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Whole-Genome Metalloproteases in the Wheat Sharp Eyespot Pathogen Rhizoctonia cerealis and a Role in Fungal Virulence

Rhizoctonia cerealis is the causal agent of sharp eyespot, a devastating disease of cereal crops including wheat. Several metalloproteases have been implicated in pathogenic virulence, but little is known about whole-genome metalloproteases in R. cerealis. In this study, a total of 116 metalloproteases-encoding genes were identified and characterized from the R. cerealis Rc207 genome. The gene expression profiles and phylogenetic relationship of 11 MEP36/fungalysin metalloproteases were examined during the fungal infection to wheat, and function of an upregulated secretory MEP36 named RcFL1 was validated. Of 11 MEP36 family metalloproteases, ten, except RcFL5, were predicted to be secreted proteins and nine encoding genes, but not RcFL5 and RcFL2, were expressed during the R. cerealis infection process. Phylogenetic analysis suggested that MEP36 metalloproteases in R. cerealis were closely related to those of Rhizoctonia solani but were remote to those of Bipolaris sorokiniana, Fusarium graminearum, F. pseudograminearum, and Pyricularia oryzae. Expression of RcFL1 was significantly upregulated during the infection process and induced plant cell death in wheat to promote the virulence of the pathogen. The MEP36 domain was necessary for the activities of RcFL1. Furthermore, RcFL1 could repress the expression of wheat genes coding for the chitin elicitor receptor kinase TaCERK1 and chitinases. These results suggest that this MEP36 metalloprotease RcFL1 may function as a virulence factor of R. cerealis through inhibiting host chitin-triggered immunity and chitinases. This study provides insights on pathogenic mechanisms of R. cerealis. RcFL1 likely is an important gene resource for improving resistance of wheat to R. cerealis through host-induced gene silencing strategy.


Introduction
Bread wheat (Triticum aestivum L.) is one of the most important staple crops worldwide [1]. However, yield and grain quality of wheat often are negatively impacted by fungal diseases [2]. The necrotrophic fungus Rhizoctonia cerealis van der Hoeven [3] is the major causal agent of sharp eyespot, a destructive stem-base disease of wheat [4,5]. Sharp eyespot can reduce yields of wheat in many regions of the world, including Asia, Oceania, Europe, North America, and Africa [6][7][8][9]. In addition, the fungus R. cerealis can also infect other important economical crops and bio-energy plans, including barley (Hordeum vulgare), rye (Secale cereale), sugar beet (Beta vulgaris), cotton (Gossypium spp.), potato (Solanum tuberosum), and turfgrasses [10,11]. To control wheat sharp eyespot, it is vital to identify host resistance genes, mine important virulence factors of the pathogen

Genome-Wide Characterization of Metalloproteases Based on R. cerealis Genome Assembly
The recently-constructed proteases database MEROPS currently contains over 4000 entries, and MEROPS classification is an excellent tool for categorizing proteases deduced from genome annotations and predicting functions of validated proteases [20,25]. Herein, the MEROPS database (release 12.4) [20] was deployed to identify genome-wide metallopro-teases from the high-quality genome assembly of R. cerealis Rc207. The results showed that the Rc207 genome contained a total of 116 genes encoding metalloproteases, belonging to 24 families (Table S1). In these 24 metalloprotease families, MEP24, MEP28, and MEP36 families are particularly richer in R. cerealis Rc207. Based on the presence of a typical signal peptide, 34 metalloproteases in R. cerealis Rc207 genome belong to secretory proteins. Among them, 3,3,9,8,10, and 1 secretory proteins belong to the MEP1, MEP14, MEP28, MEP35, MEP36, and MEP64 families, respectively (Table S1). Furthermore, in R. cerealis Rc207, 32 metalloproteases are also annotated as pathogen-host interaction (PHI) proteins, which may play significant roles in pathogen infection (Table S1). Although certain researchers annotated candidate effectors with a signal peptide-containing protein pipeline [26], experimentally-validated effectors/virulance factors tend to be cysteine-rich (≥4) [27]. Our analysis indicated that these 116 metalloproteases included 93 cysteine-rich (the number of cysteine ≥ 4) metalloproteases (Table S1). These secreted, cysteine-rich metalloproteases likely function as virulence factors.
To further investigate the sequence similarity between metalloproteases in the R. cerealis Rc207 genome, OrthoFinder v2.5.1 software (https://github.com/davidemms/OrthoFinder/ releases, accessed on 30 November 2020) was used to group all metalloprotease sequences into ortholog groups (OGs). These 116 metalloproteases from R. cerealis Rc207 were clustered in 65 OGs by OrthoFinder (Table S1). Then, we analyzed the OG distribution of the metalloproteases in the R. cerealis Rc207 genome, and results showed that 41 OGs are specific, containing only one metalloprotease, and the largest OG present in the data set is OG6_103356 with nine metalloproteases from the MEP28 family, which are able to release a variety of N-terminal amino acids (Table S1).

