A R2R3 MYB Transcription Factor, TaMYB391, Is Positively Involved in Wheat Resistance to Puccinia striiformis f. sp. tritici

A biotrophic fungus, Puccinia striiformis f.sp. tritici (Pst), which causes stripe rust disease in wheat is the most yield-limiting factor in wheat production. Plants have complex defense mechanisms against invading pathogens. Hypersensitive response (HR), a kind of programmed cell death (PCD) at the infection site, is among these defense mechanisms. Transcription factors (TFs) play a crucial role in plant defense response against invading pathogens. Myeloblastosis (MYB) TFs are among the largest TFs families that are involved in response to both biotic and abiotic stresses. However, little is known about the mechanisms of MYB TFs during the interaction between wheat and the stripe rust fungus. Here, we identified an R2R3 MYB TF from wheat, designated as TaMYB391, and characterized its functional role during wheat–Pst interaction. Our data indicated that TaMYB391 is induced by Pst infection and exogenous application of salicylic acid (SA) and abscisic acid (ABA). TaMYB391 is localized in the nucleus of both wheat and Nicotiana benthamiana. Transient overexpression of TaMYB391 in N. benthamiana triggered HR-related PCD accompanied by increased electrolyte leakage, high accumulation of reactive oxygen species (ROS), and transcriptional accumulation of SA defense-related genes and HR-specific marker genes. Overexpression of TaMYB391 in wheat significantly enhanced wheat resistance to stripe rust fungus through the induction of pathogenesis-related (PR) genes, ROS accumulation and hypersensitive cell death. On the other hand, RNAi-mediated silencing of TaMYB391 decreased the resistance of wheat to Pst accompanied by enhanced growth of the pathogen. Together our findings demonstrate that TaMYB391 acts as a positive regulator of HR-associated cell death and positively contributes to the resistance of wheat to the stripe rust fungus by regulating certain PR genes, possibly through SA signaling pathways.


Introduction
Wheat (Triticum aestivum) is one of the most important stable crops globally. Its production and productivity, however, are influenced by both biotic and abiotic stresses. Stripe rust disease which is caused by a biotrophic fungus called Puccinia striiformis f. sp. tritici (Pst) is the most yield-limiting factor in wheat production. The disease occurs in more than 60 countries worldwide, and stripe rust epidemics occur more frequently and cause annual crop losses of 5-10% in several countries [1].
Plants have complex defense mechanisms against invading pathogens. Upon invasion by pathogens, pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) are triggered by the recognition of pathogen-associated molecular patterns (PAMPs) and effectors, respectively [2]. Both PTI and ETI lead to the activation of multiple defense signal transduction pathways, involving salicylic acid (SA), reactive oxygen species (ROS), and mitogen-activated protein kinase (MAPK) [3]. These defense responses involve the rapid transcriptional reprogramming mediated by transcription factors (TFs), such as the upregulation of defense genes encoding antimicrobial proteins [4]. These eventually result in plant resistance against plant pathogens [2,4]. Several TFs have been reported to play a crucial role in wheat defense responses to Pst. For instance, TaBZR2, TaLOL2, TabZIP74, TaAP2-15, TaWRKY10 and TaCBF1 act as positive regulators in wheat stripe rust resistance [5][6][7][8][9][10]. In addition, previous studies have reported that wheat TFs such as TaWRKY62 and TaWRKY70 were positively involved in high-temperature seedling plant resistance to Pst [11,12].
Myeloblastosis (MYB) TFs are among the largest TF families. The conserved MYB DNA-binding domain comprises 1-4 tandem incomplete repeats of MYB repeats, and each incomplete repeat contains 50-53 amino acid residues [13]. Based on the number of adjacent imperfect repeats of these domains, MYB TFs are classified into four categories: 1R-MYB/MYB-related, R2R3-MYB, 3R-MYB and 4R-MYB [14]. In recent years, MYB TF genes have been identified and characterized in the genomes of many plant species, including Arabidopsis, Gossypium hirsutum, Dendrobium officinale, Oryza sativa, Arachis hypogaea, Rhododendron delavayi and Triticum aestivum [13,15]. There are some differences in the number of different types of MYB in plants, and most of the identified MYB proteins belong to the R2R3-MYB subgroup [13]. Many MYB proteins have been reported to play significant roles in diverse biological processes and defense responses to environmental stresses [13,14,16]. Some of the reported MYB proteins are involved in defense response against invading pathogens. For instance, overexpression of AtMYB96, an R2R3-MYB gene, increased disease resistance to pathogen infection in Arabidopsis [17]. In Arabidopsis, disease resistance in myb15 mutant plants was reduced after inoculation with the avirulent bacterial pathogen, Pseudomonas syringae pv. tomato DC3000 (AvrRpm1) [18]. Overexpression of MYB115 from poplar showed increased resistance to the fungal pathogen, Dothiorella gregaria [19]. In rose, the silencing of RcMYB84 and RcMYB123 increased the susceptibility of plants to Botrytis cinerea [20]. Ectopic expression of the Thinopyrum intermedium TiMYB2R-1 in wheat significantly improved wheat resistance to take-all disease, caused by Gaeumannomyces graminis [21]. In Einkorn wheat, decreased expression of TuMYB46L, a R2R3-MYB gene, induced an elevated function of the 1-aminocyclopropane-1-carboxylic acid oxidase gene TuACO3 and promoted wheat defense against Blumeria graminis f.sp. tritici (Bgt) [22]. In wheat, overexpression of two MYB genes, TaPIMP1 [23] and TaPIMP2 [24] significantly increased resistance to the fungal pathogen Bipolaris sorokiniana. Overexpression of TaRIM1, a R2R3-MYB gene from wheat, positively modulated wheat defense response to Rhizoctonia cerealis [25]. The silencing of three MYB genes, TaMYB4, TaMYB29 and TaLHY from wheat increased susceptibility to Pst [26][27][28]. Although several MYB transcription factors involved in defense response have been identified in wheat, the functions of MYB genes during the interaction between wheat and Pst need to be studied in depth.
A number of phytohormones have been reported to play a significant role in mediating defense responses. Any change in the status of these hormones, their perception and signaling results in the alteration of defense responses to pathogens [29,30]. Upon invasion by pathogens, plants accumulate high amounts of SA that are involved in the activation of defense responses, such as the induced expression of pathogenesis-related (PR) genes [31]. SA plays a significant role in the plant-pathogen interactions. Mutants of an isochorismate synthase gene, ICS1, which is a key enzyme for pathogen-induced SA biosynthesis, showed enhanced susceptibility to pathogen infection and were unable to accumulate high SA levels, failing to develop systemic acquired resistance (SAR) [32]. In plants, most SA exists in a glucose-conjugated inactive form. Thus, the defense response was enhanced by inhibiting the conversion of SA to SA-O-β-D-glucoside [30,33]. Mutants of salicylic acid 5-hydroxylase gene, S5H, which encodes a key enzyme that catalyzes the conversion of SA to 2,5-dihydroxybenzoic acid (2,5-DHBA), exhibit higher SA content and a constitutive defense response [34]. In plants, SA-related signaling is required for PTI and ETI, and is involved in controlling cell death during ETI [32]. 3 of 18 ETI is usually correlated with ROS accumulation, which ultimately leads to the activation of various groups of defense-related genes [2]. ROS as the signaling molecules of the secondary signaling process are well known for their roles in response to environmental stresses [35]. ROS molecules have been shown to play an important role in triggering the HR [36]. HR, where necrotic lesions develop at the spot where the pathogen enters the host, is the most visible phenomenon of the ETI [37]. Precise regulation of organelle-specific ROS is essential for a successful immune response [38].
In this study, we reported the functional role of a wheat R2R3 MYB TF, TaMYB391, during the interaction between wheat and the stripe rust fungus. We found that TaMYB391 is highly induced upon Pst inoculation and exogenous hormone treatment. The sub-cellular localization assays revealed that TaMYB391 is localized to the nucleus of both wheat and Nicotiana benthamiana. Agrobacterium-mediated transient overexpression of TaMYB391 in N. benthamiana induced programmed cell death accompanied by increased ROS accumulation and transcriptional induction of HR-specific and defense-related marker genes. Results obtained from both overexpression and silencing (RNAi) experiments demonstrated that TaMYB391 is positively involved in the defense responses of wheat against Pst infection. Our findings provide new insights into understanding the molecular mechanisms of MYB TFs.

