Manipulation of the plant microbiome: antimicrobial peptide expression in nicotiana attenuata

Endophytic bacteria are believed to be important for plant growth, plant health and plant resistance. But the exploration of these roles in nature is difficult. To be able to manipulate endophytic bacteria of Nicotiana attenuata, I made 11 different genotypes for the constitutive expression of antimicrobial peptides under a 35S promoter. Several plant lines showed gene expression loss and a promoter methylation analysis by bisulfite sequencing revealed epigenetic gene silencing. The augmentation of the silencing occurred independently from a generational change and plants developed epigenetic changes solely during normal plant development with rapid methylation increase in somatic cells. Lines with low promoter methylation and trans-generational stable gene expression where further analyzed in peptide abundance. The peptide accumulation of each genotype was directly analyzed in the apoplast by a new and versatile nanoUPLC-MSE method suitable for the absolute quantification of peptides allowing the selection of lines with high peptide levels. The in planta activities against 19 different bacterial strains were examined by leaf infiltration and confirmed in vivo activity only for a single genotype, the so called ICE line, expressing a knottin from the common ice plant. This line showed high taxa specific activity against most Bacilli but did not affect proteobacteria and most actinobacteria. The different native Bacillus isolates showed distinct susceptibilities against the peptide indicating a high heterogeneity within wild isolates, which allows bacterial communities to compensate for antimicrobial activity by diversity. Plants were further used for comparative growth studies in the native environment, showed no reduction in growth performance and harbored a similar bacterial community, observed by high throughput pyrosequencing