The wheat R2R3-MYB transcription factor TaRIM1 participates in resistance response against the pathogen Rhizoctonia cerealis infection through regulating defense genes

Abstract

The necrotrophic fungus Rhizoctonia cerealis is a major pathogen of sharp eyespot that is a devastating disease of wheat (Triticum aestivum). Little is known about roles of MYB genes in wheat defense response to R. cerealis. In this study, TaRIM1, a R. cerealis-induced wheat MYB gene, was identified by transcriptome analysis, then cloned from resistant wheat CI12633, and its function and preliminary mechanism were studied. Sequence analysis showed that TaRIM1 encodes a R2R3-MYB transcription factor with transcription-activation activity. The molecular-biological assays revealed that the TaRIM1 protein localizes to nuclear and can bind to five MYB-binding site cis-elements. Functional dissection results showed that following R. cerealis inoculation, TaRIM1 silencing impaired the resistance of wheat CI12633, whereas TaRIM1 overexpression significantly increased resistance of transgenic wheat compared with susceptible recipient. TaRIM1 positively regulated the expression of five defense genes (Defensin, PR10, PR17c, nsLTP1, and chitinase1) possibly through binding to MYB-binding sites in their promoters. These results suggest that the R2R3-MYB transcription factor TaRIM1 positively regulates resistance response to R. cerealis infection through modulating the expression of a range of defense genes, and that TaRIM1 is a candidate gene to improve sharp eyespot resistance in wheat.

Introduction

Common wheat (Triticum aestivum L.) is one of most important staple crops in the world and plays a fundamental role in food security. Wheat production often is negatively affected by various fungal diseases. The sharp eyespot disease, mainly caused by the soil-borne fungus Rhizoctonia cerealis van der Hoeven (teleomorph: Ceratobasidium cereale D. Murray & L.L. Burpee), is a devastative disease of wheat production in the world^1,2,3. The sharp eyespot symptoms in wheat include dark-bordered lesions on stem bases and based sheaths of adult-plants³. The sharp eyespot disease can destroy the transport tissues in stems of plants, and cause pre- and post-emergence damping off and premature spike senescence or ripening (white heads), leading to yield losses of ~10–30%. The environmentally safe and efficient way to protect wheat from sharp eyespot is to breed resistant wheat varieties. However, the resistances in partially-resistant wheat lines are controlled by multiple quantitative loci^1,4. Currently, breeding sharp eyespot-resistant wheat cultivars using traditional methods is difficult since none of sharp eyespot-immune wheat cultivars/lines is available. Thus, to improve wheat resistance to R. cerealis, it is necessary to identify important genes in the defense response and clarify their defense roles.

In order to defend attack of pathogens, plants have evolved comprehensive and complicated defense system. The transcription factor families play an important role in the plants responses against different stresses. The myeloblastosis (MYB) transcription factors are characterized by a DNA-binding MYB domain. The MYB domain is composed of approximately 52 amino acid residues that adopt a helix-turn-helix conformation that intercalates into the major groove of DNA^5,6. Since the first plant MYB gene COLORED1 (C1) being involved in anthocyanin biosynthesis was identified in maize⁷, a large number of MYB proteins have been identified in different plant species. Based on the number of adjacent MYB repeats, MYB proteins can be divided into 4 classes: 1R-MYB, 2R-MYB (R2R3-MYB), 3R-MYB and 4R-MYB⁶. Numerous MYB proteins have been implicated in diverse biological processes, including cell cycle regulation, cell wall biosynthesis, development and reproduction, and defense responses to abiotic and biotic stresses^{6,8,9,10,11,12,13,14}. To date, most of the identified MYB genes belong to R2R3-MYB subfamily, and a number of R2R3-MYB transcriptional factors have been evidenced to play different important roles in various plant species^{6,15,16,17,18,19,20,21}. For example, in Arabidopsis, BOS1 (BOTRYTIS-SUSCEPTIBLE1), an R2R3-MYB gene (AtMYB108), is required for restricting the spread of 2 necrotrophic pathogens Botrytis cinerea and Alternaria brassicicola, and involved in the tolerance to osmotic and oxidative stresses²². Overexpression of an Arabidopsis R2R3-MYB AtMYB96 could enhance tolerance to drought stress²³ and increase resistance to bacteria pathogens²⁴. In barley, the MYB transcription factor HvMYB6 functions as positive regulator of basal and MLA-mediated immunity responses to Blumeria graminis²⁵. Ectopic expression of the wheat MYB gene TaMYB33 that was induced by NaCl and PEG stresses increased salt and drought tolerance in Arabidopsis plants¹⁷. The ectopic expression of TaMYB73 improved salt tolerance of transgenic Arabidopsis plants¹⁵. Overexpression of the wheat pathogen-induced MYB gene TaPIMP1 in transgenic wheat could significantly enhance resistance to the fungal pathogen Bipolaris sorokiniana and drought stresses²⁶. Ectopic expression of a Thinopyrum intermedium MYB gene TiMYB2R-1 could significantly increase resistance of transgenic wheat lines to take-all caused by Gaeumannomyces graminis²⁷. Silencing of a wheat R2R3-MYB gene TaMYB4 in wheat impaired the resistance to Puccinia striiformis f. sp. tritici⁵. However, none of MYB genes being involved in defense response to R. cerealis infection has been reported yet.

