Bin Chen,Yan Zhang,Jun Yang,Man Zhang,Qingming Ma,Xingfen Wang*,Zhiying Ma*

State Key Laboratory of North China Crop Improvement and Regulation/Key Laboratory for Crop Germplasm Resources of Hebei,Hebei Agricultural University,Baoding 071001,Hebei,China

ABSTRACT Verticillium wilt,a devastating disease in cotton caused by Verticillium dahliae,reduces cotton quality and yield.Heterotrimeric GTP-binding proteins,consisting of Gα,Gβ,and Gγ subunits,transducers of receptor signaling,function in a wide range of biological events.However,the function of Gα proteins in the regulation of defense responses in plants is largely unexplored,except for a few reports on model species.In the present study,a cotton G-protein α-subunit-encoding gene (GhGPA) was isolated from Verticillium wilt-resistant Gossypium hirsutum (upland cotton) cv.ND601. GhGPA transcription was up-regulated under V.dahliae stress,with higher expression in tolerant than in susceptible cotton cultivars.Subcellular localization revealed GhGPA to be located in the plasma membrane. GhGPA shows high(85.0%)identity with Arabidopsis AT2G26300(AtGPA1),and AtGPA1 gpa1-4 mutants displayed susceptibility to V.dahliae.Ectopic expression of GhGPA successfully restored the resistance of Arabidopsis gpa1-4 mutants to Verticillium wilt and made them more resistant than the wild type.Overexpression of GhGPA in Arabidopsis markedly increased the resistance and resulted in dramatic up-regulation of pathogenesis-related(PR)genes and increased in H2O2 accumulation and salicylic acid(SA)and jasmonic acid (JA) contents.However,suppressing GhGPA expression via virus-induced gene silencing (VIGS)increased susceptibility to Verticillium wilt,down-regulated the expression of PR and marker genes in SA and JA signaling pathways,and reduced H2O2 content.The contents of SA and JA in Arabidopsis gpa1-4 and VIGS cotton were lower than those in the wild type and empty-vector control.However,GhGPA-overexpressing Arabidopsis contained more SA and JA than the wild type when inoculated with V.dahliae.Thus, GhGPA plays a vital role in Verticillium wilt resistance by inducing SA and JA signaling pathways and regulating the production of reactive oxygen species.These findings not only broaden our knowledge about the biological role of GhGPA,but also shed light on the defense mechanisms involving GhGPA against V.dahliae in cotton.

Keywords:Gossypium hirsutum GhGPA Verticillium wilt Pathogenesis-related genes ROS

1.Introduction

Upland cotton (Gossypium hirsutum) accounts for more than 90% of total cotton yield worldwide,providing natural fibers for the global textile industry.Verticillium wilt (VW),caused by the soilborne fungusV.dahliae,is one of the main threats to cotton quality and production worldwide [1].No efficient management strategy has been developed to protect cotton fromV.dahliaeinfection.Developing new resistant cotton cultivars is an economical,efficient,and environmentally friendly method for controlling VW in cotton [2].The identification of novel genes conferring VW resistance in cotton is thus desirable for improving cotton molecular breeding.A growing number of resistance genes in cotton have been characterized,including chitinase 28 (Chi28),Cysrich repeat protein 1 (CRR1) [3],glutathione S-transferase (GhGST)[4],laccase (GhLAC15) [5],cytochrome P450 fatty acid xhydroxylase (GbCYP86A1-1) [6],and hybrid proline-rich protein(GbHyPRP1) [7].

