The national standard strain of R. solani AG1 IA was precultured in potato sucrose agar (PSA) medium (per liter: 200 g boiled potato extract, 20 g sucrose, and 15 g agar powder), and then small pieces of agar block with fresh R. solani mycelia were transferred into potato sucrose broth (PSB) medium (200 g/L boiled potato extract and 20 g/L sucrose) to amplify the mycelia. Mycelia were collected after 2 days and immediately frozen using liquid nitrogen then stored at −80°C until use.

Seeds of N. benthamiana and Arabidopsis were sown on soil and placed in a chamber at 25°C with a 16-h illumination cycle per day under 60–80% humidity. After 2 weeks, tobacco seedlings were used for virus-induced gene silencing (VIGS) experiments. Mature leaves of 4-week-old plants were used for effector bioassays and gene expression analysis. Sterilized seeds of the rice cultivar Nipponbare were sown on half-strength Murashige Skoog medium (Murashige and Skoog, 1962) supplemented with 2% sucrose, 1% agar, and vitamins and placed in a chamber under dark conditions at 25°C. After approximately 2 weeks, rice leaves were used for protoplast preparation. Oryza sativa subsp. indica R600, a rice variety applied by Sichuan Agricultural University and certified by China, is classified as a moderately susceptible variety following inoculation with Magnaporthe grisea (the rice blast fungus). R600 was cultivated in the summer season in a rice paddy field of the Wenjiang experimental field of Sichuan Agricultural University, located in Huihe village (Chengdu, Sichuan Province, China; 104.06°E, 30.67°N). Before rice plants reached the tilling stage, leaf blades were detached, and R. solani AG1 IA mycelia were inoculated on the surface of the detached leaves for gene expression assays.

R. solani mycelia were powdered in liquid nitrogen using a mortar and pestle, then total RNA was isolated using a Spin Column Fungal Total RNA Purification Kit (Sangon, Shanghai, China) according to the manufacturer’s protocol. Tobacco and rice tissues were also powdered in liquid nitrogen using a mortar and pestle, and then total RNA was extracted using a Spin Column Plant Total RNA Purification Kit (Sangon) according to the manufacturer’s instructions.

cDNAs of candidate SCR effectors were amplified from R. solani AG1 IA mycelia by RT-PCR. Briefly, the first-strand cDNA was synthesized using a Transcriptor First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland) according to the manufacturer’s protocol, and cDNA fragments were then amplified using Phusion High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA, United States) and gene-specific primers (Supplementary Table 1). The resulting PCR products were cloned into a T-vector, and the cDNA inserts were sequenced.

For validation of effector candidates, full-length coding regions of the cDNAs were directionally subcloned into the BamHI and SalI site of the GATEWAY binary vector pMDC32 (Curtis and Grossniklaus, 2003) using ClonExpress II One Step Cloning Kit (Vazyme Biotech Co., Ltd, Nanjing, China). pMDC32 allows overexpression of a cloned gene in plant cells driven by a CaMV35S promoter. In these constructs, 2 × FLAG peptide tags (DYKDDDDK) were fused downstream of the inserted gene by a linker peptide, allowing immune detection of transiently expressed proteins. For the SCR gene RsSCR10, a deletion constructs RsSCR10^ΔSP (lacking the SP region) was generated using another primer set (Supplementary Table 1) and was used for functional analysis of the SP. An overexpression construct of the green fluorescent protein (GFP) acted as a negative control, and both a mouse proapoptotic Bcl-2 family member (BAX) and INF1 constructs were prepared as positive controls.

Constructed plasmids were transformed into Agrobacterium tumefaciens GV3101 using the freeze-thaw method (An et al., 1989). Agrobacterium clones harboring the plasmid constructs were cultured in YEP medium (0.5% yeast extract, 1% tryptone, and 1% NaCl) overnight at 28°C, and bacterial cultures were harvested and resuspended in infiltration buffer [10 mM MES-Tris (pH 5.6), 10 mM MgCl₂, and 100 μM acetosyringone] at an optical density of 600 nm (OD₆₀₀) of 0.5. The Agrobacterium solutions were pressure infiltrated into tobacco leaves using needleless syringes. A total of 20 leaves from different plants were infiltrated for each control. Cell-death status on tobacco and Arabidopsis leaves were monitored and photographed at 3–5 days after agroinfiltration (Wei et al., 2020).

