Capping Protein Modulates Actin Remodeling in Response to ... · Capping Protein Modulates Actin...

12
Capping Protein Modulates Actin Remodeling in Response to Reactive Oxygen Species during Plant Innate Immunity 1[OPEN] Jiejie Li, Lingyan Cao, and Christopher J. Staiger * Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907-2064 (J.L., L.C., C.J.S.); and The Bindley Bioscience Center and Department of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana 47907 (C.J.S.) ORCID IDs: 0000-0002-2906-1566 (J.L.); 0000-0003-2321-1671 (C.J.S.). Plants perceive microbe-associated molecular patterns and damage-associated molecular patterns to activate innate immune signaling events, such as bursts of reactive oxygen species (ROS). The actin cytoskeleton remodels during the rst 5 min of innate immune signaling in Arabidopsis (Arabidopsis thaliana) epidermal cells; however, the immune signals that impinge on actin cytoskeleton and its response regulators remain largely unknown. Here, we demonstrate that rapid actin remodeling upon elicitation with diverse microbe-associated molecular patterns and damage-associated molecular patterns represent a conserved plant immune response. Actin remodeling requires ROS generated by the defense-associated NADPH oxidase, RBOHD. Moreover, perception of g22 by its cognate receptor complex triggers actin remodeling through the activation of RBOHD- dependent ROS production. Our genetic studies reveal that the ubiquitous heterodimeric capping protein transduces ROS signaling to the actin cytoskeleton during innate immunity. Additionally, we uncover a negative feedback loop between actin remodeling and g22-induced ROS production. In plant cells, the rst layer of innate immunity is initiated by the detection of various danger signals by pattern recognition receptors (PRRs; Boller and Felix, 2009; Dodds and Rathjen, 2010). PRRs directly sense conserved microbe-associated molecular patterns (MAMPs), such as bacterial agellin and elongation factor-Tu, as well as chitin from fungal cell walls. In addition to MAMPs, PRRs also perceive endogenous damage-associated molecular patterns (DAMPs), in- cluding plant cell wall fragments released by patho- gen lytic enzymes or peptides synthesized de novo during pathogen infection (Boller and Felix, 2009). Activation of PRRs leads to pattern-triggered immu- nity (PTI), including initiation of mitogen-associated and calcium-dependent protein kinase cascades; bursts of calcium and reactive oxygen species (ROS); uxes in phospholipid production and turnover; transcriptional reprogramming; and cellular responses such as cell wall fortication by callose deposition (Boller and Felix, 2009; Tena et al., 2011; Monaghan and Zipfel, 2012). In Ara- bidopsis (Arabidopsis thaliana), the best-characterized PTI pathway involves the perception of bacterial agellin, or its immunogenic peptide g22, by the plasma-membrane associated receptor kinase FLAGELLIN SENSING 2 (FLS2). Upon perception, FLS2 associates with another receptor-like kinase BRI1-ASSOCIATED KINASE (BAK1) and the cytoplasmic kinase BOTRYTIS- INDUCED KINASE (BIK1) to activate downstream defense signaling (Gómez-Gómez and Boller, 2000; Chinchilla et al., 2006, 2007; Heese et al., 2007; Lu et al., 2010; Zhang et al., 2010). Emerging evidence demonstrates a critical role for the actin cytoskeleton during plant innate immunity (Li et al., 2015a; Porter and Day, 2016). In Arabidopsis epidermal cells, the density of cortical actin lament arrays increases within minutes after infection with pathogenic and nonpathogenic bacteria as well as elic- itation by diverse MAMPs (Henty-Ridilla et al., 2013b, 2014; Li et al., 2015b). This actin remodeling is required for defense responses such as callose deposition in the cell wall, transcriptional reprogramming of defense genes, and subsequently contributes to plant resistance against virulent and avirulent microbes (Henty-Ridilla et al., 2013b, 2014; Li et al., 2015b). Quantitative anal- yses of actin dynamics in live cells have uncovered the potential molecular mechanisms that contribute to in- creased lament abundance. These include reduction in lament disassembly via inhibition of actin depoly- merizing factors (ADF4 or ADF1; Henty-Ridilla et al., 2014) and increases in free barbed ends for lament 1 This work was supported by the National Science Foundation (grant no. IOS-1021185 to C.J.S.). * Address correspondence to [email protected]. The author responsible for distribution of materials integral to the ndings presented in this article in accordance with the policy de- scribed in the Instructions for Authors (www.plantphysiol.org) is: Christopher J. Staiger ([email protected]). J.L. and L.C. performed the experiments and analyzed the data; J.L. and C.J.S designed the experiments and wrote the manuscript. [OPEN] Articles can be viewed without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.16.00992 Plant Physiology Ò , February 2017, Vol. 173, pp. 11251136, www.plantphysiol.org Ó 2017 American Society of Plant Biologists. All Rights Reserved. 1125 www.plantphysiol.org on May 1, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.

Transcript of Capping Protein Modulates Actin Remodeling in Response to ... · Capping Protein Modulates Actin...

Page 1: Capping Protein Modulates Actin Remodeling in Response to ... · Capping Protein Modulates Actin Remodeling in Response to Reactive Oxygen Species during Plant Innate Immunity1[OPEN]

Capping Protein Modulates Actin Remodeling inResponse to Reactive Oxygen Species during PlantInnate Immunity1[OPEN]

Jiejie Li, Lingyan Cao, and Christopher J. Staiger*

Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907-2064 (J.L., L.C., C.J.S.);and The Bindley Bioscience Center and Department of Botany and Plant Pathology, Purdue University, WestLafayette, Indiana 47907 (C.J.S.)

ORCID IDs: 0000-0002-2906-1566 (J.L.); 0000-0003-2321-1671 (C.J.S.).

Plants perceive microbe-associated molecular patterns and damage-associated molecular patterns to activate innate immunesignaling events, such as bursts of reactive oxygen species (ROS). The actin cytoskeleton remodels during the first 5 min of innateimmune signaling in Arabidopsis (Arabidopsis thaliana) epidermal cells; however, the immune signals that impinge on actincytoskeleton and its response regulators remain largely unknown. Here, we demonstrate that rapid actin remodeling uponelicitation with diverse microbe-associated molecular patterns and damage-associated molecular patterns represent a conservedplant immune response. Actin remodeling requires ROS generated by the defense-associated NADPH oxidase, RBOHD.Moreover, perception of flg22 by its cognate receptor complex triggers actin remodeling through the activation of RBOHD-dependent ROS production. Our genetic studies reveal that the ubiquitous heterodimeric capping protein transduces ROSsignaling to the actin cytoskeleton during innate immunity. Additionally, we uncover a negative feedback loop betweenactin remodeling and flg22-induced ROS production.

In plant cells, the first layer of innate immunity isinitiated by the detection of various danger signals bypattern recognition receptors (PRRs; Boller and Felix,2009; Dodds and Rathjen, 2010). PRRs directly senseconserved microbe-associated molecular patterns(MAMPs), such as bacterial flagellin and elongationfactor-Tu, as well as chitin from fungal cell walls. Inaddition to MAMPs, PRRs also perceive endogenousdamage-associated molecular patterns (DAMPs), in-cluding plant cell wall fragments released by patho-gen lytic enzymes or peptides synthesized de novoduring pathogen infection (Boller and Felix, 2009).Activation of PRRs leads to pattern-triggered immu-nity (PTI), including initiation of mitogen-associatedand calcium-dependent protein kinase cascades; burstsof calcium and reactive oxygen species (ROS); fluxes inphospholipid production and turnover; transcriptionalreprogramming; and cellular responses such as cell wallfortification by callose deposition (Boller and Felix, 2009;

Tena et al., 2011; Monaghan and Zipfel, 2012). In Ara-bidopsis (Arabidopsis thaliana), the best-characterized PTIpathway involves the perception of bacterial flagellin, orits immunogenic peptideflg22, by the plasma-membraneassociated receptor kinase FLAGELLIN SENSING 2(FLS2). Upon perception, FLS2 associates with anotherreceptor-like kinase BRI1-ASSOCIATED KINASE(BAK1) and the cytoplasmic kinase BOTRYTIS-INDUCED KINASE (BIK1) to activate downstreamdefense signaling (Gómez-Gómez and Boller, 2000;Chinchilla et al., 2006, 2007; Heese et al., 2007; Luet al., 2010; Zhang et al., 2010).