Heterotrimeric guanine-nucleotide-binding proteins (G proteins)are composed of alpha(Gα),beta(Gβ)and gamma(Gγ)subunits.Diverse roles have been reported for G proteins in plants,including in plant growth,development,and response to various biotic and abiotic stresses [8].Gα proteins self-activate and bind GTP in the absence of guanine nucleotide exchange factors,and then interact with components of signaling pathways [9].However,the function of Gα proteins,especially canonical Gα,in the regulation of defense responses against pathogens in plants is largely unexplored.TheArabidopsisgenome encodes only one canonical Gα,AtGPA1,which was the first to be cloned in plants [10].AtGPA1plays a positive role in plant defense against bacterial pathogens (Pseudomonas) by mediating stomatal closure,a response observed in a deficient mutant Gα(gpa1-4)and Gα-overexpressingArabidopsis[11,12].AtGPA1can constitutively interact with NADPH oxidase (RbohD) to potentiate a flg22-induced reactive oxygen species (ROS) burst independent of the receptor-like cytoplasmic kinase Botrytis-induced kinase 1 (BIK1),which mediates pathogen-associated molecular pattern-triggered immunity signaling [13,14].SilencingGα by virus-induced gene silencing(VIGS) impaired the hypersensitive response (HR) and ROS accumulation inNicotiana benthamianatreated with an effector harpin fromXanthomonas oryzaeand reduced the expression of several defense genes,includingPR2b,enhanced disease susceptibility 1(EDS1),NbrbohAandNbrbohB[15].The mutant ricedwarf1(d1)gene,encoding the non-functional rice heterotrimeric G protein alpha subunit(RGA1),results in a highly reduced HR,deferred activation of PR genes,and lowered H2O2production upon plant infection by rice blast (Magnaporthe grisea),compared with that in the wild type (WT) [16].These findings suggest that Gα proteins play crucial roles in abiotic stresses in model species.To date,no functional investigation of cotton Gα proteins has been reported,in particular with respect to disease resistance.

A comparative proteomic analysis by two-dimensional electrophoresis combined with MS/MS analysis in a previous study[17] identified a G-protein α subunit,designated asGhGPA,which is potentially involved in cotton resistance toV.dahliae.The purpose of the present study was to investigate the role ofGhGPAin the resistance of cotton toV.dahliae.

2.Materials and methods

2.1.Plant materials and the V.dahliae strain

The resistant cotton cultivar ND601 was used for gene cloning.Twelve cotton cultivars including six tolerant and six susceptible cultivars (Table S1) were selected from a core collection of cotton comprising 419 accessions [18].Cotton seedlings were grown in a growth chamber under a 16/8 h light/dark cycle and at 27°C(day)/23°C (night).Wild-typeArabidopsis(Col-0) and theArabidopsis GhGPAorthologue geneAtGPA1mutant (gpa1-4,SALK_001846) were grown in a growth chamber under a 16/8 h light/dark cycle at a constant temperature of 22°C.

A highly aggressive defoliatingV.dahliaestrain (LX2-1) was used for the identification of disease resistance.The preparation of conidial suspensions (107conidia mL-1for cotton;106conidia mL-1forArabidopsis)and inoculation were performed as described previously[7].Control plants were inoculated with distilled water using the same method.

2.2.Gene cloning,sequence analyses,and subcellular localization

Total RNA from the upland cotton ND601 was extracted using an EASYspin Plus Plant RNA Kit (Aidlab Biotechnologies,Beijing,China) according to the manufacturer’s instructions.The RNA(5 μg) was used as a template to synthesize first-strand cDNA using the PrimeScript 1st Strand cDNA Synthesis Kit (TaKaRa Biomedical Technology,Beijing,China).The open reading frame(ORF)ofGhGPAwas cloned into the donor vector pDONR207 using GATEWAY BP Clonase Enzyme Mix (Invitrogen,Gaithersburg,MD,USA),transferred toEscherichia coliDH5α cells,and sequenced.The ExPASy program (http://www.expasy.org/) was used to compute protein parameters,including molecular weight,theoretical isoelectric point (pI),and amino acid composition.Proteins with sequence identity were identified and downloaded from the NCBI protein database ((http://www.ncbi.nlm.nih.gov/).Multiple sequence alignments were performed using DNAMAN 8 software(Lynnon Biosoft Company,Quebec,Canada).

Preparation of protoplasts fromArabidopsisand PEG–calcium transfection were performed as previously described [19].The ORF without the termination codon was amplified and inserted into the pGreen vector[20]between theClaI andSpeI sites to generate pGreen-GhGPA-GFP,which is a fusion protein with green fluorescent protein(GFP)at its C terminal.pGreen-GFP was used as a control.For subcellular localization,15 μg of recombinant plasmids and the plasma membrane marker AtPIP2A-mCherry [21]were co-transfected into 105protoplasts,and fluorescence signals were observed at 12 h after transfection,using a confocal laser scanning microscope(TCS SP8,Leica,Wetzlar,Germany)at specific excitation and emission wavelengths (GFP:488 and 510–550 nm,respectively;mCherry:587 and 600–640 nm,respectively).

