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    TaARPC5 is required for wheat defense signaling in response to infection by the stripe rust fungus

    2022-02-19 09:31:20JiGuoHunPengTuoQiSndingXuMdAshrfulIslmBrdDyQingZhenshengKngJunGuo
    The Crop Journal 2022年1期

    Ji Guo,Hun Peng,Tuo Qi,Snding Xu,Md Ashrful Islm,Brd Dy,Qing M,Zhensheng Kng,Jun Guo,*

    a State Key Laboratory of Crop Stress Biology for Arid Areas and College of Plant Protection, Northwest A&F University, Yangling 712100, Shaanxi, China

    b Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing, MI 48824, USA

    Keywords:ARPC5 Cytoskeleton Wheat Puccinia striiformis f.sp. tritici Virus-induced gene silencing (VIGS)

    ABSTRACT Numerous studies using a combination of confocal microscopic- and pharmacological-based approaches have demonstrated that the actin cytoskeleton dynamically responds to pathogen infection.Here, we observed that phalloidin treatment induced actin nucleation, resulting in enhanced resistance of wheat against the stripe rust pathogen Puccinia striiformis f.sp. tritici (Pst).To define the mechanism underpinning this process,we characterized a family of conserved actin-binding proteins,the actin related protein(ARP) family, which controls actin polymerization.Specifically, we identified and characterized a wheat ARPC gene(TaARPC5),which encodes a 136-amino acid protein containing a P16-Arc domain,the smallest subunit of the ARP2/3 complex.TaARPC5 mRNA accumulation was induced following the infection of plants with the avirulent Pst strain, and following the elicitation with flagellin (e.g., flg22) as well.Subcellular localization analysis revealed that TaARPC5 is primarily localized to the cortical actin cytoskeleton, and its precise cellular localizations suggest the proximity to processes correlated with the actin-organelle interface.Upon treatment with virulent Pst, TaARPC5-knockdown plants exhibited a significant reduction in the expression of PTI-specific mRNAs.Conversely, we observed enhanced induction of reactive oxygen species(ROS)accumulation and a decrease in TaCAT1 expression following infection with an incompatible Pst isolate.Together with yeast complementation assays, the current study demonstrates a role for TaARPC5 in resistance signaling in wheat against Pst infection by regulating the host actin cytoskeleton.

    1.Introduction

    The plant cytoskeleton, comprised of actin microfilaments and microtubules, is highly dynamic and versatile and required for a suite of cellular processes, including those associated with responses to endogenous and external stimuli.As a key regulator of cellular response to the environment, the actin cytoskeleton has been demonstrated to be essential for the perception, activation,and signaling in response to abiotic and biotic stimuli,including pathogen infections[1-6].Data also support a role for the actin cytoskeleton during multiple stages, and the activation, of host defenses during infection, suggesting that the function and dynamic nature of the host cytoskeleton are key regulators of immunity [7].

    Plant defense signaling is generally classified based on the activation of two temporally distinguishable and yet interlinked nodes.The first tier, pattern-triggered immunity (PTI), is widely considered to function as a broad-based host immune response elicited by the recognition of conserved pathogen-associated molecular patterns (PAMPs; e.g., chitin, flagellin, and lipopolysaccharide) [8,9].Following the perception of PAMPs by host pattern recognition receptors(PRR),rapid,and often transient,responses are activated, including the oxidative burst response,callose deposition, phytoalexin accumulation, actin cytoskeleton rearrangement, and activation of defense genes.Of the PAMPPRR interactions, the perception of chitin, a major component of fungal cell walls, by the PRRs CHITIN ELICITOR BINDING PROTEIN(CEBiP) and CHITIN ELICITOR RECEPTOR-LIKE KINASE 1 (CERK1)has been demonstrated to be an early and critical component of fungal pathogen perception and the activation of defense signaling in wheat[10].Similarly,the recognition of bacterial pathogen flagellin(i.e.,flg22)by the PRR FLAGELLIN-SENSING 2(FLS2)has been demonstrated to initiate a suite of downstream signaling processes hypothesized to halt bacterial pathogen growth and proliferation[11,12].

