Hongzhe Cao,Kang Zhang,Wei Li,Xi Pang,Pengfei Liu,Helong Si,Jinping Zang,Jihong Xinga,,*,Jingao Donga,,*

a State Key Laboratory of North China Crop Improvement and Regulation,Hebei Agricultural University,Baoding 071000,Hebei,China

b Hebei Key Laboratory of Plant Physiology and Molecular Pathology,Hebei Agricultural University,Baoding 071000,Hebei,China

Keywords:Maize Fusarium graminearum ZmMYC7 ZmERF147

ABSTRACT The jasmonic acid(JA)signaling pathway is involved in plant growth,development,and response to abiotic or biotic stresses.MYC2,a bHLH transcription factor,is a regulatory hub in the pathway.The function of ZmMYC7,a putative MYC2 ortholog,in jasmonate-signaled defense responses of maize has not been reported.In this study,we found that ZmMYC7 possesses JID,TAD,bHLH and Zip domains and essential characteristics of transcription factors:a nuclear location and transactivation activity.The ZmMYC7 mutants showed markedly increased sensitivity to Fusarium graminearum and Setosphaeria turcica.The expression levels of the defense-associated genes ZmPR1,ZmPR2,ZmPR3,ZmPR5,ZmPR6,and ZmPR7 in response to F.graminearum infection were downregulated in ZmMYC7 mutants,while ZmPR4 and ZmPR10 were up-regulated.ZmMYC7 interacted with members of the ZmJAZ family,including ZmJAZ8,ZmJAZ11,and ZmJAZ12.ZmMYC7 physically interacted with G-box cis-elements in the ZmERF147 promoter in vitro and transcriptional activation of ZmERF147 by ZmMYC7 was inhibited by ZmJAZ11 and ZmJAZ12.ZmERF147 mutants were more susceptible to F.graminearum infection than inbred line B73 with concomitant down-regulation of all defense-associated ZmPRs except ZmPR4.These findings indicate that ZmMYC7 functions in maize resistance to F.graminearum and sheds light on maize defense responses to pathogenic fungi via the JA signaling pathway.

1.Introduction

Maize(Zea mays L.)is a global food and feed crop and a source of energy and industrial raw material.Growing maize is often infected by various pathogenic fungi,such as Fusarium graminearum and Setosphaeria turcica,resulting in severe yield losses[1,2].

In natural environments,plants are continuously exposed to various stresses,including abiotic stresses(cold,heat,salinity,drought,etc.)and biotic stresses(pathogen infection and mechanical damage by insects).Plants have evolved intricate mechanisms to acclimate to a variety of stress conditions.Jasmonates(JAs)play essential roles in plant defense responses to abiotic and biotic stresses[3].In the absence of a stimulus,JA-responsive gene expression is inhibited by the action of JA ZIM-domain(JAZ)proteins,which act as transcriptional repressors of associated basic helix-loop-helix(bHLH)transcription factors.ERF-associated amphiphilic repression(EAR)motif-containing JAZ proteins directly recruit the co-repressor TOPLESS(TPL),whereas JAZ proteins without the EAR motif recruit TPL via Novel Interactor of JAZ(NINJA),which contains an EAR motif[4].Consequently,the transcription of JA-responsive genes is suppressed via the action of histone deacetylases(HDACs)[5,6].In response to biotic and abiotic stresses,JA-Ile is rapidly synthesized in plant tissue[7,8].Upon JA-Ile perception,COI1-based SCF complexes recruit JAZ proteins for ubiquitination and degradation by the 26S proteasome[9],thereby releasing JA-responsive genes from JAZ-mediated suppression to participate in essential JA responses.

In the JA signaling pathway,the primary signal transduction processes following JA perception are basically associated to bHLH transcription factors[6,10].Of these,MYC2 is considered[10]a regulatory hub in the JA signaling pathway and has been intensively studied.MYC2 fine-tunes the JA signaling pathway by regulating the expression of JAZ genes and also the expression of transcriptional activators that function downstream from MYC2.MYC2 regulates the expression of early JA-responsive genes that include JA-responsive transcription factors involved in the regulation of specific branches of the JA signaling pathway[11].Based on JA-responsive gene expression analyses and functional tests,it was found[12]that in general,MYC2 negatively regulates pathogen defense and secondary metabolism,but positively regulates wound response,insect defense,flavonoid metabolism,and oxidative stress during JA signaling.During JA signaling,MYC2 negatively regulates some JA-responsive pathogen defense genes including PDF1.2,CHIB,and PR4,but activates the defense genes AP2/ERF transcription factors ERF1 and ORA59[13–15].

