Project Details
Projekt
Cross kingdom exRNA transfer in the Ustilago maydis/corn pathosystem
Applicant
Professor Dr. Michael Feldbrügge
Subject Area
Plant Breeding and Plant Pathology
Term
since 2020
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 433194101
Studying the U. maydis/corn pathosystem, we identified a distinct set of full-length ex-mRNAs that were enriched in EVs from cultured infectious hyphae. mRNAs encode, for example, enzymes involved in thiamine biosynthesis, such as Thi4, which are upregulated during infection. Interestingly, Thi4 contains a predicted chloroplast targeting signal suggesting transfer to host organelles in order to reprogramme plant metabolism during infection. For verification, we have established a fluorescence-assisted cell sorting approach purifying protoplasts from infected plants devoid of fungal hyphae. Pilot sequencing experiments revealed fungal transcripts in the sorted protoplasts. Currently, we are increasing the sequencing depth to obtain high confidence candidates. In a more targeted approach, we succeed in demonstrating that Thi4 mRNA and the encoded protein were present in EVs. This suggests that it might be important for this example to deliver mRNA and protein effector simultaneously for perfect spatiotemporal control. Finally, we established the syntaxin SNARE protein Sso1 as a generic EV marker. Thus, we aim to get cleaner, high confidence protein inventory of EVs in the future, especially for identifying RBPs relevant for cross-kingdom RNA warfare. In the 2nd funding period, we will address how and why mRNAs are transferred from U. maydis to the host plant maize during infection. We follow our successful approach in studying delivery of ex-mRNAs via EVs as key transport vehicles. We will apply syntaxins as EV marker protein in collaboration with B2 Kehr to optimize EV purifications resulting in high quality mRNA and protein inventories. In order to identify components of the EV maturation and loading machinery, we will follow a candidate gene approach. Comparable to A4 Weiberg, we investigate defined mutants in membrane trafficking affecting, for example, formation of endosomes or multivesicular bodies. The possible involvement of RNA-binding proteins such as Annexin will be addressed together with B2 Kehr and B5 Meister. The potential translation of fungal mRNAs in planta will be analyzed in collaboration with A2 Panstruga applying plant-based in vitro translation systems. To demonstrate translation during infection we will carry out TRAP-Seq experiments using antibodies specific for plant ribosomes in collaboration with B5 Meister and A5 Gutjahr. Gene deletion mutants of candidate translated mRNAs will provide insights in the biological function of the respective translation products. In essence, we will clarify the mechanism of ex-mRNA transfer and its functional consequences, paving the road for identification of novel drug targets.
DFG Programme
Research Units