In this study, we identified and functional characterized a R. cerealis-induced MYB gene in wheat, named TaRIM1. Toward R. cerealis infection, the gene expression goes higher level. The sequence analysis and bio-molecular assays proved that TaRIM1 protein is a R2R3-type MYB transcription factor. It is localized in the nucleus and can bind to MYB binding site cis-elements. Through generation of TaRIM1- silencing and overexpression wheat plants and assessment of their defense responses following R. cerealis inoculation, the functional dissection results indicated that TaRIM1 positively modulated wheat defense response to R. cerealis. Further investigation suggested that TaRIM1 might activate the expression of a range of defense-related genes, resulting in enhanced resistance to R. cerealis infection.

Results

Identification and cloned sequence of TaRIM1 induced by R. cerealis infection

To identify wheat genes being involved in defense response to R. cerealis, we performed transcriptomic analysis through Deep RNA-seq on 3 resistance lines of the recombinant inbred lines (RILs, being derived from the cross of sharp eyespot-resistant wheat line Shanhongmai and sharp eyespot-susceptible wheat cultivar Wenmai 6) at 4 and 10 d post inoculation (dpi) with R. cerealis high-virulence strain WK207 (Unpublished). Among the up-regulated sequences, the expression of the sequence with no. Traes_6BL_E5A9546C9, being homologous to the wheat MYB gene TaMYB33 sequence, was up-regulated in the resistant wheat lines after R. cerealis inoculation. It showed a 4.18-fold at 4 dpi or a 10.23-fold at 10 dpi transcriptional increase than the mocked (Fig. 1a). Quantitative RT-PCR (qRT-PCR) analysis showed that the transcriptional levels of this gene were induced after R. cerealis inoculation (Fig. 1b), and the expression tendency by experimental qRT-PCR was in agreement with the RNA-Seq data. This gene was designated as TaRIM1 and was suggested to be involved in wheat defense response to R. cerealis infection.

Figure 1: Transcriptonal analyses of TaRIM1 in Rhizoctonia cerealis-inoculated wheat.

The RNAs are isolated from pooled stems of 3 resistant lines in recombinant inbred lines of cross of R. cerealis-resistant wheat Shanhongmai and susceptible wheat cultivar Wenmai 6 at 4, or 10 days post inoculation (dpi) with R. cerealis strain WK207 or mocked ones. (a) RNA-Seq data of the sequence Traes_6BL_E5A9546C9 being corresponding to TaRIM1. (b) qRT-PCR verification of the transcription of TaRIM1. The transcriptional level in the mocked one is set to 1. The transcriptional levels with different letters are significantly different from each other based on statistically significant analysis on the results of three replications (t-test, **P < 0.01).

The full-length cDNA sequence (with 1028 bp, Fig. 2a, NCBI accession no. KU864997) of TaRIM1 was obtained from R. cerealis-infected stem cDNA of the resistance wheat line CI12633 by RACE and nest RT-PCR. It includes the complete ORF with 732-bp, 5′-untranslated region (UTR) with 98-bp, and 3′-UTR with 198-bp. The genomic DNA sequence of TaRIM1 was amplified from CI12633 genomic DNA. The comparison of the genomic and cDNA sequences indicated that no intron existed in genomic transcription unit of TaRIM1, at least in the amplification region. The deduced protein TaRIM1 contains 243 amino acids with a predicted molecular weight of 26.61 KD and predicted PI of 6.09. As shown in Fig. 2a, the TaRIM1 protein sequence possesses two conserved MYB DNA-binding domains [one (R2) located at amino acids 13-63 and another (R3) at amino acids 66-114], a putative nuclear localization signal (NLS, located at amino acids110-136), and an acidic region (amino acids 138-191) possibly acting as a transcription activation domain²⁸. The reconstructed phylogenetic tree analysis showed that this protein TaRIM1 was clustered into the R2R3-MYB subfamily (Fig. 2b). Thus, TaRIM1 most likely is a R2R3-MYB transcription factor with transcription-activation activity.

Figure 2: The sequence and phylogenetic tree analyses of TaRIM1.

(a) The nucleotide sequence and deduced amino acid sequence of TaRIM1. Green part represents R2 domain, yellow space indicates R3 domain, the nucleus localization signal is marked by red lines, and blue dotted line represents the acidic region. (b) Phylogenetic analysis of the TaRIM1 protein. The phylogenetic tree including TaRIM1 protein and 16 known-function MYB proteins is constructed using neighbor-joining phylogeny of MEGA 5.0, and is showed in bootstrapped manner.

