Xianpeng Xiong,Shichao Sun,Yanjun Li,Xinyu Zhang,Jie Sun*,Fei Xue*

Key Laboratory of Oasis Eco-agriculture,College of Agriculture,Shihezi University,Shihezi 832003,Xinjiang,China

Keywords:WRKY transcription factors Gossypium hirsutum Verticillium dahliae SA JAA B S T R A C T WRKY transcription factors(TFs)play im portant roles in the regulation of biotic and abiotic stresses.How ever,the functions of m ost WRKY TFs in upland cotton(Gossypium hirsutum)are still unknow n.In this study,w e functionally identified a group IIIWRKY transcription factor,GhWRKY70,in upland cotton.Reverse transcription-quantitative PCR analysis show ed that GhWRKY70 expression w as induced by Verticillium dahliae,salicylic acid(SA)and m ethyl jasm onate.Virus-induced gene silencing of GhWRKY70 increased the resistance of cotton to V.dahliae.Specifically,jasm onic acid(JA)response-associated genes w ere upregulated and SA-related genes were downregulated in GhWRKY70-silenced cotton plants.Overexpression of GhWRKY70 reduced tolerance to V.dahliae in Arabidopsis thaliana.Transgenic Arabidopsis plants show ed increased expression of SA-associated genes and reduced expression of JA response-associated genes.These results suggest that GhWRKY70 negatively regulates tolerance to V.dahliae in at least tw o w ays:(i)by upregulating the expression of SA-associated genes and(ii)by reducing the expression of JA-associated genes.

1.Introduction

Cotton(Gossypium hirsutum)is a m ajor fiber and an im portant econom ic crop w orldw ide.Its grow th and yield are constrained by various abiotic and biotic stresses.Verticillium w ilt,a vascular disease caused by the soilborne fungus Verticillium dahliae,affects＞40%of cotton-grow ing areas in China[1-4].Use of resistant cultivars is a cost-effective and environmentally friendly method of preventing yield losses caused by Verticillium w ilt.How ever,no G.hirsutum cultivars show high resistance to Verticillium w ilt[5,6].The m olecular mechanisms of cotton resistance to Verticillium infection are not w ell understood.

Three w ell-know n plant horm ones,salicylic acid(SA),jasm onic acid(JA),and ethylene(ET),act as central m odulators of plant im m une signaling netw ork.SA plays a role m ainly in defense signaling against biotrophic pathogens,w hereas JA and ET act m ainly in defense signaling against necrotrophic pathogens[7-9].SA,JA,and ET are the key hormones mediating the response to V.dahliae[10].In several studies,JA m ediated resistance to V.dahliae in cotton,e.g.by m odulating GbWRKY1[11],GhCYP82D[12],GhLac1[13],and GhJAZ2[14].Several resistance genes in SA pathways have been reported to be involved in the defense response against

V.dahliae,including GhPAO[15],GhSPMS[16],GhSAMDC[16],and GaRPL18[17].These studies have im proved our understanding of the innate defense m echanism s of cotton against Verticillium w ilt.How ever,additional research is needed to elucidate the com plex interactions betw een cotton and the pathogen.

WRKY transcription factors(TFs)are plant-specific transcription factors,containing both a zinc finger m otif at their C-term inus and a highly conserved WRKY dom ain[18].Many WRKY genes regulate stress-induced signaling under biotic and abiotic stress[18].In Arabidopsis,m ost WRKY group III genes,including AtWRKY70,are responsive both to pathogen infection and to SA[19].Most studies of WRKY protein function have been focused on m odel plants,and little is known about the role of WRKY IFs in cotton.Recently,several studies have show n that III WRKY transcription factors play im portant roles in cotton response to biotic and abiotic stresses[20-26].Overexpression of GhWRKY34 in Arabidopsis increased expression of salt stress-associated genes and enhanced plant tolerance to salt stress[20].Overexpression of GhWRKY41 increased salt and drought tolerance in transgenic Nicotiana benthamiana plants[21].GhWRKY27a overexpression in N.benthamiana reduced tolerance to salt stress and resistance to Rhizoctonia solani infection[22].A recent study show ed by virus-induced gene silencing (VIGS)that GhWRKY59 is an important transcription factor that regulates the drought stress response in cotton[23].Moreover,Overexpression of GhWRKY40 in N.benthamiana increased susceptibility to Ralstonia solanacearum and R.solani,respectively[24].In contrast,overexpression of GhWRKY39-1 and GhWRKY39 enhanced the response to R.solani and R.solanacearum[25,26].

