Deshui Yu,Renchun Fn,Ling Zhng,Pengy Xue,c,Liing Lio,Meizhen Hu,Ynjun Cheng,Jine Li,c,Ting Qi,Shojun Jing,Qiuyun Wng,Arvind Bhtt,Qin-Hu Shen,c,*

a Lushan Botanical Garden,Chinese Academy of Science,Jiujiang 332900,Jiangxi,China

b State Key Laboratory of Plant Cell and Chromosome Engineering,Institute of Genetics and Developmental Biology,Chinese Academy of Sciences,Innovation Academy for Seed Design,Beijing 100101,China

c CAS Center for Excellence in Biotic Interactions,University of Chinese Academy of Sciences,Beijing 100049,China

Keywords:HvWRKY2 Basal immunity Chromatin immunoprecipitation(ChIP)HvCEBiP

ABSTRACT Plants use a sophisticated immune system to perceive pathogen infection and activate immune responses in a tightly controlled manner.In barley,HvWRKY2 acts as a repressor in barley disease resistance to the powdery mildew fungus,Blumeria graminis f.sp.hordei(Bgh).However,the molecular features of HvWRKY2 in its DNA-binding and repressor functions,as well as its target genes,are uncharacterized.We show that the W-box binding of HvWRKY2 requires an intact WRKY domain and an upstream sequence of～75 amino acids,and the HvWRKY2 W-box binding activity is linked to its repressor function in disease resistance.Chromatin immunoprecipitation(ChIP)-seq analysis identified HvCEBiP,a putative chitin receptor gene,as a target gene of HvWRKY2 in overexpressing transgenic barley plants.ChIP-qPCR and Electrophoretic Mobility Shift Assay(EMSA)verified the direct binding of HvWRKY2 to a W-boxcontaining sequence in the HvCEBiP promoter.HvCEBiP positively regulates resistance against Bgh in barley.Our findings suggest that HvWRKY2 represses barley basal immunity by directly targeting pathogen-associated molecular pattern(PAMP)recognition receptor genes,suggesting that HvCEBiP and likely chitin signaling function in barley PAMP-triggered immune responses to Bgh infection.

1.Introduction

Plants rely on a two-tiered innate immune system to protect them against pathogens[1–3].In this immune system,pathogen or microbe-associated molecular patterns(PAMPs or MAMPs)and damage-associated molecular patterns(DAMPs)are recognized via cell surface-localized pattern-recognition receptors(PRRs)and trigger PAMP-triggered immunity(PTI)[4–7].PRRs belong mainly to two types:receptor-like proteins(RLP)and receptor-like kinases(RLK)[2,4,6].Successful pathogens secrete various effectors that,when delivered into host plant cells or the apoplast,disturb or suppress host immunity,thereby promoting pathogen virulence[8].Plants have evolved numerous intracellular nucleotide-binding site/leucine-rich repeat(NLR)receptors to directly or indirectly recognize pathogen effectors and launch effector-triggered immunity(ETI),which is more potent and usually accompanied by localized host-cell death(hypersensitive response,HR)[3,9,10].PTI and ETI can converge in signaling pathways and lead to dynamically differing but similar and interdependent immune responses[11–18].

Chitin is a major component of the fungal cell wall,and chitin elicitors serve as PAMPs to trigger immune responses in both plants and animals[19].In plants,chitin oligomers are perceived by a plasma membrane-localized receptor complex composed of multiple lysine motif(LysM)-containing receptor-like kinases(LysM-RLKs)or receptor-like proteins(LysM-RLPs)[20].In rice,the LysM-RLP OsCEBiP is the major high-affinity chitin receptor[21].Chitin elicitation induces homodimerization of OsCEBiP itself and heterodimerization between OsCEBiP and OsCERK1,and OsCERK1 as a LysM receptor-like kinase is indispensable for intracellular chitin signaling in rice[22–25].Barley HvCEBiP,a homolog of OsCEBiP,is involved in barley basal defense against appressorium-mediated infection by Magnaporthe oryzae,and this basal defense is likely triggered by chitin oligosaccharides derived from M.oryzae[26].