Characterics of MEP36 Metalloproteases and Their Encoding Genes' Structures
We focused on MEP36 domain-containing metalloproteases, possessing HEXXH motifs characteristic of Zn-binding metalloproteases, since almost all MEP36 metalloproteases in R. cerealis Rc207 genome are predicted as secreted proteins, each with a signal peptide in the N-terminal. In previous studies, MEP36 metalloproteases have been named as fungalysins [17,22]. Thus, these 11 MEP36 genes in R. cerealis are designated as RcFL1-RcFL11 (Table 1). By means of analysis using DNAMAN software, we found that the metalloprotease gene RcFL4 possesses the shortest coding sequence size with 1788 bp, and the coding sequence sizes of RcFL8 and RcFL10, with 2346 bp, are the longest. Multiple sequence alignment results showed that the identity among the coding sequences of the 11 RcFLs genes was 56.95% ( Figure S1). All the 11 MEP36 metalloprotease genes in R. cerealis had different intron-exon patterns in relation to both the position and number of introns. Based on the number of introns, these metalloproteases were segregated into four patterns, pattern1 (RcFL8 and RcFL10), pattern 2 (RcFL1, RcFL3, and RcFL4), pattern 3 (RcFL2, RcFL5, and RcFL11), and pattern 4 (RcFL6, RcFL7, and RcFL9), containing 6, 7, 8, and 9 introns, respectively. Furthermore, significant differences in size (28-875 bp) between the exons were observed. These results suggested that these MEP36 metalloprotease genes had variable and complex gene structures ( Figure 1).
The Pfam software was used to predict the domains of these RcFLs proteins, and the results showed that all the 11 RcFL proteins, RcFL1-RcFL11, contained the MEP36 domain. With the exception of RcFL5, 10 remaining RcFLs all contained a signal peptide with a length of 19-23 amino acid (aa) residues and a Fungalysin/Thermolysin Propeptide Motif (FTP) with a length of 32-50 aa residues ( Figure 2). Thus, expect for RcFL5, the 10 remaining R. cerealis metalloproteases, namely RcFL1-RcFL4 and RcFL6-RcFL11, were predicted to be secreted proteins based on the presence of a typical signal peptide (Table 1). Using DNAMAN software, we found that the shortest metalloprotease protein, RcFL4, consisted of 595 aa residues, and the longest metalloprotease proteins, RcFL8 and RcFL10, had 781 aa residues. The lengths of the MEP36 domains across RcFL1-RcFL11 ranged from 175 aa (RcFL7) to 417 aa (RcFL8 and RcFL10) residues ( Figure 2). The predicted molecular weights of all the metalloprotease proteins ranged from 63.38 kD to 86.13 kD and their isoelectric points (pIs) ranged from 5.20 to 6.68, respectively (Table 1). Multiple sequence alignment results showed that the identity among the amino acid sequences encoded by the 11 RcFLs genes was 49.70% ( Figure S2).   The Pfam software was used to predict the domains of these RcFLs proteins, and the results showed that all the 11 RcFL proteins, RcFL1-RcFL11, contained the MEP36 domain. With the exception of RcFL5, 10 remaining RcFLs all contained a signal peptide with a length of 19-23 amino acid (aa) residues and a Fungalysin/Thermolysin Propeptide Motif (FTP) with a length of 32-50 aa residues ( Figure 2). Thus, expect for RcFL5, the 10 remaining R. cerealis metalloproteases, namely RcFL1-RcFL4 and RcFL6-RcFL11, were predicted to be secreted proteins based on the presence of a typical signal peptide (Table  1). Using DNAMAN software, we found that the shortest metalloprotease protein, RcFL10, had 781 aa residues. The lengths of the MEP36 domains across RcFL1-RcFL11 ranged from 175 aa (RcFL7) to 417 aa (RcFL8 and RcFL10) residues ( Figure 2). The predicted molecular weights of all the metalloprotease proteins ranged from 63.38 kD to 86.13 kD and their isoelectric points (pIs) ranged from 5.20 to 6.68, respectively (Table 1). Multiple sequence alignment results showed that the identity among the amino acid sequences encoded by the 11 RcFLs genes was 49.70% ( Figure S2).
The resulting phylogenetic tree divided these MEP36 metalloproteases into five groups ( Figure 3). The Group I (grey) was constituted by RcFL4, RcFL6, RcFL8, RcFL9, RcFL10, and RcFL11 from R. cerealis Rc207 with seven MEP36 metalloproteases from R. solani AG-8 (KDN45325. 1  . Phylogenetic relationships among MEP36 metalloproteases from R. cerealis and other fungal plant-pathogens. The phylogenic tree was constructed by Mega 7.0 using the neighbor-joining method (parameters: 1000 bootstraps). Grey represents group I; Blue represents group II; Green represents group III; Yellow represents group IV; Red represents group V. These stars represent RcFLs proteins from R. cerealis.
The resulting phylogenetic tree divided these MEP36 metalloproteases into five groups ( Figure 3). The Group I (grey) was constituted by RcFL4, RcFL6, RcFL8, RcFL9, RcFL10, and RcFL11 from R. cerealis Rc207 with seven MEP36 metalloproteases from R. solani AG-8 (KDN45325. 1  . Phylogenetic relationships among MEP36 metalloproteases from R. cerealis and other fungal plant-pathogens. The phylogenic tree was constructed by Mega 7.0 using the neighbor-joining method (parameters: 1000 bootstraps). Grey represents group I; Blue represents group II; Green represents group III; Yellow represents group IV; Red represents group V. These stars represent RcFLs proteins from R. cerealis.

