The Arabidopsis Rho of Plants GTPase AtROP6 Functions in ......The Arabidopsis Rho of Plants GTPase...

The Arabidopsis Rho of Plants GTPase AtROP6Functions in Developmental andPathogen Response Pathways1[C][W][OA]

Limor Poraty-Gavra2, Philip Zimmermann, Sabine Haigis, Paweł Bednarek, Ora Hazak,Oksana Rogovoy Stelmakh, Einat Sadot, Paul Schulze-Lefert, Wilhelm Gruissem, and Shaul Yalovsky*

Department of Molecular Biology and Ecology of Plants, Tel Aviv University, Tel Aviv 69978, Israel (L.P.-G.,O.H., S.Y.); Department of Biology, Eidgenössisch Technische Hochschule, Zurich CH–8092, Switzerland (P.Z.,W.G.); Department of Plant Microbe Interactions, Max Planck Institute for Plant Breeding, Cologne D–50829,Germany (S.H., P.B., P.S.-L.); and Department of Ornamental Horticulture, Agricultural ResearchOrganization, Volcani Center, Bet Dagan 50250, Israel (O.R.S., E.S.)

How plants coordinate developmental processes and environmental stress responses is a pressing question. Here, we show thatArabidopsis (Arabidopsis thaliana) Rho of Plants6 (AtROP6) integrates developmental and pathogen response signaling. AtROP6expression is induced by auxin and detected in the root meristem, lateral root initials, and leaf hydathodes. Plants expressing adominant negative AtROP6 (rop6DN) under the regulation of its endogenous promoter are small and have multiple inflorescencestems, twisted leaves, deformed leaf epidermis pavement cells, and differentially organized cytoskeleton. Microarray analyses ofrop6DN plants revealed that major changes in gene expression are associated with constitutive salicylic acid (SA)-mediateddefense responses. In agreement, their free and total SA levels resembled those of wild-type plants inoculated with a virulentpowdery mildew pathogen. The constitutive SA-associated response in rop6DN was suppressed in mutant backgrounds defectivein SA signaling (nonexpresser of PR genes1 [npr1]) or biosynthesis (salicylic acid induction deficient2 [sid2]). However, the rop6DN npr1and rop6DN sid2 double mutants retained the aberrant developmental phenotypes, indicating that the constitutive SA response canbe uncoupled from ROP function(s) in development. rop6DN plants exhibited enhanced preinvasive defense responses to ahost-adapted virulent powdery mildew fungus but were impaired in preinvasive defenses upon inoculation with anonadapted powdery mildew. The host-adapted powdery mildew had a reduced reproductive fitness on rop6DN plants,which was retained in mutant backgrounds defective in SA biosynthesis or signaling. Our findings indicate that both themorphological aberrations and altered sensitivity to powdery mildews of rop6DN plants result from perturbations that are independentfrom the SA-associated response. These perturbations uncouple SA-dependent defense signaling from disease resistanceexecution.

Rho of Plants (ROPs), also known as RACs (forclarity, the ROP nomenclature will be used throughoutthis article), comprise a plant-specific group of Rhofamily small G proteins. Like other members of the Rassuperfamily of small G proteins, ROPs function as

molecular switches, existing in a GTP-bound “on”state and a GDP-bound “off” state. In the GTP-boundstate, ROPs interact with specific effectors that trans-duce downstream signaling or function as scaffolds forinteraction with additional effector molecules (Berkenand Wittinghofer, 2008). Conserved point mutations inthe G1 (P loop) Gly-15 or the G3 (switch II) Gln-64,which abolish GTP hydrolysis, or the G1 Thr-20 orG4 Asp-121 that compromise GDP/GTP exchange, canform either constitutively active or dominant negativemutants, respectively (Feig, 1999; Berken et al., 2005;Berken and Wittinghofer, 2008; Sorek et al., 2010).Primarily based on studies with neomorphic mutants,ROPs have been implicated in the regulation of cyto-skeleton organization and dynamics, vesicle traffick-ing, auxin transport and response, abscisic acid (ABA)response, and response to pathogens (Nibau et al.,2006; Yalovsky et al., 2008; Yang, 2008; Lorek et al.,2010; Wu et al., 2011; and refs. therein).

In Arabidopsis (Arabidopsis thaliana), there are 11ROP proteins (Winge et al., 1997). Assigning specificfunctions to individual members of this family isdifficult, however, because ROPs are functionally

1 This work was supported by the Deutschland Israel Program(grant no. H.3.1 to S.Y and P.S.-L.), the Israel Science Foundation (grantno. 1244/11), the U.S.-Israel Binational Science Foundation (grant no.2009309), the Manna Center for Plant Biology (to S.Y.), the Deut-sche Forschungsgemeinschaftand (DFG SPP1212 to P.S.L.), and theEidgenössisch Technische Hochschule (to W.G.).

2 Present address: Evogene, Inc., Rehovot 76121, Israel.* Corresponding author; e-mail [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:Shaul Yalovsky ([email protected]).

[C] Some figures in this article are displayed in color online but inblack and white in the print edition.

[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a subscrip-

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redundant. A ROP10 loss-of-function mutant wasreported to be ABA hypersensitive (Zheng et al., 2002),displaying enhanced expression of tens of genes inresponse to ABA treatments (Xin et al., 2005). How-ever, in the absence of exogenous ABA, gene ex-pression in the rop10 mutant was similar to that inwild-type plants (Xin et al., 2005). Loss of leaf epider-mis pavement cell polarity was reported for rop4 rop2-RNAi (for RNA interference) double mutant plants (Fuet al., 2005). Mild changes in pavement and hypocotylcell structure and microtubule (MT) organization werereported for a rop6 loss-of-function mutant (Fu et al.,2009).The involvement of ROPs in auxin-regulated de-

velopment has been addressed in several studies (Wuet al., 2011). Ectopic expression of a dominant negativeROP2 (rop2DN) mutant under regulation of the 35Spromoter resulted in a loss of apical dominance anda reduction in the number of lateral roots. In con-trast, ectopic expression of constitutively active ROP2(rop2CA) caused an increase in the number of lateralroots and an enhanced decrease in primary root lengthin response to auxin. Consistent with these findings,the expression of a constitutively active NtRAC1 intobacco (Nicotiana tabacum) protoplasts induced theexpression of auxin-regulated genes in the absence ofauxin and promoted the formation of protein nuclearbodies containing components of the proteasome andCOP9 signalosome (Tao et al., 2002, 2005; Wu et al.,2011). The ROP effector ICR1 (for interactor of consti-tutively active ROP1) regulates polarized secretion andis required for polar auxin transport (Lavy et al., 2007;Bloch et al., 2008; Hazak et al., 2010; Hazak andYalovsky, 2010). In the root, local auxin gradients in-duce the accumulation of ROPs in trichoblasts at thesite of future root hair formation (Fischer et al., 2006).Recently, it was shown that interdigitation of leaf ep-idermis pavement cells depends on Auxin-BindingProtein1 (ABP1)-mediated ROP activation (Xu et al.,2010). Taken together, these data indicate that ROPsare involved in both mediating the auxin responseand facilitating directional auxin transport. It is stillunclear, however, which ROPs function in theseprocesses.ROP function was linked to plant defense responses

in several studies. In rice (Oryza sativa), OsRAC1 isa positive regulator of the hypersensitive response,possibly through interactions with the NADPH oxi-dase RbohB, Required for Mla12 Resistance, and HeatShock Protein90 (Ono et al., 2001; Thao et al., 2007;Wong et al., 2007). Interestingly, other members of therice ROP family, namely RAC4 and RAC5, are neg-ative regulators of resistance to the rice blast patho-gen Magnaporthe grisea (Chen et al., 2010). Similar torice, when expressed in tobacco, dominant negativeOsRAC1 suppressed the hypersensitive response(Moeder et al., 2005). In barley (Hordeum vulgare),several constitutively active ROP/RAC mutants and aMT-associated ROPGAP1 loss-of-function mutant en-hanced susceptibility to the powdery mildew Blumeria

graminis f. sp. hordei (Bgh). The activated ROP-enhancedsusceptibility to Bgh was attributed to disorganizationof the actin cytoskeleton and was shown to depend onMildew Resistance Locus O (MLO; Schultheiss et al.,2002, 2003; Opalski et al., 2005; Hoefle et al., 2011). Inbarley, three ROP proteins, HvRACB, HvRAC1, andHvRAC3, were linked to both development and path-ogen response (Schultheiss et al., 2005; Pathuri et al.,2008; Hoefle et al., 2011).

We have analyzed the function of the ArabidopsisAtROP6 (ROP6) by characterizing its expression pat-tern and its regulation by auxin and the phenotypeof plants that express rop6DN under the regulation ofits endogenous promoter. The utilization of the domi-nant negative mutant overcame functional redundancy,while expression under the regulation of the endoge-nous promoter enabled the analysis of ROP6 functionin a developmental context. Phenotypic and gene ex-pression analyses indicate that ROP6 functions indevelopmental, salicylic acid (SA)-dependent, and SA-independent defense response pathways.

RESULTS

ROP6 Expression Pattern and Subcellular Localization

Previously, we analyzed lipid modifications, mem-brane interaction dynamics, and the function of ROP6in the regulation of cell polarity using the constitu-tively active rop6CA mutant expressed under the reg-ulation of the cauliflower mosaic virus 35S promoter.Because of the functional redundancy of ROPs, therop6 mutant phenotype is inseparable from that ofwild-type plants. To overcome this difficulty and elu-cidate the specific function of ROP6, its promoter wasisolated (Supplemental Fig. S1) and subcloned into theLhG4/pOp transcription transactivation system (Mooreet al., 1998) to drive the expression of GUS, DsRed,GFP-ROP6, GFP-rop6CA, and GFP-rop6DN. To verifythe expression pattern and phenotypes, three inde-pendent transgenic lines were analyzed for each ex-pression construct.

In rosette leaves, ROP6 expression was detected atthe tip and in the hydathodes at the leaf margin (Fig. 1,A–C). In cauline leaves, however, expression wasnot confined to the leaf margins, and leaf epidermispavement cells expressing a constitutively active GFP-rop6CA (rop6CA) were rectangular with few and shal-low lobes (Fig. 1F). No expression could be detected inthe inflorescence meristem and in young flowers (Fig.1D). In mature flowers, strong expression was detectedin stamen filaments, pollen grains, and stigma andweak expression was detected in ovules (Fig. 1E). Inthe primary root, expression was detected in the roottip, was absent from the cell division zone, and in-creased in the stele of the transition zone. In the rootdifferentiation zone, expression increased in lateralroot initials (LRI) and was already detected in lateralroot founder cells in the pericycle (Fig. 1, G–I). The

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expression pattern of ROP6 in the root was furtherconfirmed by whole-mount RNA in situ hybridization.Similar to expression detected with reporter genesfused to the ROP6 promoter, following hybridizationwith an antisense probe, ROP6 RNA was detected inthe stele, LRI, and the tip of the emerging lateral root(Fig. 2).

