Genome-wide investigation of defensin genes in peanut (Arachis hypogaea L.) reveals AhDef2.2 conferring resistance to bacterial wilt

Kai Zhao,Rui Ren,Xingli Ma,Kunkun Zhao,Chengxin Qu,Di Cao,Qian Ma,Yingying Ma,Fangping Gong,Zhongfeng Li,Xingguo Zhang,Dongmei Yin*

College of Agronomy &Center for Crop Genome Engineering,Henan Agricultural University,Zhengzhou 450046,Henan,China

Keywords:Peanut Bacterial wilt Defensin Expression analysis Resistance

ABSTRACT Peanut(Arachis hypogea L.)bacterial wilt(BW)is a devastating disease caused by Ralstonia solanacearum that results in severe yield and quality losses.Plant defensins are short cysteine-rich peptides with antimicrobial activity.The role of defensin genes(AhDef)in peanut is unclear.A genome-wide investigation of AhDef genes was undertaken,and 12 identified AhDef genes were classified into two groups containing the gamma-thionin domain formed by four disulfide pairs:Cys1-Cys8,Cys2-Cys5,Cys3-Cys6,and Cys4-Cys7.In silico analysis revealed that AhDef genes showed highly conserved architectural features and contained cis-elements associated with phytohormone signaling and defense responses.A highly resistant cultivar,H108 (R) and a susceptible accession,H107 (S) were tested by R.solanacearum inoculation.H108(R)showed fewer symptoms than H107(S)owing to inhibition of bacterial reproduction and spread in the vascular bundles of roots and stems.In a transcriptomic expression profile,AhDef genes,particularly AhDef1.6 and AhDef2.2,were up-regulated in H108(R)compared with H107(S)under R.solanacearum infection and phytohormone treatment.Subcellular localization showed that the AhDef1.6 and AhDef2.2 proteins were both expressed specifically on the plasma membrane.Overexpression of protein fusion AhDef2.2-YFP in Nicotiana benthamiana and peanut leaves increased resistance to R.solanacearum,suggesting its role in response to BW infection.AhDef2.2 may be valuable for peanut resistance breeding.

1.Introduction

Peanut (Arachis hypogaea L.) is a major oil and food crop.More than 95% of the peanut cultivation area is in Asia and Africa [1].Peanut plants are subject to various bacterial,fungal and viral diseases throughout their growth,development,and storage.Bacterial wilt(BW)is one of the most destructive soil-borne diseases affecting peanut production [2,3].Peanut BW is caused by the Gramnegative bacterium Ralstonia solanacearum,which results in plant wilt,severely reducing yield and quality [4,5].BW is widespread and easily transmissible,and threatens peanut production in China and worldwide [6].

The use of cultivars with durable broad-spectrum resistance is the most efficient,economical,and sustainable strategy for crop disease management.However,identification and deployment of genes conferring peanut resistance to BW have not been successful to date.Quantitative trait loci(QTL)contribute to peanut resistance to BW.The main QTL have been mapped [2,7] to a region rich in resistance genes (R genes) with nucleotide binding site–leucine rich repeat (NBS-LRR) motifs on peanut chromosome B02.Two BW R genes,ERECTA [8] and RRS1-R [9],have been cloned in Arabidopsis thaliana,and encode a receptor-like protein kinase (RLK)and an NBS-LRR protein,respectively.Homologs of RRS1-R and ERECTA have been cloned in peanut as AHRRS5 [3] and AHRLK1[5],respectively,and transgenic tobacco lines overexpressing AHRLK1 and AHRRS5 showed increased resistance to R.solanacearum.These studies suggested that peanut BW resistance genes were likely to be typical R genes,such as those encoding RLK and NBS-LRR proteins.However,R genes usually exhibit race-specific resistance and have a narrow resistance spectrum [10].

Defensins,small peptides with a broad spectrum of antimicrobial activities,are widespread in plant and animals as essential components of their immune system [10].Plant defensins (PDFs)were first isolated from seeds of wheat,barley and carrot [11,12]and shown to inhibit the growth of fungi.Thereafter,numerous PDF genes were cloned from various monocotyledonous and dicotyledonous plants(Table S1),and were shown[13]to function in plant defense response to various fungal and bacterial pathogens.Most PDFs function as strong growth inhibitors of fungal pathogens,such as Botrytis cinerea,Fusarium culmorum,Colletotrichum lindemuthianum,and C.sphaerospermum [10].Other PDFs inhibit bacterial growth,though the inhibitory effect is not as strong as that against fungal growth [13].For example,the spinach (Spinacia oleracea) PDFs So-D2 and So-D7 show highly efficient broad-spectrum antimicrobial activities against bacterial pathogens such as R.solanacearum [14].PDFs can also increase plant resistance to insects by inhibiting the activities of αamylase and trypsin in the gut of insects such as Spodoptera liturata,and have been used in the genetic engineering of plant insect resistance [15–17].PDFs can also block ion channels and participate in plant adaptation to adverse external stresses,such as drought [18],zinc [19] and the heavy metal cadmium [20].

PDFs with broad-spectrum disease resistance have been successfully applied in molecular breeding to increase disease resistance in crops including rice [21],wheat [22],soybean [23],and cotton [24].However,there are few studies of the isolation and functional identification of PDFs in A.hypogaea.Recently,we identified by germplasm screening a peanut cultivar,H108 that is highly resistant to R.solanacearum.The objective of the present study was to identify the role of defensin genes in resistance to R.solanacearum in H108.

