Fungal strains and growth conditions

M. oryzae P2 strain, a Japanese rice blast pathogenic isolate, was used as the wild-type strain in this study. For preservation, P2 and derived strains were cultured on oatmeal agar medium containing 5.0% oatmeal (Quaker Oats Company, Chicago, IL, USA), sucrose (Nacalai Tesque, Kyoto, Japan), and 1.5% agar (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) at 28 °C. For induction of conidiation, aerial hyphae were removed with a sterilised brush and stationary-cultured under blacklight blue lamps (FL20S, 20 W; Toshiba Co. Ltd, Tokyo, Japan) at 28 °C for 2–3 days. Conidia were brushed off into sterile water and used in subsequent experiments.

Appressorium formation assay

The percentages of germinated conidia and appressorium formation were determined by means of an appressorium formation assay as described previously54. Chloramphenicol (Wako) was dissolved in ethanol (Nacalai Tesque) or dimethyl sulfoxide (DMSO; Wako) and a stock solution (10 mg ml−1) was prepared. The stock solutions were diluted to the appropriate concentrations with each solvent. Before placing drops of conidial suspension on the surface of hydrophobic polyvinyl chloride (Thermo Fisher Scientific, Inc., Waltham, MA, USA), diluted Cm solution was mixed with conidial suspensions at various concentrations in 0.1% ethanol or 0.5% DMSO.

Phage display method

For screening we used the T7Select® Phage Display System (Novagen, Madison, WI, USA) following the manufacturer’s protocol. For the T7 phage display method, a M. oryzae genomic DNA library was used, which was constructed as previously reported15. As bait, Bio-Cm was immobilised on 5 µg NeutrAvidin™ Protein (Pierce, Rockford, IL, USA) placed on the sensor chip of an AFFINIX Q QCM apparatus (Initium, Kanagawa, Japan). Non-immobilised avidin was removed using an air duster (Sanwa Supply, Tokyo, Japan). Bio-Cm dissolved in 20 µl QCM buffer (10 mM Tris-Cl, 200 mM NaCl; Nacalai Tesque) with 10% DMSO was placed on the avidin-immobilised chip and left at room temperature for 80 min. Non-immobilised Bio-Cm was removed using an air duster. The chip was set up for the QCM apparatus with the cuvette containing 8 ml QCM buffer. An aliquot (80 µl) of the T7 phage display library was injected into the cuvette. The frequency changes, which were caused by binding between phages and Bio-Cm on the sensor chip, were monitored for 10 min. The chip was detached from the apparatus and air-dried, then bound phages were recovered by applying a 10 µl drop of host Escherichia coli (BLT5615). The isolated phage DNA sequence was amplified by PCR and sequenced.

Extraction of DNA and RNA from M. oryzae

For induction of hyphal growth to extract genomic DNA, M. oryzae strains were cultured in 20 ml YG liquid medium containing 0.5% yeast extract and 2% glucose (Nacalai Tesque) and incubated at 28 °C on a rotary shaker at 150 rpm for 2 days. The fungal mycelium samples were inoculated into 100 ml YG liquid medium and incubated at 28 °C on a rotary shaker at 150 rpm for 1 day. The fungal fluid was concentrated by centrifugation at 2000 × g at room temperature for 10 min. After freeze-fracturing with liquid nitrogen, genomic DNA was extracted using a previously described method15. The extraction of RNA at the conidial germling stage was performed following a previously described method54.

