Attenuation of ethylene signaling increases cotton resistance to a defoliating strain of Verticillium dahliae

Tinyi Wng,Muhmm Shbn,Junhui Shi,Wirn Wng,Shiming Liu,Xinhui Ni,Yu Yu,Ji Kong,Stvn J.Klostrmn,Xinlong Zhng,Aliu Airxi,*,Longu Zhu,*

a National Key Laboratory of Crop Genetic Improvement,Huazhong Agricultural University,Wuhan 430070,Hubei,China

b Department of Plant Breeding and Genetics,University of Agriculture Faisalabad,Sub-Campus Burewala,Punjab 61010,Pakistan

c Institute of Economic Crops,Xinjiang Academy of Agricultural Sciences,Kuqa 842000,Xinjiang,China

d Key Laboratory of Oasis Eco-agriculture of the Xinjiang Production and Construction Crops,College of Agronomy,Shihezi University,Shihezi 832000,Xinjiang,China

e Xinjiang Academy of Agricultural & Reclamation Sciences,Shihezi 832000,Xinjiang,China

f United States Department of Agriculture,Agricultural Research Service,Salinas,CA 93905,USA

Keywords:Cotton Verticillium dahilae Ethylene Defoliation

ABSTRACT The severity of Verticillium wilt on cotton caused by defoliating strains of Verticillium dahliae has gradually increased and threatens production worldwide.Identification of the molecular components of leaf defoliation may increase cotton tolerance to V.dahliae.Ethylene,a major player in plant physiological processes,is often associated with senescence and defoliation of plants.We investigated the cotton–V.dahliae interaction with a focus on the role of ethylene in defoliation and defense against V.dahliae.Cotton plants inoculated with V.dahliae isolate V991,a defoliating strain,accumulated more ethylene and showed increased disease symptoms than those inoculated with a non-defoliating strain.In cotton with a transiently silenced ethylene synthesis gene(GhACOs)and signaling gene(GhEINs)during cotton–V.dahliae interaction,ethylene produced was derived from cotton and more ethylene increased cotton susceptibility and defoliation rate.Overexpression of AtCTR1,a negative regulator in ethylene signaling,in cotton reduced sensitivity to ethylene and increased plant resistance to V.dahliae.Collectively,the results indicated precise regulation of ethylene synthesis or signaling pathways improve cotton resistant to Verticillium wilt.

1.Introduction

Cotton supports the economies of many developing countries,but is affected by diseases that lead to yield and fiber quality losses.Verticillium wilt,caused by the soilborne fungus Verticillium dahliae,is highly destructive[1].The pathogen infects the roots,spreads upward in the xylem,and causes leaf wilting,chlorosis,and necrosis[2].Depending upon the severity and type of the V.dahliae strain infecting the plant,the disease may result in complete leaf defoliation and plant death[3].The proportion of infections that cause defoliation of cotton has increased yearly[4],prompting study of the physiological basis of V.dahliae-induced leaf defoliation.

Strains of V.dahliae infecting cotton are classified as either defoliating or non-defoliating strains.Defoliating strains typically cause a more rapid onset of disease symptoms,defoliation,and ultimately higher yield losses than non-defoliating strains[5].Recent population genomics analysis[6]has revealed a lineage-specific region of DNA found only in the genomes of defoliating strains of V.dahliae and associated with production of N-acylethanolamine(NAE)12:0,which can induce defoliation.The mechanism by which NAE promotes defoliation may involve altering sensitivity to abscisic acid or other hormones.Among thess implicated in V.dahliae-induced leaf defoliation,levels of plant-derived ethylene production have been correlated with the severity of Verticillium wilt in cotton[7].

Ethylene is a multi-functional gaseous hormone implicated in plant–pathogen interactions,and is often associated with defoliation,chlorosis,or necrosis[8].The biosynthesis of ethylene in higher plants is initiated with the conversion of Sadenosylmethionine into 1-aminocyclopropane-1-carboxylic acid(ACC)by the enzyme ACC synthase(ACS).ACC is converted to ethylene by the action of enzyme ACC oxidase(ACO)[9].Some microorganisms also have the ability to synthesize ethylene autogenously,in one of three ways.Most fungi and some bacteria synthesize ethylene primarily through the KMBA(2-keto-4-methylbutyric acid)pathway[10].

