Prout, J. | bioRxiv Science

bioRxiv2025

Common symbiotic signalling pathway not essential for formation of functional mutualisms with endophytic fungi

Most plants form mutualistic symbioses with soil fungi, including arbuscular mycorrhizal (AM) fungi. These fungi usually transfer soil nutrients to plants and assimilate carbon from host plant photosynthesis. Recently, Mucoromycotina fine root endophytes (MFRE) were identified as nutritionally mutualistic and widespread fungal symbionts of plants, establishing MFRE as a new class of mycorrhizal fungi. However, the regulatory mechanisms for MFRE symbioses are completely unknown. Other symbionts, like AM fungi, use the Common Symbiotic Signalling Pathway (CSSP) to establish symbiosis. To explore whether MFRE interactions also involve this pathway, we cultured MFRE with CSSP mutants of Medicago truncatula which show impaired AM symbioses and tracked carbon and nutrient transfers using isotope tracers. Results show no differences in root colonization or nutrient exchange, suggesting MFRE symbioses are regulated by different molecular mechanisms. This finding highlights the unique nature of MFRE symbiosis, broadening our understanding of diverse fungal symbioses and their evolutionary significance.

microbiology↗

bioRxiv2023

Genome-wide assays to characterize rAAV integration into human genomic DNA in vivo

Adeno-associated viral (AAV) vectors are used to treat genetic diseases, expressing therapeutic genes from both extrachromosomal episomes and payloads that integrate into the host genome. Assays were developed to evaluate HR-mediated on-target integration and the potential occurrence of off-target integration. While many studies have addressed elements of these processes, proper characterization requires long-read sequencing to ensure that integrated viral DNA is examined and not the more prevalent episomes. We used Oxford Nanopore to characterize integrated DNA and scan the whole genome for off-target integrations. These assays were applied to cell-based and in vivo models to study vectors that correct phenylketonuria (PKU), caused by loss of phenylalanine hydroxylase (PAH). Administration of the human-specific vector in a humanized-liver mouse xenograft model resulted in stable, nuclease-free integration into PAH. Because detection of rare integration events in a much larger pool of episomal DNA is subject to artifacts, careful assay validation was required. A long-read, genome-wide assay capable of detecting on- and off-target vector integrations showed no evidence of off-target integration. Artifactual false positive events were below the limit of blank. These data support rAAV as an investigational therapeutic for genetic diseases and reinforce the need for characterization of integration assays to avoid artifacts.

genomics↗