Emerging evidence demonstrates a critical role forthe actin cytoskeleton during plant innate immunity (Liet al., 2015a; Porter and Day, 2016). In Arabidopsisepidermal cells, the density of cortical actin filamentarrays increases within minutes after infection withpathogenic and nonpathogenic bacteria as well as elic-itation by diverse MAMPs (Henty-Ridilla et al., 2013b,2014; Li et al., 2015b). This actin remodeling is requiredfor defense responses such as callose deposition in thecell wall, transcriptional reprogramming of defensegenes, and subsequently contributes to plant resistanceagainst virulent and avirulent microbes (Henty-Ridillaet al., 2013b, 2014; Li et al., 2015b). Quantitative anal-yses of actin dynamics in live cells have uncovered thepotential molecular mechanisms that contribute to in-creased filament abundance. These include reduction infilament disassembly via inhibition of actin depoly-merizing factors (ADF4 or ADF1; Henty-Ridilla et al.,2014) and increases in free barbed ends for filament

1 This work was supported by the National Science Foundation(grant no. IOS-1021185 to C.J.S.).

* Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Christopher J. Staiger ([email protected]).

J.L. and L.C. performed the experiments and analyzed the data;J.L. and C.J.S designed the experiments and wrote the manuscript.

[OPEN] Articles can be viewed without a subscription.www.plantphysiol.org/cgi/doi/10.1104/pp.16.00992

Plant Physiology�, February 2017, Vol. 173, pp. 1125–1136, www.plantphysiol.org � 2017 American Society of Plant Biologists. All Rights Reserved. 1125 www.plantphysiol.orgon May 1, 2020 - Published by Downloaded from

Copyright © 2017 American Society of Plant Biologists. All rights reserved.

Page 2: Capping Protein Modulates Actin Remodeling in Response to ... · Capping Protein Modulates Actin Remodeling in Response to Reactive Oxygen Species during Plant Innate Immunity1[OPEN]

assembly by negative regulation of the heterodimericcapping protein (CP; Li et al., 2015b). CP is a ubiquitousregulator of actin filaments (Cooper and Sept, 2008). Itbinds the barbed end of actin filaments to inhibit ac-tin polymerization and filament-filament annealingin vitro (Huang et al., 2003; Cooper and Sept, 2008).Genetic evidence from various organisms demonstratesthat the availability of barbed ends for actin assembly isinversely correlated with cellular CP levels (Kim et al.,2004; Li et al., 2012, 2014a). Moreover, CP has beenimplicated in the crosstalk of signaling events frommembrane to actin cytoskeleton via phospholipidbinding (Pleskot et al., 2013). Plant CP is unique in theability to bind the stress signaling phospholipid,phosphatidic acid (PA), which inhibits capping activityand uncaps filament ends in vitro and in vivo (Huanget al., 2006; Li et al., 2012; Pleskot et al., 2013). Thepathways for actin remodeling during innate immunesignaling are both parallel and convergent, and poten-tially tissue-specific. In hypocotyl epidermal cells, forexample, ADF4 regulates dynamics in response to elf26(a peptide mimic of bacterial elongation factor-Tu) butis not required for chitin signaling, whereas CP andADF1 are implicated in both pathways (Henty-Ridillaet al., 2014; Li et al., 2015b). These data reveal thecomplexity of molecular machinery that senses andtransduces immune signaling to actin rearrangements.

In Arabidopsis, actin remodeling in response toMAMPs is, to our knowledge, a new early hallmark ofsignaling that requires cognate PRR complexes (Henty-Ridilla et al., 2013b, 2014; Li et al., 2015b). Upon per-ception, other rapid signaling events, such as increasedcytosolic Ca2+, mitogen-associated protein kinasephosphorylation, accumulation of ROS, and fluxes incertain signaling phospholipids, occur within secondsto minutes (Boller and Felix, 2009; Canonne et al., 2011).Data from a recent study suggest that phospholipase D(PLD)-dependent PA signaling is required for actin re-modeling upon perception of MAMPs (Li et al., 2015b).Moreover, PA mediates actin remodeling via its inhib-itory effect on CP (Li et al., 2012, 2015b). In addition toPA, the entire complement of immune signals that im-pinge on actin cytoskeleton remodeling and its re-sponse regulators need to be further investigated.

The rapid production of ROS is a conserved signalingoutput during immunity across kingdoms and species.ROS contributes to plant defense responses by rein-forcing the cell wall and exerting cytotoxic effects toblock pathogen invasion, or by acting as a secondmessenger to trigger additional immune responses,such as gene expression or stomatal closure (Suzukiet al., 2011; Nathan and Cunningham-Bussel, 2013).However, a role for ROS signaling to actin dynamicsduring innate immunity has not been established foreither plant or animal cells. The interaction betweenROS and actin cytoskeleton has long been recognized(Wilson and González-Billault, 2015). In mammaliancells, ROS regulate actin rearrangements associatedwith cellular processes such as cell adhesion, migration,wound repair, as well as neurite outgrowth (Chiarugi

et al., 2003; Fiaschi et al., 2006; Munnamalai and Suter,2009; Sakai et al., 2012; Taulet et al., 2012; Xu andChisholm, 2014). In plant cells, ROS signaling is requiredfor actin remodeling during the self-incompatibility re-sponse in pollen tubes and abscisic acid-induced sto-matal closure (Wilkins et al., 2011; Li et al., 2014c). InArabidopsis, flg22-induced stomatal closure also re-quires NADPH oxidase, RBOHD. This provides an in-direct link between ROS and actin dynamics (Mersmannet al., 2010). Furthermore, several cytoskeletal targets forROS have been found in both plant and animal cells.Actin itself is modified by ROS via glutathionylationduring neutrophilmigration (Sakai et al., 2012). Oxidant-induced apoptosis requires oxidation of cofilin (Klamtet al., 2009). A recent study suggests that Arp2/3 com-plex participates in the response to ROS signaling duringstomatal closure (Li et al., 2014c). Here, we demonstratethat, after elicitation by diverse MAMPs and DAMPs,ROS serves as a common upstream signal that mediatesactin remodeling. Additionally, we provide genetic evi-dence that CP is an intermediary in ROS signaling to theactin cytoskeleton during innate immunity.

RESULTS

Cortical Actin Arrays Respond Rapidly to a Diverse Arrayof Danger Signals

Plant innate immunity is triggered by the detection ofvarious danger signals, including MAMPs and DAMPs.Previously, we found that the cortical actin array in epi-dermal pavement cells from Arabidopsis rearrange aftertreatment with MAMPs, and this actin reorganizationplays an important role during plant innate immunity(Henty-Ridilla et al., 2013b). Here, we extend our find-ings by applying DAMPs to Arabidopsis rosette leavesexpressing the actin reporter comprising GFP fusedwiththe second actin-binding domain of Arabidopsis FIM-BRIN1 (GFP-fABD2; Sheahan et al., 2004). The elicitorswere syringe-infiltrated into rosette leaves and the cor-tical actin cytoskeleton of epidermal pavement cellswas monitored with spinning disk confocal microscopy(SDCM).

Similar to our previous study, a more dense actinarray was observed in wild-type cells treated withMAMPs (flg22 and chitin) compared to mock treatedleaves (Fig. 1A). Moreover, we found that this actinrearrangement occurred faster than observed previ-ously (Henty-Ridilla et al., 2013b); it could be detectedas early as 5 min to 15 min after treatment (Fig. 1A). Inaddition, similar changes to actin filament arrays wereobserved when cells were treated with DAMPs such asArabidopsis Pep1 (a 23-amino acid peptide processedfrom PROPEP1; Huffaker et al., 2006) and oligoga-lacturonides (OGs; D’Ovidio et al., 2004), whereas an-other bacterial MAMP, elf26, was indistinguishablefrommock control (Fig. 1A). Organizational changes ofthe cortical actin array were quantified with a set ofmetrics called skewness and density to estimate theextent of actin filament bundling and the percentage of

1126 Plant Physiol. Vol. 173, 2017

Li et al.

www.plantphysiol.orgon May 1, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.

Page 3: Capping Protein Modulates Actin Remodeling in Response to ... · Capping Protein Modulates Actin Remodeling in Response to Reactive Oxygen Species during Plant Innate Immunity1[OPEN]

occupancy of actin filaments, respectively (Higaki et al.,2010; Henty-Ridilla et al., 2013b). As shown in Figure1B, actin filament abundance in wild-type cells wassignificantly elevated after treatment with all elicitorstested, except for elf26, when compared with mock-treated cells. No significant changes in the extent offilament bundling were detected among treatments(Fig. 1C). Taken together, these data suggest that actinfilament abundance in epidermal pavement cells increasesrapidly after treatment with MAMPs and DAMPs; thislikely represents a broad-based cytoskeletal responseduring plant innate immunity.

Lack of the NADPH Oxidase, RBOHD, Abrogates theActin Response to MAMPs and DAMPs

One of the common signaling events triggered byMAMPs and DAMPs is the production of apoplasticROS; this occurs within minutes and is mediated by theArabidopsis NADPH oxidase, RBOHD (SupplementalFig. S1; Torres et al., 2006). Thus, we predict that ele-vated levels of ROS are required for the rapid changesto cortical actin arrays in response to MAMPsand DAMPs. To test this, we applied elicitors to thehomozygous knockout mutant for rbohD expressing

GFP-fABD2, and compared actin architecture quanti-tatively. As shown in Figure 1, cortical actin arrays inrbohD mutant epidermal cells showed no significantdifferences compared with mock-treated, wild-typecells. Moreover, all MAMPs and DAMPs failed toelicit an actin architecture change in rbohD as they didwhen applied to wild-type cells (Fig. 1), suggesting thatRBOHD-mediated ROS generation is upstream of actinreorganization in response to MAMPs or DAMPs.