2.3.Expression analysis of GhGPA in different tissues and under V.dahliae stress in cotton

The seeds of cotton varieties were covered with quartz sand to approximately 1 cm depth and soaked with sterile distilled water.After 7 days of growth,the seedlings were cultivated in 1/2 Hoagland nutrient solution for another 15 days.They were then inoculated with LX2-1 following Zhang et al.[22].Tissues (root,stem,and leaf) from at least five plants for each treatment were harvested at 0,6,12,24,36,and 48 h post-inoculation (hpi) and quickly frozen in liquid nitrogen.Total RNA was extracted as described above,and cDNA was generated using the PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa Biomedical Technology,Beijing,China).Gene-specific primers were used to analyze gene expression,and cottonUBQ14(GhUBQ14) was used as a reference gene for normalization.qRT-PCR was performed in a 10 μL reaction system using the ABI 7500 Real-Time PCR System(Applied Biosystems,Foster City,CA,USA) with three biological replicates of each sample and three technical replicates.The AugeGreen qPCR Master Mix(US Everbright Inc.,Suzhou,China)was used.The cycle threshold (CT) was used to calculate the relative expression level using the comparative 2-ΔΔCTmethod [23].

2.4.Generation of transgenic Arabidopsis

The entry vector pDONR207-GhGPAwas recombined into the destination vector pGWB414 [24] using the LR reaction mix II(Invitrogen,Carlsbad,CA,USA) to obtain the expression vector pGWB414-GhGPA,which was transformed intoAgrobacterium tumefaciensstrain GV3101.TransgenicArabidopsisplants were obtained using the floral dip method [25].The transformants (T0,T1,and T2seeds)were screened on half-strength MS medium containing 50 mg L-1kanamycin.T3transgenic lines were identified by PCR,qRT-PCR,and Western blotting and then used in subsequent experiments.

Protein samples fromArabidopsisleaves were separated on 12%SDS-PAGE gels.Western blotting was performed according to a previously described method[26]with Anti HA-Tag mouse monoclonal antibody and goat anti-mouse IgG,AP conjugated (CWBIO,Beijing,China).Twenty-day-oldArabidopsisplants were inoculated withV.dahliae.After 20 d post-inoculation (dpi),symptoms were scored.The degree of VW resistance was graded from 0 to 4 according to the extent of leaf chlorosis [27].The disease index(DI) was calculated as follows:DI=[(∑disease grades×number of infected plants)/ (total number of scored plants×4)]×100.

2.5.Cotton VIGS and identification of disease resistance

Cotton VIGS was performed according to a previously described procedure [28].A 386-bp fragment ofGhGPAwas amplified and inserted into the tobacco rattle virus (TRV) binary vector pTRV2 between theBamHI andSacI sites.Thecloroplastos alterados 1(CLA1) gene was used as a marker to monitor silencing reliability.The experiments were repeated three times independently with more than 35 plants used for each treatment.At 25 dpi,the symptoms of seedlings were classified into five grades(0,1,2,3,and 4)according to the symptoms on the leaves [29].The DI was calculated as described above.

2.6.Fungal recovery assay and biomass quantification

Fungal recovery assays forV.dahliaefrom infected cotton stems were performed as described previously[30].Colonies ofV.dahliaewere observed after three days of culture.qRT-PCR was performed to quantifyV.dahliaeDNA using aV.dahliae-specific primer pair(Vd-ITS-F and Vd-ITS-R),which was designed to complement the internal transcribed spacer (ITS) region.Total DNA was extracted from the first true leaves of cotton andArabidopsisplants using a Plant DNA Mini Kit(Aidlab Biotechnologies,Beijing,China).CottonGhUBQ14andArabidopsis AtTUB2were used as reference genes for the normalization of real-time quantitative PCR (qRT-PCR) data.

2.7.Expression analysis of defense marker genes

Leaf tissues ofArabidopsisand cotton infected withV.dahliaewere harvested at 2 dpi and frozen in liquid nitrogen for RNA extraction.Control plants were treated similarly with distilled water.Primers specific forPR1,PR2,PR3,PR4,andPR5were used to detect these defense marker genes inArabidopsisand cotton.Expression of the SA pathway-associated genesEDS1,PAD4,andNPR1and JA pathway-associated genesAOC,AOS1,andMYC2was detected in cotton.All qRT-PCR assays were repeated three times.

