The Cyclin-Dependent Kinase Inhibitor KRP6 InducesMitosis and Impairs Cytokinesis in Giant Cells Inducedby Plant-Parasitic Nematodes in ArabidopsisW

Paulo Vieira,a,1,2 Annelies De Clercq,b,c,1 Hilde Stals,b,c,1 Jelle Van Leene,b,c Eveline Van De Slijke,b,c Gert VanIsterdael,b,c Dominique Eeckhout,b,c Geert Persiau,b,c Daniël VanDamme,b,c Aurine Verkest,b,c JoséDijair Antoninode Souza, Júnior,d Nathalie Glab,e Pierre Abad,a Gilbert Engler,a Dirk Inzé,b,c,3 Lieven De Veylder,b,c,3 Geert DeJaeger,b,c,3 and Janice de Almeida Englera,3,4

a Institut National de la Recherche Agronomique, UMR 1355 ISA/Centre National de la Recherche Scientifique, UMR 7254 ISA/Université de Nice-Sophia Antipolis, UMR ISA, 400 route des Chappes, 06903 Sophia-Antipolis, FrancebDepartment of Plant Systems Biology, Flanders Institute for Biotechnology, 9052 Gent, BelgiumcDepartment of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Gent, Belgiumd Laboratório de Interação Molecular Planta-Praga, Embrapa Recursos Genéticos e Biotecnologia, Brasília, 70770-900 DistritoFederal, Brazile Institut de Biologie des Plantes, Centre National de la Recherche Scientifique Unité Mixte de Recherche 8618, Université Paris-Sud,Saclay Plant Sciences, 91405 Orsay Cedex, France

In Arabidopsis thaliana, seven cyclin-dependent kinase (CDK) inhibitors have been identified, designated interactors of CDKsor Kip-related proteins (KRPs). Here, the function of KRP6 was investigated during cell cycle progression in roots infected byplant-parasitic root-knot nematodes. Contrary to expectations, analysis of Meloidogyne incognita–induced galls of KRP6-overexpressing lines revealed a role for this particular KRP as an activator of the mitotic cell cycle. In accordance, KRP6-overexpressing suspension cultures displayed accelerated entry into mitosis, but delayed mitotic progression. Likewise,phenotypic analysis of cultured cells and nematode-induced giant cells revealed a failure in mitotic exit, with the appearanceof multinucleated cells as a consequence. Strong KRP6 expression upon nematode infection and the phenotypic resemblancebetween KRP6 overexpression cell cultures and root-knot morphology point toward the involvement of KRP6 in themultinucleate and acytokinetic state of giant cells. Along these lines, the parasite might have evolved to manipulate plantKRP6 transcription to the benefit of gall establishment.

INTRODUCTION

The establishment and maintenance of the nematode feedingsite by root-knot nematodes (RKNs) is a complex process as-sociated with significant changes in gene expression of both theplant and the plant-parasitic nematode (Jammes et al., 2005;Caillaud et al., 2008; Gheysen and Mitchum, 2009). Previousefforts have aimed to enhance our understanding of the mo-lecular mechanisms driving feeding cell development inducedby these sedentary nematodes (Meloidogyne spp). The com-plexity of plant–RKN interaction is well demonstrated by thenumber of host genes involved, and transcriptional data

showing extensive crosstalk between the various host molecularpathways (reviewed by Gheysen and Fenoll, 2002; Gheysen andMitchum, 2009). Because RKN infection leads to the formationof multinucleated giant cells through synchronous nuclear divi-sions in the absence of cytokinesis, the specific involvement ofthe host cell cycle machinery appears to be a major driver for theformation of these specialized feeding cells (de Almeida Engleret al., 1999; de Almeida Engler and Gheysen, 2013). Therefore,knowledge of the plant cell cycle machinery is fundamental forunderstanding comparable events occurring during nematodefeeding site development in plant roots. Previously, infection ofArabidopsis thaliana reporter lines carrying cell cycle markerslike cyclin-dependent kinases (CDKs) and their regulatory cyclinsubunits (CYCs) illustrated that there is early stimulation of thehost cell cycle machinery at the nematode feeding site (Niebelet al., 1996; de Almeida Engler et al., 1999). More recently,a detailed characterization of plant genes directly involved inendocycle, such as CCS52A, CCS52B, DEL1, and RHL1, hasbeen reported for RKN, as well as for cyst nematodes (deAlmeida Engler et al., 2012; Vieira et al., 2013a). Giant cells makeuse of this alternative cell cycle process during which mitosis isrepressed in favor of continued DNA replication, resulting inpolyploid cells. The involvement of cell cycle and endocyclegenes in proper nematode feeding site establishment illustrates

1 These authors contributed equally to this work.2 Current address: NemaLab/ Instituto de Ciências Agrárias e AmbientaisMediterrânicas, Universidade de Évora, Núcleo da Mitra, Ap. 94, 7002-554 Évora, Portugal.3 These authors share senior authorship.4 Address correspondence to janice.almeida-engler@sophia.inra.fr.The authors responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) are: Geert De Jaeger(geert.dejaeger@psb.vib-ugent.be) and Janice de Almeida Engler (janice.almeida-engler@sophia.inra.fr).W Online version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.114.126425

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the need for both cell cycle types for successful RKN re-production (de Almeida Engler et al., 2012).

Different types of CDK/CYC complexes account for the cor-rect temporal and unidirectional ordering of cell cycle events(Inzé and De Veylder, 2006). The model plant species Arabi-dopsis encodes up to 12 CDKs and 49 CYCs that have beencategorized into different classes according to their sequencesimilarity (Vandepoele et al., 2002; Wang et al., 2004; Mengeset al., 2005). Plants possess six types of CDKs. The A-type CDKsare the most closely related to the mammalian CDK1 and CDK2because they contain the characteristic PSTAIRE amino acidsequence in their cyclin binding domain. In association with theD-type cyclins (CYCDs), the CDKA/CYCD complexes are believedto regulate the G1-to-S transition through phosphorylation of theretinoblastoma-related protein (De Veylder et al., 2007). TheG2-to-M transition most probably requires A- and plant-specificB-type CDKs, as well as A- and B-type cyclins to form the mitoticCDK/CYC complexes (Inzé, 2005; De Veylder et al., 2007).

