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Virulence Activities of the Metalloprotease Effector AVR-Pita from the rice blast fungus Magnaporthe oryzae

Periodic Reporting for period 1 - VAMEAP (Virulence Activities of the Metalloprotease Effector AVR-Pita from the rice blast fungus Magnaporthe oryzae)

Reporting period: 2021-01-04 to 2023-01-03

Magnaporthe oryzae is a fungal pathogen that can cause tremendous losses on staple cereals crops such as rice and wheat. Like most pathogens, M. oryzae uses effector proteins to manipulate host cell processes and overcome plant immune reactions. These small proteins are secreted by the pathogen during the infection and can remain in the apoplast or can be transferred inside the host cell. In some cases, effector proteins are detected by the host plant which triggers a quick and strong immune response. Effectors therefore play a crucial role in determining the outcome (disease or resistance) of the interaction.
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.
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.
The tools and knowledge generated by the VAMEAP project will help to bring a better understanding to the general field of plant-pathogen interactions. This knowledge is critical to design new management strategies for a more sustainable crop production. The understanding of how plant immunity works and how pathogen can evade this immune system is important to evaluate the durability of resistant varieties and for the rational engineering of new genetic resistance. More specifically, the characterization of AVR-Pita in the VAMEAP project will yield essential knowledge on fungal metalloprotease effectors and their virulence activities. Once completed, the localization studies initiated during the action, will be very informative to better understand the recognition of AVR-Pita by the host. The current published model, which is being challenged, states that AVR-Pita needs to be translocated to directly interact with a cytoplasmic resistance protein. Our preliminary data suggest that AVR-Pita remains in the apoplast which imply different recognition mechanisms. Beyond the academic impacts of the project, the participation to public events such as science fairs helped to raise societal awareness about the challenges of plant pathology and the role of public research.
Rice epidermal cells infected by Magnaporthe oryzae