Wheat Elongator subunit 4 is required for epigenetic regulation of host immune response to Rhizoctonia cerealis

Ki Wng, Wei Rong, Yuping Liu, Hui Li, Zengyn Zhng,

aThe National Key Facility for Crop Gene Resources and Genetic Improvement, Key Laboratory of Biology and Genetic Improvement of Triticeae Crops, Ministry of Agriculture, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China

bInstitute of Cereal and Oil Crops, Hebei Academy of Agriculture and Forestry Sciences, Shijiazhuang 050035, Hebei, China

ABSTRACT Wheat (Triticum aestivum) is necessary for global food security. The necrotrophic fungus Rhizoctonia cerealis is the causal agent of sharp eyespot, a devastating disease of wheat.Although the Elongator complex,composed of six subunits,has been implicated in growth,development, and innate immunity in Arabidopsis, little is known about its functions in wheat or the involvement of Elongator subunit 4 in histone acetylation. In this study, we identified the Elongator subunit 4-encoding gene TaELP4 in wheat resistance response to R.cerealis, and verified that TaELP4 increased histone acetylation in regions of defenseassociated genes and regulated immune response to R. cerealis. TaELP4 was more highly expressed in resistant than in susceptible wheat cultivars and was induced in resistant wheat after infection by R.cerealis.Silencing of TaELP4 in wheat not only impaired resistance to R. cerealis, but also repressed both histone acetylation levels and the expression of a subset of defense-associated genes, including TaAGC1, TaCPK7-D, TaPAL5, Defensin, and Chitinase2. Ectopic expression of TaELP4 in Arabidopsis increased histone acetylation levels in coding and promoter regions of defense genes and increased their transcription,leading to increased resistance to infection by the pathogen Botrytis cinerea. These results suggest that TaELP4 positively regulates innate immune responses of wheat and Arabidopsis to R.cerealis and B. cinerea by increasing histone acetylation levels of defense-associated genes and increasing their transcription.This study has shed light on the involvement of TaELP4 in histone acetylation and resistance response against R.cerealis.TaELP4 may potentially be used to improve wheat resistance against sharp eyespot.

1. Introduction

Wheat (Triticum aestivum L.) is a staple food crop that plays a fundamental role in global food security. The necrotrophic fungus Rhizoctonia cerealis is the major causal agent of the sharp eyespot disease of major food crops including wheat, barley,oats,and rye[1-3].R.cerealis infection can occur in the root and basal stem tissues of wheat plants at any time during the growing season. Sharp eyespot reduces wheat yields in many regions of Asia, Oceania, Europe, North America, and Africa[4,5]. Since the late 1990s, China has become the largest epidemic region in the world [2,6]. Breeding resistant wheat cultivars is an effective and environmentally safe approach to controlling disease. The efficiency of breeding depends on the availability of resistance sources and on an understanding of resistance mechanisms. However, it is difficult to breed sharp eyespot-resistant wheat cultivars by conventional methods,because no sharp eyespot-immune wheat cultivars and few typical resistance genes have been identified [2,6,7]. To meet this challenge,it is vital to isolate genes that mediate resistance responses to R.cerealis and to identify their functional roles.

Plants, as sessile organisms, have evolved elaborate and effective innate immune systems to cope with diverse pathogenic microorganisms [8]. Plants recognize pathogenor microbe-associated molecular patterns(PAMPs) using cellsurface pattern-recognition receptors or sense pathogen effectors by intracellular resistance-gene products, resulting in PAMP-triggered immunity (PTI) and effector-triggered immunity(ETI),respectively[8,9].The two immune responses are similar and involve ion fluxes, phosphorylation of target proteins, generation of reactive oxygen species, deposition of callose, and large-scale transcriptional reprogramming of genes [9-11]. Numerous transcriptional regulators, including the Elongator complex, have been shown to modulate transcriptional reprogramming[12].