WRKY transcription factors(TFs)constitute one of the largest TF families in plants and function in diverse developmental and physiological processes[27–29].WRKY TFs comprise either one or two WRKY domains of about 60 conserved amino acids that serve as the DNA binding domain,which contains a highly conserved WRKYGQK motif followed by a zinc-finger motif of C2H2 or C2HC type[30,31].WRKY TFs are classified into groups I,II,and III and each group is further divided into subgroups according to the numbers of WRKY domains and the zinc-finger structure[31].Previous studies have shown that WRKY TFs bind specifically to the W-box(TTGAC-C/T)DNA sequence in the promoters of target genes[30–36].

Plant WRKY transcription factors play important roles in regulating defense transcription in response to pathogen infection[27,37].The WRKY-IIa subgroup members from Arabidopsis,barley,and rice act mainly as repressors of plant immunity to diverse pathogens[38–45].The Arabidopsis Atwrky18wrky40 double mutant showed increased resistance to Golovinomyces orontii and

Pseudomonas syringae[41,42].Resistance in Atwrky18wrky40

plants is accompanied by massive transcriptional reprogramming and imbalance in salicylic acid(SA)and jasmonic acid(JA)signaling,altered Enhanced Disease Susceptibility1(EDS1)expression,and accumulation of the phytoalexin camalexin[40].AtWRKY40 binds directly to the promoter region of EDS1,JAZ8,and RRTF1(an AP2/ERF-type transcription factor),as demonstrated by chromatin immunoprecipitation(ChIP)[40].Genome-wide binding analysis revealed that AtWRKY18 and AtWRKY40 target more than 1000 gene loci and act as negative regulators of flg22-induced PTI responses[38].In barley,we have shown that HvWRKY1/2/3,homologs of AtWRKY18/40/60,are all repressors of barley basal immunity to powdery mildew fungus[41,46].The Mla locus in barley encodes～30 allelic CC-NB-LRR-type MLA receptors that each confer isolate-specific disease resistance against the Bgh fungal pathogen[41,47,48].Upon activation,MLA receptors interact with HvWRKY1/2 in the nucleus,derepressing and potentiating PAMPtriggered immunity[41].MLA receptors also interact with HvMYB6,a positive regulator of barley immunity,to stimulate the DNA-binding activity of HvMYB6 and increase barley immunity to the Bgh fungus[49].We have recently identified a conserved sucrose non-fermenting-related kinase 1(SnRK1)in barley that specifically targets HvWRKY3 for phosphorylation and promotes its proteasomal degradation to also derepress barley immunity against Bgh[46].It is not yet fully understood how different types of TFs,including negative and positive regulators,dynamically associate with one another to regulate defense gene expressions,and whether they target the same or different genes for immune responses.

In this study,we show that the activity of HvWRKY2 W-box binding requires an intact WRKY domain and the upstream sequence of～75 amino acids,which is associated with its immune repressor function in barley resistance against the Bgh fungus.ChIP-seq analysis using barley transgenic plants overexpressing HvWRKY2 identified a putative chitin receptor gene HvCEBiP as a potential target gene.ChIP-qPCR and EMSA further showed that HvWRKY2 binds directly to the W-box fragment in the promoter of HvCEBiP.Functional analyses showed that HvCEBiP positively regulates resistance against Bgh in barley.Our findings suggest that HvWRKY2 represses barley basal immunity by directly targeting PAMP-recognition receptor genes,indicating that HvCEBiP is part of PTI signaling in barley immunity to the Bgh fungus.

2.Materials and methods

2.1.Primers and bacterial strains

All primers used in this study were synthesized by Invitrogen,Life Technologies(Beijing,China)and are listed in Table S1.Escherichia coli strains Trans5a(for normal vectors)and TransDB3.1(for constructs harboring the ccdB gene)competent cells were purchased from TransGen Biotech(Beijing,China).

2.2.HvWRKY2-3×HA overexpressing transgenic barley plant generation and analysis

The HvWRKY2-3×HA overexpression vector was generated using overhanging primers(Yu01:Yu03,Yu01:Yu04,Yu01:Yu05,and Yu01:Yu06)in four cycles of PCR,fusing the 3×HA epitopetag codon sequence and attB recombination sites with HvWRKY2,and then cloning it into pWBvec8+Ga-b(with the pUbi promoter)using Gateway technique(Invitrogen).HvWRKY2-3×HA overexpressing transgenic plants were generated by Agrobacteriummediated barley transformation following a previously developed protocol[50],using immature embryos of Golden Promise(GP)barley.