Ethylene signaling can be initiated at membrane-bound ethylene receptors on the endoplasmic reticulum.Five ethylene receptors have been identified in Arabidopsis:ETR1 and its paralogous gene products ERS1,ERS2,ETR2,and EIN4[11–14].ETR1 is a homodimer and is dependent on the copper ion transporter RAN1 for ethylene binding[15].Ethylene receptors interact directly with Constitutive Triple Response 1(CTR1)and regulate its activity.CTR1 is a serine/threonine protein kinase that resembles animal Raf kinases,is predicted to act in a mitogen-activated protein kinase(MAPK)cascade,and negatively regulates ethylene signaling[16].In the absence of ethylene,CTR1 inhibits ethylene responses,whereas in the presence of ethylene,CTR1 becomes inactivated and(ethylene-insensitive 2)EIN2,a positive regulator of ET signaling,activates an ethylene signaling pathway[17].Genetic studies[18,19]have shown that EIN2 acts directly downstream of CTR1.In the absence of ethylene,EIN2 is phosphorylated at the C-terminal domain(C-END)by CTR1[20],whereas in the presence of ethylene,CTR1 is inactivated and EIN2 in turn triggers its cleavage and movement to the nucleus[21].Downstream of EIN2,a signaling cascade regulates the expression of multiple genes[22].Ethylene response factors(ERF)are secondary transcription factors downstream of ethylene signaling and are reported to activate the expression of PR genes[23].

Ethylene functions in various physiological and developmental responses in plants,including microtubule function[24],hypocotyl elongation[25],ripening,and shedding of leaves,fruits,and other organs[26].Ethylene plays a variety of roles in regulation of plant immunity;it not only is involved in increasing host resistance[27,28]but contributes to host susceptibility to numerous pathogens in multiple plant species[29–31].As in other plant–pathogen interactions,ethylene is involved in signaling during cotton–V.dahliae interactions and some components in ethylene signaling positively regulate cotton resistance to V.dahliae[32,33].Although several ethylene-signaling-associated genes have been reported in transcriptomic studies during cotton–V.dahliae interaction[34,35],the role of ethylene during cotton–V.dahliae interaction remains ambiguous.The objective of this study was to functionally characterize key genes in ethylene signaling and those involved in its synthesis in cotton,with the aim of investigating the effects of compromised ethylene signaling on the rate of defoliation and resistance to V.dahliae.

2.Materials and methods

2.1.Plant and fungal materials

The upland cotton(Gossypium hirsutum)cultivar YZ1 was used in this study.AtCTR1-overexpressing plants were generated using the modified pCAMBIA2300S vector.This vector was obtained from Center for the Application of Molecular Biology to International Agriculture(CAMBIA),Australia and modified by integrating the CaMV35S promoter into the polyclonal sites following Li et al.[36].The pTRV1 and pTRV2 vectors were used to knock down the expression of GhACOs and GhEINs using Virus induced gene silencing(VIGS)following Gao et al.[37].The highly virulent defoliating V.dahliae strain V991 was provided by Jian Guiliang from Institute of Plant Protection,Chinese Academy of Agricultural Sciences,Beijing.The non-defoliating V.dahliae strain 1cd3-2 was kindly provided by Professor Li Guoqing,Huazhong Agricultural University.

2.2.Genetic transformation,procedure for VIGS and plant growth conditions

Using the Gateway cloning system(Invitrogen,Carlsbad,CA,USA),an AtCTR1-overexpression vector was constructed and transferred into cotton(YZ1)by Agrobacterium tumifaciens(strain GV3101)-mediated transformation following Tian et al.[38].Cotton seedlings were grown in one of two ways for experiments.For growth in soil,seeds were germinated in an incubator at 28°C for two days and seeds with the same germination characteristics were selected and placed in moist soil at 25 °C with water.For growth in hydroponic conditions,seeds were soaked in warm water for 6 h,moved to a wet towel,and incubated at 28 °C for 2 days.Seeds with similar growth were moved to Hoagland’s solution.Alternatively,seeds were sterilized,and sown in seedling medium.One week after germination,seedlings were moved to Hoagland’s solution.All seedlings were grown at 28 °C with a 16 h light/8 h dark period and 75% relative humidity.

VIGS vectors(TRV:GhEINs),(TRV:GhACO)and(TRV:00)were constructed following Gao et al.[37],electroporated into Agrobacterium(strain GV3101),and infiltrated into two fully expanded cotyledons of 10-day-old cotton seedlings following Gao et al.[37].

