Gabriel Mendoza-Rojas
Resource allocation during biotic stress: Nutrition of fungal pathogens in plants
Plants tightly control nutrient uptake and allocation for optimal growth, but pathogen infections disturb this fine-tuned balance. Therefore, an overarching goal is to unravel the molecular interaction of pathogens with the plant vasculature, i. e. the highways of water (xylem) and nutrient transport (phloem). In my project, my aim is to understand how smut fungi use some effectors to modify host nutrient allocation at the cellular level and the phloem for their own benefit. In the context of vasculature, T. thlaspeos shows a systemically growth and is now a promising smut fungi model where new research questions arise, i.e. different virulence factors (like Pit1 or Tue1) and their molecular function can show us how these fungi use their own molecular tools to survive. Combining genetics, infection assays and biochemistry with state-of-the art biosensors I will show how metabolic re-routing is achieved with the help of fungal membrane factors to answer 3 central questions:
- Which factors are used by the fungus to modify host nutrient allocation?
- What are the molecular mechanisms underlying nutrient rerouting by these factors?
- How does the metabolic re-routing pathways work?
As a starting point for my qualification year, I´ve been studding the soluble fungal effector Tue1 from T. thlaspeos, to unravel the mechanisms behind its function, that remain enigmatic. Previous studies in the lab showed that Tue1 may interact at the Jasmonate signaling pathway (JA) a key plant response to stress conditions and pathogen infection. Using pull-down assay and Surface Plasmon Resonance Spectroscopy for detailed kinetics analysis of the binding events, I want to confirm this interaction. On the plant cell biology side, the development of Tue1-deletion strain will help to reveal changes in the phenotype (if there are) in the plant infection assays. Understanding the translocation of the effector to the plant cell using a Tue1-GFP11 system also will apport information in which part of the cell the effector works.
In my thesis, I will focus on the well-known virulence factor Pit1. Comparative genetics and structure prediction by AlphaFold between U. maydis and T. thlaspeos suggest that Pit1 may be a transceptor involved in Mg-signalling/transport. To uncover the function of Pit1, I will produce the heterologous protein Pit1 in S. cerevisiae using the Pdr1-3 system. Once the purified protein is ready, I will use microscale-thermophoresis to analyse the potential binding to magnesium or other nutrients and perform transport assays. Using the TurboID system in U. maydis I will furthermore identify Pit1 interaction partners that might be part of the intracellular signaling cascade. Finally, I will use the purified protein to prepare samples for CryoEM aiming to solve its structure to unravel mechanistic and structural details from both pathosystems.
Starting date: 01.10.2022 / Qualification Fellow
Thesis committee members: Michael Feldbrügge, Vera Göhre, Florian Altegoer, tba
Publications:
Mendoza-Rojas, G., Sarabia-Vega, V., Sanchez-Castro, A., Tello, L., Cabrera-Sosa, L., Nakamoto, J. A., Peñaranda, K., Adaui, V., Alcántara, R., & Milón, P. (2021). A low-cost and open-source protocol to produce key enzymes for molecular detection assays. STAR Protocols. doi: 10.1016/j.xpro.2021.100899
Alcántara, R., Peñaranda, K., Mendoza-Rojas, G., Nakamoto, J. A., Martins-Luna, J., del Valle-Mendoza, J., Adaui, V., & Milón, P. (2021). Unlocking SARS-CoV-2 detection in low- and middle-income countries. Cell Reports Methods. doi: 10.1016/j.crmeth.2021.100093
Alcántara, R., Peñaranda, K., Mendoza-Rojas, G., Nakamoto, J. A., Dueñas, E., Alvarez, D., Adaui, V., & Milón, P. (2021). UnCovid: A versatile, low-cost, and open-source protocol for SARS-CoV-2 RNA detection. STAR Protocols. doi: 10.1016/j.xpro.2021.100878