Southern blotting was used to detect the insert gene copy number in genomic DNA from transgenic barley plants.About 5 g of barley leaves from each sample were harvested for isolation of genomic DNA,which was digested with HindIII or XhoI(NEB).The digested DNA was then separated by electrophoresis in 0.8%(w/v)agarose gel,transferred onto a positively charged nylon membrane,and kept in an oven at 80 °C for 2 h to crosslink DNA to the membrane.The membrane was then hybridized with probes.p32-dCTP labeled probes were prepared using a Prime-a-Gene Labeling System(Promega Catalog number:U1100)with a hygromycin resistance gene in plasmid pWBvec8+Ga-b used as a template,and following the product manual.The hybridized probe DNA was exposed to a phosphor screen and the images were developed by Typhoon trio(GE Healthcare).

Western blotting was performed for HvWRKY2-3×HA fusion protein accumulation analysis in barley transgenic plants.Leaf samples were collected 4 h post infection(hpi)with powdery mildew fungus BghA6,from one-week old plants or from mockinfected plants.Total protein extraction and Western blotting were performed as in our previous study[51].

Microcolony formation rate analyses of transgenic GP lines overexpressing HvWRKY2-3×HA were performed as in previous studies[46,52].

2.3.Single-cell transient gene expression assay

Expression vectors used in single-cell transient gene expression assays were constructed as in previous studies[47,49],with the exception of the HvWRKY2-3×HA fusion gene expression vector.HvWRKY2-3×HA fusion gene transient expression used the same plasmid as that used in the HvWRKY2-3×HA overexpression stable transformation experiment.Single-cell transient gene expression assays were performed as previously described[46,47,49,53,54].A plasmid containing a β-glucuronidase(GUS)reporter driven by the ubiquitin promoter was co-expressed with tested gene expression plasmids at a molar ratio of 1:1 and then delivered into barley leaf epidermal cells by particle bombardment(Bio-Rad,Model PDS-1000/He).The leaves were infected with the powdery mildew fungus BghA6 4 h after bombardment.Transformed cells were stained with GUS staining solution,and the fungal haustorium index was scored under a microscope 48 h after inoculation with fungal spores.Each assay was repeated three times.Transiently induced gene silencing(TIGS)assay performed as previously described[49].

2.4.Yeast one-hybrid assay

First,a 3×W-box or 3×mW-box was inserted into yeast vector pHisi-1(using site-directed mutagenesis as in the previous study[55],to construct pHisi-1–3×W-box(primers Yu13:Yu14)and pHisi-1–3×mW-box(primers Yu15:Yu16),followed by linearizing and recombinant into the yeast strain YM4271 genome,generating two yeast strains:YM4271:3×W-box and YM4271:3×mW-box.The full-length cDNA of HvWRKY1 was subcloned into the yeast expression vector pGADT7 at EcoR I and HindIII cut sites,and HvWRKY2 and the HvWRKY2 fragments were subcloned into the yeast expression vector pGADT7 at EcoR I and Xho I sites.Yeast expression vectors pGADT7-HvWRKY2Q193Kand pGADT7-HvWRKY2ΔWRKYwere generated based on pGADT7-HvWRKY2 using site-directed mutagenesis with primers Yu19:Yu20/Yu21:Yu22,and then transformed into the two yeast strains YM4271:3×W-box and YM4271:3×mW-box,respectively.The transformed yeast was grown on SD/L-H-medium at 30 °C for 5 days.Binding activity was tested by growing the positive yeast transformants on SD/L-H-medium containing 60 mmol L-13-amino-1,2,4-triazole(3AT)as described previously[55].

2.5.ChIP assays

Chromatin immunoprecipitation assays were performed using HvWRKY2-3×HA overexpressing transgenic plants following the protocol described previously[56].The precipitated DNA fragments were used to generate Illumina sequencing libraries following the manufacturer’s instructions.Sequencing and bioinformatic analysis were performed by the DNA Sequencing and Bioinformatics Platform of Beijing Institute of Genomics,Chinese Academy of Sciences.ChIP-qPCR was performed according to a previously published article[57],using the ABI Step-One Real-Time PCR system,with the Actin promoter used as control.

