Yutin Go,Xuejun Tin,Weidong Wng,Xingru Xu,Yuqing Su,Jitin Yng,Shuonn Dun,Jinlong Li,Mingming Xin,Huiru Peng,Qixin Sun,Chojie Xie,Jun M,

a College of Agronomy and Biotechnology,China Agricultural University,Beijing 100193,China

b Institute of Biotechnology and Food Science,Hebei Academy of Agriculture and Forestry Sciences,Shijiazhuang 050051,Hebei,China

Keywords: Fusarium crown rot (FCR)Wheat Hormone Root TaOPR3

ABSTRACT Fusarium crown rot(FCR)is a soilborne disease causing severe yield losses in many wheat-growing areas of the world.Diseased plants show browning and necrosis of roots and stems causing white heads at maturity.Little is known about the molecular processes employed by wheat roots to respond to the disease.We characterized morphological,transcriptional and hormonal changes in wheat seedling roots following challenge with Fusarium pseudograminearum (Fp),the main pathogen of FCR.The pathogen inhibited root development to various extents depending on plants’resistance level.Many genes responsive to FCR infection in wheat roots were enriched in plant hormone pathways.The contents of compounds involved in biosynthesis and metabolism of jasmonic acid,salicylic acid,cytokinin and auxin were drastically changed in roots at five days post-inoculation.Presoaking seeds in methyl jasmonate for 24 h promoted FCR resistance,whereas presoaking with cytokinin 6-benzylaminopurine made plants more susceptible.Overexpression of TaOPR3,a gene involved in jasmonic acid biosynthesis,enhanced plant resistance as well as root and shoot growth during infection.

1.Introduction

Fusarium crown rot(FCR)is a severe soilborne wheat disease in many arid and semiarid regions [1].It can be caused by variousFusariumspecies,mainlyF.pseudograminearumandF.culmorum[2,3].The typical symptoms of disease-infected plants include brown discoloration on stem bases and roots,which interfered the translocation of water and nutrients in plants and caused the formation of white heads containing no or shriveled grain [4].FCR has caused large yield losses in Australia [5],USA [2],Turkey[6] and Iran [7].In recent years,the incidence and severity of the disease have rapidly increased in major wheat-producing regions in China.FCR has become a new threat to local wheat production[8,9].

The plant root system absorbs water and nutrient from the soil.Every extra millimeter of water extracted from the soil by the roots during the grain-filling stage increases wheat production by an estimated 55 kg ha-1[10].The FCR pathogens could invade roots through physical contact and cause brown necrosis.The necrosis then extends rapidly along the roots,causing collapse of distal root tissue as early as 96 h after infection [11].Reductions in root length,fresh root weight,and root diameter caused by FCR have been reported[12,13].However,because only a few cultivars were used in those studies,it was unclear whether root growth inhibition by FCR was cultivar-dependent.The molecular responses of wheat roots to FCR infection have also remained largely unexplored.It has been suggested [14] that the defense responses of roots to Fusarium pathogens are both similar to and distinct from those of aboveground organs in the model plantArabidopsis thaliana.

To date,complete resistance to FCR has not been observed in wheat globally,and only some partially resistant genotypes are available (such as Sunco,2-49,CSCR6 and EGA-Wylie) [1].Many QTL affecting FCR resistance have been identified by linkage mapping or genome-wide association mapping studies,including loci on chromosome 2D,3B,and 5D identified in multiple genetic backgrounds [3,9,15-23].However,only two candidate genes for FCR resistance have been identified.Yang et al.[22]reported that a loss of function of the dirigent geneTaDIR-B1increased resistance of wheat plants to FCR.A Fusarium head-blight(FHB)resistance geneFhb7that detoxified the toxins produced byFusariumspecies during infection also conferred resistance to FCR [23].

