Transmission of Mycoviruses in Fungal Protoplasts

Fungal mycelium was grown on cellophane-covered PDA plates and 10 g of fungal mass was used for TkTV1 particles purification and transmission electron microscopic (TEM) visualization as previously described by Boine et al. (2012) (link). TkTV1-free protoplasts were prepared from TkTV1-free isolates of T. koningiopsis (isolate TkF) and C. rosea (isolate CrF) using Glucanex and β-Glucuronidase as cell wall lytic enzymes (Sigma-Aldrich) as described by Shi-Wang et al. (2007) (link) and Sun et al. (2017) (link), respectively. For protoplast transfection, purified particles were passed through 0.45-μm syringe filters and used in PEG-mediated transfection tests of the previously prepared TkTV1-free protoplasts as described by Hillman et al. (2004) (link). Transfected protoplasts were regenerated on regeneration medium for 4–5 days and 25 colonies for each fungus were randomly picked and transferred to PDA plates. DsRNA and total RNA were extracted as above and TkTV1 transmission was verified by RT-PCR amplification of an 879 bp-long fragment using specific primer pair TVF5 (5′-GTAAAGTAGGAGCCGTCC-3′) and TVR6 (5′-CTTCCAATTCGAGTGTTTCC-3′).