TaRIM1 localizes to the nucleus and binds to MYB-binding site cis-elements

As the TaRIM1 protein sequence contains the NLS sequence (Fig. 2a), the p35S:TaRIM1-GFP (green fluorescent protein) fusion expressing-vector was prepared for investigating the subcellular localization of TaRIM1. The p35S:TaRIM1-GFP and control p35S:GFP construct DNAs were separately introduced into and transiently expressed in both wheat mesophyll protoplasts and onion epidermal cells. Confocal imaging of the transient expression showed that TaRIM1-GFP localized in the nucleus in both the wheat mesophyll protoplasts and onion epidermal cells (Fig. 3a,b), whereas the fluorescence of the control GFP was distributed throughout the cell (Fig. 3a,b). These results indicated that the expressing TaRIM1 protein localizes in the nuclear.

Figure 3: Subcellular localization of the TaRIM1 protein in either the wheat mesophyll protoplasts or onion epidermal cells.

The 35S:TaRIM1-GFP and 35S:GFP constructs were separately introduced into and transiently expressed in both the wheat mesophyll protoplasts (a) and onion epidermal cells (b). The transient expressing green-fluorescence proteins (GFPs) were observed and photographed under 488 nm in Confocal Laser Scanning Microscopy (Zeiss LSM 700, Germany). Auto-fluorescence of the wheat chloroplast was observed and photographed under 639 nm in Confocal Laser Scanning Microscopy (Zeiss LSM 700, Germany).

The amino acid sequence of TaRIM1 contains R2 and R3 DNA-binding domains. To investigate if TaRIM1 binds to MYB-binding site (MBS) cis-elements, the glutathione S-transferase (GST)-TaRIM1 recombinant protein was prepared, expressed, and purified. Electrophoretic mobility shift assay (EMSA) was used to examine the DNA binding ability of TaRIM1 with MBS. Here, the tested 5 MBS cis-elements include ACI, MBS1-Bz, MBS1-w, RT1, and St1R that were bound by known functional R2R3-MYB, StMYB1R-1 and OsMYB3R-2 transcription factors²⁶ (Fig. 4a, Table S1). The EMSA results showed that the GST-TaRIM1 protein could bind to all the tested 5 MBS probes, especially showing the strongest binding to ACI, but not bind to the GCC-box cis-element that is specifically bound by ERF transcription factors, whereas GST failed to bind with the MBS cis-element ACI and the GCC-box cis-element (Fig. 4b). These data proved that the protein TaRIM1 can bind to these five MBS elements.

Figure 4: Electrophoretic mobility shift assay on binding activity of TaRIM1 to MBS cis-element.

MBS indicates MYB-binding site. (a) The retarded band of binding of GST-TaRIM1 fusion protein to MBS probe and free probe are indicated by arrows. (b) Commassie blue staining shows the position of GST protein and GST-TaRIM1 fusion protein by arrows.

Silencing of TaRIM1 impairs wheat resistance to R. cerealis

Virus-induced gene silencing (VIGS) is an efficient reverse-genetic tool for rapidly analyzing functions of genes in plants. Barley stripe mosaic virus (BSMV)-based VIGS is extensively used for investigating functions of interest genes in barley and wheat^29,30,31. To explore whether TaRIM1 plays an important role in wheat resistance response against R. cerealis, we used BSMV-based VIGS method to down-regulate transcriptional levels of TaRIM1 in the resistant wheat line CI12633. At 15 dpi with the virus, the transcript of BSMV coat protein (cp) gene was readily detected (Fig. 5a), suggesting that BSMV successfully infected these wheat plants. Importantly, the transcript levels of TaRIM1 were significantly reduced in CI12633 plants infected by BSMV:TaRIM1 compared to BSMV:GFP infected CI12633 plants (control plants) (Fig. 5a,b), suggesting that TaRIM1 transcript was successfully down-regulated in BSMV:TaRIM1 infected plants, hereafter TaRIM1-silenced plants represented BSMV:TaRIM1 infected ones. The TaRIM1-silenced and BSMV:GFP infected CI12633 plants were further inoculated with R. cerealis. Subsequently, the infection types (ITs) by the fungus were evaluated. At 45 dpi with R. cerealis, TaRIM1-silenced CI12633 plants showed more susceptible to the sharp eyespot disease caused by R. cerealis (ITs: ~2.8–3.8; Fig. 5c), whereas BSMV:GFP infected CI12633 plants showed more resistance of sharp eyespot (IT: 1.2, Fig. 5c). These results suggested the down-regulation of TaRIM1 compromised the resistance to R. cerealis in CI12633, and that TaRIM1 is required for host resistance response to R. cerealis.

Figure 5: VIGS-based functional analysis of TaRIM1 in response of wheat to Rhizoctonia cerealis.

