The Pathogen-Induced MATE Gene TaPIMA1 Is Required for Defense Responses to Rhizoctonia cerealis in Wheat

The sharp eyespot, mainly caused by the soil-borne fungus Rhizoctonia cerealis, is a devastating disease endangering production of wheat (Triticum aestivum). Multi-Antimicrobial Extrusion (MATE) family genes are widely distributed in plant species, but little is known about MATE functions in wheat disease resistance. In this study, we identified TaPIMA1, a pathogen-induced MATE gene in wheat, from RNA-seq data. TaPIMA1 expression was induced by Rhizoctonia cerealis and was higher in sharp eyespot-resistant wheat genotypes than in susceptible wheat genotypes. Molecular biology assays showed that TaPIMA1 belonged to the MATE family, and the expressed protein could distribute in the cytoplasm and plasma membrane. Virus-Induced Gene Silencing plus disease assessment indicated that knock-down of TaPIMA1 impaired resistance of wheat to sharp eyespot and down-regulated the expression of defense genes (Defensin, PR10, PR1.2, and Chitinase3). Furthermore, TaPIMA1 was rapidly induced by exogenous H2O2 and jasmonate (JA) treatments, which also promoted the expression of pathogenesis-related genes. These results suggested that TaPIMA1 might positively regulate the defense against R. cerealis by up-regulating the expression of defense-associated genes in H2O2 and JA signal pathways. This study sheds light on the role of MATE transporter in wheat defense to Rhizoctonia cerealis and provides a potential gene for improving wheat resistance against sharp eyespot.


Introduction
Common wheat (Triticum aestivum) is an important staple crop for global human consumption [1]. Various diseases cause great yield losses of wheat worldwide [2]. Sharp eyespot is a damaging soil-borne disease of wheat in many regions of world [3]. This disease is caused by the necrotrophic fungal pathogen Rhizoctonia cerealis, which mainly infects plant stems and sheaths [3,4]. China is the largest epidemic region, where at least 6.67 million hectares of wheat plants have been infected with R. cerealis every year since 2005 [5,6]. R. cerealis also infects other cereal crops (such as barley, oats, and rye), and sugar beet, cotton, and potato [3,7,8]. To date, no completely resistant wheat germplasm to R. cerealis has been identified, and chemical control is still limited [9]. Various studies have shown that using important resistance-associated genes would generate wheat with better resistance to sharp eyespot [5,6,[9][10][11][12][13][14]. Therefore, exploration of effective resistance genes is a viable strategy to breeding wheat with resistance to sharp eyespot.
To ward off invading pathogens, plants have evolved a two-tiered innate immune system: pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI), and effectortriggered immunity (ETI) [15]. By using an Arabidopsis-Pseudomonas syringeae pathosystem, studies revealed that PTI and ETI are initiated by distinct activation mechanisms and involve different early signaling cascades [16,17]. Both produce reactive oxygen species of AtDTX18, a MATE transporter controlling the extracellular accumulation of coumaroylagmatine, improves resistance of transgenic potato to Phytophthora infestans and Botrytis cinerea [40]. Conversely, ADS1 negatively regulates Arabidopsis against P. syringeae (PstDC3000) by down-regulating the accumulation of SA and the expression of PR1 homolog genes [39]. Similarly, ectopic expression of OsMATE1 and OsMATE2 in Arabidopsis negatively regulates resistance to PstDC3000 and affects plant growth and development [52]. In a previous study, RNA-seq data showed that in wheat, a MATE gene with sequence number TraesCS2B01G296000 was upregulated and might be associated with defense to Fusarium graminearum [53]. However, the functional roles of MATEs in defense of R. cerealis remain unknown in wheat.
In this study, we identified a pathogen-induced MATE transporter named TaPIMA1 from a set of wheat RNA-seq transcriptome data and revealed that TaPIMA1 participated in resistance responses to R. cerealis in wheat. The expression of TaPIMA1 was induced by R. cerealis, H 2 O 2 , and JA. Virus-Induced Gene Silencing (VIGS) and disease assessment results showed that TaPIMA1 was required for wheat resistance to sharp eyespot. TaPIMA1 positively regulated the expression profiles of at least 4 PR genes.