Fig.1.Characterization of GhGPA.(A) Schematic structure of GhGPA.Blue triangles denote conserved functional sites.(B) Subcellular localization of GhGPA in transiently transformed Arabidopsis mesophyll protoplasts.Images were taken using bright field,fluorescence,and merging.Scale bars represent 10 μm.(C)Expression of GhGPA in roots of ND601 after V.dahliae infection.(D)Expression of GhGPA in six tolerant and six susceptible upland cotton cultivars upon infection with V.dahliae.Values are means with standard deviation(SD)(n=3 biological replicates).Error bars represent the SD of three biological replicates.Asterisks indicate statistically significant differences according to Student’s t-test (two-tailed) (**, P<0.01).All experiments were repeated at least three times.

2.8.Measurement of SA and JA

The SA and JA measurement assay was performed by Triploid Biotech.(Wuhan,China).To measure the endogenous concentrations of SA and JA,approximately 500-mg leaf samples ofArabidopsisand cotton were harvested at 2 dpi and extracted with acetonitrile.Internal standards were added to each sample,and the phytohormones were measured using ESI-HPLC-MS/MS and a standard curve.

2.9.3,3′-diaminobenzidine (DAB) staining for ROS detection

Leaves fromArabidopsisand cotton plants were incubated in staining solution (1 mg L-1DAB,10 mmol L-1sodium phosphate at pH 7.0,and 0.05% v/v Tween-20) under gentle vacuum in darkness at 25°C for 3 h,decolorized in 95% ethanol until the green color faded to a yellowish color,and observed under a stereomicroscope.Leaves from at least five plants were sampled for each treatment.

2.10.Determination of H2O2 content and NADPH oxidase (NOX) and SOD activities

Fresh leaves were ground into powder in liquid nitrogen,extracted(0.1 g)in 1.0 mL of 0.1 mol L-1potassium phosphate buffer (pH 7.0),and centrifuged at 10,000×gfor 5 min at 4°C.The supernatant was used for H2O2quantification and for performing NOX and SOD activity assays using commercial kits (Jian Cheng Bioengineering Institute,Nanjing,China).Quantification of total protein in the supernatant was performed using a Micro BCA Protein Assay Kit (Thermo Scientific,Shanghai,China) with bovine serum albumin as a standard.All measurements were performed independently in triplicate.

2.11.Primers for gene cloning,vector construction,and expression analysis

All primers used in this study are listed in Table S2.

2.12.Statistical analysis

Experiments were repeated at least three times.Statistical analyses were performed using SPSS 20.0 (SPSS Inc.,Chicago,IL,USA).Three biological replicates were used to calculate the SD.Statistical significance of differences between treatment groups and controls was evaluated by one-way ANOVA.

3.Results

3.1.GhGPA is involved in the resistance response to V.dahliae

Fig.2.Overexpression of GhGPA increased Arabidopsis resistance to V.dahliae.The twenty-day-old T3 homozygous transgenic lines were inoculated with V.dahliae LX2-1.(A)Transcriptional level of GhGPA in transgenic lines and wild type by qRT-PCR.(B)GhGPA protein detection in transgenic lines and WT using Western blotting.The membranes were stained with Coomassie brilliant blue (CBB) as loading control.(C) VW symptoms of wild type and two homozygous transgenic plants (OE1,OE2) at 25 dpi.(D)Assessment of resistance level based on disease index at 25 dpi.(E) Quantification of V.dahliae biomass in middle-to-upper rosette leaves by qRT-PCR at 10 dpi.Values are means with SD(n=3 biological replicates).Error bars represent the SD of three biological replicates.Asterisks indicate statistically significant differences by Student’s t-test(two-tailed) (**, P<0.01).All experiments were repeated at least three times.

The ORF of the G protein α subunit,designated asGhGPA,cloned from the resistant upland cotton cv.ND601,contained 1179 nucleotides and encoded 392 amino acid residues.GhGPA had a molecular weight of 45.69 kDa and a theoretical pI of 5.85.Several conserved functional sites were found in GhGPA,including the GTP/Mg2+binding site,GoLoco binding site,adenylyl cyclase interaction site,beta-gamma complex interaction site,two switch regions (I and II),putative receptor binding site,and five G boxes(Fig.1A).Multiple amino acid sequence alignment showed thatGhGPAwas completely conserved among resistant and susceptible cotton cultivars(Fig.S1)and showed high identity with GPAs fromTheobroma cacao(93.9%),Vitis vinifera(88.6%),Populus trichocarpa(88.0%),Glycinemax(88.0%),Nicotiana tabacum(85.2%),Arabidopsis thaliana(85.0%) andOryza sativa(74.30%) (Fig.S2).These results suggest thatGhGPAperforms functions similar to those of other plant G-protein α subunits.In the subcellular localization analysis,the GhGPA-GFP fusion protein was transiently co-expressed with the plasma membrane marker mCherry (Fig.1B),indicating that GhGPA was localized in the plasma membrane.