The Elongator complex was originally identified in yeast as a protein interacting with hyperphosphorylated RNA polymerase II (RNAPII) [13,14], and subsequently purified from human [15] and Arabidopsis thaliana cells [16]. The Elongator complex activates RNAPII-mediated transcription and is composed of six subunits (ELP1 to ELP6), with ELP1-ELP3 forming the core subcomplex and ELP4-ELP6 forming the accessory subcomplex [13-19]. ELP1 and ELP2 are WD40 proteins and serve as scaffolds for the complex assembly,while ELP3 is the catalytic subunit, harboring a C-terminal GNAT-type histone acetyltransferase(HAT)domain and an Nterminal iron -sulfur radical S-adenosylmethionine (SAM)domain [12,16-20]. Each ELP4-ELP6 forms a RecA-ATPase-like fold and ELP4-ELP6 together assemble into a hexameric ringshaped structure [21,22]. The Elongator complex has been implicated in multiple cellular processes, including histone modification, tubulin acetylation, exocytosis, genome stability maintenance, DNA methylation or demethylation, tRNA modification, and microRNA biogenesis [16-19,23,24]. All six subunits are required for Elongator’s cellular functions.In the model plant A. thaliana, mutations of the Elongator subunits AtELP1-AtELP6 result in pleiotropic effects including hypersensitivity to abscisic acid, resistance to oxidative stress,severely aberrant auxin phenotypes, disease susceptibility,altered cell cycle progression, and defective root stem cell maintenance[12,16,25-29].AtELP2 and AtELP3 are required for basal immunity and ETI to multiple pathogens [12,28-30].AtELP3 possesses HAT activity and radical SAM domains,which are essential for maintaining the levels of basal or normal histone acetylation levels and are required for regulatory roles of AtELP3 in leaf and primary root growth and plant immunity [16,26,28]. AtELP2 is an epigenetic regulator required for pathogen-induced transcriptome reprogramming via alteration of methylation levels in defense genes and increasing histone acetylation levels in coding regions of several defense genes during innate immune responses [29,30]. In recent reports [31,32], Fragaria vesca and tomato(Solanum lycopersicum) genomes both contained all six Elongator subunits, and AtELP4-overexpressing transgenic F.vesca and tomato plants displayed enhanced resistance to anthracnose crown rot (caused by the hemibiotrophic fungus Colletotrichum gloeosporioides), powdery mildew (caused by the obligate biotrophic fungus Podosphaera aphanis), and tomato bacterial speck(caused by the Pseudomonas syringae pv.tomato strain J4).However,little is known about the role of Elongator in wheat or the involvement of ELP4 in histone acetylation during plant immune response.

In this study, to determine whether and how wheat Elongator regulates host immune responses to R. cerealis, a wheat Elongator subunit 4-encoding gene designated as TaELP4, participating in wheat responses to R. cerealis infection, was identified through RNA-Seq-based transcriptomics,and its defense role was dissected by means of virus-induced gene-silencing (VIGS), transgenic plants and disease test, as well as expression of defense-associated genes and their histone acetylation levels were investigated by RT-qPCR and chromatin immunoprecipitation-qPCR analyses. We found that TaELP4 is required for the epigenetic regulation of host immune responses against R. cerealis by increasing histone acetylation levels in coding or promoter regions of a subset of defense-associated genes, and elevating their transcription levels. This is the first investigation of ELP4-triggered transcriptome reprogramming by histone acetylation in plant innate immunity.

2. Materials and methods

2.1.Plant and fungal materials, primers, and treatments

Six wheat cultivars (cvs.), including R. cerealis-resistant CI12633 and Shanhongmai, moderately resistant Shannong 0431 and Niavt 14,and highly-susceptible cvs.Wenmai 6 and Yangmai 9 [6,33], were used to investigate TaELP4 transcription profiles. F9recombinant inbred lines (RILs) derived from the cross Shanhongmai × Wenmai 6 were provided by Prof.Jizeng Jia (ICS, CAAS). The resistant wheat cv. CI12633 was used in a virus-inducing gene silencing (VIGS) experiment.The A. thaliana Columbia-0 (Col-0) ecotype was used for ectopic expression of TaELP4-7B and for further study of the immune role of TaELP4.

Rhizoctonia cerealis isolate WK207, which is highly virulent in north China, was provided by Prof. Jinfen Yu, Shandong Agricultural University,China.Botrytis cinerea t-8 was provided by Prof.Bingyan Xie in the Institute of Vegetables and Flowers,Chinese Academy of Agricultural Sciences,China.

All primers used are listed in Table S1.

All wheat plants were grown in a greenhouse under 23°C/14 h light and 10 °C/10 h dark. At the tillering stage, the stem base of each plant was inoculated with toothpick fragments harboring well-developed mycelia of R.cerealis.The inoculated sites were covered with wet cotton to increase the humidity,which promotes R. cerealis infection [33]. Inoculated plants were grown at 90% relative humidity for four days.Arabidopsis thaliana plants were grown in a greenhouse under 22 °C/14 h light and 22 °C/10 h dark. Samples at several times postinoculation or from several tissues were harvested and stored at ?80°C prior to total RNA extraction.At 4-and 10-days postinoculation (dpi) with R. cerealis WK207 or mock inoculation,RNAs derived from three resistant and three susceptible lines from the RILs were individually subjected to deep sequencing and comparative transcriptomic analysis.