2.Materials and methods

2.1.Plant materials and R.solanacearum inoculation

Arachis hypogea H108 is highly resistant to bacterial wilt caused by R.solanacearum,whereas Arachis hypogea H107 is susceptible.The two cultivars were bred by Professor Yin,Henan Agricultural University.They were planted in plastic boxes (40 × 60 cm) in a greenhouse with environmental conditions.The tobacco(Nicotiana benthamiana) plants were grown in plant growth rooms at 24 °C under a 16 h light (15,000 lx)/8 h dark cycle with 60% humidity.

A virulent R.solanacearum isolate was obtained from the Institute of Plant Protection,Henan Academy of Agricultural Sciences.The bacterial pathogen was streaked on triphenyl tetrazole chloride (TTC) agar medium containing 0.5 g L-12,3,5-triphenyltetrazolium chloride,5.0 g L-1peptone,0.1 g L-1casein hydrolysate,2.0 g L-1D-glucose and 15.0 g L-1agar [25].Colonies of R.solanacearum were picked with sterile toothpicks and incubated in TTC broth in a shaker at 200 r min-1at 28 °C for 2 days.Inoculum was prepared by adjusting the concentration of bacterial cells to an optical density of OD600nm=0.5 corresponding to approximately 108CFU mL-1with a Nanodrop 2000c spectrophotometer (Thermo Fisher Scientific,San Jose,CA,USA).

Inoculation of peanut seedlings with R.solanacearum employed a root-tip cutting method with minor modification[4].Root tips of 3-week-old peanut seedlings were excised and the wounded plants were inoculated with a solution containing approximately 108-CFU mL-1R.solanacearum in a growth chamber maintained at 28°C under a 16 h light/8 h dark cycle with 60%humidity.Tobacco and peanut leaves transiently overexpressing candidate genes were inoculated with R.solanacearum for estimation of resistance following Zhang et al.[3].For each leaf,100 μL of inoculum with three bacterial concentrations (108,107,and 106CFU mL-1) was infiltrated using a syringe without a needle.Mock inoculations with TTC broth were used as controls.

2.2.Pathological section analysis

Pathological section analyses were performed by hand-slicing primary roots of peanut seedlings,10 days post inoculation (dpi).Longitudinal root sections were visualized and photographed with a Stemi-305 stereomicroscope (Carl Zeiss,Inc.,Jena,Germany).Histological analysis of cellulose accumulation in root tip cell walls was performed using toluidine blue(TB)staining[26].Briefly,root tips were cut into an appropriate size (1–3 mm) and embedded with acetone and resinene (HMDS,Ted Pella Inc.,Redding,CA,USA) following fixation and dehydration at 28 °C.The resin blocks were cut to 1.5 μmol L-1thin slices using a Leica semi-thin slicer(Leica Microsystems GmbH,Wetzlar,Germany)and the slices were stained with toluidine blue solution (0.1%,weight/volume [w/v]).After dehydration in ethanol,the tissues were sealed and placed onto microscope slides.The samples were observed and photographed with the stereomicroscope.R.solanacearum-infected root tissues were also treated with acetone and EMBed reagent(Servicebio,Wuhan,Hubei,China) for ultrathin sectioning,as described previously [27].The samples were observed and photographed under a transmission electron microscope (Hitachi,Pleasanton,CA,USA).

Bacterial pathogens causing peanut wilt were isolated and identified as described previously[28].First,root sections of the wilted plants were examined to check whether whitish bacterial ooze was released in water.Root sections were collected,surface-disinfected with 75%(volume/volume[v/v])ethanol for 20 s,and washed three times with sterile water,and each section was ground in 2 mL Tris buffer (pH 7.0).Samples of 50 μL of the 100-fold diluted supernatant of the grinding fluid were processed for bacterial isolation by plating on TTC agar medium.Following incubation at 28 °C for 2 days,the bacterial colonies were observed and photographed under the stereomicroscope.The R.solanacearum colonies on the TTC agar medium were treated as the positive control.

2.3.Illumina RNA-seq and comparative transcriptome analysis

The H108(R)and H107(S)seedlings were used for comparative transcriptome analysis of peanut responses to R.solanacearum.Peanut seedlings mock-inoculated and infected with R.solanacearum were collected at 0,1,and 7 dpi.Each sample was independently collected with three biological replicates and stored at-80 °C immediately after freezing in liquid nitrogen.Total RNA of each sample was extracted using an RNA Simple Total RNA Kit(Tiangen,Beijing,China).RNAs from root,stem,and leaf were quantified with a Nanodrop 2000c spectrophotometer (Thermo Fisher Scientific,Waltham,MA,USA)and were mixed in equal proportions.Approximately 3 μg of each RNA mixture was used for RNA-seq library construction with the NEB Next Ultra RNA Library Prep Kit for Illumina(New England Biolabs,Beijing,China).An Illumina Hi-Seq X(Illumina,San Diego,CA,USA)RNA-sequencing system was employed for transcriptome analysis.RNA-seq data processing,gene annotation,differential gene expression,gene ontology (GO,http://geneontology.org/) and Kyoto Encyclopedia of Genes and Genomes (KEGG,https://www.kegg.jp/) pathway enrichment analyses were performed as described previously[29].The expression patterns of AhDef genes in tissues were determined from peanut transcriptomic data available in PeanutBase(https://peanutbase.org/).Tissue-specific expression profiles of these AhDef genes were determined from RNA-seq raw data of leaves,roots,flowers,petals,stamens,and fruits.Log10(TPM+1)normalization was performed on the expression data.The standardized data were drawn with R software,and the results were visualized using TBtools (https://github.com/CJ-Chen/TBtools/releases).