qRT-PCR analysis indicated thatGhGPAwas expressed in roots,stems,and leaves,with highest expression in leaves(Fig.S3).Compared to mock-inoculated plants,GhGPAquickly responded toV.dahliaeinfection,maintaining high expression levels from 12 to 48 hpi,with a peak at 24 hpi (Fig.1C);thus,GhGPAwas involved in the defense againstV.dahliae.Furthermore,GhGPAshowed higher expression in all six tolerant cultivars than in the six susceptible cultivars at 48 hpi (Fig.1D),supporting its involvement in resistance.

3.2.Overexpression of GhGPA in Arabidopsis increased VW resistance

To better understand the function ofGhGPAin VW resistance,GhGPAwas transferred intoArabidopsisto generate transgenic lines driven by the CaMV35S promoter.The transgenic lines (T3) were confirmed using a kanamycin plate,qRT-PCR,and Western blotting(Fig.2A,B).VW resistance was assessed in two overexpression(OE)lines (OE1 and OE2).OE1 and OE2 displayed slight VW symptoms compared to the WT at 20 dpi(Fig.2C).The DI of the WT was 56.4,whereas those of the OE1 and OE2 were dramatically lower,at 35.2 and 28.6,respectively (Fig.2D).Moreover,the fungal biomass in the transgenic lines was significantly lower than that in the WT(Fig.2E).Thus,transgenicArabidopsislines overexpressingGhGPAshowed increased resistance againstV.dahliae.

3.3.GhGPA restores Arabidopsis mutant gpa1-4 defect in resistance to V.dahliae

Fig.3.The performance of different Arabidopsis lines response to V.dahliae.Arabidopsis AT2G26300 mutant gpa1-4 resistance to VW was complemented by GhGPA.GhGPA was ectopically expressed in an Arabidopsis AT2G26300 mutant.(A,B) Identification of homozygous mutants and ectopic expression in Arabidopsis by triple primer method and Western blotting,respectively.The left border primer of the T-DNA insertion and forward and reverse primers of AT2G26300 were used to amplify DNA of the mutants.(C)The VW symptoms of wilt-type,mutant gpa1-4,EC line and OE2 line at 20 dpi.(D)The disease index at 20 dpi.(E)Biomass of V.dahliae from the infected seedlings was quantified by qRT-PCR in middle-to-upper rosette leaves.Values are means with SD(n=3 biological replicates).Error bars represent the SD of three biological replicates,Lowercase letters represent statistically significant differences(P<0.05)according to Tukey’s honest significant difference(HSD)test.All experiments were repeated at least three times.

Given thatGhGPAshowed high identity withAtGPA1(At2G26300),GPAmight also be involved in VW resistance inArabidopsis.To test this speculation,a T-DNA insertion mutantgpa1-4(SALK_001846) was used for ectopic expression (EC) ofGhGPA(Fig.3A,B).After infection withV.dahliae(20 dpi),gpa1-4showed more severe disease symptoms (Fig.3C),with higher DI (Fig.3D) and more fungal biomass in stems (Fig.3E)than the WT.These results were in accord with the downregulation ofGPAexpression.WhenGhGPAwas expressed ingpa1-4,the EC plants displayed weaker disease symptoms thangpa1-4and almost identical phenotypes to those of OE2(Fig.3C).There was no statistically significant difference between EC and OE2 plants in either DI or fungal biomass(Fig.3D,E),suggesting thatGhGPAsuccessfully restored the resistance of thegpa1-4mutants toV.dahliae.

