Exploring the cross-kingdom RNA interference landscape in the barley-powdery mildew interaction

• Applicant: Professor Dr. Ralph Panstruga
• Subject Area: Organismic Interactions, Chemical Ecology and Microbiomes of Plant Systems ; Plant Breeding and Plant Pathology ; Plant Cell and Developmental Biology
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 433194101

Powdery mildew fungi are obligate biotrophic phytopathogens that are agronomically relevant and engage in intimate relationships with their plant hosts. The interaction between barley (Hordeum vulgare) and its adapted powdery mildew pathogen, Blumeria hordei, is the focus of our research activities. In the 2nd funding period of FOR5116, we will build upon the results achieved by studying communication via extracellular RNAs between barley and Blumeria hordei in the 1st funding period. A key finding from the 1st funding period is the discovery of site-specific occurrence of specific small RNAs (sRNAs) in fungal hyphae and haustoria, infected barley epidermis, a vesicle-enriched fraction of the epidermis, and extracellular vesicles. Apart from canonical sRNAs from the host plant and the fungal pathogen, we discovered unexpectedly an enrichment of specific fragments of barley 5.8S ribosomal RNA in extracellular vesicles and infected epidermis, as well as particular B. hordei transfer RNA fragments in haustoria. Overall, these observations point to cross-kingdom shuttling of sRNAs in the course of the barley-powdery mildew interaction. In the 2nd funding period, we wish to explore the biological significance of these findings. To this end, we already initiated a more comprehensive sRNA profiling experiment, also including mRNA and degradome sequencing to identify putative sRNA target genes in both interaction partners. We will further overexpress and sequester (using short-tandem-target-mimic (STTM) technology) selected sRNAs in the context of the interaction and study their gene silencing capacity with a dedicated reporter system. Additionally, we will investigate the ability of the found transfer RNA and ribosomal RNA fragments to interfere with the process of translation. As robust extracellular vesicle markers are crucial for any cell biological studies, we will perform an advanced proteomic analysis of enriched and purified extracellular vesicle fractions. We finally wish to explore the RNA-binding proteins associated with the sRNAs we discovered. As Argonaute proteins are obvious candidates, we will follow up their enrichment, e.g. via the novel TraPR and AGO-APP approaches. We will also continue studying the RNase-like effector protein BEC1015/CSEP0064, which is a member of a large superfamily in B. hordei and a promising candidate for sRNA binding. On the one hand, we will test in a targeted manner the in vitro RNA-binding capacity of this effector protein. On the other hand, we will use advanced Cross-Linking and Immuno Precipitation (CLIP) methods for the enrichment of RNAs bound to this effector protein in vivo. In summary, these experimental approaches promise to shed further light on the role of cross-kingdom kRNA transfer in the course of the barley-powdery mildew interaction.

• DFG Programme: Research Units
• Subproject of: FOR 5116: “exRNA”: Plant-microbe communication through extracellular RNA