Dh5a competent cells

The DENV3 viral RNA standard was prepared by amplifying a 464-bp fragment of DENV3 strain (GI:163644368) with primers 5′-ACAGTTTCGACTCGG-3′ (corresponding to genome position 29 to 43) and 5′-CTTTCATTCTTCCCC-3′ (corresponding to genome position 492 to 478). The resulting product was ligated into pGEM-T Easy vector (Promega) and transformed in DH5a competent cells (Invitrogen). Minipreps of isolated plasmid DNA were then prepared (Promega). RNA transcripts were generated using the Megascript kit according to the manufacturer’s specifications, and the RNA concentration was determined by UV spectrophotometry. Primers (DV3-qF 5′-AACCGTGTGTCAACTGGATCA-3′ and DV3-qR 5′-TTAATGGCCCCCGACTTCTT-3′) were designed to target the DENV pre-membrane region (prM) within the viral RNA standard that was previously designed. Real-time PCR was conducted using SYBRGreen I Mastermix (Applied Biosystems) on a Bio-Rad CFX96 detection instrument. Amplification conditions were 95 °C for 30 s, and 40 cycles of 95 °C for 5 s, 55 °C for 20 s, and 72 °C for 30 s. Viral genome copy numbers were calculated from a standard curve generated by an in vitro transcribed RNA standard.

A study of the cloning of the recombinant protein has previously been published [18 (link),19 (link)]. Briefly, the prokaryotic expression system pEcoli-Cterm 6xHN Linear (Clontech^®, Mountain View, CA, USA) was chosen to ligate the sIFNAR2 insert. DH5a Competent Cells™ (Invitrogen^®, Carlsbad, CA, USA) were transformed with the plasmid and the colony, forming isolated units. After plasmid purification (PureYield™ (Promega^®, Madison, WI, USA)), BL21 (DE3) (Invitrogen^®) bacteria were transformed to produce the recombinant sIFNAR2. This was purified on high-capacity Ni⁺²-iminodiacetic acid resin columns, detected by Western Blot using anti-IFNAR2 Human MaxPab antibody (Abnova^®, Taipei, Taiwan), and its identity was confirmed by the matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry. The recombinant protein had 239 amino acids and a molecular mass of 29 kDa, and its sequence corresponded to IFNAR2.3 (uniprot: P48551-3). Supplementary Figure S1 shows the identification of the protein by Western blot and by mass spectrometry.

Cenococcum geophilum isolates originating from different sites (Supplementary Table S1) were kept in Petri dishes (100 × 20 mm) containing Cenococcum medium, a modified MMN medium containing casein (Trappe, 1962 ), at 25°C and transferred to new culture medium every 20 days. Escherichia coli (subcloning efficiency DH5a competent cells; Invitrogen, Carlsbad, CA, U.S.A.) and Agrobacterium tumefaciens (electrocompetent strain GV3101) were conserved at −80°C and they were grown in LB and YEPD medium at 37 and 28°C, respectively.