3.4.Silencing of GhGPA reduced cotton VW resistance

The involvement ofGhGPAin cotton resistance toV.dahliaewas tested by use of VIGS to specifically silenceGhGPA.At approximately 10 days post-infiltration,the first true leaves of infiltrated plants withAgrobacteriumcarryingGhCLA1displayed an albino phenotype (Fig.4A),indicating that the silencing was successful.The expression level ofGhGPAin theTRV:GhGPAplants was significantly lower than that in the empty-vector control(TRV:00)plants(Fig.4B),indicating thatGhGPAwas effectively silenced in the cotton plants.The plants were then inoculated withV.dahliae.Compared to theTRV:00plants,the silenced cotton plants displayed more severe VW symptoms at 25 dpi,in the form of wilted and etiolated leaves(Fig.4C).Most of the silenced plants showed a higher disease grade (1,2,or 3);however,the majority of theTRV:00plants presented a disease grade of 0,1,or 2 (Fig.4D).The DI of the silenced plants (50.3) was significantly higher than that of theTRV:00plants(27.9)(Fig.4E).More fungal colonies were recovered from the stems of the silenced plants(Fig.4F),and more fungal biomass was detected from the leaves of the silenced plants(Fig.4G).These results showed thatGhGPAplays a positively role in cotton VW resistance.

3.5.GhGPA affected the expression of PR genes and regulated the SA and JA signaling pathways in Arabidopsis and cotton

To investigate howGhGPAfunctions in the process of resistance toV.dahliae,the expression of several PR genes was measured inArabidopsisand cotton.As shown in Fig.5,upon inoculation withV.dahliae,PR genes displayed significantly higher expression inGhGPA-overexpressingArabidopsisthan in the WT;however,the expression of PR genes was lower in thegpa1-4plants than in the WT,and their transcriptional levels were rescued by ectopic expression ofGhGPA.Similarly,PR genes were significantly down-regulated in theTRV:GhGPAplants compared to the control.Thus,GhGPAincreased the expression of PR genes.

To determine whetherGhGPAis involved in hormone-mediated signaling defense response,we measured the expression of marker genes associated with the SA and JA signaling pathways in cotton.As shown in Fig.S4,the transcriptional levels of these marker genes were significantly down-regulated in theTRV:GhGPAplants under normal conditions compared to those in theTRV:00plants.Moreover,after inoculation withV.dahliae,these genes were significantly suppressed in theTRV:GhGPAplants,suggesting thatGhGPAmight affectV.dahliaeresistance by regulating the SA and JA signaling pathways in cotton.

To further confirm the expression results of the marker genes and investigate the function ofGhGPAinArabidopsisand cotton,we measured SA and JA after plant inoculation withV.dahliae.In agreement with the expression of marker genes in SA and JA signaling pathways after treatment withV.dahliae(Fig.6),the SA content was lower in thegpa1-4mutants andTRV:GhGPAplants than in the WT andTRV:00plants,respectively.However,the SA content was significantly increased in the transgenicArabidopsisplants compared to that in the WT plants.The JA content was significantly lower in thegpa1-4andTRV:GhGPAplants.Thus,GhGPAactivated defense responses via the SA and JA signaling pathways to confer VW resistance.

Fig.4.Silencing of GhGPA in cotton cv.ND601 increased the susceptibility of the plants to V.dahliae.(A) Albino phenotype of TRV:GhCLA1 at 15 days after infiltration.(B)Detection of GhGPA silencing efficiency by qRT-PCR.(C)The VW symptoms of silenced and TRV:00 plants at 25 dpi.(D)Statistics of the seedling number with different diseasegrade at 25 dpi.(E)Disease index of seedlings at 25 dpi.(F)V.dahliae isolation from the infected stems.(G)Detection of fungal biomass from the first true leaves by qRT-PCR.Values are means with SD(n=3 biological replicates).Error bars represent the SD of three biological replicates.Asterisks indicate statistically significant differences according to Student’s t-test (two-tailed) (**, P<0.01).All experiments were repeated at least three times.

Fig.5.Expression of PR genes in Arabidopsis and cotton.(A–E),qRT-PCR analysis of the expression of PR genes in wild-type,transgenic Arabidopsis lines,mutant gpa1-4,and EC plants,respectively.(F–J),qRT-PCR analysis of the expression levels of PR genes in silenced plants and control.Values are means with SD(n=3 biological replicates).Error bars represent the SD of three biological replicates,Lowercase letters represent statistically significant differences (P<0.05) according to Tukey’s HSD test.All experiments were repeated at least three times.

3.6.GhGPA contributed to the production of ROS

Most defense reactions in plant–pathogen interactions are associated with the rapid production of ROS [31].To determine whetherGhGPAis involved in ROS production,leaves fromArabidopsisand VIGS cotton plants inoculated withV.dahliaewere stained with DAB.Leaves from thegpa1-4mutant plants andTRV:GhGPAplants showed a weaker intensity of brown deposits than those from WT andTRV:00plants,respectively,but the leaves from the OE2 showed a stronger intensity than those from WT plants(Fig.7A,B).Furthermore,the intensity in leaves from EC plants was similar to that in leaves from the WT plants and higher than that in leaves fromgpa1-4plants (Fig.7A).The deposits in leaves of theTRV:GhGPAplants showed a weaker intensity than those in the leaves ofTRV:00plants under water treatment.These results suggest thatGhGPAis involved in ROS production during plant immune responses toV.dahliae.