Identification of TaPIMA1 by Transcriptomic Analysis
To identify resistance-related genes to R. cerealis in wheat, we analyzed RNA-seq data of the sharp eyespot resistant and susceptible recombinant inbred lines (RILs, derived from Shanhongmai×Wenmai6) [11]. In previous studies, some MATE transporters, e.g., AtEDS5, AtADS1, OsMATE1, OsMATE2, were shown to play important roles in disease resistance responses in Arabidopsis and rice [38,39]. Herein, we focused on mining wheat MATE transporters that were up-regulated in the resistant RILs than in the susceptible RILs. As a result, a MATE gene with sequence number TraesCS3B02G563500.1, named TaPIMA1, showed much higher expression levels in resistant RILs (RIL-R) compared with susceptible RILs (RIL-S) ( Figure 1A). Furthermore, qRT-PCR analysis showed that TaPIMA1 expression was significantly induced by R. cerealis in the resistant wheat cultivars Shanhongmai and CI12633 from 1 to 10 days post-inoculation (dpi) ( Figure 1B). Subsequently, we investigated the expression profiles of TaPIMA1 in different wheat cultivars at 10 dpi with R. cerealis. As expected, the transcript level of TaPIMA1 was significantly higher in the resistant cultivars (Shanhongmai and CI12633) than in susceptible cultivars (Yangmai16 and Wenmai6), and the highest expression level was detected in Shanhongmai ( Figure 1B,C). Moreover, at the filling stage of CI12633 plants, TaPIMA1 was highly expressed in roots and stems, where sharp eyespot mainly occurred ( Figure 1D). Taken together, the above results suggested that TaPIMA1 might function in defense responses to R. cerealis in wheat.

Identification of TaPIMA1 by Transcriptomic Analysis
To identify resistance-related genes to R. cerealis in wheat, we analyzed RNA-seq data of the sharp eyespot resistant and susceptible recombinant inbred lines (RILs, derived from Shanhongmai×Wenmai6) [11]. In previous studies, some MATE transporters, e.g., AtEDS5, AtADS1, OsMATE1, OsMATE2, were shown to play important roles in disease resistance responses in Arabidopsis and rice [38,39]. Herein, we focused on mining wheat MATE transporters that were up-regulated in the resistant RILs than in the susceptible RILs. As a result, a MATE gene with sequence number TraesCS3B02G563500.1, named TaPIMA1, showed much higher expression levels in resistant RILs (RIL-R) compared with susceptible RILs (RIL-S) ( Figure 1A). Furthermore, qRT-PCR analysis showed that TaPIMA1 expression was significantly induced by R. cerealis in the resistant wheat cultivars Shanhongmai and CI12633 from 1 to 10 days post-inoculation (dpi) ( Figure 1B). Subsequently, we investigated the expression profiles of TaPIMA1 in different wheat cultivars at 10dpi with R. cerealis. As expected, the transcript level of TaPIMA1 was significantly higher in the resistant cultivars (Shanhongmai and CI12633) than in susceptible cultivars (Yangmai16 and Wenmai6), and the highest expression level was detected in Shanhongmai ( Figure 1B,C). Moreover, at the filling stage of CI12633 plants, TaPIMA1 was highly expressed in roots and stems, where sharp eyespot mainly occurred ( Figure  1D). Taken together, the above results suggested that TaPIMA1 might function in defense responses to R. cerealis in wheat.

