detect the presence of genetically modified pollen in white clover honey. The results presented in this paper describe the development and application of PCR-based techniques to detect the Alfalfa mosaic virus coat protein gene (AMV CP) and the neomycin phosphotransferase 2 selectable marker gene (npt2) in genetically modified white clover pollen, whether this pollen is collected fresh, from honey bees that have been foraging white clover, or from honey. Further research and development will be required to develop ‘field-ready’ tools for the detection and quantification of these transgenes in pollen and honey products. However, this paper demonstrates prospects and principles in pollen and honey from honeybees foraging transgenic white clover.

Recovery of virus-infected Dendrobium orchids by constitutive expression of the cymbidium mosaic virus coat protein gene

A common practice in the production of viral disease-resistant plant varieties is the transfer of the viral coat protein (CP) gene to a wide range of healthy plants. This is the first report on the effect of a bombarded CP transgene on the replication of cymbidium mosaic virus (CymMV) in naturally infected Dendrobium orchids. Nine months after transformation, two naturally CymMV-infected transgenic Dendrobium orchids exhibited a gradual decrease in CymMV-CP transcript and coat protein levels as detected by RT-PCR and western blot hybridization, respectively. At 18 months, the transgenic lines were completely free of endogenous CymMV contamination and were protected from CymMV re-infection following inoculation. Six weeks after inoculation with purified CymMV, these virus-free transgenic lines contained no viral coat protein transcript, replicase transcript or particle accumulation from the re-invading CymMV, as detected by RT-PCR and Immunoelectron microscopy assays, respectively. Additionally, no small RNAs derived from viral CymMV-CP were detected, but the transcribed CymMV-CP positive-strand derived from the transgene appeared in these transgenic lines.

Application of RNA interference methodology to investigate and develop SCMV resistance in maize.

Specific fragments of the sugarcane mosaic virus (SCMV) coat protein gene (cp) were amplified by reverse transcriptionpolymerase chain reaction and used to construct a marker free small interfering RNA complex expression vector against SCMV. In planta transformation was performed on maize (Zea mays) inbred line 8112 mediated by Agrobacterium tumefaciens. PCR and Southern blot analyses demonstrated successful integration of the cp segment into the 8112 genome. The in planta transformation frequency was 0.1%, and the cotransformed frequency with the cp and bar genes was 0.034%. Real-time quantitative PCR of samples from different transgenic plant organs showed that the expression of the cp gene fragment in transgenic plants was variable and that the highest expression level occurred in the tassels and leaves and the lowest expression occurred in the roots. Real-time quantitative PCR was also used to measure how gene expression in transgenic T2 generation plants inoculated with SCMV changes over time. The results showed that the hairpin RNA structure transcribed from the cp gene interfered with SCMV infection and transgenic maize lines were not equally effective in preventing SCMV infection. Our findings provide a valuable tool for controlling plant viruses using RNA interference and the posttranslational gene silencing approach.

Prokaryotic expression of a Thunberg fritillary mosaic virus CP gene and antiserum preparation

Wei, C. B., Wu, Q. Y., Yang, Y. and Liu, C. Y. 2012. Prokaryotic expression of a Thunberg fritillary mosaic virus CP gene and antiserum preparation. Can. J. Plant Sci. 92: 495–500. Fritillaria anhuiensis is a valued traditional Chinese medicinal plant. Thunberg fritillary mosaic virus (TFMV) was found to cause mosaic symptoms. Virus particles of the Lu'an isolate were purified and its RNA genome was extracted. cDNA was synthesized from the RNA and specific PCR primers were used to amplify the virus coat protein (CP) gene. It shared 96.8% nucleotide identity and 97.6% amino acid identity with the sequences of CPs from the other TFMV isolates. The CP gene was inserted into pSBET and expressed in Escherichia coli BL21 (DE3) plys S strain. The expressed protein was first purified by 12% (wt/vol) SDS-PAGE and subsequently by 5–20% (wt/vol) gradient SDS-PAGE. Antiserum against the CP was raised in mouse and its specificity was confirmed by Western blot analysis suggesting that it may be useful for detecting the virus.