According to prediction results from the website https://predictprotein.org/, RsSCR10 has 10 cysteine residues that can form five disulfide bonds. We used splicing overlap extension PCR for site-directed mutagenesis. Two DNA fragments of RsSCR10 were amplified from the cDNA of R. solani AG1 IA, and the PCR product was cloned into 35S-PMDC32 vectors. The primer sequences used were listed in Supplementary Table 1.

For detection of dead cells, tobacco leaves were stained with trypan blue (Fernández-Bautista et al., 2016). Briefly, agroinfiltrated leaves at 5–7 days postinfiltration (dpi) were collected and immersed in ethanol: glacial acetic acid solution (3:1, v/v) for 24 h to fix the cell tissues and destain chlorophyll. The treated leaves were then soaked in a solution containing trypan blue dye [10 ml lactic acid, 10 ml glycerin, 10 ml deionized water, 10 g phenol, and 20 mg trypan blue (Sigma-Aldrich, St. Louis, United States)] for 24 h. Tissue images were taken and decolorization was performed. Samples were decolorized in 1.25 g/ml chloral hydrate solution for 3 days. Triplicated bioassays were performed to confirm the reproducibility of the effector function. Oxygen burst status in tobacco leaves was monitored by detecting accumulation of H₂O₂ using 3,3′-diaminobenzidine (DAB) according to the method of Thordal-Christensen et al. (1997). Briefly, agroinfiltrated tobacco leaves were detached at 72 h postinfiltration (hpi), soaked in 1 mg/ml DAB solution, and incubated at 25°C for 8 h. Leaf tissues were then boiled in 95% ethanol until all chlorophyll pigments were entirely bleached. For further removal of the background color, the bleached samples were soaked in 1.25 g/ml trichloroacetic aldehyde, and the leaves were photographed. Callose deposition was monitored at 48 h after agroinfiltration of N. benthamiana. All experiments were repeated at least three times.

Frozen tobacco leaves were ground in liquid nitrogen using a mortar and pestle, and 1 g powdered sample was mixed with 2 ml extraction buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 1% Tergitol-type NP-40] containing fresh “plant-specific protease inhibitor mixture” at 1 mM final concentration and 1 mM phenylmethylsulfonyl fluoride. The sample mixture was placed in an ice bath for 30 min with vortexing every 10 min. After ultrasonication for 5 min, the sample was centrifuged at 12,000 × g, 4°C for 20 min, and the supernatant was collected as a crude extract. Soluble protein concentration was determined with the Easy II Protein Quantitative Kit (BCA) TransGen Biotech Co., Ltd, Beijing, China) and 150 μg soluble protein was then denatured by urea and electrophoresed in a urea polyacrylamide gel. Proteins in the gel were electrotransferred onto a polyvinylidene fluoride membrane, and the FLAG tag of transiently expressed proteins was detected using an anti-FLAG antibody (ABclonal, Inc., Wuhan, China). The immunoblotting signal was detected using chemiluminescence technology (Vigorous Biotechnology Beijing Co., Ltd., Beijing, China) and X-ray film exposure to obtain an autoradiograph.

The tobacco rattle virus (TRV)-mediated method of gene silencing (Liu et al., 2002) uses two types of expression vector: the pTRV1 binary vector, which harbors an expression cassette of a cDNA clone of TRV RNA1, and the pTRV2-LIC-based vector, in which the target genes to be silenced are inserted via the multicloning site. These binary vectors were purchased from BioVector NTCC Inc. (Beijing, China), and cDNA clones of RAR1 and Hsp90 in N. benthamiana (GenBank ID: LC314308 and KT726860, respectively), NbPDS (GenBank ID: EU165355), and GFP were cloned into the pTRV2 vector. pTRV1 and the pTRV2-LIC-based vectors were transformed into A. tumefaciens GV3101. Overnight cultures of transformants for these constructs were prepared, and pTRV1 and one of the pTRV2-LIC-based clones of Agrobacterium cells were collected and mixed with MMA buffer [10 mM MgCl₂, 10 mM MES-Tris (pH 5.6), and 100 μM acetosyringone] to an OD₆₀₀ of 0.5. After incubation at room temperature for 3 h, the resultant bacterial mixture was injected into a few primary leaves of tobacco using a needle-free syringe. Three weeks later, Agrobacterium transformed with pMDC32-based constructs for RsSCR10 from R. solani AG1 IA or INF1 from Phytophthora cactorum (Genbank ID: KF893299) were infiltrated onto at least three leaves of tobacco plants. A total of 27 leaves from different plants were infiltrated for each control. Gene silencing status was verified by real-time quantitative RT-PCR (qRT-PCR).