(a) RT-PCR analysis of the transcription levels of TaRIM1 and the barley stripe mosaic virus (BSMV) CP gene in wheat plants infected by BSMV:GFP or BSMV:TaRIM1 for 15 d. (b) qRT-PCR analysis of relative transcript level of TaRIM1 in stems of wheat CI12633 plants infected by BSMV:TaRIM1 or BSMV:GFP at 45 dpi with R. cerealis strain WK207. The relative transcript level of TaRIM1 in BSMV:TaRIM1-infected wheat plants is relative to that in BSMV:GFP-infected plants. Statistically significant differences between BSMV:GFP-infected and BSMV:TaRIM1-infected (TaRIM1-silencing) CI12633 plants were determined based on three replications using Student’s t-test (**P < 0.01). (C) Typical sharp eyespot phenotypes of TaRIM1-silencing CI12633 plants and BSMV:GFP-infected CI12633 plants after inoculation with R. cerealis strain WK207 for 45 days. IT indicates infection type of wheat plants to R. cerealis infection.

Overexpression of TaRIM1 enhances wheat resistance to R. cerealis

To further investigate the defense role of TaRIM1 in wheat, we constructed the TaRIM1 overexpression vector pUbi:myc-TaRIM1 (Fig. 6a), in which the expression of the fused protein gene myc-TaRIM1 of a c-myc epitope tag and TaRIM1 was driven by a maize ubiquitin (Ubi) promoter and terminated by the terminator of the Agrobacterium tumefaciens nopaline synthase gene (Tnos) in a modified monocot transformation vector pAHC25 ^31,32. The pUbi:myc-TaRIM1 vector DNA was bombarded by gene gun into immature embryos of the spring wheat cultivar Yangmai 16 for generating transgenic wheat plants. The presence of TaRIM1 transgene cassette was detected by the desired PCR product (374 bp) using the primer pairs specific to TaRIM1-Tnos transgene (Fig. 6b). Based on results of PCR detection in 3 successive generations of T₀-T₂, five stably transgenic lines (MO1-MO5) containing Ubi:myc-TaRIM1 transgene were selected. qRT-PCR assays showed that the transcriptional levels of TaRIM1 in these five TaRIM1-overexpressing transgenic lines were significantly elevated compared with non-transformed (wild type, WT) recipient wheat Yangmai 16 (Fig. 6c). Western blotting analysis indicated that the introduced myc-TaRIM1 gene was translated into the myc-TaRIM1 protein in these 5 overexpressing transgenic lines (MO1-MO5), but not in WT Yangmai 16 (Fig. 6d). Following inoculation with R. cerealis for ~47 d, all the 5 TaRIM1-overexpressing wheat lines in successive two (T₁-T₂) generations showed significantly enhanced-resistance to sharp eyespot compared with susceptible WT wheat Yangmai 16 (Table 1). These results indicated that TaRIM1 positively contributed to wheat defense response to R. cerealis infection.

Figure 6: The transformation vector and molecular characterization of TaRIM1-overexpressing transgenic wheat plants.

(a) The TaRIM1-overexpression transformation vector pUbi:myc-TaRIM1. The arrow indicates the fragment amplified in the PCR detection of the transgene. (b) PCR patterns of T₀-T₂ plants. P: indicates pUbi:myc-TaRIM1 plasmid as the positive control; MO1-MO5 indicate 5 TaRIM1 transgenic wheat lines. WT indicates non-transformation wheat (recipient) Yangmai 16. (c) qRT-PCR analysis of TaRIM1 transcriptional levels in these 5 transgenic wheat lines. The relative transcript level of TaRIM1 in these transgenic lines is relative to that in the WT plants. Three biological replicates each line were averaged and statistically treated (t-test; **P < 0.01). Bars indicate standard error of the mean. (d) Western blot pattern of these 5 TaRIM1-overexpressing transgenic lines and WT Yangmai 16 using an anti-myc antibody.

Table 1 Sharp eyespot responses of TaRIM1-overexpression and wild-type wheat lines ^a .

TaRIM1 positively regulates the expression of defense genes in wheat

To explore the putative mechanism of TaRIM1 in the resistance response to R. cerealis infection, we analyzed the transcript levels of 5 wheat defense genes by qRT-PCR in TaRIM1- silencing and overexpression wheat plants as well their controls after the pathogen inoculation. The examined genes include Defensin (NCBI accession no. CA630387), PR10 (NCBI accession no. CA613496), PR17c (NCBI accession no. TA65181), nsLTP1 (NCBI accession no. TC411506), and chitinase1 (Chit1, NCBI accession no. CA665185). As shown in Fig. 7, the transcription levels of all the 5 defense genes were significantly decreased in susceptible TaRIM1-silenced wheat plants than that in BSMV:GFP infected control plants. These results suggested that the expression level of TaRIM1 was correlated with the transcriptional levels of these defense genes. As expected, the transcription levels of these 5 defense genes were significantly elevated in resistant TaRIM1-overexpressing transgenic wheat lines compared to those in susceptible WT wheat Yangmai 16 plants (Fig. 8). These results indicated that TaRIM1 positively regulated, most likely activated, the expression of these defense genes in wheat.

Figure 7: qRT-PCR analysis of the relative transcript level of TaRIM1 and five defense genes in TaRIM1-silencing and BSMV:GFP-infected CI12633 plants inoculated with R. cerealis for 45 d.

