Controlling Endophyte Colonization to Reduce TSNA in Tobacco Leaves

Tremendous interests have been focusing on decreasing accumulation of tobacco-specific nitrosamines (TSNA) during curing, processing and aging of tobacco. How to suppress the biosynthesis of the most carcinogenic TSNA, 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (nitrosonicotine ketone, NNK), is currently the top priority in harm reduction for tobacco breeder, biologist and chemist. Nitrosation of nicotine oxidation product by nitrite released by microorganisms is the crucial step for NNK production. It was well documented that reducing microbe populations on or in tobacco by chemical treatments resulted in decreased TSNA including NNK. However, the endophytic bacterial or fungi cannot be controlled by chemicals. Understanding the molecular mechanisms underlying colonization by endophyte in tobacco during growth and especially curing will provide potential for engineering tobacco plants to decrease NNK accumulation. How the endophytes infect and transduce in tobacco plants and how they persist under the curing conditions remains unaddressed. Tantalizing evidence revealed that colonization in plants by endophytic bacterial was affected by the signaling pathway related to nodulation and mycorrhizal symbiosis. Given that the significant contribution of endophytic microorganisms to TSNA production, we hypothesize that the reduced level of TSNA, including NNK, can be achieved by restriction of endosymbiosis. Here, we are proposing to identify genes involving in endosymbiosis and to generate endophyte-free mutants by using CRISPR/Cas-mediated targeted mutation.