Gold-FISH enables targeted NanoSIMS analysis of plant-associated bacteria.

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 in situ 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-based in situ hybridization with correlative NanoSIMS imaging of incorporated stable isotopes, indicative of metabolic activity. We incubated Kosakonia strain DS-1-associated, gnotobiotically grown rice plants with N-N gas to detect in situ N fixation activity. Bacterial cells along the rhizoplane showed heterogeneous patterns of N enrichment, ranging from the natural isotope abundance levels up to 12.07 at% N (average and median of 3.36 and 2.85 at% N, respectively, n = 697 cells). The presented correlative optical and chemical imaging analysis is applicable to a broad range of studies investigating plant-microbe interactions. For example, it enables verification of the in situ metabolic activity of host-associated commercialized strains or plant growth-promoting bacteria, thereby disentangling their role in plant nutrition. Such data facilitate the design of plant-microbe combinations for improvement of crop management.