Gene Expression Analysis of Metalloproteases during R. cerealis Infection Process to Wheat
To investigate how the metalloprotease genes of fungal transcriptional reprogramming occurs during R. cerealis infection to wheat, we performed RNA-sequencing to investigate transcript dynamics of the metalloprotease genes in the R. cerealis Rc207 strain at five infection time-points (18,36,72,96, and 240 h after inoculation, hai) to wheat and in culture (vitro) mycelia. The transcriptomes showed that 82, 82, 98, 116, and 116 genes were expressed at 18, 36, 72, 96, and 240 hai, suggesting there was a temporal sequence leading to full expression of all genes (Table S2, Figure S3). Compared with in vitro, a total of 6, 4, 13, 19, and 19 genes were significantly upregulated (log 2 fold-change > 1, FDR P < 0.05) at 18, 36, 72, 96, and 240 hai, respectively (Table S2, Figure S3). Furthermore, we investigated the gene expression profiles of the 11 MEP36 metalloproteases in R. cerealis. Their RNA-sequencing results were shown in the heat map, and nine encoding genes were expressed during R. cerealis infection process to wheat ( Figure 4A). Among them, the transcript levels of the RcFL1 (Rc_11192.1) were significantly up-regulated during R. cerealis infection process (18,72,96, and 240 hai) compared to in vitro fungal mycelia ( Figure 4A). The RT-qPCR analysis indicated that the transcript abundances of the RcFL1 (Rc_11192.1) were markedly upregulated during the R. cerealis infection process (18,36,72,96, and 240 hai) compared to in vitro fungal mycelia ( Figure 4B), peaked at 72 hai. Hereafter, we further investigated the functional role of the RcFL1 in R. cerealis infection to wheat.
Furthermore, we investigated the gene expression profiles of the 11 MEP36 metalloproteases in R. cerealis. Their RNA-sequencing results were shown in the heat map, and nine encoding genes were expressed during R. cerealis infection process to wheat ( Figure  4A). Among them, the transcript levels of the RcFL1 (Rc_11192.1) were significantly up-regulated during R. cerealis infection process (18,72,96, and 240 hai) compared to in vitro fungal mycelia ( Figure 4A). The RT-qPCR analysis indicated that the transcript abundances of the RcFL1 (Rc_11192.1) were markedly upregulated during the R. cerealis infection process (18,36,72,96, and 240 hai) compared to in vitro fungal mycelia ( Figure  4B), peaked at 72 hai. Hereafter, we further investigated the functional role of the RcFL1 in R. cerealis infection to wheat.