2.4.Genome-wide identification of peanut AhDef genes

For the genome-wide identification of AhDef genes,the A.hypogaea protein dataset was retrieved from PeanutBase.A hidden Markov model (HMM) profile of the defensin domain (PF00304) was used to identify peanut AhDef proteins using the HMM from HMMER3.2.1 software (http://hmmer.org/) with default parameters.After redundancies were removed,domain verifications of candidate genes were performed in the NCBI-CDD database(https://www.ncbi.nlm.nih.gov/cdd/). Putative amino acid sequences of AhDef genes were verified by submission to PFAM(http://pfam.xfam.org/) and SMART (http://smart.embl-heidelberg.de/) to identify the conserved defensin domain at an E-value threshold of 0.01.AhDef proteins without conserved PDF domains were removed.The confirmed AhDef gene family members were further characterized by their biophysical properties:aa length,molecular weight (MW),and theoretical protein isoelectric point(pI),with ExPASy online software (http://cn.expasy.org/tools).The chromosome location information of AhDef gene family members was extracted from the A.hypogaea genome annotation file(https://www.peanutbase.org/),and the chromosome location map of the AhDef genes was drawn with MG2C software (http://mg2c.iask.in/mg2c_v2.0/).Motif analysis of AhDef proteins was performed with MEME (https://meme-suite.org/meme/index.html).

Phylogenetic analysis and sequence alignments of AhDef genes were performed with the full-length deduced aa sequences of AhDef proteins and their homologs in other plant species(Table S1).The phylogenetic tree was constructed based on the results of sequence alignment using MEGA 7.0 (https://www.mega.com/) with the neighbor-joining method and a bootstrap value of 1000 replicates.Based on the phylogenetic tree,homologs in other species showing close phylogenetic relationships were aligned and analyzed for protein identities using Genius Prime software (BioMatters,Ltd.,Auckland,New Zealand).The structure of AhDef peptides was constructed by homology modeling method SWISS-MODEL server(https://swissmodel.expasy.org).To predict a structural model with an accuracy equal to that of a crystallographic structure,a template with ＞30%sequence identity to a target protein was used for model building.First,loop structures of templates were applied to the amino acid sequence of plant defensin for loop modeling.Then side chains were computed and generated using a backbone-dependent rotamer library.Finally,we mapped these structures with PyMOL (https://pymol.org/2/).

The promoter sequence (～2000 bp) of peanut AhDef genes was obtained from PeanutBase.Possible cis-acting elements were predicted with PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) and visualized with TBtools.

2.5.Expression profiling of AhDef genes by qRT-PCR

The expression profiles of AhDef genes in response to R.solanacearum infection and phytohormone induction were determined by qRT-PCR.Root,stem,and leaf tissues of mock-inoculated (with double-distilled water) and R.solanacearum infected H108(R) and H107 (S) seedlings were collected at 0,0.5,1,and 7 dpi.Threeweek-old seedlings of H108 (R) and H107 (S) were used for salicylic acid (SA),methyl jasmonic acid (MeJA),and abscisic acid(ABA) treatments.The optimal concentrations of phytohormones were determined from those used for other plants.Peanut seedlings were sprayed with 3 mmol L-1SA,100 mmol L-1MeJA,and 10 μg mL-1ABA,as well as double-distilled water used as control.Leaves of treated peanut seedlings were collected at 0,0.5,1.0,and 7.0 dpi.Each sample was independently collected with three biological replicates and stored at -80 °C immediately after freezing in liquid nitrogen.

Total RNA was extracted as described in Section 2.5 and reverse-transcribed using the PrimeScript RT reagent Kit with gDNA Eraser(Takara,Dalian,Liaoning,China)following the manufacturer’s instructions.The resulting first-strand cDNA was subjected to quantitative real-time PCR (qRT-PCR) as described previously [30].Gene-specific primers were designed from transcript sequences of AhDef genes using Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) (Table S2).The gene AhActin7 (XM_025826875) was used as an internal reference control to normalize the total amount of cDNA in each reaction(Table S2).PCR was conducted,and only primers that amplified a single product (approximately 150–250 bp) were selected for qRT-PCR.The total reaction system of qRT-PCR was performed in a 20 μL reaction volume comprising 2.0 μL of 5-fold diluted firststrand cDNA (approximately 20 ng),0.8 μL of each forward and reverse primer(10.0 μmol L-1),10.0 μL of 2×SYBR Green I Master Mix (Takara) and 6.4 μL of sterile distilled H2O.All reactions were performed in 96-well reaction plates using a Bio-Rad CFX-96 Realtime PCR System (Bio-Rad,Hercules,Canada) with three technical replicates.The following PCR conditions were used:95 °C for 3 min,followed by 40 cycles at 95 °C for 15 s,58 °C for 30 s and 72 °C for 30 s,and then at 68 °C for 5 min.The expression of candidate genes was quantified using the relative quantification(2-ΔΔCT) method as described previously [31].