The AP2/ERF superfamily is one of the largest transcription factor families in plants and includes four subfamilies(AP2,ERTFF,DREB,and RAV)[16,17].The ethylene response factor(ERF)subfamily contains a single AP2 domain and an EAR motif.The AP2 domain consists of about 60 amino acid residues,forming a three-dimensional structure with one α-helix and three β-sheets,which can bind to the GCC-box(5′-AGCCGCC-3′)of the target gene promoter.The EAR motif mediates interactions with repressor TPL and TOPLESS-RELATED(TPR)proteins to jointly inhibit the transcription of target genes[18].The ERF transcription factors function in plant response to abiotic[19,20]and biotic stresses[21–24].JAs and ethylene(ET)often act cooperatively and function in regulating plant defense against pests and pathogens[25].Synergistic regulation of downstream genes by MYC2 and ERFs has been reported[26–29].Overexpression of AtERF4 in Arabidopsis thaliana reduced the expression of defense-associated genes PDF1.2 and increased susceptibility to F.oxysporum[30].AtERF4 is a direct downstream target gene of AtMYC2[11].

The function of MYC2 has been studied mainly in Arabidopsis[31,32]and other dicotyledons such as tomato[33],apple[29]and tobacco[34,35].Relatively little is known about the function of MYC2 orthologs or homologs in monocotyledonous crops.Research into the mechanism of MYC2 regulation of ERFs affecting downstream genes has focused on the accumulation of plant metabolites and the role of this regulatory mechanism in plant resistance to fungal diseases remains relatively unexplored.

The purpose of the present study was to investigate the functions of ZmMYC7,a putative MYC2 ortholog,in JA-signaled defense responses of maize and the underlying molecular mechanisms.A bioinformatic analysis was used to identify ZmMYC7 as an ortholog of MYC2.Subcellular localization and transcriptional activation of ZmMYC7 were performed to determine whether ZmMYC7 possesses essential characteristics of a transcription factor.ZmMYC7 mutants were inoculated with F.graminearum and S.turcica to investigate the function of ZmMYC7 in maize resistance to fungal pathogens.Using yeast two-hybrid(Y2H)assay and bimolecular fluorescence complementation(BiFC)assay,interaction between ZmMYC7 and JAZ family members in maize was identified.Yeast one-hybrid(Y1H)assay and electrophoretic mobility shift assay(EMSA)were used to identify ZmERF147,the AtERF4 ortholog in maize,as a downstream target gene regulated by ZmMYC7.The phenotypes of ZmERF147 mutants in response to F.graminearum were inspected.

2.Materials and methods

2.1.Plant materials and pathogenic fungal strains

The maize inbred line B73 and pathogenic fungus S.turcica strain Et28A were obtained from the Mycotoxin and Molecular Plant Pathology Laboratory,Hebei Agricultural University.The ZmMYC7 ethyl methanesulfonate(EMS)mutagenized mutants myc7-e1(EMS3-008b80)and myc7-e2(EMS3-008b76)were obtained from Maize EMS induced Mutant Database(MEMD)(https://elabcaas.cn/memd/)[36].The ZmERF147 Mu insertion mutant erf147-m1(Chr3,Insertion site 191580900,V4.0)was obtained from ChinaMu Project(https://chinamu.jaas.ac.cn/Default.html)[37].F.graminearum strain PH-1 was provided by Prof.Mingguo Zhou at Nanjing Agricultural University.All maize seeds were soaked in sterile water for about 24 h,sown and grown in the experimental field during the natural growing season at the Hebei Agricultural University(Hebei province,China).

2.2.Bioinformatics analysis of ZmMYC7

The protein sequence of ZmMYC7 from maize was retrieved from MaizeGDB(https://www.maizegdb.org/).The protein sequences of AtMYC2,AtMYC3,and AtMYC4 from A.thaliana were retrieved from TAIR(https://www.arabidopsis.org).The protein sequence of OsMYC2 from Oryza sativa was retrieved from RAPDB(https://rapdb.dna.affrc.go.jp).The protein sequences of other MYCs were retrieved from NCBI(https://www.ncbi.nlm.nih.gov/),including AaMYC2 from Artemisia annua,NbbHLH1 and NbbHLH2 from Nicotiana benthamiana,NtMYC1a,NtMYC1b,NtMYC2a,and NtMYC2b from N.tabacum,SlMYC1 from Solanum lycopersicum,TcJAMYC1 from Taxus cuspidata,and VvMYC2 from Vitis vinifera.These protein sequences were multiply aligned with ClustalX 1.83(https://www.clustal.org/)and imported to MEGA11(https://www.megasoftware.net/)for phylogenetic analysis using the neighbor-joining(NJ)method and 1000 bootstrap resamplings.The conserved domain of ZmMYC7 was identified by Pfam(https://pfam.xfam.org)and SMART(https://smart.embl-heidelberg.de)and plotted with IBS 1.0.3(https://ibs.biocuckoo.org/).The in silico 3D model of AtMYC2,OsMYC2,and ZmMYC7 were modeled online with SWISS-MODEL(https://www.swissmodel.expasy.org).The promoter region of ZmERF147 was identified by PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html).

2.3.Subcellular localization and transactivation activity analysis of ZmMYC7

For subcellular localization assays,the ZmMYC7 coding sequence was amplified by PCR with ZmMYC7-specific primers(Table S1)and inserted into the pEarlyGate103 vector to construct a binary vector 35S:ZmMYC7-GFP.The recombinant plasmid 35S:ZmMYC7-GFP and the empty pEarlyGate103 vector were then transformed into tobacco(N.benthamiana)leaves by the Agrobacterium-mediated method.Fluorescence signals were excited at 488 nm and detected under a laser scanning confocal microscope(Carl Zeiss AG,Oberkochen,Germany)using a 500–530 nm emission filter,with 4′,6-diamidino-2-phenylindole(DAPI)used as a nuclear localization marker.