The expression pattern of ROP6 in rosette leaves andprimary and lateral roots resembled the expressionpattern of the auxin-sensitive synthetic promoter DR5(Sabatini et al., 1999; Aloni et al., 2003; Benková et al.,2003), suggesting that ROP6 expression might be in-duced by auxin. Furthermore, the ROP6 promotercontains a TGTCTC auxin response element. To ex-amine whether ROP6 expression is induced by auxin,5-d-old seedlings were transferred to control or auxin-containing media. The GFP-rop6CA signal was detectedin some pericycle cells as early as 3 h following transfer

to an auxin-containing medium (10 mM naphthalene-acetic acid [NAA]; Fig. 1J). Six hours after transfer toauxin-containing medium, GFP-rop6CA was detectedin many pericycle cells, whereas on control plates, itwas detected only in a few cells (Fig. 1K). GFP-rop6CA

was visible in many dividing pericycle cells 24 h aftertransfer to auxin (Fig. 1L). The results of the auxininduction experiments confirm that ROP6 expressionis quickly induced by auxin during lateral root devel-opment. Furthermore, the identical expression pattern,which was detected when using GUS, DsRed, GFP-ROP6, GFP-rop6CA, or GFP-rop6DN (Figs. 1 and 3),and the fast (3-h) response to auxin treatments make itunlikely that the activation status of the transgenicROP protein affected the AtROP6 expression patternand the preliminary response to auxin.

To further explore possible function(s) of ROP6 inlateral roots, we examined its subcellular localization

Figure 1. Expression patterns of AtROP6in the shoot and the root. A, B, D, E, G,and H, pROP6..GUS. C, F, J, K, and L,pROP6..GFP-rop6CA. I, pROP6..DsRed. A to C and F, In rosette leaves,ROP6 expression is primarily restrictedto hydathodes, whereas in cauline leaves,it is detected throughout the epider-mis. D, ROP6 expression could not bedetected in the inflorescence meristemand young flowers. E, In mature flow-ers, expression is detected in stamenfilaments and pollen grains, stigma,style, and weakly in ovules. G, In theroot, expression is detected in the rootcap, in and around the quiescent cen-ter; it is absent from the cell divisionand transition zone, and it reappears inthe stele in the differentiation zone.H and I, Expression then drops againand reappears in LRI and founder cells.The arrow denotes a protoxylem file. J,Expression of ROP6 is induced byauxin and can be detected in pericyclecells as early as 3 h after induction. K,After 6 h, expression is spread to morecells in the pericycle. L, Twenty-fourhours after induction, expression isspread throughout the pericycle andextensive cell divisions are detected.Bars = 100 mm (A), 50 mm (B, D, E, andG–L), and 20 mm (C and F). For moreinformation, see Supplemental Figure S2.

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in the developing LRI. In lateral root founder cells inthe pericycle, either GFP-ROP6 or GFP-rop6CA waslocalized in the cell at the side that is closer to thexylem pole (Fig. 3, A, B, I, and J). At later stages oflateral root development, GFP-ROP6 and GFP-rop6CA

were localized such that they aligned the cell divisionplanes (Fig. 3, C–P) and were absent from the side ofthe cell pointing toward the lateral root tip (Fig. 3, E–H, M, and N). This polar localization suggests thatROP6 relays a special signal in LRI cells. Similarly, ithas been found that during stomata development inmaize (Zea mays), the type I ROPs ROP2 and ROP9enhance the polarization of asymmetric cell divisions(Humphries et al., 2011). Together, these data suggestthat ROP-dependent polarization could be critical forasymmetric cell divisions. In contrast to GFP-ROP6and GFP-rop6CA, the dominant negative GFP-rop6DN

accumulated in the cytoplasm and nuclei in root andleaves (Fig. 3, Q–S), resembling the subcellular distri-bution of the nonprenylated GFP-rop6C195S mutant(Fig. 3T). Hence, nucleotide exchange is required forthe recruitment of ROPs to the plasma membrane.

The Phenotype of rop6DN Plants

Next, we examined the phenotype of the pROP6..GFP-rop6DN (rop6DN) plants (Fig. 4). The rop6DN plants

were small and developed multiple inflorescencestems (Figs. 4, A and B, and 7, D and E). The rosetteleaves of the rop6DN plants were curled, and theircauline leaves were twisted (Fig. 4, A–F). The strongereffect of rop6DN on the structure of cauline leavescompared with rosette leaves was closely correlatedwith its expression levels in the two leaf types (Fig. 1,A and F). Analysis of the cell structures in rosette (Figs.4, G and H, and 7, H and I) and cauline leaves (Fig. 4,I and J) revealed that pavement cells in the rop6DN

leaves had fewer lobes and indentations. Quantitativeanalysis of cell structure, using a value designated theaverage polarity score, which is based on the ImageJtools “circularity” and “Skeleton end points” (Soreket al., 2010, 2011), shows that the rop6DN cells are signif-icantly less polar compared with wild-type cells (P #0.001, ANOVA, Tukey-Kramer test; Fig. 7, L–N).

Organization of the Cytoskeleton in Wild-Type androp6DN Plants

ROPs have been implicated in cytoskeleton organi-zation and dynamics, suggesting that the phenotype ofrop6DN plants could be associated with impaired actinand MT functions. Therefore, we examined the orga-nization of MT and actin in wild-type and rop6DN plantsby immunostaining the cytoskeleton in rosette leaves(Fig. 5). In wild-type pavement cells, F-actin was orga-nized in thin filaments and a few thicker bundles thatwere oriented at different angles. In addition, diffusefluorescence likely corresponding to fine F-actin orG-actin could be seen (Fig. 5A; Supplemental Fig. S3, A–C). In comparison, in the rop6DN pavement cells, F-actinwas primarily organized in thick bundles, many ofwhich were arranged parallel to the long cell axis, andlittle diffuse fluorescence could be seen (Fig. 5B;Supplemental Fig. S3, E–H). The MTs were arranged atdifferent angles in wild-type pavement cells (Fig. 5C;Supplemental Fig. S3, I–L), while in rop6DN pavementcells, they were arranged mainly in parallel arrays, per-pendicular to the long axis of cells (Fig. 5D; SupplementalFig. S3, M–P). Taken together, the immunostains dem-onstrate that both actin and MT were differentially orga-nized in the rop6DN plants.

rop6DN-Associated Changes in Gene Expression

The obvious developmental perturbations of rop6DN

plants prompted us to examine whether the pheno-type of the rop6DN plants is associated with changes ingene expression. Gene expression in rop6DN, wild-type, pROP6..GFP-ROP6 (ROP6), and pROP6..GFP-rop6CA (rop6CA) plants (Supplemental Fig. S2)was analyzed using microarrays. Entire seedlingswere used in the microarrays. To identify differen-tially expressed genes, we used the “rank product”method. The rank product analysis gives an estimateof false discovery rate while providing flexible means

Figure 2. Expression of ROP6 in the roots determined by whole-mountin situ hybridization. A, Hybridization with a ROP6 gene-specificsense probe, negative control. B to D, An antisense ROP6 gene-specific probe. Expression was detected in the stele (B, arrow, and D),in the LRI (C), and in the meristematic region of the mature lateral root(D). B, Lateral root founder cells prior to the first anticlinal division. C,Lateral root primordia emerging from the primary root. D, Young lat-eral root. Bars = 20 mm. [See online article for color version of thisfigure.]












Figure 3. Subcellular localization of GFP-ROP6, GFP-rop6CA, and GFP-rop6DN during lateral root development. Subcellularlocalizations of GFP-ROP6 (A–H) and GFP-rop6CA (I–P) were observed in developing lateral roots in pROP6..GFP-ROP6 andpROP6..GFP-rop6CA seedlings. A, B, I, and J, Lateral root founder cells. GFP-ROP6 and GFP-rop6CA were localized along theside of the cell adjacent to the xylem pole (J, arrow). C, D, K, and L, Stage I/II LRI. The majorities of the GFP-ROP6 and GFP-rop6CA proteins moved and were localized along anticlinal cell borders (arrows). E, F, M, and N, Stage II/III LRI. The majority ofGFP-ROP6 and GFP-rop6CA were localized along anticlinal and periclinal cell borders (arrows) separating the newly formedouter and inner layers. G, H, O, and P, Emerged and mature lateral roots with organized cell files. The majority of the GFP-ROP6 and GFP-rop6CA proteins were concentrated on proximal anticlinal cell borders (arrows). In the meristematic zone, theprotein was distributed more or less evenly around the cells. Q to S, GFP-rop6DN was observed in pROP6..GFP-rop6DN

seedlings in lateral root founder cells (Q), stage II/II LRI cells (R), and leaf epidermis pavement cells (S). The recruitment of GFP-rop6DN to the plasma membrane is compromised, and it accumulates in the cytoplasm and nuclei in roots (arrows). T, Non-prenylated GFP-rop6CA mutant accumulates in cytoplasm and nuclei (arrows) but, unlike GFP-rop6DN, does not affect cellpolarity. Bars = 20 mm.




to assign a significance level to each gene (Breitlinget al., 2004). Differentially expressed genes are listed inSupplemental Tables S1 to S5. Pairwise plots show-ing relative gene expression levels revealed that theexpression of many genes was changed in rop6DN

seedlings relative to wild-type, ROP6, and rop6CA

plants (Fig. 6A). Gene expression data were analyzedusing hierarchical (Fig. 6B) and K-means (Fig. 6, C–H)clustering algorithms to identify genes with similar ex-pression patterns. The clustering analyses revealed agroup of 14 genes that were strongly induced in rop6DN

plants (Fig. 6, C and F), a group of 24 genes that weremildly up-regulated (Fig. 6, D and G), and a group of 30genes that were suppressed in rop6DN plants relativeto wild-type, ROP6, or rop6CA transgenic plants (Fig. 6,E and H). Functional categorization of the differentially

expressed genes by Gene Ontology (Martin et al., 2004)revealed that many of the up-regulated genes in therop6DN plants were reminiscent of SA-dependent defenseresponses (Supplemental Tables S1–S5; SupplementalFig. S4). SA-mediated disease resistance is a mechanismof induced defense that confers protection against abroad spectrum of microorganisms. SA-mediatedpathogen defense requires the accumulation of SA,which serves as a signaling molecule leading to theinduction of pathogenesis-related proteins that con-tribute to pathogen resistance (Durrant and Dong,2004; Conrath, 2006). The frequency of SA-associatedgenes among the up-regulated genes is statisticallysignificant (P # 0.05) compared with their randomup-regulation (Supplemental Table S4). Furthermore,expression analysis of the SA-associated genes in

Figure 4. Phenotype of pROP6..GFP-rop6DN plants. A and B, A rop6DN

plant with folded rosette leaves. Notethe loss of apical dominance in therop6DN plant, characterized by thedevelopment of many short adventi-tious inflorescence stems. Folded cau-line leaves are highlighted (arrow). Cand D, Rosette leaves of wild-type (C)and rop6DN (D) plants. E and F, Caulineleaves of wild-type (E) and rop6DN (F)plants. G to J, Scanning electron mi-croscopy images of epidermal cellsfrom rosette leaves (G and H) andcauline leaves (I and J) from wild-type(G and I) and rop6DN (H and J) plants.Note the loss of lobes in the epidermalcells of rop6DN plants. Bars = 1 cm (Aand B), 0.5 cm (C–F), and 10 mm (G–J).[See online article for color version ofthis figure.]










