Multiple Disease Resistance Loci Affect Soilborne Disease Resistance in Tobacco (Nicotiana tabacum).

Phytophthora nicotianae and Ralstonia solanacearum are two of the most important pathogens affecting tobacco worldwide. Greater insight regarding genetic systems controlling resistance to these two soilborne pathogens, as well as identification of DNA markers associated with genomic regions controlling this resistance, could aid in variety development. An evaluation of 50 historical tobacco lines revealed a high positive correlation between resistances to the two pathogens, preliminarily suggesting that some genomic regions may confer resistance to both pathogens. A quantitative trait loci (QTL) mapping experiment designed to investigate the genetic control of soilborne disease resistance of highly resistant 'K346' tobacco identified four QTL significantly associated with resistance to P. nicotianae (explaining 60.0% of the observed phenotypic variation) and three QTL to be associated with R. solanacearum resistance (explaining 50.3% of the observed variation). The two QTL with the largest effect on Phytophthora resistance were also found to be the QTL with the greatest effects on resistance to Ralstonia. This finding partially explains previously observed associations between resistances to these two pathogens among U.S. current cultivars and within breeding populations. Further study is needed to determine whether these relationships are due to the same genes (i.e., pleiotropy) or favorable coupling-phase linkages that have been established over time.

Identification of CYP82E21 as a functional nicotine N-demethylase in tobacco flowers.

In the tobacco plant, nicotine N-demethylase enzymes (NND) belonging to the cytochrome P450 family catalyse the conversion of nicotine to nornicotine, the precursor of the carcinogenic tobacco-specific N-nitrosamine, N-nitrosonornicotine. To date three demethylase genes, namely CYP82E4, CYP82E5 and CYP82E10, have been shown to be involved in this process, while the related CYP82E2 and CYP82E3 genes are not functional. We have identified a further gene named CYP82E21 encoding a putative nicotine N-demethylase closely related to the CYP82E genes. The CYP82E21 gene was found in all Nicotiana tabacum cultivars analysed and originates from the tobacco ancestor Nicotiana tomentosiformis. We show that, in contrast to all other previously characterized NND genes, CYP82E21 is not expressed in green or senescent leaves, but in flowers, more specifically in ovaries. The nicotine N-demethylase activity of CYP82E21 was confirmed by ectopic expression of the coding sequence in a tobacco line lacking functional CYP82E4, CYP82E5 and CYP82E10 genes, resulting in an eightfold increase of nicotine demethylation compared to the control plants. Furthermore, nornicotine formation can be reduced in ovaries by introducing a CYP82E21-specific RNAi construct. Together, our results demonstrate that the CYP82E21 gene encodes a functional ovary-specific nicotine N-demethylase.