Sequence and Phylogenetic Analyses of TaPIMA1
The cDNA and genomic sequences of TaPIMA1 were cloned from wheat cultivar CI12633 and determined by Sanger sequencing. Sequence alignment showed that the cDNA sequence of TaPIMA1 displayed 100% identity with the reference sequence TraesCS3B02G563500.1. In addition, the genomic sequence of TaPIMA1 contained six introns and seven exons, which was transcribed into a 1500 bp-length coding (CD) sequence ( Figure 2A). The TaPIMA1 protein has 499 amino acid (aa) residues, and its molecular weight is predicted to be 53.50 kDa. Moreover, the TaPIMA1 protein includes two MatE domains (no. 50-210 aa and no. 271-434 aa, respectively) and 12 transmembrane helices (TMHs, no. 445-464 aa) ( Figure 2B).

Subcellular Localization of TaPIMA1 Protein
To investigate the subcellular location of TaPIMA1 in wheat, we performed the protein transient expression assay in wheat protoplasts. The TaPIMA1 was introduced into the PH16318 construct that was driven by the 35S CaMV promoter. Then, the PH16318-TaPIMA1-GFP and PH16318-GFP (control) construct DNAs were introduced into wheat mesophyll protoplasts and expressed, respectively. Confocal microscopic observation showed that the TaPIMA1-GFP fusion protein distributed in the cytoplasm and in plasma membrane, while the control GFP was expressed throughout the cell ( Figure 3). Therefore, the results suggested that TaPIMA1 localized in wheat cytoplasm and plasma membrane. MATEs, including OsMATE1, OsMATE2, AtADS1, and TraesCS2B01G296000, were clustered into the group I ( Figure 2C). TaPIMA1 was closely related to the anthocyanidins transporters Arabidopsis AtTT12 and G. hirsutum GhTT12 ( Figure 2C). The full-length of TaPIMA1 shared 47.35% and 46.47% identities with GhTT12 and AtTT12, respectively ( Figure S1). These preliminary analyses suggest that the MATE protein TaPIMA1 might be involved in defense and/or anthocyanidin transport.

Subcellular Localization of TaPIMA1 Protein
To investigate the subcellular location of TaPIMA1 in wheat, we performed the protein transient expression assay in wheat protoplasts. The TaPIMA1 was introduced into the PH16318 construct that was driven by the 35S CaMV promoter. Then, the PH16318-TaPIMA1-GFP and PH16318-GFP (control) construct DNAs were introduced into wheat mesophyll protoplasts and expressed, respectively. Confocal microscopic observation showed that the TaPIMA1-GFP fusion protein distributed in the cytoplasm and in plasma membrane, while the control GFP was expressed throughout the cell ( Figure 3). Therefore, the results suggested that TaPIMA1 localized in wheat cytoplasm and plasma membrane.

Knock-Down of TaPIMA1 Reduced Resistance to Sharp Eyespot in Wheat
To specifically knock-down TaPIMA1, a 216 bp cDNA fragment specific to TaPIMA1 was sub-cloned in an antisense orientation into the multi-clone site of the γ chain of Barley stripe mosaic virus (BSMV), generating the recombinant γ-TaPIMA1 ( Figure S2A,B). The BSMV α, β, γ-TaPIMA1 and γ-GFP construct DNA was individually transcribed into RNA in vitro ( Figure S2C). Subsequently, these virus RNAs (BSMV α, β, and γ-TaPIMA1