Genevestigator (http://www.genevestigator.ethz.ch;Hruz et al., 2008) showed that their expression is pri-marily associated with biotic defense responses andnot with other stress responses. Examination of the geneexpression patterns of all the genes that were differen-tially expressed in rop6DN plants revealed that most ofthem are subject to regulation by biotic stress. Hence,the microarray study suggested that expression ofthe dominant negative rop6DN induces a constitutiveSA-mediated defense response and that most if not allof the observed changes in gene expression can beexplained by activated SA signaling.

The microarray data required validation of the geneexpression pattern by an independent method and raisedquestions that required additional experimental work.The questions that we asked were as follows. (1) Are thelevels of SA higher in the rop6DN plants compared withthe controls, as would be expected from the geneexpression analysis? (2) Is the developmental phenotypeof the rop6DN plants associated with a constitutive sys-temic acquired resistance response? (3) Are the rop6DN

plants more resistant to pathogens as a result of theconstitutive systemic acquired resistance response?

rop6DN and the SA Response

Validation of the microarray data was carried outby real-time quantitative reverse transcription PCR

(qPCR) analysis of three genes that displayed strongdifferential expression in the rop6DN plants and areoften used as markers for SA-dependent defenseresponses: (1) NIMIN1 (for nonexpresser of PRgenes1/noninducible immunity1 interacting pro-tein1; AT1G02450), (2) Pathogenesis-Related Protein5(PR-5; AT1G75040), and (3) PR-1 (AT2G14610). TheqPCR results show that NIMIN1, PR-5, and PR-1 ex-pression levels are 4-, 12-, and 2,000-fold higher, re-spectively, in rop6DN relative to wild-type plants (Fig. 7,A–C), confirming the microarray results. Importantly,the expression levels were the same when RNA wasextracted from 14-d-old seedlings (Supplemental Fig.S2), like the plants analyzed by microarrays or frommature flowering plants (Figs. 4 and 7).

To examine whether the changes in gene expressionin the rop6DN plants are indeed associated with SA, wemeasured the levels of total and free SA in wild-type,ROP6, rop6CA, and two groups of rop6DN plants. Onegroup is characterized by strong expression of the rop6DN

protein, similar to the plants shown in Figures 3, 4,and 7, while the second group is characterized by verylow expression levels of rop6DN and has a mild de-velopmental phenotype (these plants are resistant toboth kanamycin and basta, confirming that bothtransfer DNA (T-DNA) inserts of the transactivationexpression system are present). In each of the lines,SA levels were compared with those in plants thatwere inoculated with spores of Golovinomyces orontii,

Figure 5. Immunostaining showing actin and MTorganization in Col-0 wild-type and GFP-rop6DN

plants. Actin (red) and MTs (green) are shown inwild-type Col-0 (A and C) and GFP-rop6DN (B andD) leaf epidermal cells. Bars = 10 mm. [See onlinearticle for color version of this figure.]


Poraty et al.


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a host-adapted virulent powdery mildew fungus ofArabidopsis (Spanu et al., 2010). As expected from themicroarray and qPCR analyses, the levels of both totaland free SA in noninoculated plants is markedly el-evated in rop6DN plants (Fig. 6, I and J). In addition,noninoculated and G. orontii-infected rop6DN plantswith high transgene expression accumulate similarSA levels, but these levels are considerably highercompared with pathogen-inoculated wild-type plants.

In contrast, in wild-type, ROP6, and rop6CA plants,SA levels only increase following inoculation withG. orontii. SA levels are also increased in the rop6DN

plants with low transgene expression levels but atlower levels compared with rop6DN plants with highexpression levels of the transgene (Fig. 6, I and J).The higher SA levels confirmed that the changes ingene expression detected in rop6DN plants are indeedassociated with the SA-induced defense response.

To examine whether the developmental phenotype ofthe rop6DN plants is linked to the constitutive SA-mediatedresponse, the rop6DN plants were crossed to mutantsin NPR1, which is required for SA-mediated signaling(Cao et al., 1997), and Salicylic Acid Induction Deficient2(SID2)/Enhanced Disease Susceptibility16 (EDS16), whichis required for pathogen-inducible SA biosynthesis(Nawrath and Métraux, 1999; Supplemental Figs. S4and S5). It has been shown that both npr1 and sid2plants fail to induce the expression of PR genes anddisplay enhanced disease susceptibility to pathogens(Cao et al., 1997; Nawrath and Métraux, 1999; Kinkemaet al., 2000; Stein et al., 2006). The expression ofNIMIN1, PR-5, and PR-1 is suppressed in the rop6DN

npr1 and rop6DN sid2 double mutant plants, resem-bling their expression in naive wild-type plants (Fig. 7,A–C). The mutant analysis confirmed that the ex-pression of PR genes in the rop6DN mutant plantsrequires SA biosynthesis and NPR1 function, as isknown to occur in authentic pathogen-induced SA-mediated defense responses (Durrant and Dong,2004).

The macroscopic phenotype of both rop6DN npr1and rop6DN sid2 double mutants resembles the rop6DN

single mutants. The plants were small, the rosette andcauline leaves were uneven and twisted, and the plantslost their apical dominance and developed numerousadventitious inflorescence stems (Fig. 7, D–G). Qualita-tive (Fig. 7, H–K) and quantitative (Fig. 7, L–N)analyses of leaf epidermis pavement cell structureshowed that cells of rop6DN npr1 and rop6DN sid2 dou-ble mutants resembled the rop6DN single mutant andare significantly (P # 0.001, ANOVA, Tukey-Kramertest) different from wild-type plants. The phenotypicanalysis of the rop6DN npr1 and rop6DN sid2 doublemutant plants indicates that the developmental phe-notype of the rop6DN plants can be uncoupled from theconstitutive SA-dependent defense response. Hence,the overall small plant size, loss of apical dominance,twisted leaves, and deformed cells observed in therop6DN plants are not associated with the constitutiveSA-dependent defense response.

Pathogen Response of rop6DN Plants

Mutants in SA biosynthesis or signaling enhancedisease susceptibility to pathogens, while the induc-tion of SA-dependent defense responses reducespathogen growth, especially against biotrophic patho-gens such as powdery mildews (Durrant and Dong,

Figure 6. Microarray analysis of gene expression in pROP6..GFP-rop6DN seedlings and SA measurements. A, Pairwise plots showingrelative gene expression levels in rop6DN versus the wild type (WT),rop6DN versus rop6CA, and rop6DN versus ROP6. The dashed red linesdenote 2-fold change in gene expression levels. B, Hierarchical clus-tering showing the pattern of expression in rop6CA versus the wild type,rop6CA versus ROP6, ROP6 versus the wild type, rop6DN versus thewild type, rop6DN versus rop6CA, and rop6DN versus ROP6. C to H,K-means clustering showing a cluster of 14 genes that were stronglyinduced (C and F), a cluster of 24 genes that were mildly induced (Dand G), and a cluster of 30 genes that were suppressed (E and H).Yellow color indicates an increase in gene expression, and blue indi-cates a decrease. I and J, Free (I) and total (J) SA levels in control andG. orontii-inoculated (Go) plants 2 and 4 d post spore inoculation. 6DN,GFP-rop6DN plants; 6[DN], GFP-rop6DN plants with low expressionlevels and weak phenotype; FW, fresh weight. Note the high levels offree and total SA in noninoculated rop6DN plants.







Figure 7. PR gene expression and phenotypes of rop6DN npr1 and rop6DN sid2 double mutants. A to C, Expression analysis ofNIMIN1 (A), PR-5 (B), and PR-1 (C) by qPCR in wild-type (WT), rop6DN, rop6DN npr1, and rop6DN sid2 plants. For each gene, itsrelative expression level in the wild type was taken as 1. Note the suppression of expression of each of the three genes to wild-type levels in the rop6DN npr1 and rop6DN sid2 double mutants. D to G, Plants of the wild type (D), rop6DN (E), rop6DN npr1 (F),and rop6DN sid2 (G). H to K, Abaxial epidermis pavement cells. Bars = 20 mm. L to N, Quantitative parameters of epidermal cellstructure. L, Number of lobes. M, Circularity. N, Average polarity score. Note that the rop6DN single mutant and the rop6DN npr1and rop6DN sid2 double mutants have the same plant and cell structures. [See online article for color version of this figure.]