2.3.Fungus culture and plant inoculation

Defoliating(V991)or non-defoliating(1cd3-2)strains of V.dahliae were maintained on potato dextrose agar(PDA)medium for sporulation at 25 °C for 4 days in the dark.Highly active hyphae were selected on the plate and placed into Czapek liquid medium or PDA liquid medium and incubated at 25 °C with shaking at 170 r min-1for 4 days.After filtration,spores were counted with a hemocytometer and diluted with sterile water to desired concentrations.For inoculation of cotton seedlings grown in soil,root dip inoculation was used.Seedlings with similar size and growth were carefully removed from soil,avoiding root damage,rinsed with clean water,and dipped for 1 min into a spore suspension of V.dahliae having a concentration of 2×105spores mL-1[39].After inoculation,seedlings were replanted in fresh soil pots.For inoculation of seedlings grown under hydroponic conditions,fungal spores were cultured in Czapek or PDA liquid medium.The suspension was centrifuged at 2000 r min-1for 10 min,the supernatant was decanted,and the spores were adjusted to a final concentration of 107spores mL-1in sterile water for inoculation[40].For plant inoculation,Hoagland’s solution was replaced with fungal suspension solution for a 12–24 h incubation period followed by a switch to Hoagland’s solution for growth at 25 °C,16 h light/8 h dark,with a light source of 120 μmol m-2s-1.The disease index and rate of defoliation were calculated as follows:

where n represents the level of disease from 0 to 4.

The rate of defoliation(%)=number of leaves shed/total number of leaves investigated×100.

Disease symptoms of cotton plants were scored as described by Xu et al.[39].

2.4.Exogenous treatments with ethylene

Three concentrations of ethephon solution:0.5,2.5,and 12.5 μg mL-1,were prepared.After inoculation with V991,100 mL of ethephon solution of each concentration was sprayed on leaves of 24 cotton seedlings in a sealed container.Two hours after treatment,the sealed container was removed and conditions described above were maintained for growth of the plants.

For ethylene treatment of etiolated seedlings,seeds were germinated for two days in the dark,and uniform small seedlings were selected and placed in soil.They were sprayed with 50 mL of ethephon solution(20 μg mL-1concentration).After three days in a sealed container,followed by two days without containment,hypocotyl length was measured.

2.5.Ethylene production

Ethylene measurement followed Wang et al.[41].Stem sections of 1cm in length just above the cotyledonary node of young seedlings were collected,weighed,and sealed into air-tight 12 mL vials in light for 24 h at room temperature.Then,1 mL of gas was extracted from the vial with an air-tight syringe and injected into a gas chromatograph(Agilent Technologies,Palo Alto,CA,USA).A standard curve was obtained with ethylene concentrations of 1,2,3,and 5 μL L-1with the equation y=12.51x-0.914;r2=0.998(x means the measured value of the gas chromatograph,y means the ethylene concentration).

2.6.Histocytological analyses

For histocytological analyses,tissue sections containing the abscission area(petioles and stem segments near the abscission layer)were taken.The tissues was placed in 70%formaldehyde alcohol acetic acid fixative solution,placed under vacuum for 15 min followed by slow deflating,repeated three times,and changing the fixative solution for overnight fixation.Dehydration,transparent embedding,and sectioning followed Jin et al.[42].Three biological repeats at least were employed.In each independent biological repeat,12 samples were prepared from stem sections of both control and treated plants.Images were captured with a light microscope(Zeiss,Oberkochen,Baden-Wurttemberg,Germany).

2.7.Fungal recovery assays

Fungal recovery assays followed Zhang et al.[43].Following inoculation with V.dahliae,cotton plants were randomly selected.Stem sections between the second and third nodes were cut into segments of 3 mm in length,placed on PDA culture plates,and incubated at 25 °C for 3–5 days in the dark to observe V.dahliae growth.

2.8.Southern blotting and expression analysis

For Southern blotting,total DNA was extracted using the Plant Genomic DNA Extraction Kit,DP305(Tiangen Biotech,Beijing,China)following the manufacturer’s protocol.After quality confirmation by gel electrophoresis,15–20 μg of genomic DNA was digested with HindIII enzyme(NEB,USA).Further steps of electrophoresis and blotting followed Li et al.[44].PCR fragments of the neomycin phosphotransferase(NPTII)gene were used as probe.Labeling and detection of probe were performed with a DIG Probe DNA Labeling and Detection Starter Kit II(Roche,Basel,Switzerland)following the manufacturer’s instruction.