The relative transcript level of the tested genes in TaRIM1-silencing wheat CI12633 plants is relative to that in BSMV:GFP-infected CI12633 plants. The examined genes include Defensin (NCBI accession no. CA630387), PR10 (NCBI accession no. CA613496), PR17c (NCBI accession no. TA65181), nsLTP1 (NCBI accession no. TC411506), and chitinase1 (Chit1, NCBI accession no. CA665185). Statistically significant differences between TaRIM1-silencing wheat plants and BSMV:GFP-infected plants were determined based on three replications using Student’s t-test (**P < 0.01).

Figure 8: qRT-PCR analysis of the expression of TaRIM1 and five defense genes in TaRIM1-overexpressing transgenic and WT lines inoculated with R. cerealis for 47 d.

The relative transcript level of the tested genes in transgenic lines is relative to that in the WT plants. The examined genes include Defensin (NCBI accession no. CA630387), PR10 (NCBI accession no. CA613496), PR17c (NCBI accession no. TA65181), nsLTP1 (NCBI accession no. TC411506), and chitinase1 (Chit1, NCBI accession no. CA665185). Statistically significant differences between transgenic lines with WT were determined based on three replications using Student’s t-test (**P < 0.01).

MYB proteins can regulate the expression of defense- and stress-related genes following by interaction with MBS cis-elements. Our above EMSA results showed that the TaRIM1 protein could bind to 5 MBS motif sequences (Fig. 4). Furthermore, to address how TaRIM1 activates the afore-tested 5 defense genes, we obtained the promoters’ sequences of these 5 defense genes from International Wheat Genome Sequencing Consortium (http://www.wheatgenome.org/), and then searched MBS cis-elements in −2000 to −1 bp promoter sequences upstream of ATG of all of these genes using PLACE (https://sogo.dna.affrc.go.jp/cgi-bin/sogo.cgi?lang=en&pj=640&action=page&page=newplace) and the tested 5 MBS motif sequences. As shown in Table 2, the promoters of Defensin and PR10 contain 4 MBS motif sequences, including ACI, MBS1, RT1, and St1R, respectively. The promoter of Chit1 contains 3 MBS motif sequences, including ACI, MBS1, and RT1. The promoter of PR17c and nsLTP1 contain 2 MBS cis-elements, including MBS1 and St1R. These data implied that TaRIM1 may interact with these MBS cis-elements in the promoters of these defense genes.

Table 2 MBS sequences in promoters of defense genes regulated by TaRIM1.

Discussion

The sharp eyespot disease, caused primarily by the necrotrophic fungal pathogen R. cerealis, seriously limits the wheat production worldwide. The wheat defense response to R. cerealis is complicated and involves expression changes of a series of defense-related genes^31,33. Identification of important genes in wheat defense response to R. cerealis is critical for developing wheat varieties with resistance to sharp eyespot. In plants, MYB transcription factors play important roles in development and defense responses against abiotic and biotic stresses. Many MYB genes are induced after various stress stimuli. For example, the Arabidopsis R2R3-MYB gene BOS1 showed significant induction after B. cinerea, bos1 mutant displayed more sensitivity to 2 necrotrophic pathogens, osmotic and oxidative stresses than WT plants²². In wheat, the R2R3-MYB gene TaPIMP1 was induced after B. sorokiniana infection and drought stress, and positively regulated resistance responses to B. sorokiniana and drought stresses²⁶. TaLHY, a wheat 1R-MYB gene, was induced by the infection of stripe rust pathogen strain CYR32. VIGS-based functional analysis suggest that TaLHY may positively participate in wheat defense response to the biotrophic fungal pathogen CYR32 ¹³. However, no MYB gene being involved in resistance to the necrotrophic pathogen R. cerealis has been identified yet.

In this study, through RNA-Seq-based transcriptomic analyses, TaRIM1, a wheat MYB gene induced by R. cerealis in wheat, was identified, and cloned from R. cerealis-resistant wheat CI12633. In fact, some stress-induced genes play important roles in defense responses to the stresses, whereas other stress-induced genes do not. To explore the functional role of TaRIM1 in wheat defense response to R. cerealis, we generated the TaRIM1- silencing and overexpression wheat plants. Through molecular characterization and assessment of resistance responses of TaRIM1- silencing and overexpression as well their control wheat plants after inoculation with the fungal pathogen, the functional dissection results displayed that silencing of TaRIM1 did obviously impair resistance to R. cerealis in CI12633, TaRIM1-overexpression wheat lines exhibited significantly enhanced-resistance compared with susceptible WT recipient Yangmai16. These data suggest that TaRIM1 is required for defense response against R. cerealis in wheat, at least for the resistant wheat line CI12633, and positively contributes to wheat defense response to R. cerealis infection. Moreover, the transgenic wheat produced in this study will provide potential wheat germplasm for enhancing resistance to sharp eyespot disease. To our knowledge, TaRIM1 is the first reported member of MYB family positively participating in resistance response to R. cerealis.

