Isolation and characterization of a novel wheat cysteine-rich receptor-like kinase gene induced by Rhizoctonia cerealis

Cysteine-rich receptor kinases (CRKs) belong to the receptor-like kinase family. Little is known about CRK genes in wheat. We isolated a wheat CRK gene TaCRK1 from Rhizoctonia cerealis-resistant wheat CI12633 based on a differentially expressed sequence identified by RNA-Sequencing (RNA-Seq) analysis. TaCRK1 was more highly expressed in CI12633 than in susceptible Wenmai 6. Transcription of TaCRK1 in wheat was induced in CI12633 after R. cerealis infection and exogenous abscisic acid (ABA) treatment. The deduced TaCRK1 protein contained a signal peptide, two DUF26 domains, a transmembrane domain, and a serine/threonine protein kinase domain. Transient expression of a green fluorescence protein fused with TaCRK1 in wheat and onion indicated that TaCRK1 may localize to plasma membranes. Characterization of TaCRK1 silencing induced by virus-mediated method in CI12633 showed that the downregulation of TaCRK1 transcript did not obviously impair resistance to R. cerealis. This study paves the way to further CRK research in wheat.

Moreover, PvPK20-1, a CRK gene isolated from roots of the common bean, is also differentially regulated by pathogens, symbionts, and nodulation factors 23 . These studies suggested that some CRK proteins are involved in plant responses to biotic and/or abiotic stresses.
Wheat (Triticum aestivum) is one of the most important staple crops in the world and plays a fundamental role in food security. Sharp eyespot, mainly caused by soil-borne fungal pathogen Rhizoctonia cerealis, is one of the most devastating diseases of wheat 24,25 . In infected wheat plants, R. cerealis may destroy the transport tissues in stems and sheaths of host plants, causing blocked transportation of substances required for nutrition, lodging, and even dead spikes 24 . Previous studies have demonstrated several genes in wheat defense response to R. cerealis, such as TaERF3 26 and TaGluD 27 ; however, very little is known about the roles of RLKs in wheat defense response to R. cerealis. To explore whether RLK proteins function in wheat defense response to R. cerealis, we used RNA-Sequencing (RNA-Seq) to study transcript patterns of RLK genes in resistant and susceptible wheat genotypes toward R. cerealis infection.
In this paper, a novel CRK gene in wheat, TaCRK1, was isolated based on a differentially expressed sequence. The expression of TaCRK1 was markedly induced after infection with R. cerealis and by exogenously applied ABA in resistant line CI12633. We investigated the subcellular localization of the TaCRK1 protein and also analyzed the function of TaCRK1 in wheat defense response to R. cerealis.

Results
TaCRK1 was induced by R. cerealis infection in resistant line CI12633. To identify wheat RLK genes in response to R. cerealis infection, RNA-Sequencing (RNA-Seq) analysis was used to compare transcriptome differences of R. cerealis-resistant line CI12633 and R. cerealis-susceptible cultivar Wenmai 6 under R. cerealis inoculation. Based on RNA-seq data, gene ontology (GO) analyses, and pathway analyses, wheat cDNA clone AK330939 (GenBank accession no.AK330939) was identified to show twofold increase in transcriptional level in the R. cerealis-inoculated CI12633 relative to the mock-treated CI12633. Moreover, at 4 days post inoculation (dpi) with R. cerealis, the transcriptional level of AK330939 was elevated 2.6-fold in the resistant wheat CI12633 compared with the susceptible wheat Wenmai 6. This gene, hereafter designated as TaCRK1, showed homologous to the genes encoding cysteine-rich receptor-like protein kinases in plants. The transcriptional levels of TaCRK1 in CI12633 and Wenmai 6 were further evaluated by real-time quantitative reverse RT-PCR (qRT-PCR). The result of qRT-PCR assay (Fig. 1a) was consistent with the RNA-Seq analysis. As shown in Figure 1a, the transcriptional level of TaCRK1 was elevated 2.2-fold in R. cerealis-inoculated CI12633 relative to mock-treated CI12633, but down-regulated 2.2-fold in R. cerealis-inoculated Wenmai 6 compared with mock-treated Wenmai 6. The expression abundance of the gene was significantly higher in CI12633 than in Wenmai 6 at 4 dpi with R. cerealis. These results suggested that TaCRK1 may be involved in wheat defense response to R. cerealis infection.