Knock-Down of TaPIMA1 Reduced Resistance to Sharp Eyespot in Wheat
To specifically knock-down TaPIMA1, a 216 bp cDNA fragment specific to TaPIMA1 was sub-cloned in an antisense orientation into the multi-clone site of the γ chain of Barley stripe mosaic virus (BSMV), generating the recombinant γ-TaPIMA1 ( Figure S2A,B). The BSMV α, β, γ-TaPIMA1 and γ-GFP construct DNA was individually transcribed into RNA in vitro ( Figure S2C). Subsequently, these virus RNAs (BSMV α, β, and γ-TaPIMA1 or α, β, and γ-GFP) were mixed and inoculated into the emerged third leaves of the resistant wheat cultivar CI12633 plants to execute the BSMV-mediated VIGS (BSMV-VIGS). At 10 dpi with BSMV:TaPIMA1 and BSMV:GFP, the BSMV symptom was exhibited on the new leaves of both BSMV:GFP-infected and BSMV:TaPIMA1-infected plants ( Figure 4A). Additionally, the BSMV coat protein (CP) gene was detected in BSMV-infected plants by RT-PCR ( Figure 4B). Moreover, the qRT-PCR showed that the mRNA levels of TaPIMA1 were significantly decreased in BSMV:TaPIMA1-infected CI12633 plants compared with the BSMV:GFP-infected CI12633 plants ( Figure 4C). These results indicated, TaPIMA1 was successfully knocked-down in BSMV:TaPIMA1-infected CI12633 plants. or α, β, and γ-GFP) were mixed and inoculated into the emerged third leaves of the resistant wheat cultivar CI12633 plants to execute the BSMV-mediated VIGS (BSMV-VIGS). At 10 dpi with BSMV:TaPIMA1 and BSMV:GFP, the BSMV symptom was exhibited on the new leaves of both BSMV:GFP-infected and BSMV:TaPIMA1-infected plants ( Figure  4A). Additionally, the BSMV coat protein (CP) gene was detected in BSMV-infected plants by RT-PCR ( Figure 4B). Moreover, the qRT-PCR showed that the mRNA levels of TaPIMA1 were significantly decreased in BSMV:TaPIMA1-infected CI12633 plants compared with the BSMV:GFP-infected CI12633 plants ( Figure 4C). These results indicated, TaPIMA1 was successfully knocked-down in BSMV:TaPIMA1-infected CI12633 plants.  Next, we assessed disease severity of BSMV-infected wheat plants after inoculation with R. cerealis. At 10 dpi with R. cerealis, the sharp eyespot symptoms exhibited on the leaf sheaths and stems of BSMV-infected CI12633 plants, while the lesions of BSMV:TaPIMA1infected plants were larger than those of control plants ( Figure 4D). Furthermore, the fungal biomass, measured by the transcriptional level of R. cerealis Actin, was significantly higher in BSMV:TaPIMA1-infected plants (more than 29.8-fold) than that in control plants ( Figure 4E). At~30 dpi with R. cerealis, there were more serious lesions on the stems of BSMV-infected CI12633 plants, where the lesions were significantly larger in BSMV:TaPIMA1-infected plants compared with BSMV:GFP-infected plants ( Figure 4F,G). The average necrotic length and width of BSMV: TaPIMA1 Table S2). These results indicated that silencing of TaPIMA1 significantly reduced the wheat resistance to sharp eyespot, and suggested that TaPIMA1 is required for wheat resistance to R. cerealis.

Knock-Down of TaPIMA1 Decreased the Expression of PR Genes
Previously studies have shown that the defense-associated even PR genes (including PR1.2, PR10, Chitinase3, and defensin) are involved in resistance responses to R. cerealis infection in wheat [5,22,[25][26][27]. To investigate the regulatory pathway of TaPIMA1 in response to R. cerealis infection, we examined the expression profiles of several PR genes in TaPIMA1-silenced CI12633 plants. As shown in Figure 5, the transcriptional levels of PR1.2, PR10, Chitinase3, and defensin were significantly downregulated in TaPIMA1-silenced plants compared with the control (BSMV:GFP) plants ( Figure 5A-D). These results indicated that the TaPIMA1 positively regulated the expression of PR genes, resulting in enhanced resistance to R. cerealis. of the BSMV-infected CI12633 plants in two batches. dpi, days post inoculation. The significant differences determined by one-way ANOVA (** p < 0.01). Error bars indicates standard deviation.
Next, we assessed disease severity of BSMV-infected wheat plants after inoculation with R. cerealis. At 10 dpi with R. cerealis, the sharp eyespot symptoms exhibited on the leaf sheaths and stems of BSMV-infected CI12633 plants, while the lesions of BSMV:TaPIMA1-infected plants were larger than those of control plants ( Figure 4D). Furthermore, the fungal biomass, measured by the transcriptional level of R. cerealis Actin, was significantly higher in BSMV:TaPIMA1-infected plants (more than 29.8-fold) than that in control plants ( Figure 4E). At ~30 dpi with R. cerealis, there were more serious lesions on the stems of BSMV-infected CI12633 plants, where the lesions were significantly larger in BSMV:TaPIMA1-infected plants compared with BSMV:GFP-infected plants ( Figure 4F,G). The average necrotic length and width of BSMV: TaPIMA1 Table S2). These results indicated that silencing of TaPIMA1 significantly reduced the wheat resistance to sharp eyespot, and suggested that TaPIMA1 is required for wheat resistance to R. cerealis.

