A FAMILY OF SMALL SECRETED PROTEINS FROM RHIZOPHAGUS IRREGULARIS CONTRIBUTE TO ARBUSCULAR MYCORRHIZA SYMBIOSIS

Arbuscular mycorrhizal (AM) fungi are able to colonize more than 80% of all land plants, providing several benefits to their host, such as an increased nutritious status or an increased resistance to pathogens. The molecular mechanisms underlying plant colonization are still unknown, due to the lack of molecular tools to genetically modify the fungus. To date, only two secreted proteins from Rhizophagus irregularis have been described and known to participate in the symbiosis. However, based on recent transcriptomic data, more secreted proteins have arisen as potential participating candidates in development of the symbiosis. This project aimed at mining the predicted secretome of R. irregularis to discover additional secreted proteins contributing to the development and establishment of the AM symbiosis. In this study, a bioinformatic approach, associated with expression assays, led to the identification and characterization of twenty-four candidate effectors from the AM fungus R. irregularis. Two candidate effectors were exclusively targeting the plant nucleus and nucleolus. Both of these candidates showed a potential effect on the oomycete plant pathogen Phytophthora palmivora when transiently expressed in planta. Overexpression of the other three candidate effectors in Nicotiana benthamiana roots showed evidence for an increase in AM symbiosis. Amongst them, one candidate effector, R5, showed expression evidence in R. irregularis when colonizing N. benthamiana. The gene was found to be part of a gene family containing 15 members, most of them showing a strong and in planta restricted expression. BLASTp analysis has revealed sequence similarity with a characterized effector from Fusarium oxysporum, secreted in xylem 6 (Six6). Yeast-two-hybrid assay revealed a vascular plant one-zinc- finger (VOZ1) transcription factor as a potential plant target to R5. However, in vivo validation of the interaction failed at confirming the association. Additionally, preliminary data seem to indicate that other members of the family might participate in the symbiosis, as an increased level of AM colonization was observed in Medicago truncatula hairy roots upon expression of some of the genes. Future work would be needed to validate these results together with expanding the search for plant targets for other members of the family.