In Arabidopsis,WRKY70 proves to play key roles in the antagonistic interaction betw een SA and JA[27].OsWRKY45 and AtWRKY70 are hom ologs[28],and their expressions can be activated by SA but repressed by JA[27,29-31].In this study,w e identified GhWRKY70 in upland cotton and analyzed its expression patterns in response to SA,m ethyl jasm onate(MeJA),and V.dahliae infection.The function of GhWRKY70 w as determ ined by VIGS and transgenic Arabidopsis lines overexpressing GhWRKY70.The results w ill enrich our know ledge on the mechanism of GhWRKY70 regulating resistance against V.dahliae resistance.

2.Materials and m ethods

2.1.Plant materials

Gene expression analysis under stress w as conducted using a V.dahliae-susceptible cultivar,G.hirsutum cv.Xinluzao 7.Both G.hirsutum cv.Xinluzao 7 and a V.dahliae-resistant cotton variety,G.hirsutum cv.Zhongzhim ian 2 w ere used for VIGS experim ents and disease assays.Plants w ere grow n in a grow th cham ber w ith a 16-h-light/8-h-dark cycle at 23°C.Arabidopsis thaliana(ecotype Colum bia-0)plants w ere grow n in a greenhouse at 23°C w ith a 16-h-light/8-h-dark cycle.All plants w ere w atered w eekly w ith Hoagland's nutrient solution.

2.2.V.dahliae infection of cotton and Arabidopsis plants

A highly aggressive strain of V.dahliae,strain V991,w as grow n on potato dextrose agar(PDA)m edium at 25°C for 7-10 days.Colonies were then cultured in Czapek medium for 10 days at 25°C w ith shaking(150 r m in-1).Spore suspensions(106conidia m L-1)w ere prepared and then inoculated(10 m L per treatm ent)into the soil of pots containing cotton seedlings and Arabidopsis plants.Cotton roots w ere harvested at 0,6,12,24,and 48 h after inoculation.The roots of the Arabidopsis plants w ere collected 48 h after inoculation,quickly frozen in liquid nitrogen,and stored at-80°C for transcription analysis.

2.3.SA and MeJA treatments

Cotton seedlings(20-day-old,tw o-leaf stage)raised in a greenhouse w ere sprayed w ith either 100μm ol L-1MeJA or 1 m m ol L-1SA.Plants in the control treatm ent w ere sprayed w ith w ater.Roots w ere harvested at 0,6,12,24,and 48 h after treatm ent,quickly frozen in liquid nitrogen,and stored at-80°C.

2.4. Gene cloning, phylogenetic analysis, and plant transformation

The ORF of GhWRKY70 w as am plified w ith the prim ers listed in Table S1.The c DNA template w as prepared from roots of G.hirsutum cv.Zhongzhim ian 2.PCR products w ere cloned into the p MDT-19 vector and three clones w ere selected for sequencing by Sangon Biotech(Shanghai)Co.,Ltd.(Shanghai,China).Multiple sequence alignments of GhWRKY70 w ere perform ed using ClustalX 1.83[32].A phylogenetic tree w as constructed w ith MEGA 7.0[33]using the neighbor-joining method.The ORF w as cloned into an expression vector,p GWB17,containing the hygrom ycin B resistance gene.The resulting plasm id pGWB17-GhWRKY70 w as introduced into Agrobacterium tumefaciens strain GV3101.Transform ation of Arabidopsis plants w as perform ed using the floral-dip method[34].