2.6.Electrophoretic mobility shift assays

EMSAs were performed to detect the binding activity of HvWRKY2 to the CEBiP promoter.The GST-HvWRKY2 fusions genes were cloned into pGEX-4T-1 and the fusion protein expressed in E.coli(BL21)and then purified according to the manual(GST gene Fusion System Handbook,Cytiva/GE).A pair of oligonucleotides,the CEBiP promoter region harboring the W-box motif,and that mutated in the W-box,were synthesized and labeled with biotin at the 3′end(or left without biotin label).Gel-shift assays were performed as previously described[58].

3.Results

3.1.W-box binding of HvWRKY2 requires an intact WRKY domain

Plant WRKY transcription factors regulate transcription by binding the W-box(TTGACC/T)cis-element in the promoter of the target genes[31,36,59].To test the W-box binding activity of HvWRKY1 and HvWRKY2,we performed a yeast one-hybrid assay.cDNA of HvWRKY1 and HvWRKY2 was subcloned into the pGADT7 vector and fused in frame to an activation domain(AD),and the plasmids were transformed into yeast strain YM4271:3×W-box or YM4271:3×mW-box carrying a plasmid to express a HIS3 gene driven by a promoter sequence containing the 3×W-box or 3×mW-box,respectively(Fig.1A,top).As shown in Fig.1A,when pGADT7-HvWRKY1(AD-WRKY1)or pGADT7-HvWRKY2(AD-WRKY2)was transformed into the yeast strain YM4271:3×W-box,yeast cells grew well on medium lacking histidine but supplemented with 60 mmol L-1of 3-AT(3-amino-1,2,4-triazole,a competitive inhibitor of HIS3 gene expression)(Fig.1A,upper half),whereas yeast strain YM4271:3×mW-box carrying the mutated W-box in the HIS3 gene promoter was unable to grow(Fig.1A,lower half).Yeast cells transformed with the pGADT7 empty vector(EV)were unable to grow.This result confirmed that HvWRKY1 and HvWRKY2 bound specifically to the wild-type,but not the mutated,W-box ciselement.

To further identify sequences in HvWRKY2 that contribute to W-box binding or interaction,a set of constructs were generated with pGADT7 to express fusions of HvWRKY2 full-length or fragments or mutants for yeast one-hybrid analysis(Fig.1B,left).The plasmid expressing fusion of wildtype HvWRKY2(1–319)enabled yeast to grow on SD media,as expected.The C-terminus deleted fragment HvWRKY2(1–242),including the WRKY domain(182–242,60 aa),still enabled yeast growth,although weaker growth than the full-length protein(Fig.1B,second panel).However,both the N-terminal half 1–175 fragment(without a WRKY domain)and the C-terminal half 176–319 fragments(with WRKY domain)reduced yeast growth(Fig.1B,third and fourth panels),suggesting that the WRKY domain and some N-terminal sequence upstream of the WRKY domain are essential to W-box binding.Indeed,HvWRKY2(107–319)fragment with more sequence up to the nuclear localization signals(NLS)restored yeast growth(Fig.1B,fifth panel),in contrast to the C-terminal half 176–319 fragment,indicating that both WRKY domain and the immediate Nterminal sequence up to the NLS are essential for W-box binding.A mutated full-length HvWRKY2,mHvWRKY2(Q193K),with Gln193 mutated to Lys in the typical WRKY motif WRKYGQK,did not support yeast growth,nor did the WRKY domain-deleted mutant,ΔHvWRKY2(Fig.1B,bottom two panels).

These results indicated that barley HvWRKY1 and HvWRKY2 bind to the W-box cis-element,and that HvWRKY2 W-box binding requires an intact WRKY domain and the immediate upstream 75 amino acids with an NLS.

3.2.W-box binding activity of HvWRKY2 is associated with its function in repressing barley immunity

