Quantification of the Volume and Surface Area of Symbiosomes and Vacuoles of Infected Cells in Root Nodules of Medicago truncatula

Abstract

Legumes are able to form endosymbiotic interactions with nitrogen-fixing rhizobia. Endosymbiosis takes shape in formation of a symbiotic organ, the root nodule. Medicago truncatula (M. truncatula) nodules contain several zones representing subsequent stages of development. The apical part of the nodule consists of the meristem and the infection zone. At this site, bacteria are released into the host cell from infection threads. Upon release, bacteria are surrounded by a host cell–derived membrane to form symbiosomes. After release, rhizobia grow, divide, and gradually colonize the entire host cell of the fixation zone of root nodules. Therefore, mature infected cells contain thousands of symbiosomes, which remain as individual units among other organelles. Visualization of the organization and dynamics of the symbiosomes as well as other organelles in infected cells of nodules is essential to understand mechanisms regulating the development of endosymbiosis between plants and rhizobia. To examine this highly dynamic developmental process we designed a useful imaging technique that is based on confocal scanning microscopy combined with different fluorescent dyes and GFP-tagged proteins (Gavrin et al., 2014). Here, we describe a protocol for microscopic observation, 3D rendering, and volume/area measurements of symbiosomes and other organelles in infected cells of M. truncatula root nodules. This protocol can be applied for monitoring the development of different host-microbe interactions whether symbiotic or pathogenic.