Cell-specific reprogramming of legume roots for endosymbiotic infection
Plant Cell and Developmental Biology
Final Report Abstract
Land plants are able to adapt to nutrient-limiting environments by establishing beneficial associations with soil microbes. The symbiotic association between legumes and nitrogen-fixing rhizobia bacteria results in the formation of an elaborated new organ, the root nodule, hosting the microbial partner intracellularly for efficient nitrogen acquisition. Early host recognition and microbial entry are crucial early stages controlled by host-driven signalling pathways to provide intracellular microbial accommodation. Although essential symbiosis plant genes have been identified through genetic approaches, the precise cellular and molecular events underlying bacterial colonization of the root remain poorly understood. The COME-IN project has investigated the signalling and cellular events that take place in host cells engaged in rhizobia infection. We have combined genetics and cell-specific molecular and in vivo technologies in Medicago truncatula and Lotus japonicus model systems to decipher the reprograming of plant cells for endosymbiotic root colonization, and how major symbiotic regulators (CYCLOPS, Ethylene Response Factor Required for Nodulation1/2 (ERN1/2), NIN) contribute to the spatial-temporal regulation of this crucial entry process. By developing cutting-edge cell imaging strategies, we were able to monitor the dynamics of transcription factor (TFs), calcium (Ca2+) and remodeling responses in legume cells undergoing infection. Notably, new sensitive GECO Ca2+ sensors allowed to measure subtle changes in intracellular Ca2+ levels in plant cellular compartments and together with other markers demonstrated the in vivo spatiotemporal dynamics of host infection cellular reprogramming. New sensitive fluorescence FRET pairs enabled us to visualize, by live fluorescence lifetime imaging microscopy (FLIM), phosphorylation-dependent conformational changes of the crucial symbiotic regulator CYCLOPS and in that way obtain crucial information on the regulation of CYCLOPS complex formation with the CCaMK kinase and a new repressor interacting factor. Additionally, we have established FRET-FLIM in vivo that allowed us to analyse conformational changes of the CCaMK/CYCLOPS complex in root hair cells upon stimulus application. We also identified a conserved infection-associated cis-regulatory element driving CYCLOPS expression and discovered the transcription factor (TF)-encoding ERN1 gene as a new target of this factor. Further mutant and RNA profiling analyses revealed the key roles of ERN1/ERN2 regulators for the spatio-temporal control of root infection and their associated candidate targets. Finally, the successful development of INTACT (Isolation of Nuclei TAgged in specific Cell Types) in M. truncatula has recently allowed us to get access by RNA profiling to the small subpopulation of host cells undergoing infection reprogramming, which now opens exciting perspectives in the elucidation of associated rhizobia-induced transcriptional cellular changes.
Publications
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(2016). The symbiosis-related ERN transcription factors act in concert to coordinate rhizobial host root infection. Plant Physiol. 171: 1037-54
Cerri, M.R., Frances, L., Kelner, A., Fournier, J., Middleton, P.H., Auriac, M.C., Mysore, K.S., Wen, J., Erard, M., Barker, D.G., Oldroyd G.E., de Carvalho-Niebel, F.
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(2017). The ERN1 transcription factor gene is a target of the CCaMK/CYCLOPS complex and controls rhizobial infection in Lotus japonicus. New Phytol. 215:323-337
Cerri, M.R., Wang, Q., Stolz, P., Folgmann, J., Frances, L., Katzer, K., Li, X., Heckmann, A.B., Wang, T.L., Downie, J.A., Klingl, A., de Carvalho-Niebel, F., Xie, F., Parniske, M.
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(2018). A simple Agrobacterium tumefaciens-mediated transformation method for rapid transgene expression in Medicago truncatula root hairs. Plant Cell Tiss. Organ Cult. 132: 181–190
Remblière, C., Fournier, J., de Carvalho-Niebel, F., Chabaud, M.
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(2018). Callose-regulated symplastic communication coordinates symbiotic root nodule development. Curr. Biol. 28: 3562-3577
Gaudioso-Pedraza, R., Beck, M., Frances, L., Kirk, P., Ripodas, C., Niebel, A., Oldroyd, G.E.D., Benitez-Alfonso, Y., de Carvalho-Niebel, F.
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(2018). Dual color sensors for simultaneous analysis of calcium signal dynamics in the nuclear and cytoplasmic compartments of plant cells. Front. Plant Sci. 9: 245
Kelner, A., Leitão, N., Chabaud, M., Charpentier, M., de Carvalho-Niebel, F.
