Nicotiana Bigelovii Research Articles

Structure and Genome Organization of a Novel Fiji Strain of Sweet Potato Vein Clearing Virus Identified by High-Throughput Sequencing.

ABSTRACTThe complete genome of a Sweet potato vein clearing virus (SPVCV) isolate infecting a quarantined sweet potato accession from Fiji was determined. Sequence comparisons revealed the highest nucleotide sequence identity of 94.6% with that of the SPVCV type species, an isolate from the Dominican Republic. The virus was mechanically transmitted to Nicotiana bigelovii plants.

A putative antiviral role of plant cytidine deaminases

Background: A mechanism of innate antiviral immunity operating against viruses infecting mammalian cells has been described during the last decade. Host cytidine deaminases ( e.g., APOBEC3 proteins) edit viral genomes, giving rise to hypermutated nonfunctional viruses; consequently, viral fitness is reduced through lethal mutagenesis. By contrast, sub-lethal hypermutagenesis may contribute to virus evolvability by increasing population diversity. To prevent genome editing, some viruses have evolved proteins that mediate APOBEC3 degradation. The model plant Arabidopsis thaliana genome encodes nine cytidine deaminases ( AtCDAs), raising the question of whether deamination is an antiviral mechanism in plants as well. Methods: Here we tested the effects of expression of AtCDAs on the pararetrovirus Cauliflower mosaic virus (CaMV). Two different experiments were carried out. First, we transiently overexpressed each one of the nine A. thaliana AtCDA genes in Nicotiana bigelovii plants infected with CaMV, and characterized the resulting mutational spectra, comparing them with those generated under normal conditions. Secondly, we created A. thaliana transgenic plants expressing an artificial microRNA designed to knock-out the expression of up to six AtCDA genes. This and control plants were then infected with CaMV. Virus accumulation and mutational spectra where characterized in both types of plants. Results: We have shown that the A. thaliana AtCDA1 gene product exerts a mutagenic activity, significantly increasing the number of G to A mutations in vivo, with a concomitant reduction in the amount of CaMV genomes accumulated. Furthermore, the magnitude of this mutagenic effect on CaMV accumulation is positively correlated with the level of AtCDA1 mRNA expression in the plant. Conclusions: Our results suggest that deamination of viral genomes may also work as an antiviral mechanism in plants.

A DNA cytophotometric study of salt adaptation in Allium cepa and Nicotiana bigelovii

Abstract Nuclear DNA was measured by Feulgen cytophotometry in NaCl-exposed Allium cepa roots and Nico-tiana bigelovii calli cultured in vitro. Analysis of the differentiation zone of A. cepa roots grown in water showed that only 2.8% of the nuclei had DNA contents higher than 4 C, where 2 C nuclei were predominant. In roots grown in salt water, 2 C nuclei were less numerous than 4 C nuclei and the DNA content was greater than 4 C in 19.2% of the cells, with 1.4% of the cells having a DNA content of 16 C. Two C, 4 C, 8 C, and a few 16 C nuclei, together with many nuclei with intermediate DNA content values, occurred in both the control and salt-treated calli of N. bigelovii. About 49.1% of the nuclei had DNA values around 2 C in the controls, while the percentage of cells with nuclei with higher DNA content values increased in calli as the NaCl concentration in the medium increased. Fifteen per cent of the nuclei had DNA values around 16 C in calli grown on media with 10 gr l−1 NaCl added, compared to 2.8...

Potato latent virus: a proposed new species in the genus Carlavirus

A previously undescribed carlavirus, potato latent virus (PotLV), was found infecting the potato cultivar Red La Soda imported from the USA. The particles were filamentous and slightly curved, with modal lengths of 530 and 670 nm. The 11 kDa protein encoded downstream from the coat protein contained a ‘zinc‐finger’ motif characteristic of carlaviruses, and RT–PCR using a carlavirus‐specific primer gave a PCR product of 857 bp. Antibodies produced to PotLV did not detect other carlaviruses when used in ELISA and the coat‐protein nucleic acid sequence of PotLV showed < 67% similarity with the other carlaviruses tested. The closest similarity was with the Andean strain of potato virus S. Unusually for a carlavirus, PotLV systemically infected Nicotiana bigelovii, N. glutinosa, N. rustica, N. tabacum and Physalis floridana.