Treatment with H2O2 Mimics the Increase in ActinFilament Abundance Induced by MAMPs and DAMPs

To dissect the signaling cascades and response reg-ulators that elicit this actin rearrangement in greaterdetail, we focused on flg22-induced innate immunesignaling. In Arabidopsis, the molecular componentsinvolved in this pathway have been well characterized(Monaghan and Zipfel, 2012). Flg22 is perceived by aPRR complex comprising receptor kinase FLS2, co-receptor BAK1, and the cytoplasmic kinase BIK1. Theperception of flg22 by FLS2 receptor complex furtheractivates defense responses including ROS production(Monaghan and Zipfel, 2012). Several recent studiesdemonstrate that RBOHD is a component of the FLS2

Figure 1. Increased actin filament abundance triggered by MAMPs or DAMPs requires RBOHD-dependent ROS production. A,Representative images of epidermal pavement cells fromwild-type and rbohDmutant rosette leaves treatedwith various MAMPsorDAMPs for 5 to 15min. Bar = 10mm. B andC, Actin architecturewasmeasured onwild-type and rbohDmutant epidermal cellsafter treatment with MAMPs or DAMPs. B, Percentage of occupancy, or density, is a measure of the abundance of actin filamentsin the cortical array. C, Skewness is a measure of the extent of actin filament bundling. No significant changes in actin organi-zation were observed in the rbohDmutant after treatment with MAMPs or DAMPs, whereas wild type had increased density afterflg22, chitin, Pep1, and OG treatment. Values given are means 6 SE (n = 50 cells for each treatment and genotype; a, P , 0.01between mock and treatment of the same phenotype; b, no significant difference compared to mock control of the same phe-notype; c, no significant difference compared to wild type treated with mock; t test; results from two additional biological rep-licates are shown in Supplemental Fig. S12).

Plant Physiol. Vol. 173, 2017 1127

Actin Dynamics and ROS Signaling in Plant Immunity

www.plantphysiol.orgon May 1, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.

Page 4: Capping Protein Modulates Actin Remodeling in Response to ... · Capping Protein Modulates Actin Remodeling in Response to Reactive Oxygen Species during Plant Innate Immunity1[OPEN]

receptor complex (Kadota et al., 2014; Li et al., 2014b).Upon flg22 perception, RBOHD can rapidly associateand be phosphorylated by BIK1. Moreover, RBOHDphosphorylation by BIK1 is critical for ROS productionduring MAMP signaling.

When flg22 was applied to wild-type plants for 5 to15min, we detected a dose-dependent increase in actinfilament abundance (Supplemental Fig. S2A); how-ever, no change in the extent of filament bundling wasobserved at any concentration tested (SupplementalFig. S2B). It has been reported previously that flg22-induced actin remodeling requires the perception offlg22 by its cognate PRR (Henty-Ridilla et al., 2013b).In the homozygous PRR mutants fls2, bik1, as well asbak1-5, expressing GFP-fABD2, the cortical actin ar-ray was completely unresponsive to flg22 treatment,which is consistent with previous results (Fig. 2A;Henty-Ridilla et al., 2013b). Additionally, loss ofRBOHD in Arabidopsis inhibited the actin remodelingin response to flg22 (Figs. 1, A and B, and 2A). In thePRR mutants and rbohD mutant, flg22-induced ROSproduction was inhibited partially or completely(Supplemental Fig. S3). These data suggest that uponflg22 perception ROS production via RBOHD is nec-essary to induce actin remodeling. To test this hy-pothesis, we performed exogenous H2O2 treatment onwild-type plants, the PRR mutants, as well as therbohD mutant. In wild-type plants, treatment withH2O2 was sufficient to elicit a dose-dependent increasein actin filament abundance, mimicking the actin re-sponse that occurs afterMAMP treatment (SupplementalFig. S4). Moreover, exogenous H2O2 treatment recapitu-lated the flg22-induced actin remodeling in mutants ofrbohD and the PRR complex in the absence of MAMPs(Fig. 2C). Except for fls2, the extent of filament bundlingshowed no significant differences in any genotype testedafter H2O2 treatment (Fig. 2D). These data confirmed thatelevated ROS levels in cells are required for actin re-modeling in response to flg22. In support of this, we usedpharmaceutical agents, such as diphenyleneiodonium(DPI; Wilkins et al., 2011; Ben Rejeb et al., 2015) to inhibitNADPH oxidase activity, or the ROS scavengerTEMPOL (Scott and Logan, 2008; Wilkins et al., 2011)to decrease ROS level in wild-type cells. We found thatboth treatments blocked the increase in actin filamentabundance induced by flg22 in a dose-dependentmanner (Supplemental Figs. S5 and S6). Collectively,these results demonstrate that upon MAMP percep-tion ROS production via RBOHD is required for actinremodeling during innate immune signaling.

The Actin Filament CP Is an Intermediary in ROSSignaling to the Actin Cytoskeleton during the InnateImmune Response

We previously demonstrated that negative regula-tion of CP is required for actin remodeling during elf26and chitin signaling in Arabidopsis hypocotyl epider-mal cells (Li et al., 2015b). In addition, CP enhances

plant resistance to bacterial pathogens; specifically,leaves from cp knockdown mutants supported greatergrowth of Pseudomonas syringae pv. tomato DC3000compared to wild type, whereas a CP overexpression(OX) line was somewhat resistant to bacterial growth(Supplemental Fig. S7; Li et al., 2015b). Here, to testwhether flg22-triggered actin reorganization also in-volves CP, we applied flg22 to knockdown and over-expression mutants of CP, and compared actinarchitecture with wild-type leaf epidermal pavementcells quantitatively. In the absence of flg22 treatment,actin arrays in cells from two cp knockdown mutant al-leles were denser and less bundled compared withmock-treated wild-type cells (Fig. 3, A–C; SupplementalFig. S8). Actin arrays in CP OX#1 cells showed the op-posite phenotype to cpmutants (Fig. 3, A–C), which wasconsistent with our previous findings (Li et al., 2012,2014a). After flg22 treatment, a significant increase in thedensity of actin filament arrays was observed in wild-type cells (Fig. 3, B and C). The organization of actinarrays in cpb-1 or cpb-3 mutants treated with flg22 didnot show any significant differences when compared tomock treatment (Fig. 3, B and C; Supplemental Fig. S8),even when high concentrations of flg22 were used(Supplemental Fig. S9). In CP OX#1 cells, the actinfilament density after MAMP treatment increasedsignificantly, but to a lesser extent when comparedwith wild type (Fig. 3B). Collectively, these data sug-gest that, in addition to elf26 and chitin (Li et al.,2015b), CP is also necessary for actin remodeling inresponse to flg22 signaling.

To investigate whether CP acts downstream of ROSproduction, exogenous H2O2 was applied to wild typeand CP mutants. As shown in Fig. 3, A and D, an in-crease in actin filament density occurred in wild-typecells treated with H2O2. However, H2O2 treatmentfailed to elicit actin remodeling in the cpb-1mutant, butdid restore the increase in actin filament density inCP OX#1 cells to a level comparable to wild type (Fig.3D). The extent of bundling, on the other hand, de-creased after H2O2 treatment inwild type andCPOX#1,not in cpb-1 mutant (Fig. 3E). Even when 10 mM H2O2was applied to cpb-1mutant cells, no significant changein actin filament density or extent of bundling wasobserved (Supplemental Fig. S9). In addition, the in-hibitory effect of DPI on flg22-induced actin remodel-ing was completely abrogated in cpb-1 mutant whencompared with wild type (Fig. 4). Taken together, thesefindings suggest that CP regulates flg22-inducedactin remodeling by responding to ROS signaling inArabidopsis.

Perturbation of Actin Dynamics Enhances flg22-triggeredROS Production

During plant innate immunity, actin remodeling isrequired for a variety of cellular defense responses.These include transcriptional reprogramming of defense-responsive gene networks and fortification of the cell

1128 Plant Physiol. Vol. 173, 2017

Li et al.

www.plantphysiol.orgon May 1, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.