For expression analyses,total RNA from cotton roots or leaves(approximately 0.1 g,precise weight was noted)was extracted and purified using the modified guanidine thiocyanate method[45].Total RNA(0.5–3 μg)was reverse transcribed to synthesize cDNA using the SuperScript first strand synthesis system(Invitrogen,Carlsbad,CA,USA).The reaction procedure for RT-PCR was as follows:pre-denaturation at 94 °C for 5 min;then 25–35 cycles(94°C for 30 s;58°C for 30 s;72°C for 30 s);72°C for 7 min,after which the PCR product was stored at 4 °C.Parallel PCR reactions and homogeneity analysis of samples were performed using ubiquitin(GhUBQ7,DQ116441)as an internal reference gene.After the reaction,electrophoresis was performed on a 1% agarose gel,and PCR amplification and electrophoresis results were examined using a gel imaging system(BioRad,Hercules,CA,USA).RT-qPCR was performed on an ABI 7500 Real-Time PCR Sequence Detection System(Applied Biosystems,Foster City,CA,USA)using SYBR Green PCR Master Mix(Applied Biosystems).The PCR cycling conditions included a denaturation step at 95 °C for 2 min followed by 40 amplification cycles(with denaturation at 95 °C for 10 s,and extension at 58/60°C for 35 s).The specificity of the amplified product after 40 cycles was determined by dissociation curve analysis.Relative expression levels of genes were determined using the 2-ΔΔCT method[46]with UBQ7(DQ116441)as an internal reference for normalization.Three technical and biological repeats were performed and for each independent biological repeat,samples were collected from at least 4–8 plants of each line or treatment.All the primers used in this study were designed with Primer Premier 5.0(Premier Biosoft,San Francisco,CA,USA)and all the primers were listed in Table S1.

3.Results

3.1.The defoliating strain V991 of V.dahliae triggered increased ethylene accumulation in cotton

To investigate the roles of ethylene in defoliation during cotton–V.dahliae interaction,we inoculated cotton with the highly virulent defoliating strain V991 and the non-defoliating strain 1cd3-2.As shown in Fig.1A,cotton plants inoculated with V991 were more susceptible to Verticillium wilt than those inoculated with the 1cd3-2 strain.The difference in disease indices of cotton plants inoculated with V991 and 1cd3-2 at both 10 days postinoculation(dpi)and 13 dpi was highly significant(Fig.1B).Development of disease symptoms occurred more rapidly in cotton plants inoculated with V991,indicating the greater virulence of strain V991 than strain 1cd3-2.A highly significant difference was observed between the rates of defoliation of cotton plants inoculated with strain V991 and those inoculated with strain 1cd3-2,at both 10 and 13 dpi.The rates of defoliation of cotton plants inoculated with strains V991 and 1cd3-2 were respectively 10% and 1% at 13 dpi(Fig.1C).Thus,V991 caused rapid and severe defoliation in cotton,in contrast to the non-defoliating strain 1cd3-2.

Inoculation with either strain V991 or 1cd3-2 led plants to accumulate more ethylene than mock controls in all time intervals examined.However,plants inoculated with strain V991 accumulated highly significantly greater levels of ethylene relative to those inoculated with strain 1cd3-2 at 7–11 dpi.Though plants inoculated with strain 1cd3-2 accumulated more ethylene than the mock control,the difference was not significant(Fig.1D).Thus,inoculation with strain V991 or strain 1cd3-2 both induced ethylene production in cotton plants,but more ethylene was produced following inoculation with defoliating V.dahliae strain V991.

To assess the roles of ethylene during cotton interaction with V.dahliae,we used the commercially available ethylene-releasing chemical ethephon to treat cotton seedlings.Three concentrations of ethephon(0.5,2.5,and 12.5 μg mL-1)were prepared and sprayed on cotton leaves immediately after inoculation with V.dahliae(V991).Compared with mock treatment,exogenous ethephon treatments of all three concentrations compromised cotton resistance to V.dahliae(Fig.2A).There was a highly significant difference between the disease indices of mock-and ethephontreated cotton plants at 12,14,and 16 dpi(Fig.2B).

3.2.Ethylene production during cotton–V.dahliae interaction comes mainly from cotton

Fig.1.Inoculation with a defoliating Verticillium dahliae isolate V991 triggers more ethylene production in cotton seedlings.(A)The phenotype of cotton cultivar YZ1 inoculated with V991 and 1cd3-2 at 18 days post-inoculation(dpi)with V.dahliae.(B)The disease index at the 10 and 13 dpi with V.dahliae.(C)The defoliation rate at 10 and 13 dpi with V.dahliae.(D)Quantification of ethylene production at 0,3,7,9,and 11 dpi with V.dahliae.All experiments were repeated three times,values are means of three biological repeats,and bars indicate standard deviations(*,P＜0.05;**,P＜0.01;Student’s t-test).