To elucidate the host-pathogen interactions and identify candidate genes,the responses of wheat stems to FCR have been examined at the transcriptomic,proteomic,and metabolomic levels[24].FCR-responsive genes have been found [25] to be involved mainly in antimicrobial defense,oxidative stress and signaling,and primary and secondary metabolism.Proteomic profiles of FCR-infected wheat revealed [12,26] that proteins involved in phenylpropanoid biosynthesis,plant hormone signal transduction,MAPK signaling pathway-plant,and photosynthesis were differentially expressed at multiple time points during FCR infection.Combined gene expression and metabolite analysis suggested[24,27,28] that genes and metabolites associated with pathogen sensing and signaling and benzoxazinoid biosynthesis were involved in FCR resistance.

In this study,two wheat cultivars,one susceptible and the other partially resistant to FCR,were used to investigate morphological,transcriptional,and hormonal responses of wheat roots to FCR infection.The effects of several major plant hormones on FCR resistance were investigated via exogenous application.

2.Materials and methods

2.1.Plant materials,pathogen inoculation,and growth conditions

Two Chinese wheat cultivars with known resistance to FCR:Yangao 21 (YG21,partially FCR-resistant) and Xinmai 26 (XM26,FCR susceptible) [17],were used to investigate the responses of wheat seedling roots to FCR.A panel of 20 Chinese wheat cultivars was assembled to validate the effects of FCR on root and shoot development.A second panel of 10 Chinese wheat cultivars was used to investigate the effects of exogenous phytohormone treatment on FCR resistance(Table S1).Previously described[29]overexpression lines (OE1,OE2 and OE3) of the wheat gene 12-oxophytodienoate reductase 3 (TaOPR3) involved in jasmonic acid(JA) biosynthesis were also used.

TheFpisolate NL5,kindly provided by Prof.Haiyan Hu,Henan Institute of Science and Technology,was used for inoculation.Inoculum preparation followed Jin et al.[17].NL5 was first incubated on potato dextrose agar plates for seven days at room temperature.Then about 20 pieces of mycelial discs (5 mm) were taken from the margins of the plates and transferred to a 250-mL conical flask containing 150 mL of liquid carboxymethylcellulose medium.The flask was placed in a shaking incubator at 180 r min-1and 25 °C for approximately 4 days.The spores were harvested and the spore suspensions were diluted with autoclaved water to a final concentration of 1 × 106spores mL-1and stored at -20 °C until inoculation.Tween 20 was added to the spore suspension to a final concentration of 0.1% (v/v) prior to inoculation.

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Wheat seeds were first selected by removal of small and shriveled kernels.The selected kernels were surface sterilized and germinated in Petri dishes at room temperature.The newly germinated seedlings (0.5-1.0 cm in length) were immersed in spore suspension(for inoculation withFp)or water(for mock inoculation)for 1 min.The seedlings were then wrapped and grown in a single piece of moist paper towel following Yang et al.[30].The paper towels were transferred to a container with 1/2 Hoagland solution in a glasshouse at 25°C/18°C,day/night;16 h/8 h,light/-dark;60% relative humidity;and 1200 lm light intensity (BN058C LED11/WW L1200,CW L1200;Signify Luminaires (Shanghai) Co.,Ltd.,Shanghai,China).

2.2.Measurement of FCR severity and morphological traits

Plants were first evaluated for FCR severity with a scale of 0(no obvious symptoms) to 5 (the whole plant severely to completely necrotic)following Li et al.[31].A disease index(DI)was calculated for each cultivar as (∑nX/5N) × 100,whereXis the scale value of each plant,nis the number of plants in the category,andNis the total number of plants scored for each cultivar.After disease assessment,theFp-inoculated and mock-inoculated seedlings of YG21 and XM26 were measured for maximum root length (RL),root fresh weight (RFW),root dry weight (RDW),shoot length(SL),shoot fresh weight(SFW),shoot dry weight(SDW),total fresh weight (TFW),and total dry weight (TDW) at 5,10 and 15 days post-inoculation(dpi).RL and SL were measured with a ruler.Samples were dried to constant weight at 80 °C for 48 h before dry weight measurement.The value of TFW was the sum of RFW and SFW.The value of TDW was the sum of RDW and SDW.RFW,RL,SFW,SL,TFW,and DI of the 20 cultivars were recorded at 15 dpi.Three replicates,with 10 seedlings per cultivar in each replicate,were used at each time point.