The transcriptional level of the TaCRK1 gene was also investigated via qRT-PCR analyses in the stems of seven wheat lines/cultivars with different levels of resistance and susceptibility to R. cerealis at 4 dpi. The experimental wheat lines/cultivars include resistant lines CI12633 and Shanhongmai; moderate-resistant lines Xifeng, shan-nong0431, and Navit14; moderate susceptible line Yangmai 158; highly-susceptible line Wenmai 6, whose disease indexes after R. cerealis infection were shown in Supplementary Table 1. As shown in Fig. 1b, the transcriptional level of TaCRK1 was the highest in moderate-resistant line Xifeng and the lowest in highly susceptible cultivar Wenmai 6. However, the transcriptional levels of TaCRK1 were not consistent with the resistance degrees in other tested wheat lines/cultivars. For example, compared with the susceptible line Yangmai 158, the relative transcriptional level of TaCRK1 was lower in more resistant Shanhongmai. These results suggested that the expression levels of TaCRK1 in the seven wheat lines/cultivars at 4 dpi were not associated with their resistance degrees.
TaCRK1 encodes a cysteine-rich receptor-like protein kinase. The 39 un-translated region (UTR) of TaCRK1 was cloned by 39 rapid amplification of cDNA ends (RACE), and the full open reading frame (ORF) sequence was amplified from R. cerealis-infected stem cDNA of CI12633. The cDNA sequence of TaCRK1 with 2330-bp length was obtained through analyzing the overlaid sequences and deposited in the public GenBank database (GenBank accession no. KC818618). Sequence analysis showed that the cDNA of TaCRK1 includes an ORF consisting of 2043 nucleotides (from 19 to 2061 nucleotides) (Fig. 2). BLAST analysis showed that the nucleotide sequence of this gene was highly similar to those of   predicted receptor-like protein kinases from Brachypodium distachyon (GenBank accession no. XM_003560070) (83% identity) and rice (GenBank accession no. AK111650) (77% identity). We compared the nucleotide sequence and amino acid sequence of TaCRK1 with that of AK330939. The nucleotide sequences of TaCRK1 and AK330939 share 95.7% identity. The deduced amino acid sequence of TaCRK1 shares 96.9% identity with that of AK330939 ( Supplementary Fig. 1). The predicted TaCRK1 protein sequence exhibits 18 amino acid substitutions and one insertion compared with the deduced AK330939 protein. Among these, six amino acid substitutions occur in protein kinase catalytic domain. These results suggested that TaCRK1 and AK330939 were homologous, but not identical.
The deduced TaCRK1 protein contains 680 amino acid residues with a molecular weight of 74.93 kD and a pI of 6.01. The search for protein conserved domain using InterPro-Scan web indicated that the TaCRK1 protein contains a signal-peptide domain, two DUF26 domains, a transmembrane domain, and a serine/threonine protein kinase catalytic domain that includes 11 subdomains (Fig. 2). The predicted result using Smart software was consistent with that from InterPro-Scan.
TaCRK1 protein was likely to be localized to the plasma membrane in planta. To study the subcellular location of TaCRK1, p35S:TaCRK1-green fluorescence protein (GFP) fusion expressing vector was generated and introduced into onion epidermal cells or wheat protoplasts, using p35S:GFP construct as the control. Confocal microscopic observations showed that the transient expression of p35S:TaCRK1-GFP localized to cell periphery both in the onion epidermal cells and in the wheat protoplasts, whereas GFP protein alone was distributed in the entire cytoplasm and nucleus ( Fig. 4a-b), suggesting that TaCRK1 seems to be a plasma membrane protein in planta. To further confirm this localization, a cyan fluorescent protein (CFP)-labeled plasma membrane marker AtPIP2A 28 construct and the TaCRK1-GFP construct were co-transformed into wheat protoplasts and then co-expressed. AtPIP2A was an Arabidopsis plasma membrane intrinsic protein, which is often used as a plasma membrane targeted marker. The co-expression of AtPIP2A-CFP together with other plant protein was used to study the subcellular localization of the plant proteins, including barley (Hordeum vulgare L.) 29 , Medicago truncatula 30 , and Oncidium Gower Ramsey 31 . Here, the AtPIP2A-CFP fusion protein localized to plasma membrane in wheat protoplast from 15 h to 18 h after transformation (Fig. 4c), similar to the plasma membrane localization pattern of AtPIP2A in Arabidopsis and Medicago truncatula. TaCRK1-GFP also exhibited a plasma membrane localization pattern in wheat protoplast from 15 h to 18 h after transformation (Fig. 4c). The merging images obtained from the GFP and CFP channels showed that the TaCRK1-GFP and AtPIP2A-CFP fluorescence proteins co-localized to the plasma membrane (Fig. 4c), suggesting that TaCRK1 protein was likely to be a the plasma membrane protein in wheat. These results were consistent with those of RLKs that typically function in the cellular membrane.