2.6.Subcellular localization

Recombinant plant expression vectors were obtained by inserting the full length coding sequence (CDS) of AhDef genes without termination codons between the Cauliflower mosaic virus 35S promoter and yellow fluorescent protein (YFP) gene of the vector pCambia1300-YFP.Specific primers with overlapping ends were designed from the full-length CDS of AhDef genes using Primer Premier 5.0 software (Premier,Palo Alto,CA,USA) (Table S2).The fragment of the AhDef genes was then amplified with the cDNA and the specific primers using the PrimerSTAR Max DNA Polymerase (Takara).PCR products were purified and then cloned into the linearized pCambia1300-YFP vector using the Seamless Assembly Cloning Kit (CloneSmarter,Houston,TX,USA) following the manufacturer’s instructions.Vectors were transformed into Escherichia coli DH5α competent cells (Tiangen,Beijing,China)according to the manufacturer’s instructions.Each single E.coli colony was picked and inoculated in 50 mL Luria–Bertani broth[tryptone (1.0%,w/v),yeast extract (0.5%,w/v),and NaCl (1.0%,w/v),pH 7.0] containing 50 ng mL-1of kanamycin.Bacterial cells were pelleted by centrifugation of cultures at 3000g for 10 min following culturing at 37 °C with 200 r min-1shaking for 12–16 h.Following mass replication of the bacterium,plasmid DNA was extracted using the Endo-Free Plasmid Maxi Kit (Omega Bio-tek,Norcross,GA,USA).Plasmid DNA was concentrated(about 2.0 μg μL-1) for protoplast transfection and subcellular localization.

Arabidopsis protoplast isolation and transfection were conducted following Yoo [32–33] with minor modification.Leaves of 3-week-old A.thaliana plants were cut in 0.5–1.0 mm strips and transferred to freshly prepared enzyme solution[1.0%(weight/volume,w/v) Cellulase R10,0.5% (w/v) (Yakult,Japan),Macerozyme R10 (Yakult),500 mmol L-1D-mannitol,20 mmol L-1KCl,and 20 mmol L-12-morpholinoethanesulfonic acid (Solarbio) (pH 5.7),10 mmol L-1CaCl2,0.1% (w/v) Albumin Bovine V (Solarbio)].After incubation at 28 °C in the dark with rotations of 30 r min-1for 5–6 h,the released protoplasts were purified and adjusted to a suitable density.Plasmid DNA was transfected into protoplasts by the PEG CaCl2-mediated method.After incubation in the dark at 23 °C for 12 to 24 h,the fluorescence of YFP or YFP-protein fusions was viewed under a LSM710 confocal laser scanning microscope (Carl Zeiss).For subcellular localization,red chlorophyll fluorescence was used to indicate the intracellular location of chloroplasts.

2.7.Transient overexpression of AhDef genes in tobacco and peanut leaves

AhDef genes were transiently overexpressed in tobacco and peanut leaves through the Agrobacterium-mediated method.Briefly,the recombinant vectors and the empty vector pCambia1300-YFP were transfected into Agrobacterium tumefaciens strain EHA105,individually.A.tumefaciens were grown on lysogeny broth agar plates with the appropriate antibiotics for 16–18 h,after which a single colony from the plate was used to inoculate 1 mL LB broth containing 50 ng mL-1of kanamycin,and further grown for 18 h at 28 °C.Bacterial cells were pelleted by centrifuging cultures at 3000g for 10 min following culturing at 37 °C and 200 r min-1shaking for 12–16 h.The overnight culture (50–100 μL) was used to inoculate 50 mL of freshly prepared infiltration media that included 10 mmol L-1MgCl2,5 mmol L-1MES and 5 μmol L-1acetosyringone(AS;Solarbio Life Sciences Co.,Ltd,Beijing,China)with 50 ng mL-1kanamycin.The culture was further incubated in a shaker at 37°C at 200 r min-1for 12–16 h.After the concentration of bacterial cells reached a density of OD600nm=0.5–0.7,they were pelleted by centrifugation at 2500×g for 5 min.The bacterial cells were resuspended and adjusted to an optical density of OD600nm= 0.7–1.0 with the appropriate media containing 100 mmol L-1MgCl2,200 mmol L-1MES and 100 μmol L-1AS.The bacterial culture was incubated at 28 °C for 2–3 h,and was then used for leaf infiltration.

Healthy fully expanded tobacco leaves were infiltrated using a syringe without a needle.Infiltrated tobacco plants were incubated in the dark for 2 days.Similarly,transient overexpression of AhDef genes in peanut leaves was conducted following the leaf disc infiltration method with minor modifications [34].Expanded leaves of 3-week-old peanut seedlings were collected and immediately placed in the freshly prepared bacterial culture in 100 mL flasks.This was followed by vacuum infiltration of peanut leaves for 25 min at a pressure of 0.8 MPa,with 2–3 abrupt breaks.Finally,the leaves were incubated in the same bacterial culture in the dark for 30 min at 28 °C on a horizontal shaker (40 r min-1).Following incubation,the leaves were washed 3–4 times in PBS and incubated on moistened sterile filter paper at 28 °C for 24 h.Finally,the leaves were washed with PBS containing 250 μg mL-1cefotaxime (Solarbio Life Sciences Co.,Ltd,Beijing,China) to remove Agrobacterium and incubated on wet paper at 28 °C for another 24 h.Plasmolysis of N.benthamina cells was performed by immersing pieces of leaves in 1 mol L-1NaCl for 10 min under the confocal laser scanning microscope).