Inhibitory proteins regulate CDK/CYC activity. KRP proteins area family of CDK inhibitors identified in plants. They are distantlyrelated to the Kip/Cip family of animal CDK inhibitors, designatedKip-related proteins (KRPs) (De Veylder et al., 2001) or interactorsof Cdc2 kinases (Wang et al., 1997, 1998; Lui et al., 2000). Theyare generally believed to specifically interact with and inhibitA-type CDKs and CYCDs (Wang et al., 1998; De Veylder et al.,2001), although some family members might interact with B-typeCDKs as well (Nakai et al., 2006; Pettkó-Szandtner et al., 2006).The level of inhibition of this KRP family of proteins seems to beconcentration dependent (Verkest et al., 2005a), differentially af-fecting the cellular DNA content. Low KRP2 levels increase DNAcontent, while high levels decrease DNA content (Verkest et al.,2005b; Weinl et al., 2005). Sequence alignment and specifictemporal and spatial expression patterns during cell cycle pro-gression and plant development (Menges and Murray, 2002;Ormenese et al., 2004; Menges et al., 2005) point to a functionaldifference among the various members of the KRP family (Kimet al., 2008; Jégu et al., 2013; Jun et al., 2013; Wen et al., 2013).Recently, KRPs have been linked to different physiological pro-cesses. KRP6 and KRP7 have been reported to be involved in thecontrol of Arabidopsis male gametogenesis (Kim et al., 2008).Guérinier et al. (2013) have shown that recombinant KRP6 andKRP7 can be phosphorylated by SNF1-Related protein Kinase-1,providing a possible connection between energy sensing and cellproliferation. KRP2 influences lateral root density in an auxin-dependent manner, whereas KRP5 appears to be limiting forprimary root growth (Sanz et al., 2011; Wen et al., 2013). In ad-dition to their role in CDK/CYC inhibition, some KRPs, like KRP5,may serve other functions regulating gene transcription involvedin cell wall organization (Jégu et al., 2013).

Although the link between RKN infection and cell cycle activityfor nematode feeding site formation is well recognized, themolecular mechanisms employed by nematodes to exploit thehost plant remain ambiguous. Recent work has shown thatectopic KRP1, KRP2, and KRP4 expression led to a drastic re-duction in gall size by inhibiting mitosis (Vieira et al., 2012,2013b). Here, we found that KRP6 is highly expressed duringnematode feeding site ontogenesis and studied the role of KRP6during cell cycle progression in Arabidopsis and nematode

feeding site development induced by RKN. Unexpectedly, wefound that overexpression of KRP6 leads to multinucleated cellsin Arabidopsis cell cultures and root cells, therefore stimulatingand not inhibiting mitotic activity, as seen for other ArabidopsisKRP genes. We further show that overexpression of KRP6increases mitotic activity in the nematode-feeding site, yetstrongly interfered with nematode reproduction. Collectively,these findings point toward the involvement of KRP6 in themultinucleate and acytokinetic state of RKN-induced giant cells.

RESULTS

KRP6 Expression in Developing Galls Induced byMeloidogyne incognita

During our analysis of promoter activity of the seven KRP genesin Arabidopsis, we observed that KRP6 was strongly expressedin galls induced by RKN (Vieira et al., 2013b). Therefore, weanalyzed a b-glucuronidase (GUS) reporter line in detail andperformed in situ transcript localization of KRP6 in galls inducedby M. incognita at early, intermediate, and later developmentalstages (7, 14, and 21 d after nematode inoculation [DAI], re-spectively) (Figure 1). At 7 DAI, galls are in the mitotic phase,whereas at 14 DAI, nuclei in giant cells enlarge and show intenseDNA biosynthesis, most likely via endoreduplication. At laterdevelopmental stages (21 up to 60 DAI, when the nematode lifecycle ends with egg laying), no mitotic activity is observed eitherin giant or neighboring cells and DNA biosynthesis is signifi-cantly decreased or not occurring in giant cells (de AlmeidaEngler et al., 1999). Our analysis showed KRP6 expression in theroot apex and the vascular tissue of noninfected roots (Figures1A and 1B) and in galls at different developmental stages (Fig-ures 1C to 1F). High promoter activity was observed in cells ofthe root vascular tissue showing a dense cytoplasm surroundingthe nematode during migration and during the initial feeding siteactivation (Figure 1C). At 7 DAI, high KRP6 transcript levels weredetected in giant and in neighboring cells (Figure 1G), whichcorresponds to high mitotic activity during gall development.Throughout gall expansion (up to 14 DAI) KRP6 expressiondecreased in giant cells but persisted in mitotic neighboringcells (Figures 1E and 1H), and at 21 DAI the promoter activity(Figure 1F) and transcript levels (Figure 1I) decreased in thewhole gall.

GFP-KRP6 Localization and Dynamics during NematodeFeeding Site Development

Having analyzed promoter activity and performed transcript lo-calization, additional localization and dynamics in the 35S:GFP-KRP6 line (hereafter named KRP6OE) was followed in galls byconfocal microscopy. GFP-KRP6 was detected in nuclei of bothgiant and neighboring cells at early stages (2 DAI) of gall de-velopment (Figure 2A), while at later stages (21 to 40 DAI),fluorescence was weak (Figure 2B) or below the detection levelin giant cells (Figure 2C). Free GFP lines (35S:GFP) were used ascontrols and showed constant fluorescence intensity levelsthroughout gall development (Supplemental Figure 1).

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GFP-KRP6 Localizes to the Nucleus during Interphase andIts Overexpression Leads to Multinucleated Cells inArabidopsis Cell Cultures

To study the dynamics of KRP6 protein localization during mi-totic progression, we generated Arabidopsis cell cultures co-expressing 35S:GFP-KRP6 and 35S:RFP-TUA2, a microtubulemarker (Figure 3). During time-lapse analysis throughout mitosis,GFP-KRP6 was localized within the nucleus in interphase andearly preprophase cells (Figure 3A). The GFP signal dispersedinto the cytoplasm from late prophase (Figure 3B) until meta-phase (Figures 3C and 3D) and anaphase (Figure 3E). Duringtelophase, when the phragmoplast is formed, and the follow-ing cytokinesis, GFP-KRP6 relocalized to the newly formeddaughter nuclei (Figures 3F to 3J). Similar observations for GFP-KRP6 localization during mitosis were made in root cells ofKRP6OE stable transgenic seedlings (Supplemental Figure 2).

Strikingly, in ;5% of the cells, overexpression of GFP-KRP6led to the formation of polynucleate cells with up to 20 unequally

sized nuclei (Figure 4; Supplemental Movie 1). Such nuclei werenever seen in Arabidopsis control lines that overexpressed GFPwith a nuclear localization signal or RFP-TUA2 alone.To investigate the formation of these multinucleate cells

coexpressing GFP-KRP6 and RFP-TUA2, we followed thecourse of cytokinesis using four-dimensional confocal time-lapse microscopy. Figure 5 presents a single projection of aZ-stack of three cells that divide synchronously with two nucleiin metaphase and two nuclei in anaphase (Figure 5A, asterisks).Originally, two nuclei were present in the upper left cell and onesingle nucleus in the other two cells (Figure 5A). The microtu-bular organization of the different spindles and phragmoplastsas visualized by RFP-TUA2 appeared normal in these cells(Figures 5A to 5D, asterisks). However, upon nuclear reforma-tion, it became apparent that nuclear material was unevenlydistributed relative to the expanding phragmoplast and that in-stead of two daughter nuclei, multiple nuclei formed accumu-lating GFP-KRP6 (Figures 5C to 5L). Often, some of thesemicronuclei fused to form larger nuclei (Figures 5D to 5G, 5K,

Figure 1. Expression Pattern of KRP6 in M. incognita–Induced Galls in Arabidopsis Roots.