Stability of habituation in Nicotiana bigelovii var. bigelovii shoots regenerated from habituated callus

In Nicotiana bigelovii var. bigelovii the formation of callus habituated for auxin and cytokinin can be induced with high frequency after a very short treatment of the hypocotylar tissues with 2,4-dichlorophenoxyacetic acid (2,4-D). Some habituated callus lines exhibit the capacity to regenerate normal shoots for a period of several months. In this study we report that stem and leaf explants, when excised from N. bigelovii var. bigelovii shoots regenerated from 10 habituated callus lines, were able to dedifferentiate and grow, in hormone-free medium and with a very high frequency, as habituated calluses. The developmental process during the shoot regeneration from the previously habituated callus using 2,4-D in this variety of N. bigelovii, seems to imply the possible irreversibility of the habituated state through the regeneration phase.

Temporal and Spatial Appearance of Recombinant Viruses Formed Between Cauliflower Mosaic Virus (CaMV) and CaMV Sequences Present in Transgenic Nicotiana bigelovii

Cauliflower mosaic virus (CaMV) strain CM1841 is able to recombine with a CaMV transgene sequence present in Nicotiana bigelovii. In the present study we have characterized the temporal and spatial appearance of recombinant viruses formed between CM1841 and the transgene within individual transgenic plants. CM1841 infections were initiated by mechanical inoculation and by agro-inoculation to nontransformed N. bigelovii and transgenic N. bigelovii that expressed the gene VI product of CaMV strain D4. In agroinoculated transgenic plants, inoculated leaf tissue turned necrotic around the point of agroinocu-lation, while chlorotic lesions appeared in the leaves inoculated with CM1841 virions. The first systemic symptoms in both agroinoculated and mechanically inoculated transgenic N. bigelovii consisted of necrotic patches. The predominant type of virus recovered from the inoculated and first systemically infected leaves was the wild-type CM1841 rather than a recombinant. As the infection progressed in the transgenic plants, symptoms changed from necrosis in the lower leaves to a chlorotic mosaic in the upper leaves. This shift in symptom type was associated with the recovery of recombinant viruses, indicating that the recombinants predominated only in later stages of pathogenesis.

Manganese toxicity to different growth processes in vitro in Nicotiana

Manganese toxicity to germination, callus induction and shoot regeneration was studied on three cultivars of Nicotiana tabacum: BEL W3, Burley 21, Bright NC 944. All materials were cultured on MS solid medium containing 0.1 (control), 2, 5, 10, 15 and 20 mM Mn, to which 5 µM NAA and 5 µM kinetin were added for callus induction and shoot regeneration. Mn toxicity to callus growth was tested using habituated callus of Nicotiana bigelovii var. bigelovii grown on MS medium without growth regulators. Mn concentrations higher than 2 mM were toxic for germination, and concentrations higher than 5 mM were toxic for callus induction, shoot regeneration and callus growth. Among the cultivars examined, Bright tobacco appeared more tolerant to high Mn concentrations during callus formation and shoot regeneration. However, many regenerated plants capable of growing in vitro in the presence of 2 and 5 mM Mn were obtained.

A chimeric and truncated mitochondrial atpA gene is transcribed in alloplasmic cytoplasmic male-sterile tobacco with Nicotiana bigelovii mitochondria