2.8.Diaminobenzidine (DAB) and trypan blue staining

Inoculated tobacco leaves were soaked in DAB staining solution with 50 mg DAB powder (Solarbio),90 mL sterile water,10 mL 10× phosphate buffer (Solarbio),25 μL Tween 20 (Solarbio),pH 3.0,and vacuum infiltrated at a pressure of 0.8 MPa for 2 min.Leaves were placed at 28°C in the dark overnight,and then boiled in 90% anhydrous ethanol for 10 min until the green leaves faded completely to colorless.Fading was observed with the stereomicroscope,photographed,and recorded.

Leaves were immersed in Trypan blue staining solution(Solarbio),stained in a boiling water bath for 2 min,allowed to cool,and stained overnight at 28 °C.Stained leaves were placed in chloral hydrate solution (1.25 g mL-1),placed at 25 °C and shaken at 50 r min-1.The decolorization solution was changed every 3 h until the leaves were completely clear.Cell death was observed with the stereomicroscope and photographed.

3.Results

3.1.Pathological analysis of peanut resistance to R.solanacearum

To investigate the mechanism of peanut resistance to BW,pathological analyses were conducted following R.solanacearum inoculation of resistant H108 (R) and susceptible H107 (S) peanut cultivars.Infection by R.solanacearum led to peanut root rot,ultimately resulting in dwarfed and wilted seedlings.H108(R)showed fewer symptoms than H107 (S).Most R.solanacearum-infected H107 (S) plants withered at 21 dpi,whereas all H108 (R) plants survived (Fig.1A).Pathological section analysis showed that the vascular bundles in the roots and stems of H107 (S) were blocked by viscous bacteria at 21 dpi,while those of H108(R)were almost healthy and were only slightly influenced by infection (Fig.1B).Trypan blue staining of the root cross sections further verified that the degree of blockage in the vascular bundle in H108(R)was more severe than that in H107 (S) at 3 dpi (Fig.1C).These results suggested that peanut resistance to R.solanacearum was due to inhibition of bacterial reproduction and spread in the vascular bundles of roots and stems.

To confirm that peanut wilt was caused by R.solanacearum,the bacterial pathogens in the roots of the susceptible plants were isolated and identified.The suspension of diseased roots was applied to TTC agar medium,and red bacterial colonies were observed on the plates following 2 days culture (Fig.1D).All colonies were round,pink in the center,and milky white on the edge (Fig.1E).Using scanning electron microscopy,R.solanacearum particles were viewed as short rod-shaped bacteria (Fig.1F).These finding indicated R.solanacearum as the causative agent of disease.

3.2.Identification of the AhDef genes involved in peanut resistance to R.solanacearum

To investigate the molecular mechanism of peanut resistance to BW,high-throughput transcriptomic RNA-seq of A.hypogaea in response to R.solanacearum infection was conducted (Fig.S1A).RNA-seq showed that genes involved in ABA,SA,JA,and ethylene(Eth)signaling pathways,and genes responding to heat,wounding,oxides and fungal pathogens,were(P ＜0.05)up or down-regulated(Fig.S1Bv.Among them,multiple AhDef genes were identified to be(P ＜0.05) up-regulated in H108 (R) and H107 (S) under R.solanacearum infection.This finding suggested that AhDef genes were involved in peanut resistance to R.solanacearum.Twelve genes were identified as putative AhDef genes in A.hypogaea (Table S3).The numbers of amino acids encoded by these genes ranged from 66 to 88.The isoelectric point (pI) and molecular weight of AhDef proteins ranged from 6.03 to 9.77 and from 7.41 to 9.96 kDa,respectively.

In the phylogenetic tree,the 12 putative AhDef proteins were assigned to two distinct groups (A and B) with 83 other PDF proteins (Fig.2;Table S1).These AhDef proteins were designated as AhDef1.1–AhDef1.6 and AhDef2.1–AhDef2.6.Group A was composed of two subgroups (I and II),and group B of four subgroups(III,IV,V,and VI).AhDef1.1 was classified into subgroup I and was most closely related to HsAFP1 [35],CtAMP1 [36],DmAMP1[36],and AtPDF1.4 [37],having excellent anti-fungal activities.AhDef1.2,AhDef1.3,AhDef1.4,AhDef1.5,and AhDef1.6 were grouped together in subgroup II with ZmESR6 [38],MsDef1 [39],and CaDef1 [40],which possess both anti-fungal and antibacterial activities.The defensin proteins AhDef2.1,AhDef2.2,AhDef2.3,AhDef2.4,and AhDef2.5 were all classified into subgroup VI with various antimicrobial peptides (AMPs),including OsDEF8[41],NaD2 [42],CaDef2 [40],Ec-AMP-D1 [43],AtPDF2.1 [44],MtDef4.2 [45] and Tad1 [46].The remaining AhDef2.6 was clustered into subgroup V with the AMPs VuDEF2[47]and ZmES1[48],metal tolerance proteins AtPDF2.4,AtPDF2.5,and AtPDF2.6[37,44],and proteins functioning as α-amylase and trypsin inhibitors,including VuDEF1[49],ZmES1[48],and SbDEF homologs[50].These results suggested that AhDef proteins are involved in peanut stress tolerance and resistance to R.solanacearum and other pathogens.