RcFL1 Induces Necrosis and Plant-Cell Death in Leaves of Wheat and Nicotiana benthamiana
To investigate role of RcFL1 in virulence of R. cerealis, we examined whether RcFL1 full-length protein and the truncated version containing MEP36 domain (no. 349-749 aa, RcFL1-MEP36) can induce the cell death of wheat or N. benthamiana leaves. To obtain purified RcFL1 and RcFL1-MEP36 proteins, the full RcFL1 or the RcFL1-MEP36 peptideencoding sequences were separately sub-cloned in fusion to the His-TF tag of the pCOLD Trigger Factor (TF) vector, generating two recombinant protein vectors pHis-TF-RcFL1 and pHis-TF-RcFL1-MEP36. After induction by 0.5 mM isopropyl-β-d-thiogalactoside at 16 • C, the resulting pHisTF-RcFL1 and pHis-TF-RcFL1-MEP36 as well as the control His-TF proteins were highly expressed in E. coli DE3, respectively. After purification using Ni+ resin and elution with imidazole buffer, these proteins were examined through SDS-PAGE ( Figure S4). Subsequently, the purified His-TF-RcFL1, His-TF-RcFL1-MEP36, or His-TF proteins, each 25 µL (concentration 5 µM/L), were individually infiltrated into detached leaves of the R. cerealis-susceptible wheat cv. Wenmai 6. As a result, compared to His-TF (CK), the His-TF-RcFL1 (expressing the full RcFL1 protein) and His-TF-RcFL1-MEP36 (expressing the M36 domain of RcFL1) were able to trigger more obvious necrosis and plant cell death on the infiltrated leaves of susceptible wheat cv. Wenmai 6 ( Figure 5A). Additionally, the results of trypan blue staining, which detects plant cell death, showed that, compared with those treated by His-TF control protein, the stain sizes in wheat leaves with His-TF-RcFL1 were larger and more marked ( Figure 5B). These results suggest that RcFL1 is able to induce plant cell death and that the MEP36 domain is required for the cell death-inducing activity of this metalloprotease. Cell death induction by His-TF-RCFL1 was reproduced also in tobacco leaves ( Figure 5C).
6. As a result, compared to His-TF (CK), the His-TF-RcFL1 (expressing the full RcFL1 protein) and His-TF-RcFL1-MEP36 (expressing the M36 domain of RcFL1) were able to trigger more obvious necrosis and plant cell death on the infiltrated leaves of susceptible wheat cv. Wenmai 6 ( Figure 5A). Additionally, the results of trypan blue staining, which detects plant cell death, showed that, compared with those treated by His-TF control protein, the stain sizes in wheat leaves with His-TF-RcFL1 were larger and more marked ( Figure 5B). These results suggest that RcFL1 is able to induce plant cell death and that the MEP36 domain is required for the cell death-inducing activity of this metalloprotease. Cell death induction by His-TF-RCFL1 was reproduced also in tobacco leaves ( Figure 5C).