To detect transactivation activity of ZmMYC7,the normal coding sequence(CDS)and the single nucleotide mutated CDS of ZmMYC7 were amplified and cloned into the yeast vector pGBKT7 to obtain the BD-ZmMYC7,BD-myc7-e1 and BD-myc7-e2 construct,respectively.BD-ZmMYC7,BD-myc7-e1,BD-myc7-e2,and the empty vector pGBKT7 were separately transformed into the yeast strain AH109.Transformed colonies were selected on yeast synthetic dropout(SD)glucose medium lacking Trp.Transformed single colonies(2 mm diameter)grown on selection medium for 3 days were resuspended in 100 μL of autoclaved distilled H2O,and 10 μL of resuspended cells were plated on SD/-Trp/-His,SD/-Trp/-His/-Ade,SD/-Trp/-His/X-α-Gal and SD/-Trp/-His/-Ade/X-α-Gal medium for 2–3 days.The primers used in transactivation activity analyses are listed in Table S2.

2.4.Pathogen inoculation

Fusarium graminearum was grown on carboxymethylcellulose(CMC)medium and incubated at 28°C for 5 days.Conidia were collected and suspended in autoclaved distilled H2O to a concentration of 106mL-1.A 50-μL aliquot of the conidial suspension was inoculated into the first internode in the stalk of B73 and the myc7 and erf147 mutants at the sixth-leaf stage.Similarly,50 μL of autoclaved distilled H2O was inoculated into B73 as control.Inoculated plants were held in a growth chamber with high humidity for 24 h and then transferred to normal conditions.Maize stalks treated with F.graminearum were randomly sampled at 0 h,6 h,12 h,1 d,2 d and 7 d to record measurements of the lesion area.Lesion sites were excised and immediately frozen in liquid nitrogen and stored in a-80 °C refrigerator for RNA extraction.

The conidial suspension of S.turcica was diluted in autoclaved distilled H2O containing 0.1%(v/v)Tween-20 with a spore concentration of 105mL-1.Approximately 10 μL of the S.turcica conidial suspension was inoculated into injured leaves of B73 and the myc7 mutants at the sixth-leaf stage.After inoculation,the maize plants were moisturized at 25°C for 24 h in the dark and then incubated at 25°C with a photoperiod of 12 h.Lesion formation was recorded at 7 days post-inoculation.

2.5.RNA extraction and qRT-PCR analysis of gene expression

Total RNA of maize was extracted using the E.Z.N.A.Plant RNA Kit(Omega Biotek Inc.,Norcross,GA,USA)and reversetranscribed to cDNA using a Reverse Transcription and cDNA Synthesis Kit(Clontech Laboratories Inc.,Mountain View,CA,USA),both according to the manufacturer’s instructions.Gene expression was measured by quantitative real-time PCR(qRT-PCR)with SYBR Premix(Takara Biotechnology Co.,Dalian,Liaoning,China)using a Real-time PCR System(Bio-Rad Laboratories Inc.,Hercules,CA,USA).All PCR amplifications were performed in 96-well optical reaction plates with 45 cycles of denaturation for 15 s at 95 °C,annealing for 20 s at 56°C,and extension for 45 s at 72°C.Expression levels were normalized to ubiquitin 9(UBQ9).Primers used for qRT-PCR are listed in Table S3.Each qRT-PCR was repeated three times independently.

2.6.Y2H assay

To test for protein–protein interactions,full-length cDNAs of ZmMYC7 and ZmJAZs genes were amplified by reverse transcription-PCR(RT-PCR)from the maize inbred line B73 and cloned into the Y2H vectors pGADT7 and pGBKT7 to obtain prey and bait gene constructs,respectively.These constructs were cotransformed into the yeast strain AH109.Co-transformed colonies were selected on yeast SD-dropout medium lacking Leu and Trp.For ZmMYC7 and ZmJAZs interactions,a co-transformed single colony(2 mm diameter)grown on selection medium for 3 days was resuspended in 100 μL of autoclaved distilled H2O,and 10 μL of the re-suspended cells was plated on SD/-Trp/-Leu,SD/-Trp/-Leu/-His and SD/-Trp/-Leu/-His/-Ade for 3–4 days.Primers used in the Y2H assay are listed in Table S4.

2.7.BiFC assay

To verify the interaction between ZmMYC7 and ZmJAZs,the ORFs of ZmMYC7 and ZmJAZs were amplified by RT-PCR and cloned into the BiFC vectors pX-nYFP and pX-cYFP to generate ZmMYC7-nYFP,ZmMYC7-cYFP,ZmJAZs-nYFP and ZmJAZs-cYFP vectors.Combinations of vectors ZmMYC7-nYFP and ZmJAZs-cYFP or of vectors ZmMYC7-cYFP and ZmJAZs-nYFP were co-transformed into tobacco leaves by the Agrobacterium-mediated method.YFP fluorescence was visualized and imaged with the laser scanning confocal microscope.The primers for BiFC are listed in Table S5.