It was demonstrated by use of a single-cell transient gene expression assay that overexpression of HvWRKY2 represses barley immunity against powdery mildew fungus[41].We conducted a similar assay in barley line P01 by overexpressing two HvWRKY2 fragments and a HvWRKY2 mutant.The fragments were the 1–242 and 107–319 fragments,which conferred respectively weaker or similar W-box binding,as well as the Q193K mutant variant,which eliminated W-box binding(Figs.2A,1B).The HvWRKY2-expressing constructs were co-delivered with a GUS reporter into barley leaf epidermal cells by particle bombardment.Following fungal spore inoculation of a compatible isolate BghA6,haustorium formation rate was scored in transformed cells as haustorium index(HI%),which represents the susceptibility level[60].Overexpression of HvWRKY2 led to almost doubled HI%:80%,as compared to～40%for the EV control(Fig.2A,1–2 column).Overexpression of HvWRKY2(1–242)and HvWRKY2(107–319)also significantly increased HI%,to～60% and～70%,respectively(Fig.2A,3–4 column).It appeared that the W-box binding activity of the fragments was associated with the immune-suppressing activity.Overexpression of the HvWRKY2Q193Kmutant with fully eliminated W-box binding activity had no effect on fungal HI%,similar to that of the empty vector(Fig.2A,columns 1 and 5).These data indicate that W-box binding of HvWRKY2 is associated with its function in repressing barley basal immunity to the Bgh fungus,and that an intact WRKY domain is essential for the immune suppression function.

3.3.HvWRKY2 suppresses barley immunity in stable transgenic plants

Fig.1.HvWRKY2 binds DNA with W-box element.(A)Yeast one-hybrid assay showing that barley HvWRKY1 and HvWRKY2 binds the 3× W -box DNA fragment.Three Wboxes and the mutated W-boxes are shown at top and mutated nucleotides are highlighted in red.Yeast strain of YM4271 containing 3×W-box was used and grown on SD media as indicated.(B)Yeast one-hybrid assay using HvWRKY2 full-length,N-or C-terminal deleted fragments,and mutated HvWRKY2 as effectors.pGADT7 construct expressing different HvWRKY2 fragments was transformed into YM4271:3×W-box yeast cells.A single transformant colony was inoculated in SD/L-H-liquid culture for 24 h and then dropping transferred on SD/L-H-plates supplemented with 60 mmol L-1 3-AT after dilution to OD600=0.5(1×),and further 10×and 100×dilution.

In order to identify HvWRKY2 target genes in barley,we generated barley transgenic plants overexpressing HvWRKY2-3×HA fusion under the control of the maize ubiquitin promoter by Agrobacterium-mediated transformation.We first investigated the function of the HvWRKY2-3×HA fusion by single-cell transient gene expression assay.Transient overexpression of HvWRKY2-3×HA led to significantly increased HI% in leaf epidermal cells of barley P01 as compared to EV control,and similar to the wild type HvWRKY2(Fig.2B),indicating that the HvWRKY2-3×HA fusion retained full function in repressing barley immunity.We further generated barley stable transgenic lines overexpressing the Hv-WRKY2-3×HA fusion by Agrobacterium-mediated transformation,and obtained two independent transgenic lines:OE1(OE1-HvWRKY2-3×HA)and OE2(OE2-HvWRKY2-3×HA),each of them with a single-copy insertion,verified by Southern blotting(Fig.S1A).Highly increased accumulation of HvWRKY2 transcripts was verified by qRT-PCR in two overexpressing transgenic lines as compared to the GP recipient(Fig.S1B).Fusion protein accumulation in healthy and Bgh inoculated transgenic lines was detected by Western blotting analysis(Fig.S1C).The two transgenic lines OE1 and OE2 were inoculated with the compatible isolate BghA6,and the frequency of fungal microcolonies(microcolony index,MI%)was scored at 48 h post infection.We observed significantly increased MI% by respectively～20% and～30% in the OE1 and OE2 lines,in comparison with the GP recipient(Fig.2C),in agreement with the results from the transient gene expression analysis(Fig.2B).The transgenic barley plants overexpressing HvWRKY2-3×HA fusion also provide materials for further identification of potential HvWRKY2 targets in vivo.

3.4.HvWRKY2 bound directly to the promoter of the barley HvCEBiP gene

We performed ChIP-seq to identify genome-wide binding sites of HvWRKY2 in barley using OE1 transgenic plants.Leaf materials of OE1 BghA6 infected plants were collected at 4 hpi for ChIP assay,a time point when the endogenous HvWRKY2 gene was induced by Bgh infection and reached its peak point,determined by a timecourse qRT-PCR analysis in compatible GP and BghA6 interaction(Fig.S2).Analysis of the ChIP-seq data indicated an enrichment of the promoter fragments of a barley chitin elicitor-binding protein(CEBiP)[26](Fig.3A,left,top panel),suggesting that HvWRKY2 might bind to the promoter of the HvCEBiP gene.We amplified a 2 kb upstream regulatory sequence of HvCEBiP from GP(1 kb upstream sequence of HvCEBiP is shown in Fig.S3),which contained two W-box/W-box-like elements at the sites of HvWRKY2-3×HA enriched positions(Fig.3A,left,bottom panel),suggesting the possibility that WRKY2 binds the promoter region of HvCEBiP.Indeed,ChIP-qPCR analysis resulted in a clear enrichment of a Wbox-containing fragment in the HvCEBiP promoter by～30-fold,as compared to the input(Fig.3A,right).