Protoplast fusions were performed between two sexually produced alloplasmic male-sterile tobacco cultivars, with cytoplasms from Nicotiana bigelovii [Nta (big)S] and N. undulata[Nta(und)S], both of which exhibit homeotic-like phenotypes affecting the petal and stamen whorls. Among the fusion products obtained, both novel male-sterile and pollen-producing cybrid plants were identified. Of the pollen-producing cybrid plants, all of which were indehiscent, some had flowers with stamens that appeared normal when compared to male-fertile tobacco plants. Other hybrid plants were incompletely restored as they exhibited petaloid structures on the anther-bearing pollen-producing stamens. In this study, gel-blot analyses with mitochondrial geneprobes were conducted comparing the mitochondrial DNA of cybrids and male-sterile parents. It was found that the flower morphology typical of the Nta(big)S parental plants, as well as of the novel male-sterile cybrids, coincided with the presence of a chimeric atpA gene copy where an open reading frame of unknown origin was found to be linked in-frame to the 3'-end of a truncated atpA gene. RNA gel-blot hybridizations revealed the presence of atpA transcripts in the malesterile parent Nta(big)S and novel male-sterile cybrids, but which were absent in cybrids capable of pollen production.

RNA editing of the tobacco mitochondrial orf38/220.

An alloplasmic, male-sterile tobacco line obtained by sexual hybridization, resulting in introgression of the Nicotiana bigelovii cytoplasm into the Nicotiana tabacum species, is characterized by male-sterile flowers that have filaments with flat-fringed ends instead of normal stamens, and a split corolla (Burk, 1960; Chaplin, 1964). A mitochondrially encoded chimeric gene, orf38/220, corresponding to a fusion of an orf coding for 38 amino acids and the sequence coding for the carboxy-terminal 220 amino acids of the ATPase a subunit (atpA), was found to be associated with the cytoplas'mic male-sterile phenotype (P. Bergman, W. Kofer, G. Hakansson, and K. Glimelius, unpublished data). Our studies show that this chimeric gene is a target for posttranscriptional editing in which seven cytosines are replaced by uridines in the mitochondrial mRNA (Table I). At the protein level, three Pro codons (codons 180,221, and 246) were changed to Leu codons, as previously described for sugar beet atpA mRNAs (Senda et al., 1993). We have identified four additional sites of editing, codons 96 and 115 in which the Pro codon is edited to Ser, codon 142 in which Ser becomes a Leu codon, and codon 155 in which the Leu codon is transformed into a Phe codon. Eight independent clones were sequenced; three of them were fully edited, whereas in five of the clones codon 115 was unedited. Furthermore, in one of these clones codon

Expansion of Viral Host Range through Complementation and Recombination in Transgenic Plants.

We have shown previously that gene VI of cauliflower mosaic virus (CaMV) strain D4 governs systemic infection of Nicotiana bigelovii and that transgenic N. bigelovii expressing the D4 gene VI product can complement at least one CaMV isolate for long-distance transport. We have now found that DNA of two other isolates of CaMV recombine with the gene VI coding sequence present in the transgenic plants. The formation of recombinant viruses occurs as a consequence of CaMV replication, involving two template switches during reverse transcription of the CaMV RNA to DNA. The first template switch occurs at the 5[prime] end of the 35S RNA to the gene VI mRNA produced by the transgenic plants. A second switch occurs at the 5[prime] end of the gene VI mRNA back to the 35S RNA. We also demonstrate that CaMV can acquire sequences from transgenic plants that alter the symptomatology and host range of the virus, an observation that may have important risk assessment implications for strategies using pathogen-derived resistance to protect plants against virus diseases.

Identification of Domains within Gene VI of Cauliflower Mosaic Virus That Influence Systemic Infection of Nicotiana bigelovii in a Light-Dependent Manner

