Pes membrane

OF-sEVs were obtained from the isthmus of ipsilateral oviducts from pregnant and non-pregnant groups according to the isolation protocol previously used by da Silveira et al. (53 (link)), Alminaña et al. (32 (link)), and Ávila et al. (54 (link)). Briefly, the oviductal flushing was centrifuged at 300 × g for 10 min to remove live cells, 2,000 × g for 10 min to remove cellular debris, 16,500 × g for 30 min to remove large vesicles, and the remaining supernatant was placed at −80°C until further use. The supernatant was filtered through a 0.20 μm sterile syringe filter (PES membrane; Corning) in order to remove any remaining large EVs. For sEVs isolation, filtered oviductal flushing was centrifuged twice at 119,700 × g for 70 min at 4°C (Optima XE-90 Ultracentrifuge; rotor 70 Ti; Beckman Coulter). After this procedure, the supernatant was discarded, and the sEVs pellets were resuspended in 20 μL of phosphate-buffered saline (1 × Ca²⁺/Mg²⁺ free PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄) and used for further analysis. Due to the small volume of oviductal flushing obtained from a single animal, the protocol to isolate OF-sEVs was validated using female reproductive tracts obtained from slaughterhouses, as described in Supplementary Data 1.

To isolate dsRNA, 10 ml cultures of the 55 T. marneffei isolates in the mycelial phase were filtered using a Corning bottle top vacuum filter (150 ml) with a pore size of 0.22 µm and a polyethersulfone (PES) membrane (Corning, Corning, NY), and the mycelial mass was collected and subjected to total RNA extraction using RiboPure yeast (Ambion, USA) as described previously (31 (link), 33 (link)) followed by treatment with 8 units of DNase I (Ambion)–5 µg/µl RNase A (Qiagen, Hilden, Germany)–2× SSC buffer (Ambion; 1× SSC buffer is 0.3 M NaCl, 30 mM sodium citrate) for 1 h at 37°C, separated by electrophoresis through 1% agarose gels, and visualized under UV light after staining with ethidium bromide, as described previously (79 (link), 80 (link)). The isolated dsRNA was purified using a PureLink RNA minikit (Ambion) and stored at −80°C before use.

The sEVs were isolated as previously described [25 (link)]. Briefly, 200 µL of iFF or cFF were centrifuged at 300 x g for 10 min, 2,000 x g for 10 min, and at 16,500 x g for 30 min, to remove live cells, cellular debris, and large vesicles, respectively. The remaining supernatant was placed in freezer at −80°C for further analysis. Upon use, supernatant was filtered through a 0.2 µm filter (PES membrane; Corning) and ultracentrifuged (Optima XE-90 Ultracentrifuge; rotor 70 Ti; Beckman Coulter) at 119.700 x g for 70 min twice to obtain an enriched pellet of sEVs. All centrifugation steps were performed at 4°C. The pellet was resuspended in 50 µL of phosphate-buffered saline (1× PBS Ca²⁺/Mg²⁺-free; 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄) and used for further analysis.