Fig.2.HvWRKY2 suppresses barley immunity to the Bgh fungus.(A)Single-cell transient gene expression analysis of HvWRKY2 fragments and mutants.Barley leaves of P01 were bombarded with gold particles coated with DNA plasmids to express indicated HvWRKY2 proteins.Relative susceptibility is shown by fungal haustorium index in leaf epidermal cells coexpressing a plasmid vector and a GUS reporter after inoculation with spores of the compatible B.graminis isolate BghA6.Fungal haustoria were microscopically scored at 48 h after inoculation.At least 50 GUS-expressing cells were examined in one experiment,and the values shown are means of three independent experiments.Student’s t-tests were performed to calculate statistical significance(*,P＜0.05;**,P＜0.01).(B)Functional analysis of HvWRKY2-3×HA fusion in transient expression assay.Experiments were performed as above(A).(C)Functional analysis of HvWRKY2 fusion in stable transgenic barley plants.Bgh microcolony index(MI%)was compared between a Golden Promise(GP)recipient and two transgenic lines expressing HvWRKY2-3×HA fusion(OE1 and OE2).Mean values of MI% were microscopically scored for at least 600 interaction sites after inoculation with BghA6 at 48 h.Values shown are from three independent experiments.Student’s t tests were performed to calculate statistical significance(**,P＜0.01).

We further performed EMSA to verify HvWRKY2 binding to this fragment.Recombinant protein GST-HvWRKY2 was obtained from E.coli and incubated with a biotin-labeled W-box-containing fragment derived from the CEBiP promoter(the probe).Indeed,GSTHvWRKY2 formed a DNA–protein complex when the wildtype probe was used in the incubation(Fig.3B,lane 1–2),and this signal of DNA–protein complex was eliminated by an unlabeled wildtype sequence as a competitor,but not by the mutated W-box sequences as a competitor(mCompetitor)(Fig.3B,lane 3–4).These findings confirmed that HvWRKY2 directly binds to the W-box containing region derived from the HvCEBiP promoter.

Together,these results suggested that HvWRKY2 binds directly to the promoter region of the barley HvCEBiP.

3.5.HvCEBiP positively regulates barley immunity against the Bgh fungus

HvCEBiP was shown to positively regulate basal immunity against Magnaporthe oryzae in barley[26].We investigated the potential function of HvCEBiP in barley immunity to powdery mildew using a single-cell transient gene expression assay.Overexpression of HvCEBiP markedly reduced fungal HI% to～30% in a compatible interaction,as compared to HI%of～40%for the EV control(Fig.4A).Moreover,we employed a transiently-induced gene silencing(TIGS)technique[61]to silence the HvCEBiP gene in barley leaf epidermal cells.Delivery of the TIGS-HvCEBiP vector to barley leaf cells led to a more than doubled fungal HI% upon inoculation with the compatible isolate BghA6,in comparison with the EV control(Fig.4B).

Taken together,these findings indicated that HvCEBiP positively regulates immunity against Bgh fungus in barley.