Full-length RsSCR10 cDNA was subcloned into 2^×35S: pHB-YFP digested with HindIII and BamHI to generate the construct pHB-YFP-RsSCR10. This plasmid was separately transformed into A. tumefaciens GV3101. Agroinfiltrated tobacco leaves were excised into small pieces and imaged using a confocal laser scanning microscope (Nikon A1; Tokyo, Japan). Excitation and emission wavelengths for yellow fluorescent protein (YFP) were set at 514–527 nm.

Cotransfection assays in rice protoplasts were performed as previously described (Zhang et al., 2011; Chen et al., 2014). Namely, rice protoplasts were isolated and transfected using a plant protoplast preparation and transformation kit (Real-Times Biotechnology Co., Beijing, China) according to the manufacturer’s protocol. The pHB-YFP-RsSCR10 plasmid was purified from Escherichia coli clones using an E.Z.N.A. FastFilter Plasmid Maxi Kit (Omega Bio-tek, Inc., Norcross, GA, United States) and used for polyethylene glycol (PEG)-mediated transfection.

A functional test of the SP of RsSCR10 was performed using the method of Jacobs et al. (1997). Namely, the cDNA fragment corresponding to the predicted SP of RsSCR10 was amplified using a gene-specific primer set (Supplementary Table 1) and subcloned into the vector pSUC2 (BioVector NTCC Inc.), which generated an in-frame fusion with an invertase gene. The resultant construct pSUC2-RsSCR10^SP was transformed into the yeast Saccharomyces cerevisiae (S. cerevisiae) YTK12, and positive selection was performed on CMD-W medium, which lacks tryptophan. Positive colonies were inoculated on plates of YPRAA media for invertase secretion. YTK12 transformed with pSUC2-Avr1b^SP was used as a positive control, while YTK12 transformed separately with pSUC2-Mg87^SP or the empty pSUC2 vector were used as negative controls.

R. solani AG1 IA was inoculated onto leaf blades of R600, and mycelia were collected at 12, 24, 36, 48, and 60 hpi. Four-week-old tobacco plants were subjected to agroinfiltration as described above, and infiltrated leaves were harvested after 12, 24, 36, 48, and 72 hpi. Tobacco plants under the VIGS experiments were maintained for an additional 3 weeks, then primary leaves were harvested for quantitative gene expression analysis.

qRT-PCR was performed using a previously described method (Wu et al., 2010) and SYBR premix ex Taq (TaKaRa Co., Ltd., Tokyo, Japan). Real-time PCR reactions were conducted using the advanced Universal SYBR Green Supermix (Bio-Rad, Hercules CA, United States). cDNAs were diluted 10-fold, and 1 μl of each dilution was used for one assay. Gene-specific primers were designed for actin, NbPR1b (Genbank ID: X17680), NbPR2b (GenBank ID: M60460), NbLOX (GenBank ID: KC585517), and ethylene-responsive factor 1 (NbERF1; GenBank ID: GQ859157) in N. benthamiana and RsSCR10 and the 18S ribosomal RNA (rRNA) gene in R. solani (Supplementary Table 1). All PCR products were confirmed to be a single peak in melting curve analyses. Actin and the 18S rRNA gene were used as internal references to determine relative expression levels of genes by the ΔΔCT method (Livak and Schmittgen, 2002). qRT-PCR assays included biological triplicates and technical quadruplicates.