White lines delimit individual giant cells in both GUS and in situ hybridization micrographs.(A) and (B) KRP6pro:GUS expression in non-infected roots. Root apical meristem (A) and root vascular tissue (B).(C) to (F) KRP6pro:GUS expression in roots infected with M. incognita, during nematode migration at 2 DAI (C) and during gall development at 7 (D), 14(E), and 21 (F) DAI. Dark-field images illustrate GUS staining in red.(G) to (I) KRP6mRNA in situ localization in galls at 7 (G), 14 (H), and 21 (I) DAI. Sections were hybridized with 35S-labeled antisense KRP6 RNA probes,and hybridization signal is visible as white dots under dark-field optics.Asterisks, giant cell; n, nematode; NCs, neighboring cells. Bars = 50 mm.

KRP6 Is Involved in Giant Cell Multinucleation 3 of 15

and 5L, arrowheads). Finally, the overexpression of GFP-KRP6led to the formation of multinucleated cells with nuclei of un-equal size (Supplemental Movie 2). The formation of micronucleicould result from the observed lagging chromosomes duringanaphase (Figure 6A, upper arrows; Supplemental Movie 3)and/or the unequal separation of chromosomes during telo-phase (Figure 6B).

Overexpression of KRP6 Accelerates Entry into Mitosis, yetDelays Mitosis Progression and Exit

To assess the consequences of overexpressing KRP6 on cellcycle progression, we synchronized the GFP-KRP6–over-expressing Arabidopsis culture by aphidicolin treatment, whichblocks cell cycle progression specifically at early S phase. Afterremoval of the inhibitor, cell cycle progression was monitored bycounting the number of cells entering S phase through bromo-deoxyuridine (BrdU) labeling of de novo–synthesized DNA (BrdUlabeling index) and by scoring the metaphase/anaphase index(M/A index) (Figure 7A). Cells that overexpressed GFP-KRP6entered S phase earlier than the control cells (PSB-D), as shown

by the BrdU labeling index that peaked 1 h earlier in cells ec-topically expressing KRP6 (Figure 7A). Entry into mitosis came2 h earlier than in the control cells, as shown by the M/A index thatpeaked at 13 h, instead of 15 h in untransformed cells (Figure7A). Also, the G2-M phase CDKB1;1 protein marker accumu-lated more rapidly in the 35S:GFP-KRP6 cell line than in thewild-type cells, while the CDKA;1 protein levels remained con-stant in both lines (Figure 7B). Although cells entered mitosismore rapidly, progression through mitosis was transiently ar-rested after metaphase-to-anaphase transition, as indicated bya slower decay in M/A index than that in the wild type (Figure7A). These observations indicate that KRP6 overexpressionleads to a faster entry into mitosis, but delays mitosis pro-gression and exit. Similar observations of a shift in cell cycleprogression, accompanied by a faster increase of CDKB1;1protein level, were observed in a second cell culture over-expressing KRP6, obtained independently from the first culture(Supplemental Figure 3).

KRP6 Overexpression Leads to Increased Mitotic Activity inGall Tissues, Strongly Interfering with NematodeReproduction

To evaluate the effects of ectopic KRP6 expression on M.incognita–induced gall ontogeny, independent KRP6OE Arabi-dopsis lines were generated (Supplemental Figure 4), showinga serrated leaf phenotype and the expected nuclear localizationof the KRP6 protein. For RKN infection, one out of the 10KRP6OE generated lines was chosen to perform a detailed mi-croscopic analysis during different feeding site developmentalstages (7, 14, and 40 DAI). Remarkably, overexpression of KRP6resulted in more nuclei in giant cells (Supplemental Figure 5A),as well as proliferation of the neighboring cells (Figures 8A to 8Ccompared with wild-type galls in Figures 8D to 8F). Therefore,ectopic KRP6 expression promotes mitotic activity in galls.Furthermore, in ;10% of the gall sections, we observed theformation of multiple aggregated micronuclei in giant cells(Supplemental Figures 5C to 5D,D’), whereas in the wild typesuch nuclear organization was not detected (Figures 8D to 8F;Supplemental Figure 5B). Subsequently, 3D confocal projec-tions of serial optical sections of whole KRP6OE galls at differentdevelopmental stages (7, 14, and 40 DAI; Figures 8G to 8I) weregenerated and compared with wild-type galls (Figures 8J to 8L).These observations clearly show the large number of nuclei ineach giant cell and the increased number of neighboring cells,confirming augmented nuclear mitotic activity (Figures 8G and8H). At later stages of gall development, KRP6OE nuclei weremostly clustered in all giant cells (Figure 8I). Electron microscopyanalysis of gall sections confirmed the increased nuclei numberin giant cells of the KRP6OE line (Supplemental Figure 6). Fur-thermore, nuclei in giant cells of the KRP6OE appeared morelobed compared with the wild type (Supplemental Figures 6Dand 6B).Statistical analysis revealed that the giant cells in the KRP6OE

line were significantly smaller than those of the wild-type giantcells, at each time point after nematode infection (Figure 9A).Simultaneously, nuclear surface measurements in giant cells ofthe KRP6OE line revealed that nuclei were smaller than in the

Figure 2. GFP-KRP6 Protein Accumulation Is Regulated in Giant Cellsduring M. incognita–Induced Gall Development.

In vivo localization shows that GFP-KRP6 (green) is present in nuclei ofgiant cells (arrows in [A]) and neighboring cells 2 DAI, decreases in giantcells at 21 DAI (arrows in [B]), and is nearly absent in giant cells at 40 DAI(arrows in [C]). Asterisks, giant cell; n, nematode. Bars = 20 mm.

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wild type (Figure 9B). Due to the convoluted shape and clus-tering of nuclei in older galls (40 DAI; Figures 8I and 8L),measurements were not performed at this stage. The nuclearsize in wild-type giant cells gradually increased, whereas nucleiin giant cells of the KRP6OE line remained small (Figure 9B).

Ultimately, long-term infection tests showed that ectopic ex-pression of KRP6 affected nematode development and re-production (Figures 9C and 9D). Although there were not moregalls in the KRP6OE line compared with the wild type, nema-todes clearly showed a delay in development (Figure 9D). Con-sequently, a significant decrease in reproduction was observed,as seen by the strong decrease in the number of egg masses(Figure 9C).

Ectopic KRP6 Expression Affects Gall Ploidy Levels

To evaluate the effect of KRP6 overexpression on the nuclearploidy levels, flow cytometric measurements were performed onnoninfected roots (Supplemental Figure 7), gall-less roots (Fig-ure 9E), and on galls (Figure 9F) of the wild type and KRP6OE.Ploidy levels in gall-less roots in wild-type plants ranged from 2Cto 16C, whereas few nuclei above 8C were detected in com-parable root samples of the KRP6OE line (Figure 9E). Ploidylevels in wild-type galls ranged from 2C to up to 64C (Figure 9F)and more than 50% of the nuclei showed 2C DNA content, most

likely due to the large number of cells neighboring the giant-feeding cells. Ploidy levels of nuclei in wild-type galls above 16Cmost likely represent nuclei derived from giant cells. In gallsoverexpressing KRP6, the number of nuclei with a 16C and 32Cploidy level was strongly reduced, and no nuclei with a 64C DNAcontent were detected (Figure 9F). These data demonstrate thatthe ploidy levels are decreased in the KRP6OE line.

