The VAMEAP project was initiated in January 2021 and terminated in October 2022, two months prior the end of the action, due to the recruitment of the fellowship recipient Maël Baudin at the University of Angers (France).
We first focused on the sub-cellular localization of AVR-Pita during the infection of rice leaves by M. oryzae. To do so, we generated chimeric constructions of AVR-Pita fused to the fluorescent protein mCherry and expressed them under the native AVR-Pita promoter in the M. oryzae Guy11 strain. The transformed strain and appropriate control were spot inoculated on rice and barley and imaged using confocal microscopy. We did not observe an obvious translocation of AVR-Pita inside the host cell which suggest that this effector remains in the apoplast. However, the expression level of the chimeric proteins were very low, therefore additional experiments are required to reach a final conclusion.
To evaluate the role of AVR-Pita during rice infection, we used avr-pita mutants of M. oryzae in the Guy11 genetic background. The mutants and appropriate controls were inoculated on a panel of susceptible rice varieties. The lesions size and number were quantified 7 days post inoculation by image analysis. We carried out four independent biological replicates but could not observe a significant impact of AVR-Pita deletion on M. oryzae virulence in our conditions.
Biochemical approaches were used to characterize the enzymatic activities and host targets of AVR-Pita. We were not successful in the production and purification of AVR-Pita recombinant protein in E. coli. However, a collaborator succeeded in this task at the end of the action and this material permits further investigation. To assess the metalloprotease activity of AVR-Pita, we collaborated with Prof. Renier Van der Hoorn at the University of Oxford to perform activity-based labeling. Finally, putative host targets of AVR-Pita were identified using yeast-two-hybrid to screen a rice cDNA library. We identified several strong candidates including one previously published AVR-Pita-interacting proteins.
Regarding the dissemination of the work performed during the VAMEAP project, a total of three scientific presentations in conferences have been performed by the researcher. The project will lead to two publications in high impact journals and a review article on M. oryzae. We also participated in the “fête de la science” events in 2021 and 2022 which aim to explain science and research activities to the general public. We generated posters and a simplify game to explain the virulence activities of pathogens. While no website has been developed for the project specifically, we designed and updated of a public website gathering the research projects and activities of the CIME (Cereal Immunity and Magnaporthe Effectors) research team that hosted the action.