Page 5: Capping Protein Modulates Actin Remodeling in Response to ... · Capping Protein Modulates Actin Remodeling in Response to Reactive Oxygen Species during Plant Innate Immunity1[OPEN]

wall by callose deposition (Henty-Ridilla et al., 2014; Liet al., 2015b). Here, we examined whether ROS produc-tion, as one hallmark feature of innate immunity, requiresCP and actin remodeling.To test this, we compared the flg22-induced ROS

production between wild-type plants and CP mutants.In wild-type leaves treated with flg22, a robust ROSproduction was triggered. A significantly enhancedROS production after flg22 treatment occurred in bothcp knockdown and CP overexpression mutants com-pared to wild type (Fig. 5A; Supplemental Fig. S10).Moreover, enhanced ROS production in the cpb-1mutant depends completely on RBOHD, because thecpb-1 rbohD double mutant failed to exhibit an ROSresponse after elicitation with flg22 (Supplemental Fig.S11). These data suggest that CP-dependent actin

remodeling negatively regulates ROS production inresponse to flg22. Furthermore, disrupting actin arrayswith the actin polymerization inhibitor, LatB, alsoresulted in a significant increase in flg22-induced ROSproduction (Fig. 5B). Treatment with LatB alone, how-ever, did not trigger ROS production in the absence ofMAMP. In the rbohD mutant, no ROS production wasdetected among any of the treatments tested (Fig. 5B).These data further confirm that actin remodeling playsa role in ROS production during flg22 signaling, and theregulation of flg22-induced ROS production by actindynamics is completely RBOHD-dependent. Interest-ingly, we found that LatB treatment of CP mutants didnot enhance flg22-induced ROS production even fur-ther; on the contrary, it diminished the increased ROSproduction in these mutants after flg22 treatment

Figure 2. Actin rearrangement upon flg22 perception requires ROS signaling through the cognate PRR. Actin architecturemeasurements were performed on epidermal pavement cells from wild-type, rbohD, fls2, bik1, and bak1-5mutants treated withmock and 1 mM flg22 (A and B) or 1 mM H2O2 (C and D) for 5 min to 15 min. A and B, Treatment with 1 mM flg22 significantlyincreased the actin filament abundance in wild-type cells, whereas actin arrays in rbohD and PRR mutants failed to remodel inresponse to the stimuli (A). No significant differences in the extent of filament bundling were observed in all genotypes testedexcept for bik1 (B). C and D, A significant increase in actin filament abundance occurred in wild-type, rbohD, and PRR mutantstreated with H2O2 (C). No significant differences in the extent of filament bundling were observed in all genotypes tested exceptfor fls2 (D). Values given aremeans6 SE (n = 50 cells for each treatment and genotype; a, P, 0.05 betweenmock and treatment ofthe same phenotype; b, no significant difference between treatment within the same genotype; c, no significant differencecompared to wild type treated with mock; d, P, 0.05 when compared with mock-treated wild type. Student’s t test; results fromtwo additional biological replicates are shown in Supplemental Fig. S13).

Plant Physiol. Vol. 173, 2017 1129

Actin Dynamics and ROS Signaling in Plant Immunity

www.plantphysiol.orgon May 1, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.

Page 6: Capping Protein Modulates Actin Remodeling in Response to ... · Capping Protein Modulates Actin Remodeling in Response to Reactive Oxygen Species during Plant Innate Immunity1[OPEN]

(Fig. 5C; Supplemental Fig. S10). The distinct effect ofLatB on wild type and CP mutants suggest that precisecontrol of actin architecture and dynamics is essentialfor cells to accurately execute defense responses duringinnate immunity.

DISCUSSION

In this study, we show that the cortical actin array inplant cells responds to a diverse array of danger signals.Within minutes after treatment with diverse MAMPsand DAMPs, a significant increase in actin filamentabundance occurs in Arabidopsis epidermal pavementcells. Actin remodeling requires a common innate im-mune signal, ROS, generated by the defense-associatedNADPH oxidase, RBOHD. Moreover, ROS-inducedactin remodeling in response to flg22 requires activa-tion of RBOHD through the cognate FLS2 receptorcomplex. By combining advanced live-cell imagingwith genetic dissection, we find that actin in mutants ofCP fails to respond to flg22 and H2O2, suggesting thatCP acts downstream of ROS during PTI. In addition,chemical and genetic perturbation of actin dynamicsenhances flg22-induced ROS production, revealing

negative feedback regulation between these two pro-cesses (Fig. 5D).

The actin cytoskeleton in Arabidopsis epidermal cellshas been shown to respond to diverse microbes andexternal elicitors (Henty-Ridilla et al., 2013a, 2013b).Here, we observed the occurrence of actin remodelingnot only in response to various MAMPs, but also toendogenous DAMP signaling. Our data further supporta role for actin cytoskeleton as basal defense machineryduring innate immunity. Consistent with Henty-Ridillaet al. (2013b), we failed to detect actin remodeling aftertreatment with a peptide mimic of the bacterial elon-gation factor EF-Tu, elf26. This may result from: (1) thecortical actin array may only respond to particularMAMPs/DAMPs; (2) the receptor for elf26, EFR, maybe not expressed in epidermal pavement cells of rosetteleaves but be present in other cell types in this tissue,which leads to a lack of detectable actin response inepidermal cells, whereas other PTI responses can stilltake place in the whole tissue; (3) the actin response toelf26 does not occur as rapidly as to other MAMPs/DAMPs, and is undetectable by SDCM over the time-scale (5min to 15min)wemonitored. BothMAMPs andDAMPs elicit an increase in actin filament abundance,which likely represents a conserved PTI response in

Figure 3. CP is required for ROS-induced actin remodeling during flg22 signaling. A, Representative images of epidermalpavement cells fromwild type, cpb-1mutant, and CPOX#1 rosette leaves treatedwith mock, 1 mM flg22 as well as 1 mMH2O2 for5 min to 15 min. Bar = 10 mm. B to E, Actin architecture measurements were performed on epidermal pavement cells from wild-type, cpb-1, and CPOX#1mutants treatedwith mock, 1 mM flg22 (B and C) or 1 mMH2O2 (D and E) for 5 min to 15 min. The actinfilament abundance was significantly increased in wild-type and CP OX#1 cells after flg22 or H2O2 treatment. However, nosignificant changes in actin filament abundancewere observed in cpb-1mutant cells after flg22 or H2O2 treatment (B and D). Theextent of filament bundling did not change in all genotypes after flg22 treatment (C). Decreased actin filament bundling occurredin wild type and CPOX#1 mutant upon treatment with H2O2 (E). Values given are means6 SE (n = 50 cells per treatment for eachgenotype; a, P, 0.05 between mock and treatment of the same phenotype; b, P , 0.05 when compared with wild type treatedwith mock; c, no significant difference between treatment within the same genotype; t test; results from two additional biologicalreplicates are shown in Supplemental Fig. S14).

1130 Plant Physiol. Vol. 173, 2017

Li et al.

www.plantphysiol.orgon May 1, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.

Page 7: Capping Protein Modulates Actin Remodeling in Response to ... · Capping Protein Modulates Actin Remodeling in Response to Reactive Oxygen Species during Plant Innate Immunity1[OPEN]

plants. Actin remodeling associates with a variety ofcellular defense responses during innate immunity. Ac-tin rearrangements are required for receptor-mediatedendocytosis of ligand including the flagellin receptorFLS2, as vesicle dynamics are significantly reduced aftertreatment with the actin polymerization inhibitor LatB(Robatzek et al., 2006; Beck et al., 2012). In addition,genetic disruption of MAMP-induced actin remodelingleads to impaired callose deposition, transcriptionalreprogramming of defense genes (Henty-Ridilla et al.,2014; Li et al., 2015b), as well as generation of ROS (thisstudy). These data suggest that the actin cytoskeletonintegrates innate immune signaling to coordinate broaddefense responses. The actin cytoskeleton responds notonly to diverse MAMPs and DAMPs, but it can alsobe remodeled by bacterial effectors. During infectionof Arabidopsis by P. syringae DC3000, increased ac-tin filament bundling occurs at late time points, andis dependent on both T3SS and bacterial T3SS ef-fectors (Henty-Ridilla et al., 2013b). The enhancedactin filament bundling is suggested to associatewith effector-triggered susceptibility and cell death(Henty-Ridilla et al., 2013b). Shimono et al. (2016)have recently demonstrated that a P. syringae T3SSeffector, HopG1, is involved in the increase in actinfilament bundling via its interaction with an Arabi-dopsis mitochondria-localized kinesin. In addition,HopW1 from P. syringae pv. maculicola directly tar-gets and disrupts actin filaments in vitro and in vivo(Kang et al., 2014). Collectively, these data furthersupport the argument that the actin cytoskeleton isan essential component of host defense machineryinvolved in multilayered responses during dynamicplant-microbial interaction.

The conserved actin-binding protein CP participatesin multiple MAMP signaling pathways in differenttissues and cell types. It was reported previously that inhypocotyl epidermal cells, CP regulates actin dynamicsin response to elf26 and chitin (Li et al., 2015b). WhetherCP is required for flg22 signaling remained unclear,because flg22 fails to activate actin response in this tis-sue due to lack of its receptor FLS2 (Ma et al., 2005;Henty-Ridilla et al., 2014). In this study, we provideevidence that CP is involved in flg22-induced actin re-modeling in epidermal pavement cells from leaves. Ourresults further demonstrate that CP is a convergencepoint that transduces multiple MAMP signaling eventsto actin dynamics.