KRP6 Is Required for Normal Gall Development

To analyze the need for KRP6 expression during gall de-velopment, we analyzed gall morphology in two available T-DNAinsertion lines (krp6KO). The first has an insertion in the third exon(SAIL_54B_B03) and the second a T-DNA insertion just beforethe start codon (ATG) (SALK_142997) (Supplemental Figure 8).Both lines were devoid of KRP6 transcripts (SupplementalFigure 8C). Nematodes were able to induce galls and reproducein krp6KO roots (Supplemental Figures 8D and 8E). However,clear cellular abnormalities were observed during gall de-velopment (7, 14, and 21 DAI) as observed in both mutant linesby the increased cell wall stub frequency in giant cells comparedwith the wild type (Figure 10; Supplemental Figures 8G to 8I).Likewise, mature giant cells in krp6KO lines were significantlysmaller than those in wild-type giant cells (Figure 10; SupplementalFigure 8F) and contained fewer and more dispersed nuclei, rather

Figure 3. Localization of GFP-KRP6 in Arabidopsis Cell Suspensions during Cell Cycle Progression.

Single-plane time lapse (interval 10 min) of a mitotic Arabidopsis suspension cell expressing both RFP-TUA2 and GFP-KRP6. GFP-KRP6 localizeswithin the nucleus during preprophase (A), when the RFP-TUA2–labeled preprophase band is visible. The green nuclear GFP-KRP6 signal is dispersedin the cytoplasm during late prophase (B), early and late metaphase ([C] and [D]), and anaphase (E). During telophase, when the phragmoplast forms,until cytokinesis, the green GFP-KRP6 signal reaccumulates into the separated daughter nuclei ([F] to [J]). Bar = 10 mm.

KRP6 Is Involved in Giant Cell Multinucleation 5 of 15

than clustered nuclei as in mature wild-type giant cells. Thesedata demonstrate that KRP6 function is important for giant celldevelopment.

DISCUSSION

Strong KRP6 Expression Is Associated with the MitoticState in Galls

Targeting cellular mechanisms through modulation of host cellcycle components is a common feature adopted by severalplant pathogens (Doonan and Sablowski, 2010; Wildermuth,2010). The RKN Meloidogyne spp induces the formation ofmultinucleated giant cells through activation of host cell cyclemachinery and inhibition of cytokinesis (de Almeida Engler et al.,1999, 2012). Here, we show that KRP6 promoter activity andhigh transcript levels are present mainly at early stages of gallformation (up to 14 DAI) associated with their mitotic activityphase. Consistent with this, GFP-KRP6 protein accumulates innuclei of giant cells during this stage of high mitotic activity.GFP-KRP6 dynamics studies revealed that once the mitoticphase ended (21 to 40 DAI), GFP-KRP6 fluorescence becameweak or below the detection levels within the giant cell nuclei.KRP6 protein levels remained high in cells neighboring giant-feeding cells until later stages (21 DAI) of gall development,possibly associated with their proliferative status during gallexpansion. By contrast, localization and dynamics studies ofGFP-KRP2 in galls illustrated that at an early phase of gall de-velopment, protein levels were low in giant cell nuclei (Vieiraet al., 2013b), most likely allowing ongoing mitosis of giant andneighboring cells. This mitotic phase would rely on the presenceof proteins like KRP6 to stimulate mitosis and possibly inhibitcytokinesis. Once the mitotic activity phase ceased, nucleusexpansion takes place in giant cells and KRP2 fluorescenceincreased, while the KRP6 signal faded. This new gall statuspossibly reflects a shift from the mitotic phase to the endocyclephase. We observed that this transition involves giant cell nucleienlargement and slowing of neighboring cell division. Althoughpromoter activity of KRP2 and KRP6 is high in galls, the

observed differences in protein levels suggest that nematodesare able to regulate their expression during parasitism. Addi-tionally, it is possible that KRP2 and KRP6 levels might affect thecell cycle in a different way in giant cells.

KRP6 Overexpression Leads to Acceleration of G1-to-S andG2-to-M Transitions

The KRP gene family has been associated with inhibition of celldivision and endoreduplication (Verkest et al., 2005b; Wanget al., 2007). Apparently in contrast to its supposed role as cellcycle inhibitor, KRP6 overexpression led to accelerated G1-to-Sand G2-to-M transitions in cell cultures. Previously, KRP1 hasbeen found to mediate the transport of CDKA;1 into plant nuclei(Zhou et al., 2006), reminiscent of the situation in mammals,where Cip/Kip proteins have been suggested as assembly andnuclear targeting factors of D-type cyclin complexes to the nu-cleus (LaBaer et al., 1997; Cheng et al., 1999). In addition,p21Cip1 promotes nuclear accumulation of CYCD1 by binding toits phosphorylated form to prevent its nuclear export (Alt et al.,2002). Analogously, the accelerated G1-S and G2-M transitionin KRP6-overexpressing cultures may be explained if KRP6functions through activation of CDK/CYCD complexes by pro-moting their assembly or stability, or their targeting to the nu-cleus, resulting in more efficient phosphorylation of targetproteins. In accordance with a role as a nuclear targeting factor,KRP6 specifically localizes in the nucleus and might facilitate thetransport of CYCDs into the nuclear compartment. There isevidence that increased CYCD/CDKA;1 activity may indirectlytrigger G1-S phase transition and mitosis. D-type cyclins wereshown to accelerate the cell cycle in cultured tobacco cellsthrough the enhancement of G1/S entry and progressionthrough S and G2 phases (Koroleva et al., 2004). A tripleCYCD3KO mutant displays decreased mitosis and increasedendoreduplication in leaves (Dewitte et al., 2007), whereasoverexpression of CYCD3;1 in developing trichomes leads todivision of the normally unicellular trichomes (Schnittger et al.,2002).

KRP6 Expression in Giant Cells Is Needed forMultinucleation and Inhibition of Cytokinesis

Together, our analyses of cell cultures and gall sections illus-trated that the increased CDK activity mentioned above mightbe linked with the occurrence of multinucleate cells. Also, ec-topic KRP6 expression in nematode feeding sites triggered anincrease of mitotic activity. Cells overexpressing KRP6 enteredmitosis earlier than normal cells, although progression throughmitosis was slowed down. These effects were revealed by theincreased number of nuclei in Arabidopsis suspension cellsas well as in giant cells and by the abnormal proliferation ofneighboring cells in galls. As a result, galls within the KRP6OE

line were bigger than in KRP1-, KRP2-, or KRP4-overexpressinglines, which exhibited a reduced giant cell size and neighboringcell number severely affecting gall size (Vieira et al., 2012,2013b). This differential gall morphology between the previouslyanalyzed KRPOE lines and KRP6OE suggest a distinct function ofthe latter during feeding site development.

Figure 4. Multinucleate Cells Result from the Overexpression of GFP-KRP6 in Arabidopsis Suspension Cells.

Single-plane confocal section (A) and confocal Z-projection ([B]; eightimages, 18.68 mm) of interphase cells expressing GFP-KRP6 and RFP-TUA2 containing multiple nuclei of variable sizes that accumulate GFP-KRP6. Bars = 10 mm.