RcFL1 Protein Is Required for R. cerealis Infection to Wheat
To further examine the virulence roles of RcFL1 protein or its MEP36 domain-containing peptide (RcFL1-MEP36), the control His-TF, RcFL1, or RcFL1-MEP36 proteins were individually infiltrated into leaves of a R. cerealis-susceptible wheat cv. Wenmai 6 for six hours, and then the level surfaces were further inoculated with liquid hypha of R. cerealis Rc207. After three days, the water-soaking (disease lesion) areas in these wheat leaves were assessed. As a result, the larger disease lesion sizes were present in His-TF-RcFL1 and His-TF-RcFL1-MEP36 infiltrated leaves compared to those infiltrated with the His-TF control ( Figure 6A). The statistical analysis also indicated the significantly different lesions and R. cerealis biomass occurred between the His-TF-RcFL1/

RcFL1 Protein Is Required for R. cerealis Infection to Wheat
To further examine the virulence roles of RcFL1 protein or its MEP36 domain-containing peptide (RcFL1-MEP36), the control His-TF, RcFL1, or RcFL1-MEP36 proteins were individually infiltrated into leaves of a R. cerealis-susceptible wheat cv. Wenmai 6 for six hours, and then the level surfaces were further inoculated with liquid hypha of R. cerealis Rc207. After three days, the water-soaking (disease lesion) areas in these wheat leaves were assessed. As a result, the larger disease lesion sizes were present in His-TF-RcFL1 and His-TF-RcFL1-MEP36 infiltrated leaves compared to those infiltrated with the His-TF control ( Figure 6A). The statistical analysis also indicated the significantly different lesions occurred between the His-TF-RcFL1/ His-TF-RcFL1-MEP36 and the His-TF ( Figure 6B). These results suggested that both RcFL1 and its MEP36 domain peptides contribute to virulence of R. cerealis and the fungal infection to wheat.

RcFL1 Represses the Expression of TaCERK1 (a Chitin Elicitor Receptor Kinase) and Chitinases in Wheat
To explore if RcFL1 functional role is related to the chitin elicitor pathway or chitinases in wheat, RT-qPCR was deployed to examine transcript levels of TaCERK1 (TraesCS6D01G403200.1), a wheat chitin elicitor receptor kinase CERK1, and two chitinase-coding genes (TaChit3 and TaChitIV) in the susceptible wheat cv. Wenmai 6 leaves infiltrated with His-TF-RcFL1 or the His-TF control. The results showed that the expression levels of TaCERK1, TaChit3 and TaChitIV were significantly decreased by the RcFL1 protein in the His-TF-RcFL1 infiltrated wheat leaves compared to His-TF infiltrated wheat leaves and healthy wheat leaves (Figure 7), suggesting that RcFL1 repressed the expression of the kinase gene TaCERK1 and chitinases (TaChit3 and TaChitIV) in wheat. His-TF-RcFL1-MEP36 and the His-TF ( Figure 6B). These results suggested that both RcFL1 and its MEP36 domain peptides contribute to virulence of R. cerealis and the fungal infection to wheat.