2.8.Y1H assay

The Matchmaker Gold Yeast One-Hybrid System(Clontech)was used following the manufacturer’s instructions to perform the Y1H assay for investigating the interaction of ZmMYC7 with target gene promoters.The full-length promoter fragments of the candidate target gene ZmERF147 containing either the full G-box,a G-box mutation,or a G-box deletion were PCR-amplified with specific primers(Table S6)and ligated separately into the pAbAi vector as reporters.The reporter vectors were linearized at the XhoI and KpnI sites as described in the user manual and transformed into the yeast strain Y1H Gold.The full-length cDNA of ZmMYC7 was cloned into the pGADT7 vector as an effector and transferred into the reporter strains.The co-transformation yeast strains were plated on SD/-Leu/Aureobasidin A plates for 3–4 days.The Y1H assay was performed according to the manufacturer’s protocol.

2.9.EMSA

The full-length CDS sequence of ZmMYC7 was amplified and inserted into the pGEX-4T-1 vector.GST-ZmMYC7 was induced,expressed,and purified from the E.coli strain BL21.DNA probes with biotin labeling were synthesized using a Biotin 3′End DNA Labeling Kit(Thermo Fisher Scientific Inc.,Waltham,MA,USA).DNA gel-shift assays were performed using a LightShift Chemiluminescent EMSA Kit(Thermo Fisher Scientific,Waltham,MA,USA)following the manufacturer’s instructions.The primers used for EMSA are listed in Table S7.

2.10.Dual-luciferase reporter gene(dual-LUC)assay

The promoter sequence of ZmERF147 was PCR-amplified and ligated into the pGreenII-0800-LUC vector as a reporter.The fulllength CDS sequence of ZmMYC7 and ZmJAZs were ligated into pCAMBIA1307 and pEarlyGate203 vectors,respectively,as effectors.Reporter and effector vectors were co-transformed into tobacco leaves by the Agrobacterium-mediated method.Firefly luciferase(LUC)and Renilla luciferase(REN)activity were measured with a Dual Luciferase Reporter Gene Assay Kit(Beyotime Biotechnology,Shanghai,China)and the Spark Multimode Microplate Reader(Tecan Trading AG,Mannedorf,Switzerland).The primers for dual-LUC are listed in Table S8.

3.Results

3.1.Identification and sequence analysis of ZmMYC7

The full-length CDS sequence of ZmMYC7 was cloned from the leaves of maize inbred line B73.ZmMYC7 has one open reading frame(ORF)of 2118 bp encoding a protein of 705 amino acids.ZmMYC7 possessed domains of JAZ interaction(JID),transcriptional activation(TAD),basic helix-loop-helix(bHLH)and a leucine zipper region(Zip)(Fig.1A,B),suggesting that ZmMYC7 has high DNA-binding affinity and could form protein–protein interactions.A phylogenetic tree showed that ZmMYC7 has high similarity with OsMYC2 of O.sativa and AtMYC2 of A.thaliana(Fig.1C).The in silico 3D model of ZmMYC7 also suggested significant structural similarity with MYC2 of O.sativa and A.thaliana(Fig.S1).These results suggested that ZmMYC7 is a putative MYC2 ortholog and would share similar functions with OsMYC2 and AtMYC2.

Fig.1.Domain and phylogenetic analysis of ZmMYC7.(A)Domain architecture analysis of ZmMYC7.Conserved domains of ZmMYC7 were identified by Pfam and SMART.Different domains are indicated by different colors.The lengths and location of the domains are proportional to the complete amino acid sequence of ZmMYC7.JID,JAZ interaction domain;TAD,transcriptional activation domain;bHLH,basic helix-loop-helix domain;Zip,leucine zipper region.(B)Comparison of the amino acid sequences of ZmMYC7 with those of MYCs in other species.The comparison alignment was performed using DNAMAN(https://www.lynnon.com/dnaman.html).The outlined red boxes indicate conserved domains.(C)Phylogenetic tree of MYC amino acid sequences of multiple species.The tree was generated using the NJ method with 1000 bootstrap replicates using MEGA11.ZmMYC7 is indicated in red.

3.2.ZmMYC7 possesses essential characteristics of a transcription factor

Green fluorescent signals were observed in transformed tobacco cells that overlapped with the nucleus-specific dye DAPI(Fig.2A).These results indicated that ZmMYC7 was localized in the nucleus.

The transactivation activity analysis showed that the yeast transformed with BD-ZmMYC7 grew on SD/-Trp/-His,SD/-Trp/-His/-Ade,SD/-Trp/-His/X-α-Gal,and SD/-Trp/-His/-Ade/X-α-Gal medium and showed alpha-galactosidase activity,whereas the negative control pGBKT7 grew only in SD/-Trp medium(Fig.2B).Thus,ZmMYC7 showed transactivation activity in yeast cells.

3.3.ZmMYC7 is involved in maize resistance to F.graminearum and S.turcica

The expression of ZmMYC7 was maximal 12 h after F.graminearum inoculation,with a 30×fold-change relative to the control(0 h),after which expression declined(Fig.S2).These results suggested that the expression of ZmMYC7 is induced by pathogenic fungal infection.