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High mitotic activity in giant cells within KRP6OE roots wasalso prolonged compared with wild-type feeding cells, possiblyfacilitating their multinucleate status. The observation of accel-erated G1-S and G2-M transitions in cells with ectopic KRP6expression as well as the increased CDKB1;1 protein levelsprovide further evidence that mitotic activity is enhanced in plantcells upon KRP6 overexpression. In addition, ectopic KRP6expression led to the formation of micronuclei of unequal sizesin cultured cells as well as in nematode-induced giant cells. Theaugmented CDK activity during mitosis could lead to an accel-erated nuclear division followed by inhibited cell division as seenby the later decelerated mitotic activity. This effect might be trueas well for giant cells, given the high KRP6 expression.

Metaphase-to-anaphase transition requires the removal ofcohesins that glue chromosomes together, a process thatinvolves the activation of Aurora kinases. Recently, treatment oftobacco (Nicotiana tabacum) Bright Yellow 2 with the Aurorakinase inhibitor hesperadin has been found to induce the

formation of micronuclei because of lagging chromosomesduring the metaphase-to-anaphase transition (Kurihara et al.,2006). Multinucleate cells with two or more nuclei of unequalsize, as observed here in KRP6OE cultured cells, and in nematode-infected root cells, has been similarly reported to occur upon theexpression of indestructible CYCB1;1 in tobacco Bright Yellow2 cells (Criqui et al., 2001; Weingartner et al., 2004). Destructionof both cohesins and CYCBs is essential to proceed into anaphaseand is controlled through the activation of the anaphase-promotingcomplex/cyclosome (APC/C) at the spindle checkpoint (Peters,2006). The resemblance of the phenotype of the KRP6 over-expression lines to that of hesperadin-treated and CYCB1;1-stabilized cells strongly suggests a link between KRP6 andAPC/C activity. APC/C activity is at least in part regulated byCDK phosphorylation of several of its subunits (Harper et al.,2002; Peters, 2006). Therefore, increased KRP6 levels mightinterfere with CDK-dependent APC/C activation, delaying mi-tosis progression and inhibiting cytokinesis.

Figure 5. KRP6 Overexpression Interferes with Proper Daughter Nuclei Formation.

Confocal Z-stack projections (14 images, 15.98 mm) of the nuclear material labeled with GFP-KRP6 (green signal) together with microtubule labelingwith TUA2-RFP (red signal). The yellow signal is the overlap of green and red. Overexpression of GFP-KRP6 causes the formation of abnormal cellularphenotypes with cells containing multi- or micronuclei of unequal sizes. Time-lapse recording of three cells with one cell containing two nuclei (t = 0) (A).Microtubule organization of spindles and phragmoplasts appears normal as indicated with asterisks ([A] to [D]), while the nuclear material dividesunequally and several micronuclei become visible after reformation of the nuclear membranes ([B] to [D]). Some of the micronuclei fuse together to formone large nucleus, as indicated by arrowheads ([D] to [G], [K], and [L]). Cells subsequently undergoing mitosis are indicated with asterisks ([G] to [J]).Bar = 10 mm.

KRP6 Is Involved in Giant Cell Multinucleation 7 of 15

The assessment of KRP6 loss-of-function lines showed that,although giant cells were smaller, nematode reproduction wassimilar to the wild type. The induction of more cell wall stubsbetween nuclei as observed in a fraction of giant cells of thekrp6KO lines suggests a stimulation of cytokinesis. Nematodesecretions combined with the lack of KRP6 protein might pre-vent formation of complete cell walls between nuclei in giantcells. Combining our observations of the effects of KRP6 over-expression and loss of function, our data indicate that KRP6expression on the one hand might stimulate nuclear division andon the other hand inhibits cytokinesis, both needed to stimulatemultinuclear giant feeding cell formation in galls.

Ectopic KRP6 Expression Disrupts Gall and NematodeDevelopment and Affects Ploidy in Nematode Feeding Sites

It is likely that a balance of the mitotic cycle followed by anincrease in ploidy during nematode infection ultimately supportgiant cell fate and maintenance, and consequently nematodedevelopment and reproduction (de Almeida Engler et al., 2012).A fluctuation in expression of KRP genes is recognized to affectthe endoreduplication process in plant cells (De Veylder et al.,2001; Zhou et al., 2003; Roeder et al., 2010). A decline of nuclearsize was detected in giant cells overexpressing KRP6. Bycomparing the DNA content of galls of the KRP6OE line (2C to32C) with the wild type (2C to 64C), a noteworthy reduction ofgiant cell nuclei ploidy levels was observed in gall tissue over-expressing KRP6. It is tempting to suggest that lower ploidydramatically influenced giant cell size. Indeed, giant cells, thesole food source for the feeding nematode, were undersized inthe KRP6OE line compared with the wild type. Nevertheless, theglobal gall size in the KRP6OE line was similar to the wild typedue to the elevated number of neighboring cells, which ulti-mately determined gall size and shape. The extension of thismitotic status most likely delayed the onset of the endocycle in

the small-sized giant cells of the KRP6OE line, thus also affectingthe completion of RKN life cycle. These data confirm that themultinucleate state in giant cells is not sufficient to drive giantcell expansion and nematode reproduction (de Almeida Engleret al., 1999, 2012). A phase governed by the endocycle seems tobe essential for giant cell expansion (de Almeida Engler et al.,2012; Vieira et al., 2013a).

Concluding Remarks

This work points to an unexpected role for KRP6 during mitosis,suggesting that not all KRPs regulate the cell cycle in the samemanner. Whereas KRPs such as KRP1 and KRP2 are associatedwith inhibition of mitosis and consequently cell division, here weillustrate that upon its overexpression, KRP6 can accelerateentry into mitosis, delaying mitosis progression and exit. Theseobservations are supported by plant cells presenting an ampli-fied cell cycle, like giant-feeding cells induced by plant-parasiticnematodes. We found that KRP6 expression parallels the in-duction of a mitotic state in plant and giant cells prompting their

Figure 6. Micronuclei Form upon Overexpression of GFP-KRP6 inArabidopsis Suspension Cells.

(A) Confocal Z-projection (18 images, 21.15 mm) of a telophase cell. Thenuclear envelope reforms around the separated daughter nuclei andaround lagging chromosomes (upper arrows). Lagging chromosomeslikely result in the formation of the micronuclei (lower arrows).(B) Single-plane confocal section of a telophase cell overexpressingGFP-KRP6 and TUA2-RFP. A ring-like phragmoplast indicated by theRFP-labeled microtubule separates reforming micronuclei at either sideof the forming cell plate.Bars = 10 mm.

Figure 7. Effect of Ectopic Expression of KRP6 on Cell Cycle Pro-gression of Cultured Arabidopsis Cells.

(A) BrdU labeling index (dashed lines) and M/A index (solid lines) in-dicating the percentage of cells that are in S phase and in metaphaseand anaphase after aphidicolin synchronization of PSB-D (blue lines) and35S:GFP-KRP6 (green lines) cell cultures.(B) Total protein extracts of PSB-D and transgenic Arabidopsis culturesoverexpressing GFP-KRP6 after release from the aphidicolin block, an-alyzed hourly by immunoblot analysis with antisera against CDKA;1 andCDKB1;1.

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Figure 8. Ectopic KRP6 Expression Increases Mitotic Activity and Affects the Size of M. incognita–Induced Giant Cells.