2.5.RNA isolation and expression pattern analysis

Total RNA w as extracted from cotton roots using a RN09-EASYspin RNA Plant Mini Kit(Aidlab Biotechnologies Co.Ltd.,Beijing,China)according to the m anufacturer's instructions.The RNA w as then treated with DNase I(TaKaRa Biotechnology(Dalian)Co.,Ltd.,Dalian,China)to rem ove genom ic DNA.A 2-μg aliquot of total RNA w as used for first-strand c DNA synthesis using the Prim eScript II 1st Strand cDNA Synthesis Kit(TaKaRa Biotechnology(Dalian)Co.,Ltd.).The primer pairs used for Reverse transcription-quantitative PCR(RT-q PCR)analysis w ere designed w ith Prim er3 Plus(http://prim er3plus.com/cgi-bin/dev/prim er3plus.cgi)from the GhWRKY70 sequences in G.hirsutum.The am plified fragm ent lengths w ere set to 80-200 bp.The annealing tem perature w as controlled at 60°C.The cotton UBQ7(DQ116441.1)gene w as used as the reference gene for RT-qPCR.Quantitative real-time PCR(q PCR)am plification reactions w ere perform ed on a Light Cycler 480II(Roche,Rotkreuz,Sw itzerland)using SYBRGreen(Roche)w ith three technical replicates.The amplification parameters w ere as follow s:denaturation at 95°C for 10 m in,40 cycles of denaturation at 95°C for 15 s,annealing at 60°C for 15 s,and extension at 72°C for 15 s.Expression data from three biological independent experim ents w ere analyzed and presented as m eans±SD.The prim ers used are listed in Table S1.

2.6.VIGSand pathogen inoculation

Tobacco rattle virus(TRV)-based vectors w ere used for VIGS[35],pTRV2:GhCHLI(encoding magnesium chelatase subunit I)as positive control[36].A fragment of GhWRKY70 c DNA w as cloned into the p TRV2 plasmid using Eco RIand Kpn Ito construct the pTRV2:GhWRKY70 VIGS vector.Recom binant plasm ids w ere transformed into A.tumefaciens GV3101 as described previously[37,38].Cotton gene silencing w as perform ed as described previously[38].Cotyledons of 10-day-old seedlings w ere injected w ith a m ixture(1:1 ratio,v/v)of p TRV1 and pTRV2:GhWRKY70 Agrobacterium(OD600=1.0).The seedlings w ere incubated for 12 h in darkness and then transferred to the greenhouse.RNA w as extracted from the cotton leaves 10 days after infiltration to m easure GhWRKY70 expression.Tw o-weekold VIGS-silenced cotton plants w ere inoculated by root irrigation w ith 10 m Lspore suspensions(106conidia m L-1).

Fig.1-Alignm ent of the am ino acid sequences and p hylogenetic analysis of Gh WRKY70 hom ologs.(A)Sequence alignm ent of the WRKY70s hom ologs of Durio zibethinus(XP_022764430.1),Theobroma cacao(XP_017985156.1),Camelina sativa(XP_010504547.1),Paeonia lactiflora(AMW 90776.1),Arabidopsis thaliana(NP_191199.1).(B)Phylogenetic analysis of Gh WRKY70 in relation to Arabidopsis and cotton WRKY TFs.The phylogenetic tree w as constructed using the m axim um likelihood m ethod in MEGA 7.0.

2.7.Recovery of V.dahliae from the infected stem of cotton

The first stem node(from the bottom of plants)w as collected from eight plants in each treatm ent group 15 days after inoculation w ith V.dahliae.The nodes w ere sterilized in 75%hypochlorite solution for 3 m in,rinsed three tim es w ith sterile w ater,and p lated on PDA containing cefotaxim e(400 m g L-1)and incubated at 25°C.Fungal biom ass w as quantified as fungal DNA present in infected plant extracts via q PCR.The fungus-sp ecific prim ers ITS1-F and ST-Ve1-R w ere used to m easure fungal colonization[39].Prim ers for cotton UBQ7 and Arabidopsis EF-la(Table S1)w ere used as endogenous control.