Fig.1.Pathological analysis of peanut resistance to R.solanacearum.(A)Phenotypes of peanut resistant H108(R)and susceptible H107(S)seedlings following R.solanacearum infection at 21 dpi.(B)Root and stem section analysis of H108(R)and H107(S)following R.solanacearum infection at 3 dpi.(C)Trypan blue staining of root cross sections of H108 (R) and H107 (S) following R.solanacearum infection at 3 dpi.(D) Red bacterial colonies of R.solanacearum isolated from diseased roots on TTC agar medium.(E)Microstructures of the R.solanacearum colonies isolated from diseased roots.(F) R.solanacearum bacteria viewed in the infected peanut root using scanning electron microscopy.

3.3.In silico characterization of AhDef gene family members

The 12 AhDef genes were unevenly distributed on six chromosomes (1,3,8,11,16,and 18) (Fig.S2A).AhDef1.2,AhDef1.4,AhDef1.6,AhDef2.3,and AhDef2.6 were clustered on the end of chromosome 8;AhDef1.3,AhDef1.5,and AhDef2.4 were located on chromosome 18;and AhDef 2.1,AhDef 2.5,AhDef2.2 and AhDef1.1 were identified to lie on chromosomes 1,3,11,and 16,respectively.The exon and intron structures of the AhDef genes are shown in Fig.S2B.Two introns are present in the open reading frame of AhDef2.1 and AhDef2.5,whereas each of the remaining AhDef genes contains only one intron.Eleven conserved motifs were identified and were fairly uniformly distributed,and all family members contained the gamma-thionin domain(Fig.S2C).The disulfide pairs of the 12 defensin genes in peanut were Cys3-Cys6 and Cys4-Cys7,with the Cys1-Cys8,Cys2-Cys5 pairs being the most conserved in plant defensins (Fig.3A) [51].With the help of four pairs of disulfide bonds,a very stable βαββ structure was formed.Based on the amino acid structure of previous studies [48,51],we selected the corresponding peanut defensin genes from the first and the second group,predicted the three-dimensional structure (Fig.3B).The overall structure of the two groups of defensin genes was conserved,with some differences apparent.

The 2000-bp upstream promoter sequences of AhDef genes were searched for plant cis-elements.Various cis-elements were unevenly distributed on the promoter sequences (Fig.3C).These cis-elements were associated mainly with hormone signaling,including the CGTCA motif responsive to JA,the TCA element responsive to SA,the TGA box involved in auxin responses,the TATC box and P-box responsive to gibberellin,the ABRE motif involved in ABA response and stress responses including the defense and stress-responsive TC rich repeats,wound-responsive WUN motif,the anaerobic responsive element ARE,and MYB binding sites involved in drought response and flavonoid biosynthesis.These results suggested that AhDef genes function in peanut response to various hormones and stresses.

3.4.Expression profiling of AhDef genes

Fig.2.Phylogenetic analyses of AhDef genes and their homologs in various plant species.The deduced amino acid sequences of AhDef homologs were used to construct a phylogenetic tree using the neighbor-joining method with the 1000 bootstrap values indicated.

To characterize the expression profiles of AhDef genes,RNA-seq data of peanut plants in response to R.solanacearum infection were analyzed(Fig.3D).Expression of these genes varied in magnitude.AhDef1.3,AhDef1.4,AhDef1.5,AhDef1.6,AhDef2.1,and AhDef2.2 showed higher expression than other family members.In particular,expression of AhDef1.6,AhDef2.1 and AhDef2.2 was up-regulated (P ＜ 0.05) in H108 (R) and H107 (S) following R.solanacearum infection.However,expression of AhDef1.1 in both cultivars was relatively low at all time points.Tissue-specific expression patterns of AhDef genes were identified from the RNAseq data (Fig.3E).The expression profiles of AhDef genes differed among tissues from one another (Fig.3F).The expression of AhDef2.1 and AhDef2.2 was relatively high in all tissues,while that of AhDef1.1 was low.The two genes AhDef1.5 and AhDef1.6 showed similar expression patterns that were higher in the leaf,the perianth (flower),the pistil,and the seed.The expression of the four genes AhDef1.5,AhDef1.6,AhDef2.1,and AhDef2.2,was markedly up-regulated under R.solanacearum infection and in various tissues,suggesting that they function in peanut resistance to R.solanacearum.

Four genes:AhDef1.5,AhDef1.6,AhDef2.1,and AhDef2.2,were selected for further functional identification.Their expression profiles under R.solanacearum infection were verified by qRT-PCR.Their relative expression in root,stem and leaf of H108 (R) and H107 (S) seedlings at 0,0.5,1.0,and 7.0 dpi was measured(Fig.4A).Both AhDef1.6 and AhDef2.2 were up-regulated by approximately 1.5–5.0 fold in the root,stem,and leaf of H108(R),whereas their expression was relatively unchanged in H107(S).Though the relative expression of AhDef1.5 was up-regulated in the root,no difference (P ＜0.05) was seen between H108 (R)and H107 (S) after inoculation.The expression of AhDef2.1 was suppressed in H108 (R),while that in H107 (S) remained almost unchanged.These findings suggested that AhDef1.6 and AhDef2.2 might confer peanut resistance to R.solanacearum.