Fig.2.Ethephon impairs cotton resistance to V.dahlia.(A)The phenotype of cotton cultivar YZ1 at day 12 after inoculation with V.dahliae(V991)and spraying with ethephon(0.5,2.5,and 12.5 μg mL-1)or with water(0 μg mL-1)as mock treatment.For every treatment,100 mL of ethephon solution with corresponding concentration was sprayed on the leaves of 20 cotton seedlings.(B)Disease indices at 10,12,14,and 16 days post-inoculation with V.dahliae.Values are means of three biological replicates,each with 20 plants.Bars indicate standard deviation(*,P＜0.05;Student’s t-test).

Fig.3.Knockdown of ethylene signaling increases cotton resistance to V.dahliae.(A)Relative expression analysis of GhACOs in response to agroinfiltration of cotton cultivar YZ1 with the tobacco rattle virus constructs TRV:00 and TRV:GhACOs.The analysis was performed at 15 days after infiltration.(B)Measurement of ethylene production at 7 days post-inoculation with V.dahliae.(C)Relative expressions of GhEINs in response to agroinfiltration of cotton seedlings with TRV:00 and TRV:GhEINs at 15 days after infiltration,and the phenotype of TRV:00 and TRV:GhEINs at 14 days post-inoculation with V.dahliae(n=16).(D)Fungal biomass analysis of V.dahliae after inoculation in TRV:00 and TRV:GhEINs seedlings based on V.dahliae internal transcribed spacer(ITS)DNA levels by quantitative PCR.(E)Fungal recovery assays were conducted using stem sections from plants taken at 18 days post-inoculation with V.dahliae.(F)Disease indices of TRV:00 and TRV:GhEINs cotton plants.(G)Defoliation rates of TRV:00 and TRV:GhEINs.All experiments were replicated three times with similar results.Values presented are means of three biological repeats and bars indicate standard deviations(*,P＜0.05;**,P＜0.01;Student’s t-test).

To investigate the ethylene produced during cotton–V.dahliae interactions is derived from the host or from V.dahliae,we chose ACO as target genes via virus-induced gene silencing(VIGS)to inhibit cotton ethylene synthesis.There are four members of ACO in Arabidopsis named ACO1,ACO2,ACO3,and ACO4.According to the proteins encoded by ACOs in Arabidopsis,the Cottongen(https://www.cottongen.org/)tool was employed to find possible ACO genes in cotton.Further screening based on high conservation among the ACO proteins of Arabidopsis enabled us to select 22 candidate genes in cotton(Dataset 1),which were collectively designated as GhACOs.These 22 genes were divided into six groups according to differences in nucleotide sequences,and six corresponding VIGS vectors named GhACO1 to GhACO6 were constructed and transferred into Agrobacterium.The target regions of the six vectors are shown in Dataset 1.All the six VIGS Agrobacterium strains were mixed equally and infiltrated into cotyledons to coinhibit the expression of target genes.Fifteen days after infiltration,the expression of target genes was measured by reverse transcription PCR(RT-PCR)(Fig.3A).The targeted gene-silenced plants(TRV:GhACOs)and control plants(TRV:00)were inoculated with V991 and the amounts of ethylene were measured at 7 dpi.The results shown that ethylene production was significantly reduced in TRV:GhACOs plants compared with control plants(Fig.3B)and TRV:GhACOs plants showed more resistance than controls(Fig.S1).The increased production of ethylene upon V.dahliae infection was thus derived from the plants.

3.3.Knockdown of GhEINs increased cotton resistance to V.dahliae

Given that mutation of a single EIN2 in Arabidopsis results in complete insensitivity to ethylene[18],we systematically studied the role of cotton EINs(GhEINs)to further investigate the role of ethylene during cotton-V.dahliae interactions.A total of 10 candidate GhEIN homologs were identified by alignment with the AtEIN2 sequences(Fig.S2A).Of these,seven clustered with OsEIN2,AtEIN2,and LeEIN2(Fig.S2B).Based on the protein similarity scores of＞50% as the screening standard,six genes were obtained(The six genes and their similarities are provided in Dataset 2)while the remaining four showed only about 10% similarity.The six sequences shared high identity,owing to the genomic synteny of the A and D subgenomes between Gh_A04G0622 and Gh_D04G1080,Gh_A09G1399 and Gh_D09G1403,and Gh_A11G2892 and Gh_D11G3278.Three VIGS vectors were constructed using Gh_A04G0622,Gh_A09G1399,and Gh_A11G2892 as templates and transferred into Agrobacterium with the following names:TRV:GhEINs-4,TRV:GhEINs-9,and TRV:GhEINs-11(Dataset 2).

