Understanding the mechanisms by which legumes are infected by symbiotic, nitrogen-fixing bacteria (rhizobia) may prove crucial in the pursuit of bio-engineering nitrogen-fixation in current food crops, and as a result, reducing reliance on harmful nitrogen fertilisers. Despite the importance of infection to the establishment of the symbiosis, the physical and biochemical changes that roots undergo to entrap rhizobia and drive the infection process, remain poorly understood. In this study, we sought to test whether fluorescence lifetime imaging of autofluorescence within root hair cell walls undergoing infection could reveal new information in the details of the infection process.
The process of symbiotic infection in the model legume Medicago truncatula begins when a rhizobia cell makes contact with the growing tip of a root hair cell [ 3]. The root hair then curls around the bacteria, trapping it between the cell walls which then soften to form a tube called the infection thread that carries the rhizobial cells into the root [ 2]. Fournier, et al. [ 1] noted an intriguing accumulation of autofluorescent material in a defined area of the root hair cell wall adjacent to the trapped bacteria ( Figure 1A), and that the infection thread itself was characterized by a lack of autofluorescence. In our study, we examined these autofluorescence phenomena via fluorescence lifetime imaging (FLIM) to determine whether the changes were the product of unique compounds. To achieve this, live M. truncatula roots were imaged via Leica’s FALCON on a STELLARIS 8 confocal at three timepoints post inoculation with rhizobia. The resulting FLIM data revealed that the accumulation of autofluorescence material noted by Fournier, et al. [ 1] is distinct from the surrounding cell wall ( Figure 1B, double arrowhead), and therefore must be the product of the accumulation of a distinct compound.
Fig. 1.
Lifetime imaging of Medicago truncatula root hair cells infected with Sinorhizobium meliloti reveals distinct autofluorescent components. (A) Original observation of autofluorescence accumulation by Fournier, et al [ 1]. Double arrowhead points out accumulation of autofluorescence in root hair cell wall adjacent to the infection chamber (dashed arrow). Black arrowhead shows an mCherry tagged rhizobia. (B-C) Lifetime imaging of autofluorescence in infected root hairs reveals a distict, shorter lifetime component in root hair cell wall (double arrowhead), and infection chamber (dashed arrow), and a longer lifetime component associated with the infection thread. Colour shows fluorescence lifetime scale of 0 ns – 2 ns. Scale bar = 10 µm.
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