2.2. RNA extraction and RT-(q) PCR

Total RNA was extracted from samples collected from wheat plants of all cultivars using Trizol reagent (Invitrogen, USA).RNA was purified and then checked for integrity. For RT-PCR or real-time quantitative PCR (RT-qPCR), total RNA was reverse-transcribed to cDNA using the FastQuant RT Kit(Tiangen, China). Specific primers were used to measure the expression levels of TaELP4, TaAGC1, TaCPK7-D, TaPAL5,

Defensin, Chitinase 2 in wheat and a barley yellow dwarf virus(BSMV)coat protein(CP) gene. RT-qPCR was performed on an ABI 7500 instrument (Applied Biosystems, USA) using a SYBR Premix Ex Taq kit (TaKaRa, Japan). Reactions were programmed with the following thermal cycling profile: 95 °C for 30 s followed by 40 cycles of 95°C for 5 s,58°C for 30 s and 72°C for 34 s.The PCR products were loaded onto 1.5% agarose gels and visualized under UV after staining with ethidium bromide. Each experiment was replicated three times. The relative expression of target genes was calculated using the 2?ΔΔCTmethod [34], with the wheat actin gene TaActin or Arabidopsis actin gene AtActin used as internal reference genes.

2.3. Cloning and sequence analyses of TaELP4

The full-length open reading frame (ORF) sequence of TaELP4 was amplified using primer TaELP4-7B-F/R from cDNA of CI12633 stems inoculated with R. cerealis four days. The untranslated region (UTR) of TaELP4 was amplified with a SMARTer RACE 5′/3′ Kit (Clontech, USA). The PCR products were cloned into pMD18-T vector (TaKaRa, Japan) and then sequenced. The predicted protein sequence was analyzed with the Compute pI/Mw tool (http://web.expasy.org/compute_pi/) to determine the theoretical pI (isoelectric point) and Mw (molecular weight), interPro-Scan (http://www.ebi.ac.uk/interpro/) to identify domains, and Smart software (http://smart.embl-heidelberg.de/) to predict conserved motifs. A phylogenetic tree was constructed using a neighbor-joining method implemented in MEGA 6.0 software(https://www.megasoftware.net/) after alignment with other ELP4 protein sequences using Clustal IW software (https://www.genome.jp/tools-bin/clustalw).

2.4. Functional assay of TaELP4-mediated defense against R.cerealis in wheat

Barley stripe mosaic virus (BSMV)-mediated VIGS has been successfully utilized to study gene function in barley and wheat [7,35,36]. To generate a BSMV:TaELP4 recombinant construct, a fragment 272 bp long matching the coding sequence of TaELP4-7A, and TaELP4-7D (nucleotides 414 to 686 in the TaELP4-7B sequence), was subcloned in antisense orientation into the Nhe I restriction site of the RNA γ of BSMV(Fig.S1).Following Holzberg et al.[35]and Zhou et al.[36],the tripartite cDNA chains of BSMV:TaELP4 or the control BSMV:GFP virus genomes were separately transcribed into RNAs,mixed, and used to infect CI12633 seedlings at the three-leaf stage.At 15 days after virus infection,the fourth leaves of the inoculated seedlings were collected to monitor BSMV infection and to evaluate the transcript change of TaELP4. At～20 days after BSMV infection, the BSMV-infected CI12633 plants were inoculated with R. cerealis isolate WK207.

In each batch,10-20 plants of TaELP4-silenced wheat or the control BSMV:GFP-infected wheat were inoculated with small toothpicks (about 3 cm) harboring well-developed mycelia of R. cerealis WK207. At 10 and 40 dpi with R. cerealis, infection types (ITs) of these wheat plants were scored from 0 to 5 as previously described [37]. At 40 dpi with R. cerealis, sharp eyespot disease indexes of TaELP4-silenced or control BSMV:GFP-infected wheat lines were scored.

2.5. Generation and defense test of Arabidopsis ectopically expressing TaELP4-7B

The full-length coding sequence of TaELP4-7B was amplified and used to generate the p35S:TaELP4-7B-GFP vector. The resulting p35S:TaELP4-7B-GFP DNA was then introduced into Arabidopsis Col-0 plants by Agrobacterium-mediated transformation [38]. Positive transformants were selected based on their resistance to hygromycin and by PCR detection. Homozygous T3transgenic seedlings were selected through RT-PCR and western blotting, and used to investigate the defense response to B. cinerea. B. cinerea inoculation and lesion size measurement followed Pre et al.[39]and Zhang et al.[40].