    Pathogens have evolved unique strategies to suppress host defense activation and signaling.Among these, the delivery of the secreted virulence factors, such as type-III secreted effectors(T3Es), is arguably the best characterized pathogen virulence mechanism to evade the host immune system by activation of the second tier of the plant immune system, effector-triggered immunity (ETI) [8].Moreover, using pathogen effector molecules and cellular probes, numerous studies have begun to uncover the physiological mechanisms that plants use to sustain immune signaling and the virulence strategies that pathogens use to dampen these responses [13].As a platform for defense signaling and the regulation of the host immune system,recent studies have demonstrated the role of the plant actin cytoskeleton during pathogen infection [3,5,6,14].As a primer for much of this work, the earlier studies, using a suite of pharmacological-based approaches,revealed that the plant actin cytoskeleton was rapidly rearranged during infection by fungal pathogens [15].Besides, actininteracting drugs such as cytochalasins, latrunculin, phalloidin,and jasplakinolide have been reported as important tools to dissect the regulation of actin cytoskeletal organization during plantpathogen interactionsin vivo.In total,these approaches have built the foundation for describing the relationship(s) between cytoskeletal organization and resistance/susceptibility [16-19].In addition to demonstrating the correlations among the filament bundling, depolymerization, and stabilization, pharmacologicalbased approaches have also illuminated our understanding of the roles actin filament organization play in regulating guard cell aperture[20],papillae formation[15],and the early defense responses,such as inducing gene expression and activating the pathogenesis related (PR) proteins [21].

    The actin-related protein(Arp)2/3 complex was first discovered inAcanthamoebaand is largely conserved across all eukaryotes[22].In short, the Arp2/3 complex is a multifunctional actin organizer,which promotes the actin assembly and nucleation and regulates filament cross-linking and branched actin nucleation[23].In amoebas,yeast,and mammals,the Arp2/3 complex has seven subunits named Arp3, Arp2, p41-Arc, p34-Arc, p21-Arc, p20-Arc, and p16-Arc.InArabidopsis,WURM,DISTORTED1,DISTORTED2andCROOKEDencode the ARP2, ARP3, ARPC2, and ARPC5 subunits,respectively.Mutations in Arp2/3 complex lead to disordered microfilament organization, resulting in various phenotypes,including trichome, root hair, and hypocotyl deformation [24,25].In addition to the association with processes controlling developing and cell architecture, ARPC4 and ARPC5 have been demonstrated to play key roles in mediating actin dynamics associated with ABA- and H2O2-induced guard cell actin dynamics [26].

    Wheat stripe rust, caused by an obligate parasitic fungus,Puccinia striiformisf.sp.tritici(Pst), causes tremendous yield losses on wheat production in cool and temperate regions, especially in China[27,28].Stripe rust-resistant wheat cultivars are usually race specific and their resistances are mediated by gene-for-gene interactions[29].As such,the use of fungicides for disease management is generally ineffective.Therefore, it is crucial to define the signaling mechanisms underpinning resistance toPstas an approach that will lead to potential strategies for engineering durable resistance in wheat.In the current study, we identified and characterized a wheat P16-Arc homolog,TaARPC5, and characterized the role of this Arp2/3 subunit duringPstinfection of wheat.Using a combination of genetic-, cellular-, and pharmacological-based approaches,we herein demonstrate thatTaARPC5is required for resistance signaling againstPstinfection, and moreover, its functions in this capacity are performed by regulating actin filament organization during infection.

    2.Materials and methods

    2.1.Plant materials and fungal isolates

    Wheat (Triticum aestivum) cultivar Suwon11 andPuccinia striiformisf.sp.tritici(Pst) isolates CYR23 (incompatible) and CYR31/CYR32 (compatible) were involved in this study.The urediniospores were collected from the inoculated wheat cultivar MX169.Wheat seedlings were grown in an environmentally controlled growth chamber at 16 °C with a 16 h/8 h (light/dark) photoperiod and 60% relative humidity (RH).

    2.2.Phalloidin treatment

    Phalloidin(Sigma-Aldrich,https://www.sigmaaldrich.com)was dissolved in ddH2O to the final concentration of 0 μmol L-1(DMSO), 1 μmol L-1, 5 μmol L-1, and 10 μmol L-1.Previous study has reported that concentrations of DMSO up to 0.5%(v/v)does not inhibit the germination, growth, and/or differentiation of urediniospores.The phalloidin solution (2 mL) was transferred into the apoplast under gentle pressure using a needleless syringe as previously described [30].All the inoculated wheat leaves were rinsed three times with ddH2O before inoculating with fresh urediniospores.Two wheat cultivars Suwon 11 and Fielder were inoculated with twoPstisolates, CYR31 and CYR32, respectively.Each treatment consisted of at least 30 leaves.