Identification and Sequence Analysis of TaMYB391
The amino acid sequences of an Arabidopsis gene, AtMYB44, which is involved in resistance response to biotrophic pathogen and regulates the expression of WRKY70 [39] were used as a query search, and we found a homologous protein (TraesCS7D02G514800) in wheat, which was previously named as TaMYB391 [38]. TaMYB391 was then isolated from wheat cv. Su11 infected with Pst isolates. The coding sequence (CDS) is 1077 bp in length. The predicted ORF of TaMYB391 encodes 358 deduced amino acids with a molecular weight of 38.85 kDa and an isoelectric point (pI) of 8.07. Multisequence alignment indicated that TaMYB391 is highly conserved with Oryza sativa (GenBank accession No. XP_015641187.1), Brachypodium distachyon (GenBank accession No. XP_024318409.1), Hordeum vulgare (GenBank accession No. KAE8776319.1) and Sorghum bicolor (GenBank accession No. XP_002451692.1) ( Figure S1a). Domain feature analysis revealed that TaMYB391 contains two MYB DNA binding domains: R2 (amino acid 73-118) and R3 (amino acid 125-167) ( Figure S1a). Eight MYB amino acid sequences from different plant species were downloaded from GenBank, and a phylogenetic tree was constructed using the neighbor-joining analysis method. Phylogenetic analysis indicated the homology similarity between TaMYB391 and MYB proteins from other plant species. As indicated in Figure S1b, TaMYB391 is closely related to OsMYB77 and HvMYB44.