Based on genetic evidence herein, CP acts down-stream of ROS in response to flg22; however, it isunclear how ROS signaling impacts on CP activity.Phosphatidic acid (PA) is an abundant membranephospholipid increasingly recognized as a signalingintermediate during innate immunity (Zhao, 2015). Inrice (Oryza sativa) and tomato (Solanum lycopersicum)suspension-cultured cells, PA accumulates within a fewminutes upon treatment with various MAMPs (van derLuit et al., 2000; Yamaguchi et al., 2003, 2005; Bargmannet al., 2006; Laxalt et al., 2007; Lanteri et al., 2011; Rahoet al., 2011). Nod factors have also been reported toinduce PA production in vivo (den Hartog et al., 2001).Recognition of pathogen effectors leads to increased PAlevel in host cells (de Jong et al., 2004; Andersson et al.,2006; Kirik and Mudgett, 2009). Pharmacologicalstudies suggest that actin remodeling upon perceptionof elf26 and chitin requires PLD-dependent PA signal-ing (Li et al., 2015b). Moreover, the negative regulationof CP by PA is essential for MAMP-induced actin

Figure 4. The NADPH oxidase inhibitor DPI blocks actin remodeling in wild type after flg22 treatment. A, Percentage of oc-cupancy or density wasmeasured on epidermal pavement cells of wild type and cpb-1mutant treatedwith 50mMDPI for 10min,and followed by treatment with 1 mM flg22 for 5 min to 10 min. The actin abundance in wild-type cells treated with DPI failed toincrease after flg22 treatments. By contrast, actin filament abundance in the cpb-1mutant was not altered by either treatment. B,No significant differences in the extent of filament bundlingwere observed inwild-type and cpb-1mutant cells after flg22, DPI, orboth treatments. Values given are means 6 SE (n = 50 cells for each treatment and genotype; a, P , 0.01 between mock andtreatment of the same phenotype; b, P , 0.01 when compared with wild type treated with mock; c, no significant differencecompared to mock control of the same genotype. Student’s t test; results from two additional biological replicates are shown inSupplemental Fig. S15).

Plant Physiol. Vol. 173, 2017 1131

Actin Dynamics and ROS Signaling in Plant Immunity

www.plantphysiol.orgon May 1, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.

Page 8: Capping Protein Modulates Actin Remodeling in Response to ... · Capping Protein Modulates Actin Remodeling in Response to Reactive Oxygen Species during Plant Innate Immunity1[OPEN]

reorganization (Li et al., 2015b). The activation of PLD/PA signaling by ROS has been documented in plantcells. Treatment with H2O2 induces rapid PA accumu-lation in rice suspension cells, which is required forelicitor-induced biosynthesis of phytoalexin (Yamaguchiet al., 2004). Arabidopsis PLDd is activated in response toH2O2, resulting in increased cellular PA levels (Zhanget al., 2003). Thus, ROSmay regulate CP activity throughPLD/PA signaling. Another possible mechanism is theredoxmodification of CP by ROS. ROS is likely involvedin redox signaling during plant defense, mainly throughregulation of protein activities via glutathionylation ofprotein Cys thiol residues (Lehmann et al., 2015). Bothsubunits of human CP are glutathionylated during oxi-dative stress (Lind et al., 2002). Whether CP undergoes

redox modification by ROS and its physiological rele-vance in plant innate immunity needs to be investigatedin the future.

Actin remodeling participates in flg22-induced ROSproduction, but the underlying mechanism remainsunknown. In mammalian cells, the activity of NADPHoxidase is directly regulated by actin dynamics. Neu-trophil NADPH oxidase binds to actin in vitro (Tamuraet al., 2000a, 2000b, 2006). Actin polymerization acti-vates NADPH oxidase, whereas depolymerizing actindeactivates it. These data suggest a simple correlationbetween NADPH oxidase activity and the state of actindynamics (Morimatsu et al., 1997; Tamura et al., 2000a,2000b). In vivo, however, actin polymerization anddepolymerization both activate NADPH oxidase. Close

Figure 5. Perturbation of actin remodeling enhances ROS production in response to flg22 treatment. Apoplastic ROS productionwas measured on 4-week-old leaf disks using a luminescence assay. A, Compared to wild type, ROS production was significantlyincreased in cpb-1mutant and CPOX#1 line after elicitation with 1 mM flg22. B, Pretreatment with 1 mM LatB for 30 min resultedin enhanced flg22-dependent ROS production in wild-type leaves. C, By contrast, in cpb-1mutant and CPOX#1 lines, the ROSproduction induced by flg22was significantly decreased by LatB treatment. D, Amodel for negative feedback regulation betweenROS signaling and actin dynamics during flg22 signaling. Rapid actin rearrangement upon flg22 perception requires ROS sig-naling through the cognate PRR complex. CP is required for ROS-induced actin remodeling in response to flg22. CP-dependentactin remodeling further regulates flg22-triggered ROS production (left right arrows: direct interaction; solid arrows: activation ofsignaling cascades; dashed arrows: potential regulation; bars: inhibitory effect). Values given are means6 SE (n = 24 leaf disks foreach treatment and genotype; a, P, 0.05 between mock and treatment in wild type; b, P, 0.05 when compared with wild typetreated with flg22; c, no significant difference compared to mock control of the same genotype; d, P , 0.05 between flg22 andtreatment of the same genotype. t test; results from two additional biological replicates are shown in Supplemental Fig. S16).

1132 Plant Physiol. Vol. 173, 2017

Li et al.

www.plantphysiol.orgon May 1, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.

Page 9: Capping Protein Modulates Actin Remodeling in Response to ... · Capping Protein Modulates Actin Remodeling in Response to Reactive Oxygen Species during Plant Innate Immunity1[OPEN]

examination of actin dynamics, such as F/G-actin ratioas well as actin turnover rates further disprove thein vitro correlation (Rasmussen et al., 2010). In ourstudy, disruption of actin dynamics by up- and down-regulation of CP, or by LatB treatment, all lead to anenhancement of flg22-induced ROS production, sug-gesting that the repertoire of mechanisms that controlsNADPH oxidase activation by actin remodeling ismorecomplex than expected.Successful immunity often requires dynamic mem-

brane trafficking and cytoplasmic rearrangements toprecisely localize immune receptors and defense com-pounds, these processes are dependent on the actin cy-toskeleton (Schmidt and Panstruga, 2007; Yang et al.,2014). A hallmark of PTI inArabidopsis is ligand-inducedendocytosis of the flagellin receptor FLS2 (Robatzek et al.,2006). FLS2 endocytosis has been implicated in flg22-signal attenuation, because impaired FLS2 internaliza-tion often correlates with altered downstream defensesignaling (Robatzek et al., 2006; Salomon and Robatzek,2006; Chinchilla et al., 2007). A recent study showed thatloss of Dynamin-Related Protein 2B inhibits FLS2 endo-cytosis, leading to enhancedROSproduction (Smith et al.,2014). Pharmacological studies imply that actin dynamicsis required for FLS2 receptor internalization and/or ves-icle trafficking, as vesicle dynamics are reduced aftertreatment with either LatB or the actin stabilizer endo-sidin1 (Robatzek et al., 2006; Beck et al., 2012). Thus, it ispossible that the impaired flg22-induced ROS productionmay result from the involvement of actin remodeling inFLS2 internalization.During plant interactions with fungi and oomycetes,

the recruitment of plasma membrane-localized NADPHoxidase toward the penetration site and its subsequentactivation involve an intact actin cytoskeleton (Hardhamet al., 2007; Schmidt and Panstruga, 2007). A study fromHao et al. (2014) indicated that the RBOHD enzymaticactivity is closely related to its dynamics at the plasmamembrane. Treatment with flg22 significantly increasesthemobility and aggregation of surface-localizedRBOHD(Hao et al., 2014). However, whether changes in RBOHDlocalization are associated with flg22-induced ROSproduction, and the involvement of actin remodeling inthis process, remain to be examined.

CONCLUSION

During innate immunity, production of ROS is anearly hallmark of cellular signaling. Similarly, remod-eling of the network of filaments or cytoskeleton is animportant facet of recognition and response to signalsfrom microbial invaders. However, whether ROS sig-naling impinges on cytoskeletal rearrangements and itsunderlying molecular mechanisms remains poorly un-derstood. Using high performance, live-cell imagingapproaches, we find that epidermal pavement cellsfrom Arabidopsis plants respond to a diverse array ofMAMPs and DAMPs, by significantly elevating thedensity of actin filament arrays. Additionally, theseactin rearrangements requireNADPHoxidase-dependent

ROSproduction and can be recapitulatedwith exogenousaddition of H2O2. We further demonstrate that the con-served heterodimeric, actin filament CP is a key down-stream intermediary of ROS signaling during innateimmunity. Finally, disrupting actin dynamics by LatB orregulation of in vivo CP levels leads to enhanced ROSproduction, suggesting a negative feedback loop betweenROS signaling and actin remodeling during innate im-munity. Collectively, our data provide compelling geneticevidence that CP is a key transducer of ROS signaling intochanges of actin dynamics during plant immunity.

