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.

NtTPN1: a RPP8-like R gene required for Potato virus Y-induced veinal necrosis in tobacco.

Potato virus Y (PVY) is one of the most damaging viruses of tobacco. In particular, aggressive necrotic strains (PVYN ) lead to considerable losses in yield. The main source of resistance against PVY is linked to the va locus. However, va-overcoming PVY isolates inducing necrotic symptoms were observed in several countries. In this context, it is important to find va-independent protection strategies. In a previous study, the phenotyping of 162 tobacco varieties revealed 10 accessions that do not carry the va allele and do not exhibit typical PVYN -induced veinal necrosis. Despite the absence of necrotic symptoms, normal viral accumulation in these plants suggests a va-independent mechanism of tolerance to PVYN -induced systemic veinal necrosis. Fine mapping of the genetic determinant(s) was performed in a segregating F2 population. The tolerance trait is inherited as a single recessive gene, and allelism tests demonstrated that eight of the 10 tolerant varieties carry the same determinant. Anchoring the linkage map to the tobacco genome physical map allowed the identification of a RPP8-like R gene, called NtTPN1 (for Nicotiana tabacum Tolerance to PVY-induced Necrosis1), with the same single-nucleotide polymorphism in the eight tolerant accessions. Functional assays using homozygous NtTPN1 EMS mutants confirmed the role of NtTPN1 in the tolerance phenotype. PVYN -induced systemic veinal necrosis in tobacco likely represents an inefficient defense response with hypersensitive response-like characteristics. The identification of NtTPN1 opens breeding options to minimize the impact of emerging and so far uncontrolled va-breaking necrotic PVY isolates.

Parental transposable element loads influence their dynamics in young Nicotiana hybrids and allotetraploids.

The genomic shock hypothesis suggests that allopolyploidy is associated with genome changes driven by transposable elements, as a response to imbalances between parental insertion loads. To explore this hypothesis, we compared three allotetraploids, Nicotiana arentsii, N. rustica and N. tabacum, which arose over comparable time frames from hybridisation between increasingly divergent diploid species. We used sequence-specific amplification polymorphism (SSAP) to compare the dynamics of six transposable elements in these allopolyploids, their diploid progenitors and in corresponding synthetic hybrids. We show that element-specific dynamics in young Nicotiana allopolyploids reflect their dynamics in diploid progenitors. Transposable element mobilisation is not concomitant with immediate genome merger, but occurs within the first generations of allopolyploid formation. In natural allopolyploids, such mobilisations correlate with imbalances in the repeat profile of the parental species, which increases with their genetic divergence. Other restructuring leading to locus loss is immediate, nonrandom and targeted at specific subgenomes, independently of cross orientation. The correlation between transposable element mobilisation in allopolyploids and quantitative imbalances in parental transposable element loads supports the genome shock hypothesis proposed by McClintock.

Deep sequencing of the ancestral tobacco species Nicotiana tomentosiformis reveals multiple T-DNA inserts and a complex evolutionary history of natural transformation in the genus Nicotiana.

Nicotiana species carry cellular T-DNA sequences (cT-DNAs), acquired by Agrobacterium-mediated transformation. We characterized the cT-DNA sequences of the ancestral Nicotiana tabacum species Nicotiana tomentosiformis by deep sequencing. N. tomentosiformis contains four cT-DNA inserts derived from different Agrobacterium strains. Each has an incomplete inverted-repeat structure. TA is similar to part of the Agrobacterium rhizogenes 1724 mikimopine-type T-DNA, but has unusual orf14 and mis genes. TB carries a 1724 mikimopine-type orf14-mis fragment and a mannopine-agropine synthesis region (mas2-mas1-ags). The mas2' gene codes for an active enzyme. TC is similar to the left part of the A. rhizogenes A4 T-DNA, but also carries octopine synthase-like (ocl) and c-like genes normally found in A. tumefaciens. TD shows a complex rearrangement of T-DNA fragments similar to the right end of the A4 TL-DNA, and including an orf14-like gene and a gene with unknown function, orf511. The TA, TB, TC and TD insertion sites were identified by alignment with N. tabacum and Nicotiana sylvestris sequences. The divergence values for the TA, TB, TC and TD repeats provide an estimate for their relative introduction times. A large deletion has occurred in the central part of the N. tabacum cv. Basma/Xanthi TA region, and another deletion removed the complete TC region in N. tabacum. Nicotiana otophora lacks TA, TB and TD, but contains TC and another cT-DNA, TE. This analysis, together with that of Nicotiana glauca and other Nicotiana species, indicates multiple sequential insertions of cT-DNAs during the evolution of the genus Nicotiana.