Knock-Down of TaPIMA1 Decreased the Expression of PR Genes
Previously studies have shown that the defense-associated even PR genes (including PR1.2, PR10, Chitinase3, and defensin) are involved in resistance responses to R. cerealis infection in wheat [5,22,[25][26][27]. To investigate the regulatory pathway of TaPIMA1 in response to R. cerealis infection, we examined the expression profiles of several PR genes in TaPIMA1-silenced CI12633 plants. As shown in Figure 5, the transcriptional levels of PR1.2, PR10, Chitinase3, and defensin were significantly downregulated in TaPI-MA1-silenced plants compared with the control (BSMV:GFP) plants ( Figure 5A-D). These results indicated that the TaPIMA1 positively regulated the expression of PR genes, resulting in enhanced resistance to R. cerealis.

TaPIMA1 and Its Regulated PR Genes Were Induced by Exogenous H 2 O 2 and JA Stimuli
The ROS, JA and SA, play important roles in plant defense responses to pathogens [17,33,34]. Thus, we analyzed the expression profiles of TaPIMA1 in wheat CI12633 plants treated by exogenous H 2 O 2 , JA, or SA. After H 2 O 2 treatment, the expression level of TaPIMA1 was dramatically elevated from 0.5 h to 12 h and peaked at 3 h (~25.64-fold over non-treatment) ( Figure 6A). Upon MeJA stimulus, the transcript level of TaPIMA1 was induced from 0.5 h to 6 h and reached a peak at 0.5 h (~2.19-fold) ( Figure 6B). However, TaPIMA1 was barely responsive to exogenous SA stimulus ( Figure S3). plants treated by exogenous H2O2, JA, or SA. After H2O2 treatment, the expression level of TaPIMA1 was dramatically elevated from 0.5 h to 12 h and peaked at 3 h (~25.64-fold over non-treatment) ( Figure 6A). Upon MeJA stimulus, the transcript level of TaPIMA1 was induced from 0.5 h to 6 h and reached a peak at 0.5 h (~2.19-fold) ( Figure 6B). However, TaPIMA1 was barely responsive to exogenous SA stimulus ( Figure S3).
Further, the expression profiles of PRgenes that are regulated by TaPIMA1 were examined in CI12633 plants treated by H2O2 or JA. Compared with mock-treatment, the transcription levels of PR1.2, PR10, and Chitinase3 were significantly increased after treatment with H2O2 for 0.5 h and 3 h ( Figure 6C-E). Similarly, PR1.2, PR10, and Chi-tinase3 were significantly up-regulated by MeJA ( Figure 6F-H). These data indicated that TaPIMA1 and its regulated PR genes were significantly induced by H2O2 and JA.  (mock) at the four-leaf stage, respectively. The significant differences were determined by Student's t-test (* p < 0.05, ** p < 0.01). Error bars indicate standard deviation. TaActin was used as internal control.
Further, the expression profiles of PRgenes that are regulated by TaPIMA1 were examined in CI12633 plants treated by H 2 O 2 or JA. Compared with mock-treatment, the transcription levels of PR1.2, PR10, and Chitinase3 were significantly increased after treatment with H 2 O 2 for 0.5 h and 3 h ( Figure 6C-E). Similarly, PR1.2, PR10, and Chitinase3 were significantly up-regulated by MeJA ( Figure 6F-H). These data indicated that TaPIMA1 and its regulated PR genes were significantly induced by H 2 O 2 and JA.