RcFL1 Represses the Expression of TaCERK1 (a Chitin Elicitor Receptor Kinase) and Chitinases in Wheat
To explore if RcFL1 functional role is related to the chitin elicitor pathway or chitinases in wheat, RT-qPCR was deployed to examine transcript levels of TaCERK1 (TraesCS6D01G403200.1), a wheat chitin elicitor receptor kinase CERK1, and two chitinase-coding genes (TaChit3 and TaChitIV) in the susceptible wheat cv. Wenmai 6 leaves infiltrated with His-TF-RcFL1 or the His-TF control. The results showed that the expression levels of TaCERK1, TaChit3 and TaChitIV were significantly decreased by the RcFL1 protein in the His-TF-RcFL1 infiltrated wheat leaves compared to His-TF infiltrated wheat leaves and healthy wheat leaves (Figure 7), suggesting that RcFL1 repressed the expression of the kinase gene TaCERK1 and chitinases (TaChit3 and TaChitIV) in wheat.   Heterologously-expressed protein, His-TF-RcFL1, His-TF-RcFL1-MEP36, or His-TF; each was infiltrated in 25 µL (concentration 5 µM/L). Pictures of the lesions were taken at three days post inoculation with the fungus and the lesion area was measured; (B) the lesion area/infection was measured after R. cerealis Rc207 liquid mycelia inoculation for three days on leaves infiltrated with heterologously-expressed RcFL1, RcFL1-MEP36 in 25 µL (concentration 5 µM/L). Six leaves were used in each experiment and repeated three times. Asterisk * indicates significant difference between His-TF-RcFL1 or His-TF-RcFL1-MEP36 treatment and His-TF treatment (t-tests, p < 0.05).

RcFL1 Represses the Expression of TaCERK1 (a Chitin Elicitor Receptor Kinase) and Chitinases in Wheat
To explore if RcFL1 functional role is related to the chitin elicitor pathway or chitinases in wheat, RT-qPCR was deployed to examine transcript levels of TaCERK1 (TraesCS6D01G403200.1), a wheat chitin elicitor receptor kinase CERK1, and two chitinase-coding genes (TaChit3 and TaChitIV) in the susceptible wheat cv. Wenmai 6 leaves infiltrated with His-TF-RcFL1 or the His-TF control. The results showed that the expression levels of TaCERK1, TaChit3 and TaChitIV were significantly decreased by the RcFL1 protein in the His-TF-RcFL1 infiltrated wheat leaves compared to His-TF infiltrated wheat leaves and healthy wheat leaves (Figure 7), suggesting that RcFL1 repressed the expression of the kinase gene TaCERK1 and chitinases (TaChit3 and TaChitIV) in wheat. The wheat Actin gene was used as an internal control to normalize the data. SE was calculated Figure 7. The RcFL1 protein treatment decreases the transcripts of wheat chitinases and receptor-like kinase in infiltrated leaves. RNAs were extracted from wheat leaves at 3 d post treatment with His-TF-RcFL1 or the His-TF (CK) in 25 µL at a concentration of 5 µM/L and none-treatment. The wheat Actin gene was used as an internal control to normalize the data. SE was calculated based on three technical replicates. ** indicates significant differences (p < 0.01, t-test) between His-TF-RcFL1 and the His-TF infiltrated leaves.

