Gold‐FISH enables targeted NanoSIMS analysis of plant‐associated bacteria

Summary

Bacteria colonize plant roots and engage in reciprocal interactions with their hosts. However, the contribution of individual taxa or groups of bacteria to plant nutrition and fitness is not well characterized due to a lack of inￂﾠsitu evidence of bacterial activity.

To address this knowledge gap, we developed an analytical approach that combines the identification and localization of individual bacteria on root surfaces via gold￢ﾀﾐbased inￂﾠsitu hybridization with correlative NanoSIMS imaging of incorporated stable isotopes, indicative of metabolic activity.

We incubated Kosakonia strain DS￢ﾀﾐ1￢ﾀﾐassociated, gnotobiotically grown rice plants with 15N￢ﾀﾓN2 gas to detect inￂﾠsitu N2 fixation activity. Bacterial cells along the rhizoplane showedￂﾠheterogeneous patterns of 15N enrichment, ranging from the natural isotope abundance levels up to 12.07 at% 15N (average and median of 3.36 and 2.85 at% 15N, respectively, n￢ﾀﾉ=￢ﾀﾉ697 cells).

The presented correlative optical and chemical imaging analysis is applicable to a broad range of studies investigating plant￢ﾀﾓmicrobe interactions. For example, it enables verification of the inￂﾠsitu metabolic activity of host￢ﾀﾐassociated commercialized strains or plant growth￢ﾀﾐpromoting bacteria, thereby disentangling their role in plant nutrition. Such data facilitate the design of plant￢ﾀﾓmicrobe combinations for improvement of crop management.