Fig. 3. Altered disease resistance of ZmMYC7 mutants against F. graminearum and S. turcica. (A) Stalks of B73 and ZmMYC7 mutants at the sixth-leaf stage were inoculated with 50 μL F. graminearum conidia suspension (×106 mL-1) for 7 days. Scale bar, 1 cm. (B) Measurement of lesion areas of B73 and ZmMYC7 mutants inoculated with F. graminearum, shown in panel A. The necrotic lesion areas were measured with ImageJ (https://imagej.nih.gov/ij/index.html). (C) The leaves of B73 and ZmMYC7 mutants at the sixth-leaf stage were inoculated with 10 μL S. turcica conidia suspension (105 mL-1) for 7 days. Scale bar, 1 cm. (D) Measurement of the lesion area of B73 and ZmMYC7 mutants inoculated with S. turcica, shown in panel C. myc7-e1-9 and myc7-e1-12 are two lines of the ZmMYC7 EMS mutagenized mutant, myc7-e1. myc7-e2-8 is a single line of the ZmMYC7 EMS mutagenized mutant, myc7-e2. The experiments were repeated three times with similar results. Error bars indicate mean ± standard deviation. Asterisks indicate significant difference by Student’s t-test (*, P ＜ 0.05; **, P ＜ 0.01).

To investigate whether the mutation of ZmMYC7 affected maize resistance to pathogenic fungi,the response to F.graminearum and S.turcica in WT B73 was compared to that in the ZmMYC7 mutants,myc7-e1 and myc7-e2.The mutation sites of myc7-e1 and myc7-e2

Fig.2.Subcellular localization(A)and transactivation activity(B)of ZmMYC7.Scale bar,20 μm.

are indicated in Fig.1A and were confirmed from the sequencing of their PCR products.ZmMYC7 gene expression levels in the mutant backgrounds were significantly downregulated relative to WT B73(Fig.S3A).As shown in Fig.3A,B,the lesion area in myc7 mutants were significantly larger than that in B73 7 d after stem inoculation with F.graminearum.Similarly,the myc7 mutants also developed larger leaf lesions than WT B73 7 d after inoculation with S.turcica(Fig.3C,D).These findings indicated that the ZmMYC7 gene is involved in maize resistance to F.graminearum and S.turcica.

The mutations of ZmMYC7 above affected the transactivation activity and the expression of genes downstream of ZmMYC7.After the alteration of one amino acid in the conserved domain of ZmMYC7,the transactivation activities of BD-myc7-e1 and BD-myc7-e2 were significantly decreased(Fig.S3B).The expression levels of ZmPR1,ZmPR2,ZmPR3,ZmPR5,ZmPR6,and ZmPR7 were markedly lower in the myc7 mutants than in B73.In contrast,higher expression levels of ZmPR4 and ZmPR10 in myc7 mutants were observed(Fig.S4).These results indicated that the increased susceptibility to F.graminearum infection in ZmMYC7-deficient maize occurred with a reduced mobilization of defense genes.

3.4.ZmMYC7 interacted with some members of the ZmJAZ family

In a Y2H assay,ZmJAZ11 and ZmJAZ12 showed relatively strong interaction with ZmMYC7,while that of ZmJAZ8 was weaker(Fig.4A).In a BiFC assay,fluorescence in nuclei was detected when ZmMYC7 was co-expressed with ZmJAZ8,ZmJAZ11,or ZmJAZ12 but not with an empty vector(Fig.4B).

3.5.ZmMYC7 binds to G-box cis-elements in the ZmERF147 promoter

The ZmERF147 promoter sequence contained the G-box element(5′-CACGTG-3′)(Fig.5A),suggesting that ZmMYC7 might directly bind to the promoter of this gene to regulate its expression.In qRT-PCR analyses of the expression levels of ZmERF147 in myc7 mutants inoculated by F.graminearum,compared with B73,the expression levels of ZmERF147 were significantly downregulated in the myc7 mutants(Fig.5B).These results suggested that ZmERF147 might be one of the downstream target genes of ZmMYC7.

A Y1H assay was performed to investigate whether ZmMYC7 physically interacts with the promoter of ZmERF147.The generation of full-length(AD-ZmMYC7)effectors and reporters of the WT ZmERF147 promoter pERF147-pAbAi and that with a G-box mutation(pERF147(m)-pAbAi)or G-box deletion(pERF147(ΔG-box)-pAbAi)are schematically described in Fig.5C.Whereas co-transformation of Y1H Gold yeast cells with the effector AD-ZmMYC7 and the reporter pERF147-pAbAi could grow on SD/-Leu/AbA medium,the reporters with altered G-box sequences(pERF147(m)-pAbAi and pERF147(ΔG-box)-pAbAi)could not(Fig.5C).These results suggest that ZmMYC7 interacts with the G-box element of the ZmERF147 promoter in yeasts.