Gene VI of cauliflower mosaic virus strains D4 and W260 is an important determinant of systemic infection in solanaceous species (Qiu and Schoelz, 1992). To investigate whether D4 and W260 share any sequences within gene VI that determine their solanaceous host range, we characterized more completely the regions of gene VI involved in systemic infection of Nicotiana bigelovii. We found that two domains within gene VI, which corresponded approximately to the 5' third and middle third of gene VI, influenced systemic infection of N. bigelovii. Exchange of these domains between D4, W260, and CaMV strain CM1841, a strain which is unable to systemically infect any solanaceous plant, revealed different virus combinations that could specify systemic infection of N. bigelovii when plants were grown under two different lighting conditions. Systemic infection of N. bigelovii by D4/CM1841 chimeric viruses required only the 5' third (domain 1) of gene VI of D4. In contrast, systemic infection of N. bigelovii by W260/CM1841 chimeric viruses required both the domain 1 and middle third of gene VI (domain 2) of W260, as well as two other regions primarily containing W260 genes I and II, and gene IV. The genetic requirements for systemic infection by chimeric viruses were not as stringent when plants were grown under low light conditions. Specifically, domain 2 of gene VI of D4 contained sequences sufficient for D4/CM1841 chimeric viruses to systemically infect N. bigelovii at low light intensity. We sequenced gene VI of strain W260 and compared differences in the deduced amino acid sequences between W260 and the previously published sequences of D4 and CM1841. There was only one amino acid position in domain 1 of gene VI, and no sites in domain 2, in which W260 and D4 agreed with each other and differed from CM1841. Consequently, the host range studies and sequence information indicate that different sequences within gene VI of CaMV strains W260 and D4 are responsible for systemic infection of N. bigelovii.

Three regions of cauliflower mosaic virus strain W260 are involved in systemic infection of solanaceous hosts

We have identified regions of CaMV strain W260 involved in systemic infection of Nicotiana bigelovii and Datura stramonium by constructing chimeric viruses between W260 and CM1841, a strain that is unable to systemically infect any solanaceous host. All of the chimeric viruses systemically infected turnips, demonstrating the viability of the chimeric viruses in a host that is susceptible to both CM 1841 and W260. Three regions of W260, containing primarily genes I, IV, and VI, influenced the ability of that virus to induce systemic symptoms in the solanaceous hosts. The involvement of the regions containing gene I, and to a lesser extent gene IV, were affected by environmental conditions. When infected plants were grown under conditions of low light, low temperatures (18°), and short days (9.5-hr day), the source of genes I and IV no longer influenced whether a chimeric virus moved systemically. As light intensity and day length were increased, the genetic requirements became more stringent and genes I and IV, as well as gene VI, had to be derived from W260.

Isolation of glycolipids from the surface lipids of Nicotiana bigelovii and their distribution in Nicotiana species.

Isolation of glycolipids from the surface lipids of Nicotiana bigelovii and their distribution in Nicotiana species.

Host range control of cauliflower mosaic virus

Studies with recombinant genomes of cauliflower mosaic virus (CaMV) strains D4, CM1841, and Cabb-B have shown that a host range determinant of CaMV is encoded within the first half of region VI, a gene which codes for P62, an inclusion body protein. In order to further study the host specificity of CaMV, a fourth CaMV strain, W260, was chosen that has a host range that is intermediate between D4 and CM1841. To determine which portion of the W260 genome controls systemic spread, recombinant viruses made between this strain and CM 1841 and D4 were tested for their ability to systemically infect several solanaceous plants ( Datura stramonium, Nicotiana edwardsonfi, and Nicotiana bigelovii). The first half of gene VI specified the type of local lesions and systemic spread of recombinant strains in D. stramonium. In N. edwardsonii, it was found that the first half of gene VI controlled the type of local lesion formed but systemic spread was dependent on the whole of gene VI. In N. bigelovii the number of genes that determined systemic spread of CaMV varied with the strain of CaMV. Systemic spread of D4 in N. bigelovii was dependent on the first half of gene VI. In contrast, systemic spread of W260 in the same host was dependent on the whole of gene VI and another locus which mapped within genes I-V. Consequently, it appears that other viral proteins may interact with P62 or that P62 may function well in some hosts only in combination with compatible forms of other viral proteins.

Region VI of cauliflower mosaic virus encodes a host range determinant.