Expression of TaCRK1 was induced by exogenous ABA stimuli. Certain RLKs have implicated in hormone signal transduction. To determine if the transcript of TaCRK1 is induced by phytohormones including abscisic acid (ABA), jasmonic acid (JA), ethylene (ET), and salicylic acid (SA), qRT-PCR was used to investigate the transcriptional patterns of TaCRK1 in R. cerealis-resistant wheat CI12633 across a time course taken at 0, 1, 3, 6, 12 and 24 h after treatment with the exogenous hormones. As shown in Fig. 5a, the transcriptional level of TaCRK1 increased at 1-6 hours post-treatment (hpt), reached a peak at 3 hpt (more than three-fold over that of 0 hpt) and then decreased at 12-24 hpt. Upon MeJA treatment, the expression of TaCRK1 decreased at 1-6 hpt, but slightly increased at 12-24 hpt (Fig. 5b). Upon ET treatment, the transcriptional level of TaCRK1 decreased from 1 to 24 hpt (Fig. 5c). Upon SA treatment, the expression of TaCRK1 decreased from 1 to 12 hpt, but at 24 hpt it increased close to non-treated level (Fig. 5d). To understand the putative molecular basis of TaCRK1 in these responses, we analyzed cis-elements in the 1899-bp upstream of the start codon of TaCRK1. Analysis showed that the promoter contained six ABAresponsive elements (ABRE) (core sequence, PyACGTGG/TC) 32 , among which the box between 21753 and 21746 (CACGTGTC, in trans orientation) is a typical ABRE (shown in Supplementary Table 3 and Supplementary Fig. 2). These results suggested that TaCRK1 may be involved in the ABA signaling pathway.
Down-regulation of TaCRK1 transcript did not obviously impair R. cerealis resistance. To investigate whether TaCRK1 plays an important role in wheat resistance to R. cerealis, TaCRK1 transcript level was knocked down in resistant wheat CI12633 using a virus-induced gene silencing (VIGS) technique. VIGS was developed with barley stripe mosaic virus (BSMV) and demonstrated to be an effective reverse genetics tool for investigating the functions of some genes in barley and wheat [33][34][35][36][37] . The RNAc cDNA clone of BSMV can be manipulated to accommodate the transcription of non-viral sequences in infected barley or wheat plants 34 . In this study, a 298bp fragment comprising the 39 end of the ORF and part of the 39 UTR sequence was inserted in an antisense orientation into NheI restriction site of the RNAc to generate the BSMV:TaCRK1 construct. Semi-quantitative RT-PCR analyses showed that the transcript of the BSMV CP gene was detected in both BSMV:GFP-and BSMV: TaCRK1-inoculated CI12633 plants, but not in the mock (bufferinoculated) plants (Fig. 6a). As expected, the TaCRK1 transcript level was substantially reduced in CI12633 plants infected by BSMV: TaCRK1 (Fig. 6a-b), proving that the TaCRK1 expression was suppressed in these CI12633 plants infected by BSMV:TaCRK1.
At the tillering stage, the 4 th sheaths in the mock CI12633 plants and those infected with the recombinant BSMV viruses were further inoculated with mycelia of R. cerealis. As positive control for successful R. cerealis inoculation, the 4 th sheath of Wenmai 6 was also infected with mycelia of R. cerealis. At 2 weeks post inoculation with R. cerealis, a dark-brown margin (an early symptom of sharp eyespot disease) was present at the 4 th sheaths of susceptible Wenmai 6 but www.nature.com/scientificreports SCIENTIFIC REPORTS | 3 : 3021 | DOI: 10.1038/srep03021  absent in BSMV:TaCRK1-inoculated, BSMV:GFP-inoculated, and mock CI12633 plants (Fig. 6c). Furthermore, until the mature stage, no sharp eyespot symptom was observed at 4 th sheaths and stems of BSMV:TaCRK1-inoculated, BSMV:GFP-inoculated, and mock CI12633 plants, but the obvious symptoms were present at 4 th sheaths and stems of Wenmai 6 plants. These results suggested that TaCRK1 silencing did not directly compromise the wheat resistance to R. cerealis in CI12633.