    2.3.Cloning and identification of TaARPC5

    Brachypodium distachyonactin related protein complex subunit 5 gene sequence was submitted to ensemble plant website against the wheat IWGSC v1.0 genome by blastP.Three 408-nucleotide sequences were found in wheat chromosome 2A, 2B, and 2D(TraesCS2A01G106100.2, TraesCS2B01G123100.1, and TraesCS2D01G105700.1), respectively.The structures of those three genes were predicted by the IWGSC genome.Phylogenetic tree analysis was performed with ARPC5 from 17 species by MEGA 6.1 [31], and named those genes asTaARPC5-2A,TaARPC5-2B, andTaARPC5-2C.Based on those data,TaARPC5(GenBank accession number # KF606978.1) was cloned from the wheat cv.Suwon 11 by a special primer designed using primer5 (Table S1).The amino acid sequence of protein TaARPC5 was analyzed with InterProScan(http://www.ebi.ac.uk/InterProScan) and PROSITE (https://prosite.expasy.org/)for conserved domain identification and signal peptide prediction.Multiple sequence alignment was performed via the CLC Sequence Viewer 6 and DNAMAN6.0 (Lynnon BioSoft,USA).

    2.4.Quantitative RT-PCR analysis

    To identify the relative expression levels ofTaARPC5duringPstinfection,leaf samples were harvested at 0,6,12,18,24,36,48,72,120 h post inoculation(hpi).For flg22 treatment,10-day-old wheat seedlings were sprayed with 500 nmol L-1flg22 and sampled at 0,0.5,0.75,1,3,6,12,24,and 48 h post treatment(hpt).Roots,stems,and leaves of 10-day-old wheat seedlings were sampled to assess mRNA accumulation in different tissues.

    Total RNA of the samples mentioned above was extracted using the BiozolTM (Invitrogen, Carlsbad, CA).Total RNA (2.5 μg) was used for first strand cDNA synthesis using the Promega RT-PCR system(Promega,Madison,WI,USA)with the oligo(dT)18 primer.As the three homologs of TaARPC5 are too conserved(above 99%identity) to be distinguished, the qRT-PCR ofTaARPC5was performed for the total transcript levels of three homeologs with the primers specifically designed forTaARPC5(Table S1) by using the BioRad CFX sequence detection system (BioRad, USA) [32].The transcript level (i.e., mRNA accumulation) ofTaARPC5was analyzed by the comparative 2-ΔΔCTmethod [33], and the transcript level of TaEF-1α(GenBank accession number Q03033)was used as the reference for normalization (Table S1).The relative expression levels ofTaARPC5were calculated from three independent biological replicates.

    2.5.Virus induced gene silencing (VIGS)

    A specific fragment(208 bp)designated asTaARPC5-as was used for silencing theTaARPC5.This fragment was constructed into the BMSV-γ genome with the primers shown in Table S1 and designated as γ-TaARPC5-as.TaPDS-as.This fragment worked well as positive control in the VIGS system.The BMSV genomes with α,β, γ, γ-TaARPC5-as, and γ-TaPDS-as were generatedin vitrowith the Message T7in vitrotranscription kit (Ambion, Austin, TX,USA.) following the manufacturer’s instruction.The RNA of the BSMV genomes α, β with γ, or γ-TaARPC5-as, or γ-TaPDS-as were mixed in a 1:1:1 ratio and mechanically rubbed onto the second leaves of wheat seedlings with FES buffer (0.5:0.5:0.5:8.5).The wheat seedlings were cultured in a growth chamber at 25 °C.Ten days after inoculation, the phenotypes of inoculated leaves were observed.TheTaPDS-as-silencing leaves, which expressed photobleaching phenotypes served as the positive control; leaves exhibiting similar photo-bleaching symptoms were noted as the successfully silenced materials.All the silenced wheat leaves were inoculated withPstisolate CYR23 or CYR31,respectively.The inoculated leaves were collected at 0, 24, 48, and 120 hpi for qRT-PCR assays and histological observations.The phenotype ofPstinoculated leaves was photographed at 14 dpi.

    For histological observation, the samples were stained with wheat germ agglutinin (WGA), and the infection sites and the infection hyphae length were subsequently observed with an Olympus BX-51 microscope (Olympus Corp., Tokyo, Japan).The necrotic areas around infection sites were observed under the auto-fluorescence.At least 50 infection sites were examined for each sample, and all the experiments included three biological replicates.

    2.6.Subcellular localization of TaARPC5

    The recombinant plasmid pJIT163:TaARPC5-GFP was constructed and transferred into wheat protoplasts using PEG4000 as previously described for subcellular localization in wheat [34].The transformed wheat protoplasts were cultured at 23 °C for 12 to 48 h.For the localization inNicotiana benthamiana,the recombinant plasmid pCAM1302:TaARPC5-GFP was constructed and infiltrated into tobacco cells withAgrobacterium tumefaciensstrain GV3101.F-actin was stained by TRITC-phalloidin following the manufacturer’s instructions (YEASEN, USA).The fluorescence was photographed with a confocal laser scanning microscope(Olympus microscope FV3000, Tokyo, Japan).GFP was used as control.