A normalized rice leaf cDNA library was constructed for yeast two-hybrid assays. Total RNA was extracted using TRIzol (Invitrogen Corp., Carlsbad, CA, United States), and poly (A)⁺ RNAs were purified using an Oligotex mRNA kit (Qiagen GmbH, Geschäftsführer, Germany). The integrity of the RNA fractions was analyzed by agarose gel electrophoresis. cDNAs were normalized, then ligated into the plasmid pGADT7 and transformed into the yeast S. cerevisiae Y2H gold strain. Total plasmids of the cDNA library were extracted for the screening of interacting proteins of interest. RsSCR10 cDNA was subcloned into the bait vector pGBKT7, cotransformed with the rice cDNA plasmids into Y₂H gold, and cultured on a synthetic dropout medium without leucine and tryptophan (SD/-Leu/-Trp) at 28°C for 3–5 days. Amplified yeast clones were transferred onto the synthetic dropout medium supplemented with aureobasidin A and X-α-Gal but lacking leucine, tryptophan, adenine, and histidine (SD/-Leu/-Trp/-Ade/-His/ + Aba/ + X-α-Gal) at 28°C for 3–5 days. Every colony obtained was then analyzed by PCR to determine the insert sequence. After repeating the screening at least six times, 30 proteins demonstrated a reproducible interaction with RsSCR10 and were collected as positive clones. These positive clones were separately tested in the yeast two-hybrid system to retain five candidate clones.

The cDNA insert of RsSCR10 was subcloned into the vector pXY106 (N-terminal split-nYFP) (Tao et al., 2019), which contains the partial coding sequence of YFP, and this subcloning yielded the fusion protein gene YN-RsSCR10. Full-length cDNAs of five rice genes without the stop codon were separately cloned into the pXY104 (C-terminal split-cYFP) vector from Tao et al. (2019), generating YC-OsP21, YC-PsbO, YC-OsHyPRP8, YC-OsALDH2B1, and YC-OsBcat-3. The different pairs of plasmids containing the split nYFP- and cYFP-tagged protein were transiently coexpressed in leaf epidermis cells of N. benthamiana by using the method with agroinfiltration described above. A total of 45 leaves from different plants were infiltrated for each control. YFP signals were detected using a Nikon A1 confocal scanning microscope after 48 h of agroinfiltration.

The cDNA fragments of RsSCR10 and five rice genes were subcloned into the SacI/SalI site of pCAMBIA1300-nLuc or the KpnI/BamHI site of pCAMBIA1300-cLuc (Chen et al., 2008). The resulting recombinant plasmids were, respectively transformed into A. tumefaciens GV3101 and used for agroinfiltration into leaves of 1-month-old tobacco plants. A total of 45 leaves from different plants were infiltrated for each control. Leaf samples were harvested within 48–72 h after agroinfiltration and smeared with a solution of D-Luciferin sodium salt (BioVision Inc., San Francisco, CA, United States) to detect fluorescence by luciferase activity.

Prediction of effector proteins was performed using EffectorP 2.0 (Sperschneider et al., 2018). Prediction of SP sequences was performed using SignalP 4.1 (Petersen et al., 2011) with the default setting parameters. Subcellular localization of rice proteins was predicted using TargetP version 1.1 (Emanuelsson et al., 2000).

Student’s t-tests were performed using Microsoft Excel software. The R statistical package¹ was used for checking the equality of variance and conducting a two-way analysis of variance (ANOVA).

In a previous study on the R. solani genome (Zheng et al., 2013), a total of 103 SCR-secreted proteins were predicted as potential effectors. The present study used a machine-learning method for the prediction of effector proteins (Sperschneider et al., 2018) to select 10 SCRs to focus on (Supplementary Table 1). cDNAs for these genes were successfully cloned from the national standard strain of R. solani AG1 IA, and their molecular functions were examined by agroinfiltration-based bioassays in model plant N. benthamiana. Infiltration of tobacco leaves with two of these genes, AG1 IA_09956 and AG1 IA_10375, resulted in the observation of cell death with transiently expressed protein in the agroinfiltrated leaves (Figure 1A). To determine whether these two genes induced the defense response in other plants, the two constructs were transiently expressed into Arabidopsis leaves by agroinfiltration-based bioassays and the phenotype was examined (Figure 1B). Only AG1 IA_09956 resulted in the cell death phenotype in the non-host plant Arabidopsis leaves (Figure 1B). Hence, the cloned cDNA of AG1 IA _09956 was designated RsSCR10 and was a transcript variant from the gene AG1 IA_09956. Consequently, RsSCR10 was selected as the object of subsequent research. There was more accumulated hydrogen peroxide (H₂O₂) and callose deposition in the agroinfiltrated leaves than in control leaves (Supplementary Figure 1), accompanied by distinct gene expression patterns for the effector compared with the GFP control (Supplementary Figure 2). These findings suggested that RsSCR10 stimulated basal host immunity in tobacco, resulting in oxidative burst and callose deposition.