Transcriptional Responses of TaMYB391 to Pst
To gain insight into whether TaMYB391 participates in the wheat response to Pst infection, the transcript levels of TaMYB391 in infected leaves at different time points were analyzed using RT-qPCR. As the cultivar Suwon11 was reported to have a resistant gene, YrSu, and be resistant to CYR23 (incompatible) and highly susceptible to CYR31 (compatible) [40], the transcript levels of TaMYB391 were analyzed in both compatible and incompatible interactions. In the incompatible interaction, transcript levels were upregulated at 12, 18, 72, 120 and 216 h post-inoculation (hpi) as compared to the control. Upon infection with the avirulent race CYR23, the transcript levels were induced as early as 12 hpi. The highest transcription accumulation was obtained at 18 hpi which was~10-fold higher than the control. The next highest transcript accumulation was attained at 216 hpi which was~8-fold higher compared to the control (Figure 1a). The transcript profiles of TaMYB391 in the compatible interaction were also significantly upregulated at 12, 18, 24 and 48 hpi as compared to the control (Figure 1a). These results suggested that TaMYB391 may be involved in wheat resistance response against Pst during their interaction.
with the avirulent race CYR23, the transcript levels were induced as early as 12 hpi. The highest transcription accumulation was obtained at 18 hpi which was ~10-fold higher than the control. The next highest transcript accumulation was attained at 216 hpi which was ~8-fold higher compared to the control (Figure 1a). The transcript profiles of TaMYB391 in the compatible interaction were also significantly upregulated at 12, 18, 24 and 48 hpi as compared to the control (Figure 1a). These results suggested that TaMYB391 may be involved in wheat resistance response against Pst during their interaction. . Two-week-old wheat seedlings were used in this experiment. Wheat leaves treated with distilled water or 0.1% ethanol were included as a control. TaEF-1α was used as an internal reference. The relative quantity of expression of TaMYB391 was computed via the comparative threshold (2 −ΔΔCt ) method. The transcript levels were quantified by RT-qPCR and the values were standardized to that of TaEF-1α and presented as relative changes to the control. The expression level of TaMYB391 at time 0 h was normalized as 1. Error bars represent the variation among three independent replicates. Statistical variations were analyzed using Student's t-test. *, p < 0.05, **, p < 0.01.

Transcriptional Response of TaMYB391 to Exogeneous Hormones
The expression patterns of TaMYB391 in response to four hormones (ABA, Eth, MeJA and SA) were investigated using RT-qPCR. As illustrated in Figure 1b, the expression levels of TaMYB391 after treatment with ABA were induced ~7-fold as early as 0.5 h posttreatment (hpt). As compared to the controls, maximum transcript levels of TaMYB391 (~7.5-fold) were obtained at 12 hpt. After treatment with SA, the transcript levels of TaMYB391 were significantly upregulated at 12 hpt which was ~4.5-fold compared to control plants. On the other hand, when treated with MeJA and Eth, the expression of TaMYB391 was repressed at most of the time points analyzed (Figure 1b). These results suggest that TaMYB391 might participate in diverse hormone signal transduction pathways. TaEF-1α was used as an internal reference. The relative quantity of expression of TaMYB391 was computed via the comparative threshold (2 −∆∆Ct ) method. The transcript levels were quantified by RT-qPCR and the values were standardized to that of TaEF-1α and presented as relative changes to the control. The expression level of TaMYB391 at time 0 h was normalized as 1. Error bars represent the variation among three independent replicates. Statistical variations were analyzed using Student's t-test. *, p < 0.05, **, p < 0.01.

Transcriptional Response of TaMYB391 to Exogeneous Hormones
The expression patterns of TaMYB391 in response to four hormones (ABA, Eth, MeJA and SA) were investigated using RT-qPCR. As illustrated in Figure 1b, the expression levels of TaMYB391 after treatment with ABA were induced~7-fold as early as 0.5 h post-treatment (hpt). As compared to the controls, maximum transcript levels of TaMYB391 (~7.5-fold) were obtained at 12 hpt. After treatment with SA, the transcript levels of TaMYB391 were significantly upregulated at 12 hpt which was~4.5-fold compared to control plants. On the other hand, when treated with MeJA and Eth, the expression of TaMYB391 was repressed at most of the time points analyzed (Figure 1b). These results suggest that TaMYB391 might participate in diverse hormone signal transduction pathways.

TaMYB391 Is Localized in the Nucleus
We verified the sub-cellular localization of TaMYB391 in both wheat mesophyll protoplasts and N. benthamiana cells. We transiently expressed the constructs: p16318hGFP: TaMYB391 and pCAMBIA1302:TaMYB391-GFP (green fluorescence protein) or empty vector p16318hGFP as a control in wheat mesophyll protoplasts and N. benthamina through PEGand Agrobacterium tumefaciens-mediated transformation, respectively. As illustrated in Supplementary Figure S2a, TaMYB391-GFP fusion protein was localized in the nucleus of wheat mesophyll cells, while the GFP protein produced by protoplasts transformed with the empty vector was detected throughout the cell. Similar results were obtained in N. benthamiana ( Figure S2b). These results indicated that TaMYB391 is a nuclear-localized protein.

Transient Overexpression of TaMYB391 in N. benthamiana Triggered HR-Related Programmed Cell Death
In plant immunity, PCD plays a significant role against invading pathogens and abiotic stress [37]. To determine whether TaMYB391 is involved in producing PCD, we transiently overexpressed TaMYB391 in N. benthamiana using a PVX vector. Four-to six-week-old tobacco leaves were agroinfiltrated with the following recombinants: PVX: TaMYB391, PVX: Pst322 (positive control), GFP and empty vector (EV) as negative controls. After seven days, obvious cell death was observed on PVX: TaMYB391and PVX: Pst322-infiltrated leaves (Figure 2a), suggesting their role in cell death induction, whereas there was no cell death on GFP-or empty vector-infiltrated leaves. The cell death was further confirmed by electrolyte leakage. A high percentage of electrolyte leakage was obtained from PVX: TaMYB391-infiltrated leaves compared to control leaves ( Figure 2b). These results strongly suggested that TaMYB391 might be involved in producing PCD.

