Live-tracking rhizobial switch from rhizosphere to plant-confined colonization modes – Live-Switch

Live-Switch aims to address the largely unexplored question of how rhizobia cells reprogram during the switch from a free-living to a confined host-environment, and how this reprogramming interconnects with specific plant responses. By taking advantage of complementary model legumes systems, Medicago and Lotus, Live-Switch aims to track with cellular resolution the early colonization of host roots by bacteria using transcellular or intercellular entry routes. Using the transcellular-infection Medicago model plant, the project particularly aims to address the spatio-temporal dynamics of bacterial gene or protein fusions (related to signalling, cell proliferation and motility) during infection thread development. Taking advantage of a previously established method, where the plant root system is grown under a plastic film compatible with repeated microscopic observations, we set up suitable conditions for in vivo monitoring bacterial and plant fluorescent fusions at early stages of infection in root hairs. This required the engineering of bacterial genes or proteins (fusions of interest) fused to fluorescent proteins in combination with a reference control for co-dynamic studies in conjunction or not with plant markers. The fusions of interest and the reference control carry fluorescent proteins with non-overlapping emission spectra, allowing the relative quantification of the fluorescent emission signal of the fusions of interest by confocal microscopy. These observations are being carried out in both WT and infection-deficient mutant backgrounds, and are complemented by in vivo co-dynamic analyses of relevant plant fluorescent fusions, to dissect the living plant-bacteria interaction in the infected root hair. To further our understanding of how bacterial motility is regulated during early colonization stages, we are currently analysing the infection phenotype of bacterial motility mutants and exploiting the in vivo data we are currently generating in the project to build an integrated mathematical model of rhizobia progression in the plant infection compartment.
Live-Switch also aims to determine how bacterial reprogramming takes place in two contrasting infection modes (intercellular vs transcellular) and proposes to do this by conducting a comparative study in Lotus plant species establishing these two types of infection. We have therefore adapted plant growth conditions, previously established for in vivo studies in Medicago, to Lotus plant species. These are used to dissect the dynamics of fusions of Lotus genes, selected after transcriptomic studies, during transcellular and intercellular infection modes.

The study of the in vivo dynamics of bacterial genes or protein fusions of interest within the Medicago infection compartment required the design and engineering of suitable constructs. In this frame, we have succeeded in developing a novel expression system allowing live dynamic visualisation of bacterial fluorescent reporter genes or protein fusions, at overall native levels, in single bacterial cells in both free-living and in planta compartments. We were also able to establish brighter fluorescence reference controls expressed under regulated conditions, but still offering sufficient sensitivity to trace individual bacterial cells within the infection thread. Co-imaging analyses of yellow/red fluorescent fusions together with improved cyan reference controls validated the suitability of our system to follow the progression of the bacteria within the plant infection thread. We also showed that this system is reliable for concomitantly quantifying the relative expression of fluorescent fusions in single bacterial cells, inside and outside plant cells. In this context, fluorescent fusions of interest related to bacterial signal production or cell division status have been generated and their dynamics are being analysed in vivo in bacterial cells present in the rhizosphere or within the infection thread. On the plant side, we have demonstrated, using the plant cell wall-associated fluorescent fusion ENOD11, that transient changes in the cell wall interface occur during infection thread maturation and that these changes are defective in a specific infection-defective plant mutant. Specific bacterial markers also accumulate transiently during infection thread maturation and ongoing work will help determine if these plant and bacterial responses are functionally linked.
To elucidate the regulation of bacterial movement we have initiated the phenotypic analyses of bacterial motility mutants. We generated non-motile flagella mutant strains and demonstrated that flagella motility is not essential for Medicago root colonization, although it partially affect its efficiency. In parallel, we have created a mathematical model, based on live imaging data of colonizing rhizobia, to reproduce rhizobia progression within the infection thread. This provided relevant information on the speed and motion of bacterial progression within the thread. Adding future data should help reducing the experimental noise of the model.
Finally, the in vivo confocal microscopy system used for Medicago was adapted to Lotus plant species for imaging the early stages of root colonization during contrasting modes of colonization by transcellular (Mesorhizobium loti MAFF 303099) and intercellular (R. leguminosarum Norway)-infecting rhizobia strains. We generated fluorescent bacterial strains and used them to study early stages of root colonization and the expression profiles of GUS or fluorescent reporters of genes that were selected on the basis of available transcriptomic studies.