4.Discussion

Plants cope with various pathogen attacks using a complex immune system that is tightly controlled at transcriptional and posttranscriptional levels for transcriptional reprogramming[37,54,62–64].WRKY TFs play a key role in plant immune regulation[27,37].Previously,we have identified three WRKYs TFs,HvWRKY1/2/3,and a R2R3 MYB transcription factor HvMYB6 that play important roles in suppressing or increasing barley immunity against the powdery mildew fungus[41,46,49].Our findings[41,49]also revealed that barley MLA immune receptors trigger ETI upon Bgh infection and that this involves MLA activation and interaction with HvWRKY1/2 and HvMYB6,but not HvWRKY3,in the nucleus[41,49].However,how these transcription factors regulate barley immunity and what the target genes are remain largely uncharacterized.In the present study,we conducted structure and functional analysis of HvWRKY2,and attempted to identify potential target genes of HvWRKY2.Our results reveal that the WRKY domain as well as some extra upstream sequence are essential for HvWRKY2 W-box binding.We further identified HvCEBiP,a putative chitin receptor gene in barley,as a target gene of HvWRKY2.HvCEBiP acts as a positive regulator in barley immunity against Bgh fungus.Our findings suggest that HvWRKY2 binds the promoter region of a potential chitin-receptor to repress barley immunity,most likely to avoid unspecific immune gene expression and defense activation that is harmful for plant growth.The finding that HvWRKY2 expression is induced at very early stages of Bgh infection is in accord with this notion(Fig.S2)[41].

Fig.3.HvWRKY2 binds to the promoter region of HvCEBiP.(A)HvWRKY2 binds the promoter of barley CEBiP.The promoter region of the barley CEBiP gene and the binding sites of HvWRKY2 are shown schematically at top,with the specific sequence containing a W-box(TTGACC)and a W-box like element(TTGACG)at the indicated position highlighted below(left).Data were obtained and analyzed from ChIP-seq experiments showing that HvWRKY2 binding sites are highly enriched around this position in the HvCEBiP promoter(right).For ChIP-qPCR,OE1 leaves were inoculated with BghA6 for 4 hpi.Input DNA before immunoprecipitation(Input)and coimmunoprecipitated DNA using an anti-HA(ChIP)were analyzed by qPCR employing gene-specific primer pairs and are expressed as fold enrichment relative to a promoter fragment of HvActin.(B)EMSA confirmed that HvWRKY2 binds a W-box-containing promoter sequence of HvCEBiP.The probe and competitor probes containing W-box or mutated W-box are shown at top.GST-HvWRKY2 fusion proteins were expressed and purified from E.coli and incubated with various DNA fragments.Lane 2 GST-HvWRKY2 fusion proteins incubated with biotin-labeled oligo fragments of HvCEBiP promoter,Lanes 3 and 4 illustrate competition in the presence of unlabeled HvCEBiP promoter fragments and fragments mutated in W-box/W-box-like elements.

Most reported WRKY TFs regulate gene expression by binding to the W-box in the promoters of target genes.The WRKY domain,containing a highly conserved WRKYGQK core sequence motif,confers binding activity[30–32,35,36,59,65].Alanine replacement of each of the amino acid residues in the WRKYGQK sequence reduces the W-box binding activity[32,65].Three-dimensional structures of the carboxyl-terminal WRKY domains of several Arabidopsis WRKYs have strongly confirmed that the conserved WRKYGQK residues are directly involved in W-box binding[32,66–68].However,previous studies have also shown that the N-terminal WRKY domain of groupⅠWRKY TFs has no W-box binding activity[35,59,65,69],suggesting that the WRKY domain may not be sufficient for W-box binding.Our yeast-one hybrid results verified that both HvWRKY1 and HvWRKY2 bind to the W-box sequence in yeast(Fig.1A).Further deletion and mutation analyses showed that an intact WRKY domain was indispensable for HvWRKY2-W-box binding,given that either deletion of the WRKY domain(HvWRKY2ΔWRKYfragment)or one-amino-acid replacement in the WRKYGQK motif(mHvWRKY2)resulted in loss of W-box binding(Fig.1B).Although the HvWRKY2176-319fragment showed no W-box binding activity,the HvWRKY2107-319fragment restored W-box binding as well as suppression of barley immunity(Figs.1B,2A).Our results confirm that a conserved WRKY domain is essential but not sufficient for in vivo W-box binding of HvWRKY2 and that the immediately upstream 75 amino acids are also essential for W-box binding and for immune suppression in barley.