A probable SP was predicted in the N-terminus of RsSCR10 via a search using SignalP 4.1 (Petersen et al., 2011). To validate the function of the SP of RsSCR10, a signal sequence trap system in yeast (Jacobs et al., 1997) was employed. As expected, the fusion protein of invertase with the SP from RsSCR10 was secreted from the transformed yeast YTK12 strain (Figure 2), indicating that RsSCR10 is trafficked via the classical secretory pathway. Furthermore, in the agroinfiltration bioassays, there were differences in bioactivity between the full-length RsSCR10 and the partial-length RsSCR10 that lacked the SP. When the SP of elicitin (encoded by the INF1 gene) was substituted for the SP of RsSCR10, the fusion protein induced cell death in tobacco (Figure 2B). These results indicated that the SP encoded by the gene RsSCR10 has a secretory function and is necessary for RsSCR10 to induce cell death into tobacco.

To investigate whether RsSCR10 activates the immune response in N. benthamiana, qRT-PCR was used to evaluate the expression of several genes related to activation of the immune response in N. benthamiana. Gene markers for host defense signaling, pathogenesis-related protein 1B (PR1b), pathogenesis-related protein 2B (PR2b), ethylene-responsive transcription factor 1 (ERF1), and linoleate 9S-lipoxygenase 5 (LOX), which were relevant for salicylic acid, ethylene, and jasmonic acid signaling pathways, respectively (Wang et al., 2000; Lorenzo et al., 2003; Lee et al., 2013), exhibited different expression patterns between agroinfiltrated samples for RsSCR10 and GFP control samples. These differences in gene expression were statistically significant. Specifically, compared with the control samples, NbPR1b, NbPR2b, and NbERF1 exhibited higher expression levels at 36 hpi, while Nb-9-LOX exhibited higher expression at 24 hpi. These findings support that RsSCR10 has elicitor activity (Figure 3).

RsSCR10 encodes an 84-aa protein that contains a predicted N-terminal SP (19 aa) and 10 conserved cysteines (Cys) residues. To identify the key amino acid residues of RsSCR10 that were required for induction of cell death, RsSCR10 sequences alignment in 25 strains of R. solani AG1 IA isolated from different areas of China were analyzed (Supplementary Figure 3; Wei et al., 2020). Among the 25 strains, cysteine residues were particularly conserved in RsSCR10 (Supplementary Figure 3). The functionality of Cys residues in RsSCR10 was assessed by mutating Cys sites to Alanine in RsSCR10 using overlap-PCR, and transient expression constructs containing the mutations in RsSCR10 were infiltrated into tobacco leaves. RsSCR10^C23A and RsSCR10^C49A could not induce tobacco cell death (Figure 4A), but cell death was induced in N. benthamiana leaves by RsSCR10 containing the other Cys mutations (Figures 4A,B). Western blotting revealed that all mutated proteins of RsSCR10 were expressed in the infiltrated leaves (Figure 4C). These results indicate that mutations of residues 23 and 49 prevented RsSCR10 from triggering cell death in N. benthamiana and therefore these sites might be key amino acid residues for the function of RsSCR10.

Effector genes in filamentous phytopathogens are often transcriptionally induced during infection (Stergiopoulos and De Wit, 2009; Xia et al., 2017). To explore whether RsSCR10 was regulated differently after inoculation onto plant hosts exhibiting variations in sheath blight resistance, gene expression patterns of RsSCR10 in R. solani AG1 IA inoculated on the susceptible rice variety R600, expression of RsSCR10 was significantly increased at 12, 24, 48, and 60 hpi, relative to the 18S rRNA gene, and the highest expression among the samples analyzed were detected at 12 hpi (at 1% level in Student’s t-test) (Figure 5). After inoculating rice leaves with rice sheath blight AG1 IA, a small number of disease spots were found 12 h later. After 24 h, the disease spots increased and gradually expanded into moiré, with gray white spots in the middle. At 60 h, the disease of leaves was serious and becoming yellows. Increased expression of RsSCR10 at 12 hpi on R600 may be necessary to modulate host immunity. This result shows that RsSCR10 is possibly involved in the preprocess of pathogenesis by R. solani.