The levels of H2O2and the enzyme activities of NADPH oxidase(NOX)and superoxide dismutase(SOD)were measured,which are considered primary regulators of ROS production.After inoculation withV.dahliae,the H2O2content and the activities of both NOX and SOD in theArabidopsisWT (Fig.8A–C) and cottonTRV:00plants(Fig.8D–F) were significantly increased compared with those in plants under water treatment.GhGPAdid not significantly increase the H2O2content(Fig.8A)and enzymatic activity(Fig.8B,C)under normal conditions (H2O).However,the OE plants showed higher H2O2content and enzymatic activity than the WT plants after inoculation withV.dahliae.TheTRV:GhGPAplants showed lower H2O2contents (Fig.8D) and enzymatic activity (Fig.8E,F) than theTRV:00plants,indicating thatGhGPAplays a central role in the production of H2O2and the regulation of NOX and SOD activities.

These findings indicate thatGhGPAis involved in plant defense againstV.dahliaevia H2O2accumulation and increase in activities of NOX and SOD.

4.Discussion

Fig.6.Measurement of SA and JA content in Arabidopsis and cotton plants.Arabidopsis seedlings at 20 days post-infiltration(dpi)and cotton seedlings at 10 dpi were inoculated by V.dahliae and leaves at 2 dpi from each line were used for SA and JA detection.(A)Measurement of SA content in mutant gpa1-4,transgenic Arabidopsis,and wild type.(B)Measurement of SA content in GhGPA-silenced cottons and the control.(C)Measurement of JA content in the mutant gpa1-4,transgenic Arabidopsis and wildtype.(D)Measurement of JA in GhGPA-silenced cottons and the control.Values are means with SD(n=3 biological replicates).Error bars represent the SD of three biological replicates,Lowercase letters represent statistically significant differences(P<0.05)according to Tukey’s HSD test.All experiments were repeated at least three times.

In all eukaryotes,G protein complexes,comprising one Gα,one Gβ,and one Gγ subunit,are key signal transducers between upstream receptors and downstream events (effectors).G protein activation depends on the release of GDP by Gα and its binding to GTP [8].In our previous study,GhGPAwas differentially expressed in response toV.dahliae[17],indicating that it is potentially important for cotton resistance.In the present study,we clonedGhGPAand studied its role in cotton defense againstV.dahliae.There were several conserved functional sites in GhGPA,and GhGPA showed high amino acid sequence identity with GPAs from other plants.GhGPA possessed all the signature sequences required for releasing GDP,binding GTP,and accumulating in the active state without a regulatory protein,similarly to other known plant Gα proteins [9].

G proteins mediate multiple processes in plants,including immune responses to pathogens [8,32,33].In this study,the expression ofGhGPAwas up-regulated in cotton roots underV.dahliaestress andGhGPAwas more strongly expressed in tolerant than in susceptible cultivars.Thus,GhGPAresponds toV.dahliaeinfection.Furthermore,GhGPAoverexpression and mutant recovery withGhGPAinArabidopsisandGhGPA-VIGS in cotton clearly demonstrated thatGhGPApositively regulates resistance toV.dahliaeinArabidopsisand cotton.

Plant immune responses are initiated mostly by the recognition of pathogen invasion by immune receptors.Pattern recognition receptors that detect pathogen-associated molecular patterns,consisting mainly of various defense-related receptor-like protein/kinases (RLP(K)s),are usually localized on the plasma membrane[34,35].Some RLP(K)s interacting with G protein,such as chitin elicitor receptor kinase 1 (CERK1),brassinosteroid insensitive 1(BRI1),BRI1-associated kinase (BAK1),BAK1-interacting receptor 1 (BIR1),and FLS2 are called G-protein-coupled receptors (GPCRs)[8,36–38].In the present study,GhGPA was localized to the plasma membrane,suggesting its potential role in the signal transduction pathway.GPCRs and the downstream targets ofGhGPAawait further study.