    2.7.Yeast mutant complementation assay

    The yeastSaccharomyces cerevisiaediploid mutant strain YIL062c (BY4743; MATa/MATα; ura3Δ0/ura3Δ0; leu2Δ0/leu2Δ0;his3Δ1/his3Δ1; met15Δ0/MET15; LYS2/lys2Δ0; YIL062c/YIL062c::kanMX4)and WT strain BY4741(MATa;his3D1;leu2D0;met15D0;ura3D0)were obtained from EUROSCARF(Table S2).The recombinant plasmid pDR195:TaARPC5was constructed and transferred into the mutant strain YIL062c by electroporation.The colonies were selected on SC-U medium at 37 °C.To examine the complementation yeast under different stress conditions, yeast cells with pDR195 (Δarc15+ empty), pDR195-TaARPC5(Δar-c15+ TaARPC5) and WT-1 were cultured on SC medium and treated with 0.3 mol L-1NaCl,0.1 mol L-1CaCl2,2 mol L-1Sorbitol,and 0.05 mmol L-1H2O2,respectively.Then,the yeast cells(OD6000.2)were stained with 4′,6-diamino-2-phenylindole(DAPI)to examine their nuclei under a fluorescence microscope with a blue filter.

    3.Results

    3.1.Phalloidin treatment enhances resistance of wheat against Pst infection.

    Phalloidin stabilizes actin and effectively prevents the depolymerization of actin fibers.To assess the impact of increased actin filament polymerization on the response of wheat toPst, we inoculated two wheat cultivars,Suwon 11 and Fielder,with twoPstisolates, CYR31 and CYR32, under treatment with different concentrations of phalloidin (0 μmol L-1(DMSO), 1 μmol L-1,5 μmol L-1, and 10 μmol L-1) (Fig.S1A, B).Urediniospores of CYR31 showed no significant differences with respect to germination and differentiation (Fig.S1C), which is consistent with the observation previously reported by Tucker and colleagues forUromyces[35].At 11 dpi,the plants treated with 1 μmol L-1phalloidin showed no significant change compared with the mock-treated plants in both Suwon 11 and Fielder inoculated with CYR31 and CYR32, whereas in the plants treated with 5 μmol L-1phalloidin,more than 50% of the leaves showed a mild disease phenotype compared with mock-treated plants.Meanwhile, in both Suwon 11 and Fielder plants treated with 10 μmol L-1phalloidin more than 90% and 60% expressed mild disease symptoms caused by CYR31 and CYR32, respectively (Fig.S1D).However, after 16 dpi,disease symptoms were not significantly different compared to mock-treated leaves.These results suggest that actin fibers may be involved in resistance of wheat toPst.

    3.2.Structural and phylogenetic analysis of TaARPC5

    The ARP2/3 complex consists of seven subunits of different molecular weights and was first discovered inAcanthamoeba castellanii[22].The complex is an enhancer of actin nucleation and polymerization.As our previous study showed, TaARPC3 contributes to resistance againstPst[46].To investigate the involvement of other subunits of the ARP2/3 complex, genes containing the P16-Arc domain were up-regulated duringPstinfection(shown in Section 3.3).Three genes were found in wheat chromosome 2A,2B, and 2D (TraesCS2A01G106100.2, TraesCS2B01G123100.1, and TraesCS2D01G105700.1).Phylogenetic analysis revealed that these genes cluster with BdARPC5 named asTaARPC5-2A,TaARPC5-2B,andTaARPC5-2C(Fig.1A).TaARPC5-2A,TaARPC5-2B, andTaARPC5-2Cshowed similar gene structure with three introns and phase 0 or 1 (Fig.1B) and the predicted putative 135-amino acid proteins were highly conserved (above 99% identity) (Fig.1C).TaARPC5was cloned from wheat cultivar Suwon 11 (GenBank accession number KF606978.1)with a predicted molecular weight of approximately 15 kDa and a predicted P16-Arc domain (Figs.1D, S2).