To further confirm whether ZmMYC7 physically binds to the G-box of the ZmERF147 promoter,we expressed and purified the GST-tagged ZmMYC7 fusion protein in E.coli and performed the EMSA.The GST-ZmMYC7 fusion protein was able to bind to the labeled DNA probes containing the G-box of the ZmERF147 promoter(biotin probe),but failed to bind to the biotin-(m)probe.Increasing the concentrations of unlabeled probes(to 10×and 100×)in the binding reactions led to much weaker combined bands(Fig.5D).These results suggested that ZmMYC7 physically interacts with the G-box of ZmERF147 promoter in vitro.

The regulatory effect of ZmMYC7 on the promoter of ZmERF147 was further tested by a dual-LUC assay.The ratio of LUC/REN with effector ZmMYC7 was significantly higher than with an empty vector,indicating that ZmMYC7 could increase the expression of ZmERF147.The transactivation activity of ZmMYC7 on the ZmERF147 promoter was inhibited when ZmJAZ11 and ZmJAZ12 was co-expressed(Fig.5E).These results indicated that ZmERF147 is a direct downstream target gene of ZmMYC7 and that the transactivation activity of ZmMYC7 was inhibited by ZmJAZ11 and ZmJAZ12.

3.6.ZmERF147 affected maize resistance to F.graminearum

To investigate the role of ZmERF147 in maize resistance to pathogenic fungi,we performed further tests with the ZmERF147 mutant,erf147-m1(Fig.S5).This mutation was confirmed by PCR and the mutant was shown by qRT-PCR to have reduced expression relative to WT B73.The lesion areas developed in the erf147-m1 mutant after inoculation with F.graminearum indicated a greater susceptibility to infection than WT B73(Fig.6),confirming a contribution of ZmERF147 to the maize resistance to F.graminearum.Furthermore,relative to the WT,the expression levels of all ZmPRs but ZmPR4 were reduced 1 d after inoculation with F.graminearum in the erf147 mutants(Fig.S6).These results showed that the ZmERF147 gene affected maize resistance to F.graminearum.

Fig.4.ZmMYC7 interacted with ZmJAZ8,ZmJAZ11,and ZmJAZ12.(A)Y2H assay of ZmMYC7 interactions with ZmJAZ8,ZmJAZ11,and ZmJAZ12.ZmMYC7 and ZmJAZs were fused with the DNA binding domain(BD)in pGBKT7 and the activation domain(AD)in pGADT7,respectively.Transformed yeast was grown on SD-dropout medium lacking Leu(-L),Trp(-T),His(-H)and Ade(-A)to test for protein interactions.The empty pGBKT7 and pGADT7 vector were used as negative controls.(B)BiFC assay of ZmMYC7 interactions with ZmJAZ8,ZmJAZ11,and ZmJAZ12.ZmMYC7 and ZmJAZs were fused with the BiFC vectors pX-nYFP and pX-cYFP,respectively.Combinations of transformed vectors were co-transformed into tobacco and YFP fluorescence was captured by laser scanning confocal microscopy.The empty pX-nYFP and pX-cYFP vectors were used as negative controls.

Fig.5.ZmMYC7 interacts with the G-box of the ZmERF147 promoter.(A)Schematic representation of the ZmERF147 promoter.The G-box is indicated in red.(B)Relative expression levels of ZmERF147 in ZmMYC7 mutants after inoculation with F.graminearum.dpi,days post inoculation.Each qRT-PCR assay represents the mean of three independent replicates.Error bars indicate mean±standard deviation.Asterisks indicate significant difference according to Student’s t test(**,P＜0.01).(C)Y1H assays of the interaction of ZmMYC7 with native and altered G-box fragments of the ZmERF147 promoter.pERF147,promoter fragment of ZmERF147 containing the normal G-box;pERF147(m),promoter fragment of ZmERF147 with G-box containing an A/G substitution(indicated above panel);pERF147(ΔG-box),promoter fragment of ZmERF147 with G-box deletion.(D)The G-box of the ZmERF147 promoter is recognized by ZmMYC7 in an EMSA assay.Unlabeled probes(10×and 100×)were used to competitively bind GSTZmMYC7.The biotin-(m)probe contains the mutated G-box sequence(with CACGTG replaced by CTGACG).(E)A dual-LUC assay verified the transactivating activity of ZmMYC7 on the ZmERF147 promoter.This activity was inhibited with co-expression of ZmJAZ11 and ZmJAZ12.Asterisks indicate significant difference by Student’s t-test(*,P＜0.05;**,P＜0.01).

