PhD in Biological Sciences and Applied Biotechnologies

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Supervisor

Phd thesis

The arbuscular mycorrhizal symbiosis (AMS) is an ancient association between plants and fungi that is present in over 80% of all land plants. In exchange for difficult-to-obtain mineral nutrients taken up by the fungus, plants provide photosynthates that allow the fungus to complete its life cycle. This symbiotic relationship is a very intimate interaction, as the fungus grows inside the plant and produces highly branched hyphal structures called arbuscules that serve as the interface for nutrient exchange. But how can such an intimate association form? How is a fungus able to grow inside of a plant without triggering an immune response from the plant? Some form of communication between these two distantly related organisms must be at play for such a relationship to exist.

Communication in biological systems is incredibly layered and complex, even between and within single cells. One layer of this communication is the post-transcriptional silencing of mRNA by small RNAs. Small RNAs (sRNAs) are short non-coding RNA molecules that regulate gene expression in eukaryotes via RNA interference (RNAi). RNAi begins with the expression of precursor sRNAs which are then processed into mature double stranded sRNA (dsRNA) by Dicer-like (DCL) proteins. Half of the dsRNA then binds to an Argonaute (AGO) protein, forming the RNA-induced silencing complex, or RISC. The RISC in turn uses the sRNA as a guide and binds to complementary target mRNA sequences in the cytoplasm. Once target mRNA is bound to the RISC, it can be cleaved by the enzyme, leading to its degradation and preventing its translation entirely.

sRNAs are now known as important signaling molecules, not only in intercellular environments but in different inter-species, and even inter-kingdom, interactions. Indeed, all eukaryotic cells secrete RNAs and mobile sRNAs can be transferred from “donor” to “receiver” organisms where they regulate host gene expression by exploiting the host’s molecular RNAi machinery. This process, known as cross-kingdom RNAi, has been principally studied in plant-pathogen interactions, where sRNAs have been shown to move between organisms via extracellular vesicles.

In analogy to pathogenic interactions, cross-kingdom RNAi may also occur in the most common symbiotic interaction between plants and fungi, that is the arbuscular mycorrhizal (AM) symbiosis. To form this symbiosis, there must be some form of communication between the two symbionts to allow such an intimate interaction to take place without the activation of plant immune responses. Indirect evidence such as the identification of extracellular vesicles at the plant-fungus interface sheds light on the possibility of cross-kingdom RNAi in the AM symbiosis.

Recently, the sRNA population of the AM fungus Rhizophagus irregularis was characterized during the symbiosis with the host plant Medicago truncatula. Through in silico predictions, dozens of fungal sRNAs with potential for host gene regulation through cross-kingdom RNAi were identified. Most notably, the R. irregularis sRNA 2216 (Rir-2216) was found to have multiple targets in the M. truncatula genome including a WRKY transcription factor whose homolog in Arabidopsis thaliana is associated with fungal defense.

My PhD thesis is to explore the role of Rir-2216 in cross-kingdom RNAi in the AM symbiosis and to characterize its target as well as investigate the potential mechanism of sRNA delivery.