2.3.Root transcriptome analysis

The roots ofFp-and mock-inoculated seedlings of YG21 and XM26 were collected at 5 and 10 dpi.Three biological replicates,with 10 seedlings in each replicate,were collected for each cultivar at each time point and immediately frozen in liquid nitrogen.The samples were stored in dry ice and sent to Berry Genomics Co.,Ltd.(Beijing,China)for RNA-seq analysis.The libraries were sequenced on an Illumina NovaSeq 6000 platform.Cleaned reads were aligned to the wheat reference genome (Chinese Spring IWGSC RefSeq v2.1) using HISAT2 [32] and transformed into fragments per kilobase of transcript per million fragments mapped(FPKM)using featureCounts [33].

Differentially expressed genes(DEGs)were identified using the edgeR package [34] with a false discovery rate (FDR) ＜0.05 and |log2(fold change)|＞1 as thresholds.To characterize genes responsive to FCR infection,four comparison groups were designed: R5(R5M vs.R5F),S5 (S5M vs.S5F),R10 (R10M vs.R10F),and S10(S10M vs.S10F).The symbols are M for mock inoculation,F forFpinoculation,5 and 10 to indicate dpi,R for Yangao 21,and S for Xinmai 26.TopGO R package [35] and KOBAS software [36]were used for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathway analysis.FDR ＜0.05 was used as the threshold for significance.The Benjamini and Hochberg approach [37] was used for controlling the FDR of theP-values.

2.4.Gene expression analyses by qRT-PCR

Total RNA was extracted from samples using the TRIzon reagent(CW0580S;CWBIO,Beijing,China).Total RNA was reverse transcribed using the FastKing RT Kit (With gDNase) (KR116,Tiangen,Beijing,China).qRT-PCR was performed using a SuperReal PreMix Plus (SYBR Green) kit (FP205,Tiangen) on a CFX96 Real-Time PCR Detection System (Bio-Rad,Hercules,CA,USA) according to the manufacturer’s instructions.The β-tubulingene(U76895)was used as a wheat internal reference gene [38].Analysis of relative gene expression data was performed using the 2-ΔΔCT method [39].Three biological replicates,each consisting of three technical replicates,were performed.All primers are listed in Table S2.

2.5.Root hormone quantification and exogenous hormone treatment

Roots from inoculated and mock-inoculated seedlings of YG21 and XM26 were collected at 5 dpi and stored at -80 °C for further analysis.The contents of five major phytohormones including auxin,JA,salicylic acid (SA),cytokinin(CK),abscisic acid and their related compounds were quantified by MetWare Biotechnology Co.,Ltd.(Wuhan,Hubei,China) based on the AB Sciex QTRAP 6500 LC-MS/MS platform.A fold-change of either ≥1.5 or ≤0.5 was used to identify changed metabolites.Three replicates were conducted in the control and inoculation treatment,with 10 seedlings per cultivar in each replicate.

Based on the data from hormone quantification,an experiment was conducted to investigate the effects of several hormones on FCR resistance and root development.A total of 10 Chinese cultivars were used in the experiment.Briefly,methyl jasmonate(MeJA,M8640,Solarbio,Beijing,China),3-indoleacetic acid (IAA,I8020,Solarbio),and SA(S7080,Solarbio)were dissolved in absolute ethanol,and cytokinin 6-benzylaminopurine (BAP,A8170,Solarbio)was dissolved in 1 mol L-1NaOH to make a stock solution of 50 mmol L-1.Seeds were presoaked for 24 h in Petri dishes on one layer of filter paper saturated with MeJA,SA,IAA,or BAP(pH=7,adjusted with hydrochloric acid) solutions diluted with water to a final concentration of 100 μmol L-1.Control seeds were presoaked in Petri dishes with solvents of 0.2% ethanol or 2 mmol L-1NaOH(pH=7,adjusted with hydrochloric acid),depending on which solvent was used to dissolve a given hormone.The methods for plant growth,inoculation,disease assessment,and measurement of five seedling traits (RL,RFW,SL,SFW,TFW) were as described above.Three replicates were conducted in theFpinoculation treatment,with 14 seedlings per cultivar in each replicate.