3.Results

3.1.Sequence alignment and phylogenetic analysis ofGh WRKY70

The 918 bp full-length GhWRKY70 ORF sequence(Gen Bank accession num ber:MH607118)w as cloned by RT-PCR.Sequence analysis show ed that the ORF of GhWRKY70 encoded 305 amino acids w ith a predicted molecular w eight of 34.65 k Da.Multiple alignm ents show ed that GhWRKY70 contained a conserved WRKY and a C2HC zinc finger dom ain and shared high identity w ith conserved functional regions in other plant species(Fig.1-A).The phylogenetic tree suggested that GhWRKY70 is a m em ber of WRKY group III(Fig.1-B).

3.2.Analysis of Gh WRKY70 expression pattern

The transcription level of GhWRKY70 in G.hirsutum cv.Xinluzao 7 w as greater in roots than in either stem s or leaves(Fig.2-A).Com pared w ith the control,V.dahliae-inoculated plants show ed low er GhWRKY70 expression at 6,24 and 48 h after infection but higher expression at 12 h (Fig.2-B).Treatment w ith SA reduced GhWRKY70 expression after 6,12,and 48 h but increased it after 24 h(Fig.2-C).Treatm ent w ith MeJA reduced GhWRKY70 expression(Fig.2-D).

3.3.Silencing of Gh WRKY70 in cotton increases tolerance to V.dahliae infection

Fig.2-Ex pression profiles of Gh WRKY70 ind uced by V.dahliae,SA,and MeJA.Total RNA w as extracted from roots of 14-d ayold seedlings 0-48 h after treatm ent.Error bars indicate the standard deviation of three biological rep licates.Statistical significance betw een m ock and treatm ent w as d eterm ined using Student's t-test(*P＜0.05;**P＜0.01).(A)Exp ression p atterns of Gh WRKY70 in various cotton organs(G.hirsutum cv.Xinluzao 7).Roots,stem s,and leaves w ere sam pled from 14-day-old seedlings grow n in a greenhouse.Different letters indicate significant d ifferences(P＜0.05)using the Duncan's m ultiple range test.(B)Exp ression of Gh WRKY70 after inoculation w ith V.dahliae.Total RNA w as ex tracted from roots of 14-day-old seed lings 0-48 h after inoculation.The m ock treatm ent w as w ater.(C-D)Accum ulation of Gh WRKY70 transcrip ts in cotton roots after treatm ent w ith SA(C)and MeJA(D).The m ock treatm ent w as w ater.

Fig.3-Increased resistance of GhWRKY70-silenced plants after V.dahliae infection in G.hirsutum cv.Xinluzao 7.Error bars represent the stand ard deviation of three biological replicates.Statistical significance w as d eterm ined using Student's t-test(*P＜0.05;**P＜0.01).(A)Relative transcript levels of Gh WRKY70 in plants infiltrated w ith TRV:00,TRV:Gh WRKY70(n=3).Total RNA w as extracted from leaves at 10 days post-infiltration.Transcript levels w ere determ ined by RT-q PCRusing Gh UBQ7 as control.(B)Disease symp toms on leaves from TRV:00 plants and Gh WRKY70-silenced plants infected by V.dahliae.(C)Disease index of TRV:00 and Gh WRKY70-silenced p lants at 15 and 20 DPI(n=30).(D)Disease sym p tom s in infected stem s of TRV:00 and TRV:Gh WRKY70 plants inoculated w ith V.dahliae.(E)Fungal biomass d eterm ined by q PCRin Verticillium-inoculated cotton p lants at 15 DPI.(F)Stem sections w ere collected at 15 DPI,plated on PDA m edium,and incubated at 25°C.Photos w ere taken after culturing for 10 days.