2.6.Chromatin immunoprecipitation with acetylated histone 3 lysine 9/14, and qPCR

Chromatin immunoprecipitation (ChIP) was performed following Gendrel et al. [41]. Briefly, ChIP was performed with acetylated histone H3K9/K14 (histone H3 acetylation at lysines 9 and 14) an antibody (Santa Cruz Biotechnology), the immune complex was collected on Dynabeads protein G(Invitrogen, USA) and DNA fragments were recovered by the phenol-chloroform method [41]. The amount of precipitated DNA corresponding to a specific gene region was determined by qPCR and normalized by both input DNA and a constitutively expressed gene (TaActin or AtActin) as described by Mosher et al. [42,43]. The resulting values represented the levels of histone H3K9/14ac in specific gene regions.

3. Results

3.1. Identification and sequence characterization of TaELP4 in wheat

By comparative analysis of RNA-seq data from three resistant and three susceptible lines of RILs from the cross Shanhongmai × Wenmai 6 inoculated with R. cerealis WK207,the gene sequence with ID TraesCS7B02G439900 was identified as being significantly upregulated in the resistant relative to the susceptible RILs following mock inoculation and 4 and 10 dpi with R. cerealis. RNA-seq showed 4.1-, 5.6-, and 2.1-fold transcriptional increases in resistant RILs relative to susceptible RILs following mock inoculation and 4 and 10 dpi (Table S2). BlastP (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and sequence analyses indicated that TraesCS7B02G439900 was homologous to the Arabidopsis Elongator subunit 4 (The Arabidopsis Information Resource accession no. AT3G11220),encoded an Elongator subunit 4, and was located on wheat chromosome 7B. The gene TraesCS7B02G439900 was accordingly designated as TaELP4-7B. Phylogenetic tree construction using TaELP4 and ELP4 proteins from various plant species indicated that all the ELP4s of monocots clustered on the same branch, including Triticum aestivum ELP4, TaELP4-7B(A0A1D6BP38),TaELP4-7A(A0A1D6BP38),TaELP4-7D(A0A1D6CV37), Aegilops tauschii ELP4 (M8C139), Hordeum vulgare subsp. vulgare ELP4 (A0A287XX36), Brachypodium distachyon ELP4 (I1GVD6), Setaria italica ELP4 (K4ABE7), Oryza sativa subsp. japonica ELP4 (Q67WD2), Zea mays ELP4(A0A1D6P418), and Sorghum bicolor ELP4 (C5Z6M5). TaELP4-7B protein was closely related to Aegilops tauschii ELP4 (M8C139,with 76.6% identity). All the ELP4s of dicots clustered into another branch, including Solanum lycopersicum ELP4(K4D5Q3), Corchorus capsularis ELP4 (A0A1R3JJG1), Medicago truncatula ELP4 (A0A072TKF7), Arabidopsis thaliana ELP4(Q9C778), Brassica oleracea ELP4 (A0A0D3CL87), and Brassica rapa subsp. pekinensis ELP4 (M4F1B2) (Fig. 1-A). The full deduced protein sequence of TaELP4-7B shared 56.10% identity with that of A. thaliana ELP4 (Q9C778). These results indicated that TraesCS7B02G439900 encodes TaELP4 and that distinct alterations in ELP4 proteins occurred during monocotdicot divergence.

Fig.1-Phylogenetic tree and conserved-domain and gene-structure analysis of TaELP4-7B.(A)A phylogenetic tree of TaELP4-7B and 16 other ELP4 members from 8 monocots and 6 dicots.The bootstrapped phylogenetic tree was constructed using the neighbor-joining method(MEGA 6.0).The red circle indicates the position of TaELP4-7B. (B)Schematic diagram of the TaELP4 protein,Red box means RecA-ATPase-like domain(49-112 aa). (C)Gene structure of TaELP4-7B,White boxes indicate UTRs,black boxes exons,and black lines introns.

The full cDNA sequence of TaELP4-7B was cloned from stem cDNA of the resistant wheat cv. CI12633. The cloned cDNA sequence contained an open reading frame (ORF) with 1155 bp length,a 5′-untranslated region(UTR)with 61 bp,and a 3′-UTR with 234 bp(Fig.S2).The predicted TaELP4-7B protein consisted of 385 amino acid(aa)residues with a predicted Mw of 41.22 kDa and theoretical pI of 8.97. The deduced protein contains a conserved RecA-ATPase-like domain (49-112 aa)that is a characteristic of ELP4 (Fig. 1-B). BLAST search of TaELP4-7B against the public wheat genome database (http://plants.ensembl.org/index.html) revealed two additional homoeologous genes located on chromosomes 7A and 7D,designated as TaELP4-7A (TraesCS7A02G522900) and TaELP4-7D(TraesCS7D02G512100),respectively.TaELP4-7B,TaELP4-7A,and TaELP4-7D all contain 8 exons and 7 introns (Fig. 1-C).Pairwise comparison of these TaELP4 protein sequences showed that TaELP4-7B shared 95.8% and 96.4% identity with TaELP4-7A (385 aa) and TaELP4-7D (384 aa), respectively.These three copies of TaELP4 all contained the conserved RecA-ATPase-like domain, suggesting that these three proteins might have redundant functions.