MATERIALS AND METHODS

Plant Material and Growth Conditions

The homozygous fls2 (SALK_062054), bik1 (SALK_005291), rbohD (Torreset al., 2002), as well as cpb-1 (SALK_014783) and cpb-3 (SALK_101017) singlemutants were crossed to wild-type Arabidopsis (Arabidopsis thaliana) Col-0expressing the GFP-fABD2 reporter (Sheahan et al., 2004; Staiger et al., 2009)and homozygotes were recovered from F2 populations. The homozygous bak1-5mutant (Schwessinger et al., 2011) and CP OX#1 mutant (Li et al., 2014a) wastransformed with a binary vector for the actin reporter GFP-fABD2 by the floral-dip method (Zhang et al., 2006) and selected on plates containing antibiotics. T3plants were used for analysis of actin organization. The expression levels of CP incpmutants andCPOX#1 linewere examined previously by quantitative real-timePCR and semiquantitative immunoblotting (Li et al., 2012, 2014a). The transcriptsand protein levels for bothCP subunitswere decreased approximately 2-fold in cpmutants, and increased up to 5-fold in CP OX#1 line compared with wild-typeplants (Li et al., 2012, 2014a). Seeds were sown onto soil and stratified at 4°C for3 d. Flats were transferred to a growth chamber and plants grown under long-dayconditions (16 h light, 8 h dark) at 21°C for 3 to 4 weeks.

Plant Treatments with MAMPs and DAMPs

Before use, flg22, elf26, Pep1 (NeoBioSci), chitin (Sigma-Aldrich), or OG (Dr.Bruce Kohorn, Bowdoin College) were diluted in 13 PBS at various concen-trations. Immediately before imaging, the abaxial surface of rosette leaves from3- to 4-week-old plants was infiltrated with MAMPs and DAMPs using a 1-mLneedleless syringe until the intercellular space was filled with solution (;200 to300 mL per leaf). As a negative control, plants were treated with solvent used todilute the elicitors.

Image Acquisition and Quantitative Analyses of CorticalActin Array Architecture

The extent of actin filament bundling (skewness) and the percentage of oc-cupancy (density) were measured as described in Henty-Ridilla et al. (2013b).The cortical actin array in epidermal pavement cells was imaged using SDCMby collecting 15 to 20 steps of 0.5 mm each starting at the plasma membrane.SDCM was performed using a Yokogawa CSU-X1 mounted on an OlympusIX83 motorized microscope equipped with a 1003/1.4 NA objective. Illumi-nation was from a solid-state 50-mW, 488-nm laser with AOTF control overexcitation wavelength. The emission was captured with an Andor iXON ultra888 EMCCD camera. The SDCM was operated using the software MetaMorph(V. 7.1; Molecular Devices). A fixed exposure time and gain settings were se-lected for all the genotypes or treatments and their respective controls. Imageswere analyzed with Image J (National Institutes of Health) using the methodsdescribed in Henty-Ridilla et al. (2013b). Each experiment was repeated threetimes independently. The data presented are from one replicate. Data of twoadditional replicates are shown in Supplemental Figures S12 to S15. All imagecollection and data analyses were performed as double-blind experiments.

Measurement of ROS Production

Leaf disks (4-mm diameter) from 3- to 4-week-old plants were floated onwaterovernight ina96-wellplate (onediskperwell).ApoplasticROSreleasedbythe leaf tissuewere measured by a luminol-dependent assay (Heese et al., 2007).

Plant Physiol. Vol. 173, 2017 1133

Actin Dynamics and ROS Signaling in Plant Immunity

www.plantphysiol.orgon May 1, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.

Page 10: Capping Protein Modulates Actin Remodeling in Response to ... · Capping Protein Modulates Actin Remodeling in Response to Reactive Oxygen Species during Plant Innate Immunity1[OPEN]

Before detection, the water was replaced with 200 mL of a solution containing34 mg/mL luminol (Santa Cruz Biotechnology), 20 mg/mL horse radish per-oxidase (Sigma-Aldrich) and elicitors. Luminescence over time was recordedwith a Synergy 2 plate reader system (Biotek). For LatB treatment, leaf diskswere pretreated by floating in water containing 1 mM LatB for 30 min beforesubsequent flg22-elicitation. Each experiment was repeated three times inde-pendently. The data presented are from one replicate. Data of two additionalreplicates are shown in Supplemental Fig. S16.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL datalibraries under accession numbers AT5G47910 (RBOHD), AT5G46330 (FLS2),ÚT2G39660 (BIK1), AT4G33430 (BAK1), At3g05520 (AtCPA), and At1g71790(AtCPB).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. RBOHD is required for ROS production in re-sponse to various stimuli.

Supplemental Figure S2. The increase in actin filament abundance trig-gered by flg22 treatment is dose-dependent.

Supplemental Figure S3. ROS production in response to flg22 is inhibitedby mutants in the cognate PRR complex.

Supplemental Figure S4. Exogenously applied H2O2 increases actin fila-ment abundance in a dose-dependent manner.

Supplemental Figure S5. Diphenyleneiodonium treatment blocksflg22-triggered actin remodeling.

Supplemental Figure S6. Actin remodeling induced by flg22 treatment isinhibited by the ROS scavenger TEMPOL.

Supplemental Figure S7. CP contributes to plant resistance against bacte-rial pathogens.

Supplemental Figure S8. Actin architecture in cpb-3 mutant cells fails torespond to flg22 treatments.

Supplemental Figure S9. Actin remodeling in cpb-1 mutant cannot beelicited with high concentrations of flg22 or H2O2.

Supplemental Figure S10. The flg22-dependent ROS production is signif-icantly enhanced in cpb-3 mutant.

Supplemental Figure S11. The increase in flg22-induced apoplastic ROS inthe cpb-1 mutant depends on RBOHD.

Supplemental Figure S12. Results from two additional replicates for theexperiment shown in Figure 1.

Supplemental Figure S13. Results from two additional replicates for theexperiment shown in Figure 2.

Supplemental Figure S14. Results from two additional replicates for theexperiment shown in Figure 3.

Supplemental Figure S15. Results from two additional replicates for theexperiment shown in Figure 4.

Supplemental Figure S16. Results from two additional replicates for theexperiment shown in Figure 5.

ACKNOWLEDGMENTS

The seeds for bak1-5 were generously provided by Cyril Zipfel (TheSainsbury Laboratory) and oligogalacturonides from Bruce Kohorn (Bow-doin College). We thank Antje Heese (University of Missouri) for advice onthe ROS assay and Zhao-Qing Luo (Purdue University) for access to a platereader. We thank Hongbing Luo for excellent care and maintenance of plantmaterials.

Received June 24, 2016; accepted November 30, 2016; published December 1,2016.

LITERATURE CITED

Andersson MX, Kourtchenko O, Dangl JL, Mackey D, Ellerström M(2006) Phospholipase-dependent signalling during the AvrRpm1- andAvrRpt2-induced disease resistance responses in Arabidopsis thaliana.Plant J 47: 947–959

Bargmann BO, Laxalt AM, Riet BT, Schouten E, van Leeuwen W, DekkerHL, de Koster CG, Haring MA, Munnik T (2006) LePLDb1 activationand relocalization in suspension-cultured tomato cells treated with xy-lanase. Plant J 45: 358–368

Beck M, Zhou J, Faulkner C, MacLean D, Robatzek S (2012) Spatio-temporal cellular dynamics of the Arabidopsis flagellin receptor revealactivation status-dependent endosomal sorting. Plant Cell 24: 4205–4219

Ben Rejeb K, Benzarti M, Debez A, Bailly C, Savouré A, Abdelly C (2015)NADPH oxidase-dependent H2O2 production is required for salt-induced antioxidant defense in Arabidopsis thaliana. J Plant Physiol 174:5–15

Boller T, Felix G (2009) A renaissance of elicitors: perception of microbe-associated molecular patterns and danger signals by pattern-recognitionreceptors. Annu Rev Plant Biol 60: 379–406

Canonne J, Froidure-Nicolas S, Rivas S (2011) Phospholipases in actionduring plant defense signaling. Plant Signal Behav 6: 13–18

Chiarugi P, Pani G, Giannoni E, Taddei L, Colavitti R, Raugei G, SymonsM, Borrello S, Galeotti T, Ramponi G (2003) Reactive oxygen species asessential mediators of cell adhesion: the oxidative inhibition of a FAKtyrosine phosphatase is required for cell adhesion. J Cell Biol 161: 933–944

Chinchilla D, Bauer Z, Regenass M, Boller T, Felix G (2006) The Arabi-dopsis receptor kinase FLS2 binds flg22 and determines the specificity offlagellin perception. Plant Cell 18: 465–476