Pathogen PAMP recognition by plant cell-surface localized PRRs triggers the first immune response to pathogens[1,6,70].Chitin is one of the major components of fungal cell wall and can be hydrolyzed by plant chitinases to release chitin oligomers.Chitin oligomers are recognized as PAMP molecules by plant chitin receptors at the plasma membrane at early stages of fungal infection[20,70,71].In rice,chitin-triggered plant immunity is conferred by two interacting proteins,OsCEBiP and OsCERK1,and intracellular downstream signaling[21,22,71–73].HvCEBiP,as a homolog of OsCEBiP,was reported to contribute to basal immunity against Magnaporthe oryzae[26].Here we have shown that HvCEBiP also positively regulates resistance against the B.graminis fungal pathogen.Given that chitin is a major component of the haustorial cell wall of powdery mildew fungi[74],it is reasonable to speculate that HvCEBiP perceives chitin elicitors derived from the haustorial cell wall and induces defense signaling with the help of co-receptor(s)such as HvCERK1.It is still unclear whether HvCEBiP and HvCERK1 cooperate in conferring disease resistance against fungal pathogen by forming a receptor complex.HvCERK1 confers resistance against Fusarium graminearum in barley,most likely as a receptor for chitin elicitation and/or recognition[75].This example and our findings strongly suggest that barley chitin receptor(s)function in plant perception of chitin elicitors derived from powdery mildew and other fungal pathogens.This suggestion is also supported by the identification of a lytic polysaccharide monooxygenase that is expressed in the haustorium of cucurbit powdery mildew and suppresses chitin-triggered immunity in cucurbits[76].

Fig.4.HvCEBiP positively regulates barley immunity against Bgh.(A)Single-cell transient gene expression showing that barley CEBiP positively regulates basal immunity to Bgh in barley.Transient HvCEBiP overexpression in barley leaves epidermal cells reduced haustoria formation compared with empty vector control(shown by HI% analysis).(B)Transiently induced gene silencing(TIGS)assay in barley epidermal cells showing that HvCEBiP is essential for basal immunity to Bgh.Transiently induced gene silencing of HvCEBiP was achieved by particle bombardment using a silencing construct harboring an antisense fragment of the HvCEBiP gene in barley leaf epidermal cells.Haustorial formation rate(HI%)was scored as in Fig.2 and described in the Methods section.*and**indicate significant differences at P＜0.05 and P＜0.01,respectively.

Fig.5.Putative model of HvWRKY2 suppressing barley immunity to powdery mildew fungus.When powdery mildew fungus infects barley,chitin as a PAMP molecule from the fungus is recognized by barley HvCEBiP,activating PTI.HvWRKY2 acts as a repressor of HvCEBiP-triggered PTI by binding to the W-box element in the promoter region to suppress the transcription of HvCEBiP.

5.Conclusions

We have shown that the W-box binding of HvWRKY2 requires an intact WRKY domain and the upstream sequence of～75 amino acids.We identified HvCEBiP as a potential target gene of HvWRKY2 in barley by ChIP-seq analysis,and confirmed binding of HvWRKY2 to the W-box-containing sequence in the HvCEBiP promoter.HvCEBiP positively regulates resistance against Bgh in barley.Based on these findings,we propose a model in which HvWRKY2 negatively regulates barley basal immunity(PTI)by directly targeting PAMP-recognition receptor genes,such as HvCEBiP,and repressing their expression(Fig.5).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

CRediT authorship contribution statement

Deshui Yu:Visualization,Methodology,Writing–original draft,Writing–review & editing,Funding acquisition,Formal analysis.Renchun Fan:Conceptualization,Formal analysis.Ling Zhang:Visualization,Methodology.Pengya Xue:Visualization,Methodology.Libing Liao:Visualization,Methodology.Meizhen Hu:Visualization,Methodology.Yanjun Cheng:Visualization,Methodology.Jine Li:Visualization,Methodology.Ting Qi:Conceptualization,Formal analysis.Shaojuan Jing:Conceptualization,Formal analysis.Qiuyun Wang:Conceptualization,Formal analysis.Arvind Bhatt:Writing–original draft.Qian-Hua Shen:Conceptualization,Supervision,Visualization,Writing–original draft,Writing–review & editing,Funding acquisition,Formal analysis.

Acknowledgments

This work was supported by National Key Research and Development Program of China(2018YFD1000703,2018YFD1000700),Strategic Priority Research Program of the Chinese Academy of Sciences(XDB11020400),National Program on Research and Development of Transgenic Plants(2016ZX08009-003-001),Startup Fund for Advanced Talents of Lushan Botanical Garden,Chinese Academy of Science(2020ZWZX03 and 2020ZWZX05),and the‘‘Double Hundred and Double Thousand”Talent Project of Jiujiang City(jjsbsq2020026).

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2022.05.010.