A domain of cauliflower mosaic virus (CaMV) which controls systemic spread in two solanaceous hosts (Datura stramonium and Nicotiana bigelovii) was mapped to the first half of open reading frame 6. Whereas ordinary strains of CaMV are unable to infect solanaceous species except to replicate locally in inoculated leaves, a new CaMV strain (D4) induces chlorotic local lesions and systemically infects both D. stramonium and N. bigelovii. To determine which portion of the CaMV genome controls systemic spread of the virus in solanaceous hosts, nine recombinant genomes constructed between D4 and two ordinary strains of the virus were tested for their ability to infect solanaceous hosts. A 496-base-pair DNA segment comprising the first half of open reading frame 6 specified the type of local lesions and systemic spread of the virus in solanaceous hosts. Exchange of this segment of the genome between strains of CaMV converted a compatible host reaction to an incompatible (hypersensitive) one in response to infection. This suggests that the gene VI protein interacts with the plant to suppress hypersensitivity, the normal response of solanaceous hosts to CaMV infection.

Region VI of cauliflower mosaic virus encodes a host range determinant.

A domain of cauliflower mosaic virus (CaMV) which controls systemic spread in two solanaceous hosts (Datura stramonium and Nicotiana bigelovii) was mapped to the first half of open reading frame 6. Whereas ordinary strains of CaMV are unable to infect solanaceous species except to replicate locally in inoculated leaves, a new CaMV strain (D4) induces chlorotic local lesions and systemically infects both D. stramonium and N. bigelovii. To determine which portion of the CaMV genome controls systemic spread of the virus in solanaceous hosts, nine recombinant genomes constructed between D4 and two ordinary strains of the virus were tested for their ability to infect solanaceous hosts. A 496-base-pair DNA segment comprising the first half of open reading frame 6 specified the type of local lesions and systemic spread of the virus in solanaceous hosts. Exchange of this segment of the genome between strains of CaMV converted a compatible host reaction to an incompatible (hypersensitive) one in response to infection. This suggests that the gene VI protein interacts with the plant to suppress hypersensitivity, the normal response of solanaceous hosts to CaMV infection.

Expression of hormone-autotrophy in tissues of plantlets regenerated from habituated callus of Nicotiana bigelovii

Experiments presented in the literature show that habituation, a phenomenon characterized by a newly acquired capacity of in vitro cultured plant cells to produce growth regulatory substances, occurs at high rates, leaves the heritably altered cells totipotent and is regularly reversible.

Host plant reactions, physical properties and serology of three isolates of Andean potato latent virus from Peru

SUMMARYThree isolates of Andean potato latent virus (APLV) (Caj, Hu, Ay) each infected twenty‐seven species of plants in the families Amaranthaceae, Chenopodiaceae, Cucurbitaceae and Solanaceae but differed somewhat in the symptoms they induced. Nicotiana bigelovii and N. clevelandii proved the most useful diagnostic hosts. Symptoms were sometimes produced by all three isolates in cultivated and wild potatoes. In sap from systemically infected N. bigelovii and N. clevelandii leaves, all three isolates remained infective when diluted to 10‐6 and when stored at room temperature for at least 3 wk. The thermal inactivation points were 65–70 °C for Hu and Ay, but 75–80 °C for Caj. All three isolates differed serologically from Col, the original isolate of APLV, forming spurs in gel diffusion tests. No serological difference was found between Hu and Ay, but both formed spurs in reciprocal reactions with Caj. The data from light absorption, particle morphology and protein molecular weight for Caj, Hu and Ay are similar to those reported for other tymoviruses. APLV was found widespread in Andean countries.

Beetle, contact and potato true seed transmission of Andean potato latent virus

SUMMARYA species of the flea beetle (Epitrix sp.) transmitted Andean potato latent virus (APLV) from Datura stramonium to D. stramonium, Nicandra physalodes and potato, and from N. physalodes to N. physalodes and D. stramonium. Although the acquisition and inoculation feeding times used were varied, this transmission always occurred at low efficiency. By contrast, APLV was very readily transmitted in tests in which infected plants of D. stramonium, N. physalodes, Nicotiana bigelovii and potato were brushed against healthy plants of one or more of these species. A low level of seed transmission of APLV occurred in potato.

Habituation in Nicotiana bigelovii tissue cultures: Different behaviour of two varieties1

Habituation in Nicotiana bigelovii tissue cultures: Different behaviour of two varieties1