Fig.4-Increased resistance of Gh WRKY70-silenced plants after V.dahliae infection in G.hirsutum cv.Zhongzhim ian 2.Error bars represent the stand ard deviation of three biological replicates.Statistical significance w as d eterm ined using Stud ent's ttest(*P＜0.05;**P＜0.01).(A)Relative transcript levels of Gh WRKY70 in plants infiltrated w ith TRV:00,TRV:Gh WRKY70(n=3).Total RNA w as extracted from leaves at 10 days post-infiltration.Transcript levels w ere determ ined by RT-q PCRusing Gh UBQ7 as the control.(B)Disease sym ptom s on leaves from TRV:00 plants and Gh WRKY70-silenced plants infected by V.dahliae.(C)Disease index of TRV:00 and Gh WRKY70-silenced p lants at 15 and 20 DPI(n=30).(D)Disease sym p tom s in infected stem s of TRV:00 and TRV:Gh WRKY70 plants inoculated w ith V.dahliae.(E)Fungal biom ass d eterm ined by q PCR in Verticilliuminoculated cotton plants at 15 DPI.(F)Stem sections w ere collected at 15 DPI,plated on PDA medium,and incubated at 25°C.Photos w ere taken after culturing for 10 days.

To investigate the function of GhWRKY70 in cotton resistance to V.dahliae,TRV-based VIGS was used to generate GhWRKY70-knockdown plants.GhCHLI was used as the positive control.As expected,cotton leaves exhibited yellowing at 10 days after agroinfiltration w ith the GhCHLI construct(Fig.S1).This result show ed that the VIGSsystem w orked w ell under our experim ental conditions.RT-q PCR analysis show ed that the GhWRKY70 transcripts w ere significantly reduced in the gene-silenced plants in comparison w ith the vector control plants(P＜0.01)(Fig.3-A),indicating that GhWRKY70 was effectively silenced in these plants.Control plants displayed obvious disease symptoms 15 days post-inoculation(DPI),w ith many w ilted leaves at the bottom of the plants(Fig.3-B).Control plants w ere m ore severely affected than GhWRKY70-silenced plants.The disease index w as significantly higher in control plants than in GhWRKY70-silenced plants at 15 and 20 DPI(Fig.3-C).The phenotype in vascular tissue of GhWRKY70-silenced plants turned brow n at 15 DPI.How ever,control plants show ed greater vascular brow ning than GhWRKY70-silenced plants(Fig.3-D).There were more fungal colonies in control plants than in GhWRKY70-silenced plants(Fig.3-E,F),indicating that GhWRKY70 silencing increased V.dahliae resistance.Silencing GhWRKY70 in the resistant variety,G.hirsutum cv.Zhongzhim ian 2,also increased V.dahliae resistance(Fig.4).

3.4.Silencing of Gh WRKY70 in cotton activates the JAassociated genes and suppresses SA-associated genes

The expression levels of signaling pathw ay-associated genes,including JA response(GhPDF1.2 and GhPR3)and SA response(GhNPR1 and GhPR1)in G.hirsutum cv.Xinluzao 7 after inoculation w ith w ater or V.dahliae w ere m easured.The expression of SA-associated genes w as significantly reduced in TRV:GhWRKY70 com pared w ith TRV:00 under both the w ater(m ock)and V.dahliae treatments(Fig.5-A).However,GhWRKY70 silencing show ed no significant effect on GhNPR1 expression in the m ock treatm ent(Fig.5-B).The expression of JA-associated genes w as significantly upregulated in TRV:GhWRKY70 relative to TRV:00(Fig.5-C,D).