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2004). Hence, it was expected that the rop6DN plantswould display increased resistance to G. orontii due tothe high levels of SA and PR gene expression thatgreatly exceed the SA levels seen in wild-type plantsafter G. orontii infection (Fig. 6, I and J). To test this,wild-type, ROP6, rop6CA, rop6DN, rop6DN WT (plantsthat phenotypically resembled wild-type plants andin which no visible GFP-rop6DN expression could bedetected, although they were resistant to both the

kanamycin and basta selection markers), eds1-2, sid2-1,sid2-2, rop6DN sid2-1, and rop6DN npr1-2 plants wereinoculated with G. orontii. Five days after conidiosporeinoculation, patches of sporulating powdery mildewcolonies were macroscopically detectable on the leafsurface of all lines (Fig. 8; Supplemental Fig. S6).Consistent with this, similar growth of epiphyticfungal hyphae was observed microscopically onleaves of wild-type, rop6CA, high-expressing, and

Figure 8. Susceptibility to the powdery mildew fungus. A, Wild-type (WT), GFP-rop6DN (ROP6DN), GFP-rop6DN with low GFP-rop6DN expression and weak phenotype (ROP6DN wild-type), SA biosynthesis mutants eds1-2, sid2-1, and sid2-2, and doublemutants of GFP-rop6DN with sid2-1 (ROP6DN sid2-1) and with the SA signaling mutant npr1-2 (ROP6DN npr1-2) plants aresusceptible to the powdery mildew fungal pathogen G. orontii. B, Fungal entry rates of G. orontii on GFP-rop6DN (6DN) andinto GFP-rop6DN plants with low expression levels and weak phenotype (6DN WT) are lower compared with the entry rate onwild-type, GFP-ROP6 (R6), and GFP-rop6CA (6CA) plants. C, Fungal entry rates of Bgh on GFP-rop6DN (6DN) and on GFP-rop6DN plants with low expression levels and weak phenotype (6DNWT) are higher compared with the entry rate on wild-type,GFP-ROP6 (R6), and GFP-rop6CA (6CA) plants. The entry rates of Bgh on the GFP-rop6CA plants are higher than on wild-typeand GFP-ROP6 plants. D, G. orontii conidiospore formation was significantly lower on GFP-rop6DN (6DN), GFP-rop6DN sid2-1(6DN sid2-1), and GFP-rop6DN npr1-2 (6DN npr1-2) compared with wild-type and GFP-rop6DN WT (6DN WT) plants. Con-idiospore formation was strongly enhanced compared with the wild type on eds1-2 and slightly enhanced on sid2-1 and sid2-2SA biosynthesis mutants. E, Treatments with the SA analog BTH (black bars) reduced G. orontii conidiospore formation andfurther enhanced resistance of GFP-rop6DN plants. Gray bars show mock-treated (water) plants.






low-expressing rop6DN plants at early stages of patho-genesis (48 h post inoculation; Supplemental Fig. S7).As expected, enhanced disease susceptibility wasdetected in the eds1-2mutant that lacks a key regulatorynode of plant immune responses known to limit thegrowth of host-adapted virulent pathogens (Fig. 8A;Wiermer et al., 2005).

To obtain a more accurate account of infectionphenotypes on the different lines, fungal entry intoplant cells from each of the different lines was deter-mined (as percentage entry at single plant-powderymildew interaction sites). The results of this quantita-tive analysis show that 48 h after inoculation, thefungal entry rates into the rop6DN plants were around65% compared with 90% in wild-type, ROP6, androp6CA plants. The fungal entry rates on rop6DN plantswith low expression of the transgene were around80%, slightly lower compared with wild-type, ROP6,and rop6CA plants and higher compared with rop6DN

plants with high transgene expression levels (Fig. 8B).These data indicate that the constitutive SA-dependentdefense response in the rop6DN plants results in amoderate increase in preinvasive defense responses tothe powdery mildew fungus, despite even higherconstitutive SA levels than in wild-type plants foundafter pathogen challenge (Fig. 6, I and J).

We also determined preinvasion infection pheno-types after inoculation with Bgh, a nonadapted pow-dery mildew that fails to colonize Arabidopsis dueto effective extracellular (preinvasive) resistance re-sponses at early stages during fungal pathogenesis(Collins et al., 2003). At 48 h post inoculation, the entryrates of Bgh sporelings into wild-type Columbia (Col-0) leaf epidermal cells were around 10% (Fig. 8C). Incomparison, the entry rates of virulent G. orontii at 48 hpost inoculation were approximately 90% (Fig. 8B).The entry rates of Bgh into epidermal cells expressing aconstitutively active rop6CA were higher, averaging be-tween 16% and 20% (Fig. 8C). Interestingly, in acompatible interaction in barley expressing constitu-tively active ROP mutants, entry rates of Bgh were alsohigher compared with the wild type (Schultheiss et al.,2002, 2003; Opalski et al., 2005; Hoefle et al., 2011).Significantly, Bgh entry rates of rop6DN epidermal cellswere even higher, reaching approximately 30% (Fig.8C). Taken together, these data suggest that the dis-ruption in cytoskeleton organization impaired extra-cellular disease resistance responses of the rop6DN

plants toward Bgh, albeit the SA levels in these plantswere constitutively elevated.

Next, we quantified the reproductive fitness of thehost-adapted G. orontii powdery mildew by countingfungal conidiospores at a late stage after pathogenchallenge (7 d after inoculation) in wild-type,rop6DN, rop6DN WT (low levels of rop6DN expression),sid2-1, eds1-2, sid2-2, rop6DN sid2-1, and rop6DN npr1-2plants. As expected, G. orontii reproductive fitnesson eds1-2 plants was greatly increased compared withthe wild type and was moderately increased on sid2-1and sid2-2 plants (Fig. 8D; Supplemental Fig. S8). In

contrast, G. orontii reproductive fitness on rop6DN plantswas significantly lower compared with the wild type(Fig. 8D; Supplemental Fig. S8). Importantly, repro-ductive fitness of G. orontii remained significantly lowerthan wild-type levels in rop6DN sid2-1 and rop6DN

npr1-2 double mutants and was comparable to or onlyslightly higher than in rop6DN single mutants (Fig. 8D;Supplemental Fig. S8). To further substantiate thesefindings, we tested G. orontii reproductive fitness fol-lowing pretreatments with the SA analog benzothia-diazole (BTH). The BTH treatments caused decreasedG. orontii reproductive fitness in wild-type, ROP6,rop6CA, rop6DN, and rop6DNWT plants, demonstrating that SAcan confer enhanced disease resistance to G. orontii inArabidopsis. In rop6DN plants, BTH treatments had anadditive effect, further reducing the already lower re-productive fitness of the fungus (Fig. 8E; SupplementalFig. S9). Taken together, the extended data set pro-vides evidence that rop6DN mediates an enhanceddisease resistance phenotype to host-adapted G. orontiiindependently of the SA-mediated defense pathway.

DISCUSSION

Plant Development and Hormonal Responseand Transport

In the absence of other families of signaling smallGTPases in plants, ROPs were suggested to function asregulators of diverse signaling pathways (Yang, 2002).The results in our work are consistent with earlierfindings and implicate ROP6 in both developmentaland pathogen response regulation. The expressionpattern of ROP6 is highly specific and closely resem-bles that of auxin-regulated genes both in leaves androots. Correspondingly, the small plant size, loss ofapical dominance, and twisted leaf phenotypes ofplants expressing a dominant negative rop6DN under theregulation of the ROP6 promoter resemble mutantplants that are affected in auxin signaling, transport,and biosynthesis. Because the phenotype of rop6T-DNA knockout mutants is indistinguishable fromwild-type plants, it is likely that the rop6DN mutant affectedfunctions of other ROPs, which are expressed in thesame tissue as ROP6. Hence, by expressing rop6DN

under the regulation of it native promoter, we over-came functional redundancy between ROPs whilemaintaining specificity and analyzing ROP function ina developmental context.

It has recently been proposed that ROPs are acti-vated by auxin through a mechanism that involvesABP1 (Xu et al., 2010). Activated ROPs undergotransient S-acylation and partition into lipid rafts, aprocess that is required for their function (Soreket al., 2007, 2010) and that induces localized inhibi-tion of endocytosis (Bloch et al., 2005). In lipid rafts,ROPs interact with their effector ICR1 (Sorek et al.,2010), which regulates the recruitment of PIN auxinefflux transporters to polar domains in the plasma


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membrane and is required for directional auxintransport (Hazak et al., 2010). ICR1 transcription isinduced by auxin, and similar to ROP6, it is alsoexpressed in LRI (Hazak et al., 2010). Our results (Fig. 3,Q–S) indicate that the dominant negative rop6DN

accumulates in the cytoplasm and nuclei, where itpresumably interacts with ROPGEFs, inhibiting theirfunction in ROP activation (Feig, 1999; Berken et al.,2005; Berken and Wittinghofer, 2008). In addition, thealtered cytoskeleton organization in the rop6DN plants(Fig. 5) may interfere with auxin transport. It has beendemonstrated that actin is required for PIN recycling(Geldner et al., 2001) and MTs are required for polarPIN localization (Boutté et al., 2006; Kleine-Vehn et al.,2008; Heisler et al., 2010). Hence, the expression ofrop6DN could have inhibited ROP function in regulat-ing auxin transport.

Epidermal Cell Structure and Growth

The changes in rosette leaf pavement cell morphol-ogy, which were detectable in independent transgeniclines, suggest that although ROP6 is primarilyexpressed at the leaf tip and hydathodes (Fig. 1, A–C),it may also be expressed in pavement cells at levelsthat are below detection with GUS and GFP reportergenes. Alternatively, the dominant negative rop6DN

may affect pavement cell morphology indirectly. Forexample, it has recently been shown that perturbationsin auxin biosynthesis or distribution affect pavementcell morphology (Xu et al., 2010). Because ROP6 ex-pression in rosette leaves corresponds to sites of highauxin response, it is possible that suppression of ROPfunction at these sites affects auxin response and/ordistribution. Furthermore, the overall small size ofthe rop6DN plants and the development of multipleinflorescence stems indicate that the dominant nega-tive rop6DN has non-cell-autonomous effects.The rosette and cauline leaves of the rop6DN plants

have a wavy or twisted appearance (Fig. 4), likelyowing to uneven cell growth rates in different parts ofthe leaf. Analysis of pavement cell growth dynamics indeveloping Arabidopsis cotyledons showed that cellgrowth is coordinated between groups of neighboringcells, and it was suggested that this coordination isrequired for the maintenance of flattened leaf surface(Zhang et al., 2011). Close comparison of the scanningelectron microscopy images of wild-type and rop6DN ro-sette and cauline leaf epidermal cells shows that thepavement cells are flatter in the wild type comparedwith rop6DN (Fig. 4, compare G with H and I with J).Furthermore, the cells of the rop6DN plants range in sizeand shape: some cells are rectangular or cubical, whileother cells develop more lobes (Fig. 7). The bulging ofthe rop6DN epidermal cells probably occurred due toisotropic and uncoordinated cell growth that may havealtered the mutual pressures that cells exert on theirneighbors. Pavement cells of plants expressing con-stitutively active ROP mutants also display isotropic

growth, but they are usually more rectangular andeven in their size. The leaf margins of the ropCA mu-tants usually bend downward, but the leaves are notwavy or twisted (Li et al., 2001; Fu et al., 2002; Blochet al., 2005; Sorek et al., 2010). This suggests that theisomorphic cell growth cannot fully explain the unco-ordinated cell growth that leads to leaf waviness andtwisting. It could be that the expression of rop6DN

disrupted a non-cell-autonomous mechanism that isresponsible for coordinating cell growth in leaves.