    3.3.TaARPC5 is differentially induced by both Pst infection and flg22 treatment

    To analyze the roles ofTaARPC5in incompatible and compatible interactions,we evaluated the changes inTaARPC5mRNA accumulation by quantitative real-time PCR (qPCR).As shown in Fig.2A,the expression level ofTaARPC5was found to be up-regulated at 18 hpi, a time point regarded as an early stage forPstpenetration.However, at 96 hpi, mRNA accumulation ofTaARPC5was significantly increased in the incompatible interaction over that in the compatible interaction (Fig.2A).To explore the possibility thatTaARPC5is involved in PTI, we analyzed the gene expression induced by flg22 in wheat leaves,TaARPC5was induced at 0.5 and 24 hpt (Fig.2B).In addition,TaARPC5was highly induced in stems and leaves, with expression levels approximately 4- and 2-fold more than those in roots, respectively (Fig.2C).These results support the hypothesis thatTaARPC5participates in flg22-triggered PTI and may also be associated with broader, basal defense signaling processes.

    Fig.1.Sequence analysis of TaARPC5 and other ARPC5 proteins of higher plants.(A)Phylogenetic analysis of ARPC5 genes from monocots,eudicots,animals,and fungi.(B)The structure of the TaARPC5-2A,TaARPC5-2B,and TaARPC5-2C.The green box represents the exon and the black lines represent introns.The numbers 0 or 1 indicates phase.(C)Multiple protein sequence alignment of TaARPC5-2A, TaARPC5-2B, and TaARPC5-2C (D) Multiple amino acid sequence alignment of TaARPC5 with ARPC5 proteins from Setaria italica (SiARPC5), Brachypodium distachyon (BdARPC5), Zea mays (ZmARPC5), Sorghum bicolor (SbARPC5), and Oryza sativa (OsARPC5).The P16-Arc domains are underlined.

    Fig.2.The transcript level of TaARPC5 in different tissues in response to Pst infection and flg22 treatment.(A) qRT-PCR analysis of wheat leaves inoculated with Pst race CYR23(avirulent)or CYR31(virulent).Samples were collected at 0,12,18,24,36,48,72,96,and 120 h post inoculation(hpi).(B)Transcript levels of TaARPC5 in wheat leaves treated with flg22 for 0, 0.5, 0.75, 1, 3, 6, 12, 24, and 48 h.(C) qRT-PCR analysis of TaARPC5 mRNA accumulation in different wheat tissues.Asterisks indicate significant difference (P < 0.05) from 0 hpi or root using Student’s t-test, respectively.

    3.4.TaARPC5-GFP fusion protein is located in cytoplasm, nucleus, and the network of actin in both wheat protoplasts and N.benthamiana

    To dissect the function of TaARPC5,we investigated the cellular localization of TaARPC5-GFP through transient expression in wheat protoplasts andN.benthamiana.As shown in Figs.3A and B,S3,the green fluorescent signal was predominantly localized in the cytoplasm, nucleus, and the network of actin.

    3.5.TaARPC5 partially complements the function of the gene in Saccharomyces cerevisiae.

    Previous studies have utilized the heterologous expression of Arp2/3 subunits inSaccharomyces cerevisiaeto infer the gene/protein,including their roles in cell organization,cell division,as well as growth and development.To evaluate the function of TaARPC5,we constructed a yeast complementation expression system(pDR195) and expressed TaARPC5 inS.cerevisiaestrain YIL062c.TheS.cerevisiaecells carrying the empty pDR195 vector, as well as the wild type (WT) yeast strain BY4741, were used as controls.First,we tested theS.cerevisiaeYIL062c mutant(Δarc15),the wildtype strain BY4741, ΔARPC5carrying the empty pDR195 vector(Δarc15+ empty vector), and the complemented strain carrying the recombinant pDR195-TaARPC5plasmid (Δarc15+ TaARPC5)for growth on SC-U medium (lacking uracil and containing a variety of carbon sources).As shown in Fig.4, the Δaprc5mutant did not grow on SC-U medium.Next, yeast cell morphology was observed after transforming and expressing pDR195-TaARPC5.As shown in Fig.4,the P16-Arc yeast mutant showed an obvious morphological defect, indicating a loss-of-function mutant of Arp2/3.Fluorescence microscopy of 4′,6-diamino-2-phenylindole (DAPI)-stained cells revealed that complementation withTaARPC5restored the loss-of-function mutation (Fig.4).Additionally, complementation with pDR195-TaARPC5also complemented the lossof-function mutation, restoring cell growth on SC mediaum supplemented with 0.3 mol L-1NaCl (Fig.S4).Yeast cells expressing pDR195 and pDR195-TaARPC5grew more slowly than WT-1 cells on stress media (i.e., containing with 0.3 mol L-1NaCl, 0.1 mol L-1CaCl2,2 mol L-1sorbitol,or 0.05 mmol L-1H2O2).As expected,the complemented strain containing pDR195-TaARPC5grew faster than the strain carrying only the empty vector (e.g., pDR195;Fig.S4).These data not only demonstrate thatTaARPC5can functionally complement the loss of ARP in yeast,but also indicate that TaARPC5 can enhance the stress tolerance of the host plant.

