Organization of the TC and TE cellular T-DNA regions in Nicotiana otophora and functional analysis of three diverged TE-6b genes.

Nicotiana otophora contains Agrobacterium-derived T-DNA sequences introduced by horizontal gene transfer (Chen et al., 2014). Sixty-nine contigs were assembled into four different cellular T-DNAs (cT-DNAs) totalling 83 kb. TC and TE result from two successive transformation events, each followed by duplication, yielding two TC and two TE inserts. TC is also found in other Nicotiana species, whereas TE is unique to N. otophora. Both cT-DNA regions are partially duplicated inverted repeats. Analysis of the cT-DNA divergence patterns allowed reconstruction of the evolution of the TC and TE regions. TC and TE carry 10 intact open reading frames. Three of these are TE-6b genes, derived from a single 6b gene carried by the Agrobacterium strain which inserted TE in the N. otophora ancestor. 6b genes have so far only been found in Agrobacterium tumefaciens or Agrobacterium vitis T-DNAs and strongly modify plant growth (Chen and Otten, 2016). The TE-6b genes were expressed in Nicotiana tabacum under the constitutive 2 × 35S promoter. TE-1-6b-R and TE-2-6b led to shorter plants, dark-green leaves, a strong increase in leaf vein development and modified petiole wings. TE-1-6b-L expression led to a similar phenotype, but in addition leaves show outgrowths at the margins, flowers were modified and plants became viviparous, i.e. embryos germinated in the capsules at an early stage of their development. Embryos could be rescued by culture in vitro. The TE-6b phenotypes are very different from the earlier described 6b phenotypes and could provide new insight into the mode of action of the 6b genes.journal articleresearch support, non-u.s. gov't2018 042018 03 23importe

Transgene Silencing and Transgene-Derived siRNA Production in Tobacco Plants Homozygous for an Introduced AtMYB90 Construct

Transgenic tobacco (Nicotiana tabacum) lines were engineered to ectopically over-express AtMYB90 (PAP2), an R2–R3 Myb gene associated with regulation of anthocyanin production in Arabidopsis thaliana. Independently transformed transgenic lines, Myb27 and Myb237, accumulated large quantities of anthocyanin, generating a dark purple phenotype in nearly all tissues. After self-fertilization, some progeny of the Myb27 line displayed an unexpected pigmentation pattern, with most leaves displaying large sectors of dramatically reduced anthocyanin production. The green-sectored 27Hmo plants were all found to be homozygous for the transgene and, despite a doubled transgene dosage, to have reduced levels of AtMYB90 mRNA. The observed reduction in anthocyanin pigmentation and AtMYB90 mRNA was phenotypically identical to the patterns seen in leaves systemically silenced for the AtMYB90 transgene, and was associated with the presence of AtMYB90-derived siRNA homologous to both strands of a portion of the AtMYB90 transcribed region. Activation of transgene silencing in the Myb27 line was triggered when the 35S::AtMYB90 transgene dosage was doubled, in both Myb27 homozygotes, and in plants containing one copy of each of the independently segregating Myb27 and Myb237 transgene loci. Mapping of sequenced siRNA molecules to the Myb27 TDNA (including flanking tobacco sequences) indicated that the 3′ half of the AtMYB90 transcript is the primary target for siRNA associated silencing in both homozygous Myb27 plants and in systemically silenced tissues. The transgene within the Myb27 line was found to consist of a single, fully intact, copy of the AtMYB90 construct. Silencing appears to initiate in response to elevated levels of transgene mRNA (or an aberrant product thereof) present within a subset of leaf cells, followed by spread of the resulting small RNA to adjacent leaf tissues and subsequent amplification of siRNA production

Etude du comportement agronomique de tabacs industriels transgeniques présentant une activité nitrate réductase élevée

National audienc

Co-suppression of nitrate reductase host genes and transgenes in transgenic tobacco plants

International audienc

Cadmium partitioning in transgenic tobacco plants expressing a mammalian metallothionein gene

International audienc

Functional analysis of HMA genes in tobacco and production of low-cadmium tobacco lines

International audienc

Cadmium in transgenic tobacco plants expressing a mammalian metallothionein gene

International audienc

A Eukaryotic Translation Initiation Factor 4E (eIF4E) is responsible for the “va” tobacco recessive resistance to potyviruses

Potato virus Y (PVY), the type member of the genus Potyvirus, is transmitted by aphids and can cause severe damage in several solanaceous family crops. In Nicotiana tabacum, a large genome deletion conferring resistance to PVY, the va gene, is commonly used. This resistance is unfortunately associated with lower tobacco quality parameters, potentially due to the presence of several other important genes in the deleted region. In the present study, we have used next-generation sequencing to analyze the transcriptome of a dozen of tobacco F7 recombinant inbred lines (RILs) segregating for PVY resistance. After comparison with a reference transcriptome, genes differentially expressed between resistant and susceptible plants were identified. About 30 candidate sequences were selected, including a sequence annotated as encoding an eukaryotic translation initiation factor 4E (eIF4E), which was strongly expressed in susceptible plants but not in resistant ones. Other differentially expressed candidates are mostly related to photosynthesis. A complete correlation between susceptibility and expression of this eIF4E sequence was confirmed by amplification in 91 F8 RILs and in a segregating F2 population. The gene was mapped on chromosome 21 of the tobacco genetic map and corresponds to an eIF4E isoform derived from the N. sylvestris parent of tobacco. Final confirmation of the identification of the va gene came from the analysis of two tobacco lines with missense mutations in the eIF4E gene and which correspondingly showed resistance to PVY infection. Screening of a large collection of tobacco accessions revealed a strong correlation between the status of this eIF4 gene and PVY resistance, but the identification of a few resistant accessions with an apparently intact gene suggests the possible existence of alternative resistance sources. The identification of the va gene and of molecular markers linked to it or to the large deletion associated with it opens the way to breeding efforts aimed at breaking the linkage drag associated with this valuable resistance gene

Distribution of the Tnt1 retrotransposon family in the amphidiploid tobacco (Nicotiana tabacum) and its wild Nicotiana relatives

40 ref.International audienceTransposable elements can generate considerable genetic diversity. Here we examine the distribution of the Tnt1 retrotransposon family in representative species of the genus Nicotiana. We show that multiple Tnt1 insertions are found in all Nicotiana species. However, Tnt1 insertions are too polymorphic to reveal species relationships. This indicates that Tnt1 has amplified rapidly and independently after Nicotiana speciation. We compare patterns of Tnt1 insertion in allotetraploid tobacco (N. tabacum) with those in the diploid species that are most closely related to the progenitors of tobacco, N. sylvestris (S-genome donor) and N. tomentosiformis (T-genome donor). We found no evidence for Tnt1 insertion sites of N. otophora origin in tobacco. Nicotiana sylvestris has a higher Tnt1 content than N. tomentosiformis and the elements are distributed more uniformly across the genome. This is reflected in tobacco where there is a higher Tnt1 content in S-genome chromosomes. However, the total Tnt1 content of tobacco is not the sum of the two modern-day parental species. We also observed tobacco-specific Tnt1 insertions and an absence of tobacco Tnt1 insertion sites in the diploid relatives. These data indicate Tnt1 evolution subsequent to allopolyploidy. We explore the possibility that fast evolution of Tnt1 is associated with ‘genomic-shock’ arising out of interspecific hybridization and allopolyploidy