3.2. TaELP4 transcription is associated with wheat resistance to R.cerealis

Further RT-qPCR results showed that TaELP4 transcriptional levels were significantly greater in resistant lines than in susceptible lines of the RIL population,with respectively 3.22-fold,8.40-fold,and 2.87-fold transcriptional increases in mock and 4 and 10 dpi with R. cerealis (Fig. 2-A), in agreement with the trend in the microarray results. Furthermore, after infection with R. cerealis, TaELP4 transcription was significantly induced in stems of the resistant wheat cv.CI12633 but not in those of the susceptible wheat cv. Wenmai 6 (Fig. 2-B).As shown in Fig. 2-B, the transcription of the gene was elevated 2.03-4.73-fold in CI12633 relative to Wenmai 6 at 2,4,7,10,and 14 dpi with R.cerealis.Notably,TaELP4 transcription was the highest in resistant wheat cv.Shanhongmai,and the lowest in the highly susceptible cv. Yangmai 9 (Fig. 2-C),suggesting that the transcriptional abundance of TaELP4 corresponded to the degree of resistance of the wheat cultivars. Tissue expression analysis showed that at 4 dpi with R. cerealis, the greatest induction appeared at the stems of CI12633(Fig.2-D),a finding consistent with the occurrence of sharp eyespot symptoms mainly in wheat stems.

Fig.2-TaELP4 responding to Rhizoctonia cerealis in wheat.(A)The transcriptional level of TaELP4 was significantly higher in the resistant lines(RIL-R)of the RIL population than in the susceptible lines(RIL-S)at mock and 4 and 10 dpi with R.cerealis,as measured by RT-qPCR.The expression level of TaELP4 in RIL-S plants at mock was set to 1. (B) Transcription of TaELP4 was higher in the stems of the resistant wheat cultivar CI12633 than in those of the susceptible wheat cultivar Wenmai 6 at 2,4,7,10,and 14 dpi with R.cerealis.The expression level of TaELP4 in Wenmai 6 at 0 was set to 1.(C)Expression patterns of TaELP4 in six wheat cultivars with different resistance degrees at 4 dpi with R. cerealis.DI indicates disease index of sharp eyespot.Transcript abundances with different letters were significantly different from one another based on statistical comparisons at the same time point(D)Expression pattern of TaELP4 in stems,leaves and ears of CI12633 at 4 dpi with R.cerealis or mock.The transcriptional level of TaELP4 in stems with mock treatment was set to 1.TaActin was used as the internal control. t-Test:*P ＜0.05; **P ＜0.01.Bars indicate standard error of the mean.

3.3. TaELP4 is required for wheat immune response to R.cerealis

Fig.3- Silencing of TaELP4 impairs resistance of the resistant wheat cv.CI12633 to R.cerealis.(A)Typical symptom of BSMV in fourth leaves of wheat plants infected by BSMV:GFP or BSMV:TaELP4 at 15 dpi with BSMV and RT-PCR analysis of the transcription of BSMV CP gene.(B)RT-qPCR analysis of the relative transcript levels of TaELP4 in wheat plants infected by BSMV:GFP or BSMV:TaELP4 before and at 10 dpi with R.cerealis.The transcriptional level of TaELP4 in BSMV:GFP-infected wheat CI12633 plants was set to 1.(C)Sharp eyespot symptoms of BSMV:TaELP4-and BSMV:GFP-inoculated CI12633 plants at 10 dpi with R.cerealis.Bar represents 1 cm.(D)RT-qPCR analysis of RcActin gene in stems of TaELP4-silencing and BSMV:GFP-infected wheat CI12633 plants at 10 dpi with R.cereali.RcActin transcription represents the relative biomass of R.cerealis.The expression level of RcActin in BSMV:GFP-infected wheat CI12633 plants was set to 1.(E)Sharp eyespot symptoms of the BSMV:GFP-and BSMV:TaELP4-inoculated CI12633 plants at 40 dpi with R.cerealis.IT indicates sharp eyespot infection type of each wheat plant.(F)Mean infection types of CI12633 plants infected by BSMV:GFP or BSMV:TaELP4 in three independent batches.Significant differences were determined based on three independent replications(t-test:**P ＜0.01).Bars indicate standard error of the mean.