In this report, Blast and phylogenetic analyses showed that the deduced protein TaRIM1 is a member of the R2R3-MYB subfamily. The sequence of the TaRIM1 protein possesses R2 and R3 MYB DNA-binding domains, a NLS and a putative transcription-activation domain, implying that the TaRIM1 protein is an activator-type R2R3-MYB transcription factor. Our biochemical and molecular-biological experiment results reveal that TaRIM1 is localized in the nuclear and can bind to all the test 5 MBS cis-elements. These biochemical properties are in agreement with the sequence characteristics of TaRIM1, and are necessary for the active function of MYB transcription factors⁵.

In plants, activator-type transcription factors have been implicated in defense responses through activating the expression of defense-related genes^{10,26,27,28,33,34,35}. Defense genes positively contribute to resistance to pathogens in plants^24,26. For example, transgenic wheat plants overexpressing a barley chitinase gene or a radish defensin gene RsAFP2 or a wheat LTP gene showed enhanced-resistance to fungal pathogens^36,37,38. To explore the putative mechanism of TaRIM1 in wheat resistance response, we analyzed the transcriptional levels of 5 wheat defense genes, including Defensin, PR10, PR17c, nsLTP1, and Chit1, in TaRIM1-silencing and TaRIM1-overexpression wheat plants and their control plants. The results showed that the expression levels of these 5 defense genes were lower in TaRIM1-silenced wheat than in the control plants, whereas the transcriptional levels of the 5 genes in TaRIM1-overexpression wheat plants were elevated compared with non-transformed recipient wheat, suggesting that TaRIM1 positively regulates the transcriptional levels of the above-tested 5 defense genes. These results suggest that overexpression of TaRIM1 up-regulates, most likely activates, the expression of a subset of, at least the above-tested 5, defense-related genes. Many papers document that transcription factors firstly bind to specific DNA sequences (cis-acting elements) in target genes, and then modulate the transcription levels of these genes^{10,26,27,28,33,34,35,39}. To address how TaRIM1 up-regulates the expression of the 5 afore-tested defense genes, we analyzed the promoter sequences of the examined 5 defense genes in wheat. The promoter sequences of these 5 defense genes, including Defensin, PR10, PR17c, nsLTP1, and Chit1, contain 2–4 kinds of MBS cis-acting elements. Our EMSA results prove that TaRIM1 indeed binds to these MBS cis-acting elements (Fig. 4). Thus, we deduce that the TaRIM1 transcription factor interacts with the promoters of these defense genes and then activates the expression of these genes. Consequently, the expression change of a range of wheat defense-related genes regulated by TaRIM1 probably results in enhanced resistance in transgenic wheat to sharp eyespot caused by R. cerealis infection.

In conclusion, TaRIM1, a wheat MYB gene induced by R. cerealis infection, was identified and its functional role was dissected. TaRIM1 encodes an activator-type R2R3-MYB transcription factor TaRIM1. TaRIM1 positively contributes to wheat resistance response to R. cerealis infection through regulation of the expression of a range of defense-related genes. TaRIM1 is a candidate gene for breeding wheat varieties with resistance to sharp eyespot caused by R. cerealis infection. This study provides novel insight into characteristics and functional roles of the MYB members in plants’ defense responses.

Methods

Plant and fungal materials, and treatments

R. cerealis-resistant wheat line CI12633 was used in cloning and VIGS analysis. The spring wheat cultivar Yangmai16 displaying susceptible was used as transformation recipient. Three resistant lines of RILs (cross of resistant wheat cultivar Shanhongmai and highly-susceptible wheat cultivar Wenmai 6), provided by Prof. Jizeng Jia in our Institute, were used in RNA-Sequencing.

The fungus R. cerealis Jiangsu-prevailing strain R0301 and North-China high-virulence strain WK207 were provided by Profs Huaigu Chen and Shibin Cai (Jiangsu Academy of Agricultural Sciences, China,) and Prof. Jinfen Yu (Shandong Agricultural University), respectively.

Wheat plants were grown in a 15 h light (~22 °C)/9 h dark (~10 °C) regime. The treatments were conducted according to the protocol by Zhu et al.³¹.

RNA extraction and cDNA synthesis

Total RNA was extracted using TRIZOL reagent (Qiagen, China) and subjected to DNase I (Takara, Japan) digestion and purification. The first-strand cDNA was synthesized using 2-μg purified RNA, AMV reverse transcriptase and AP primer (5′-GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT-3′) and Oligo dT primer according to the manual (Takara, Japan).

Cloning of cDNA and genomic DNA full-length sequences of TaRIM1

After inoculation with R. cerealis WK207 for 4 or 10 d, RNAs extracted from the infected base stems and sheaths of three resistant lines of the RILs were deeply sequenced. The further transcriptiomic analyses were performed by Bioinformatics to identify up-regulated genes (Data unpublished). Among them, one with no. Traes_6BL_E5A9546C9 is homologous to the TaMYB33 sequence, thus this corresponding gene was named TaRIM1.

