Neomycin

To generate the gene's replacement construct of RAM1, we amplified 1.5‐kb upstream and 1.5‐kb downstream of the gene's flanking sequences from the genomic DNA of the wild‐type strain. Both flanking sequences were fused with part of the hygromycin segment by overlap PCR. Subsequent PCR products were transformed into protoplasts of the wild‐type strain (Fig. S4, see Supporting Information). For complementation, a RAM1 gene containing a 1.5‐kb promoter region and a 0.5‐kb terminator region was amplified and cloned into pKN plasmid (Wang et al., 2018). The resulting construct pKN‐RAM1 (Table S2, see Supporting Information) was transformed into the Δram1 mutant. CM plates supplemented with 250 μg/mL hygromycin B (Roche Diagnostics, Indianapolis, IN, USA) was used to select deletion transformants, with 400 μg/mL neomycin (Amresco, Solon, OH, USA) to select complementation transformants. PCR‐mediated methods were used to confirm different transformants. All the primers used in this study were shown in Table S3 (see Supporting Information).

A split‐PCR strategy was used for gene disruption as previously described (Goswami, 2012 (link)). Left boarder and right boarder were confirmed by PCR using the Pal1LBCK/ HYG‐LBCK and Pal1RBCK/HYG‐RBCK primer pairs. Gene deletion was further confirmed by RT‐PCR of a c.900 bp internal fragment of PAL1. For complementation, a complementation vector containing 1.5 kb promoter region, the PAL1 gene‐coding region, and the adjacent 0.5 kb downstream region was transformed into the Δpal1 mutant. CM plates supplemented with 250 μg/ml hygromycin B (Roche Diagnostics) were used to select the deletion transformants, and CM plates supplemented with 400 μg/ml neomycin (Amresco) were used to select the complementation transformants.

The coding sequence of SCRE2 was amplified and then subcloned into the modified pKS vector (Park et al., 2012 (link)), by which a fusion protein with a nucleus localization sequence (NLS) and mCherry is expressed under the control of RP27 promoter (Bourett et al., 2002 (link)). The pKS-RP27::SCRE2-mCherry-NLS construct was transformed into the M. oryzae strain P131 via PEG-mediated transformation as described (Sweigard et al., 1992 (link); Yang et al., 2010 (link)). The successful transformants were screened on potato-dextrose agar plates supplemented with 400 μg/mL neomycin (Amresco). The expression of SCRE2-mCherry-NLS in M. oryzae was confirmed by fluorescence microscopy Nikon Eclipse 90i. M. oryzae conidiation was induced as described previously (Peng and Shishiyama, 1988 (link)). For barley inoculation, the conidial suspension (1 × 10⁵ conidia/mL) in 0.025% Tween 20 was spotted onto the lower epidermis of detached young barley (Hordeum vulgare) leaves and then incubated in a moist, dark chamber at 28°C. Red fluorescence was observed at 30 hpi via fluorescence microscopy.

For the deletion of Def1, a gene displacement strategy through split-PCR was used as previously described (Figure S1A) [45 (link)]. For protoplast preparation, the wild-type strain was incubated in liquid CM medium, and around 1 g of mycelium was harvested for digestion by Lysing enzyme (Sigma-Aldrich, St. Louis, MO, USA) for 2 h at 150 rpm. The digested protoplast was filtered with three-layer microscope lens papers and washed with 0.7 M NaCl and resuspended with STC buffer (1.2 M Sorbitol, 10 mM Tris [pH 7.5], 50 mM CaCl₂) and adjusted to a concentration of 1 × 10⁸/mL for transformation. For transformation, the split-PCR products were added into the protoplast (300 μL mixture) and added 2 mLPTC (60% PEG 3350, 10 mM Tris [pH 7.5], 50 mM CaCl₂) dropwise. The deletion transformants were selected by 250 μg/mL hygromycin B (Roche Diagnostics, Indianapolis, IN, USA) and confirmed by PCR using the Def1 gene-up/gene-down, LCK/HCK-up, RCK/HCK-down primer pairs (Figure S1B; Table S2). For complementation, we inserted the 1.5 kb promoter region and gene-coding region of Def1 into pKN to construct the complementation vector, pKN-Def1, which was transformed into the Def1 deletion mutant. The complementation transformants were selected by 400 μg/mL neomycin (Amresco, Framingham, MA, USA) and confirmed by PCR using Def1 gene-up/gene-down primer pair (Table S2).

The wild-type mice were pretreated with streptomycin (0.5 mg/mL; Tokyo Chemical Industry, Japan) for 5 days. The colonic feces were collected and dissolved with 1 mL PBS. PBS-diluted feces were mixed well and centrifuged at 400 g for 5 min to remove larger particles from bacteria, and then the supernatant was diluted into different concentration and plated to the Wilkins–Chalgren anaerobic agar (OXOID, USA), which contained 50 μg/mL different antibiotics [kanamycin (Solarbio, China), neomycin (Amresco, USA) or streptomycin] and cultured in anaerobic box at 37°C for 3 days. The purity of bacterial colonies was determined by qPCR analysis of 16S rDNA. Then the single colonies were streaked at least three times onto fresh agar plates with antibiotics. Culture purity was ensured by observing colony morphology. For identification and phylogenetic analysis of isolates, DNA was extracted from pure cultures, and 16S rRNA genes were amplified and sequenced. These sequences were classified and annotated by GreenGene Database (24 (link)).