The BSMV-mediated VIGS approach was used to investigate the resistance function of TaELP4 against R. cerealis infection. The recombinant BSMV:TaELP4 construct was generated (Fig. S1).After 15 days post transfection of BSMV-derived RNAs into leaves of CI12633, symptoms of BSMV infection appeared on newly emerged leaves and the transcript of BSMV coat protein(cp)was detected (Fig. 3-A), indicating that BSMV infected these wheat plants. Following use of gene-specific primers that simultaneously detected transcription of TaELP4-7A, TaELP4-7B, and TaELP4-7D, the RT-qPCR results showed that the transcriptional levels of TaELP4 were substantially decreased in BSMV:TaELP4-infected CI12633 plants compared to BSMV:GFP-infected CI12633 plants(Fig.3-B),indicating that TaELP4 was successfully silenced in BSMV:TaELP4-infected CI12633 plants (hereafter TaELP4-silenced CI12633 plants). These plants were then inoculated with R.cerealis isolate WK207.At 10 dpi with R.cerealis,the typical symptoms of sharp eyespot appeared on the sheaths of TaELP4-silenced CI12633 plants,but smaller lesions were present on the sheaths of BSMV:GFP-treated CI12633 plants (Fig. 3-C). At the same time, the biomass of R. cerealis (represented by its RcActin transcription) in TaELP4-silenced CI12633 plants was higher (by＞10-fold) than in BSMV:GFP-infected CI12633 plants (Fig. 3-D).Moreover,at 40 dpi with the pathogen,compared to BSMV:GFPinfected CI12633 plants, larger necrotic areas and significantly higher disease severity of sharp eyespot appeared on the stems of TaELP4-silenced CI12633 plants (Fig. 3-E). Based on three repetitions of VIGS and disease scoring in two years,the average ITs of TaELP4-silenced CI12633 plants were 3.18 to 3.94 and their sharp eyespot disease indexes were 63.61 to 78.82, respectively,whereas the average ITs and disease indexes of BSMV:GFPtreated CI12633 plants were 1.44 to 2.31 and 28.88 to 46.2,respectively (Fig. 3-F). These results clearly indicated that silencing of TaELP4 in resistant CI12633 compromised host resistance to sharp eyespot and suggested that functional TaELP4 expression was required for wheat immune responses to R.cerealis infection.

3.4. Silencing of TaELP4 represses transcription of defenseassociated genes in wheat

The RT-qPCR results showed that the transcription levels of TaAGC1,TaCPK7-D,TaPAL5,and Defensin,being involved in wheat resistance responses to infection by R. cerealis [7,37,44], were significantly lower in TaELP4-silenced wheat plants than in BSMV:GFP-infected(control)plants without R.cerealis inoculation(Fig. 4), suggesting that the transcription of these defenseassociated genes might be activated by TaELP4.At 10 dpi with R.cerealis, the transcriptional levels of TaAGC1, TaCPK7-D, TaPAL5,Defensin,Chitinase2,and TaELP2 were significantly downregulated in the TaELP4-silenced wheat plants in comparison with the BSMV:GFP-infected wheat plants (Fig. 4). These results suggest that the transcriptions of these defense-associated genes upregulated by TaELP4 is associated with the resistance response of wheat to R.cerealis and that the expression of TaELP4 is required for full activation of their expression during wheat resistance response to R.cerealis infection.

3.5.Silencing of TaELP4 reduces histone acetylation of defenseassociated genes in wheat

The acetylation level of histone H3 is generally associated with active transcription[29,30,45-47].To test whether TaELP4 regulates histone acetylation levels in defense genes, a combination assay of ChIP using histone H3K9/14ac (histone H3 acetylation at lysines 9 and 14) antibody and qPCR was employed to investigate histone H3 acetylation in regions of the defense-associated genes TaAGC1, TaCPK7-D, TaPAL5,Defensin, Chitinase2, and TaELP2 in TaELP4-silenced and BSMV:GFP-treated wheat leaf tissues. The results showed that histone H3K9/14ac levels in coding regions of TaAGC1,TaCPK7-D, TaPAL5, Chitinase2, and TaELP2 were significantly lower in TaELP4-silenced plants than in BSMV:GFP-infected plants, and that histone H3K9/14ac levels in partial promoter regions of Defensin, Chitinase2, and TaELP2 were significantly lower in TaELP4-silenced plants than in BSMV:GFP-infected plants (Fig. 5). These findings indicated that silencing of TaELP4 reduced histone acetylation levels in coding regions or promoters of a subset of defense-associated genes in wheat,in agreement with the transcriptional differences in these defense-associated genes between BSMV:TaELP4-infected wheat and BSMV:GFP wheat. Thus, histone H3 acetylation levels in coding regions or promoters of these defenseassociated genes were positively associated with expression of the genes.