Cytoskeleton Organization

Thick GFP-mTalin-labeled F-actin bundles havepreviously been observed in plants expressing adominant negative ROP2 (rop2DN) under the regulationof the 35S promoter. It was further shown that theabundance of diffuse F-actin was reduced followingtransient expression of rop2DN. Interestingly, the samestudy showed that the expression of rop2CA hadthe opposite effect, causing an increase in the abun-dance of diffuse F-actin (Fu et al., 2002). Using a dif-ferent experimental system, our data similarly showthat the abundance of diffuse F-actin was low and thatF-actin was primarily organized in thick bundles in therop6DN plants (Fig. 5; Supplemental Fig. S3). The effectof constitutively active rop6CA on actin in pavementcells was not studied in this work. However, previousstudies in root hairs showed that the expression ofconstitutively active rop6CA or rop11CA led to the for-mation of thick actin bundles and eliminated the zoneof fine F-actin at the root hair tip (Molendijk et al.,2001; Bloch et al., 2005, 2011). This may indicate thatactivated ROPs possibly have different effects on actinorganization in different cell types. An alternative andtempting explanation is that ROP cycling betweenactive and inactive states is required for F-actin turn-over and the formation of fine F-actin. The constitu-tively active mutants are locked in the GTP-boundstate (Berken and Wittinghofer, 2008; Sorek et al.,2010). Hence, when signaling requires the binding andrelease of effectors, it should be impaired when thedissociation of the GTPase from target proteins iscompromised. Thus, the bundling of actin filamentsmay reflect an inhibition of the ROP switch mecha-nism. How ROPs regulate actin nucleation and sta-bility is yet an open question (Hussey et al., 2006;Deeks and Hussey, 2009).

The MT analysis in this work demonstrates that inmany fully developed pavement cells, MTs were or-ganized in transverse or oblique orientation relative tothe long cell axis (Fig. 5; Supplemental Fig. S3). Earlierstudies with plants expressing rop2DN showed that thecortical MTs have transverse orientation during earlystages of pavement cell growth but are randomlyorganized in mature cells (Fu et al., 2002). The dis-crepancy between our data and the previous studycould be explained by differences in the experimentalsystem, such as the different ROPs used in each







experiment, different expression systems, the level oftransgene expression, and plant growth conditions.The observed changes in MT organization could haveresulted from changes in tissue biomechanics or fromthe direct influence by ROPs or both. In cells of theshoot apical meristem, the orientation of cortical MTswas shown to correspond to biomechanical stress fromthe surrounding tissue (Hamant et al., 2008). It wasfurther shown that both cortical MT orientation andthe localization of PIN1 auxin efflux carrier likely re-spond to the same biomechanical regulator (Heisleret al., 2010). In developing metaxylem cells, the ICR1homolog MIDD1/RIP3/ICR5 is recruited to specificplasma membrane domains by ROP11, where it in-teracts with and induces the severing of cortical MTs,leading to secondary cell wall pit formation (Oda et al.,2010; Oda and Fukuda, 2012). Interestingly, adaxialpavement cells of icr1 mutant plants have a cubicalshape lacking lobes and indentations, resembling ROPmutants (Lavy et al., 2007). During pavement cellgrowth, ROP6 is activated by auxin and stabilizes MTsvia its effector RIC1 (Fu et al., 2005; Xu et al., 2010).Hence, ROPs may modulate MTs via ICR and RICfamily proteins. Analysis of pavement cell growth dy-namics showed little correlation between MT organi-zation and orientation and cell structure (Zhang et al.,2011). Clearly, more work is required to understand thecomplex relation between ROP function, cell growth,MT organization, and cell wall formation and flexibility.

The SA Response

The microarray studies were carried out on youngseedlings before the developmental abnormalities in-duced by the expression of rop6DN became apparent(Supplemental Fig. S2). The logic behind this experi-mental design was that by looking at changes in geneexpression prior to the stage where the phenotype ofthe plant becomes apparent, it should be possible todefine whether the dominant negative ROP induceschanges in gene expression and possibly identify thekey genes whose alteration leads to the developmentalaberrations. However, the microarray analysis revealedthat the most significant changes in gene expressioninduced by rop6DN are associated with SA-dependentdefense responses. Down-regulation of lignin biosyn-thesis by silencing the expression of hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferase (HCT) inalfalfa (Medicago sativa) and Arabidopsis led to retardedplant growth, which has been associated with a con-stitutive SA response. Growth of HCT-silenced plantswas regained in HCT-RNAi sid2 but not in HCT-RNAinpr1 double mutants (Gallego-Giraldo et al., 2011a,2011b). In contrast to the HCT-silenced plants, thephenotype of the rop6DN npr1 and rop6DN sid2 doublemutants, in which the SA-dependent defense responseis blocked, resembled that of rop6DN single mutantplants, indicating that the developmental perturbationsinduced by rop6DN are unrelated to the constitutiveSA-dependent response.

ROPs and Pathogen Response

As described in the introduction, ROP function waslinked to plant defense responses in several studies.Results in this work (Figs. 6–8) suggest that the possiblesuppression of SA-dependent defense response execu-tion by activated ROPs or by loss of MT-associatedROPGAP1 function (Hoefle et al., 2011; Huesmannet al., 2011) may also contribute to the enhancedsusceptibility of the respective mutants. Loss-of-function mlo mutant alleles in Arabidopsis and barleyare resistant to host-adapted powdery mildews, andthis preinvasive resistance depends on polarized exo-cytosis regulated by PEN1/ROR2 syntaxins (Collinset al., 2003; Consonni et al., 2006; Kwon et al., 2008).Infection by the Bgh powdery mildew was shown toinduce a redistribution of MLO, ROR2, and otherproteins involved in the defense response to plasmamembrane microdomains, which could be lipid rafts,just below sites of attempted fungal ingress (Bhat et al.,2005). The actin and MT cytoskeleton are disorganizedin dominant negative ROP mutants (Fu et al., 2002; thiswork), possibly affecting directed secretion and, inturn, plant defense responses to the powdery mildewfungus. Hence, impaired extracellular resistance re-sponses of the rop6DN plants toward Bgh may haveresulted from the disorganized cytoskeleton. Consis-tent with this interpretation, pharmacological inhibi-tors and genetic interference by ectopic expression ofan actin-depolymerizing factor-encoding gene, HvADF,impaired extracellular resistance responses to Bgh entryin barley (Miklis et al., 2007), and silencing of theHvRAB- and HvRAC1-interacting protein kinaseHvRBK1 disrupted MT organization and enhancedsusceptibility to Bgh (Huesmann et al., 2012). Of note,however, preinvasive resistance responses to Bgh atearly stages during pathogenesis are essentially inde-pendent of an intact SA defense pathway (Consonniet al., 2006; Stein et al., 2006). This could explainwhy extracellular resistance responses to Bgh in rop6DN

plants are impaired despite constitutively elevated SAlevels.

The BTH treatments (Fig. 8; Supplemental Fig. S9)demonstrated that SA-dependent defense responsescan confer resistance to G. orontii in Arabidopsis.Hence, we cannot exclude a small contribution of theSA-dependent defense response to the reduced repro-ductive fitness of G. orontii seen on rop6DN plants.However, the double mutants of rop6DN with sid2 ornpr1 show that the reduced reproductive fitness ofG. orontii on rop6DN plants was largely a rop6DN-dependent and SA-independent defense response.

CONCLUSION

Our results imply that ROPs regulate highly complexdefense responses to powdery mildews that can bedetected at early and late stages of the infection pro-cess. ROPs either enhance or reduce early preinvasive


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disease resistance responses to the tested host-adaptedand nonadapted powdery mildews, presumably viatheir effects on the cytoskeleton and intracellulartrafficking. At later stages of fungal pathogenesis,interference with ROP function reduces fungal re-productive success by mechanisms that remain to beelucidated. The mechanism underlying the constitu-tive activation of SA accumulation/signaling inrop6DN plants remains unclear. One possibility is thatperturbation of the cytoskeleton or default secretionby rop6DN mimics a pathogen attack. Alternatively,similar to HCT-RNAi plants (Gallego-Giraldo et al.,2011b), release of cell wall materials or changes in cellwall integrity may activate SA responses in rop6DN

plants. Changes in cell wall integrity may have alsocontributed to the susceptibility of the rop6DN plantsto Bgh. Irrespective of this, our results implicateROPs as important regulators of plant developmentas well as in SA-dependent and SA-independentdefense execution.

MATERIALS AND METHODS

Molecular Cloning

All plasmids and primers are listed in Supplemental Tables S6 and S7,respectively. Plasmid construction was carried out using standard molecularcloning techniques. AtROP6 was cut from pSY700 with SacI and then wassubcloned to pGFP-MRC to create pSY811. PCR-based site-directed muta-genesis (QuikChange kit; Stratagene) was used to create a constitutively activemutant (Atrop6CA/DN) by substituting Gly-15 with Val and Thr-30 with Asn.pSY811was used as template with the following primer sets: SYP189 + SYP190to create pSY812 (GFP-rop6CA) and SYP602 + SYP603 to create pSY807 (GFP-rop6DN). To subclone GFP-ROP6, GFP-rop6CA, and GFP-rop6DN into the pOPeffector plasmids, pSY811, pSY812, and pSY807 were digested with XhoI toisolate the GFP-ROP6, GFP-rop6CA, and GFP-rop6DN fragments. The threefragments were, in turn, subcloned into pOP-Bj36 to create pSY836, pSY819,and pSY835, respectively. For expression in plants, pSY836, pSY819, andpSY835 were digested with NotI, and the resulting pOP-GFP-ROP6/rop6CA/rop6DN fragments were subcloned into pMLBART plant binary vector to createpSY832, pSY818, and pSY831, respectively. For construction of the ROP6promoter LhG4 driver plasmid, a 1,047-bp fragment upstream of AtROP6(pROP6) was amplified by PCR from Arabidopsis (Arabidopsis thaliana Col-0)genomic DNA using SYP615 and SYP616. The amplified AtROP6 was subcl-oned into pGEM-T (Promega) and, in turn, into pBj36 (Eshed et al., 2001) withSalI and KpnI (Acc65I) to create the pSY810 promoter LhG4 driver plasmid. Forexpression in plants, pSY810 was digested with NotI, and the LhG4-pAtROP6cassette was subcloned into a pART27 plant binary vector to create pSY813. Allfragments were fully sequenced to verify that no PCR-generated errors wereintroduced.

