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.

Root-specific expression of opine genes and opine accumulation in some cultivars of the naturally occurring genetically modified organism Nicotiana tabacum.

Previous studies have shown that Nicotiana tabacum contains three Agrobacterium-derived T-DNA sequences inherited from its paternal ancestor Nicotiana tomentosiformis. Among these, the TB locus carries an intact mannopine synthase 2' gene (TB-mas2'). This gene is similar to the Agrobacterium rhizogenes A4-mas2' gene that encodes the synthesis of the Amadori compound deoxyfructosyl-glutamine (DFG or santhopine). In this study we show that TB-mas2' is expressed at very low levels in N. tomentosiformis and in most N. tabacum cultivars; however, some cultivars show high TB-mas2' expression levels. The TB-mas2' promoter sequences of low- and high-expressing cultivars are identical. The low/high level of expression segregates as a single Mendelian factor in a cross between a low- and a high-expression cultivar. pTB-mas2'-GUS and pA4-mas2'-GUS reporter genes were stably introduced in N. benthamiana. Both were mainly expressed in the root expansion zone and leaf vasculature. Roots of tobacco cultivars with high TB-mas2' expression contain detectable levels of DFG.

A complex eIF4E locus impacts the durability of va resistance to Potato virus Y in tobacco.

Many recessive resistances against potyviruses are mediated by eukaryotic translation initiation factor 4E (eIF4E). In tobacco, the va resistance gene commonly used to control Potato virus Y (PVY) corresponds to a large deletion affecting the eIF4E-1 gene on chromosome 21. Here, we compared the resistance durability conferred by various types of mutations affecting eIF4E-1 (deletions of various sizes, frameshift or nonsense mutations). The 'large deletion' genotypes displayed the broadest and most durable resistance, whereas frameshift and nonsense mutants displayed a less durable resistance, with rapid and frequent apparition of resistance-breaking variants. In addition, genetic and transcriptomic analyses revealed that resistance durability is strongly impacted by a complex genetic locus on chromosome 14, which contains three other eIF4E genes. One of these, eIF4E-3, is rearranged as a hybrid gene between eIF4E-2 and eIF4E-3 (eIF4E-2-3 ) in the genotypes showing the most durable resistance, while eIF4E-2 is differentially expressed between the tested varieties. RNA-seq and quantitative reverse transcriptase-polymerase chain reaction experiments demonstrated that eIF4E-2 expression level is positively correlated with resistance durability. These results suggest that besides the nature of the mutation affecting eIF4E-1, three factors linked with a complex locus may potentially impact va durability: loss of an integral eIF4E-3, presence of eIF4E-2-3 and overexpression of eIF4E-2. This latter gene might act as a decoy in a non-productive virus-plant interaction, limiting the ability of PVY to evolve towards resistance breaking. Taken together, these results show that va resistance durability can in large part be explained by complex redundancy effects in the eIF4E gene family.

Evolutionary diversification of type-2 HDAC structure, function and regulation in Nicotiana tabacum.

Type-2 HDACs (HD2s) are plant-specific histone deacetylases that play diverse roles during development and in responses to biotic and abiotic stresses. In this study we characterized the six tobacco genes encoding HD2s that mainly differ by the presence or the absence of a typical zinc finger in their C-terminal part. Of particular interest, these HD2 genes exhibit a highly conserved intron/exon structure. We then further investigated the phylogenetic relationships among the HD2 gene family, and proposed a model of the genetic events that led to the organization of the HD2 family in Solanaceae. Absolute quantification of HD2 mRNAs in N. tabacum and in its precursors, N. tomentosiformis and N. sylvestris, did not reveal any pseudogenization of any of the HD2 genes, but rather specific regulation of HD2 expression in these three species. Functional complementation approaches in Arabidopsis thaliana demonstrated that the four zinc finger-containing HD2 proteins exhibit the same biological function in response to salt stress, whereas the two HD2 proteins without zinc finger have different biological function.

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.

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.