Construction of MoDullard deletion mutant

To establish a Δmodullard strain knockout vector using the AtMT method, we constructed pNR011 as the AtMT knockout vector. Restriction enzymes and calf intestinal alkaline phosphatase (New England Biolabs Inc., Ipswich, MA, USA), Ligation Convenience Kit (Wako), and QIAquick® Gel Extraction Kit (QIAGEN, Hilden, Germany) were used for vector construction. First, the vectors pBI121 and pRI910 were digested using PmeI and EcoRI. The extracted pRi replicator region (9265 bp) from pRI910 and multiple cloning site from pBI121 were ligated into the vector pNR01. The AtMT knockout vector, pNR011, was constructed by cloning PtrpC-HPH from pCSN4355 into pNR01 using SmaI. The upstream and downstream regions of MoDULLARD were amplified from genomic DNA using the primers MoDul-Up-Fwd-KpnI, MoDul-Up-Rvs-KpnI, MoDul-Down-Fwd-XbaI, and MoDul-Down-Rvs-XbaI, and each region was cloned into pNR011 digested with KpnI or XbaI. The cloned vectors were digested with RsrII, and several fragments containing the upstream or downstream region were ligated to construct the modullard knockout vector pANKO03.

To obtain Δmodullard mutants, the AtMT method was performed using a previously reported procedure27. After co-culture fungal and bacterial cells on paper filters were plated on YG agar medium supplemented with hygromycin (500 µg ml−1; Nacalai Tesque) and Merpenem (25 µg ml−1; Nacalai Tesque) at 28 °C for 4 days. Individual transformants that emerged at the edge of the paper filters were transferred onto oatmeal medium and incubated for 4 days. The deletion of modullard in the transformants was checked by PCR and Southern blot analysis.

Cloning and construction of plasmid vectors for transformation of M. oryzae

To express each gene in ∆modullard, we constructed the pBFT vector by cloning the promotor of the translation elongation factor gene (Ptef) fragment from pMK41256, amplified using the primers pTEF-Fwd-PmeI and pTEF-Rvs-BamHI, and digested with PmeI and BamHI. The pBF101 vector57 was also digested by PmeI and SmaI, then ligated with the digested PCR fragment to generate pBFT. To construct each expression vector, corresponding genes were amplified with the primers MoDUL-Fwd, MoDUL-Rvs, CTDSP1-Fwd, CTDSP1-Rvs, CTDSP2-Fwd, CTDSP2-Rvs, CTDNEP-Fwd, CTDNEP-Rvs, CTDSPL-Fwd, CTDSPL-Rvs, CTDSPL2-Fwd, and CTDSPL2-Rvs from M. oryzae cDNA (extracted from germling-stage conidia) or human cDNA (extracted from U937 cultured cells using RNAzol; Cosmo Bio Co., Ltd., Tokyo, Japan) following the manufacturer’s protocol. The amplified DNA fragments were digested by SpeI or BamHI, then cloned into pBFT to construct each expression vector. The sequence in each vector was checked by sequencing.

To obtain complementary strains harboring the inactivated DxDxT/V motif, the mutation induction vectors were constructed using improved methods for site-directed mutagenesis using the Gibson Assembly® Master Mix (New England Biolabs) following the manufacturer’s protocol. Using the primers mutDxDxT-Fwd and mutDxDxT-Rvs, the sequence of the DxDxT/V motif in the expression vectors was changed from 5′-GATCTCGACGAGACG-3′ to 5′-aATCTCaACGAGACG-3′ to mutate DxDxT to NxNxT (lower-case letters indicate the nucleotides changed from the original sequence). The sequences of the constructed vectors were checked by sequencing.