    3.6.Silencing of TaARPC5 resulted in different responses of wheat to virulent and avirulent Pst

    Fig.3.Subcellular localization of TaARPC5.(A) Localization of TaARPC5 in wheat protoplasts.(B) Localization of TaARPC5 in N.benthamiana.The green channel shows the localization of GFP and TaARPC5-GFP.Scale bars, 20 μm.

    Fig.4.Complementation of TaARPC5 in yeast mutant recovers the loss of actin morphology.Cell morphology was observed under bright field microscopy.Cells were stained with TRITC-phalloidin and DAPI.Enlarged views indicate the accumulation of F-actin.Scale bars, 10 mm.

    To investigate the function ofTaARPC5during thePstinfection,TaARPC5was knocked down in wheat using the barley stripe mosaic virus (BSMV)-mediated VIGS system.A 208-bp fragment ofTaARPC5was selected to silenceTaARPC5and the off-target situations were predicted by SiFi 4.0(Table S3).The empty vector and BSMV:γ-TaPDSwere used as additional controls.As shown in Fig.5A, BSMV-γ inoculated leaves showed mild chlorotic mosaic phenotypes, and the BSMV:γ-TaPDSshowed an obvious photobleaching,indicating the silencing system was effective.The fourth leaves of wheat plants that were inoculated with BSMV were then inoculated with thePstisolates,CYR23(avirulent)and CYR31(virulent), respectively (Fig.5A).The silencing efficiency was examined by qRT-PCR, and revealed that the mRNA abundance ofTaARPC5was reduced to 24%-68%in leaves infected with the avirulent isolate, and reduced to 16%-40% in leaves infected with the virulent isolate.At 12 dpi, necrosis was elicited on leaves by CYR23 infection in plants inoculated with BSMV:γ, as well as in leaves of plants inoculated with BSMV:TaARPC5(Fig.5A).Leaves inoculated with CYR31 produced uredinia; however, leaves previously inoculated with BSMV:TaARPC5produced more uredinia than in mock or plants inoculated with BSMV:γ (Fig.5A).These results support our hypothesis thatTaARPC5is required for host defense againstPst.

    To evaluate the histological responses associated withTaARPC5silencing,we analyzed the following symptoms:Psthyphal length,the number of hyphal branches,the formation of haustorial mother cells, and the size of colony area at infection sites.As shown in Fig.6, ROS accumulation was significantly induced after inoculation with avirulentPst.In addition, as expected, expression of the ROS marker gene (e.g.,TaCAT1) was also found to be elevated in the control leaves relative to expression in knockdown plants(Fig.S5).Interestingly,a significant change in the mRNA accumulation ofTaSODwas not observed.After infection by CYR23,the number of hyphal branches and haustorial mother cells were slightly higher than the control,while the colony size changed only slightly(Table S4;Fig.S6),whereas following the inoculation with the virulent race CYR31, the number of hyphal branches, the number of haustorial mother cells, and the colony size were significantly increased compared to the control.The colony sizes on theTaARPC5-knockdown plants were particularly noticeable, over twice that on controls (Table S4).

    TaARPC5was subsequently induced in response to flg22 perception.To determine the extent of the putative role ofTaARPC5in PTI,we evaluated the mRNA accumulation of a suite of additional PTI marker genes (Table S1).Interestingly,TaPUB23-Like,TaFLS2,TaCamBP-Like,TaROR2,TaCMPG1-Like,TaUGE2, andTaWRYK23-Likewere all significantly induced in the control leaves following inoculation with the virulent isolate CYR31 (Fig.7), whereas all these genes were down-regulated after inoculation with the avirulent race CYR23 (Fig.S7).In theTaARPC5-knockdown lines, the transcription level ofTaPUB23-Like,TaFLS2,TaCamBP-Like,TaCMPG1-Like,TaUGE2, andTaWRYK23-Likewere all reduced to about one-tenth of that observed in WT plants, whileTaPDR2andTaROR2were differentially induced.TaMPK3mRNA accumulation was not altered in either the control or silenced plants.Considering the previously published function of the ARP2/3 complex in regulating actin dynamics,our results suggest thatTaARPC5is involved in plant resistance, including both PTI and ETI, through regulating the actin cytoskeleton.

