Techniques to Study Common Root Responses to Beneficial Microbes and Iron Deficiency.

Iron (Fe) plays a central role in the vital processes of a plant. The Fe status of a plant influences growth and immunity, but it also dictates interactions of roots with soil microbiota through the production of Fe mobilizing, antimicrobial fluorescent phenolic compounds called coumarins. To adapt to low Fe availability in the soil, plants deploy an efficient Fe deficiency response. Interestingly, this Fe deficiency response is hijacked by root-colonizing microbes in the root microbiome to establish a mutually beneficial relationship. In this chapter, we describe how we cultivate plants and microbes to study the interaction between plants, beneficial rhizobacteria, and the plant's Fe deficiency response. We describe (a) how we study activity and localization of these responses by assessing gene-specific promoter activities using GUS assays, (b) how we visualize root-secreted coumarins in response to Fe deficiency and colonization by beneficial rhizobacteria, and (c) how we prepare our samples for metabolite extraction and reverse-transcriptase quantitative PCR to analyze the expression of marker genes.