Transient Overexpression of TaMYB391 in N. benthamiana Triggered HR-related Programmed Cell Death
In plant immunity, PCD plays a significant role against invading pathogens and abiotic stress [37]. To determine whether TaMYB391 is involved in producing PCD, we transiently overexpressed TaMYB391 in N. benthamiana using a PVX vector. Four-to six-weekold tobacco leaves were agroinfiltrated with the following recombinants: PVX: TaMYB391, PVX: Pst322 (positive control), GFP and empty vector (EV) as negative controls. After seven days, obvious cell death was observed on PVX: TaMYB391-and PVX: Pst322-infiltrated leaves (Figure 2a), suggesting their role in cell death induction, whereas there was no cell death on GFP-or empty vector-infiltrated leaves. The cell death was further confirmed by electrolyte leakage. A high percentage of electrolyte leakage was obtained from PVX: TaMYB391-infiltrated leaves compared to control leaves ( Figure 2b). These results strongly suggested that TaMYB391 might be involved in producing PCD.

TaMYB391 Activates Plant Immunity Responses
In plant defense response, programmed cell death, usually known as HR, is produced when plants recognize effectors and develop ETI which is usually associated with ROS accumulation and induction of defense-related genes [2,36]. To examine whether the TaMYB391-triggered cell death is associated with plant innate immunity response, ROS accumulation and transcriptional accumulation of HR-specific marker genes, NbHSR203J and NbHIN1 [41,42], were determined in N. benthamiana. At 24 h after infiltration, there was no ROS detected in the control plants, whereas obvious ROS accumulation was observed in TaMYB391-infiltrated leaves ( Figure S3a). Furthermore, the expression levels of NbHSR203J and NbHIN1 were significantly activated by TaMYB391 at different time-courses ( Figure S3b). These results suggested that TaMYB391 is involved in the activation of plant immunity responses.
To further verify whether TaMYB391-triggered immunity is related with salicylic acid (SA) signaling pathways, we analyzed the expression levels of defense-related genes, NbPR1a and NbPR2 [43] which are well known for their SA-dependent expression. The transcriptional accumulation of these genes was analyzed through RT-qPCR. Their transcriptional abundance in the TaMYB391 agroinfiltrated plants was indicative of a significant upregulation through different time points compared with the controls ( Figure S3b). These results suggested that TaMYB391 could activate innate immunity through the activation of SA-mediated defense pathways.

Overexpression of TaMYB391 in Wheat Enhances Wheat Resistance to Pst Infection
To validate the functional role of TaMYB391 during the wheat-Pst interaction, TaMYB391overexpression transgenic wheat plants were developed by using wheat cultivar Fielder as receptor material. Five successfully transformed T 1 transgenic lines (OE2, OE4, OE11, OE12 and OE15) were detected via PCR using specific primers ( Figure S4a,b and Table S1). Two transgenic lines (OE2 and OE15) were selected for further study based on their higher transcript levels analyzed through RT-qPCR ( Figure S4c). The T 2 transgenic lines (OE2 and OE15) were further validated by PCR (Figure 3a), and their defense response was evaluated following inoculation with the virulent Pst race CYR31. At 14 days post-inoculation (dpi), more uredia were observed on the wild-type (WT) Fielder plants (224 per leaf area) compared to the transgenic lines (43 and 82 per leaf area, respectively) ( Figure 3b). RT-qPCR analysis showed that the transcript levels of TaMYB391 in the transgenic lines were significantly higher than those recorded in WT controls (Figure 3c). Furthermore, biomass analysis showed that significantly lower fungal biomass was obtained in the overexpressed transgenic lines compared to the untransformed WT (Figure 3d). These results revealed that the overexpression of TaMYB391 enhanced wheat resistance to Pst infection.

RNAi-Mediated Silencing of TaMYB391 Impairs Wheat Resistance to Pst Infection
To further verify the functional role of TaMYB391 in wheat defense against Pst by loss-of-function approach, we generated transgenic wheat plants under-expressing the TaMYB391 gene via RNAi. Five successfully transformed TaMYB391-RNAi transgenic lines (Ri3, Ri4, Ri9, Ri18, Ri26) in the T 1 generation were validated by PCR using universal primers ( Figure S5a,b and Table S1), and those lines showing the lowest transcriptional accumulation (Ri4 and Ri18) were selected for further study ( Figure S5c). In the T 2 generation, these lines were again verified by PCR (Figure 4a), and their defense response was evaluated following inoculation with the avirulent Pst race CYR23. At 14 dpi, obvious HR symptoms were observed on all leaves. The two TaMYB391-RNAi lines had more uredia (80 and 89 per leaf area, respectively) than the WT plants (16 per leaf area) (Figure 4b).
RT-qPCR analysis indicated that the transcript abundance of TaMYB391 in the RNAi lines was significantly decreased compared to WT (Figure 4c). Furthermore, fungal biomass was significantly increased in TaMYB391-RNAi plants compared to WT (Figure 4d). These results suggested that TaMYB391 positively contributes to wheat resistance to Pst.