2.6.Evaluation of FCR-resistance in TaOPR3 overexpression lines

3.Results

3.1.FCR infection inhibited root and shoot growth

Differing levels of resistance were observed in YG21 and XM26 during FCR infection (Fig.1a).Although the growth of both YG21 and XM26 seedlings was inhibited by FCR infection,the XM26 seedlings appeared to suffer more severe damage than the YG21.Compared with the corresponding control plants,the inoculated seedlings of XM26 showed significant reductions in RFW,RDW,SL,SFW,SDW,TFW,and TDW at 5,10,and 15 dpi.The reductions in RL were significant at 10 and 15 dpi.For YG21,the reductions in SL and TDW were significant at all three time points.The reductions in RL,RDW,and SDW were significant at 10 and 15 dpi.Significant decreases in RFW,SFW,and TFW were observed at 15 dpi(Fig.1b-i).

Fig.1.Phenotypical parameters in the response to Fusarium pseudograminearum infection in YG21 and XM26 seedlings at 5,10,and 15 days post inoculation.Values are means±SD(n=3,10 seedlings each);*,P ＜0.05;**,P ＜0.01(two-tailed unpaired Student’s t-test).R,Yangao 21;S,Xinmai 26;5,5 dpi;10,10 dpi;15,15 dpi;Mock,mock inoculation; Fp, Fp inoculation.Dpi,days post inoculation.

Among the eight seedling traits investigated,RFW was most sensitive to FCR infection.Reductions of respectively 27.5% and 33.1% were observed in YG21 and XM26 inoculated seedlings at 15 dpi (Table S3).Similar to YG21 and XM26,the mean RL,RFW,SL,SFW and TFW of 20 randomly selected cultivars were all significantly reduced (Fig.2).The mean RFW decreased from 0.24 to 0.15 g,whereas that of SFW decreased from 0.26 to 0.15 g(Table S4).In general,in these cultivars,a positive association between DI and the FCR-caused reductions of RL,RFW,SL,SFW and TFW was observed (Fig.S1).

Fig.2.Phenotypical parameters in the response to Fusarium pseudograminearum infection in 20 wheat cultivars at 15 days post inoculation.Lines connect the same cultivar in mock-inoculated and Fp-inoculated seedlings;mean of each cultivar is plotted as a dot,with each cultivar represented by 30 seedlings;*, P ＜0.05;**, P ＜0.01 (two-tailed paired Student’s t-test).Mock,mock inoculation; Fp, Fp inoculation.

3.2.Genes responsive to FCR infection in wheat roots

A mean of 74.5 million clean reads per sample was generated,with 88.2%-93.8% of reads mapping to the reference genome of Chinese Spring IWGSC RefSeq v2.1.The Pearson correlation coefficients among the three replicates for each sample ranged from 0.985 to 0.996 (Fig.S2),indicating that the three replicates were highly consistent.Principal component analysis (PCA) indicated that the variation in gene expression was caused by sampling time point,inoculation type,and cultivar (Fig.3a).In total,respectively 1983 (1804 up-and 179 downregulated) and 2316 (2173 up-and 143 downregulated) genes were differentially expressed betweenFp-inoculated and mock-inoculated seedlings of YG21 at 5 and 10 dpi.For XM26,DEGs responsive to FCR infection numbered respectively 1566 (1278 up-and 288 downregulated) and 1434(1049 up-and 385 downregulated) (Fig.3b).The Venn diagram shows that 1279 DEGs were consistently expressed in YG21 at 5 and 10 dpi,whereas 564 DEGs were shared in XM26 across the two time points.A total of 482 DEGs were common to YG21 and XM26 seedlings at 5 and 10 dpi.While respectively 521 and 781 genes were induced specifically in YG21 at 5 and 10 dpi,483 and 514 genes were specific to XM26 at these times(Fig.3c;Table S5).