Discussion
Plant receptor protein kinases, representing the main plasma membrane receptors, play important roles in perceiving extracellular signals and triggering rapid resistance responses 38 . In this study, we isolated a wheat CRK gene, TaCRK1, from R. cerealis-resistant wheat CI12633, based on a sequence differentially expressed between resistant wheat CI12633 and susceptible wheat Wenmai 6. TaCRK1 transcript was rapidly induced by R. cerealis infection in resistant line CI12633 and was more than 2-fold higher in CI12633 than in Wenmai 6, suggesting that TaCRK1 might be involved in wheat defense responses to R. cerealis. The deduced protein possesses a signal peptide domain, two extracellular DUF26 domains (each containing one copy of CRR motif), a transmembrane domain, and a kinase catalytic domain including 11 kinase subdomains. Phylogenetic analysis revealed that TaCRK1, together with BdCRK, ZmCRK, OsCRK, and AtCRK13 fell into the CRK clade of RLK proteins. Thus, TaCRK1 is a novel member of the CRK subgroup of RLK family in wheat. Certain CRK proteins from Arabidopsis thaliana have been implicated in defense responses; for instance, overexpression of Arabidopsis AtCRK5 was correlated with enhanced leaf growth and displayed enhanced resistance to bacterial pathogen Pseudomonas syringae through induction of expression of pathogenesis-related 1 (PR1) gene 15 .
Cells of eukaryotic organisms are organized into a large number of compartments to carry out a large number of biochemical functions.
According to the intracellular localization of an uncharacterized protein, the likely functions of this protein can be infered 39 . Onion epidermal cells and wheat protoplasts were used in this study to investigate the subcellular localization of TaCRK1. In the onion cells, p35S:TaCRK1-GFP localized to the cell periphery; in wheat protoplasts, TaCRK1-GFP appeared to localize to the plasma membrane; whereas p35S:GFP localized to the entire cytoplasm and nucleus, suggesting that TaCRK1 protein was likely to targete to the plasma membrane in wheat and onion plants. AtPIP2A is a plasma membrane aquaporin and belongs to the plasma membrane intrinsic protein family 28 . Recent studies showed that AtPIP2A was an  www.nature.com/scientificreports excellent cell plasma membrane marker in Arabidopsis, barley, Medicago truncatula, and Oncidium Gower Ramsey [29][30][31]39 . For instance, a HvPHT1;6:GFP was transiently co-expressed with either the plasma membrane targeted marker, AtPIP2A:mCherry, or the vacuolar membrane marker, gTIP:mCherry. The green fluorescence of HvPHT1;6:GFP co-localized with the red fluorescence of the plasma membrane marker AtPIP2A:mCherry, separated from that of the red fluorescence of the vacuolar marker gTIP:mCherry 29 . In this study, AtPIP2A-CFP protein exhibited to a plasma membrane localization pattern in wheat protoplasts, and fluorescence from TaCRK1-GFP and AtPIP2A-CFP seemed to be co-localized to the plasma membrane. Because there is no reported research that AtPIP2A reliably localizes to the plasma membrane in wheat protoplasts, the localization results of TaCRK1-GFP need further to be proved.