TaMYB391 Positively Regulates the Expression of Pathogenesis-Related (PR) and ROS-Scavenging Genes during Pst Infection
Pathogenesis-related (PR) genes play diverse roles in host-pathogen interactions [44][45][46]. PR1 and PR2 are the marker genes for SA-mediated activation of SAR [47]. The transcriptional accumulation of TaPR1, TaPR2 and a ROS-scavenging catalase gene TaCAT3 were quantified in TaMYB391-overexpressing and TaMYB391-RNAi wheat transgenic lines using RT-qPCR. The RT-qPCR results revealed that the transcript levels of TaPR1 and TaPR2 were significantly increased in the TaMYB391-overexpressing lines compared to WT controls when inoculated with CYR31 ( Figure 5a). Conversely, the expression levels of TaCAT3 in the TaMYB391-overexpressing lines were significantly decreased compared to WT when inoculated with CYR31 (Figure 5b). On the other hand, the transcriptional accumulation of TaPR1 and TaPR2 genes in the TaMYB391-RNAi lines was significantly decreased compared to control plants when inoculated with CYR23 (Figure 5c), whereas the transcript levels of TaCAT3 were significantly increased compared to the WT when inoculated with CYR23 (Figure 5d). Together these results demonstrated that TaMYB391 is involved in the regulation of certain defense-related and ROS-scavenging genes.

TaMYB391 Positively Regulates the Expression of Pathogenesis-Related (PR) and ROS-Scavenging Genes during Pst Infection
Pathogenesis-related (PR) genes play diverse roles in host-pathogen interactions [44][45][46]. PR1 and PR2 are the marker genes for SA-mediated activation of SAR [47]. The transcrip-tional accumulation of TaPR1, TaPR2 and a ROS-scavenging catalase gene TaCAT3 were quantified in TaMYB391-overexpressing and TaMYB391-RNAi wheat transgenic lines using RT-qPCR. The RT-qPCR results revealed that the transcript levels of TaPR1 and TaPR2 were significantly increased in the TaMYB391-overexpressing lines compared to WT controls when inoculated with CYR31 ( Figure 5a). Conversely, the expression levels of TaCAT3 in the TaMYB391-overexpressing lines were significantly decreased compared to WT when inoculated with CYR31 (Figure 5b). On the other hand, the transcriptional accumulation of TaPR1 and TaPR2 genes in the TaMYB391-RNAi lines was significantly decreased compared to control plants when inoculated with CYR23 (Figure 5c), whereas the transcript levels of TaCAT3 were significantly increased compared to the WT when inoculated with CYR23 (Figure 5d). Together these results demonstrated that TaMYB391 is involved in the regulation of certain defense-related and ROS-scavenging genes.   Values represent the means ± SEs (n = 3). Significant differences between TaMYB391-overexpressing or TaMYB391-RNAi and control plants determined by Student's t-test are indicated by asterisks. *, p < 0.05; **, p < 0.01.

Pst Growth and H 2 O 2 Accumulation Were Significantly Affected in TaMYB391-Overexpressing and TaMYB391-RNAi Transgenic Lines
The phenotypic variation between the WT and TaMYB391-overexpressed or underexpressed (TaMYB391-RNAi) transgenic lines was further confirmed through histological observation. To clarify the changes in the host resistance level, H 2 O 2 accumulation and HR-associated cell death around the infection site were observed microscopically and measured by DP-BSW software. The growth of Pst was also measured by DP-BSW software. The histological observation results showed that H 2 O 2 production in the TaMYB391overexpressing transgenic lines showed a substantial increase in comparison to the WT controls (Figure 6a,b). As compared to the WT, hyphal length and infection area were significantly decreased in the TaMYB391-overexpressing lines (Figure 6c,f,g). There were no significant differences between WT and TaMYB391-overexpressing transgenic lines in the number of haustoria and haustorial mother cells (Figure 6c-e). Our data suggested that overexpression of TaMYB391 improves the resistance of wheat to Pst. On the other hand, as compared to the WT controls, H 2 O 2 accumulation was significantly decreased in the TaMYB391-RNAi lines (Figure 7a,b). Moreover, the necrotic area in the RNAi lines was significantly lower than in the WT controls (Figure 7a,c). Furthermore, the number of haustoria and haustorial mother cells, and hyphal length in the TaMYB391-RNAi lines were significantly increased compared to WT, at 48 hpi (Figure 8a-c). In addition, the infection area in the TaMYB391-RNAi line was significantly higher than in the WT controls, at 96 hpi (Figure 8a,d). Therefore, our results strongly suggest that RNAi-mediated silencing of TaMYB391 enhances wheat susceptibility to Pst.

