Deciphering the Restriction and the Mobilisation of Phenotypic Robustness By the Virulence Regulatory Network in the Plant Pathogen Ralstonia Solanacearum Using Hybrid Genome-Scale Modelling

Living organisms have to face changing environments and are subject to the inherent errors made by their internal machinery. Therefore, they need to exhibit a certain degree of robustness in order to sustain their resilience in front of these perturbations. One highly complex biological system in which robustness is paramount property is the host-pathogen interaction (Kitano & Oda, MSB 2006). Indeed, during the interaction, the pathogen hacks the host machinery in order to collect resources and counter immune system response, whereas the host reacts to maintain its own physiological functions. Since the robustness is a property emerging at network level, understanding the phenotypic robustness of a pathogen requires conducting genome-scale analysis. Numerous studies investigated robustness through metabolic networks or regulatory networks. However few addressed the question in combined analysis and with the emphases on comparing broad phenotypic responses.

On purpose to analyze the variation of the phenotypic robustness of a pathogen in front of environmental as well as genetic perturbations, we developed a methodology assessing the impact of regulatory network on the phenotypic robustness. The method relies on simulating maintenance of phenotypes demands through metabolism using constrain based modelling under the constraints coming from the regulatory network solved using discrete logical modelling. We reconstructed a cell-scale model of the world-wide devastating plant pathogen bacteria Ralstonia solanacearum which represents the first plant pathogen reconstructed so far at genome-scale level. The virtual-cell model reconstructed encompasses a high-quality genome-scale metabolic network (2085 biochemical reactions, 275 exchange reactions), a macromolecule secretion module dedicated to virulence phenotypes (363 biochemical reactions, 171 exchanged reactions) and the virulence regulatory network (1443 biochemical interactions). Among regulated genes 444 are included in the gene-to-protein-to-reaction association network of the biochemical reaction modules.

From the compendium of experimental and in silico analysis, we deciphered that the virulence regulatory network restricts the robustness of a broad range of phenotypes under environmental as well as genetic perturbations. This restriction operates on various robustness sources, i.e. versatility, genetic redundancy as well as pathway redundancy, and impacts significantly with specific patterns the virulence phenotypes compared to core-cell functions (proliferation). We demonstrate experimentally that the restriction of versatility by the virulence regulatory network can even lead the wild-type cell to harbor a drastic reduction of its fitness even in favorable environments. Hence, this observation provides a strong explanatory mechanism of the phenomenon of defective-virulence variants emergence reported within the Ralstonia solanacearum populations. In another side, we decipher that the functional redundancy within pathways involved in core-phenotypes (proliferation) which are under the control of the virulence regulatory network are specifically mobilised in the context of the host infection. This evolved overlay of core metabolic enzymes and “virulence dedicated” enzymes could support the survival of the pathogen specifically within the plant. Hence, principles, methods and tools exposed in our study may provide an interesting framework for elaborating control methods for human, animal and plant warfare thanks to a better comprehension of the phenotypes robustness of pathogens.