To obtain the R. cerealis-resistance-related sequence of TaRIM1, used as the template to the full-length cDNA of TaRIM1 was obtained from infected stems’ cDNA of resistant wheat CI12633 by two rounds of 3′RACE reactions. In 1^st round, the primers TaRIM1F-F1 (5′-GCAGCATTTACCTTCGGAC-3′) and AUAP (5′-GGCC ACGCGTCGACTAGTAC-3′) were used. The primers TaRIM1F-F2 (5′-CGTCAACA CACTGAGCAATC-3′) and AUAP (5′-GGCCACGCGTCGACTAGTAC-3′) were used in 2^nd round. Genomic sequence of TaRIM1 was obtained from CI12633 genomic DNA by 2 round PCR amplifications and 2 pairs of primers (TaRIM1F-F1: 5′-GCAG CATTTACCTTCGGAC-3′, TaRIM1F-R1: 5′-AACGATTATTGTTCCCTTCACA-3′, TaRIM1F-F2: 5′-CGTCAACACACTGAGCAATC-3′, and TaRIM1F-R2: 5′-AAACTA GCCGAGGAGCCG-3′). The purified PCR products were cloned into the pMD-18T vector (Takara) and sequenced.

Sequence blast was performed online using the website (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Protein sequence analysis was performed using SMART (http://smart.embl-heidelberg.de/). The NLS region was predicted by cNLS Mapper (http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi). Phylogenetic tree analysis was performed using MEGA 5.0.

Subcellular localization of TaRIM1 in onion epidermal cells

The TaRIM1 coding region lacking the stop codon was amplified using primers TaRIM1-GFP-Hind III-F (5′-AGTAAGCTTATGGGGAGGTCTCCTTGC-3′, underline represents the Hind III restriction site) and TaRIM1-GFP-BamHI-R (5′-GCGGGATCCAATCTGAGACAAACTCTG-3′, underline represents the BamH I restriction site). The PCR products was digested with restriction enzymes Hind III and BamH I, then sub-cloned in-frame into 5′-terminus of GFP coding region in the p35S:GFP vector, resulting in the TaRIM1-GFP fusion construct p35S:TaRIM1-GFP.

The p35S:TaRIM1-GFP or p35S:GFP alone construct was separately introduced into wheat leaf protoplasts via the PEG-mediated transfection method following the protocol of Yoo et al.⁴⁰ or transformed into onion epidermal cell by particle bombardment following Zhang et al.⁴¹. After incubation at 25 °C for 12 h, GFP signals were then observed and photographed using Confocal Laser Scanning Microscopy (Zeiss LSM 700, Germany).

VIGS-based functional analysis of TaRIM1

The specific fragment (no. 576–836 nt) of TaRIM1 was amplified from CI12633 cDNA by primers TaRIM1-NheIF (5′-ACAGCTAGCTCGTCCGCCACCGATTACT-3′, underline represents Nhe I restriction site) and TaRIM1-NheIR (5′-GGCGCTAGCCTC TGCCTAAATCTGAGACAAAC-3′ underline represents Nhe I restriction site). PCR products were digested with Nhe I, then ligated into the BSMV-γ vector, resulting in recombinant vector BSMV-γ-TaRIM1.

BSMV:TaRIM1 and control virus BSMV:GFP viruses were used to inoculate the CI12633 using protocol following Zhu et al.³¹. At 14 d after infection, the fourth leaves of the inoculated seedlings were collected to monitor BSMV infection, the transcription of the BSMV coat protein (CP) gene with the specific primers (BSMV-CPF: 5′-TGACTGCTAAGGGTGGAGGA-3′, BSMV-CPR: 5′-CGGTTGAACATCACGAAG AGT-3′) and RT-PCR was used to evaluate if BSMV inoculates wheat plants. At 20 days after BSMV infection, BSMV infected plants were inoculated with the small toothpick fragments harboring the well-developed mycelia of R. cerealis WK 207 ³⁴. At 45 dpi with R. cerealis, ITs were scored, and sharp eyespot symptoms were photographed.

EMSA on TaRIM1 binding activity to MBS Cis-elements

The coding sequence of TaRIM1 with EcoR I and Xho I restriction sites was amplified with the primers (GF: 5′-GATGAATTCAAGAGGGGGCCGTGGACG-3′, underline represents EcoR I restriction site. GR: 5′-CTACTCGAGCTGGCCGGACGTCTTGGA -3′ underline represents Xho I restriction site) and then sub-cloned in-frame into 3′-terminus of GST in pGEX-4T-1 vector. The resulting GST-TaRIM1 recombinant protein was expressed in Escherichia coli BL21 cells after induction with 0.3 mM isopropyl-β-D-thiogalactopyranose, and purified using MicroSpin module (GE Amersham).

The primers of 6 cis-element probes including 5 MBS and GCC-box were synthesized as the sequences in Table S1 ²⁶.

