Production of agrocinopine A by Ipomoea batatas agrocinopine synthase in transgenic tobacco and its effect on the rhizosphere microbial community.

Agrobacterium tumefaciens is a bacterial pathogen that causes crown gall disease on a wide range of eudicot plants by genetic transformation. Besides T-DNA integrated by natural transformation in vegetative tissues of plants by pathogenic Agrobacterium, previous reports have indicated that T-DNA sequences originating from ancestral Agrobacterium sp. are present in the genomes of all cultivated sweet potato (Ipomoea batatas) analyzed. Expression of Agrobacterium-derived agrocinopine synthase (ACS) gene was detected in leaf and root tissues of sweet potato, suggesting that the plant can produce agrocinopine, a sugar-phosphodiester opine considered to be utilized by Agrobacterium in crown gall. To validate the product synthesized by I. batatas ACS (IbACS), we introduced IbACS into tobacco under a constitutive promoter. High voltage paper electrophoresis followed by alkaline silver nitrate staining detected the production of an agrocinopine-like substance in IbACS1-expressing tobacco, and further MS and NMR analyses of the product confirmed that IbACS can produce agrocinopine A from natural plant substrates. The partially purified compound was biologically active in an agrocinopine A bioassay. 16S rRNA amplicon sequencing and meta-transcriptome analysis revealed that the rhizosphere microbial community of tobacco was affected by the expression of IbACS. A new species of Leifsonia (actinobacteria) was isolated as an enriched bacterium in the rhizosphere of IbACS1-expressing tobacco. This Leifsonia sp. can catabolize agrocinopine A produced in tobacco, indicating that the production of agrocinopine A attracts rhizosphere bacteria which can utilize this sugar-phosphodiester. These results suggest a potential role of IbACS conserved among sweet potato cultivars in manipulating their microbial community.