Nicotiana noctiflora Hook. Genome Contains Two Cellular T-DNAs with Functional Genes

Abstract

Agrobacterium (Rhizobium)-mediated transformation leads to the formation of crown galls or hairy roots on infected plants. These effects develop due to the activity of T-DNA genes, gathered on a big plasmid, acquired from agrobacteria during horizontal gene transfer. However, a lot of plant species are known to contain such sequences, called cellular T-DNAs (cT-DNAs), and maintain normal phenotypes. Some of the genes remain intact, which leads to the conclusion of their functional role in plants. In this study, we present a comprehensive analysis of the cT-DNAs in the Nicotiana noctiflora Hook. genome, including gene expression and opine identification. Deep sequencing of the Nicotiana noctiflora genome revealed the presence of two different cT-DNAs, NnT-DNA1 and NnT-DNA2, which contain the intact genes iaaM, iaaH, acs, orf13, orf13a, and orf14. According to the expression analysis results, all these genes are most active in roots in comparison with other organs, which is consistent with data on cT-DNA gene expression in other plant species. We also used genetic engineering approaches and HPTLC and HPLC-MS methods to investigate the product of the acs gene (agrocinopine synthase), which turned out to be similar to agrocinopine A. Overall, this study expands our knowledge of cT-DNAs in plants and brings us closer to understanding their possible functions. Further research of cT-DNAs in different species and their functional implications could contribute to advancements in plant genetics and potentially unveil novel traits with practical applications in agriculture and other fields.

Keywords: Nicotiana noctiflora Hook.; cT-DNA; horizontal gene transfer.

Figure 1

Maps of NnT-DNA1 (a) and NnT-DNA2 (b). Green arrows indicate sequences homologous to Agrobacterium T-DNA genes. Asterisks indicate intact ORFs. Thin blue arrows mark inverted repeats. The length of the red line corresponds to 1000 nucleotides.

Figure 2

Relative expression of genes located on NnT-DNA1 and NnT-DNA2. This figure shows the relative presence of mRNA in different organs of N. noctiflora. The expression levels are shown relative to the expression level of the gapdh housekeeping gene.