TRV:GhEINs-4,TRV:GhEINs-9,and TRV:GhEINs-11 were mixed to generate the mixture TRV:GhEINs,which was infiltrated into cotton cultivar YZ1.At 15 days after infiltration,the expression levels of the target genes were measured,proving to be decreased relative to TRV:00(Fig.3C).When all plants were inoculated with V.dahliae strain V991,knockdown of GhEINs increased resistance to V.dahliae,whereas increased disease symptoms were observed in control plants(Fig.3C).Relative fungal quantification,fungal recovery assays,and differences in disease indices between TRV:00 and TRV:GhEINs plants supported the phenotypic evaluation(Fig.3D–F).The defoliation rate in TRV:GhEINs plants was significantly lower than that observed for the control plants(Fig.3G).Thus,interruption of ethylene signaling increased cotton resistance to V.dahliae.

Fig.4.Overexpression of AtCTR1 compromises cotton sensitivity to ethylene.(A)Phenotype of etiolated seedlings of several lines of cotton cultivar YZ1 following water treatment(upper panel)and the phenotype of etiolated seedlings of lines after ethephon treatment(lower panel).Fifty milliliters of ethephon solution(20 μg mL-1)was sprayed on cotton seedlings.(B)Hypocotyl lengths of cotton lines in response to water treatment(n=15,upper panel)and the length of hypocotyls after ethephon treatment(n=15,lower panel).(C)Expression analysis of ethylene response factor(ERF)genes under water and ethephon treatment(ETH)measured by reverse transcription quantitative PCR.Values are means of three biological repeats and bars indicate standard deviation(**,P＜0.01;Student’s t-test).

3.4.Overexpression of AtCTR1 in cotton compromised cotton sensitivity to ethylene

To further test the hypothesis that reduced signaling for ethylene production increases cotton resistance to V.dahliae,CTR1(a negative regulator of the ethylene signaling pathway)was selected for further study.For this purpose,AtCTR1 was stably integrated into cotton cultivar YZ1 by Agrobacterium tumifaciens-mediated transformation.Two independent transgenic lines(C14 and C42)with high expression of AtCTR1 and single-copy insertions were selected(Fig.S3A,B).Because ethylene can inhibit hypocotyl elongation in Arabidopsis under dark conditions[25],we examined the sensitivity of transgenic cotton lines to ethylene to verify AtCTR1 function in cotton.There was no difference in hypocotyl length between either transgenic line(C14 or C42)or the wild type(WT)under mock treatment,whereas the hypocotyl length of both transgenic lines was significantly greater than that of the WT upon exogenous ethephon treatment.The hypocotyl length of both transgenic lines was slightly shorter after ethephon treatment than after mock treatment(Fig.4A,B).Thus,the C14 and C42 lines still responded to ethylene treatment and were partially insensitive to ethylene.

The RT-qPCR analysis revealed that expression of both ethylene-responsive transcription factors was significantly lower in transgenic lines(C14 and C42)than in the WT(Fig.4C).

3.5.Overexpression of AtCTR1 increased cotton resistance to V.dahliae

To further support our hypothesis that attenuation of ethylene signaling increases cotton resistance to V.dahliae,AtCTR1-overexpressing lines and WT plants were inoculated with V.dahliae(V991).As expected,both transgenic lines(C14,C42)showed increased resistance to the pathogen compared to WT(Fig.5A).Relative fungal quantification,fungal recovery assays,disease indices,and the rate of defoliation were significantly lower in transgenic lines than in the WT(Fig.5B–E).These results were similar to those in GhEINs knockdown lines(Fig.3C–G)and further demonstrate that impaired ethylene signaling promoted resistance to V.dahliae in cotton.

Fig.5.Overexpression of AtCTR1 increased cotton resistance to V.dahlia.(A)Phenotypes of wild-type(WT)and transgenic lines(C14 and C42)of cotton cultivar YZ1 at 15 days post-inoculation(dpi)with V.dahliae(n=16).(B).Relative fungal biomass analysis at 20 dpi with V.dahliae in WT and transgenic lines(C14 and C42)with the V.dahliae internal transcribed spacer(ITS)DNA levels as an inner control for fungal biomass by quantitative PCR.(C)Fungal recovery assays on potato dextrose agar with stems of plants at 20 dpi with V.dahliae.(D)Disease indices of WT and transgenic lines(C14 and C42).(E)Defoliation rates of WT and transgenic lines(C14 and C42).Values are means±SD;n=3.All experiments were replicated at least three times with similar results,and bars indicate standard deviation(*,P＜0.05;**,P＜0.01;Student’s t-test).