Following a modified EMSA protocol⁴², each probe was mixed with ~2 μg of recombinant GST-TaRIM1 or GST in a binding buffer. Each reaction mixture was incubated on ice for 6 h and loaded onto 8% polyacrylamide gel. After electrophoresis was performed at 100 V for 30 min, the gels were stained with ethidium bromide for visualization of the DNA bands.

Generation and PCR detection of TaRIM1- overexpressing transgenic wheat

ORF of TaRIM1 was amplified with the primers (TVF: 5′-CAAACTAGTATGGGGAGG TCTCCTTGC-3′, underline represents Spe I restriction site, TVR: 5′-CGTGAGCTCCT AAATCTGAGACAAACT-3′, underline represents Sac I restriction site) to sub-clone in-frame with a c-myc epitope tag in a modified pAHC25-myc vector^31,32, resulting in TaRIM1-overexpression transformation vector pUbi:myc-TaRIM1 (Fig. 6a). In predicted transformed plants, the expression of the fused protein gene myc-TaRIM1 of a c-myc epitope tag and TaRIM1 was driven by a maize ubiquitin (Ubi) promoter and terminated by the terminator of the Agrobacterium tumefaciens nopaline synthase gene (Tnos).

The resulting transformation vector pUbi:myc-TaRIM1 plasmid was transformed into 1,200 immature embryos of wheat Yangmai 16 by biolistic bombardment following Chen et al.³⁴.

The overexpressing transgene was detected by a PCR product (374 bp) specific to the transgene using specific primers (TaRIM1-TF1: 5′-CGGACAACGAGATCAA GAAC-3′, TaRIM1-TF2: 5′-AGCTTTACATCGGAGGAGTTTCAG-3′ locating in the TaRIM1 coding region, TaRIM1-TR: 5′-AAAACCCATCTCATAAATAACG-3′ locating in Tnos) with 2 round of nested amplifications.

Western blot analysis of myc-TaRIM1 protein in transgenic wheat

The protein expression of introduced c-myc-TaRIM1 was evaluated by Western blotting. Total proteins were extracted from 0.4 g of ground base stem powders. About 10 μg of total soluble proteins were separated on 12% sodium dodecyl sulfate polyacrylamide gels and transferred to PVDF membrane. The Western blots were incubated with a 2000-fold dilution of c-myc antibody (TransGen Biotech, China) at 4 °C for 12 h, and then with 1000-fold dilution of secondary antibody conjugated to horseradish peroxidase (TransGen Biotech, China) at ~25 °C for 1 h. The expressing myc-TaRIM1 proteins were visualized using the Pro-light HRP Chemiluminescent Kit (TIANGEN Biotech, China).

qRT-PCR analyses of TaRIM1 and defense genes

qRT-PCR technique was used to examine the relative transcriptional levels of TaRIM1 (TaRIM1-QF: 5′- CGACTCGTCCTCGTCCAAG-3′, TaRIM1-QR: 5′-CGACGACG GCATCGAGTAAT-3′) and five defense-marker genes in TaRIM1-silencing and overexpression wheat plants as well as their control wheat plants. These defense genes include Defensin (Defensin-Q-F: 5′-ATGTCCGTGCCTTTTGCTA-3′, Defensin-Q-R: 5′-CCAAACTACCGAGTCCCCG-3′), PR10 (PR10-Q-F: 5′-CGTGGAGGTAAACGA TGAG -3′, PR10-Q-R: 5′-GCTAAGTGTCCGGGGTAAT-3′), PR17c (PR17c-Q-F: 5′-ACGACATCACGGCGAGGT-3′, PR17c-Q-R: 5′-CACGGGGAAAGAGAGGATGA-3′), nsLTP1 (nsLTP1-Q-F: 5′-ATGCGGGTTGGCGTGAAG-3′, nsLTP1-Q-R: 5′-TGT TGCGGTGGTAGGTTGTTG-3′) and chitinase1 (Chit1-Q-F: 5′-ATGCTCTGGGACC GATACTT-3′, Chit1-Q-R: 5′-AGCCTCACTTTGTTCTCGTTTG-3′). qRT-PCR was performed (95 °C for 5 min, 41 cycles of 95 °C for 15 s and 60 °C for 31 s) using SYBR Green I Master Mix (Takara) using ABI PRISM 7300 detective system according to the manufacturer’s instruction. The relative transcript levels were calculated using the 2^−ΔΔCT method⁴³, where the wheat actin gene (NCBI accession no. BE425627) with the primers ActA: (5′-CACTGGAATGGTCAAGGCTG-3′) and ActB (5′-CTCCATGTC ATCCCAGTTG-3′) was used as the internal reference.

Assessment on wheat response to R. cerealis

At least 10 plants for each wheat line were inoculated with developed mycelia of R. cerealis following the protocol of Wei et al.⁴⁴. In T₁ generation, R. cerealis WK207 was used to inoculate plants, whereas R. cerealis isolate R0301 was used to inoculate T₂ and WT plants. ITs and disease index for wheat line were evaluated at ~47 dpi according to Wei et al.⁴⁴.