Prokaryotic expression of a Thunberg fritillary mosaic virus CP gene and antiserum preparation

Wei, C. B., Wu, Q. Y., Yang, Y. and Liu, C. Y. 2012. Prokaryotic expression of a Thunberg fritillary mosaic virus CP gene and antiserum preparation. Can. J. Plant Sci. 92: 495–500. Fritillaria anhuiensis is a valued traditional Chinese medicinal plant. Thunberg fritillary mosaic virus (TFMV) was found to cause mosaic symptoms. Virus particles of the Lu'an isolate were purified and its RNA genome was extracted. cDNA was synthesized from the RNA and specific PCR primers were used to amplify the virus coat protein (CP) gene. It shared 96.8% nucleotide identity and 97.6% amino acid identity with the sequences of CPs from the other TFMV isolates. The CP gene was inserted into pSBET and expressed in Escherichia coli BL21 (DE3) plys S strain. The expressed protein was first purified by 12% (wt/vol) SDS-PAGE and subsequently by 5–20% (wt/vol) gradient SDS-PAGE. Antiserum against the CP was raised in mouse and its specificity was confirmed by Western blot analysis suggesting that it may be useful for detecting the...

Watermelon transformation with Zucchini yellow mosaic virus coat protein gene and comparison with parental cultivar

The objective of this work was to transfer Zucchini yellow mosaic virus coat protein (ZYMV-CP) and neomycin phosphotransferase II (NPT II) genes to the watermelon 'Crimson Sweet'(CS) genome, and to compare the transgenic progenies T1 and T2 with the nontransformed parental cultivar for morphological, pomological, growth and yield characteristics. The ZYMV-CP gene was transferred by Agrobacterium tumefaciens. The presence of the gene in transgenic T0, T1 and T2 plants was determined by polymerase chain reaction, and the results were confirmed by Southern blot. Two experiments were performed, one in the winter-spring and the other in the summer-autumn. In both experiments, the hypocotyl length of transgenic seedlings was significantly higher than that of nontransgenic parental ones. In the second experiment, the differences between transgenic and nontransgenic individuals were significant concerning fruit rind thickness, flesh firmness, fruit peduncle length, size of pistil scar, and a* values for fruit stripe or flesh color. Transferring ZYMV-CP gene to CS genome affected only a few characteristics from the 80 evaluated ones. The changes in rind thickness, flesh firmness and flesh color a* values are favorable, while the increase in the size of pistil scar is undesirable. The transgenic watermelon line having ZYMV-CP gene and the parental cultivar CS are very similar.

A framework for molecular genetic assessment of a transgenic watermelon rootstock line

Transgenic plants over-expressing virus coat protein genes have attracted particular interest from researchers recently, due primarily to their tolerance to viral infection. The transgenic watermelon rootstock analyzed in this study contains the introduced cucumber green mottle mosaic virus coat protein ( CGMMV-CP) gene. The primary objective of this study was to determine the copy number, integration site, and expression level of the transgene element, in order to establish a scientific framework for the molecular genetic assessment of transgenic plant rootstocks. The results of our Southern blot analysis indicated that a single copy of the CGMMV-CP gene was inserted into the genome of a transgenic watermelon rootstock. We also identified the genomic sequences flanking the integration site of the transgene via inverse PCR analysis. In an effort to find a sequence usable as an internal positive control for the screening of the transgenic watermelons, we determined that the Sat gene appears as one copy within their genomes and is watermelon-specific. The information from the integrated site and the internal positive control sequence was utilized to establish a new event-specific PCR-based detection method. The expression of both CGMMV-CP mRNA and protein was detected in the transgenic watermelon rootstocks but not in watermelon scions, thereby suggesting that the tissues of the watermelon scions are free of the introduced gene products.

