Biology subjects

Navarro, J. A.

Publications and source records attributed to Navarro, J. A..

3 recordsLinked to original sources

bioRxiv2026

Liquid-Liquid Phase Separation of the m6A RNA ALKBH9B Demethylase: An IDR-dependent Mechanism driving the Alfalfa Mosaic Virus Infection

Biomolecular condensates play crucial role in plant-virus interactions. The plant m6A demethylase ALKBH9B acts as a proviral factor for Alfalfa mosaic virus (AMV), yet the biophysical mechanisms underlying its function remain elusive. Here, we investigate the liquid-liquid phase separation (LLPS) properties of ALKBH9B and their requirements for viral infection. Using in vitro and in vivo assays (Nicotiana benthamiana), we demonstrate that ALKBH9B assembles into highly dynamic, liquid-like cytoplasmic condensates driven primarily by weak hydrophobic interactions. Deletion and mutagenesis analysis identified a C-terminal intrinsically disordered region (IDR3), specifically its arginine-glycine (RG) motifs, as the essential molecular driver of LLPS. Importantly, this domain exhibits dual functionality, mediating both phase separation and direct binding to AMV RNA, which regulates condensate assembly and size. Disruption of the RG motifs completely abolishes ALKBH9B condensation and reduces its proviral capacity, compromising viral accumulation. Collectively, our findings reveal that the spatial compartmentalization of ALKBH9B into dynamic liquid hubs is functionally indispensable for AMV infection. This establishes LLPS as a critical regulatory checkpoint in viral pathogenesis, highlighting how plant viruses exploit the biophysical properties of host epitranscriptomic enzymes to establish specialized, protective microenvironments. HighlightsOur study establishes a direct mechanistic link between the biophysical properties of a plant m6A demethylase and viral pathogenesis, demonstrating that ALKBH9B functions as a key host factor by harnessing LLPS to regulate virus-host interactions.

plant biology↗

bioRxiv2025

Defender or accomplice? Dual roles of plant vesicle trafficking in restricting and enabling geminiviral systemic infection.

The vesicle trafficking system enables multidirectional cargo fluxes between endomembrane compartments, ensuring the viability of eukaryotic cells. However, vesicle trafficking plays dual roles during pathogen infections. In plants, the endomembrane system mediates autophagic immune responses but can also be hijacked by pathogens to facilitate successful infections. In this study, we demonstrate that vesicle trafficking machinery acts as a double-edged sword during infection by the geminivirus tomato yellow leaf curl Sardinia virus (TYLCSaV) in Nicotiana benthamiana. Virus-induced gene silencing (VIGS) of eight genes encoding key vesicle trafficking regulators revealed contrasting outcomes. Silencing of NbSAR1 and NbAP-1{gamma} significantly increased systemic geminiviral DNA accumulation, whereas silencing of Nb{delta}-COP, NbARF1, and clathrin genes almost completely abolished infection. Notably, this inhibition is hypothesized to result from direct or indirect impairment in viral movement, as replication remained unaffected by gene silencing. Furthermore, the observed effects affect other geminiviruses, including tomato yellow leaf curl virus (TYLCV) and beet curly top virus (BCTV), but not unrelated pathogens such as the RNA potato virus X (PVX) or the plant pathogenic bacterium Pseudomonas syringae. These findings suggest that while the vacuolar and autophagy branches of the vesicle trafficking system might mediate antiviral autophagic defense responses, the integrity of endocytosis and retrograde transport is essential for systemic geminiviral infection.

plant biology↗

bioRxiv2021

The S-palmitoylome and DHHC-PAT interactome of Drosophila melanogaster S2R+ cells indicate a high degree of conservation to mammalian palmitoylomes

Protein S-palmitoylation, the addition of a long-chain fatty acid to target proteins, is among the most frequent reversible protein modification in Metazoa, affecting subcellular protein localization, trafficking and protein-protein interactions. S-palmitoylated proteins are abundant in the neuronal system and are associated with neuronal diseases and cancer. Despite the importance of this post-translational modification, it has not been thoroughly studied in the model organism Drosophila melanogaster. Here we present the palmitoylome of Drosophila S2R+ cells, comprising 198 proteins, an estimated 3.5% of expressed genes in these cells. Comparison of orthologs between mammals and Drosophila suggests that S-palmitoylated proteins are more conserved between these distant phyla than non-S-palmitoylated proteins. To identify putative client proteins and interaction partners of the DHHC family of protein acyl-transferases (PATs) we established DHHC-BioID, a proximity biotinylation-based method. In S2R+ cells ectopic expression of the DHHC-PAT dHip14-BioID in combination with Snap24 or an interaction-deficient Snap24- mutant, as a negative control, resulted in biotinylation of Snap24 but not the Snap24-mutant. DHHC-BioID in S2R+ cells using 10 different DHHC-PATs as bait identified 520 putative DHHC-PAT interaction partners of which 48 were S-palmitoylated and are therefore putative DHHC-PAT client proteins. Comparison of putative client protein/DHHC-PAT combinations indicates that CG8314, CG5196, CG5880 and Patsas have a preference for transmembrane proteins, while S-palmitoylated proteins with the Hip14-interaction motif are most enriched by DHHC-BioID variants of approximated and dHip14. Finally, we show that BioID is active in larval and adult Drosophila and that dHip14-BioID rescues dHip14 mutant flies, indicating that DHHC-BioID is non-toxic. In summary we provide the first systematic analysis of a Drosophila palmitoylome. We show that DHHC-BioID is sensitive and specific enough to identify DHHC-PAT client proteins and provide DHHC-PAT assignment for ca. 25% of the S2R+ cell palmitoylome, providing a valuable resource. In addition, we establish DHHC-BioID as a useful concept for the identification of tissue-specific DHHC-PAT interactomes in Drosophila.

biochemistry↗