Fig.3.PCA analysis,differentially expressed genes(DEGs)and Venn diagram of YG21 and XM26 seedlings at 5 and 10 days post inoculation.(a)Principal component analysis(PCA) showing the divergence of the respective transcriptomes in response to cultivar,time,and inoculation type.(b) The number of DEGs responsive to FCR infection in Yangao 21 and Xinmai 26.(c-e) Venn diagrams showing the overlap of DEGs,enriched biological processes,and KEGG pathways in Yangao 21 and Xinmai 26 seedlings.R,Yangao 21;S,Xinmai 26;5,5 dpi;10,10 dpi;M,mock inoculation;F, Fp inoculation.Dpi,days post inoculation.

3.3.Functional analysis of FCR-responsive genes in wheat roots

To identify the molecular mechanisms that underpin the inhibition of root growth caused by FCR infection,the biological processes and KEGG pathways associated with the DEGs at 5 and 10 dpi were further investigated.A wide range of biological functions were interrupted by FCR infection in the two cultivars(Table S6).In total,238 and 264 biological processes were significantly enriched in YG21 inoculated seedlings at two time points,whereas 227 and 375 biological processes were enriched in XM26 seedlings.Of these enriched biological processes,151 were common to YG21 and XM26 across 5 and 10 dpi.Many of these biological processes were associated with hormone or defense responses: hormone biosynthetic process,response to jasmonic acid,response to cytokinin,response to fungus,detoxification,and others.(Fig.3d;Table S6).In addition to these common processes,10 processes were consistently and specifically enriched in YG21 at 5 and 10 dpi.They included melatonin biosynthetic process,protein phosphorylation,and amine metabolic process.Nine biological processes were enriched specifically in XM26 at 5 and 10 dpi,including response to hormone,detection of ethylene stimulus,and cellular response to reactive oxygen species.A total of 53 and 180 biological processes were specific to YG21 or XM26 at single time points.Differences in genetic backgrounds and infection time may have contributed to these cultivar-and time point-specific biological processes (Fig.3d;Table S6).

KEGG enrichment analysis showed that pathways responsive to FCR infection were highly conserved between YG21 and XM26 across two time points (Fig.3e;Table S7).Fifteen pathways,including plant hormone signal transduction,plantpathogen interaction,alpha-linolenic acid metabolism,diterpenoid biosynthesis,and phenylpropanoid biosynthesis were enriched in FCR-infected roots of YG21 and XM26 at two time points (Figs.3e,4).Of these pathways,the phenylpropanoid pathway contained 27 DEGs encoding phenylalanine ammonia lyase(PAL) which were up-regulated by FCR infection.Similarly,the enriched alpha-linolenic acid metabolism pathway contained three lipoxygenase (LOX) genes,two allene oxide synthase(AOS) and ten 12-oxophytodienoate reductase (OPR) genes.These genes are involved in JA biosynthesis and were upregulated in at least one cultivar (Table S5).The pathways of ubiquinone and other terpenoid-quinone biosynthesis were specifically up regulated in YG21 at 5 and 10 dpi,whereas linoleic acid metabolism was enriched in XM26 at both 5 and 10 dpi.Four pathways were enriched specifically in YG21 at 10 dpi,whereas three pathways were enriched in XM26 at 5 dpi (one pathway) and 10 dpi (two pathways) (Fig.3e;Table S7).

To verify the RNA-seq results,qRT-PCR analysis was performed withTaOPR3and six randomly selected DEGs.In general,the transcriptional expression pattern of these genes correlated well(R2=0.80) with the expression changes obtained from RNA-seq(Fig.S3).AlthoughTaOPR3was only significantly changed in YG21 at 10 dpi(with log2fold change ＜1)in transcriptome analysis,qRT-PCR analysis showed that this gene was upregulated in both YG21 and XM26 at 10 dpi (P＜0.05;Table S8).