Pst Growth and H2O2 Accumulation Were Significantly Affected in TaMYB391-overexpressing and TaMYB391-RNAi Transgenic Lines
The phenotypic variation between the WT and TaMYB391-overexpressed or underexpressed (TaMYB391-RNAi) transgenic lines was further confirmed through histological observation. To clarify the changes in the host resistance level, H2O2 accumulation and HR-associated cell death around the infection site were observed microscopically and measured by DP-BSW software. The growth of Pst was also measured by DP-BSW software. The histological observation results showed that H2O2 production in the TaMYB391overexpressing transgenic lines showed a substantial increase in comparison to the WT controls (Figure 6a,b). As compared to the WT, hyphal length and infection area were significantly decreased in the TaMYB391-overexpressing lines (Figure 6c,f,g). There were no significant differences between WT and TaMYB391-overexpressing transgenic lines in the number of haustoria and haustorial mother cells (Figure 6c,d,e). Our data suggested that overexpression of TaMYB391 improves the resistance of wheat to Pst. On the other hand, as compared to the WT controls, H2O2 accumulation was significantly decreased in the TaMYB391-RNAi lines (Figure 7a,b). Moreover, the necrotic area in the RNAi lines was significantly lower than in the WT controls (Figure 7a,c). Furthermore, the number of haustoria and haustorial mother cells, and hyphal length in the TaMYB391-RNAi lines were significantly increased compared to WT, at 48 hpi (Figure 8a,b,c). In addition, the infection area in the TaMYB391-RNAi line was significantly higher than in the WT controls, at 96 hpi (Figure 8a,d). Therefore, our results strongly suggest that RNAi-mediated silencing of TaMYB391 enhances wheat susceptibility to Pst. Values represent the means ± SEs. The significant difference between TaMYB391-overexpressing and control plants is indicated by asterisks, which was estimated using Student's t-test. *, p < 0.05; **, p < 0.01. with the virulent Pst race CYR31. Leaves inoculated with Pst were sampled at 48 and 96 hpi. Samples were then stained with wheat germ agglutinin (WGA) and fungal growth was detected using an Olympus BX-51 microscope (Olympus, Tokyo, Japan). SV, substomatal vesicle; HMC, haustorial mother cell; IH, infection hypha; H, haustoria. (d) The average number of haustoria per infection site. (e) Average number of haustorial mother cells per infection site. (f) Hyphal length as measured from the juncture of the hypha and substomatal vesicle to the tip of the hypha. (g) The colony area per each infection site. Data were computed from three biological replications and 50 infection sites. Values represent the means ± SEs. The significant difference between TaMYB391-overexpressing and control plants is indicated by asterisks, which was estimated using Student's t-test. *, p < 0.05; **, p < 0.01.

Discussion
Transcription factors in general, and MYB TFs in particular, play pivotal roles in plant disease resistance. Many MYB TFs have been reported for their positive roles in disease resistance in a variety of plant species. Here we reported the functional role of an R2R3 MYB TF, TaMYB391, during wheat-Pst interaction through gain-and loss-of-function approaches. RT-qPCR analysis demonstrated that the expression of TaMYB391 is associated with plant immunity against the biotrophic fungal pathogen Pst. Similarly, some MYB genes which are reported to participate in defense responses were induced after infection with different pathogens [23,[25][26][27]. Furthermore, TaMYB391 was also significantly induced by exogenous treatment of SA and ABA. Our sub-cellular localization assay indicated that TaMYB391 is a nuclear-targeted protein. In previous studies, many MYB TFs (e.g., AtMYB30, TaPIMP1, TaMYB4, TaPIMP2 and TaRIM1) have been reported to be localized in the nucleus [23][24][25][26]48].

Discussion
Transcription factors in general, and MYB TFs in particular, play pivotal roles in plant disease resistance. Many MYB TFs have been reported for their positive roles in disease resistance in a variety of plant species. Here we reported the functional role of an R2R3 MYB TF, TaMYB391, during wheat-Pst interaction through gain-and loss-of-function approaches. RT-qPCR analysis demonstrated that the expression of TaMYB391 is associated with plant immunity against the biotrophic fungal pathogen Pst. Similarly, some MYB genes which are reported to participate in defense responses were induced after infection with different pathogens [23,[25][26][27]. Furthermore, TaMYB391 was also significantly induced by exogenous treatment of SA and ABA. Our sub-cellular localization assay indicated that TaMYB391 is a nuclear-targeted protein. In previous studies, many MYB TFs (e.g., AtMYB30, TaPIMP1, TaMYB4, TaPIMP2 and TaRIM1) have been reported to be localized in the nucleus [23][24][25][26]48].
In plants, the hypersensitive response (HR), a kind of PCD, is usually associated with disease resistance. To dissect the role of TaMYB391 in HR-related PCD, we transiently overexpressed TaMYB391 in N. benthamiana. The results demonstrated that TaMYB391 is involved in inducing PCD directly. This is supported by increased electrolyte leakage. Accumulated evidence has indicated that a number of plant genes regulate the induction of HR-related PCD in plants. For instance, AtMYB30 is a well-known regulator of hypersensitive cell death [49]. Overexpression of Pti1 in tobacco accelerates HR in response to P. syringae pv. tabaci [50]. Antisense expression of hsr203 has also been shown to accelerate HR in tobacco in response to different pathogens [41]. ROS accumulation was observed in TaMYB391 agroinfiltrated samples. Meanwhile, the transcriptional accumulation of HRspecific marker genes and defense-related marker genes were significantly up-regulated. Our results indicated that HR-associated PCD triggered by TaMYB391 is correlated with the innate immune response.
In wheat, exogenous application of SA and ABA also increased the expression of TaMYB391. PR1 and PR2 are considered to be marker genes for SA-mediated activation of SAR [47]. The transcript levels of TaPR1 and TaPR2 in the TaMYB391-overexpressing plants were significantly up-regulated, whereas in the TaMYB391-RNAi lines the expression was repressed compared to WT. Plant hormones have been reported to play a significant role in plant biotic stress response [29,51]. SA is usually involved in biotrophic and hemibiotrophic pathogen defense signals [30,32]. ABA is reported to take part in both biotic and abiotic stress signals [52]. SA-mediated disease resistance against biotrophic pathogens was generally negatively regulated by ABA [53]. Some MYB TFs, for example, BOS1 [54], AtMYB96 [17,55], TaPIMP1 [23] and TaPIMP2 [24] have been proven to participate in hormone signaling networks. Taking these results together, TaMYB391 appears to activate the innate immune response via SA-mediated defense pathways.
The functional role of TaMYB391 in the wheat-Pst interaction was further validated through the gain-and loss-of-function approach. Expression analysis, phenotypic and microscopic assays indicated that TaMYB391-overexpression confers improved resistance to Pst infection, while the TaMYB391-RNAi lines showed decreased resistance to Pst compared to WT wheat plants. The degree of resistance in TaMYB391-overexpressing transgenic lines was correlated with reduced fungal growth (hyphal length and infection area), increased H 2 O 2 accumulation and enhanced expression of defense-related genes. H 2 O 2 accumulation, necrotic area and expression of defense-related genes in the RNAi lines, on the other hand, showed a significant reduction, while fungal growth was enhanced compared to WT wheat plants. These results suggested that TaMYB391 is positively involved in wheat resistance response to Pst infection. Consistent with our findings, the mutation or silencing of some MYB genes, such as TaMYB29, OsMYB30, TaPIMP1, TaMYB4, TaLHY, TaRIM1, TaPIMP2, MYB115 and AtMYB15 has been found to enhance susceptibility to different pathogens [18,19,[23][24][25][26][27][28]56].
As PR genes are reported to play a significant role in defense responses against various pathogens in different plant species [44][45][46], we analyzed the transcriptional accumulation of two PR genes (TaPR1 and TaPR2) in TaMYB391-overexpressing and TaMYB391-RNAi lines when inoculated with Pst. The expression levels of these genes in the TaMYB391overexpressing plants were significantly up-regulated, whereas in the TaMYB391-RNAi lines the expression was repressed compared to WT. These results demonstrated that TaMYB391 positively regulates the expression of certain PR genes during Pst infection. Furthermore, the expression of a ROS-scavenging gene TaCAT3 was significantly decreased in the TaMYB391-overexpressing plants, while being significantly increased in the TaMYB391-RNAi lines compared to WT plants. Previous studies have indicated that MYB TFs regulate the expression of diverse defense-related genes by binding to the respective cis-acting elements in their promoter regions [13,14,16,56,57]. In the future, we plan to identify the downstream target genes of TaMYB391 and clarify how TaMYB391 regulates the expression of TaPR1, TaPR2 and TaCAT3 during the interaction between wheat and Pst.
In conclusion, we identified an R2R3 MYB TF from wheat, TaMYB391, which acts as a positive regulator of hypersensitive response cell death. The expression of TaMYB391 was induced upon infection with Pst and exogenous application of SA and ABA. SA-dependent defense-related genes were also induced by TaMYB391 in N. benthamiana. Results obtained from overexpression and RNAi assays demonstrated that TaMYB391 positively regulates the resistance of wheat to Pst. Together, our data demonstrate that TaMYB391 acts as a positive regulator of HR and contributes to wheat resistance to Pst by modulating the defense-related genes, probably through SA-signaling pathways.