Genetic Diversity Among Viruses Associated with Sugarcane Mosaic Disease in Tucumán, Argentina

Sugarcane leaves with mosaic symptoms were collected in 2006--07 in Tucumán (Argentina) and analyzed by reverse-transcriptase polymerase chain reaction (RT-PCR) restriction fragment length polymorphism (RFLP) and sequencing of a fragment of the Sugarcane mosaic virus (SCMV) and Sorghum mosaic virus (SrMV) coat protein (CP) genes. SCMV was detected in 96.6% of samples, with 41% showing the RFLP profile consistent with strain E. The remaining samples produced eight different profiles that did not match other known strains. SCMV distribution seemed to be more related to sugarcane genotype than to geographical origin, and sequence analyses of CP genes showed a greater genetic diversity compared with other studies. SrMV was detected in 63.2% of samples and most of these were also infected by SCMV, indicating that, unlike other countries and other Argentinean provinces, where high levels of co-infection are infrequent, co-existence is common in Tucumán. RFLP analysis showed the presence of SrMV strains M (68%) and I (14%), while co-infection between M and H strains was present in 18% of samples. Other SCMV subgroup members and the Sugarcane streak mosaic virus (SCSMV) were not detected. Our results also showed that sequencing is currently the only reliable method to assess SCMV and SrMV genetic diversity, because RT-PCR-RFLP may not be sufficiently discriminating.

Evaluation of a wide range of pepper genotypes for regeneration and transformation with an Agrobacterium tumefaciens shooter strain

A regeneration protocol developed for the Agrobacterium tumefaciens-mediated transformation of pepper ( Capsicum annuum L.) was used to evaluate the potential for genetic transformation of 107 doubled haploid (DH) pepper genotypes belonging to 12 main cultivar groups. The genotypes were scored on the basis of the ratio of regenerated shoots compared to the commercial cultivar Fehérözön, which exhibited 30–70% regeneration from the total number of explants. Fifty DH genotypes responded, representing all the of the main cultivar groups, 31 of which showed regeneration frequencies similar to or better than cv. Fehérözön. By using a shooter strain with a binary vector harbouring the cucumber mosaic virus (CMV) coat protein (CP) gene and selecting regenerants on media containing kanamycin, the transformability of the pepper genotypes as well as resistance to CMV were simultaneously analyzed. The regenerated plants were selected and tested by PCR to detect the CP gene. Eighteen PCR-positive DH plants and six PCR-positive commercial Fehérözön cultivar plants were regenerated and rooted by grafting. In the case of three transgenic genotypes, presence of the CMV CP was confirmed by PCR in the T1 generation indicating the stable integration of the CP gene. In the progeny of one DH genotype of the Demre type, the presence of the transgene as well as resistance to the virus were demonstrated in the T2 generation.

Transgene expression for Gladiolus plants grown outdoors and in the greenhouse

Transgene expression was evaluated for Gladiolus plants transformed with either the CaMV 35S, double CaMV 35S, rolD, or Arabidopsis UBQ3 promoter controlling the uidA or bean yellow mosaic virus coat protein gene in either the sense or antisense orientation to determine differences in expression for plants grown in the greenhouse and outdoors for two years. There was more variability in GUS expression when plants were grown outdoors than in the greenhouse for two years. Four of the six transformed plant lines with the UBQ3, rolD, and CaMV 35S promoters grown outdoors showed significant differences in GUS expression from year to year as compared to two of the six lines with the UBQ3 and rolD promoters grown in the greenhouse. When grown the same year, two plant lines with the CaMV 35S and one line with the rolD promoter showed 2–16× higher levels of GUS expression outdoors than in the greenhouse, and one plant line with the UBQ3 promoter had 31× higher GUS expression in the greenhouse instead of outdoors. Three of six plant lines transformed with the bean yellow mosaic virus coat protein gene in either the sense or antisense orientation under control of the double CaMV 35S promoter showed obvious transgene expression as compared to three lines that did not show expression or negligible expression for both years when plants were grown both outdoors and in the greenhouse. This study verified long-term gene expression, rather than silencing, for Gladiolus plants when grown outdoors and in the greenhouse from year to year.