(A) to (F) Histological analysis of wild-type galls compared with KRP6-overexpressing lines at different stages after nematode infection (7, 14, 21, and40 DAI). Bright-field images of longitudinal gall sections stained with toluidine blue. Increased mitotic activity is observed in nuclei of giant cells, as wellas in neighboring cells ([A] to [C]) compared with the wild-type gall ([D] to [F]). Giant cell size is also reduced in the KRP6OE line.(G) to (I) KRP6OE galls at 7, 14, and 40 DAI. 3D confocal projections of serial optical sections of cleared whole-mount gall samples. Increased mitoticactivity is observed in nuclei of giant cells as well in neighboring cells.(J) to (L) Wild-type galls at 7, 14, and 40 DAI. White circles delimit giant cells. Asterisks, giant cell; n, nematode; NCs, neighboring cells. Bars = 50 mm in(A) to (F) and 20 mm in (G) to (L).

KRP6 Is Involved in Giant Cell Multinucleation 9 of 15

Figure 9. Ectopic KRP6 Expression Drastically Affects the Size and Ploidy Levels of M. incognita–Induced Giant Cells.

(A) Giant cell surface (mm2) of wild-type and KRP6OE galls was measured at different stages after nematode infection (7, 14, 21, and 40 DAI). Asterisksdenote statistical differences between the genotypes at that time point after nematode infection (P < 0.0001, ANOVA with Tukey-Kramer test).(B) Measurements (mm2) of giant cell nuclei in wild-type and KRP6OE at 7, 14, and 21 DAI. Asterisks denote statistical differences between thegenotypes at that time point after nematode infection (P < 0.0001, ANOVA with Tukey-Kramer test).(C) Infection tests byM. incognita show that the number of galls and egg masses decreased compared with the wild type. Asterisks indicate values thatwere significantly different from the wild type at P < 0.05 (Student’s t test).(D) Acid fuchsin staining show that galls in the KRP6OE line are larger even when giant cells are smaller compared with the wild type. In most galls,females have not reached maturity, consequently did not produce eggs. G, gall; n, nematode. Bars = 50 mm.

10 of 15 The Plant Cell

multinucleate and acytokinetic state. It is appealing to hypoth-esize that the irregular-sized nuclei and nuclear fusion observedin cell cultures with ectopic KRP6 expression might help toexplain the multiple nuclear sizes observed in giant cells in wild-type plants. Functional analysis shows that the lack of KRP6 innematode-induced giant cells leads to an increase in cell wallstubs, suggesting a stimulation of cytokinesis. Conversely, ec-topic KRP6 expression induced mitosis and inhibited cytokine-sis, as well as endoreduplication in plant cells, and led to smallmultinucleated giant cells. The endogenous high KRP6 ex-pression in wild-type galls might therefore stimulate mitosis ingiant cells and induce ectopic neighboring cell division. To-gether, our data support the view that KRP6 might play a role inregulating feeding cell growth, multinucleation, and the acyto-kinetic state of these giant-feeding cells. Thus, plant-parasiticnematodes might have evolved the competence to exploit plantcell cycle genes like KRP6 to the benefit of gall establishment.

METHODS

Constructs, Plant and Cell Suspension Growth, and Transformation

Arabidopsis thaliana, genotypeColumbia-0 (Col-0), was usedas thewild-typecontrol. For construction of the KRP6pro:GUS line, the intergenic region(up to maximum 2 kb) was amplified from Arabidopsis genomic DNA. Thecorresponding PCR fragment was cloned into pDONR207 entry vector (In-vitrogen, Life Technologies) by BP recombination cloning and subsequentlytransferred into the pKGWFS7 destination vector (Karimi et al., 2002) by LRcloning, resulting in a transcriptional fusion between the KRP6 promoterand the EGFP-GUS fusion gene. Single T-DNA insertion mutant lines ofKRP6 (SAIL_54B_B03 and SALK_142997) were obtained from the ABRC.

Arabidopsis cell cultures (2 d old) were stably transformed by Agro-bacterium tumefaciens cocultivation according to Van Leene et al. (2007).The 35S:GFP-KRP6-containing plant transformation vector pK7WGF2-KRP6 was obtained by Gateway LR reaction (Invitrogen) of pEntryL1L2-KRP6 with the destination vector pK7WGF2 (Karimi et al., 2002).The KRP6-coding region was amplified by PCR and cloned into theGateway pDONR221 vector (Invitrogen), resulting in pEntryL1L2-KRP6. The35S:RFP-TUA2-containing plant transformation vector was constructed asdescribed (VanDammeet al., 2004). Toobtain the double (35S:GFP-KRP6335S:RFP-TUA2) cell suspension, the transgenicGFP-KRP6 cell culture wastransformed with the pK7FWG2-35S:RFP-TUA2 plant transformationvector. PSB-D cell suspensions of Arabidopsis were maintained as de-scribed (Van Leene et al., 2007). Kanamycin-resistant T3 overexpressinglines (35S:GFP-KRP6) were evaluated based on protein localization andplant phenotype. Seeds from Arabidopsis transgenic lines and wild-typeCol-0 were surface-sterilized for 10 min in 5% NaOCl, followed by fourwasheswith 95%ethanol, and dried overnight. Seedswere kept in a growthchamberwith a 16-h-light:8-h-dark photoperiod at 21°C:18°C, respectively.

Histochemical GUS Staining and mRNA in Situ Hybridization Assays

Promoter KRP6pro:GUS activity was monitored at different time pointsafter nematode infection (7, 14, and 21 DAI) as previously described by de

Almeida Engler et al. (1999). To avoid diffusion of the GUS precipitate,galls were fixed in 2% glutaraldehyde ON and were embedded inTechnovit 7100, sectioned (3 µm), and microscopically analyzed by dark-field optics.

Gene-specific sense and antisense probes of KRP6 were generatedas previously described (de Almeida Engler et al., 2009). The in situ hy-bridization procedure was performed essentially as described by de Al-meida Engler et al. (2009). Images were taken with a digital Axiocam(Zeiss) with standard dark-field optics.

DNA and RNA Extraction and Analyses of KRP6 Knockout andOverexpressing Lines

Genomic DNA extraction and PCR for amplification of T-DNA insertions inkrp6KO mutant lines was done using the Extract-N-Amp Plant PCR kit(Sigma-Aldrich) and primers listed in Supplemental Table 1. Total RNA ofkrp6KO and KRP6OE lines was extracted from 7-d-old whole seedlings,using the RNeasy Plant Mini Kit (Qiagen), according to the manufacturer’sinstructions. The RNA was treated with RQ1 RNase-free DNase (Prom-ega) before reverse transcription. cDNA was prepared using 1 mg treatedRNA and added to RT reactions using a SuperScript III first-strandsynthesis system for RT-PCR (Invitrogen) using a mix of oligo(dT) and

Figure 9. (continued).

(E) and (F) Nuclear flow cytometry analysis of nuclei in the wild type and KRP6OE.(E) Percentage of nuclei of the indicated ploidy in gall-less root segments.(F) Percentage of nuclei of the indicated ploidy in mature galls (40 DAI). In KRP6OE, galls displayed a significant reduction in nuclei with high ploidy levels(>16C) compared with nuclei of wild-type galls.

Figure 10. Histological Analysis of the krp6KO Line of M. incognita–Induced Galls in Arabidopsis Roots.