Inactivation of two newly identified tobacco heavy metal ATPases leads to reduced Zn and Cd accumulation in shoots and reduced pollen germination.

Cadmium (Cd) is a non-essential heavy metal, which is classified as a "known human carcinogen" by the International Agency for Research on Cancer (IARC). Understanding the mechanisms controlling Cd distribution in planta is essential to develop phytoremediation approaches as well as for food safety. Unlike most other plants, tobacco (Nicotiana tabacum) plants translocate most of the Cd taken up from the soil, out of the roots and into the shoots, leading to high Cd accumulation in tobacco shoots. Two orthologs of the Arabidopsis thaliana HMA2 and HMA4 Zn and Cd ATPases that are responsible for zinc (Zn) and Cd translocation from roots to shoots were identified in tobacco and sequenced. These genes, named NtHMAα and NtHMAß, were more highly expressed in roots than in shoots. NtHMAα was expressed in the vascular tissues of both roots and leaves as well as in anthers. No visual difference was observed between wild-type plants and plants in which the NtHMAα and NtHMAß genes were either mutated or silenced. These mutants showed reduced Zn and Cd accumulation in shoots as well as increased Cd tolerance. When both NtHMA genes were silenced, plant development was altered and pollen germination was severely impaired due to Zn deficiency. Interestingly, seeds from these lines also showed decreased Zn concentration but increased iron (Fe) concentration.

Transcriptome analysis of tobacco BY-2 cells elicited by cryptogein reveals new potential actors of calcium-dependent and calcium-independent plant defense pathways.

Cryptogein is a proteinaceous elicitor secreted by the oomycete Phytophthora cryptogea, which induces a hypersensitive response in tobacco plants. We have previously reported that in tobacco BY-2 cells treated with cryptogein, most of the genes of the phenylpropanoid pathway were upregulated and cell wall-bound phenolics accumulated. Both events were Ca(2+) dependent. In this study, we designed a microarray covering a large proportion of the tobacco genome and monitored gene expression in cryptogein-elicited BY-2 cells to get a more complete view of the transcriptome changes and to assess their Ca(2+) dependence. The predominant functional gene categories affected by cryptogein included stress- and disease-related proteins, phenylpropanoid pathway, signaling components, transcription factors and cell wall reinforcement. Among the 3819 unigenes whose expression changed more than fourfold, 90% were Ca(2+) dependent, as determined by their sensitivity to lanthanum chloride. The most Ca(2+)-dependent transcripts upregulated by cryptogein were involved in defense responses or the oxylipin pathway. This genome-wide study strongly supports the importance of Ca(2+)-dependent transcriptional regulation of regulatory and defense-related genes contributing to cryptogein responses in tobacco.

Differential impact of retrotransposon populations on the genome of allotetraploid tobacco (Nicotiana tabacum).

LTR-retrotransposons contribute substantially to the structural diversity of plant genomes. Recent models of genome evolution suggest that retrotransposon amplification is offset by removal of retrotransposon sequences, leading to a turnover of retrotransposon populations. While bursts of amplification have been documented, it is not known whether removal of retrotransposon sequences occurs continuously, or is triggered by specific stimuli over short evolutionary periods. In this work, we have characterized the evolutionary dynamics of four populations of copia-type retrotransposons in allotetraploid tobacco (Nicotiana tabacum) and its two diploid progenitors Nicotiana sylvestris and Nicotiana tomentosiformis. We have used SSAP (Sequence-Specific Amplification Polymorphism) to evaluate the contribution retrotransposons have made to the diversity of tobacco and its diploid progenitor species, to quantify the contribution each diploid progenitor has made to tobacco's retrotransposon populations, and to estimate losses or amplifications of retrotransposon sequences subsequent to tobacco's formation. Our results show that the tobacco genome derives from a turnover of retrotransposon sequences with removals concomitant with new insertions. We have detected unique behaviour specific to each retrotransposon population, with differences likely reflecting distinct evolutionary histories and activities of particular elements. Our results indicate that the retrotransposon content of a given plant species is strongly influenced by the host evolutionary history, with periods of rapid turnover of retrotransposon sequences stimulated by allopolyploidy.