Discussion
Wheat provides 20% of the total daily calorie expenditure of human beings in the world, but its production is threatened by sharp eyespot [5]. Developing a resistant wheat variety with disease-resistance genes is one optimal strategy to control this disease. In this study, we provided evidence that a novel MATE gene, TaPIMA1, was required for resistance responses to R. cerealis in wheat. Here, based on the RNA-seq data and qRT-PCR analysis, we identified TaPIMA1 that was in response to R. cerealis, while the expression of TaPIMA1 was significantly higher in R. cerealis-resistant wheat genotypes compared with the susceptible wheat genotypes (Figure 1). Sequence analysis showed that the TaPIMA1 deduced protein includes two MatE domains and 12 TMHs, similar to those of two rice disease-resistant transporters, OsMATE1 and OsMATE2 [52]. A phylogenetic analysis further indicated that TaPIMA1 was classified into group I, including MATE disease-resistant transporters OsMATE1, OsMATE2 [52], AtADS1 [39], and TraesCS2B01G296000 [53], and anthocyanidins transporters Arabidopsis AtTT12 and G. hirsutum GhTT12 [46,47]. TaPIMA1 was closely related to the anthocyanidins transporters Arabidopsis AtTT12 and G. hirsutum GhTT12. Thus, the MATE protein TaPIMA1 might participate in the wheat defense and/or anthocyanidins transport. Generally, the MATE transporters are localized in the cytoplasm and plasma membrane to perform their transporting functions [67]. For instance, the pleiotropic anti-disease transporter EDS5 is localized in cytoplasm and exports the innate immune signal SA from chloroplast [49]. Herein, TaPIMA1 was confirmed to localize in the wheat cytoplasm and plasma membrane (Figure 3).
In Arabidopsis, there are 56 MATE transporters that play important roles in plant growth, development, and resistance to biotic and abiotic stresses [37,67]. However, only a few MATE transporters with disease resistance, including resistance transporter EDS5 to viral and bacterial pathogens [38,68], P. infestans and B. cinerea resistance transporter AtDTX18 [40,69], and P. syringeae susceptibility transporter ADS1 [39], have been characterized. The studies on functions of MATE transporters in plant immunity/defense are limited in crops, particularly in wheat. In this study, we reported that knock-down of TaPIMA1 significantly reduced the resistance of wheat to sharp eyespot ( Figure 4). Furthermore, the TaPIMA1 transcript level was induced by R. cerealis and higher in root and stem tissues where sharp eyespot initially appeared; importantly, the gene transcript is higher in resistance wheat genotypes than in susceptible wheat genotypes. These data indicated that TaPIMA1 was involved in wheat innate immunity responses to R. cerealis.
Some upstream immune genes substantially induce the expression of defense-related genes [23,24]. For example, heightened expression of the defense-associated genes, such as TaSTT3b-2B, TaRCR1, and TaPIE1, confer enhanced resistance to R. cerealis in transgenic wheat [6,11,23,24]. Conversely, silencing of these genes down-regulated the expression of defense genes and weakened resistance of wheat to sharp eyespot disease [10,24,36]. Herein, the transcriptional levels of PR1.2, PR10, Defensin, and Chitinase3 were significantly decreased in TaPIMA1-silenced wheat plants, which is consistent with the performance of wheat to sharp eyespot ( Figure 5). In response to ROS, plants will up-regulate defense genes, induce callose deposition, and perform hypersensitive cell death to resist pathogens [22,70]. JA and SA, acting as immune signal amplifiers, play important roles in plant responses to necrotrophic pathogens and biotrophic pathogens, respectively [29][30][31]. In this study, TaPIMA1 responded rapidly to exogenous H 2 O 2 and MeJA stimuli ( Figure 6A,B). Further, the expression of TaPIMA1-activated defense genes (PR10, PR1.2, and Chitinase3) was significantly up-regulated after H 2 O 2 and MeJA treatments ( Figure 6C-H). In Arabidopsis, EDS5 contributes to the transport and accumulation of SA, while exogenous SA stimulation induces the expression of EDS5 and enhances the resistance to biotrophic pathogens P. syringae [51]. Unlike EDS5, involved in the SA-mediated resistance pathway [38,49,51], TaPIMA1 did not response to SA but exhibited a strong inductive response to JA, similar to the MATE transporter AtDTX18 [69]. Taken together, these results suggested that H 2 O 2 and JA might participate in the TaPIMA1-mediated resistance to R. cerealis in wheat.