Morphological characterisation and genetic analysis of a bi-pistil mutant (bip) in Medicago truncatula Gaertn.

A floral organ mutant was observed in transgenic Medicago truncatula Gaertn. plants that had two separate stigmas borne on two separate styles that emerged from a single superior carpel primordium. We propose the name bi-pistil, bip for the mutation. We believe this is the first report of such a mutation in this species. Genetic and molecular analyses of the mutant were conducted. The mutant plant was crossed to a mtapetala plant with a wild-type pistil. Expression of the mutant trait in the F1 and F2 generations indicates that the bi-pistil trait is under the control of a single recessive gene. Other modifying genes may influence its expression. The mutation was associated with the presence of a T-DNA insert consisting of the Alfalfa mosaic virus (AMV) coat protein gene in antisense orientation and the nptII selectable marker gene. It is suggested that the mutation is due to gene disruption because multiple copies of the T-DNA were observed in the mutant. The bi-pistil gene was found to be independent of the male-sterile gene, tap. This novel mutant may assist in understanding pistil development in legumes.

EXPERIMENTS ON TRANSFORMATION AND IN VITRO REGENERATION OF SQUASH VARIETIES

Squash is an important species in the Cucurbitaceae family. The development of genetic transformation offers the potential of introducing valuable traits into this crop. An efficient in vitro plant regeneration system is critical for plant transformation. The regeneration ability of three Cucurbita varieties (C. pepo convar. patissoniana 'Ovari feher', C. pepo convar. giromontiina 'Black Beauty', C. maxima convar. maxima 'Nagydobosi') was tested as a first step for further transformation experiments. The cotyledon explants of the tested varieties were cultured on Murashige and Skoog medium supplemented either with indole-3-acetic-acid [IAA] (0.1-0.9 mg/L) and benzyladenine [BA] (0.6-1.2 mg/L) in twenty-five different hormone combinations. Shoot induction via organogenesis was achieved in the tested varieties. Our second objective was to develop an efficient transformation method. The cotyledon explants were cut and soaked in Agrobacterium tumefaciens strain LBA4404 suspension containing ZYMV CP gene (zucchini yellow mosaic virus coat protein gene) After co-cultivation, the explants were put on selection media, which contained the same hormones as the regeneration media, supplemented with claforan (500 mg/L) and kanamycin (100 mg/L). Leafy shoots were transferred to root induction media supplemented with kanamycin (100 mg/L) for further selection. Regenerated plantlets with roots were transferred to sterile soil. Transformation was confirmed by PCR amplification of ZYMV CP gene in transgenic plants.

Molecular detection of Cucumber mosaic virus in Chrysanthemum cultivars

Molecular detection of Cucumber mosaic virus in various chrysanthemum cultivars was attempted by RT-PCR and Southern hybridization tests using CMV specific primers and cloned probes, respectively. A pair of primers was designed from a conserved region of the Cucumber mosaic virus coat protein (CMV-CP) gene, capable of amplifying a product of ~650 bp from various CMV strains. RT-PCR using the total nucleic acid from infected leaf samples and the specific primers resulted in positive amplification of an expected size band of ~650 bp in most of the samples. The identity of the PCR amplicons was checked by Southern hybridization using the a32P-labelled DNA probes prepared from the cloned coat protein gene of a well-identified strain of CMV isolated from Amaranthus. Positive signal of hybridization of PCR products and CMV probes confirmed the identity of fected chrysanthemum samples.