Discussion
Metalloproteinases, in which zinc is an essential metal ion for the catalytic activity, have been identified with the pathogenicity and virulence of pathogenic fungi [17,[19][20][21][22][23][24][25]28]. In this study, a total of 116 metalloproteases were identified from the advanced wholegenome sequencing data of the wheat sharp eyespot pathogen R. cerealis Rc207. They were divided into 24 protein families, including 34 putative secretory metalloproteases with a predicted signal peptide. In the plant pathogen P. infestans, 91 metalloproteases were identified and divided over 21 protein families, including 25 metalloproteases with a predicted signal peptide or signal anchor [25]. Researchers reported that secreted proteins with a signal peptide often are candidate effectors or are important for virulence of pathogens [24][25][26][27]29,30]. In the R. cerealis genome, the MEP1, MEP14, MEP28, MEP35, MEP36, and MEP64 families contain 3, 3, 9, 8, 10, and 1 secretory metalloproteases, respectively. Previously, only 5 MEP35 proteins were identified from next-generation genome sequences of R. cerealis Rc207. These results suggest that the advanced whole-genome assembly of the R. cerealis Rc207 is high-quality.
Necrotrophic fungal pathogens utilize an array of effectors to induce plant cell death which may facilitate the growth of the necrotrophic pathogens [27,30]. Our previous study showed that the secretory M43 metalloprotease of R. cerealis are able to induce plant celldeath and may act as a virulence factor of R. cerealis [24]. Here, gene profile analyses showed that the transcript abundance of the fungal MEP36 gene RcFL1 was markedly upregulated during the R. cerealis infection process (18,36,72,96, and 240 hai) compared to the fungal grown in culture. Furthermore, the functional analyses demonstrated that the secretory R. cerealis MEP36 metalloprotease RcFL1 could induce plant cell-death and necrotic lesions, and promoted the fungal virulence. Moreover, our analyses suggest that the MEP36 domain is necessary for the function of the metalloprotease RcFL1 in the virulence of R. cerealis.
Chitin is a key component of the cell walls of pathogenic fungi, such as Rhizoctonia isolates [12]. Plants have evolved chitin-mediated defense responses againstpathogenic fungi, including generation of chitinases and chitin-triggered immunity [12,18,22,23,31,32]. Meanwhile, plant pathogenic fungi can generate various enzymes and proteins, such as polysaccharide deacetylase and metalloproteases, to inhibit host plant chitin-triggered immunity or directly degrade chitinases [17,18,22]. For instance, VdPDA1, a secretory polysaccharide deacetylase in the soil-borne fungus Verticillium dahlia, is required for full virulence of the pathogen through preventing host chitin-triggered immunity [18]. The fungalysin/MEP36 metalloprotease from maize pathogen F. verticilloides could completely cleave the classs IV chitinase ChitA from maize [17]. Interestingly, the current research indicated that the R. cerealis MEP36 metalloprotease RcFL1 could repress the expression of chitinases (TaChit3 and TaChitIV) and gene coding for the chitin elicitor receptor kinase TaCERK1 in host wheat. The data imply that the MEP36 metalloprotease RcFL1 functions as a virulence factor of R. cerealis possibly through preventing the host chitin-triggering immunity and reduced transcript accumulation of chitinases. It is very interesting to determine experimentally the detailed role of RcFL1 in the loss of this MAMP system in future. Taken together, the fungal-pathogen MEP36 metalloprotease RcFL1 can promote plant cell death and turn down host defense measure to help R. cerealis infect wheat.

Plant Materials, Fungal Strain, Growth Conditions, and Primers
Sharp eyespot-susceptible wheat cultivar Wenmai 6 plants were grown in a greenhouse under a 13-h light (~22 • C)/11-h dark (~10 • C) regime. Using the toothpick inoculated method, the base sheath of wenmai 6 plant was inoculated with R. cerealis Rc207 at wheat tillering stage [33]. The sheath and/or stems of Wenmai 6 at five different infection time points (18,36,72,96, and 240 hai) with R. cerealis Rc207 and in vitro mycelia were sampled for deep RAN-Sequencing.
N. benthamiana plants were grown under standard glasshouse conditions at 25 • C with a 12-h light and 12-h dark regime.
The necrotrophic fungus R. cerealis Rc207, a highly aggressive strain collected in Northern China, was isolated in Shandong [7]. This strain Rc207 was cultured on potato dextrose agar (PDA) at 25 • C for 10 days before inoculation.
All the primers and the sequences in the study are listed in Table S3.