Fig.7.Detection of ROS activities via 3,3′-diamino-benzidine staining at 2 dpi.(A)Arabidopsis.(B)GhGPA-silenced cottons and the control under normal condition and after inoculation for 2 days.Bars,100 μm.

PR genes,which encode proteins that accumulate upon pathogen challenge,have become markers of plant immune responses and pathways [39].PR1,PR2,PR3,PR4,andPR5are genes responsive to SA and JA,which contribute to the resistance of cotton toV.dahliae[40,41].SA,a key plant defense-associated hormone,plays a major role in systemic acquired resistance and broadspectrum and long-lasting disease resistance [42,43].JA plays a crucial role in plant defense responses against necrotrophic and biotrophic fungal infections [44,45].In our study,the PR genes were induced byV.dahliaeat the transcriptional level (Fig.5,ArabidopsisWT and cottonTRV:00),suggesting that they are involved in plant resistance toV.dahliae.Overexpression and mutation ofGhGPAinArabidopsisrespectively increased (OE and EC) and reduced (gpa1-4)GPAexpression,suggesting that these PR genes are regulated byGPA.As expected,expression of marker genes in the SA and JA signaling pathways and hormone content showed that the down-regulated expression ofGhGPAin the VIGS cotton inhibited SA and JA signaling pathway-mediated defense.However,overexpression ofGhGPAinArabidopsisactivated the SA and JA signaling pathways.We accordingly inferred thatGhGPAactivates the JA and SA signaling pathways in cotton uponV.dahliaeinfection.

ROS accumulate in plants after their perception of phytopathogens,and their roles in the defense responses of various plants,such as direct microbicidal activity,local and systemic signaling to activate downstream immune responses,and oxidative crosslinking to strengthen the cell wall,have been widely studied[46,47].Significantly increased ROS accumulation in roots uponV.dahliaeinfection [6,48] showed that ROS levels are associated with cotton resistance toV.dahliaeinfection.Previous studies[15,16] revealed Gα proteins fromN.benthamianaand rice to be associated with H2O2accumulation triggered by elicitors and blast fungi.Similarly,the transcription ofGhGPAwas positively associated with ROS production during plant responses toV.dahliaeinfection.Thus,ROS accumulation mediated byGPAmay be a universal defense response in plants.

Plant G proteins had been reported [8] to function in various biotic stress.BothV.dahliaeandFusarium oxysporum f.sp.vasinfectum(FOV) cause severe vascular diseases in cotton.In this study,GhGPAnot only responded toV.dahliae,but also could be induced by FOV as indicated by qRT-PCR result in multiple cotton cultivars(Fig.S5).This finding,suggesting thatGhGPAmay mediate broadspectrum resistance against vascular disease,awaits further study.

5.Conclusions

The G-protein α subunit ofGossypium hirsttumGhGPA contains conserved functional sites of the Gα protein,shows high identity with other known plant GPAs,and is localized to the plasma membrane.UnderV.dahliaestress,GhGPAwas strongly up-regulated in cotton roots and showed higher transcriptional levels in tolerant than in susceptible cultivars.GhGPApositively regulated cotton VW resistance by increasing PR gene expression and regulating SA and JA signaling pathways,H2O2accumulation,and enzymatic activity.

CRediT authorship contribution statement

Zhiying Ma and Xingfen Wang designed the research and revised the manuscript.Bin Chen wrote the manuscript.Yan Zhang and Jun Yang performed the data analysis.Bin Chen,Man Zhang,and Qingming Ma performed the experiments.All authors read and approved the final manuscript.

Fig.8.Determination of H2O2 content and NOX and SOD activities in Arabidopsis and cotton leaves at 2 dpi.(A)H2O2 content in the transgenic and wild-type Arabidopsis.(B,C)NOX and SOD activities in Arabidopsis.(D)H2O2 content in VIGS cotton.(E,F)NOX and SOD activities in VIGS cotton.Values are means with SD (n=3 biological replicates).Error bars represent the SD of three biological replicates,Lowercase letters represent statistically significant differences (P<0.05) according to Tukey’s HSD test.All experiments were repeated at least three times.

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.

Acknowledgement

We thank Xiaohong Zhang from Northwest A&F University for the kind gift of theArabidopsis gpa1-4mutant.This work was supported by the National Key Research and Development Program of China (2016YFD0101006),the China Agricultural Research System(CARS15-03),and the Outstanding Youth Found of Hebei Province(C2019204365).

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2020.09.008.