4.Discussion

JA signaling functions in many plant processes ranging from responses to abiotic and biotic stresses to growth and development[3,38].The transcription factor MYC2 is a key component of the COI1-JAZ-MYC2 conduit of JA signaling and differentially modulates diverse JA-dependent functions in Arabidopsis[39].As a regulatory hub in the JA signaling pathway,MYC2 acts as both a transcriptional repressor and a transcriptional activator in the regulation of different aspects of JA signaling[10].In this study,we investigated the role of ZmMYC7 in maize fungal resistance.The expression of ZmMYC7 was up-regulated in the maize inbred line B73 infected by F.graminearum(Fig.S2)and disruption of ZmMYC7 function led to increased sensitivity to F.graminearum and S.turcica infections(Fig.3),indicating that ZmMYC7 functions in maize resistance to infection by pathogenic fungi.In the mutant myc7-e1,an aspartic acid(Asp,D)in the TAD domain was replaced by asparagine(Asn,N),whereas in the mutant myc7-e2 a glycine(Gly,G)of the bHLH domain was replaced by serine(Ser,S)(Fig.1A).In Arabidopsis,the TAD domain is the MYC2 transactivation domain that interacts with MED25(MEDIATOR25MED25)and activates downstream target genes[40].The bHLH domain is required for heterodimerization and binding to the G-box in target gene promoters[10].Our results indicated that single amino acid changes in the conserved domains of ZmMYC7 reduce its transactivation activity(Fig.S3B).However,why the single nucleotide polymorphism(SNP)in CDS affects the transcription level(Fig.S3A)awaits further study.Among plant species,MYC2 regulates JA signals in diverse manners.In Arabidopsis,AtMYC2 activates woundresponsive genes,but represses pathogen-responsive genes[11,13,32],while in tomato,SlMYC2 functions in plant resistance to the necrotrophic pathogen Botrytis cinerea by inducing the expression of both wound-and pathogen-responsive genes[33].Overexpression of ZmMYC2,a homolog of ZmMYC7,increased resistance to B.cinerea in Arabidopsis[41].Our results with ZmMYC7 demonstrate an essential role for this MYC family member in maize resistance to F.graminearum and S.turcica.

MYC2 affects plant resistance to pathogens by directly or indirectly regulating the expression levels of downstream resistanceassociated genes.In Arabidopsis,AtMYC2 negatively regulates ERF1 and ORA59,thereby inhibiting the expression of the defense-associated genes PDF1.2 and PR4[11,42,43].In tomato,ERF.C3 is confirmed as a true MYC2-targeted transcription factor(MTF)in the JA-mediated plant response to B.cinerea infection and significantly increases the expression of the pathogenresponsive marker gene,PR-STH2[33].Our results show that ZmMYC7 up-regulates ZmPR1,ZmPR2,ZmPR3,ZmPR5,ZmPR6,and

ZmPR7,but downregulate ZmPR4 and ZmPR10(Fig.S4).However,the molecular mechanism underlying the ZmMYC7 regulation of ZmPRs is still not clear.In future work,we will further verify whether ZmMYC7 directly binds to the promoters of ZmPRs or indirectly regulates ZmPRs by regulating other transcription factors.

JAZ is a subfamily of the TIFY protein family,with two conserved domains of Jas and ZIM but no DNA binding domain.In the JA signaling pathway,JAZs inhibits the transactivation activity of MYC2 by binding to the JID domain of MYC2,and with the exception of AtJAZ7,all AtJAZ members interacted with AtMYC2 in Arabidopsis[44].Mutation of the Jas domain in AtJAZ4 suppresses its interaction and transcriptional inhibition of AtMYC2,leading to increased resistance to Pseudomonas syringae pv.tomato(Pst)DC3000[31].It has been reported[45]that OsMYC2 functions as a common and direct target of OsJAZ1,OsJAZ3,and OsJAZ6 in rice.In this study,we screened JAZ family members for their interaction with ZmMYC7 by Y2H and BiFC and found that ZmJAZ8,ZmJAZ11,and ZmJAZ12 interacted with ZmMYC7(Fig.4).In agreement with these findings,ZmJAZ14 was reported[46]to interact with AtMYC2 and AtMYC3 in Arabidopsis.ZmMYC2 has been confirmed[41]to interact directly with ZmJAZ14,ZmJAZ17,AtJAZ1 and AtJAZ9.Recent research[47]shows that ZmMYC7,coronatine sensitive 1(COI1),and ZmJAZs can form a COI-JAZ-bHLH function module in maize interactions with F.graminearum.Y2H,LUC and pulldown assays confirmed the interaction between ZmJAZ15 and ZmMYC7.The CRISPR-Cas9 knockout mutant ZmCOI1a became more resistant to F.graminearum,whereas disruption of ZmJAZ15 increased susceptibility.

MYC2 affects other downstream genes by regulating ERF subfamily genes.In tobacco,MYC2 directly and indirectly regulated the expression of genes involved in nicotine synthesis via multiple ERFs[26].MdMYC2 binds to the promoter of MdERF3 to increase its transcriptional activity,thereby activating MdACS1 expression and promoting ethylene biosynthesis in apple fruit[29].The regulatory effect of MdMYC2 on MdERF3 is also manifested in up-regulation of MdAFS,so that the downstream product,α-farnesene,is accumulated[27].TcJAZ3 interacts with TcMYC2a in Taxus chinensis,up-regulating the expression of TcERF15.TcERF15,in turn,up-regulates the key gene TASY of taxol synthesis[28].In this study,we found that ZmMYC7 directly binds to the G-box in the ZmERF147 promoter(Fig.5C,D)and after the mutation of ZmMYC7,the expression of ZmERF147 in response to F.graminearum was down-regulated(Fig.5B),indicating that ZmMYC7 has a direct regulatory effect on the expression of ZmERF17.The finding that ZmMYC7 activation of ZmERF147 expression was inhibited by co-expression of ZmJAZ11 and ZmJAZ12(Fig.5E)suggests that in order to mobilize genes involved in maize resistance to F.graminearum infection,ZmMYC7-ZmJAZ interactions must be reduced to activate downstream ZmERF147.

