Rhizophagus irregularis DAOM197198 modulates the root ubiquitinome of Medicago truncatula in the establishment and functioning of arbuscular mycorrhizal symbiosis.

The regulation of cellular protein homeostasis involves the ubiquitin-proteasome system (UPS) by selectively targeting misfolded or end-of-life proteins. The involvement of the UPS in biotic stresses has been studied mainly in plant-pathogen interactions and poorly in plant-mutualistic interactions. Here, we studied through proteomic approaches (western blot, pull-down of polyubiquinated proteins and nano-LC-MS-MS analysis), the involvement of the UPS during the establishment of the mutualistic interaction between the arbuscular mycorrhizal fungus Rhizophagus irregularis DAOM197198 and the roots of Medicago truncatula, as well as in the established symbiosis. Roots of M. truncatula seedlings were harvested 0 h, 3 h, 6 h, 9 h, 12 h, 24 h and 15 days post-inoculation. We characterized a short-time and a-long-time response of the root ubiquitinome. Some proteins as such as flotilins or involved in the translational machinery were less-ubiquitinated, suggesting the facilitation of the de novo synthesis of proteins required to the establishment of arbuscular mycorrhizal symbiosis. In contrast, other proteins as transporters involved in plant nutrition through the direct pathway (i.e., MtPT5) and some enzymes involved in the lipid biosynthesis pathways were more-ubiquitinated, highlighting their putative degradation. In addition, Cdc48 protein accumulates in roots from 9 to 24 h post-inoculation, suggesting a role of Cdc48 in the transitory immune response during plant-fungal interactions. The activity of the UPS is consequently central in the establishment and functioning of arbuscular mycorrhizal symbiosis by modulating protein ubiquitination.