Phytohormone signaling pathways operate in plant resistance to pathogens [52–53].We investigated their effect on the expression of the four genes (AhDef1.5,AhDef1.6,AhDef2.1,and AhDef2.2)by qRT-PCR.As expected,all four genes were (P ＜0.05) up-or down-regulated by the application of exogenous hormones(Fig.4B).Of the four,only AhDef2.2 was up-regulated by SA,MeJA,and ABA up to 6–20 fold in H108 (R).Both AhDef1.5 and AhDef1.6 were up-regulated by MeJA and ABA,while AhDef2.1 was upregulated by MeJA and ABA in H108 (R).Thus,AhDef1.6 and AhDef2.2 responded to R.solanacearum infection and multiple exogenous hormones in H108 (R) and not H107 (S).

3.5.Subcellular localization of AhDef1.6 and AhDef2.2

Based on the expression of AhDef genes,AhDef1.6 and AhDef2.2 were further chosen for subcellular localization.Bright green fluorescence was observed in transfected protoplasts following 12–24 h of incubation.Both the fusion proteins AhDef1.6-YFP and AhDef2.2-YFP were detected specifically on the plasma membrane,while YFP was distributed throughout the entire cell(Fig.5A,B).

When fusion proteins of AhDef1.6-YFP and AhDef2.2-YFP were transiently overexpressed in tobacco leaves,unfused YFP was expressed throughout cells,whereas the two fusion proteins were expressed mainly on the plasma membrane(Fig.S3).The results of the two subcellular localization assays were in agreement,indicating that AhDef1.6-YFP and AhDef2.2-YFP were expressed specifically on the plasma membrane.

Fig.3. In silico characterization of AhDef gene family members.(A)Phylogenetic tree and sequence alignments of AhDef peptides and their homologs in other plant species.(B) The structure of AhDef peptides was constructed by homology modeling using SWISS-MODEL (C) Possible cis-acting elements in the promoter sequence (～2000 bp) of AhDef genes were predicted with PlantCARE.(D) Expression profiles of AhDef genes under R.solanacearum infection.(E) Tissues or organs of peanut plants.(F) Tissue-or organ-specific expression profiles of AhDef genes according to transcriptomic data obtained from PeanutBase.The ratio of the experimental group to the mock group was log2 transformed.The normalization of RPKM values was performed by the min–max normalization method.Blue indicates low expression and red,high expression.

3.6.Overexpression of AhDef 2.2 increased resistance to R.solanacearum

To identify the function of AhDef1.6 and AhDef2.2,tobacco leaves transiently overexpressing these two genes were inoculated with R.solanacearum.Bright green fluorescence could be observed in almost all leaves following 24–36 h of incubation,indicating the successful transient overexpression of YFP,AhDef1.6-YFP,and AhDef2.2-YFP in tobacco leaves.Leaves overexpressing YFP showed lesions and mesophyll cell disruption at 3 dpi (the empty control),but showed only mesophyll yellowing symptoms following mock inoculation with TTC broth (the mock control) (Fig.5C).This finding indicated that the tobacco leaf-based transient expression system could be used for the identification of R.solanacearum resistance genes.

Tobacco leaves transiently overexpressing AhDef1.6-YFP showed susceptible lesions similar to those of the empty control at 3 dpi,whereas those overexpressing AhDef2.2-YFP showed no apparent difference from the mock control (Fig.5D).Trypan blue staining showed that overexpression of AhDef2.2-YFP in tobacco leaves reduced cell death and lesion development (Fig.5E).Llittle H2O2accumulated in leaves overexpressing AhDef2.2-YFP in comparison with those overexpressing AhDef1.6-YFP and the empty control (Fig.5F).This finding suggested that overexpression of AhDef2.2-YFP in tobacco leaves might directly inhibit the reproduction and spread of R.solanacearum,rather than provoking resistance via the tobacco immune system.To confirm the resistance effect of AhDef2.2 to R.solanacearum,fusion protein AhDef2.2-YFP was transiently overexpressed in peanut leaves(Fig.5G).Overexpression of AhDef2.2-YFP reduced cell death,and lesion development was also confirmed in peanut leaves by Trypan blue staining(Fig.5H).These transient expression assays suggested that AhDef2.2 increased resistance to R.solanacearum in tobacco and peanut.

Fig.4.Expression analysis of AhDef genes by qRT-PCR.(A)Expression analysis of AhDef genes in response to R.solanacearum infection at 0.5,1.0,and 7.0 dpi;(B)Expression analysis of AhDef genes in response to SA(3 mmol L-1),MeJA(100 mmol L-1),and ABA(10 μg mL-1).Y-axes indicate relative expression;significant differences were assessed by Mann-Whitney U test and indicated by asterisks;single asterisk (*)represents P ＜0.05,and double asterisk(**) represents P ＜0.01.Values are means of three biological replicates,with error bars indicating the SD.