RNA Extraction, cDNA Synthesis and Real-Time Quantitative PCR (RT-qPCR) Analysis
According to the manufacturer's instruction, total RNAs from R. cerealis Rc207 in vitro mycelia and R. cerealis-inoculated sheaths and/or stems, or RcFL1-treated leaves of the susceptible wheat cv. wenmai 6 plants were extracted using the TRIzol reagent (Invitrogen, Life Technologies, Carlsbad, CA, USA) [38]. Reverse transcription was performed by using a PrimeScriptTM RT Reagent Kit with gDNA Eraser (Takara, City, Japan).
The R. cerealis infection process in wheat, fungal, or wheat RNAs were extracted for examining transcripts of the RcFL1, TaChit3, TaChitIV, and TaCERK1 genes. Following the procedure described in Dong et al. [39], RT-qPCR was conducted with an ABI 7500 RT-PCR system (Applied Biosystems, Waltham, MA, USA). The relative expression of the target gene (RcFL1) of R. cerealis or the target genes (TaChit3, TaChitIV, and TaCERK1) of wheat were calculated using the 2 −∆∆CT method [40], where wheat Actin gene (TaActin) and the R. cerealis Actin gene (RcActin) were used as the internal reference, respectively. Three independent biological replications were evaluated.

Heterologous Expression of RcFL1 and Its MEP36 Domain RcFL1-MEP36 Peptides
The full encoding sequence of RcFL1 and its partial sequence comprising the MEP36 domain were separately subcloned in fusion to the His-TF tag of the pCOLD TF vector, generating the expression vectors pHis-TF-RcFL1 and pHis-TF-RcFL1-MEP36. The DNAs of the resulting pHis-TF-RcFL1 and pHis-TF-RcFL1-MEP36 fusion constructs as well as pCOLD TF vector were each transformed into competent cells of E. coli BL21 (DE3), respectively. After these, E. coli BL21 cells were cultured in LB medium and induced with 0.5 mM isopropyl-β-d-thiogalactoside at 16 • C at 100 rpm overnight. The recombinant proteins were purified with Ni-NTA resin and eluted [41]. Finally, the SDS-PAGE (Bio-Rad, Hercules, CA, USA) was used to examine the purified proteins His-TF-RcFL1 and His-TF-RcFL1-MEP36 as well as His-TF control protein.

Cell Death-Inducing Activities and Disease Assays of RcFL1 Protein and Its MEP36-Containing Peptide RcFL1-MEP36
Cell death-inducing activities of the RcFL1 and RcFL1-MEP36 proteins of the heterologous expressed proteins (25 µL, 5 µM/L) were assayed by infiltrating samples into detached leaves from fully expanded secondary of two-month-old wheat plants or four-week-old N. benthamiana [24,38].
Following protein infiltration for six hours, leaves were inoculated in the same location with 50 µL mycelial suspension of R. cerealis Rc207. These leaves were placed in Petri dishes containing filter paper saturated with sterile distilled water and kept under a 16-h day/8-h night regime at 25 • C. Leaf lesions were calculated by length×width and photographed at 3 days post inoculation (dpi) with R. cerealis Rc207 [24,38]. Six leaves were performed in each experiment and repeated three times.

Conclusions
In this study, a total of 116 metalloproteases were identified from the whole-genome sequences of the wheat sharp eyespot R. cerealis Rc207 and were divided over 24 protein families, including 34 secretory metalloproteases with a predicted signal peptide. All the 116 metalloproteases genes were expressed at the late stages, 96, and 240 hai, of the fungal infection to wheat. Of 11 MEP36/fungalysin family metalloproteases, 10 MEP36 were predicted to be secreted proteins and nine encoding genes were expressed during R. cerealis infection process to wheat. Phylogenetic analysis suggested that these MEP36 metalloproteases in R. cerealis were closely-related to those of R. solani but were remote to those of B. sorokiniana, F. graminearum, F. pseudograminearum, and P. oryzae. Interestingly, an MEP36 metalloprotease gene RcFL1 displayed a significantly upregulated expression during the infection process, and RcFL1 has been verified to function as a virulence factor of R. cerealis possibly through inhibiting host chitin-triggered immunity and chitinases. Thus, RcFL1 likely is a promising gene resource for improving resistance of wheat and other crop plants to R. cerealis through host-induced gene silencing strategy. This is the first systematic inventory of whole-genome metalloproteases in R. cerealis.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.