Plant and Fungus Materials, Vectors, and Primers
The resistant wheat cultivars (CI12633 and Shanhongmai) and highly susceptible wheat cultivars (Wenmai6 and Yangmai16) were used in this study. The expression profile of TaPIMA1 was studied in CI12633, Shanhongmai, Wenmai6, and Yangmai16. The CI12633 and Yangmai16 plants were used for BSMV-VIGS experiments. All above wheat varieties were planted in a greenhouse (14 h light/10 h dark, 15-23 • C, 90% relative humidity). The sharp eyespot pathogenic fungus R. cerealis strain WK207 (dominant in North China) and R0301 were used in this study.
The PH16318-GFP vector was used to express fusion protein in wheat protoplast. All BSMV vectors (α, β, γ and γ-GFP) were stored in our laboratory.
The sequences of primers are listed in Table S3.

Pathogen Infection and Plant Treatments
R. cerealis WK207 was cultured at PDA medium for 14 d. Then the R. cerealis WK207 was cultured with sterilized toothpicks and were covered with R. cerealis WK207 for 7 d at 25 • C. The leaf sheaths of wheat plants were inoculated with toothpick fragments that were covered with well-developed mycelia. The R. cerealis R0301 was cultured at PDA medium for 14 d and then inoculated to sterilized wheat seeds for 7 d at 25 • C. The wheat seeds carrying R. cerealis R0301 were inoculated into wheat roots and watered.
The CI12633 plants were grown in a greenhouse ( [11]. At the tillering stage, all resistant/susceptible RILs were inoculated with R. cerealis WK207. Leaf sheath samples of the inoculated parts were sampled at 0 d (mock), 4 d, and 10 d after inoculation with R. cerealis, while three biological replicates were analyzed.
Then, the RNAs of samples were used to deep RNA-Seq based on the HiSeq 2000/2500 platform (Illumina, CA, USA) supported by Biomarker Technologies (Beijing, China). All raw data in F ASTQ format (raw reads) were processed using in-house Perl scripts. Clean data (clean reads) were obtained by discarding low-quality reads, reads containing adapters and ploy-N (N > 10%). Screening for differentially expressed genes between resistant and susceptible RILs that have a log2 ratio greater than 1.0 (false discovery rate p < 0.05), and the expression differences between different biological replicates were consistent [11]. The number of transcript fragments per kilobase per million mapped reads for each gene was calculated based on the length of each gene and the number of fragments mapped to that gene.

Cloning and Bioinformatics Analysis of TaPIMA1
The full-length open reading frame (ORF) of TaPIMA1 was amplified by nested PCR using primers pairs (TaPIMA1-FQ-F1/R1 and TaPIMA1-FQ-F2/R2) from CI12633 plants. Then, all PCR products were cloned into T vector (Takara, Tokyo, Japan) and confirmed by sequencing. The sequence of TaPIMA1 and its homologues were referred to NCBI (https: //www.ncbi.nlm.nih.gov/ (accessed on 25 December 2021)) and Ensemble Plants database (http://plants. ensembl.org (accessed on 25 December 2021)). The Clustal Omega (https: //www.ebi.ac.uk/Tools/msa/clustalo/(accessed on 25 December 2021)) web program was used to perform the multiple sequence alignment. Additionally, MEGA 11 software was used to construct the phylogenetic tree by the maximum likelihood method.

