Gene structure of <i>OsRMC</i> and DNA fragments used for the RNAi experiment.

The OsRMC gene consists of one exon of 777 bp. DNA fragments of 311 bp in the ORF and 433 bp in the 3′-UTR region were used for RNAi experiments. (TIFF)</p

Amino acid sequence alignment among OsRMC, LOC_Os04g25650.1, and OsCBMIP-K.

(TIFF)</p

Phylogenetic analysis of CBM1-binding proteins.

Phylogenetic tree reconstructed using a single DUF26, DUF26-A and DUF26-B domains of CRRSPs and CRKs from O. sativa, A. thaliana, Selaginella moellendorffii, and M. polymorpha, and Gnk2, SiCBMIP, and AFP1. Bar, 1.5 amino acid substitutions per site.</p

Identification of CBM1-binding protein from <i>Setaria italica</i>.

(A) Protein was extracted from S. italica leaves 4 days after M. oryzae inoculation using sodium phosphate buffer (50 mM, pH 7.5) containing 150 mM NaCl. Protein preparation was incubated with (+) or without (-) MoCel6A-His for 1 h at 4°C before being further incubated with His-resin. Fractions bound to His-resin were subjected to SDS-PAGE followed by silver staining. Arrowhead indicates added MoCel6A-His. Arrow indicates a candidate protein that interacts with MoCel6A-His. (B) The candidate protein was identified as a member of the CRRSPs by LC-MS/MS. Peptide sequences obtained are underlined. The methionine residue of peptide number 7 was oxidized. CRR motifs are indicated in red. (TIFF)</p

Gel-permeation chromatography of monomers and the complex of OsRMC and MoCel10A.

MoCel10A-His, OsRMC-His, and a mixture of MoCel10A-His and OsRMC-His preincubated at 4°C for 30 min were separated on a Superdex G-75 column equilibrated with sodium phosphate buffer (50 mM, pH 7.5) and 150 mM NaCl. Proteins were detected by immunoblot analysis using anti-His antibody. Protein standard markers albumin (75 kDa), carbonic anhydrase (29 kDa), and aprotinin (6.5 kDa) were eluted in fractions 48, 60, and 72, respectively. (TIFF)</p

Binding of OsRMC to CBM1-containing proteins MoCel6A and MoCDH.

Crude protein preparations (20 μg) containing (A) Flag-tagged MoCel6A (MoCel6A-Flag) or (B) MoCDH (MoCDH-Flag) prepared from M. oryzae were incubated in sodium phosphate buffer (50 mM, pH 7.5) containing 150 mM NaCl with or without OsRMC-His (2.0 μg) for 1 h at 4°C before being further incubated with His-resin. Fractions unbound and bound to His-resin were subjected to SDS-PAGE followed by immunoblot analysis using anti-Flag antibody. (TIFF)</p

A list of amino acid sequences of DUF26 used for constructing phylogenetic tree.

(PDF)</p

Determination of qPCR conditions for evaluating the level of <i>M</i>. <i>oryzae</i> infection in rice plants.

(A) Variable amounts (0.1–10 ng per 15 mL reaction mixture) of rice genomic DNA were used to amplify the rice ACTIN gene by qPCR. Ct values decreased linearly as the amount of rice genomic DNA increased. (B) Variable amounts (0.001–10 ng) of M. oryzae genomic DNA were mixed with rice genomic DNA to a final amount of 10 ng. The mixture was used for qPCR to amplify the M. oryzae ACTIN gene. Ct values decreased linearly as the amount of M. oryzae genomic DNA increased. (TIFF)</p

Summary of the number of CBM families.

(TIFF)</p

Fractionation of OsRMC proteins expressed in <i>N</i>. <i>benthamiana</i> leaves and rice suspension cells.

(A) OsRMC-GFP expressed in N. benthamiana leaves was sequentially extracted using sodium phosphate buffer (100 mM, pH 6.0) containing 100 mM NaCl (buffer-soluble), or the same buffer containing 1% (v/v) Triton-X100 (Triton-X100-soluble) or 1% (w/v) SDS (SDS-soluble). Buffer-soluble protein from N. benthamiana leaves overexpressing GFP was used as a GFP marker. (B) Rice suspension cells overexpressing OsRMC-HA were separated into culture filtrate (extracellular) and cells. Proteins were extracted from cells using sodium phosphate buffer (100 mM, pH 6.0) containing 100 mM NaCl (buffer-soluble) or 1% (w/v) SDS (SDS-soluble). The prepared proteins (1.0 mg) were subjected to SDS-PAGE followed by immunoblot analysis using anti-GFP and anti-HA antibodies. (TIFF)</p