Plant Material

All transgenic Arabidopsis lines used in this study are listed in Supple-mental Table S8. Arabidopsis plants were grown as described previously (Lavyet al., 2002). For plant transformation, Arabidopsis plants were transformedwith Agrobacterium tumefaciens strain GV3101/pMp90 harboring the appropriateplasmids using the floral dip method (Clough and Bent, 1998). A minimumof three independent transgenic lines for each plasmid were analyzed.Transcription-transactivation lines were obtained by crossing pROP6::LhG4driver lines with pOp effector lines to obtain pROP6..GUS, pROP6..DsRed,pROP6..GFP-ROP6, pROP6..GFP-rop6CA, and pROP6..GFP-rop6DN. AnArabidopsis T-DNA insertion line (SALK_91737) in the ROP6 gene wasobtained from the Arabidopsis Biological Research Center at the University ofOhio. The existence of the insertion was verified with SYP179 and SYP163ROP6-specific primers and a T-DNA left border primer (Lba1; SYP179).

Auxin Induction

For auxin induction, seeds were germinated in the presence of the auxintransport inhibitor N-1-naphthylphthalamic acid followed by transfer togrowth medium containing 10 mM auxin (NAA), as described previously(Himanen et al., 2004). Alternatively, seeds were germinated on mediumlacking N-1-naphthylphthalamic acid and were, in turn, treated with 10 mM

NAA.

GUS Staining

GUS staining was carried out as described previously (Weigel andGlazebrook, 2002).

Whole-Mount in Situ Hybridization

Tissue preparation and whole-mount in situ hybridization were performedessentially as described previously (Brewer et al., 2006; Hejátko et al., 2006).A 244-bp fragment from the 39-untranslated region of ROP6 was amplified byPCR from Arabidopsis genomic DNA and was used as template to create gene-specific digoxigenin-labeled antisense/sense RNA probes. The digoxigenin-labeled probes were prepared by in vitro transcription according to themanufacturer’s instructions (Roche).

Fixation and Immunostaining

Fixation and immunostaining were carried out essentially as describedpreviously (Chaimovitsh et al., 2012). To stabilize actin and MT, specimenswere fixed for 5 min with 500 mM m-maleimidobenzoyl-hydroxylsuccinimideester in MT-stabilizing buffer (MTSB) containing 100 mM PIPES, pH 6.89, 5 mM

EGTA, 5 mM MgSO4$7H2O, and 1% Triton X-100 (v/v) buffer and, in turn, for1 h in freshly prepared 8% paraformaldehyde in MTSB. Fixation was carriedout at room temperature in the dark. Samples were then rinsed three times inMTSB and treated with the following enzyme mixture: 2% cellulase OnzukaR-10 and 1% (w/v) pectinase, protease inhibitor cocktail (Sigma), and 20 mM

phenylmethylsulfonyl fluoride for 10 min. After washing in MTSB for 30 min,the samples were squashed on coverslips coated with poly-L-Lys (1 mg mL21;Sigma-Aldrich). Then, the cells were retreated for 10 min with enzyme mixture(2% cellulase Onzuka R-10 and 1.5% [w/v] pectinase) with protease inhibitors.This step was followed by a rinse in phosphate-buffered saline (PBS) with 1%Triton X-100 (v/v) for 30 min and then incubation in PBS containing 1% (w/v)bovine serum albumin for 20 min. To reduce aldehyde-induced auto-fluorescence, samples were treated for 5 min with 10 mg mL21 sodium bo-rohydride in PBS and then washed in PBS for 20 min. For immunofluorescencedetection of MTs, specimens were incubated overnight at room temperaturewith primary sheep anti-ab-tubulin polyclonal antibodies (Cytoskeleton) andmouse anti-actin monoclonal (clone C4) antibodies (ICN Biomedical) at a di-lution of 1:100. Samples were then washed in PBS and incubated with anti-sheep secondary antibodies conjugated to Alexa Fluor 488 and anti-mousesecondary antibodies conjugated to Alexa 555 (Molecular Probes) diluted1:100 for 1 h at room temperature. Mounting was done in 50% glycerol/water(v/v).

Plant DNA and RNA Isolation and qPCR

Plant genomic DNA was isolated with the GenElute Plant Genomic DNAminiprep kit according to the manufacturer’s instructions (Sigma). Total RNAwas isolated with the SV Total RNA isolation kit according to the manufac-turer’s instructions (Promega). The RNA from the qPCR experiments wasextracted from both 14-d-old seedlings and mature flowering plants. Com-plementary DNA first-strand synthesis was carried out using Moloney murineleukemia virus reverse transcriptase (Promega). Quantifications with theprimer sets (Supplemental Table S7) were carried out by qPCR using an ABIPrism 7700 StepOnePlus Instrument (Applied Biosystems). Study sampleswere run in triplicate on eight-well optical PCR strips (Applied Biosystems) ina final volume of 10 mL. Primers were designed using the Roche UniversalProbe Library (https://www.roche-applied-science.com/sis/rtpcr/upl/index.jsp). The PCR cycles were run as follows: 10-min initial denaturation at95°C, followed by 40 subsequent cycles of 15 s of denaturation at 95°C, and1 min of annealing and elongation at 60°C. The specificity of the uniqueamplification product was determined by melting-curve analysis according to






https://www.roche-applied-science.com/sis/rtpcr/upl/index.jsp

https://www.roche-applied-science.com/sis/rtpcr/upl/index.jsp


the manufacturer’s instructions (Applied Biosystems). Relative quantities ofRNA were calculated by the comparative threshold cycle method (AppliedBiosystems User Bulletin 2: ABI PRISM 7700 Sequence Detection System;http://www.appliedbiosystems.com). DNA dilution series were prepared tocalculate an amplification efficiency coefficient for each gene. The relativelevels of RNA were calculated according to the amplification efficiencycoefficient and normalized against ACTIN8 and UBQ21 gene standards(Czechowski et al., 2005), the level of which was taken as 1. The stability of thestandards in each experiment was verified with the geNorm analysis tool(http://medgen.ugent.be/jvdesomp/genorm/) and was calculated as M #

0.7. The analysis was repeated with three independent biological replicates.

Array Hybridization and Evaluation

Seedlings of pAtROP6..GFP-ROP6, pAtROP6..GFP-ROP6CA, and Col-0were grown on 0.53 Murashige and Skoog medium for 14 d in growthchambers at 21°C under long-day photoperiods (16 h of light, 8 h of darkness).A total of 100 mg of plant tissue was harvested from seedlings, and RNA wasextracted using Trizol (Invitrogen) according to the manufacturer’s instruc-tions. The entire experiment was performed three times, providing indepen-dent biological replicates. Affymetrix Arabidopsis ATH1 GeneChips wereused in the experiment. Labeling of samples, hybridizations, and measure-ments were performed as described (Hennig et al., 2004). Signal values werederived from the Affymetrix *.cel files using the GCRMA algorithm (Psarroset al., 2005). Data were processed with the statistical package R (version 1.9.1).Differentially expressed genes were determined using the rank-productmethod (Breitling et al., 2004) implemented in R. Genes were considered asbeing differentially expressed at P # 0.05.

Light, Fluorescence, and Confocal Microscopy

Low-resolution imaging was performed with an SV-11 stereomicroscope(Zeiss). Wide-field fluorescence imaging was performed with an Axioplan-2Imaging fluorescence microscope (Zeiss) equipped with an AxioCam cooledCCD camera, using either 203 dry or 633 water-immersion objectives withnumerical aperture values of 0.5 and 1.2, respectively. Confocal imaging wasperformed with a Leica TCS-SL confocal laser scanning microscope equippedwith 203 multi-immersion and 633 water-immersion objectives with nu-merical aperture values of 0.7 and 1.2, respectively. GFP was visualized byexcitation with an argon laser at 488 nm, a 500-nm beam splitter, and aspectral detector set between 505 and 530 nm. DsRed was visualized by ex-citation with an argon laser set to 514 nm, a 456/514-nm double dichroic beamsplitter, and a spectral detector set between 530 and 560 nm. Scanning electronmicroscopy was carried out with JEOL JSM-840A scanning electron micro-scope. Image analysis was performed with Zeiss AxioVision, ImageJ, LeicaLCS, and Adobe Photoshop.

SA Measurements, Fungal Entry Rates, ConidiosporeCounts, and BTH Treatments

Measurements of free and conjugated SA levels were carried out by ana-lytical HPLC essentially as described previously (Bartsch et al., 2006). Golo-vinomyces orontii and Blumeria graminis f. sp. hordei conidiospore inoculationswere carried out as described previously (Consonni et al., 2006). Conidiosporecounts were carried out as described previously (Wessling and Panstruga,2012). BTH treatments were carried out essentially as described previously(Lawton et al., 1996). Briefly, 4-week-old plants were sprayed with 42.6 mgL21 water-solubilized BTH 24 h prior to inoculation by G. orontii con-idiospores. Control plants were sprayed with water (BTH mock control). Forall infection assays, plants were grown in a phytochamber with the fol-lowing settings: 65% humidity; temperature of 22°C from 9 AM to 7 PM and20°C from 7 PM to 9 AM; and 10/14-h light/dark cycles. Light intensity was100 mE m22 s21.

Supplemental Data

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

Supplemental Figure S1. Nucleotide sequence of the AtROP6 promoter.

Supplemental Figure S2. Fourteen-day-old Col-0 (wild type), rop6CA

(6CA), and rop6DN (6DN) seedlings grown on vertical-oriented plates.

Supplemental Figure S3. Immunostaining of actin and microtubule inwild-type and GFP-rop6DN leaf epidermis cells.

Supplemental Figure S4. Immunostaining of actin and microtubule inwild-type and GFP-rop6DN leaf epidermis cells.

Supplemental Figure S5. Molecular identification of rop6DN npr1 androp6DN sid2 double mutants.

Supplemental Figure S6. Susceptibility to the powdery mildew fungus.

Supplemental Figure S7. G. orontii fungal hyphae.

Supplemental Figure S8. G. orontii conidiospores formation.

Supplemental Figure S9. Susceptibility to G. orontii following SA analogBTH treatments.

Supplemental Table S1. Differentially expressed genes in GFP-rop6DN ver-sus the wild type (Col-0).

Supplemental Table S2. Differentially expressed genes in GFP-rop6DN

versus GFP-ROP6.

Supplemental Table S3. Differentially expressed genes in GFP-rop6DN ver-sus GFP-rop6CA.

Supplemental Table S4. Gene Ontology of differentially expressed genesby GoToolBox.

Supplemental Table S5. Strong differential expression of SAR associatedgenes in the GFP-rop6DN lines.