Many RLKs have been shown to be involved in hormonal signal transduction 18,40,41 . For instance, upregulation of AtCRK13 in Arabidopsis led to hypersensitive response-associated cell death and induced defense against pathogens by causing increased accumulation of salicylic acid 14 . In plants, the ABA pathway has been implicated in regulation of plant development and response to biotic and abiotic stresses [42][43][44] . A receptor-like kinase in Arabidopsi, GUARD CELL HYDROGEN PEROXIDE-RESISTANT1 (GHR1), was shown to be a critical component in ABA and H 2 O 2 signaling pathways and to be involved in stomatal movement 41 . The tomato ABA-inducible MYB transcript factor AIMI (abscisic acid-induced myb1) was suggested to function in ABA sensitivity, abiotic stress tolerance, and basal resistance against Botrytis cinerea in tomato 45 . Most ABA-inducible genes contain a conserved, ABA responsive, cis-acting element, designated as AB-RE (core sequence, PyACGTGG/TC), in their promoter regions 46 . It was found that the expression of ABA responsive gene requires multiple ABREs or the combination of an ABRE with a coupling element (CE) as a functional promoter 47,48 . In this study, qRT-PCR analyses revealed that TaCRK1 in resistant wheat CI12633 could be rapidly induced by exogenous ABA treatment. The 1899-bp promoter of TaCRK1 contained one ABRE and five ABRE-like boxes, which may partially contribute to the response of TaCRK1 to ABA stimuli. In addition, the transcript level of TaCRK1 was reduced by MeJA and ET treatments. No JA-or ethylene-or SA-responsive element was detected in the promoter of TaCRK1, suggesting that TaCRK1 indirectly regulated by MeJA or ET or SA. These results suggested that TaCRK1 might be involved in other responses regulated by ABA signaling pathway, which will be further studied in the future.
VIGS is an efficient tool for rapidly analyzing plant gene functions. In this study, the VIGS approach was utilized to investigate the function of TaCRK1 in wheat defense response to R. cerealis. Although the TaCRK1 transcript level was reduced in resistant CI12633 plants infected by BSMV:TaCRK1, down-regulation of TaCRK1 in CI12633 did not obviously impair wheat resistance to R. cerealis. Plant immunity is a complex network in which some components and network sectors interact with each other in complex ways. The function of a sector of the network can be compensated by some other sectors; consequently, functional identification of these sectors only by knocking out each of the sectors is difficult 49 . For example, BRL1 is functionally redundant with BRI1 in regulating Arabidopsis brassinosteroid signaling. The brl1-1 mutant plants did not have obvious phenotypes, but bri1-5 brl1-1 double mutants showed enhanced defective leaf phenotypes compared with the bri1-5 single mutant 50 . In this study, reducing TaCRK1 expression did not compromise CI12633 resistance to R. cerealis. The reason might be that TaCRK1 is not the major gene controlling wheat defense response to R. cerealis or that TaCRK1 is functionally redundant with some other genes. Responses of TaCRK1 in other environmental stresses will be further investigated in the future.
In summary, TaCRK1, the first DUF26-CRK gene isolated from wheat, was identified via RNA-seq and characterized. It undergoes significantly higher expression levels in resistant wheat CI12633 following R. cerealis infection and exogenous ABA stimuli. TaCRK1 encodes a cysteine-rich receptor-like protein kinase TaCRK1. The TaCRK1 protein localizes to the plasma membranes in wheat protoplasts and in onion epidermal cells. Our results give new insights into the CRK subgroup of the RLK family in plant species, and may pave the way to further study of the functions of CRKs in wheat. RNA extraction and cDNA synthesis. Total RNA was extracted using TRIzol reagent (Qiagen, China) according to the manufacturer's instructions. DNase I treatment was applied to remove contaminated genomic DNA. The first-strand cDNA was synthesized using 2 mg purified RNA, AMV reverse transcriptase, and oligo (dT 15 ) primers (TAKaRa, Japan) according to the manual.

Methods
Cloning and sequence analysis of TaCRK1. The sequence of the 39 un-translated region (UTR) was amplified from cDNA of the CI12633 stems challenged with R. cerealis for 4 days using 39-Full RACE Core Set Ver.2.0 (TaKaRa, Japan) based on the wheat cDNA clone AK330939. Then, two pairs of primers (TaCRK1-1 st -F/TaCRK1-1 st -R and TaCRK1-2 nd -F/TaCRK1-2 nd -R, Supplementary Table 2) were designed and used to amplify the full open reading frame (ORF) sequence of TaCRK1 from the cDNA of the CI12633 through two rounds of nested RT-PCR. The resulting PCR products were cloned to the pMD-18T Vector (TaKaRa, Japan) to form the positive clones. At least five positive clones were sequenced with an ABI PRISM 3130XL Genetic analyzer (Applied Biosystems, Foster City, CA). cDNA sequence data were analyzed using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/). The deduced protein sequence analyses were performed using the Compute pI/MW tool (http://web.expasy.org/compute_pi/) for computation of the theoretical iso-electric point and protein molecular weight, InterPro-Scan (http://www.ebi.ac.uk/interpro/) and Smart software (http:// smart.embl-heidelberg.de/smart/set_mode.cgi?GENOMIC51) for prediction of the conserved domains and motifs, DNAMAN software for sequence alignment, and MEGA 5.0 software for constructing a phylogenetic tree. The upstream region (1899 bp) to start codon was analyzed for detection of ABA responsive elements using the plant cis-acting regulatory DNA elements (PLACE) database 52 (http:// www.dna.affrc.go.jp/PLACE/).