3.6. Ectopic expression of TaELP4 facilitates immune responses in Arabidopsis

To investigate whether TaELP4 regulates expression of defense-related genes by modulating histone acetylation and plant immunity in A. thaliana, we generated transgenic Arabidopsis lines ectopically expressing TaELP4-7B.Two representative TaELP4-overexpressing transgenic lines (OE1 and OE2) in the T3generation were selected (Fig. S3). RT-qPCR results showed that the transcriptional levels of defenseassociated genes,including AtPAL1,AtPAL2,AtPAL3,AtPDF1.2,and Chitinase, were greater in OE1 and OE2 plants than in the wild-type (WT) plants (Fig. 6-A), suggesting that TaELP4 upregulates the expression of all of the tested genes in Arabidopsis. The transcriptional levels of all five Elongator subunits but AtELP3 were elevated in OE1 and OE2 plants compared with WT plants (Fig. 6-B), suggesting that the ectopic expression of TaELP4 can induce the expression of other Elongator subunits in Arabidopsis. Histone H3 acetylation levels in coding regions and promoter regions of AtPDF1.2, Chitinase, and AtELP2 were significantly higher in OE1 and OE2 than in WT plants(Fig.7-A).In comparison with WT plants,TaELP4-overexpressing Arabidopsis lines displayed increased resistance to B. cinerea (Fig. 7-B). Taken together,these results suggest that TaELP4 facilitates histone acetylation and the transcriptional upregulation of both defenseassociated and ELP2 genes and thereby upregulates plant immune response.

4. Discussion

RNA-seq-based transcriptome analysis revealed that TaELP4 was upregulated in resistant lines. Transcription of TaELP4 was associated with wheat resistance degree and induced after infection of R.cerealis,and transcriptional induction was marked in stems of the resistant wheat cv. CI12633. These results suggest that TaELP4 participates in wheat immune responses to R. cerealis infection. Further, three repetitions of VIGS and sharp eyespot severity scoring results indicated that silencing of TaELP4 compromised wheat resistance to R. cerealis infection,suggesting that TaELP4,acting as a functional regulator,is required for immune response of wheat to R.cerealis infection.In Arabidopsis, a loss-of-function mutant in AtELP4 is the most disease-susceptible; whereas ectopic expression of AtELP4 increased resistance of transgenic F. vesca and tomato plants to multiple pathogens[31,32].In the present study,compared with WT plants, TaELP4-overexpressing transgenic Arabidopsis lines showed increased resistance to B.cinerea,suggesting that TaELP4 upregulated innate immune response. These findings support the previous reports that ectopic expression of the single Elongator subunit TaELP4 can boost the resistance of transgenic plants, and reveal defense function of TaELP4 in dicots and monocots.

Fig.4-Transcription levels of defense-associated genes in TaELP4-silenced and BSMV:GFP-infected wheat plants without and 10 dpi with R.cerealis.The transcriptional levels of TaAGC1(A),TaCPK7-D(B),TaPAL5(C),Defensin(D),Chitinase2(E)and TaELP2(F)in the TaELP4-silenced plants are relative to those of BSMV:GFP-infected control plants.The transcription level in BSMV:GFP without R.cerealis inoculation was set to 1.Statistically significant differences were determined based on three independent replications (t-test:*P ＜0.05;**P ＜0.01).Bars indicate standard error of the mean;TaActin was used as internal control.

In Arabidopsis,a loss-of-function mutation in AtELP2 alters B. cinerea-induced transcriptome reprogramming and compromises resistance to the pathogen infection [12,29]. In previous studies [7,37,44] in our laboratory, TaAGC1, TaCPK7-D,TaPAL5,Defensin,and Chitinase2 amplified wheat resistance response to R. cerealis infection. In the present study, the transcription levels of a subset of defense-associated genes,namely TaAGC1, TaCPK7-D, TaPAL5, Defensin, and Chitinase2,were significantly decreased in TaELP4-silenced wheat plants without and with R. cerealis inoculation, suggesting that TaELP4 expression is required for elevating the transcription of these genes during the wheat immune response to R.cerealis infection. Furthermore, the transcriptional levels of a series of defense-associated genes, AtPAL1, AtPAL2, AtPAL3,AtPDF1.2,and Chitinase,were higher in TaELP4-overexpressing Arabidopsis lines than in WT Arabidopsis plants.These findings suggest that TaELP4 triggers transcriptome reprogramming and is required for activating the expression of a subset of defense genes,and thereby increases both wheat resistance to R. cerealis and the Arabidopsis immune response to B. cinerea.They agree with previous reports [12,29] that Arabidopsis AtELP2 is required for the expression of B. cinerea-induced defense genes.

