The oomycete potato pathogen Phytophthora infestans has caused devastation during the Irish Potato Famine in the 1840s. P. infestans still causes major problems in the potato industry, and is mostly controlled using agrochemicals. P. infestans manipulates its host with hundreds of secreted proteins, including secreted protease inhibitors (cystatin-like EpiC1 and EpiC2B) that specifically target extracellular host proteases.
Interestingly, tomato plants carrying the Cf-2 resistance gene are able to sense protease inhibitors that target the same host proteases. This protease inhibitor is Avr2, secreted by the fungal tomato pathogen Cladosporium fulvum. Avr2 recognition involves the Cf-2 resistance gene product. Recognition leads to the hypersensitive response (HR), which includes programmed cell death and stops further pathogen growth. Avr2 recognition by the receptor-like Cf-2 protein also requires Rcr3, a secreted papain-like protease of tomato. Rcr3 is inhibited by Avr2 and this complex is detected by Cf-2. EpiCs from P. infestans have a high affinity for the related protease C14, but inhibit Rcr3 only with very low affinity, thereby presumably escaping recognition by Cf-2.
The AIM OF THIS PROJECT is to engineer Rcr3/C14 proteases to create a high-affinity interaction with EpiCs to activate Cf-2. This increase interaction with EpiCs would result in a synthetic resistance gene against P. infestans that can be used to protect potato plants. This proof-of-concept will inspire similar strategies to engineer resistance to other pathogens that secrete protease inhibitors. Meanwhile, protease mutants with reduced affinity to EpiCs will be used to test inhibitor-resilient proteases as crop protection strategy.
Two parallel approaches will be taken: 1) transfer the high affinity EpiC-binding site from C14 into Rcr3; and 2) transfer Cf-2 interaction region of Rcr3 into C14. Both strategies are supported by our extensive knowledge on the Avr2/Rcr3/Cf-2 system, including molecular modelling of proteases and inhibitors. Experiments include molecular cloning and site-directed mutagenesis using Golden Gate cloning; transient expression of the proteases by agroinfiltration; detection of protease activity using fluorescent activity-based probes; affinity determination using competitive protease activity profiling; and Avr2/EpiC-triggered cell death (HR) and ion leakage assays by transient co-expression or protein injection into leaves carrying Cf-2.
This project builds on a long history of well-received scientific publications with a deep impact in plant immunity. The project is based on fast, transient assays and combines molecular biology with biochemistry and plant pathology.
The student should have experience in molecular cloning and some experience with protein biochemistry. We seek a team worker with strong plant immunity interests, who is able and eager to manage large experiments.
Ilyas, M., Hörger, A. C., Bozkurt, T. O., Van den Burg, H. A., Kaschani, F., Kaiser, M., Belhaj, K., Smoker, M., Joosten, M. H. A. J., Kamoun, S., and Van der Hoorn, R. A. L. (2015) Functional divergence of two secreted immune proteases of tomato. Current Biol. 25, 2300-2306.
Dong, S., Stam, R., Cano, L. M., Song, J., Sklenar, J., Yoshida, K., Bozkurt, T. O., Oliva, R., Liu, Z., Tian, M., Win, J., Banfield, M. J., Jones, A. M., Van der Hoorn, R. A. L., and Kamoun, S. (2014) Effector specialization in a lineage of the Irish potato famine pathogen. Science 343, 552-555.
Kaschani, F., Shabab, M., Bozkurt, T., Shindo, T., Schornack, S., Gu, C., Ilyas, M., Win, J., Kamoun, S., and Van der Hoorn, R. A. L. (2010) An effector-targeted protease contributes to defense against Phytophthora infestans and is under diversifying selection in natural hosts. Plant Physiol. 154, 1794-1804.
Song, J., Win, J., Tian, M., Schornack, S., Kaschani, F., Muhammad, I., Van der Hoorn, R. A. L., and Kamoun, S. (2009) Apoplastic effectors secreted by two unrelated eukaryotic plant pathogens target the tomato defense protease Rcr3. Proc. Natl. Acad. Sci. USA 106, 1654-1659.