Chinchilla D, Zipfel C, Robatzek S, Kemmerling B, Nürnberger T, JonesJD, Felix G, Boller T (2007) A flagellin-induced complex of the receptorFLS2 and BAK1 initiates plant defence. Nature 448: 497–500

Cooper JA, Sept D (2008) New insights into mechanism and regulation ofactin capping protein. Int Rev Cell Mol Biol 267: 183–206

de Jong CF, Laxalt AM, Bargmann BOR, de Wit PJGM, Joosten MHAJ,Munnik T (2004) Phosphatidic acid accumulation is an early response inthe Cf-4/Avr4 interaction. Plant J 39: 1–12

den Hartog M, Musgrave A, Munnik T (2001) Nod factor-induced phos-phatidic acid and diacylglycerol pyrophosphate formation: a role forphospholipase C and D in root hair deformation. Plant J 25: 55–65

Dodds PN, Rathjen JP (2010) Plant immunity: towards an integrated viewof plant-pathogen interactions. Nat Rev Genet 11: 539–548

D’Ovidio R, Mattei B, Roberti S, Bellincampi D (2004) Polygalacturo-nases, polygalacturonase-inhibiting proteins and pectic oligomers inplant-pathogen interactions. Biochim Biophys Acta 1696: 237–244

Fiaschi T, Cozzi G, Raugei G, Formigli L, Ramponi G, Chiarugi P (2006)Redox regulation of beta-actin during integrin-mediated cell adhesion. JBiol Chem 281: 22983–22991

Gómez-Gómez L, Boller T (2000) FLS2: an LRR receptor-like kinase in-volved in the perception of the bacterial elicitor flagellin in Arabidopsis.Mol Cell 5: 1003–1011

Hao H, Fan L, Chen T, Li R, Li X, He Q, Botella MA, Lin J (2014) Clathrinand membrane microdomains cooperatively regulate RbohD dynamicsand activity in Arabidopsis. Plant Cell 26: 1729–1745

Hardham AR, Jones DA, Takemoto D (2007) Cytoskeleton and cell wallfunction in penetration resistance. Curr Opin Plant Biol 10: 342–348

Heese A, Hann DR, Gimenez-Ibanez S, Jones AME, He K, Li J, SchroederJI, Peck SC, Rathjen JP (2007) The receptor-like kinase SERK3/BAK1 isa central regulator of innate immunity in plants. Proc Natl Acad Sci USA104: 12217–12222

Henty-Ridilla JL, Li J, Blanchoin L, Staiger CJ (2013a) Actin dynamics inthe cortical array of plant cells. Curr Opin Plant Biol 16: 678–687

Henty-Ridilla JL, Li J, Day B, Staiger CJ (2014) ACTIN DEPOLYMERIZ-ING FACTOR4 regulates actin dynamics during innate immune sig-naling in Arabidopsis. Plant Cell 26: 340–352

Henty-Ridilla JL, Shimono M, Li J, Chang JH, Day B, Staiger CJ (2013b)The plant actin cytoskeleton responds to signals from microbe-associated molecular patterns. PLoS Pathog 9: e1003290

Higaki T, Kutsuna N, Sano T, Kondo N, Hasezawa S (2010) Quantificationand cluster analysis of actin cytoskeletal structures in plant cells: role ofactin bundling in stomatal movement during diurnal cycles in Arabi-dopsis guard cells. Plant J 61: 156–165

1134 Plant Physiol. Vol. 173, 2017

Li et al.

www.plantphysiol.orgon May 1, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.

Page 11: Capping Protein Modulates Actin Remodeling in Response to ... · Capping Protein Modulates Actin Remodeling in Response to Reactive Oxygen Species during Plant Innate Immunity1[OPEN]

Huang S, Blanchoin L, Kovar DR, Staiger CJ (2003) Arabidopsis cappingprotein (AtCP) is a heterodimer that regulates assembly at the barbedends of actin filaments. J Biol Chem 278: 44832–44842

Huang S, Gao L, Blanchoin L, Staiger CJ (2006) Heterodimeric cappingprotein from Arabidopsis is regulated by phosphatidic acid. Mol Biol Cell17: 1946–1958

Huffaker A, Pearce G, Ryan CA (2006) An endogenous peptide signal inArabidopsis activates components of the innate immune response. ProcNatl Acad Sci USA 103: 10098–10103

Kadota Y, Sklenar J, Derbyshire P, Stransfeld L, Asai S, Ntoukakis V,Jones JD, Shirasu K, Menke F, Jones A, Zipfel C (2014) Direct regu-lation of the NADPH oxidase RBOHD by the PRR-associated kinaseBIK1 during plant immunity. Mol Cell 54: 43–55

Kang Y, Jelenska J, Cecchini NM, Li Y, Lee MW, Kovar DR, Greenberg JT(2014) HopW1 from Pseudomonas syringae disrupts the actin cytoskeletonto promote virulence in Arabidopsis. PLoS Pathog 10: e1004232

Kim K, Yamashita A, Wear MA, Maéda Y, Cooper JA (2004) Cappingprotein binding to actin in yeast: biochemical mechanism and physio-logical relevance. J Cell Biol 164: 567–580

Kirik A, Mudgett MB (2009) SOBER1 phospholipase activity suppressesphosphatidic acid accumulation and plant immunity in response tobacterial effector AvrBsT. Proc Natl Acad Sci USA 106: 20532–20537

Klamt F, Zdanov S, Levine RL, Pariser A, Zhang Y, Zhang B, Yu L-R,Veenstra TD, Shacter E (2009) Oxidant-induced apoptosis is mediatedby oxidation of the actin-regulatory protein cofilin. Nat Cell Biol 11:1241–1246

Lanteri ML, Lamattina L, Laxalt AM (2011) Mechanisms of xylanase-induced nitric oxide and phosphatidic acid production in tomato cells.Planta 234: 845–855

Laxalt AM, Raho N, Have AT, Lamattina L (2007) Nitric oxide is critical forinducing phosphatidic acid accumulation in xylanase-elicited tomatocells. J Biol Chem 282: 21160–21168

Lehmann S, Serrano M, L’Haridon F, Tjamos SE, Metraux J-P (2015) Re-active oxygen species and plant resistance to fungal pathogens. Phyto-chemistry 112: 54–62

Li J, Blanchoin L, Staiger CJ (2015a) Signaling to actin stochastic dynamics.Annu Rev Plant Biol 66: 415–440

Li J, Henty-Ridilla JL, Huang S, Wang X, Blanchoin L, Staiger CJ (2012)Capping protein modulates the dynamic behavior of actin filaments inresponse to phosphatidic acid in Arabidopsis. Plant Cell 24: 3742–3754

Li J, Henty-Ridilla JL, Staiger BH, Day B, Staiger CJ (2015b) Cappingprotein integrates multiple MAMP signalling pathways to modulateactin dynamics during plant innate immunity. Nat Commun 6: 7206

Li J, Staiger BH, Henty-Ridilla JL, Abu-Abied M, Sadot E, Blanchoin L,Staiger CJ (2014a) The availability of filament ends modulates actinstochastic dynamics in live plant cells. Mol Biol Cell 25: 1263–1275

Li L, Li M, Yu L, Zhou Z, Liang X, Liu Z, Cai G, Gao L, Zhang X, Wang Y,Chen S, Zhou JM (2014b) The FLS2-associated kinase BIK1 directlyphosphorylates the NADPH oxidase RbohD to control plant immunity.Cell Host Microbe 15: 329–338

Li X, Li J-H, Wang W, Chen N-Z, Ma T-S, Xi Y-N, Zhang X-L, Lin H-F, BaiY, Huang S-J, Chen Y-L (2014c) ARP2/3 complex-mediated actin dy-namics is required for hydrogen peroxide-induced stomatal closure inArabidopsis. Plant Cell Environ 37: 1548–1560

Lind C, Gerdes R, Hamnell Y, Schuppe-Koistinen I, von Löwenhielm HB,Holmgren A, Cotgreave IA (2002) Identification of S-glutathionylatedcellular proteins during oxidative stress and constitutive metabolism byaffinity purification and proteomic analysis. Arch Biochem Biophys 406:229–240

Lu D, Wu S, Gao X, Zhang Y, Shan L, He P (2010) A receptor-like cyto-plasmic kinase, BIK1, associates with a flagellin receptor complex toinitiate plant innate immunity. Proc Natl Acad Sci USA 107: 496–501

Ma L, Sun N, Liu X, Jiao Y, Zhao H, Deng XW (2005) Organ-specific ex-pression of Arabidopsis genome during development. Plant Physiol 138:80–91

Mersmann S, Bourdais G, Rietz S, Robatzek S (2010) Ethylene signalingregulates accumulation of the FLS2 receptor and is required for the ox-idative burst contributing to plant immunity. Plant Physiol 154: 391–400