    4.Discussion

    The seven-subunit protein Arp2/3 complex,the major nucleator of Y-branched actin filaments,is stable and remains intact in cells,and all the subunits are associated exclusively with the complex[36,37].In human and yeast,the subunits have been hypothesized to play regulatory roles [38] as well as maintain the structural integrity of the complex[39].In this study,we observed the effects of phalloidin,a polymerizer of actin filaments,in wheat againstPst,and characterized the smallest subunit of ARP2/3 complex that functions as a nucleator of actin filaments.TaARPC5is highly induced in the stems and leaves in response toPstinfection and flg22 treatment.ARPC5 is conserved among eukaryotic organisms,TaARPC5can partially complement the defect of the yeast mutant.Subcellular localization indicated thatTaARPC5is localized in the network of actin and may be highly related to the dynamics of the organelles.Knockdown ofTaARPC5in wheat leaves affected the ROS accumulation in the incompatible interaction and significantly reduced the expression of PTI-specific target genes in the compatible reaction.It is possible thatTaARPC5contributes to the basal layer of broad immunity as well as ROS accumulation during ETI responses.

    Fig.5.Functional characterization of TaARPC5 by the Barley Stripe Mosaic Virus(BSMV)-based virus-induced gene silencing.(A)No phenotypic changes were observed on the wheat leaves treated with 1× Fes buffer (MOCK).Mild chlorotic mosaic symptoms were observed on the leaves inoculated with BSMV:γ, BSMV:PDS or BSMV:TaARPC5.(B)Phenotypes of the fourth leaves challenged with urediniospores of the Pst avirulent race CYR23 or virulent race CYR31.Typical leaves were photographed at 14 days post inoculation(dpi).(C)Relative transcript levels of TaARPC5 in leaves of knockdown plants inoculated with CYR23 or CYR31 at 0,24,48,and 120 h post inoculation(hpi)by qRTPCR.The relative expression of each copy of TaARPC5 was calculated by the comparative threshold method (2-△△CT).Error bars represent the variations among three independent replicates.(D)Quantification of the size of infection area and(E)The number of urediniospores in BSMV:TaARPC5-inoculated wheat plants inoculated with Pst CYR31 at 14 dpi.Values represent the means±standard error of at least ten leaves.Standard differences were assessed with Student’s t-test,and asterisks indicate P<0.05.

    In plants,the actin cytoskeleton perceives and responds rapidly during the early stages of fungal penetration[40].Using pharmacological approaches, several studies previously showed that rearrangement of the plant actin cytoskeleton is correlated with the activation of defense signaling,most notably performed as the generation of reactive oxygen, changes in gene expression, and papillae formation at the site of penetration[19,41,42].In the wheat-Pstinteraction, treatment with the microtubule inhibitor, oryzalin,reduces wheat resistance to an incompatiblePstrace [42].Conversely, treatment with latrunculin-B induced depolymerization of actin filaments, which resulted in a concomitant elevation in defense responses followingPstinoculation [43].In the current study, an F-actin-stabilizing and -polymerizing agent, phalloidin,led to a reduction inPstsporulation.Previous studies described various actin binding proteins, such as TaADF3, TaADF4, TaADF7 and TaARPC3, that participate in wheat resistance.TaADF3 positively regulates wheat tolerance to abiotic stresses and negatively regulates wheat resistance toPstin an ROS-dependent manner[44].TaADF4 positively modulates wheat plant immunity via the modulation of actin cytoskeletal organization [43].TaADF7 likely contributes to wheat resistance againstPstinfection by modulating the actin cytoskeletal dynamics to influence ROS accumulation and HR [45].TaARPC3 contributes to the stability of the cytoskeleton and thereby enhances wheat resistance againstPst[46].In this study, we observed thatTaARPC5was highly induced after flg22 treatment, a process that has been previously shown to be associated with the actin filament contraction towards the nucleus and the concomitant induction of early defense responses [47].The data presented herein also show a flg22-induced response associated with the early stages ofPstinfection,an observation that,coupled withTaARPC5mRNA accumulation,provides support for a role in the basal resistance-associated signaling processes.

    Fig.6.Histological observations of wheat leaves treated with BSMV and infected with avirulent isolate CYR23.(A)H2O2 accumulation was detected by staining with DAB at infection sites observed under a microscope.SV, substomatal vesicle.Scale bars, 20 μm.(B) The amount of H2O2 production was quantified as the size of DAB-stained area.Values represent mean±standard errors of three independent assays.(C,D)Relative transcript levels of TaSOD(C)and TaCAT1(D)in TaARPC5-knockdown plants.Error bars represent the variations among three independent replicates.Asterisks indicate significant difference (P < 0.05) from BSMV:γ using Student’s t-test.