Plant Materials, Fungal Isolates and Inoculation/Treatments
Two wheat (T. aestivum L.) cultivars, Suwon11(Su11) and Fielder were used. Two Pst isolates, CYR23 and CYR31, were also used in our study. Su11, carrying a YrSu resistance gene, is susceptible to CYR31 and resistant to CYR23 [40]. Su11 was used to determine the relative transcription of TaMYB391 after Pst infection and exogenous hormones treatment. Su11 was also used to amplify the cDNA sequences of TaMYB391 and to perform the sub-cellular localization assays. Fielder is often used to introduce genes of interest for RNAi and overexpression due to its high transformation success rate [58]. Fielder, carrying Yr6 and Yr20 resistance genes, is susceptible to CYR31 and resistant to CYR23 [5,59]. In this study, Fielder was used as receptor material to generate TaMYB391-overexpressed and TaMYB391-RNAi transgenic plants. Wheat leaves challenged with the Pst isolates or water (distilled and sterile) were sampled at different time points for RNA isolation and histological study. Tobacco (Nicotiana benthamiana) was used for transient overexpression of TaMYB391 and sub-cellular localization assays.

RNA Extraction, cDNA Synthesis and Gene Expression Analysis
A Quick RNA isolation Kit (Huayueyang Biotechnology, Beijing, China) was used for total RNA extraction and was performed according to the manufacturer's instructions. A spectrometer (NanoDrop) was then used to quantify the isolated RNA. The enzyme DNase I was used to remove the DNA contamination. A RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA) was used to reverse the transcription of RNA and synthesize first-strand cDNA from 3 µg of isolated RNA. The transcriptional accumulation of TaMYB391 was then analyzed using RT-qPCR with specific primers (Table  S1). Gene expression was quantified using a ChamQ SYBR qPCR Master Mix (Vazyme, Shanghai, China) with a CFX Connect Real-Time System (Bio-Rad, Hercules, CA, USA). In wheat and N. benthamiana, the wheat elongation factor TaEF-1α (GenBank accession number: M90077) and NbActin (GenBank accession number AY179605) were used as an internal reference to quantify the transcript levels [10,[60][61][62][63][64]. The relative transcript levels of specific genes were computed using the comparative 2 −∆∆CT method. Each reaction was replicated three times.

