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.

Suspension-cultured BY-2 tobacco cells produce and mature immunologically active house dust mite allergens.

The replacement of crude allergen extracts by selected allergens currently represents a major goal for the improvement of allergy diagnosis and immunotherapy. Indeed, the development of molecularly defined vaccines would facilitate both standardization and enhance batch-to-batch reproducibility as well as treatment specificity. In this study, we have investigated the potential of tobacco plant cells to produce biologically active forms of the two major allergens from the house dust mite. A detailed characterization of these plant-made allergens has shown similar proteolytic maturation and folding as well as comparable immunoreactivity to their natural counterparts. Altogether, our results exemplify that suspension-cultured BY-2 tobacco cells represent a low cost and environmentally safe expression system suitable to produce recombinant allergens from Dermatophagoides pteronyssinus under a form appropriate for diagnostic and therapeutic purposes.