Indonesia

A study has been conducted to screen and multiply calluses and buds of tobacco transformed with the Soybean Mosaic Virus (SMV) coat protein gene and to induce them to plantlet formation by using MS medium with phytohormon treatments. The multiplication of calluses and buds was carried out by sub-culturing on MS medium using 0.3 mg/L NAA+1 mg/L BAP, followed by induction of plantlet formation on MS medium supplemented with NAA and BAP at varied concentration. The results showed that tobacco calluses transformed with SMV coat protein gene was multipliable on MS medium supplemented with NAA at a concentration of 0.3 mg/mL and BAP at 1 mg/L concentration. In this experiment kanamycin was used at a concentration of 100 I¼g/mL which resulted in the viability level of 84%. On MS medium supplemented with 0.3 mg/L NAA, 0.1 mg/L BAP, and 100 I¼g/mL kanamycin, induction of calluses to plantlet formation reached 20% level.

Transgenic Gladiolus plants transformed with the bean yellow mosaic virus coat-protein gene in either sense or antisense orientation.

Transgenic Gladiolus plants transformed with the bean yellow mosaic virus (BYMV) coat-protein (CP) gene in either sense or antisense (AS) orientation were developed using biolistics. Four of the plants were confirmed to carry the CP gene in the sense orientation of the gene and seven plants in the AS orientation. Two of the CP plant lines and all of the AS lines showed DNA rearrangements of the transgene in addition to an intact copy of the transgene. The copy number ranged from one to nine. Of the 11 lines, eight had only one to four copies of the transgene. Transcription of the transgene occurred for three of the CP lines and five of the AS lines as determined by Northern hybridization. All 11 plant lines were challenged with BYMV using controlled aphid transmission. One month following aphid transmission, the transgenic plants were examined by immunoelectron microscopy for presence of the virus. Several transgenic plant lines containing either antiviral transgene showed a lower incidence of infection (percentage of plants infected as detected by immunoelectron microscopy) than the non-transformed plants. Most of the CP- and AS-transgenic plants that did not contain BYMV 1 month after challenge were found to contain BYMV the next season. It appeared that BYMV infection was delayed in the CP- and AS-transgenic lines but that the transgenes did not prevent eventual infection of BYMV. This is the first report of developing a floral bulb crop with antiviral genes to BYMV.

Development of CMV-and TMV-resistant chili pepper: field perfermance and biosafety assessment

Both cucumber mosaic virus (CMV) and tobacco mosaic virus (TMV) coat protein (CP) genes have been transferred to chilli pepper (Capsicum annuum var. Longunt) cultivar 8212 by a modified procedure of Agrobacterium tumefaciens-mediated transformation using hypocotyl as the explant. PCR analysis revealed the presence of both CMV and TMV CP genes in at least 11 primary transformants out of 49 kanamycin-resistant chili pepper plants. Ten T1 lines from five independent transformation events were identified as putative homozygous transgenic lines based on the rooting assay of their T2 seedlings on the kanamycin-containing media. Integration and expression of CMV CP and TMV CP transgenes in one of the homozygous line, 16-13, were confirmed bySouthern blot, RT-PCR and western blot analyses. Line 16-13 was highly resistant to infection of homologous CMV and TMV strains in greenhouse conditions when successively challenged with CMV and TMV or challenged with TMV alone.Futhermore, field trials on T2, T3 and T4 progenies of Line 16-13 were performed on scales of 123, 300 and 10,000 plants, respectively, in consecutive years 1996, 1997 and 1998 with the permission of the Chinese government authority. The transgenic plants displayed delayed symptom development and significantly milder disease severity in field conditions when compared to untransformed chili pepper plants, resulting in 47 and 110% increase in pepper fruit yield in surveys conducted in 1997 and 1998 trials, respectively. Finally, quality analysis and biosafety assesment were performed on transgenic chili pepper fruit concurrently with the control fruit, and demonstrated that the transgenic chili pepper fruit is substantially equivalent to the non-transgenic pepper in terms of the quality and biosafety when consumed as a food additive.