Localization of the MAX effector AvrRvi6 during the colonization of apple leaf by Venturia inaequalis

Scientific context
During infection, fungal pathogens such as Venturia inaequalis secrete hundreds of effector proteins that can hijack the host cell machinery and promote disease (Oliveira-Garcia et al., 2023). However, plants evolved receptor proteins that can recognize pathogen effectors and induce strong and efficient defense reactions. When an effector triggers immunity, it is considered as an avirulent factor (Avr) as it usually prevents disease. Resistance genes typically encode cytoplasmic receptors from the NLR (NBS-LRR) family. Another class of resistance protein called RLP (Receptor-Like Protein) is localized at the plasma membrane and recognizes apoplastic Avr proteins (Jones et al., 2024).
The resistance gene Rvi6 which recognizes AvrRvi6 is an RLP predicted to localize at the plasma membrane. Functional studies in the heterologous system Nicotiana benthamiana showed that the Rvi6-dependent immune reaction requires the secretion of AvrRvi6 in the apoplast. Interestingly, AvrRvi6 belongs to the structural family of MAX effectors which has been only described as translocated cytoplasmic proteins recognized by NLR (Guillen et al., 2015). So far, the only information about AvrRvi6 localization is the product of functional studies in heterologous systems. It is therefore critical to better characterize the localization of AvrRvi6 during the colonization of apple leaf tissues by V. inaequalis. If validated, the apoplastic MAX effector AvrRvi6 will expend the biological functions of MAX effector and will help to better understand the role of these conserved fungal effectors.

Objectives
The goal of this training project is to gather information about AvrRvi6 protein localization. These localization studies will be performed in both heterologous and homologous systems using a wide array of complementary approaches. The main questions to answer are:
– Is AvrRvi6 secreted during infection ?
– When is AvrRvi6 produced during infection ?
– After secretion, does AvrRvi6 remain in the apoplast or is it translocated inside the host cells?
– Does AvrRvi6 co-localize with a specific cellular structure ?
Implementation
To answer these questions, several methods will be used:
– Homologous translational fusions: Before the beginning of the training period, V. inaequalis will be transformed with AvrRvi6 tagged to fluorescent reporters. The trainee will have to first characterize the transformed isolate and then perform spot inoculation before imaging the infection site using a confocal microscope. This will allow us to monitor the localization and potential translocation of AvrRvi6 during infection.
– Heterologous translational fusions: AvrRvi6 tagged to fluorescent reporter will be transiently over-expressed in N. benthamiana leaves alone or in combination of a tagged Rvi6. This will allow us to monitor the sub-cellular localization of AvrRvi6 and eventually its co-localization with Rvi6. Potential localization in plasma membrane nanodomain or internalization could be observed (Jaillais et al., 2024).
– Immuno-localization: We have developed an antibody against AvrRvi6 native protein that remains to be tested for its specificity and sensitivity. If it is suitable, Immuno-localization in infected apple leaves will be performed. Additionally, western blot analysis on apoplastic fluids or crude tissue extracts will help to pinpoint the cellular compartment where AvrRvi6 is localized. These immuno-based approaches will be complementary to the homologous translational fusions.
The diversity of approaches used will increase the chances of yielding conclusive data. As it is the first cytological study of effector secretion and localization in V inaequalis it will likely uncover important knowledge about this fungus.

References:
Guillen, K. de, Ortiz-Vallejo, D., Gracy, J., Fournier, E., Kroj, T. & Padilla, A. (2015) Structure Analysis Uncovers a Highly Diverse but Structurally Conserved Effector Family in Phytopathogenic Fungi Xu, J.-R. (Ed.). PLOS Pathogens, 11
Jaillais, Y., Bayer, E., Bergmann, D.C., Botella, M.A., Boutté, Y., Bozkurt, T.O., et al. (2024) Guidelines for naming and studying plasma membrane domains in plants. Nature Plants, 10. https://doi.org/10.1038/s41477-024-01742-8.
Jones, J.D.G., Staskawicz, B.J. & Dangl, J.L. (2024) The plant immune system: From discovery to deployment. Cell, 187, 2095–2116. https://doi.org/10.1016/j.cell.2024.03.045.
Oliveira-Garcia, E., Yan, X., Oses-Ruiz, M., Paula, S. de & Talbot, N.J. (2023) Effector-triggered susceptibility by the rice blast fungus Magnaporthe oryzae. New Phytologist.

How to apply:
We are seeking a highly motivated master student with background in molecular biology, microbiology or plant sciences. Skills in microscopy or molecular cloning will be appreciated. A PhD project on related topic is to be consider as a follow up study.
Please send resume and cover letter to:

mael.baudin@inrae.fr and sirine-aouatef.benmamar@inrae.fr