Subcelluar localization of TaCRK1. The coding region of TaCRK1 without the stop codon was amplified using gene-specific primers with PstI and XbaI restriction sites (59-GTCCTGCAGATGGCCAAACCCCACCGC-39, with underline denoting the PstI site; and 59-GCTCTAGATCTTGGCGAAAGCTCCGT-39, with underline denoting the XbaI site) and was subcloned in-frame to the 59-terminus of the GFP coding sequence in p35S:GFP vector (Dr. Daowen Wang, Chinese Academy of Sciences), generating the TaCRK1-GFP fusion construct p35S:TaCRK1-GFP.
The resulting p35S:TaCRK1-GFP or p35S:GFP alone construct was separately bombarded into white onion epidermal cells following Zhang et al 26 . The TaCRK1-GFP fusion or GFP alone construct was separately introduced into wheat protoplasts via the PEG-mediated transfection method following Yoo et al 53  cDNA with the primers (59-GACGCTAGCTCCCTTCTCTGTCCAGGC-39 and 59-CGCGCTAGCTAGCATCTTAGCAGTTCTAC-39; underlined sections are the NheI restriction sites). Then the fragment was inserted in an antisense orientation into NheI restriction site of the RNAc, resulting in the recombinant construct RNAc:TaCRK1-as. Following a previously described protocol 42 , the tripartite cDNA chains of BMSV: TaCRK1-as, or the control virus BMSV:GFP genome, were separately transcribed into RNAs and then mixed to infect CI12633 plants at the twoleaf stage. At the same time, CI12633 plants were inoculated only with the buffer containing no virus; hereafter, these plants are called mock treatments. The 4 th leaves of the inoculated seedlings were collected to monitor BSMV infection based on the transcripts of BSMV coat protein (CP) gene using BSMV-CP-F/BSMV-CP-R primers and to evaluate the transcript changes of TaCRK1 with TaCRK1-Q-F/TaCRK1-Q-R primers (Supplementary Table 2). For R. cerealis inoculation, the fungus was cultured on potato dextrose agar at 25uC for 10 days, then 1-cm 2 plugs from the edge of R. cerealis colonies were placed into liquid PDA medium and cultured at 25uC for 2 weeks to develop mycelia. At the tillering stage, the 4 th base sheath of wheat plants was inoculated with 15 ml culture of R. cerealis. Inoculated plants were grown at 90% relative humidity for 4 days. Sharp eyespot symptoms were observed at 14 days and 40 days after the fungal inoculation, when sharp eyespot symptoms were present at the infected sheaths and stems, respectively, of susceptible Wenmai 6.
RT-PCR and Real-time quantitative RT-PCR (qRT-PCR) analysis. RT-PCR was performed with the following thermal profile: initial denaturation at 94uC for 5 min; followed by 30 cycles of 30 s at 94uC, 30 s at 60uC, and 30 s at 72uC; and final extension at 72uC for 5 min. The PCR products were detected on 1.5% agarose gel. In all the semi-quantitative RT-PCR experiments, wheat elongation factor 1 alphasubunit gene (TaEF-1a) was used to normalize the cDNA contents among various samples.
qRT-PCR was performed using SYBR Green I Master Mix (TaKaRa, Japan) in a volume of 25 ml on an ABI 7300 RT-PCR system (Applied Biosystems). Reactions were set up with the following thermal profile: 95uC for 5 min, followed by 41 cycles of 95uC for 15 s and 60uC for 31s, and completed with a melting curve analysis program. All qRT-PCR reactions were repeated three times. The relative expression of the gene TaCRK1 was calculated with the 2 2DDCT method 54 , where the wheat TaActin gene was used to normalize amounts of cDNAs among the samples.
The sequences of primers were listed in Supplementary Table 2.