Now that we have a reliable experimental system in Medicago for concomitant live cell imaging analyses of both the plant and bacterial symbiotic partners, we will further investigate how key bacterial functions are regulated during the early stages of root infection. The specific questions we want to address are related to understanding how the production of symbiotic signals are dynamically regulated during infection thread development, and the way bacterial cell motility and division are regulated into the thread. These analyses will be complemented by the use of plant mutants and plant specific markers to better understand the plant-bacterial live cross talk. Finally, the live imaging data that will be generated should help to refine the mathematical model for rhizobia progression within the infection thread. Regarding the work on Lotus, stable transgenic lines expressing marker gene promoter-reporter fusions will be genotyped and characterized. Meanwhile, selected reporter fusions will be introduced in Lotus symbiont strains with either intercellular- or transcellular-infection modes, in order to study bacterial reporter gene expression at infection sites in Lotus following inoculation of the selected transgenic lines.

Communications (Conferences)
- 14th European Nitrogen Fixation Conference (Aarhus University, Denmark, virtual meeting) 2021. Impact of flagella loss during the early stages of infection in the Medicago truncatula/Sinorhizobium meliloti symbiotic interaction. Ambre Guillory, Anne Bennion, Anaïs Delers, Elizaveta Krol, Anke Becker, Joëlle Fournier and Fernanda de Carvalho-Niebel (poster and flash talk presentation by A. Guillory)
- 14th European Nitrogen Fixation Conference (Aarhus University, Denmark, virtual meeting) 2021. Signalling and cellular mechanisms in rhizobial plant cell entry. Fernanda de Carvalho-Niebel (plenary talk)
- 14th European Nitrogen Fixation Conference (Aarhus University, Denmark, virtual meeting) 2021. Dynamic cell wall modifications associated with early rhizobial infection thread development in Medicago truncatula. Joëlle Fournier, Killian Coutinho, Audrey Kelner, Marie-Christine Auriac, Lisa Frances and Fernanda de Carvalho-Niebel (poster presentation by J. Fournier)
- 14th European Nitrogen Fixation Conference (Aarhus University, Denmark, virtual meeting) 2021. Growth mode of alphaproteobacterial rhizobia generating cellular asymmetry. Elizaveta Krol, Lisa Stuckenschneider, Simon Schäper, Joana Kästle, Marcel Wagner, Heiko Wendt, Marcus Lechner, Peter L. Graumann, Anke Becker (talk by A. Becker)
- International Symposium for the 40th Anniversary of CIFN/CCG (2022) Multi-talent rhizobium - versatile adaptable symbiotic nitrogen fixer, SynBio chassis, and digital storage medium. Anke Becker (keynote by A. Becker)

Articles
-Dissecting plant cell priming mechanisms for endosymbiotic infection by rhizobia. Ambre Guillory*, Joëlle Fournier*, Audrey Kelner*, Lisa Frances, Marie-Christine Auriac, Martina Beck, Anaïs Delers and Fernanda de Carvalho-Niebel. In preparation.

Legumes establish beneficial nitrogen-fixing symbioses with rhizobia bacteria that supply nitrogen for the plant within specialized root organs, known as nodules. Early stages of this interaction require crucial molecular exchanges in the rhizosphere before the host plant reprograms for bacterial root entry. While most rhizobia species infect the roots of their host via a newly formed root hair intracellular apoplastic compartment called infection thread (IT), others colonize their host plants via alternative and less studied intercellular infection modes. In both cases, rhizobia must switch from a rhizospheric to an infecting state where they become confined within plant apoplastic compartments. Up to now, it has been technically challenging to study bacterial reprogramming within plant-confined environments during early infection, as these events are restricted to a few root cells. Live-Switch is an innovative project aimed at addressing the crucial but largely unexplored question of how rhizobia cope with the drastic transition from free living to infective within-host state. The project will take advantage of Medicago and Lotus model legume systems establishing intracellular or intercellular infection modes, to decipher at the single cell level how rhizobia cells reprogram during the switch from a free-living to a host-confined environment. The project will combine live cell imaging and mutant analyses of both bacteria and plant partners to dissect the dynamics of rhizobial signal production, cell proliferation and motility and the cross talk with the plant partner. The integration of data in a final mathematical model will permit a better understanding of the dynamics of spreading and proliferation of rhizobia in the in planta IT compartment. Live-Switch relies on an ideal integrated and collaborative frame between the French and German groups, that with their solid and complementary expertise in molecular and cell biology of both plant and bacteria partners, will be able to address the exciting question of how dynamic environments shape the in vivo switch of bacterial reprograming to in planta lifestyles.