3.4.Treatment with MeJA increased FCR resistance

Hormone quantification showed that 11 phytohormones responded to FCR infection in YG21.Most of these compounds(IPA,ICA,IAA-Asp,IAA-Glc,IAA-Phe-Me,IAA-Val-Me,and OxIAA)are related to auxin(Fig.S4).The levels of IPR and cytokinintrans-Zeatin (tZ),which belong to the group of CK,were upregulated in YG21.For XM26,the contents of three JA derivatives (OPC-4,JAVal,and JA-Ile) and IAA-Phe-Me were upregulated after inoculation.The levels of salicylic acid beta-glucoside (SAG) and IAAglucose (IAA-Glc) were decreased in both of YG21 and XM26 after inoculation (Fig.S4).

Based on the transcriptome and hormone analysis,the effects of exogenous application of MeJA,BAP,SA and IAA on FCR resistance and seedling growth were further investigated in a panel of 10 randomly selected cultivars.Treatment with MeJA significantly increased the disease resistance of the panel at 15 dpi.In contrast to control plants (DI=57.55),the mean DI of cultivars presoaked with MeJA decreased to 43.21.The RL,RFW,SFW and TFW of inoculated seedlings were also significantly increased after treatment (Fig.5).In contrast,treatment with BAP significantly increased the FCR susceptibility of 10 cultivars.Their mean DI increased from 53.44 (control plants) to 85.07 (BAP treated plants).The RL,RFW,SL,SFW and TFW were also significantly reduced compared with control plants (Fig.5).The treatment of IAA and SA did increase the disease resistance of several cultivars,but the effects were not significant when the data from 10 cultivars were used (Fig.S5).

3.5.Functional validation of TaOPR3 in wheat FCR response

No significant difference in root and shoot traits was observed between the overexpression lines and WT plants under mock inoculation (Fig.S6a,b).The abundances ofTaOPR3in OE1,OE2,and OE3 overexpression lines were all significantly higher than those in WT plants(Fig.S6c).After inoculation,FCR caused severer damage in the root system of WT than in the three overexpression lines(Fig.6a).The expression levels ofTaOPR3in OE1(5.83),OE2(4.49),and OE3 (4.10) were higher than those in WT plants (Fig.6b).The DIs of the three overexpression lines were 51.33 (OE1),48.00(OE2),and 26.33(OE3),whereas that of WT was 74.33.The RL,SL,and SFW of the three transgenic lines were also significantly improved during FCR infection (Fig.6c).The largest increase was SFW (0.060 g) compared with that of WT (0.032 g).

4.Discussion

4.1.Growth-defense tradeoffs may contribute to inhibition of root growth by FCR

As in previous studies,various levels of reduction in root (RL,RFW,and RDW) and shoot (SL,SFW,SDW) traits were observed inFp-inoculated seedlings of YG21 and XM26 [12,13,40].Decreased RL,RFW,SL and SFW was also detected in the panel of 20 randomly selected wheat cultivars at 15 days after inoculation(Fig.2;Table S4).Given the direct roles of roots in nutrient and water uptake,the damage to the root system caused by FCR may weaken the plants and facilitate the colonization and spread of the pathogens during infection.Retarded root growth during FCR infection could also increase plants’ susceptibility to drought stress,an abiotic stress condition known[41,42]to favor the occurrence and incidence of FCR.In the present study,the biological process of regulation of abscisic acid activated signaling pathway was downregulated specifically in XM26 at 10 dpi.The abscisic acid signaling pathway is known [43,44] to play a role in droughttolerance mechanisms in wheat.