Fig.5- Histone H3 acetylation levels of defense-associated genes in TaELP4-silenced and control wheat plants.Histone H3 acetylation levels in TaAGC1(A),TaCPK7-D(B),TaPAL5(C),Defensin(D),Chitinase2(E),and TaELP2(F).The position of the primers is relative to the initiation ATG codon.The relative amount of immunoprecipitated chromatin fragments(as determined by qPCR)from BSMV:TaELP4 was compared with that from BSMV:GFP.The value for the first fragment of BSMB:GFP, which was significantly lower in BSMV:TaELP4 than in BSMV:GFP,was set as 1.Statistically significant differences were identified based on three independent replications(t-test:*P ＜0.05; **P ＜0.01).Bars indicate standard error of the mean.

In eukaryotes, transcription of protein-encoding genes is strongly regulated by post-translational modifications of histones that affect accessibility of DNA by RNAPII [48]. The Elongator complex was originally identified in yeast to be a histone H3 and H4 acetyltransferase important for normal histone acetylation levels, which activates RNAPII [13,14]. In the nucleus, the acetylation of histone H3 is linked to the function of Elongator in transcription [26,47]. AtELP3 has been shown [16,26,28,32] to have histone acetylation activity,which is essential for maintaining normal histone acetylation levels at multiple genetic loci, and to be required for its regulatory roles in plant growth, development, and plant immunity. Recent studies [29,30] revealed that Arabidopsis ELP2 epigenetically regulates plant immune responses,including pathogen-induced transcriptome reprogramming,and that the elp2 mutation reduces basal histone acetylation levels in coding/promoter regions of a series of defense genes. In the present study, silencing of TaELP4 significantly reduced the histone H3K9/14 acetylation levels in the coding and promoter regions of TaAGC1, TaCPK7-D, TaPAL5, Defensin,Chitinase2, and TaELP2 in wheat, and thereby repressed their transcriptional levels. Ectopic expression of TaELP4 in Arabidopsis significantly increased histone H3K9/14 acetylation levels in the coding and promoter regions of defense genes and AtELP2, and thereby activated their transcription.These results accord with the general association[29,30,45,46,48] of histone acetylation with transcriptional activation. To our knowledge, this is the first report of upregulation by TaELP4 of transcription of defense via histone acetylation.

Fig.6- Transcription of defense genes and Elongator subunits in TaELP4-overexpressing and WT Arabidopsis Col-0 lines.(A)Transcriptional levels of AtPAL1,AtPAL2,AtPAL3,AtPDF1.2,and Chitinase in the TaELP4-overexpressing lines and WT Col-0.(B)Transcription of AtELP1-AtELP6 in TaELP4-overexpressing lines and Col-0.Transcription in WT was set to 1. Statistically significant differences were identified based on three independent replications(t-test:**P ＜0.01).Bars indicate standard error of the mean;AtActin was used as internal control.

Fig.7- Histone H3 acetylation levels of defense-associated genes and immune responses to B. cinerea in TaELP4-overexpressing and WT Arabidopsis Col-0 lines.(A)ChIP-qPCR analysis on histone H3 acetylation levels of AtPDF1.2,Chitinase and AtELP2. Position of the primers in tested genes is relative to the ATG initiation codon.The relative amount of chromatin immunoprecipitated fragments(as determined by qPCR)from TaELP4-overexpression lines were compared with that from the WT(set to 1).(B) Photographs were taken 5 days post-inoculation and BcActin/AtActin represents biomass of B. cinerea in WT and TaELP4-overexpressing lines.Transcription in WT was set to 1.Statistically significant differences were identified based on three independent replications (t-test:*P ＜0.05; **P ＜0.01).Bars indicate standard error of the mean;AtActin was used as internal control.

5. Conclusions

The wheat Elongator subunit 4-encoding gene TaELP4 was shown to be required for epigenetic regulation of host innate immune response to infection by R. cerealis.TaELP4 transcription was associated with the level of resistance of wheat and was significantly induced in resistant wheat after R. cerealis infection. TaELP4 upregulates transcriptions of a subset of defense-associated genes by elevating the histone acetylation levels of their chromatin,in turn boosting the innate immune responses of wheat and Arabidopsis to R.cerealis and B.cinerea.Thus, TaELP4 may potentially be used to improve resistance of wheat and other crop plants to R.cerealis.To our knowledge,this is the first report about the function of Elongator in monocot plant species. This study broadens the understanding of Elongator in plant immune responses to diverse pathogens.

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2019.11.005.

Declaration of competing interest

Authors declare that there are no conflicts of interest.

Acknowledgments

This work was supported by the programs of the Chinese Key Research & Development Plan Project (2016YFD0101004) and the National Transgenic Research Program of China(2016ZX08002-001-004). The authors thank Profs. Jizeng Jia,Jinfen Yu, and Bingyan Xie for kindly providing the wheat RILs and the fungal materials.