Monaghan J, Zipfel C (2012) Plant pattern recognition receptor complexesat the plasma membrane. Curr Opin Plant Biol 15: 349–357

Morimatsu T, Kawagoshi A, Yoshida K, Tamura M (1997) Actin enhancesthe activation of human neutrophil NADPH oxidase in a cell-free sys-tem. Biochem Biophys Res Commun 230: 206–210

Munnamalai V, Suter DM (2009) Reactive oxygen species regulate F-actindynamics in neuronal growth cones and neurite outgrowth. J Neuro-chem 108: 644–661

Nathan C, Cunningham-Bussel A (2013) Beyond oxidative stress: an im-munologist’s guide to reactive oxygen species. Nat Rev Immunol 13:349–361

Pleskot R, Li J, Zárský V, Potocký M, Staiger CJ (2013) Regulation ofcytoskeletal dynamics by phospholipase D and phosphatidic acid.Trends Plant Sci 18: 496–504

Porter K, Day B (2016) From filaments to function: the role of the plant actincytoskeleton in pathogen perception, signaling and immunity. J IntegrPlant Biol 58: 299–311

Raho N, Ramirez L, Lanteri ML, Gonorazky G, Lamattina L, ten Have A,Laxalt AM (2011) Phosphatidic acid production in chitosan-elicited to-mato cells, via both phospholipase D and phospholipase C/diacylglycerolkinase, requires nitric oxide. J Plant Physiol 168: 534–539

Rasmussen I, Pedersen LH, Byg L, Suzuki K, Sumimoto H, Vilhardt F(2010) Effects of F/G-actin ratio and actin turn-over rate on NADPHoxidase activity in microglia. BMC Immunol 11: 44

Robatzek S, Chinchilla D, Boller T (2006) Ligand-induced endocytosis of thepattern recognition receptor FLS2 in Arabidopsis. Genes Dev 20: 537–542

Sakai J, Li J, Subramanian KK, Mondal S, Bajrami B, Hattori H, Jia Y,Dickinson BC, Zhong J, Ye K, et al (2012) Reactive oxygen species-induced actin glutathionylation controls actin dynamics in neutro-phils. Immunity 37: 1037–1049

Salomon S, Robatzek S (2006) Induced endocytosis of the receptor kinaseFLS2. Plant Signal Behav 1: 293–295

Schmidt SM, Panstruga R (2007) Cytoskeletal functions in plant-microbeinteractions. Physiol Mol Plant Pathol 71: 135–148

Schwessinger B, Roux M, Kadota Y, Ntoukakis V, Sklenar J, Jones A,Zipfel C (2011) Phosphorylation-dependent differential regulation ofplant growth, cell death, and innate immunity by the regulatoryreceptor-like kinase BAK1. PLoS Genet 7: e1002046

Scott I, Logan DC (2008) Mitochondrial morphology transition is an earlyindicator of subsequent cell death in Arabidopsis. New Phytol 177: 90–101

Sheahan MB, Staiger CJ, Rose RJ, McCurdy DW (2004) A green fluores-cent protein fusion to actin-binding domain 2 of Arabidopsis fimbrinhighlights new features of a dynamic actin cytoskeleton in live plantcells. Plant Physiol 136: 3968–3978

Shimono M, Lu Y-J, Porter K, Kvitko BH, Henty-Ridilla J, Creason A, HeSY, Chang JH, Staiger CJ, Day B (2016) The Pseudomonas syringae typeIII effector HopG1 induces actin remodeling to promote symptom de-velopment and susceptibility during infection. Plant Physiol 171: 2239–2255

Smith JM, Leslie ME, Robinson SJ, Korasick DA, Zhang T, Backues SK,Cornish PV, Koo AJ, Bednarek SY, Heese A (2014) Loss of Arabidopsisthaliana Dynamin-Related Protein 2B reveals separation of innate im-mune signaling pathways. PLoS Pathog 10: e1004578

Staiger CJ, Sheahan MB, Khurana P, Wang X, McCurdy DW, Blanchoin L(2009) Actin filament dynamics are dominated by rapid growth andsevering activity in the Arabidopsis cortical array. J Cell Biol 184: 269–280

Suzuki N, Miller G, Morales J, Shulaev V, Torres MA, Mittler R (2011)Respiratory burst oxidases: the engines of ROS signaling. Curr OpinPlant Biol 14: 691–699

Tamura M, Itoh K, Akita H, Takano K, Oku S (2006) Identification of anactin-binding site in p47phox an organizer protein of NADPH oxidase.FEBS Lett 580: 261–267

Tamura M, Kai T, Tsunawaki S, Lambeth JD, Kameda K (2000a) Directinteraction of actin with p47(phox) of neutrophil NADPH oxidase. Bio-chem Biophys Res Commun 276: 1186–1190

Tamura M, Kanno M, Endo Y (2000b) Deactivation of neutrophil NADPHoxidase by actin-depolymerizing agents in a cell-free system. Biochem J349: 369–375

Taulet N, Delorme-Walker VD, DerMardirossian C (2012) Reactive oxy-gen species regulate protrusion efficiency by controlling actin dynamics.PLoS One 7: e41342

Tena G, Boudsocq M, Sheen J (2011) Protein kinase signaling networks inplant innate immunity. Curr Opin Plant Biol 14: 519–529

Torres MA, Dangl JL, Jones JDG (2002) Arabidopsis gp91phox homologuesAtrbohD and AtrbohF are required for accumulation of reactive oxygenintermediates in the plant defense response. Proc Natl Acad Sci USA 99:517–522

Plant Physiol. Vol. 173, 2017 1135

Actin Dynamics and ROS Signaling in Plant Immunity

www.plantphysiol.orgon May 1, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.

Page 12: Capping Protein Modulates Actin Remodeling in Response to ... · Capping Protein Modulates Actin Remodeling in Response to Reactive Oxygen Species during Plant Innate Immunity1[OPEN]

Torres MA, Jones JDG, Dangl JL (2006) Reactive oxygen species signalingin response to pathogens. Plant Physiol 141: 373–378

van der Luit AH, Piatti T, van Doorn A, Musgrave A, Felix G, Boller T,Munnik T (2000) Elicitation of suspension-cultured tomato cells triggersthe formation of phosphatidic acid and diacylglycerol pyrophosphate.Plant Physiol 123: 1507–1516

Wilkins KA, Bancroft J, Bosch M, Ings J, Smirnoff N, Franklin-Tong VE(2011) Reactive oxygen species and nitric oxide mediate actin reorga-nization and programmed cell death in the self-incompatibility responseof papaver. Plant Physiol 156: 404–416

Wilson C, González-Billault C (2015) Regulation of cytoskeletal dynamicsby redox signaling and oxidative stress: implications for neuronal de-velopment and trafficking. Front Cell Neurosci 9: 381

Xu S, Chisholm AD (2014) C. elegans epidermal wounding induces a mi-tochondrial ROS burst that promotes wound repair. Dev Cell 31: 48–60

Yamaguchi T, Minami E, Shibuya N (2003) Activation of phospholipasesby N-acetylchitooligosaccharide elicitor in suspension-cultured rice cellsmediates reactive oxygen generation. Physiol Plant 118: 361–370

Yamaguchi T, Minami E, Ueki J, Shibuya N (2005) Elicitor-induced activationof phospholipases plays an important role for the induction of defenseresponses in suspension-cultured rice cells. Plant Cell Physiol 46: 579–587

Yamaguchi T, Tanabe S, Minami E, Shibuya N (2004) Activation ofphospholipase D induced by hydrogen peroxide in suspension-culturedrice cells. Plant Cell Physiol 45: 1261–1270

Yang L, Qin L, Liu G, Peremyslov VV, Dolja VV, Wei Y (2014) Myosins XImodulate host cellular responses and penetration resistance to fungalpathogens. Proc Natl Acad Sci USA 111: 13996–14001

Zhang J, Li W, Xiang T, Liu Z, Laluk K, Ding X, Zou Y, Gao M, Zhang X,Chen S, Mengiste T, Zhang Y, et al (2010) Receptor-like cytoplasmickinases integrate signaling from multiple plant immune receptors andare targeted by a Pseudomonas syringae effector. Cell Host Microbe 7:290–301

Zhang W, Wang C, Qin C, Wood T, Olafsdottir G, Welti R, Wang X(2003) The oleate-stimulated phospholipase D, PLDd, and phosphatidicacid decrease H2O2-induced cell death in Arabidopsis. Plant Cell 15:2285–2295

Zhang X, Henriques R, Lin S-S, Niu Q-W, Chua N-H (2006) Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dipmethod. Nat Protoc 1: 641–646

Zhao J (2015) Phospholipase D and phosphatidic acid in plant defence re-sponse: from protein-protein and lipid-protein interactions to hormonesignalling. J Exp Bot 66: 1721–1736

1136 Plant Physiol. Vol. 173, 2017

Li et al.

www.plantphysiol.orgon May 1, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.