Expression and purification of proteins

To obtain purified proteins for the binding assay, we used pGEX-6p-1 (GE Healthcare UK Ltd, Amersham, UK) as an expression vector. The cDNA of interest was amplified from complementary vectors by MoD-EX-Fwd-XhoI and MoD-EX-Rvs-BamHI. Amplified fragments were digested using XhoI or BamHI and were cloned into pGEX-6p-1. The sequence of the vectors and that each cDNA was in-frame was checked by sequencing. The obtained vector was introduced to Rosetta™(DE3) Competent Cells (Merck KGaA, Darmstadt, Germany) and used as an expression host. For the expression experiment Auto Induction medium58 was used, which contains autoclaved A solution (final concentration 2.4% yeast extract and 1.2% Trypton; Nacalai Tesque), autoclaved B solution (1.1% KH 2 PO 4 , Nacalai Tesque; and 4.7% K 2 HPO 4 , Kanto Chemical Co., Tokyo, Japan), and filtered sugar solution (0.6% glycerol, 0.5% glucose, and 0.08% lactose; Nacalai Tesque). The expression hosts were harvested on LB agar medium supplemented with carbenicillin (25 µg ml−1) and the single colony was transferred to 1 ml LB liquid medium supplemented with carbenicillin (25 µg ml−1) and incubated at 37 °C overnight. An aliquot (100 µl) of the bacterial suspension was inoculated in 6 ml LB liquid medium supplemented with carbenicillin (25 µg ml−1) and incubated at 37 °C overnight. The pre-cultured suspension was injected into Auto Induction medium supplemented with carbenicillin (25 µg ml−1) to OD 600 of about 0.5 and incubated at 37 °C for 24 h. The incubated bacterial suspension was collected by centrifugation at 5000 × g for 5 min, and the pellet of E. coli cells was washed twice with 1 ml TBS buffer. The washed pellet was suspended in 1 ml sonication buffer containing Tris-Cl (pH 8.0), 150 mM NaCl, and 1 mM ethylenediamine-N,N,N,N′,-tetraacetic acid disodium salt, dehydrate (Wako). Before sonication, 1 mM phenylmethylsulfonyl fluoride (Nacalai Tesque) dissolved in DMSO was mixed and the mixture was sonicated using a Sonifier® 450 cell disrupter (Branson Ultrasonics Co., Danbury, CT, USA) at output 1 and 50% duty cycle, for three 10-second pulses. The suspension was centrifuged at 17,900 × g for 5 min. The 1 ml soluble layer was collected into a 1.5 ml tube and was frozen with liquid nitrogen then stored at −80 °C prior to the purification experiment.

The purification of sonicated soluble fractions was performed using Glutathione Sepharose® 4B (GE Healthcare) as a resin following the manufacturer’s protocol. Resin (100 µl) and 1 ml soluble fractions were mixed in a 2 ml tube and incubated at 4 °C for 16 h at 25 rpm with a RT-30mini rotator (TAITEC, Saitama, Japan). The resin was centrifuged at 500 × g for 5 min and the supernatant was discarded. The resin was washed five times with 1 ml TBS buffer. To the bound proteins 100 µl elution buffer (50 mM Tris-Cl, 10 mM reduced glutathione, pH 8.0; Nacalai Tesque) were added and the solution was incubated at 4 °C for 20 min at 25 rpm. After incubation, the resins were centrifuged at 500 × g for 5 min and the eluted fraction was collected. The elution step was repeated three times. The resulting solution was frozen with liquid nitrogen and stored at −80 °C prior to the pull-down assay.

Pull-down assay

The pull-down assay was performed as previously described59. An aliquot (40 µl) of the elution fractions of GST-fused proteins purified with GST-Sepharose was used for the pull-down assay with Streptavidin Sepharose® High Performance beads (GE Healthcare) to immobilize 40 nmol Bio-Cm or Bio-Ctrl. After mixing overnight, the resins were washed three times with 200 µl TBS buffer, mixed with SDS loading buffer containing 0.002% bromophenol blue, 5% glycerol, and 0.1% sodium dodecyl sulfate (Nacalai Tesque), and then incubated at 95 °C for 10 min. After centrifugation at 17,900 × g for 1 min, the supernatants were subjected to SDS-PAGE using 10% polyacrylamide gel. The separated proteins were blotted onto a PVDF membrane and detected by western blot analysis using Anti-Glutathione S-transferase antibody (Wako) as a primary antibody and goat anti-IgG AP (Santa Cruz Biotechnology Inc., Dallas, TX, USA) as a secondary antibody. The colorimetric detection of alkaline phosphatase activity was performed using CDP-Star (Thermo Fisher Scientific) with the ChemiDoc™ Imaging System (BIO-RAD Laboratories, Tokyo, Japan).

Homology search and alignment of amino acid sequences

Protein sequences were downloaded from the National Center for Biotechnology Information database (http://www.ncbi.nim.gov). We used the BLAST tool to search for homologous sequences and CLUSTALW to align and compare amino acid sequences60,61.