Plant defense is energy-intensive.Expression of genes and compounds involved in defense could impair agronomic traits in various crop species[45].In the present study,root traits of YG21 and XM26 were both affected by FCR to various extents at 5 and 10 dpi.Although several FCR-induced genes such asTraesCS3D03G0551000andTraesCS6A03G0689100are homologous to known genes(OsPIN3tandAt2g27230) involved in root development in rice orArabidopsis[46,47],enrichment analysis revealed that many of the enriched biological processes common to YG21 and XM26 were associated with defense and hormone responses (Table S6).Previous studies [26,28] also showed that genes and proteins involved in photosynthesis were downregulated in wheat seedling stems during FCR infection at early developmental stages.These results suggested that the redistribution of energy from plant growth to defense response may function in the inhibition of root growth at the early developmental stage during FCR infection.It is desirable to investigate the root response of adult wheat plants to FCR infection,considering that all of the current studies of the association between root inhibition and FCR were performed using wheat seedlings [11-13,40].

4.2.Genes involved in phenylpropanoid biosynthesis of may function in FCR resistance

To investigate whether similar defense responses were employed by roots and stems against FCR,we compared the FCRresponsive pathways in two organs among the present study and two previous studies[26,28].Most of the enriched KEGG pathways in roots in this study were not overrepresented in stems.The only exception was phenylpropanoid biosynthesis,which was induced by FCR infection at the transcriptomic(roots and stems),proteomic(stems),and metabolic (stems) levels [26,28].This pathway operates in plant development and plant-environment interactions[48].Phenylpropanoid compounds are involved in providing the structural and chemical barriers for resistance to pathogen infection [49].Accumulation of lignin,an end product of the phenylpropanoid biosynthesis pathway,increased FCR resistance in wheat [22].Several PAL genes such asTraesCS4A03G0998200andTraesCS6A03G0613600,which encodes the gateway enzyme for phenylpropanoid biosynthesis pathway [48,50] and was repeatedly identified in this and previous study[28],may also be potential targets to manipulate for the improvement of FCR resistance(Tables S5,S7).

4.3.CK molecules may negatively regulate plant defense responses to FCR

CK are involved in regulation of many plant processes including cellular proliferation and differentiation,control of the morphological balance between shoot and root tissue,and delay of leaf senescence [51,52].Although CK were involved in immunity against several plant diseases,they may also act as positive regulators of pathogen virulence [53-58].During infection,a large diversity of plant fungal species produce CK that are structurally similar to plant CK.These fungal-produced CK can delay senescence of the infection site on plants and thus provide more nutrients for fungal growth[59-61].In this study,the DEGs associated with the biological process ‘‘response to cytokinin” were upregulated in both YG21 and XM26 at 5 and 10 dpi (Table S6).The KEGG pathway for biosynthesis of zeatin,a plant hormone in the CK family,was upregulated in YG21 seedlings at 5 and 10 dpi and in XM26 at 10 dpi (Fig.4).The enrichment of CK-related pathway by FCR may contribute to the spread of the pathogens in plants.

Fig.4.Dot plot shows the enriched KEGG pathways of upregulated differentially expressed genes (DEGs) in YG21 and XM26 seedlings.The sizes of dots indicate the gene ratio of DEGs in each pathway.The color indicates the degrees of significance as FDR values.Only pathways showing FDR ＜0.05 are visualized.R,Yangao 21;S,Xinmai 26;5,5 dpi;10,10 dpi.Dpi,days post inoculation.

Soaking seeds in BAP solution increased FCR severity and retarded root and shoot growth of 10 randomly selected wheat cultivars at 15 dpi (Fig.5).The increased FCR susceptibility of wheat plants after treatment with BAP suggested that this compound,which is a synthetic CK and used as plant growth regulator,may negatively regulate plant resistance to FCR.In addition to BAP,hormone quantification analysis showed that the level of tZ also increased in FCR-infected roots of YG21 seedlings at 5 dpi,whereas the content of N6-isopentenyl-adenine-7-glucoside (iP7G) was reduced in XM26 (Fig.S4).Previous study showed that tZ contributed to immunity against (hemi-) biotrophic pathogens in some plant species [56-58,62].As the effects of CK molecules on resistance may vary for different diseases,the exact roles of these CK molecules in FCR resistance invite further investigation.