Bright-field images of longitudinal sections of galls stained with toluidineblue in krp6KO (SAIL_548_B03) and in wild-type lines at different stagesafter nematode infection (7, 14, and 21 DAI).(A) to (C) Galls in the krp6KO mutant line. Note the presence of cell wallstubs (red arrow) in (B) and (C).(D) to (F) Wild-type galls. Asterisks, giant cell; n, nematode. Bars =50 mm.

KRP6 Is Involved in Giant Cell Multinucleation 11 of 15

random hexamer primers. PCR amplifications were performed withspecific primer pairs (Supplemental Table 1).

Gall Morphological Analyses

Transgenic seedlings (T3) KRP6OE were used for plant-RKN studies.Transgenic and wild-type Arabidopsis Col-0 seeds were surface sterilizedfor 10 min in 5% NaOCl, followed by four washes with 95% ethanol anddried overnight. Seeds were placed into sterile Petri dishes on 1%Murashige and Skoog germination medium (Duchefa) containing 1%sucrose and 0.8% plant cell culture–tested agar (Sigma-Aldrich), sup-plementedwith the appropriate antibiotics. Plantlets were grown verticallyto allow roots to grow at the surface, with a 16-h-light/8-h-dark photo-period at 21°C/18°C respectively. Roots were infected and collected at 7,14, 21, and 40 DAI and fixed in 2% glutaraldehyde in 50 mMPIPES buffer,pH 6.9, and then dehydrated and embedded in Technovit 7100 (HeraeusKulzer), as described by the manufacturer. Embedded roots and galltissues were sectioned (5 µm) and stained in 0.05% toluidine blue andmounted in Depex (Sigma-Aldrich). Microscopy observations were per-formed using bright-field optics, and images were performed with a digitalcamera (Axiocam; Zeiss).

In Vivo Confocal Microscopy Analyses

Arabidopsis cells were applied to a chambered cover glass system (Lab-Tek) and immobilized on a thin layer of 0.8% (w/v) low melting pointagarose (Invitrogen) in MSMO medium. Cells were imaged with a Zeissconfocal fluorescence microscope 100Mwith software package LSM 510version 3.2 (Zeiss), equipped with a 633 water corrected objective (nu-merical aperture of 1.2). Dual GFP and red fluorescent protein (RFP)fluorescence was imaged in a multichannel setting with 488- and 543-nmlaser source for GFP and RFP excitation, respectively. Emission fluo-rescence was captured by sequential frame-scanning mode alternatingGFP fluorescence via a 500- to 530-nm band-pass emission filter and RFPvia a 560-nm long-pass filter. Images were recorded using 33 digitalzoom. Image analysis and Z-stack projection were done with the LSMsoftware (Zeiss). Noninfected KRP6OE T3 transformants selected onkanamycin were transferred to half-strength Murashige and Skoog platesafter selection. Roots were imaged 7 d after transfer to half-strengthMurashige and Skoog plates using an inverted confocal microscope(model LSM510 META; Zeiss).

Observation of the nuclei in the nematode feeding sites was performedin nematode infected KRP6OE line and Arabidopsis seedlings harboringfree GFP (35S:GFP) lines. Galls at various time points after infection (2 to40 DAI) were dissected from roots and embedded in 5% agar. Fresh thicksections of 50 to 100 µm (2 to 14 DAI) or 150 to 200 µm (14 to 40 DAI) wereprepared with a Microm HM650V Vibratome (Walldorf). Whole roots andfresh slices were immediately observed using an inverted confocal mi-croscope (model LSM510 META; Zeiss). GFP was excited with a 488-nmargon laser, and fluorescence was captured in lambdamode using 499- to550-nm bandwidth.

Protein Extraction, SDS-PAGE, and Immunoblotting

Cell suspension cultures were harvested by filtration on a sintered glassfilter, used immediately or snap-frozen in liquid nitrogen, and stored at270°C. Total protein extracts were prepared as described by Van Leeneet al. (2007). Equal amounts of total protein were separated on 12%SDS-PAGE gels, blotted onto Immobilon-P membranes (Millipore), andblocked in 3% skim milk in Tris-buffered saline and 0.1% Triton X-100.For immunodetection, polyclonal anti-CDKA;1 and CDKB1;1 rabbitantisera were used as primary antibodies, followed by anti-rabbit IgGcoupled to horseradish peroxidase (GE-Healthcare) as secondary

antibodies. Protein gel blots were developed by chemiluminescentdetection (Perkin-Elmer).

Synchronization of Arabidopsis Cell Cultures, BrdU Labeling, andMitotic Index Determination

For aphidicolin synchronization, 800 mL cell suspension culture wasgrown in a 2-liter Erlenmeyer flask by adding 134 mL saturated culture to670 mL MSMO medium (4.43 g/liter MSMO [Sigma-Aldrich], 30 g/litersucrose, 0.5 mg/liter a-naphthaleneacetic acid, and 0.05 mg/liter kinetin,pH 5.7, adjusted with 1 M KOH) and 5 mg mL21 aphidicolin to a finalconcentration of 6.66 mg mL21 for 21.5 h. After removal of aphidicolin bywashing cells five times with MSMOmedium, the cells were resuspendedin fresh MSMO medium. Samples were harvested hourly and cell cycleprogression was analyzed by microscopy.

For determination of the M/A index, cell samples were taken hourly,fixed 3:1 (v/v) ethanol:acetic acid mixture and washed with 70% ethanol(v/v). DNA was stained with 1 mg mL21 49,6-diamidino-2-phenylindole(Sigma-Aldrich) and observed by epifluorescence microscopy. For de-termination of BrdU incorporation, cells were immediately labeled with theBrdU-labeling reagent and incubated for 1 h at 25°C. Afterwards, cellswere fixed in 3.7% formaldehyde in PMEG buffer [50mMpiperazine-N,N’-bis(ethanesulfonic acid), 2 mMMgSO4, 5 mM EGTA, and 2% glycerol, pH6.8] and processed for immunostaining with monoclonal BrdU antibody(GE-Healthcare). For each time point, at least 400 cells were scored formitotic figures and BrdU labeling.

Giant Cell and Nucleus Surface Measurements

Two to three largest giant cells per gall were selected and analyzed atdifferent time points after nematode infection (7, 14, 21, and 40 DAI), andthe surface was measured using the AxioVisionLE (Zeiss) software. Aminimum of 60 giant cells was measured, for root sections of each timepoint studied. The nucleus surface was measured at 7, 14, and 21 DAIfrom a minimum of 80 nuclei of KRP6OE and wild-type lines, using theAxioVisionLE software.

Whole-Mount Analysis of Cleared Nuclei Stained byPropidium Iodide

Nuclear analysis of cleared feeding sites was performed as described(Vieira et al., 2012). Cleared samples were analyzed with a Zeiss LSM 510META confocal microscope. Propidium iodide excitation was performedwith the 543-nm line of a HeNe laser. Stacks were generated from ;50images of 1-mm optical slice thickness and represented as maximumbrightness projections.