To explore plant immunological pathways affected by RsSCR10, an independent VIGS assay in tobacco was designed for two genes—Hsp90, encoding the molecular chaperone heat shock protein 90 that acts to stabilize R proteins, and RAR1, which is required for Mla12 resistance and is involved in PAMP-triggered immunity (PTI), with both genes playing a role in effector-triggered immunity (ETI) (Shirasu, 2003, 2009). Tobacco plants with each gene silenced were agroinfiltrated with RsSCR10 or INF1. RsSCR10 failed to trigger cell death in Hsp90-silenced plants, whereas it induced cell death in RAR1-silenced plants (Figure 6A). Quantitative gene expression analysis confirmed the successful silencing of the two genes (Figure 6B). This was consistent with RsSCR10 not being a PAMP but being an effector protein. Furthermore, the results suggest that cell death induced by RsSCR10 is dependent on either of the R proteins stabilized by Hsp90 but not by RAR1.

To determine the subcellular localization of RsSCR10, the protein was explored by transiently expressing 2^×35S: RsSCR10-YFP in tobacco leaf epidermis cells. Transient expression of 2^×5S: YFP was used as a control. Green fluorescence was observed in multiple subcellular compartments of the infiltrated N. benthamiana cells (Figure 7). RsSCR10-YFP was coexpressed with the plasma membrane marker OsPIP2.1-mCherry and the nucleus marker PR82:2 × RFP-NLSSV40, respectively, into tobacco cells. Red fluorescence from OsPIP2.1-mCherry was partially overlapped with green fluorescence, indicating that RsSCR10 is not only localized to the membrane but also in the cytoplasm (Figures 7A–D). Meanwhile, the nucleus marker PR82:2 × RFP-NLSSV40 coexpressed with RsSCR10 further showed that RsSCR10-YFP is present in the nuclei of N. benthamiana cells (Figures 7G–J). To investigate whether the same subcellular localization occurred in different plant host cells, transient expression assays were conducted in rice protoplasts using the same YFP fusion constructs. Transient expression of RsSCR10-YFP in rice protoplasts exhibited a similar subcellular localization in the nuclei, cytoplasm, and membrane as well (Supplementary Figure 4).

Identification of protein cross-interaction between pathogens and plant host organisms could provide a hint of how RsSCR10 modulates host immunity. A yeast two-hybrid system was utilized to screen interacting partners of RsSCR10 from a normalized cDNA library constructed from rice leaf blades at the reproductive stage. Multiple-time screening retained potential positive cDNA clones for 30 proteins. Screening of multiple yeast databases identified 13 positive proteins (positive in at least three repeats) from the 30 genes and the prey and bait plasmids were then co-transformed into the Y2H gold strain. Five positive genes were obtained (Figure 8A), these were type III chlorophyll a/b-binding protein (OsP21) [MSU locus ID: LOC_Os07g37550], aldehyde dehydrogenase (OsALDH2B1) [MSU locus ID: LOC_Os06g15990], photosystem II subunit PsbO (OsPsbO) [MSU locus ID: LOC_Os01g31690], hybrid proline- or glycine-rich protein 8 (OsHyPRP8) ([MSU locus ID: LOC_ Os04g46830], and similar to branched-chain-amino-acid aminotransferase 3 (OsBcat-3) [MSU locus ID: LOC_Os03g12890]. Bimolecular fluorescence complementation (BiFC) assays in tobacco epidermal cells and luciferase complementation assays (LCAs) in tobacco leaves confirmed the cross-interactions of the above five proteins with RsSCR10 (Figures 8B, 9). These results proved that these five proteins are probable interacting partners of RsSCR10 in host plant cells. Moreover, the results indicate that the RsSCR10 is instrumental in the process of host infection and may interfere with the growth and development of the host, which is conducive to the invasion of pathogenic fungi.