Fig.5.The effects of methyl jasmonate (MeJA) and 6-benzylaminopurine (BAP) treatment on disease index and seedling growth of 10 wheat cultivars at 15 days post inoculation.Lines connected the same cultivar in mock-treated and MeJA-or BAP-treated seedlings.Means of each cultivar are plotted in dots,with each cultivar represented by 42 seedlings.*, P ＜0.05;**, P ＜0.01 (two-tailed paired Student’s t-test).

4.4.Overexpression of a key gene in JA biosynthesis increased resistance to FCR

JA and its derivatives are involved in diverse developmental processes and defense responses.Application of MeJA to the wheat seedlings has been shown [38,63] to reduce FCR severity.The biological process and KEGG pathways associated with JA were upregulated in YG21 and XM26 at 5 and 10 dpi(Fig.4;Tables S6,S7).In particular,10 12-oxophytodienoate synthase (OPR) genes,which are typical marker genes for JA biosynthesis,were induced in the roots of YG21 or XM26 during FCR infection(Table S5).Presoaking seeds in MeJA solution reduced the mean DI of 10 cultivars from 57.55 (control plants) to 43.21 (Fig.5).

Three overexpression lines ofTaOPR3showed increased resistance to FCR compared with WT plants (Fig.6) [29].The mean DI of these three overexpression lines (41.89) was much lower than that of control plants (74.33).Previous study [64] showed thatTaOPR3also conferred resistance to FHB,another disease of wheat spikes caused mainly byFusarium graminearum.Because both FHB and FCR are caused byFusariumspecies,some molecular responses of wheat plants to these two diseases could be similar [4].In fact,two toxin-detoxification genes conferring broad resistance to both diseases have been identified[23,65].However,in previous studies[8,66],cultivars with resistance to FHB did not necessarily show resistance to FCR.QTL conferring resistance to the two diseases were mapped to different chromosomes in same mapping population [66].For these reasons,caution should be taken in merging research data from these twoFusariumdiseases.

Fig.6. TaOPR3 increased resistance to FCR.(a) Overexpression of TaOPR3 increased disease resistance and seedling growth of Fp-inoculated seedlings.Scale bar,3 cm.(b)Expression of TaOPR3 in three overexpression lines (OE1,OE2,OE3) and wild-type (WT) cultivar Kenong 199 at 10 days post inoculation (dpi).(c) Mean number of disease index and five seedling traits in samples following FCR infection at 10 dpi.Values are means ± SD (n=3,10 seedlings each);*, P ＜0.05;**, P ＜0.01 (two-tailed unpaired Student’s t-test).

5.Conclusions

Infection with FCR inhibited wheat seedling root growth at the early developmental stage,with the extent of inhibition dependent on plant resistance to the disease.Combined transcriptional and hormonal analysis indicated that some hormones may function in root response to FCR.Whereas presoaking seeds in BAP made plants more susceptible to disease,treatment with MeJA increased resistance.Overexpression lines ofTaOPR3,a key gene in JA biosynthesis,showed increased resistance as well as root and shoot growth during FCR infection.

Data availability

The RNA-seq data in this study have been deposited into the National Center for Biotechnology Information (NCBI) Sequence Read Archives (SRA) with accession code PRJNA900521.

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

Yutian Gao:Writing -review &editing,Formal analysis,Writing -original draft,Investigation,Validation,Conceptualization.Xuejun Tian:Investigation,Validation.Weidong Wang:Investigation,Validation.Xiangru Xu:Investigation,Validation.Yuqing Su:Investigation,Validation.Jiatian Yang:Investigation,Validation.Shuonan Duan:Investigation,Validation.Jinlong Li:Investigation,Validation.Mingming Xin:Writing -review &editing,Resources.Huiru Peng:Writing -review &editing,Resources.Qixin Sun:Writing -review &editing.Chaojie Xie:Writing -review &editing.Jun Ma:Funding acquisition,Writing -review &editing,Formal analysis,Writing -original draft,Conceptualization.

Acknowledgments

This research was supported by the State Key Laboratory of North China Crop Improvement and Regulation,National Key Research and Development Program of China (2018YFD0300501),and National Natural Science Foundation of China (31872865).

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2023.01.006.