The ERF transcription factors function in plant growth and development and resistance to various stresses.In response to abiotic stress,overexpression of TaERF1 increased tolerance to drought,cold and salt in wheat[19].The expression of GmERF135 in soybean was up-regulated under drought stress and when the gene was overexpressed,increased drought resistance in Arabidopsis[20].In response to biological stress,some ERFs can increase resistance to pathogens.In Arabidopsis,AtERF1 is known[21]to regulate several defense-associated genes and when overexpressed,activates the expression of PDF1.2 and increases plant resistance to pathogens.Similarly,AtERF15 expression is up-regulated in response to Pst DC3000 and B.cinerea infections and is a positive regulator of genes involved in defense against both pathogens[22].In contrast,other ERFs can negatively regulate plant resistance.Overexpression of AtERF4 in Arabidopsis induced a greater susceptibility to F.oxysporum[30]and knockout of AtEFR9 caused the expression of PDF1.2 to be increased,with increased resistance to B.cinerea[24].StERF3 also negatively regulates potato resistance to Phytophthora infestans and salt stress[23].

Fig.7.Schematic model of ZmJAZs,ZmMYC7 and ZmERF147 interactions in the development of plant resistance to fungal pathogens.In the absence of JA signaling,ZmJAZs-ZmMYC7 interactions prevent ZmMYC7 from binding to the ZmERF147 promoter,thereby inhibiting ZmMYC7 transactivation of ZmERF147 and ZmERF147-mediated activation of ZmPR transcription.Upon perception of the JA signal,ZmJAZs are degraded in a ubiquitination-mediated process,allowing ZmMYC7 to transactivate the expression of ZmERF147 by binding to the G-box cis-element of the ZmERF147 promoter.Subsequently,ZmERF147 regulates the expression of defense-associated genes,including ZmPRs,to increase maize resistance to fungal pathogens.

In the maize genome,76 ERF transcription factors have been identified and classified into six groups,named B1 to B6[17].In this study,we found that under the activation of ZmMYC7,ZmERF147,belonging to group B1,has a regulatory role in the expression of ZmPRs(Fig.S6)and positively regulates the resistance of maize to F.graminearum(Fig.6).ZmERF105 and ZmERF061 have also been reported[48,49]to increase resistance to Exserohilum turcicum by activating the expression of a series of downstream disease-resistance genes.

In summary,we have identified a transcription factor,ZmMYC7,involved in the regulation of maize resistance to F.graminearum and S.turcica infection.Our results suggest a working model of ZmJAZs,ZmMYC7 and ZmERF147(Fig.7).Upon perception of the JA signal,ZmJAZs release ZmMYC7 to activate the expression of downstream ZmERF147 via its interaction with the G-box ciselement of the ZmERF147 promoter.The increased expression of ZmERF147 subsequently activates the expression of downstream defense-associated genes,including ZmPRs,thereby increasing maize resistance to pathogen infection.Future studies may reveal whether ZmERF147 directly binds to the promoter of ZmPRs to mediate plant defense against pathogen challenge.Our results may help to elucidate the role of ZmMYC7 in the JA signaling pathway,and have important implications for future maize genetic improvement programs.

Accession number

Sequence data from this study can be found under the accession numbers: ZmMYC7 (Zm00001d030028), ZmERF147(Zm00001d043205),AtMYC2(At1g32640),AtMYC3(At5g46760),AtMYC4(At4g17880),OsMYC2(Os10g0575000),AaMYC2(KP119607),NbbHLH1(GQ859152),NbbHLH2(GQ859153),NtMYC1a(GQ859158),NtMYC1b(GQ859159),NtMYC2a(GQ859160),NtMYC2b(GQ859161),SlMYC1(KF430611),TcJAMYC1(FJ608574),VvMYC2(EF636725).

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

Hongzhe Cao:Conceptualization,Formal analysis,Funding acquisition,Investigation,Visualization,Writing–original draft.Kang Zhang:Data curation,Investigation,Software,Validation.Wei Li:Data curation,Formal analysis,Investigation.Xi Pang:Investigation,Methodology.Pengfei Liu:Formal analysis,Investigation.Helong Si:Data curation.Jinping Zang:Formal analysis.Jihong Xing:Conceptualization,Funding acquisition,Project administration,Supervision,Writing–review & editing.Jingao Dong:Conceptualization,Funding acquisition,Project administration,Resources,Supervision,Writing–review & editing.

Acknowledgments

This study was supported by the State Key Laboratory of North China Crop Improvement and Regulation(NCCIR2021ZZ-14),the Natural Science Foundation of Hebei Province(C2019204246,C2019204141),the Central Government Guides Local Science and Technology Development Projects(216Z6501G,216Z6502G),the Research Project of Basic Scientific Research Business Fees in Provincial Universities of Hebei Province(KY2021043,KY2021044),and the China Agriculture Research System(CARS-02).

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2022.05.006.