To further investigate the effects of V.dahliae on the formation of the abscission layer,we characterized the expression patterns of genes involved in this physiological process by taking samples from the petiole bases of the first and third leaves at 7 days after inoculation.Expression levels of all tested genes including glucan endo-1,3-beta-glucosidase (GhE1-3B), beta-D-xylosidase 1(GhBXL1),endochitinase 1(GhCHI1),and endoglucanase 1(GhGUN1)were lower in both transgenic lines(C14,C42)than in the WT(Fig.6A).Reduction in ethylene signal transduction was thus associated with reduction in the expression level of genes involved in defoliation and might also reduce defoliation.

Cytological changes in petiole bases of WT and transgenic lines were examined after inoculation using paraffin sections prepared from the second leaf containing the abscission layer at 11 dpi.Microscopic analyses revealed that isolated cells in the WT had grown larger and become more scattered in location,whereas isolated cells of both transgenic lines(C14,C42)maintained their normal compact morphology(Fig.6B).Thus,attenuated signaling for ethylene production reduced the expression of genes involved in abscission and reduced defoliation of cotton following infection with V.dahliae.

Since ethylene also plays important roles in plant development,we examined fiber quality and development of AtCTR1-overexpressing cotton in the field.The results revealed that the fiber lengths of both transgenic lines(C14,C42)were slightly shorter and the micronaire values were higher than those observed for the WT(Fig.S4A,C).No difference in fiber strength between transgenic lines and the WT was found(Fig.S4B).Furthermore,plant heights of both transgenic lines(C14,C42)were greater than those of the WT(Fig.S4D).

4.Discussion

4.1.Ethylene production is a factor in defoliation and the severity of Verticillium wilt in cotton

Consistent with previous work[47],cotton plants inoculated with the defoliating strain of V.dahliae,V991,showed rapid onset of disease symptoms,severe defoliation,higher disease index and more ethylene production than those inoculated with nondefoliating V.dahliae 1cd3-2(Fig.1).The increased ethylene production was derived mainly from cotton plants,not V.dahliae(Fig.3B),and spraying with ethephon during inoculation aggravated the severity of Verticillium wilt(Fig.2A,B).Thus,increased ethylene levels contributed to defoliation upon V.dahliae infection,suggesting that the associated increased production of ethylene may facilitate pathogen colonization.Fusarium wilt caused by soilborne fungus Fusarium oxysporum is also a destructive disease in cotton,potato,tomato and Arabidopsis with similar disease symptoms like Verticillium wilt[48].Increased ethylene production in the Arabidopsis–F.oxysporum interaction also contributed to host susceptibility to Fusarium wilt[49].

Ethylene affects the degree of defoliation in several plant species[50–52].Ethylene signaling can induce the expression of expansion proteins that trigger cell wall relaxation,increase cell plasticity,and facilitate infection by V.dahliae[53].The findings that V991 induced more ethylene production than 1cd3-2 and that impairment of ethylene signal transduction impeded formation of the petiole abscission layer(Figs.1D,6B)suggest that ethylene is a positive regulator of defoliation in cotton–V.dahliae interactions.After colonizing the vascular bundles,V.dahliae secretes toxins leading to plant wilting[54],potentially with an as-yet unidentified compound or protein resulting in abnormal synthesis of ethylene during pathogen colonization.The secondary metabolite N-acylethanolamine(NAE)12:0,produced only by defoliating strains of V.dahliae and which induces leaf defoliation,may alter abscisic acid or other plant hormone levels[6].A linkage or synergistic activity between NAE and ethylene production in cotton is highly plausible.Stimulation of ethylene synthesis in cotton by pathogen-derived NAE invites further study.

Fig.6.Overexpression of AtCTR1 retards the formation of the abscission layer in cotton after V.dahliae inoculation.(A)Relative expression of genes involved in abscission layer formation.Glucan endo-1,3-beta-glucosidase(GhE1-3B),beta-D-xylosidase 1(GhBXL1),endochitinase 1(GhCHI1),and endoglucanase 1(GhGUN1)in cotton cultivar YZ1 at 7 dpi with V.dahliae.Values are means(±SD),n=3.(B)Paraffin sectioning of the abscission layer of different lines at 11 days after inoculation with V.dahliae.Bars indicate standard deviation(*,P＜0.05;**,P＜0.01;Student’s t-test).