Electron Microscopy of Galls

Root galls of Arabidopsis Col-0 and KRP6OE lines were dissected at 14and 21 DAI and fixed in a mixture of 1.5% glutaraldehyde and 3%paraformaldehyde in 10 mMPBS containing 150 mMNaCl (pH 7.2) for 3 hat room temperature. After several washes in PBS buffer, fixed galls wereincubated in 0.5M NH4Cl for 1 h, dehydrated in graded ethanol series,embedded in acrylic resin LR White (Sigma-Aldrich), and polymerizedovernight at 60°C. Ultrathin sections were collected on parlodion-coatednickel grids, treated with 0.1 M HCl for 10 min, and washed at least twicewith double distilled water. Samples were observed under a Philips 400Telectron microscope.

Flow Cytometry Analyses

Plant roots were chopped with a razor blade in 400 mL 45 mM MgCl2,30 mM sodium citrate, 20 mM MOPS, pH 7.0, and 0.1% Triton X-100

12 of 15 The Plant Cell

(Galbraith et al., 1991). The supernatant was filtered over 30-mm mesh,and 1 mL 49,6-diamidino-2-phenylindole from a stock of 1 mg/mL wasadded. The nuclei were analyzed with the LSRII Fortessa (BDBiosciences)flow cytometer and the BD FACSDiva software (BD Biosciences). Foruninfected roots, data were collected from ;2000 nuclei per run. Thefractions of nuclei with ploidy levels from 2C to 16C were expressed aspercentage of the total number of nuclei measured. Ploidy levels of 30galls (40 DAI) were pooled for each independent experiment. Two rep-licates and two biological repetitions were performed for each line, anddata for ;40,000 nuclei were collected per run. The mean values ofrepetitions of independent experiments were calculated, and the fractionof nuclei at ploidy levels from 2C to 64Cwas expressed as a percentage ofthe total number of nuclei measured.

Nematode Infection Tests

Three-week-old Arabidopsis wild-type and KRP6OE seedlings were in-oculated on Murashige and Skoog medium on Petri dishes, containing fiveplants per plate and six replica plates per line. Seedlings were grown ver-tically with a 16-h-light/8-h-dark photoperiod at 21°C/18°C, respectively.One root tip of each plant was inoculated with ;100 surface-sterilized,freshly hatched second stage juveniles (J2s) of Meloidogyne incognita aspreviously described (de Almeida Engler et al., 1999). Six to seven weeksafter infection, the number of galls and egg masses were counted on eachplate and comparedwith the control plants. Data shown representsmeans6 SD

from two independent biological experiments, in which a minimum of 60seedlings of each line were evaluated for nematode infection.

Acid Fuchsin

The acid fuchsin staining was performed for nematode visualization withinthe roots. The infected roots (40 DAI) were fixed and stained for 5 h ina solution of equal parts of 95% ethanol and glacial acetic acid, containing17.5 mg/L acid fuchsin. Root tissue was destained by soaking in a solutionof chloral hydrate (0.2 g/mL in water) for 16 h. After rinsing the roots severaltimes with tap water, roots containing nematodes were stored in acidifiedglycerol (6 five drops of 1.0 M HCl were added to 50 mL glycerol).

Accession Numbers

Sequence data from this article can be found in the Arabidopsis GenomeInitiative or GenBank/EMBL databases under accession number At3g19150(KRP6).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. Free-GFP Protein Expression during GallDevelopment.

Supplemental Figure 2. GFP-KRP6 Localization in Arabidopsis duringCell Division.

Supplemental Figure 3. Effect of Ectopic Expression of KRP6 on CellCycle Progression in an Arabidopsis Cell Culture IndependentlyObtained from the Transgenic Culture Used in Figure 7.

Supplemental Figure 4. GFP-KRP6 Overexpression Causes LeafSerration in Seedlings.

Supplemental Figure 5. Micronuclei Form within 10% of KRP6OE

Giant Cells.

Supplemental Figure 6. Electron Micrographs of Giant Cells of Gallsin Arabidopsis Wild-Type Plants Compared with KRP6OE Line.

Supplemental Figure 7. Nuclear Flow Cytometry Analyses of Roots inWild-Type and KRP6OE.

Supplemental Figure 8. Characterization of krp6 Loss-of-FunctionLines.

Supplemental Table 1. List of Primers.

Supplemental Movie 1. A Polynucleate KRP6OE Arabidopsis Cell.

Supplemental Movie 2. Formation of Polynucleate KRP6OE Arabi-dopsis Cells with Nuclei of Unequal Size.

Supplemental Movie 3. Lagging Chromosomes during Anaphase inKRP6OE Arabidopsis Cells.

ACKNOWLEDGMENTS

We thank Danny Geelen for constructs, Sylvie Coutuer, Evelien Mylle,and Tom Beeckman for help with microscopy, and Sophie Pagnotta forhelp with electron microscopy. This work was supported by grants fromthe Institute for the Promotion of Innovation by Science and Technologyin Flanders (IWT) (“Generisch Basisonderzoek aan de Universiteiten” no.20193). A.D.C. and A.V. are indebted to the IWT for a predoctoralfellowship. D.V.D. is a postdoctoral fellow of the Research Foundation-Flanders. L.D.V. acknowledges the Research Foundation Flanders (FWO,G.022.10N) for financial support. P.V. was supported by a doctoralscholarship from Fundação para a Ciência e Tecnologia, from Portugal(SFHR\BD\41339\2007). The work was supported by CAPES/COFECUBProject Sv 683/10. This research was funded by the InteruniversityAttraction Poles Programme (IUAP P7/29 “MARS”) initiated by theBelgian Science Policy Office.

AUTHOR CONTRIBUTIONS

P.V., J.A.E., A.D.C., H.S., J.V.L., D.V.D., A.V., L.D.V., and G.D.J.conceived and designed experiments. P.V., G.E., J.A.E., A.D.C., H.S.,J.V.L., E.V.D.S., J.D.A.S.J., G.V.I., D.E., G.P., D.V.D., and A.V. performedthe experiments. P.V., G.E., J.A.E., A.D.C., H.S., J.V.L., D.V.D., A.V., D.I.,L.D.V., and G.D.J. analyzed the data. P.A., G.J., N.G., and L.V.contributed reagents/materials/analysis tools. P.V., J.A.E., G.J., L.V.,A.D.C., H.S., D.I., L.D.V., and G.D.J. wrote the article. J.A.E. and G.D.J.supervised the research.

Received April 9, 2014; revised April 9, 2014; accepted May 28, 2014;published June 24, 2014.

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KRP6 Is Involved in Giant Cell Multinucleation 15 of 15

DOI 10.1105/tpc.114.126425; originally published online June 24, 2014;Plant Cell

Geert De Jaeger and Janice de Almeida EnglerAntonino de Souza, Júnior, Nathalie Glab, Pierre Abad, Gilbert Engler, Dirk Inzé, Lieven De Veylder,

Isterdael, Dominique Eeckhout, Geert Persiau, Daniël Van Damme, Aurine Verkest, José Dijair Paulo Vieira, Annelies De Clercq, Hilde Stals, Jelle Van Leene, Eveline Van De Slijke, Gert Van

ArabidopsisCells Induced by Plant-Parasitic Nematodes in The Cyclin-Dependent Kinase Inhibitor KRP6 Induces Mitosis and Impairs Cytokinesis in Giant

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