DNA and RNA Extraction, and qRT-PCR
Genomic DNA was extracted with CTAB. The leaves of wheat plants were harvested at 10 dpi and stored at −80 • C. Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturers operating manual. Then,~1 µg total RNA was reverse transcribed to cDNA using FastQuant RT Kit (Tiangen, Beijing, China). qRT-PCR reactions were performed on 7500 Fast Real-Time PCR Systems (Applied Biosystems, Foster City, MA, USA) using a SYBR Premix ExTaq kit (Takara, Tokyo, Japan). Additionally, each qPCR sample was run with three biological replicates and three technical replicates. The relative genes transcription levels were calculated by the 2 −∆∆CT method [71]. All primers in this study for qRT-PCR are listed in Table S3.

Subcellular Localization of TaPIMA1
The coding region of TaPIMA1 without termination codon was amplified using primers TaPIMA1-sub-F/R and sub-cloned to the PH16318-GFP vector, named PH16318-GFP-TaPIMA1. The TaPIMA1-GFP was driven by a CaMV 35S promoter. Wheat plants were grown in pots grown in a plant growth chamber after emergence for 7 days. The seedlings were collected to produce the wheat protoplasts referring to the protocol of Liu et al. [10]. Then,~10 µg plasmid DNA of PH16318-GFP (control) and PH16318-GFP-TaPIMA1 were introduced into wheat protoplasts and cultured for 20 h, respectively. Finally, the incubated protoplasts were imaged by confocal laser scanning microscope LSM 700 (Zeiss, Jena, Germany).

BSMV-VIGS in Wheat
The function of TaPIMA1 was investigated using a BSMV-VIGS in wheat as described by Holzberg [72]. Briefly, a 216 bp fragment (no. 756-955) of TaPIMA1 ORF was cloned from CI12633 plants and then subcloned into γ vector in an antisense orientation that constructed the γ-TaPIMA1 vector. The RNA of α, β, γ-TaPIMA1 and γ-GFP were transcribed in vitro by RiboMA Large Scale RNA Production System-T7 kit (Promega, Madison, WI, USA) according to the method described by Zhu [6]. All transcripts were mixed with and inoculated into the wheat plants at three-leaf stage. After 10 d, the fourth leaves of inoculated seedlings were harvested to extract the total RNA.

Assessment of Response in BSMV-VIGS Plants to R. cerealis
After 20 d infection with BSMV, the leaf sheaths of infected wheat plants were inoculated with toothpick fragments carrying R. cerealis WK207. To promote the infection of R. cerealis, the inoculated site was tied up with wet absorbent cotton that was sprayed with ddH 2 O every day. At 30 dpi with WK207, the infection types (ITs) and disease indexes (DIs) of CI12633 plants were evaluated as described previously [5,6]. The DI = ∑(d i × l i ) × 100/(L × d i max). The d i , l i , and L represent infection type, the number of plants, and the total numbers of plants for disease assessment, respectively.

Conclusions
We identified the MATE gene TaPIMA1 participating in wheat defense responses to R. cerealis. The TaPIMA1 transcript was induced by infection of R. cerealis and was higher in sharp eyespot-resistant wheat genotypes than in susceptible wheat genotypes. Functional dissection revealed that TaPIMA1, acting as a positive regulator, is required for resistance to sharp eyespot and for the expression of PR genes including PR1.2, PR10, Defensin, and Chitinase3 in wheat. TaPIMA1 and its regulated PR genes are in response to exogenous H 2 O 2 and JA stimuli, suggesting that H 2 O 2 and JA might participate in the TaPIMA1-mediated resistance to R. cerealis in wheat. This study provides insights into role of the wheat MATE in plant innate immunity to necrotrophic fungal pathogens. TaPIMA1 is a potential gene for improving resistance to sharp eyespot in wheat.