3.5.Overexpression of Gh WRKY70 increases susceptibility to V.dahliae in transgenic Arabidopsis plants

The expression of GhWRKY70 in transgenic plants w as confirm ed by RT-q PCR.Three lines(AOV1,AOV2,and AOV3)of transgenic plants w ith different expression levels of GhWRKY70 w ere chosen for further experiments(Fig.6-B).Four-w eek-old Arabidopsis plants w ere inoculated w ith V.dahliae.Fourteen days after inoculation,the Arabidopsis leaves began to yellow and w ilt,and the grow th of the plants w as stunted.Overexpression of GhWRKY70 reduced the resistance of Arabidopsis to V.dahliae com pared w ith the w ild type(WT)(Fig.6-A;Fig.S2).The transgenic plants had a significantly higher number of diseased leaves than the WT at 21 and 28 DPIw ith V.dahliae(Fig.6-C).q PCRanalysis show ed less fungal biom ass in the WT plants than in the transgenic plants(Fig.6-D),indicating that GhWRKY70 suppresses the defense response to V.dahliae.

Fig.5-RT-q PCRanalysis of SA and JA-associated genes in TRV:00 and Gh WRKY70-silenced cotton plants inoculated w ith V.dahliae or w ater.Error bars rep resent the stand ard d eviation of three biological replicates.Statistical significance w as d eterm ined using Student's t-test(*P＜0.05;**P＜0.01).(A-B)RT-q PCRanalysis of SA-associated genes(Gh PR1 and Gh NPR1)in TRV:00 and Gh WRKY70-silenced cotton plants inoculated w ith w ater(m ock)or V.dahliae at 2 DPI.(C-D)RT-q PCRanalysis of JAassociated genes(Gh PDF1.2 and Gh PR3)in TRV:00 and Gh WRKY70-silenced cotton plants inoculated w ith w ater(mock)or V.dahliae at 2 DPI.

Fig.6-Effects of overexp ression of Gh WRKY70 on the defense response of Arabidopsis lines to V.dahliae.Error bars represent the standard d eviation of three biological replicates.Statistical significance betw een WT and the transgenic line w as determ ined using Student's t-test(*P＜0.05;**P＜0.01).(A)Phenotyp es of Arabidopsis WT and Gh WRKY70-overex pressing transgenic lines(AOV1,AOV2,AOV3)after inoculation w ith V.dahliae.The m ock treatm ent w as w ater.(B)RT-q PCRanalysis of Gh WRKY70 in WT and Gh WRKY70-overexpressing transgenic Arabidopsis lines(AOV1,AOV2,AOV3).(C)Disease ind ex for WT and Gh WRKY70A-overexp ressing transgenic Arabidopsis lines(AOV1,AOV2,AOV3)at 21 and 28 DPI.(D)Fungal biom ass determ ined w ith q PCRin Verticillium-inoculated Arabidopsis plants at 21 DPI.

3.6.SA-associated genes areupregulated and JA-associated genes are downregulated in Gh WRKY70-overexpressing Arabidopsis

To investigate the role of GhWRKY70 in the resistance of Arabidopsis to V.dahliae,w e analyzed the transcript levels of Arabidopsis genes involved in the response to SA(AtNPR1,AtPR1)and JA (AtPDF1.2,AtPR3).The overexpressing Arabidopsis lines had higher transcription levels of SA-related m arker genes than the m ock.Inoculation w ith V.dahliae caused the expressions of AtPR1 and AtNPR1 to increase both in overexpressing Arabidopsis plants and in m ock(Fig.7-A,B).How ever,V.dahliae infection reduced the expressions of AtPDF1.2 and AtPR3 in overexpressing Arabidopsis lines.The expressions of JA-associated m arker genes increased significantly both in the WT and in the three transgenic lines after inoculation w ith V.dahliae(Fig.7-C,D).