4.Discussion

R.solanacearum causes BW in more than 450 plant species,including many crops such as tomato (Lycopersicon esculentum),potato (Solanum tuberosum L.),pepper (Capsicum annuum L.),eggplant (Solanum melongena L.),and peanut [6].Though multiple approaches including crop rotation,soil amendment,and fumigation have been developed to control BW,efficient and environmentally sustainable measures are still lacking for most host crops[54].Breeding of crop cultivars with durable broad-spectrum resistance is regarded [55] as the most environmentally friendly technology for disease management.However,mechanisms of plant resistance to BW have not been identified and only two BW R genes have been cloned in A.thaliana to date.Main QTL associated with resistance to BW in peanut have been mapped in a R gene-rich region on chromosome B02 [2,7],and some resistant peanut cultivars have been developed via molecular marker assisted breeding[4,56].But little is known about genetic factors that condition resistance and no peanut R genes conferring resistance to BW have been cloned.

Pathological analysis showed that the vascular bundles in roots and stems of wilted H107 (S) were blocked by viscous bacteria,whereas those of H108 (R) appeared relatively healthy and were only slightly affected by R.solanacearum infection(Fig.1).In a previous report [57],resistance to R.solanacearum was attributed mainly to the inhibition of bacterial reproduction and spread in the vascular bundles of roots and stems,with thickening of pit membranes and gum production in tomato and tobacco.In the present study,electron microscopy revealed for the first time short rod-shaped R.solanacearum bacteria in peanut vascular bundles(Fig.1F).

Fig.5.Transient overexpression and functional identification of AhDef genes during resistance to R.solanacearum.(A)Vectors used for transient expression were obtained by cloning full-length CDSs into the vector pCambia1300-YFP.(B)Subcellular localization of AhDef1.6 and AhDef2.2 in Arabidopsis protoplasts.(C)Symptoms of tobacco leaves transiently overexpressing YFP,AhDef1.6-YFP,and AhDef2.2-YFP inoculated with R.solanacearum.(D) Mesophyll cell morphology of tobacco leaves transiently overexpressing YFP,AhDef1.6-YFP,and AhDef2.2-YFP under R.solanacearum infection.(E)Trypan blue staining was employed to assess the mesophyll cell death of the lesions caused by R.solanacearum infection.(F) DAB staining was used to measure H2O2 accumulation in tobacco leaves.(G) Transient overexpression of YFP and AhDef2.2-YFP in peanut leaves.(H) Trypan blue staining of peanut leaves overexpressing YFP and AhDef2.2-YFP following R.solanacearum infection.

Histopathological analysis of roots of resistant and susceptible peanut cultivars inoculated with R.solanacearum was in general agreement with previous studies [58–59],with large numbers of bacteria found in hypocotyl vascular tissue of infested susceptible peanut roots,leading eventually to blockage of the vascular lumen.During interactions between plants and pathogens,pathogenassociated molecular patterns trigger a natural immune response(pathogen-triggered immunity)in plants,causing a burst of signaling molecules such as peroxides,changes in endogenous hormone levels,and a cascade of signaling in plants that limits the spread of pathogenic bacteria.In the present study,transcriptomic analysis showed that signaling pathways associated with ABA,responses to oxidative stress,and SA were all involved in the disease resistance mechanisms of peanut to R.solanacearum.The 2000-bp promoter of the peanut resistance gene AhDef2.2 identified in this study contained two MeJA-responsive elements,two SAresponsive elements,and three ABA-responsive elements.The expression of the AhDef2.2 gene was higher in H108 (R) than in H107 (S) to MeJA hormone responses.The above results suggest that phytohormones and peroxisomal components that regulate plant stress responses are involved in AhDef2.2-mediated resistance to BW infection in peanut (Fig.6).

PDFs are small peptides known as strong fungal growth inhibitors [10] that can inhibit or kill microorganisms such as bacteria and fungi,as well as some insects and animal and plant cells.It is not known whether PDFs also enter bacterial cells and activate downstream signaling pathways and induce apoptosis by recognizing intracellular specific receptors.In the present study,12 AhDef genes were identified and overexpression of protein fusion AhDef2.2-YFP in N.benthamiana and peanut leaves increased resistance to R.solanacearum.This finding suggests that AhDef 2.2-mediated resistance to BW needs further investigation.Our results provide genetic resources for molecular breeding of peanut with broad-spectrum disease resistance.

Fig.6.The putative AhDef2.2-mediated regulatory pathway in peanut resistance to R.solanacearum infection. Cis-acting elements in the promoter sequence of AhDef2.2 are represented by rectangles.Lines with arrows represent transcriptional regulations,and curves with arrows represent the transcription,translation and transport of AhDef2.2 peptides.

CRediT authorship contribution statement

Dongmei Yin:Conceptualizatio,Project administration,Funding acquisition.Kai Zhao:Methodology,Writing -original draft.Qian Ma:Methodology.Yingying Ma:Methodology.Kunkun Zhao:Formal analysis.Chengxin Qu:Formal analysis.Di Cao:Formal analysis.Ren Rui:Writing -original draft.Xingli Ma:Validation.Fangping Gong:Validation.Zhongfeng Li:Validation.Xingguo Zhang:Validation.All authors read and approved the manuscript.

Declaration of competing interest

Authors declare that there are no conflicts of interest.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (31471525),the Key Program of Natural Science Foundation of China-Henan United Fund(U1704232),and Key Scientific and Technological Project in Henan Province(201300111000,S2012-05-G03).We thank Zhenyu Wang,Plant Protection Institute of Henan Academy of Agricultural Sciences for the bacterial wilt strain.We are grateful to the anonymous reviewers for helpful suggestions.

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2021.11.002.