Supplemental Table S6. Plasmids used in this study.

Supplemental Table S7. Oligonucleotide primers used in this study.

Supplemental Table S8. Transgenic Arabidopsis lines used in this study.

Supplemental References S1.

ACKNOWLEDGMENTS

We thank R. Fluhr for materials.

Received December 20, 2012; accepted January 8, 2013; published January 14,2013.

LITERATURE CITED

Aloni R, Schwalm K, Langhans M, Ullrich CI (2003) Gradual shifts in sitesof free-auxin production during leaf-primordium development and theirrole in vascular differentiation and leaf morphogenesis in Arabidopsis.Planta 216: 841–853

Bartsch M, Gobbato E, Bednarek P, Debey S, Schultze JL, Bautor J, ParkerJE (2006) Salicylic acid-independent ENHANCED DISEASE SUSCEP-TIBILITY1 signaling in Arabidopsis immunity and cell death is regulatedby the monooxygenase FMO1 and the Nudix hydrolase NUDT7. PlantCell 18: 1038–1051

Benková E, Michniewicz M, Sauer M, Teichmann T, Seifertová D,Jürgens G, Friml J (2003) Local, efflux-dependent auxin gradients as acommon module for plant organ formation. Cell 115: 591–602

Berken A, Thomas C, Wittinghofer A (2005) A new family of RhoGEFsactivates the Rop molecular switch in plants. Nature 436: 1176–1180

Berken A, Wittinghofer A (2008) Structure and function of Rho-type mo-lecular switches in plants. Plant Physiol Biochem 46: 380–393

Bhat RA, Miklis M, Schmelzer E, Schulze-Lefert P, Panstruga R (2005) Recruit-ment and interaction dynamics of plant penetration resistance components in aplasma membrane microdomain. Proc Natl Acad Sci USA 102: 3135–3140

Bloch D, Hazak O, Lavy M, Yalovsky S (2008) A novel ROP/RAC GTPaseeffector integrates plant cell form and pattern formation. Plant SignalBehav 3: 41–43

Bloch D, Lavy M, Efrat Y, Efroni I, Bracha-Drori K, Abu-Abied M, SadotE, Yalovsky S (2005) Ectopic expression of an activated RAC in Arabi-dopsis disrupts membrane cycling. Mol Biol Cell 16: 1913–1927

Bloch D, Monshausen G, Singer M, Gilroy S, Yalovsky S (2011) Nitrogensource interacts with ROP signalling in root hair tip-growth. Plant CellEnviron 34: 76–88


Poraty et al.


http://www.appliedbiosystems.com

http://medgen.ugent.be/jvdesomp/genorm/




















Boutté Y, Crosnier MT, Carraro N, Traas J, Satiat-Jeunemaitre B (2006)The plasma membrane recycling pathway and cell polarity in plants:studies on PIN proteins. J Cell Sci 119: 1255–1265

Breitling R, Armengaud P, Amtmann A, Herzyk P (2004) Rank products: asimple, yet powerful, new method to detect differentially regulatedgenes in replicated microarray experiments. FEBS Lett 573: 83–92

Brewer PB, Heisler MG, Hejátko J, Friml J, Benková E (2006) In situ hy-bridization for mRNA detection in Arabidopsis tissue sections. NatProtoc 1: 1462–1467

Cao H, Glazebrook J, Clarke JD, Volko S, Dong X (1997) The ArabidopsisNPR1 gene that controls systemic acquired resistance encodes a novelprotein containing ankyrin repeats. Cell 88: 57–63

Chaimovitsh D, Rogovoy Stelmakh O, Altshuler O, Belausov E, Abu-Abied M, Rubin B, Sadot E, Dudai N (2012) The relative effect of citralon mitotic microtubules in wheat roots and BY2 cells. Plant Biol (Stuttg)14: 354–364

Chen L, Shiotani K, Togashi T, Miki D, Aoyama M, Wong HL, KawasakiT, Shimamoto K (2010) Analysis of the Rac/Rop small GTPase family inrice: expression, subcellular localization and role in disease resistance.Plant Cell Physiol 51: 585–595

Clough SJ, Bent AF (1998) Floral dip: a simplified method forAgrobacterium-mediated transformation of Arabidopsis thaliana. Plant J16: 735–743

Collins NC, Thordal-Christensen H, Lipka V, Bau S, Kombrink E, Qiu JL,Hückelhoven R, Stein M, Freialdenhoven A, Somerville SC, et al(2003) SNARE-protein-mediated disease resistance at the plant cell wall.Nature 425: 973–977

Conrath U (2006) Systemic acquired resistance. Plant Signal Behav 1:179–184

Consonni C, Humphry ME, Hartmann HA, Livaja M, Durner J, WestphalL, Vogel J, Lipka V, Kemmerling B, Schulze-Lefert P, et al (2006)Conserved requirement for a plant host cell protein in powdery mildewpathogenesis. Nat Genet 38: 716–720

Czechowski T, Stitt M, Altmann T, Udvardi MK, Scheible WR (2005)Genome-wide identification and testing of superior reference genes fortranscript normalization in Arabidopsis. Plant Physiol 139: 5–17

Deeks MJ, Hussey PJ (2009) Plant actin biology. In Encyclopedia of LifeSciences. John Wiley & Sons, Chichester, UK

Durrant WE, Dong X (2004) Systemic acquired resistance. Annu RevPhytopathol 42: 185–209

Eshed Y, Baum SF, Perea JV, Bowman JL (2001) Establishment of polarityin lateral organs of plants. Curr Biol 11: 1251–1260

Feig LA (1999) Tools of the trade: use of dominant-inhibitory mutants ofRas-family GTPases. Nat Cell Biol 1: E25–E27

Fischer U, Ikeda Y, Ljung K, Serralbo O, Singh M, Heidstra R, Palme K,Scheres B, Grebe M (2006) Vectorial information for Arabidopsis planarpolarity is mediated by combined AUX1, EIN2, and GNOM activity.Curr Biol 16: 2143–2149

Fu Y, Gu Y, Zheng Z, Wasteneys G, Yang Z (2005) Arabidopsis interdig-itating cell growth requires two antagonistic pathways with opposingaction on cell morphogenesis. Cell 120: 687–700

Fu Y, Li H, Yang Z (2002) The ROP2 GTPase controls the formation ofcortical fine F-actin and the early phase of directional cell expansionduring Arabidopsis organogenesis. Plant Cell 14: 777–794

Fu Y, Xu T, Zhu L, Wen M, Yang Z (2009) A ROP GTPase signalingpathway controls cortical microtubule ordering and cell expansion inArabidopsis. Curr Biol 19: 1827–1832

Gallego-Giraldo L, Escamilla-Trevino L, Jackson LA, Dixon RA (2011a)Salicylic acid mediates the reduced growth of lignin down-regulatedplants. Proc Natl Acad Sci USA 108: 20814–20819

Gallego-Giraldo L, Jikumaru Y, Kamiya Y, Tang Y, Dixon RA (2011b)Selective lignin downregulation leads to constitutive defense responseexpression in alfalfa (Medicago sativa L.). New Phytol 190: 627–639

Geldner N, Friml J, Stierhof YD, Jürgens G, Palme K (2001) Auxintransport inhibitors block PIN1 cycling and vesicle trafficking. Nature413: 425–428

Hamant O, Heisler MG, Jönsson H, Krupinski P, Uyttewaal M, Bokov P,Corson F, Sahlin P, Boudaoud A, Meyerowitz EM, et al (2008) Develo-pmental patterning by mechanical signals in Arabidopsis. Science 322: 1650–1655

Hazak O, Bloch D, Poraty L, Sternberg H, Zhang J, Friml J, Yalovsky S(2010) A rho scaffold integrates the secretory system with feedbackmechanisms in regulation of auxin distribution. PLoS Biol 8: e1000282

Hazak O, Yalovsky S (2010) An auxin regulated positive feedback loopintegrates Rho modulated cell polarity with pattern formation. PlantSignal Behav 5: 709–711

Heisler MG, Hamant O, Krupinski P, Uyttewaal M, Ohno C, Jönsson H,Traas J, Meyerowitz EM (2010) Alignment between PIN1 polarity andmicrotubule orientation in the shoot apical meristem reveals a tightcoupling between morphogenesis and auxin transport. PLoS Biol 8:e1000516

Hejátko J, Blilou I, Brewer PB, Friml J, Scheres B, Benková E (2006) In situhybridization technique for mRNA detection in whole mount Arabi-dopsis samples. Nat Protoc 1: 1939–1946

Hennig L, Gruissem W, Grossniklaus U, Köhler C (2004) Transcriptionalprograms of early reproductive stages in Arabidopsis. Plant Physiol 135:1765–1775

Himanen K, Vuylsteke M, Vanneste S, Vercruysse S, Boucheron E, AlardP, Chriqui D, Van Montagu M, Inzé D, Beeckman T (2004) Transcriptprofiling of early lateral root initiation. Proc Natl Acad Sci USA 101:5146–5151

Hoefle C, Huesmann C, Schultheiss H, Börnke F, Hensel G, Kumlehn J,Hückelhoven R (2011) A barley ROP GTPase activating protein asso-ciates with microtubules and regulates entry of the barley powderymildew fungus into leaf epidermal cells. Plant Cell 23: 2422–2439

Hruz T, Laule O, Szabo G, Wessendrop F, Bleuler S, Oertle L, WidmayerP, Gruissem W, Zimmermann P (2008) Genevestigator V3: a referenceexpression database for the meta-analysis of transcriptomes. Adv Bio-informatics 2008: 420747

Huesmann C, Hoefle C, Hückelhoven R (2011) ROPGAPs of Arabidopsislimit susceptibility to powdery mildew. Plant Signal Behav 6: 1691–1694

Huesmann C, Reiner T, Hoefle C, Preuss J, Jurca ME, Domoki M, Fehér A,Hückelhoven R (2012) Barley ROP binding kinase1 is involved in mi-crotubule organization and in basal penetration resistance to the barleypowdery mildew fungus. Plant Physiol 159: 311–320

Humphries JA, Vejlupkova Z, Luo A, Meeley RB, Sylvester AW, Fowler JE,Smith LG (2011) ROP GTPases act with the receptor-like protein PAN1 topolarize asymmetric cell division in maize. Plant Cell 23: 2273–2284

Hussey PJ, Ketelaar T, Deeks MJ (2006) Control of the actin cytoskeletonin plant cell growth. Annu Rev Plant Biol 57: 109–125

Kinkema M, Fan W, Dong X (2000) Nuclear localization of NPR1 is re-quired for activation of PR gene expression. Plant Cell 12: 2339–2350