4.2.Impaired ethylene signaling pathway increases cotton resistance to V.dahliae

The role of ethylene during plant–pathogen interactions is complex.Earlier it was suggested[55]that the timing of exposure of plants to ethylene and the quantity of ethylene produced during pathogen infection is critical to host resistance or susceptibility.Dissection of the roles of ethylene has been achieved in Arabidopsis and tomato.In Arabidopsis,the ethylene receptor mutant(ein4-1)and signaling mutants(ein2-1,ein6-1)in response to V.dahliae were more susceptible to Verticillium wilt[56].In contrast,an ethylene receptor mutant(etr1)showed resistance against V.dahliae[57].Similarly,during interactions of Arabidopsis with V.dahliae,ethylene-insensitive mutants etr1 and ein3 increased resistance,whereas ein2,ein4,and ein6 compromised Arabidopsis resistance to Verticillium wilt[56,57].Ethylene activates the expression of phytochrome interacting factor(PIF)genes via EIN3,which is involved in negative regulation of plant resistance to certain pathogens[58].Likewise,in tomato,the functions of ethylene during tomato–V.dahliae interaction seem to be conserved relative to those observed in the cotton system.Knockdown of the ethylene-insensitive mutant Never ripe(Nr)or an ethylene receptor(ETR4)in tomato increased plant tolerance to V.dahliae[59].Similarly,heterologous expression of a bacterial ACC deaminase(which reduces ethylene synthesis)in tomato increased resistance against V.dahliae[60].These results imply that ethylene increases host susceptibility in the tomato–V.dahliae interaction.

In our study,inhibition of the expression of GhEIN2s,an ethylene signaling pathway positive regulatory gene,or overexpression of a negative regulatory gene AtCTR1 in cotton increased resistance to Verticillium wilt(Fig.3).This finding suggests that ethylene signaling negatively regulates cotton resistance to V.dahliae.Our results are in contrast to a report that mutation of EIN2 in Arabidopsis resulted in decreased resistance to V.dahliae[56].This disparity might be explained by the functional diversification of EIN2 in different species or use of different V.dahliae strains for inoculation.The negative effects of ethylene signaling on cotton resistance to V.dahliae include the possibility that ethylene inhibits SAmediated disease resistance to favor the pathogenicity of V.dahliae,given that during cotton–V.dahliae interactions,Guo et al.[32]found that overexpression of GbERF1-like,a downstream transcription factor of ethylene and JA signaling pathways,activated the expression of PR3 and PR4 but inhibited the expression of PR1 and PR5(SA-responsive downstream genes).Similarly,ethylene can override the functions of NPR1 to mediate antagonistic cross talk between SA and JA signaling[61].Ethylene signaling genes(EIN3 and EIL1)were reported[62]to inhibit SA synthesis by targeting Salicylic Acid Induction Deficient2(SID2)gene and negatively regulating resistance to P.syringae in Arabidopsi.In tomato,ethylene increased the pathogenicity of F.oxysporum by inhibiting SA-mediated defense[49].As ethylene modulates defense responses initiated via JA/SA or other signaling pathways[63],this modulation may account for its complex roles in plant–pathogen interactions.Furthermore,ethylene also promotes fiber development and elongation in cotton[64].In our study,ethyleneinsensitive transgenic cotton showed negative effects on fiber quality(Fig.S4).These negative effects prevent the usage of ethylene-insensitive in cotton production.

In summary,our results suggest ethylene accumulation from cotton induced by V.dahliae strain V991 be a factor in defoliation and the severity of Verticillium wilt.Impairment in ethylene signaling or ethylene synthesis during V.dahliae infection can increase cotton resistance to V.dahliae.Since ethylene have multiple roles in plant growth and development,induced promoters by V.dahliae to drive the expression of AtCTR1 or specific mutant of factors involved in the abscission layer formation may create resistant germplasm to V.dahliae for cotton production.

CRediT authorship contribution statement

Tianyi Wang:Investigation,Writing–original draft.Muhammad Shaban:Investigation,Writing–original draft.Junhui Shi:Investigation.Weiran Wang:Investigation.Shiming Liu:Validation.Xinhui Nie:Resources.Yu Yu:Resources.Jie Kong:Investigation.Steven J.Klosterman:Writing–review & editing.Xianlong Zhang:Conceptualization,Funding acquisition.Alifu Aierxi:Conceptualization,Funding acquisition.Longfu Zhu:Supervision,Writing–review & editing.

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.

Acknowledgments

This work was supported by the National Key Research and Development Project of China(2018YFD0100403)and the National Natural Science Foundation of China(U1703231).

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2022.05.008.