    The seven-subunit ARP2/3 complex is an efficient modulator of the actin cytoskeleton with well-recognized roles in amoeboid locomotion and the subcellular motility of organelles and microbes[24].ARP3 was previously reported to associate with mitochondria inDictyostelium.In budding yeast,Arc15 has been demonstrated as an actin-binding peripheral mitochondrial membrane protein[48];mutations in the ARP2 and ARC15 subunits result in defective in mitochondrial movement inS.cerevisiae.In higher plants, the ARP2/3 complex has been implicated in cell motility and cell shape determination [24], while the primary function of these subunit proteins in response to fungal pathogens is largely unknown.Our data suggest that TaARPC5 may be related to organelle transport in wheat protoplasts, with a possible role in linking organelles to actin filaments.The organelles (e.g., chloroplast, peroxisome, and mitochondrial) are the major site of ROS production in plant cells[49].In this study,ROS accumulation induced by the incompatiblePstrace CYR23 was increased inTaARPC5-knockdown plants, suggesting thatTaARPC5contributes to ROS accumulation during ETI responses through the modulation of organelle transport.The phenotype ofTaARPC5-knockdown plants indicated that TaARPC5 was positively involved in the wheat defense response.As a previous study indicated, the other subunit, TaARPC3, also contributed to resistance of wheat againstPstinfection [46].According to those data, we speculate that the complex of TaARP2/3, at least the TaARPC3 and TaARPC5, could be involved in wheat-Pstinteraction to enhance wheat resistance.Future work will be performed to create overexpressing or RNAi transformation wheat plants of those subunits to reveal the molecular mechanism of wheat defense againstPstinfection and provide potential wheat materials for durable control of stripe rust.Wheat cultivar Suwon 11 contains the stripe rust resistance geneYrSu[50] and is resistant to CYR23 but highly susceptible to CYR31.The incompatible interaction is proposed to be mediated by a gene-for-gene resistance, resulting in H2O2accumulation and localized cell death at the sites of pathogen infection.We posit that this response is likely due to the inhibition of PTI, a mechanism that results in enhanced susceptibility of wheat against a compatible race.Indeed, inhibition of actin cytoskeleton-based organelle dynamics andTaCAT1expression lead to H2O2accumulation that is important for resistance during an incompatible interaction.

    TaARPC5 may have various functions in providing stability to the actin filament network, such as cross-linking and capping or free pointed ends of filaments to help them rapidly depolymerize.Moreover, effectors of the plant pathogen may also reprogram the host cytoskeleton through manipulating actin-related proteins during infection [3,6,51].While it remains to be determined ifPsteffectors function in a similar pattern,i.e.,target actin during infection, it seems that such a mechanism would have been already reported in fungi since that has been previously described in bacteria.Nevertheless, the identification of such virulence functions in fungi would shed light on the complex interaction between plant pathogens and the host actin cytoskeleton.Furthermore,we consider up-regulation ofTaARPC5expression in host plants as an alternative response to enhance host immunity and tolerance against various biotic and abiotic threats.

    CRediT authorship contribution statement

    Fig.7.Transcript levels of PTI marker genes in TaARPC5-knockdown plants during the compatible interaction.TaMPK3,TaPUB23-Like,TaFLS2,TaCamBP-Like,TaPDR2,TaROR2,TaCMPG1-Like,TaUGE2,and TaWRYK23-Like were measured by qRT-PCR.Data for Event 1 presented BSMV:γ at 0 h post inoculation(hpi).Error bars represent the variations among three independent replicates.Asterisks indicate significant difference (P < 0.05) from BSMV:γ using Student’s t-test, double asterisks indicate significant difference(P < 0.01) from BSMV:γ using Student’s t-test.

    Jia Guo:Investigation, Visualization, Writing - original draft.Huan Peng:Data curation, Investigation.Tuo Qi:Investigation,Visualization, Writing - review & editing.Sanding Xu:Investigation.Md Ashraful Islam:Visualization.Brad Day:Writing-review& editing.Qing Ma:Writing - review & editing.Zhensheng Kang:Supervision, Funding acquisition, Writing - review & editing.Jun Guo:Conceptualization, Funding acquisition, Project administration, 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 study was supported by the National Transgenic Key Project of the Ministry of Agriculture of China (2020ZX08009-15B),the National Natural Science Foundation of China (31972224),the National Key Research and Development Program of China(2018YFD0200402), the Natural Science Basic Research Program of Shaanxi (2020JZ-13), the 111 Project from the Ministry of Education of China (B07049), and the Open Project Program of State Key Laboratory of Crop Stress Biology for Arid Areas(CSBAA2020010).

    Appendix A.Supplementary data

    Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2021.01.009.

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