Kleine-Vehn J, Langowski L, Wisniewska J, Dhonukshe P, Brewer PB,Friml J (2008) Cellular and molecular requirements for polar PIN tar-geting and transcytosis in plants. Mol Plant 1: 1056–1066

Kwon C, Neu C, Pajonk S, Yun HS, Lipka U, Humphry M, Bau S, StrausM, Kwaaitaal M, Rampelt H, et al (2008) Co-option of a default secre-tory pathway for plant immune responses. Nature 451: 835–840

Lavy M, Bloch D, Hazak O, Gutman I, Poraty L, Sorek N, Sternberg H,Yalovsky S (2007) A novel ROP/RAC effector links cell polarity, root-meristem maintenance, and vesicle trafficking. Curr Biol 17: 947–952

Lavy M, Bracha-Drori K, Sternberg H, Yalovsky S (2002) A cell-specific,prenylation-independent mechanism regulates targeting of type IIRACs. Plant Cell 14: 2431–2450

Lawton KA, Friedrich L, Hunt M, Weymann K, Delaney T, Kessmann H,Staub T, Ryals J (1996) Benzothiadiazole induces disease resistance inArabidopsis by activation of the systemic acquired resistance signaltransduction pathway. Plant J 10: 71–82

Li H, Shen JJ, Zheng ZL, Lin Y, Yang Z (2001) The Rop GTPase switchcontrols multiple developmental processes in Arabidopsis. Plant Physiol126: 670–684

Lorek J, Panstruga R, Huckelhoven R (2010) The role of seven-transmembrane domain MLO proteins, heterotrimeric G-proteins, andmonomeric RAC/ROPs in plant defense. In S Yalovsky, F Baluska, AJones, eds, Integrated G Protein Signaling in Plants. Springer-Verlag,Berlin, pp 197–220

Martin D, Brun C, Remy E, Mouren P, Thieffry D, Jacq B (2004) GO-ToolBox: functional analysis of gene datasets based on Gene Ontology.Genome Biol 5: R101

Miklis M, Consonni C, Bhat RA, Lipka V, Schulze-Lefert P, Panstruga R(2007) Barley MLO modulates actin-dependent and actin-independentantifungal defense pathways at the cell periphery. Plant Physiol 144:1132–1143

Moeder W, Yoshioka K, Klessig DF (2005) Involvement of the smallGTPase Rac in the defense responses of tobacco to pathogens. Mol PlantMicrobe Interact 18: 116–124





Molendijk AJ, Bischoff F, Rajendrakumar CS, Friml J, Braun M, Gilroy S,Palme K (2001) Arabidopsis thaliana Rop GTPases are localized to tipsof root hairs and control polar growth. EMBO J 20: 2779–2788

Moore I, Gälweiler L, Grosskopf D, Schell J, Palme K (1998) A tran-scription activation system for regulated gene expression in transgenicplants. Proc Natl Acad Sci USA 95: 376–381

Nawrath C, Métraux JP (1999) Salicylic acid induction-deficient mutants ofArabidopsis express PR-2 and PR-5 and accumulate high levels of ca-malexin after pathogen inoculation. Plant Cell 11: 1393–1404

Nibau C, Wu HM, Cheung AY (2006) RAC/ROP GTPases: ‘hubs’ for signalintegration and diversification in plants. Trends Plant Sci 11: 309–315

Oda Y, Fukuda H (2012) Initiation of cell wall pattern by a Rho- andmicrotubule-driven symmetry breaking. Science 337: 1333–1336

Oda Y, Iida Y, Kondo Y, Fukuda H (2010) Wood cell-wall structure re-quires local 2D-microtubule disassembly by a novel plasma membrane-anchored protein. Curr Biol 20: 1197–1202

Ono E, Wong HL, Kawasaki T, Hasegawa M, Kodama O, Shimamoto K(2001) Essential role of the small GTPase Rac in disease resistance of rice.Proc Natl Acad Sci USA 98: 759–764

Opalski KS, Schultheiss H, Kogel KH, Hückelhoven R (2005) The receptor-likeMLO protein and the RAC/ROP family G-protein RACB modulate actinreorganization in barley attacked by the biotrophic powdery mildew fungusBlumeria graminis f.sp. hordei. Plant J 41: 291–303

Pathuri IP, Zellerhoff N, Schaffrath U, Hensel G, Kumlehn J, Kogel KH,Eichmann R, Hückelhoven R (2008) Constitutively activated barleyROPs modulate epidermal cell size, defense reactions and interactionswith fungal leaf pathogens. Plant Cell Rep 27: 1877–1887

Psarros M, Heber S, Sick M, Thoppae G, Harshman K, Sick B (2005)RACE: remote analysis computation for gene expression data. NucleicAcids Res 33: W638–W643

Sabatini S, Beis D, Wolkenfelt H, Murfett J, Guilfoyle T, Malamy J,Benfey P, Leyser O, Bechtold N, Weisbeek P, et al (1999) An auxin-dependent distal organizer of pattern and polarity in the Arabidopsisroot. Cell 99: 463–472

Schultheiss H, Dechert C, Kogel KH, Hückelhoven R (2002) A small GTP-binding host protein is required for entry of powdery mildew fungusinto epidermal cells of barley. Plant Physiol 128: 1447–1454

Schultheiss H, Dechert C, Kogel KH, Hückelhoven R (2003) Functionalanalysis of barley RAC/ROP G-protein family members in susceptibilityto the powdery mildew fungus. Plant J 36: 589–601

Schultheiss H, Hensel G, Imani J, Broeders S, Sonnewald U, Kogel KH,Kumlehn J, Hückelhoven R (2005) Ectopic expression of constitutivelyactivated RACB in barley enhances susceptibility to powdery mildewand abiotic stress. Plant Physiol 139: 353–362

Sorek N, Gutman O, Bar E, Abu-Abied M, Feng X, Running MP,Lewinsohn E, Ori N, Sadot E, Henis YI, et al (2011) Differential effectsof prenylation and S-acylation on type I and II ROPS membrane inter-action and function. Plant Physiol 155: 706–720

Sorek N, Poraty L, Sternberg H, Bar E, Lewinsohn E, Yalovsky S (2007)Activation status-coupled transient S acylation determines membranepartitioning of a plant Rho-related GTPase. Mol Cell Biol 27: 2144–2154

Sorek N, Segev O, Gutman O, Bar E, Richter S, Poraty L, Hirsch JA, HenisYI, Lewinsohn E, Jürgens G, et al (2010) An S-acylation switch ofconserved G domain cysteines is required for polarity signaling by ROPGTPases. Curr Biol 20: 914–920

Spanu PD, Abbott JC, Amselem J, Burgis TA, Soanes DM, Stüber K, VerLoren van Themaat E, Brown JK, Butcher SA, Gurr SJ, et al (2010)Genome expansion and gene loss in powdery mildew fungi revealtradeoffs in extreme parasitism. Science 330: 1543–1546

Stein M, Dittgen J, Sánchez-Rodríguez C, Hou BH, Molina A, Schulze-Lefert P, Lipka V, Somerville S (2006) Arabidopsis PEN3/PDR8, an ATPbinding cassette transporter, contributes to nonhost resistance to in-appropriate pathogens that enter by direct penetration. Plant Cell 18:731–746

Tao LZ, Cheung AY, Nibau C, Wu HM (2005) RAC GTPases in tobacco andArabidopsis mediate auxin-induced formation of proteolytically activenuclear protein bodies that contain AUX/IAA proteins. Plant Cell 17:2369–2383

Tao LZ, Cheung AY, Wu HM (2002) Plant Rac-like GTPases are activatedby auxin and mediate auxin-responsive gene expression. Plant Cell 14:2745–2760

Thao NP, Chen L, Nakashima A, Hara S, Umemura K, Takahashi A,Shirasu K, Kawasaki T, Shimamoto K (2007) RAR1 and HSP90 form acomplex with Rac/Rop GTPase and function in innate-immune re-sponses in rice. Plant Cell 19: 4035–4045

Weigel D, Glazebrook J (2002) Arabidopsis: A Laboratory Manual. ColdSpring Harbor Laboratory Press, Cold Spring Harbor, NY

Wessling R, Panstruga R (2012) Rapid quantification of plant-powdery mildewinteractions by qPCR and conidiospore counts. Plant Methods 8: 35

Wiermer M, Feys BJ, Parker JE (2005) Plant immunity: the EDS1 regulatorynode. Curr Opin Plant Biol 8: 383–389

Winge P, Brembu T, Bones AM (1997) Cloning and characterization of rac-like cDNAs from Arabidopsis thaliana. Plant Mol Biol 35: 483–495

Wong HL, Pinontoan R, Hayashi K, Tabata R, Yaeno T, Hasegawa K,Kojima C, Yoshioka H, Iba K, Kawasaki T, et al (2007) Regulation ofrice NADPH oxidase by binding of Rac GTPase to its N-terminal ex-tension. Plant Cell 19: 4022–4034

Wu HM, Hazak O, Cheung AY, Yalovsky S (2011) RAC/ROP GTPases andauxin signaling. Plant Cell 23: 1208–1218

Xin Z, Zhao Y, Zheng ZL (2005) Transcriptome analysis reveals specificmodulation of abscisic acid signaling by ROP10 small GTPase in Arabidopsis.Plant Physiol 139: 1350–1365

Xu T, Wen M, Nagawa S, Fu Y, Chen JG, Wu MJ, Perrot-Rechenmann C,Friml J, Jones AM, Yang Z (2010) Cell surface- and rho GTPase-basedauxin signaling controls cellular interdigitation in Arabidopsis. Cell 143:99–110

Yalovsky S, Bloch D, Sorek N, Kost B (2008) Regulation of membranetrafficking, cytoskeleton dynamics, and cell polarity by ROP/RACGTPases. Plant Physiol 147: 1527–1543

Yang Z (2002) Small GTPases: versatile signaling switches in plants. PlantCell (Suppl) 14: S375–S388

Yang Z (2008) Cell polarity signaling in Arabidopsis. Annu Rev Cell DevBiol 24: 551–575

Zhang C, Halsey LE, Szymanski DB (2011) The development and geom-etry of shape change in Arabidopsis thaliana cotyledon pavement cells.BMC Plant Biol 11: 27

Zheng ZL, Nafisi M, Tam A, Li H, Crowell DN, Chary SN, Schroeder JI,Shen J, Yang Z (2002) Plasma membrane-associated ROP10 smallGTPase is a specific negative regulator of abscisic acid responses inArabidopsis. Plant Cell 14: 2787–2797


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