Rapid analysis of legume root nodule development using confocal microscopy

•  A rapid method for detailed analysis of nodule formation has been developed. •  Inoculated root tissues were stained with SYTO 13, a cell-permeant fluorescent nucleic acid-binding dye, and visualized using confocal laser scanning microscopy (CLSM). Structures with high concentrations of DNA and RNA, such as plant cell nuclei and bacteria, labeled strongly. The autofluorescent properties of cell walls made it possible to use CLSM to visualize both plant and rhizobial structures and generate a three-dimensional reconstruction of the root and invading bacteria. •  This method allowed clear observation of stages and structures important in nodule formation, such as rhizobial attachment to root hairs, hair deformation, infection thread ramification, nodule primordium development and nodule cell invasion. Bacteroid structures were easily assessed without the need for fixation that might alter cellular integrity. Plant nodulation mutants with phenotypic differences in thread growth, cellular invasion and plant defense response were also documented. •  Multiple samples can be assessed using detailed microscopy without the need for extensive preparative work, labor-intensive analysis, or the generation of genetically modified samples.