4.Discussion

WRKY is a large family of transcription factors[18].WRKY70 is an im portant node of the SA and JA signaling pathw ays in plant defense responses[27].GhWRKY70 obtained in this study show ed close relationship w ith AtWRKY70 in the phylogenetic tree(Fig.1-B),and its response to JA treatm ent w as the sam e as that of AtWRKY70 and OsWRKY45.How ever,the SA-response of GhWRKY70 w as very special because the expression level of GhWRKY70 after SA treatm ent w as also repressed except at the timepoint of 12 h(Fig.2-C).We speculate that it m ight have som e relations to gene redundancy.Functional redundancy is an inherent feature of WRKY genes[40,41].As a tetraploid species,cotton has eight hom ologs of the WRKY70 gene(Fig.1-B),and GhWRKY70 m ight be functionally redundant w ith other WRKY70 or WRKY genes in cotton.This hypothesis needs to be validated w ith m ore precise experim ents.

AtWRKY70 knockout m utant experim ents show ed reduced plant resistance to the biotroph Erysiphe cichoracearum but increased resistance to the necrotroph Alternaria brassicicol.In contrast,upregulation of WRKY70 resulted in opposite effects[42].In rice,overexpression of OsWRKY45 increased plant resistance to Magnaporthe grisea and Xanthomonas oryzea pv.oryzea,but negatively modulated resistance to R.solani and Nilaparvata lugens[30-33].In this study,w e observed a negative m odulation of GhWRKY70 by reducing resistance to V.dahliae in the overexpression cotton line w hereas increasing resistance in the gene-silencing line.Based on the existing results from different experim ents,w e guess that the WRKY70 TFs m ay have diverse m echanism s tow ards different types of pathogens.

V.dahliae is a hem ibiotrophic pathogen of plant[43,44].JA has been reported recently to be able to enhance the defense responses against the infection of hem ibiotrophic pathogens[45].In Arabidopsis,JA increases the resistance to necrotrophic pathogens,such as Fusarium oxysporum[46,47].In cotton,suppression of GhLac1 and GhJAZ2 expression levels resulted in JA accum ulation and increased resistance to V.dahliae[13,14].Furthermore,overexpression of GbWRKY1,GhCYP94C1,and GhbHLH171 im proved cotton resistance to V.dahliae through increasing expressions of JA-associated genes[11,12,14].Sim ilar to GhLac1 and GhJAZ2,GhWRKY70 m ay acts as a negative regulator in cotton resistance against V.dahliae by repressing the expression of JA-responsive genes such as GhPR3 and GhPDF1.2.

Fig.7-RT-q PCRanalysis of SA and JA-associated genes in WT and Gh WRKY70-overex pressing Arabidopsis lines.Error bars rep resent the standard deviation of three biological replicates.Statistical significance betw een WT and the transgenic line w as d eterm ined using Stud ent's t-test(*P＜0.05;**P＜0.01).(A,B)RT-q PCRanalysis of SA-associated genes(AtPR1 and AtNPR1)in WT and Gh WRKY70-overexp ressing Arabidopsis lines inoculated w ith w ater(m ock)or V.dahliae at 2 DPI.(C,D)RT-q PCR analysis of JA-associated genes(AtPDF1.2 and AtPR3)in WT and Gh WRKY70-overex pressing Arabidopsis lines inoculated w ith w ater(m ock)or V.dahliae at 2 DPI.

Interestingly,it has been reported that SA-associated genes are also able to enhance cotton resistance to V.dahliae[15-17,48].The defense mechanism of plant is a complex netw ork.The pathogenic process of V.dahliae includes both biotrophic and necrotrophic stages[49,50].Our results show ed that both SA and JA are involved in the defense against V.dahliae.

Supplem entary data for this article can be found online at https://doi.org/10.1016/j.cj.2018.10.005.

Acknow ledgm ents

We thank Dr.William J.Gale(College of Agriculture,Shihezi University,China)for editing the m anuscript.This study w as supported by the National Key Research and Development Program of China(2016YFD0100200),Science and Technology Developm ent Program of Xinjiang Production and Construction Groups(2015AC007),Crops Breeding Project of Shihezi University(YZZX201704).