Psp72

Deletion mutants of Rhm1, Ddnm1, MoRad55, MoRad57, and their double deletion mutants were constructed through targeted gene disruption by HR. The gene disruction vector used was either pSP72-HPH^{32 (link)}, which carries a 1.4 kb hygromycin-resistance gene cassette between the SalI and BamHI sites in pSP72 (Promega), or pII99^{33 (link)}, which carries a geneticin-resistance gene cassette. The 5′ and 3′ flanking fragments of the target gene were amplified by PCR using sets of specific primers (Supplementary Data 1). The amplified fragments were inserted at appropriate sites in either pSP72-HPH or pII99 to establish disruption vectors. The disruption vectors were then introduced into the Br48 strain through PEG-mediated transformation. The resulting P. oryzae transformants were screening initially by PCR, and then by Southern blots analysis to confirm a HR event in them (Supplementary Figs. 2, 7).
For gene complementation experiments, a 9.2 kb DNA fragment containing the MGG_15576 and MGG_15577 loci was first PCR-amplified by the high fidelity DNA polymerase, KOD-Plus (Toyobo) with a set of primers (Supplementary Data 1) and cloned into the NotI site in pBluescript SK(-) (Stratagene) to establish pBS-MoRad51. pBS-MoRad51 was introduced into the ΔMGG_11350-22 strain by co-transformation with pII99 (pre-complementaion) or pMGY-ΔRT-G (co-complementation).

Four gene fragments of PCV2, PCV3, PPV, and PRV, generated by PCR using their respective genomes as templates, were simultaneously inserted into a cloning vector of pSP72 (Promega) in tandem and served as a standard plasmid (pSP72-PCV2 cap & PCV3 cap & PPV NS1 & PRV gE) in subsequent assays (Figure 2A). Briefly, the four gene fragments were digested with XhoI/HindIII, HindIII/BamHI, BamHI/KpnI, and KpnI/SacI(Thermo Fisher Scientific), and then purified and ligated to the vector digested with XhoI/ SacI. Finally, the ligation products were transformed into Escherichia coli DH5α competent cells (TransGen). The positive clones were cultured at 37°C for 20 h then extracted by Plasmid DNA Mini Kit (Omega) and confirmed by DNA sequencing. The resulting plasmids were quantified by ultraviolet absorbance at 260 nm and 280 nm using a NanoDrop spectrophotometer (Thermo Fisher, USA). According to the size of the standard plasmid template, the copy numbers of the purified plasmids were calculated based on the following formula: (A260 (ng/μL) × 10⁻⁹ × 6.02 × 10²³) / (DNA length×650) = copies/μL. Then, 10-fold serial dilutions of the standard plasmid, with the same starting concentration of 10⁹ copies/μL, were performed to generate four standard curves for testing the multiplex real-time PCR.