Periodic Reporting for period 1 - VAMEAP (Virulence Activities of the Metalloprotease Effector AVR-Pita from the rice blast fungus Magnaporthe oryzae)
Período documentado: 2021-01-04 hasta 2023-01-03
In the VAMEAP project, we focused on AVR-Pita, an effector protein secreted by M. oryzae during the early steps of rice infection. AVR-Pita is particularly interesting as it can be detected by rice plants that have the cognate Pita resistance locus and efficiently protect the plant. Despite the recognition and the negative impact of AVR-Pita on the fungus fitness when confronted to resistant plant, this effector is conserved in M. oryzae populations. This suggest that this protein is otherwise important for the biology of M. oryzae. In addition, AVR-Pita shows features of metalloprotease enzyme which is uncommon among fungal effectors as most of them are small proteins without any predicted enzymatic function.
Despite being identified more than 20 years ago, very little is known about the effector AVR-Pita. Next to nothing have been characterized on its virulence functions and on the metalloprotease activity. Several studies have been published on the molecular mechanisms behind the recognition of AVR-Pita by the Pita resistance locus but some of data are contradictory and the overall processes remain unclear.
With this project, we focused on the characterization of the biology and virulence activities of AVR-Pita. Our research addressed: (i) The localization of AVR-Pita protein during the infection (ii) The importance of AVR-Pita for the virulence of M. oryzae (iii) The enzymatic activity of AVR-Pita and its host targets.
The research of AVR-Pita biology will help to further characterize the resistance mechanisms underlying the Pita resistance locus. The VAMEAP project will also help to better understand fungal metalloproteases and their role in the biology of the fungus.
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.