Subcellular Localization of TaMYB391 in Wheat and N. benthamiana
The subcellular localization assay was performed to detect where TaMYB391 is located in the plant cell. Wheat protoplasts and N. benthamiana were used for this assay. The full-length CDS of TaMYB391 was subcloned into p16318hGFP or pCAMBIA:1302 vectors. Wheat protoplasts were extracted from the healthy fresh leaves of two-week-old wheat seedlings as described [65]. The p16318hGFP-TaMYB391 fusion or GFP construct were independently introduced into wheat protoplasts, as previously described [65]. The transformed wheat protoplasts were incubated in a dark chamber at 24 • C for 24-36 h and then observed with an Olympus FV1000 confocal laser microscope with a 488nm filter (Olympus, Tokyo, Japan). The localization of TaMYB391 was further confirmed by introducing pCAMBIA1302:TaMYB391 fusion into Agrobacterium tumefaciens strain GV3101 through electroporation, which was then agroinfiltrated into 4-week-old N. benthamiana leaves as described [66]. Infiltrated N. benthamiana leaves were kept in a growth chamber with a 16h/8h photoperiod at 25 • C for 2 to 3 days. Fluorescence signals were then observed and photographed with an Olympus FV1000 confocal laser microscope with a 488 nm filter (Olympus, Tokyo, Japan).

Transient Overexpression of TaMYB391 in N. benthamiana
A recombinant plasmid, PVX: TaMYB391, was constructed by cloning full length CDS without the stop codon into the pGR106 vector. The reconstructed vectors, PVX: TaMYB391, PVX: Pst322 (positive control) and PVX: GFP or empty vector (EV) (negative control), were transformed into A. tumefaciens strain GV3101 using electroporation, and then agroinfiltrated into 4-weeks-old N. benthamiana leaves as described [66]. The agroinfiltrated leaves were sampled at 0, 48, 72, 96 and 120 h post-agroinfiltration (hpa) for RNA isolation, and then analyzed by RT-qPCR. To detect ROS accumulation, the agroinfiltrated leaves were sampled at 24 hpa, and then stained with 3,3-diamino-benzidine (DAB). Symptom development was monitored at 3-8 dpa. Three biological replications were performed, and each assay consisted of three plants with three leaves.

Measurement of Electrolyte Leakage
The cell death was further confirmed via ion leakage assay, as previously described [67]. Six leaf disks, each 1 cm in diameter, were punched from PVX: TaMYB391 and GFP agroinfiltrated leaves and immersed in 5 mL distilled water for 5 h. After 5 h in distilled water, the 'value A' was obtained by measuring the conductivity of the solution with a conductivity meter (FE32 Five Easy; Mettler-Toledo, Shanghai, China). Then, the solution with the leaf disks was sealed and subjected to boiling for 20 min. After letting the solution cool, its conductivity was again measured to obtain 'value B'. Ion leakage was then computed as percent leakage; (value A/value B) × 100%. This assay was repeated three times.

Generation of Transgenic Lines
To produce TaMYB391-overexpressing transgenic wheat plants, the CDS of TaMYB391 was sub-cloned into the pCUB vector [68], and the pCUB: TaMYB391 construct was generated. The expression of TaMYB391 was driven by the maize ubiquitin promoter. pCUB: TaMYB391 was then transformed into wheat variety Fielder by an Agrobacterium-mediated wheat transformation system. Positive TaMYB391-overexpressed transgenic plants were validated through PCR using specific primers ( Figure S4a; Table S1). The wild-type (WT) wheat variety Fielder was used as a negative control during the PCR check. The second leaves of T 2 transgenic lines were challenged with the virulent Pst race, CYR31. Samples from Pst-infected leaves were collected for RNA isolation and histological observation.
To generate the TaMYB391-RNAi construct, the 200 bp specific fragment of TaMYB391 was amplified and inserted into the pC336 vector [69] using gateway cloning technology. The TaMYB391-RNAi construct contains an inverted repeat. A double-stranded (ds) RNA sequence with a hairpin structure was produced after transcription from the construct to induce the silencing of TaMYB391. TaMYB391-RNAi vector was then transformed into wheat variety Fielder by an Agrobacterium-mediated wheat transformation system. Positive transformants in the T 1 generation were detected by PCR using universal primers (Table S1). The second leaf of the T 2 generation was inoculated with the avirulent Pst race CYR23 and infected leaves were sampled for RNA isolation and histological observation.

H 2 O 2 Accumulation and Histological Observation of Fungal Growth
Wheat leaves inoculated with Pst isolates, CYR23 or CYR31, were sampled at 48 and 96 hpi and stained to detect the accumulation of H 2 O 2 and fungal structures. H 2 O 2 accumulation was examined by staining samples with DAB. Samples were then stained with wheat germ agglutinin stain (WGA) (Invitrogen, Waltham, MA, USA) for histochemical analysis [70]. During the wheat-Pst interaction, the formation of a substomatal vesicle is considered to be an effective penetration site of Pst. At least 30-50 infection sites were examined for each treatment to assess the H 2 O 2 accumulation, necrotic area and various fungal structures. Necrotic cells around the infection site, H 2 O 2 accumulation and fungal structures, such as hypha, haustoria mother cells and haustoria, and infection area were observed with a BX-51 microscope (Olympus, Tokyo, Japan), and their corresponding lengths and areas were estimated using DP-BSW software. The experiment was repeated three times.

Statistical Analysis
All data were subjected to analysis with Microsoft Excel. Microsoft Excel was used to calculate mean values and standard errors. Student's t-test was used to determine the significant differences between control and treatment groups or between time courses. Significant difference was measured by probability (p) values (p < 0.05 or p < 0.01).