LAUSR

Leguminous plants have established a mutualistic endosymbiotic interaction with nitrogen-fixing rhizobia to secure nitrogen sources in new specialised organs called root nodules. Before nodule formation, the development of early symbiotic structures is essential for rhizobia docking, internalization, targeted delivery and intracellular accommodation. We have recently reported that overexpression of stress-induced mitogen-activated protein kinase (SIMK) in alfalfa affects root hair, nodule and shoot formation, which raised the questions how SIMK may modulate these processes. In particular, detailed subcellular spatial distribution, activation and developmental relocation of SIMK during the early stages of alfalfa nodulation remain unclear. Here, we qualitatively and quantitatively characterised SIMK distribution patterns in Ensifer meliloti-infected root hairs using live-cell imaging and immunolocalization, employing alfalfa stable transgenic lines with genetically manipulated SIMK abundance and kinase activity. In the SIMKK-RNAi line, showing downregulation of SIMKK and SIMK, we found considerably decreased accumulation of phosphorylated SIMK around infection pockets and infection threads. However, this was strongly increased in the GFP-SIMK line, constitutively overexpressing GFP-tagged SIMK. Thus, genetically manipulated SIMK modulates root hair capacity to form infection pockets and infection threads. Employment of advanced light-sheet fluorescence microscopy (LSFM) on intact plants allowed gentle and non-invasive imaging of spatiotemporal interactions between root hairs and symbiotic Ensifer meliloti, while immunofluorescence detection confirmed that SIMK was activated in these locations. Our results shed new light on SIMK spatiotemporal participation in early interactions between alfalfa and Ensifer meliloti, and its internalization into root hairs, showing that local accumulation of active SIMK indeed modulates early nodulation in alfalfa.