Arabidopsis GDSL lipase 2 plays a role in pathogen defense via negative regulation of auxin signaling

Biochemical and Biophysical Research Communications 379 (2009) 1038–1042

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Arabidopsis GDSL lipase 2 plays a role in pathogen defense via negative regulation of auxin signaling Dong Sook Lee 1, Bo Kyung Kim 1, Sun Jae Kwon, Hak Chul Jin, Ohkmae K. Park * School of Life Sciences and Biotechnology, Korea University, Anam-dong, Seongbuk-gu, Seoul 136-701, Republic of Korea

a r t i c l e

i n f o

Article history: Received 26 December 2008 Available online 13 January 2009

Keywords: Arabidopsis GDSL lipase Pathogen resistance Auxin signaling GLIP2 Erwinia carotovora

a b s t r a c t GLIP1 was isolated previously from Arabidopsis, as a salicylic acid-responsive secreted GDSL lipase that functions in resistance to Alternaria brassicicola [I.S. Oh, A.R. Park, M.S. Bae, S.J. Kwon, Y.S. Kim, J.E. Lee, N.Y. Kang, S. Lee, H. Cheong, O.K. Park, Secretome analysis reveals an Arabidopsis lipase involved in defense against Alternaria brassicicola. Plant Cell 17 (2005) 2832–2847.]. To extend our knowledge of the roles played by GLIPs in Arabidopsis, we conducted functional studies of another family member, GLIP2. GLIP2 transcripts were expressed in young seedlings, as well as in the root and stem tissues of mature plants. GLIP2 transcript levels were elevated by treatment with salicylic acid, jasmonic acid and ethylene. Recombinant GLIP2 proteins possessed lipase and anti-microbial activities, inhibiting germination of fungal spores. In comparison to wild type plants, T-DNA insertion glip2 mutants exhibited enhanced auxin responses, including increased lateral root formation and elevated AUX/IAA gene expression. When challenged with the necrotropic bacteria Erwinia carotovora, glip2 mutants exhibited more susceptible phenotypes than wild type plants. These results suggest that GLIP2 plays a role in resistance to Erwinia carotovora via negative regulation of auxin signaling. Ó 2009 Elsevier Inc. All rights reserved.

GDSL lipases represent a subfamily of lipolytic enzymes, which like other members of the lipase and esterase families, possess a conserved catalytic triad (Ser, Asp, and His) [2]. However, in contrast to most lipases that contain a GxSxG motif, GDSL lipases exhibit a GDSL motif GxSxxxxG, in which the active site Ser is located near the N-terminus [3,4]. Previous studies have revealed the structures, catalytic mechanisms and other biochemical properties of microbial GDSL enzymes [5–10]. GDSL lipases have also been found in various plant species including Arabidopsis, rice and maize, and they have been implicated in plant development, morphogenesis and the defense response [3]. Enod8 is a member of the GDSL lipase/esterase family that was isolated from Medicago sativa root nodules [11]. In Brassica napus, BnLIP2 encodes a GDSL lipase; its expression was induced during germination and maintained in mature seedlings, suggesting functions in both germination and morphogenesis [12]. GDSL lipases perform critical roles in the biotic and abiotic stress responses of plants. GLIP1 is an Arabidopsis GDSL lipase that possesses anti-microbial activity and regulates pathogen resistance to Alternaria brassicicola in association with ethylene signaling [1]. In hot pepper, CaGLIP1 encodes a GDSL lipase that has been shown to modulate pathogen and wound stress resistance [13,14]. In contrast to the pathogen resistance provided

* Corresponding author. Fax: +82 2 3291 3458. E-mail address: [email protected] (O.K. Park). 1 These authors contributed equally to this work. 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.01.006

by Arabidopsis GLIP1, virus-induced gene silencing of CaGLIP1 conferred resistance to Xanthomonas campestris pv. vesicatoria. The plant hormone auxin regulates a variety of physiological processes including lateral root formation [15,16]. Exogenous auxin application increases the number of lateral roots and treatment with auxin transport inhibitors decreases their number [15–18]. Auxin rapidly induces a number of genes called early auxin responsive genes such as Aux/IAAs, SAURs, and GH3s [19,20]. Among these, the Aux/IAAs are the best characterized and appear to function as transcriptional repressors of auxin-responsive gene expression [21,22]. Recent studies show that in addition to three known defense-related hormones, salicylic acid (SA), jasmonic acid (JA), and ethylene (ET), auxin also influences pathogen resistance [23– 26]. Infection with Peudomonas syringae pv. tomato (Pst) DC3000 or expression of P. syringae type III effector AvrRpt2 in plants increased the level of indole-3-acetic acid (IAA), the most abundant plant auxin, leading to enhanced disease development [23,24]. Furthermore, disease resistance was induced by repression of auxin signaling, resulting from stabilization of AUX/IAA proteins via either microRNA miR393a- or SA-mediated negative regulation of F-box auxin receptors [25,26]. In this work, we conducted a functional analysis of Arabidopsis GLIP2, a secreted lipase containing a GDSL motif. GLIP2 was strongly expressed in roots and induced by SA, JA and ET. In contrast to wild type plants, T-DNA insertion glip2 mutants showed elevated expression of IAA genes and an increased number of lateral roots, as well as enhanced susceptibility to Erwinia carotovo-

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ra. Our results suggest that GLIP2 plays a role in pathogen resistance by suppressing the auxin response. Materials and methods Plant materials, growth conditions, and hormone treatments. The Columbia (Col-0) ecotype of Arabidopsis thaliana was grown at 24 °C under long-day condition (16-h light/8-h dark cycle) in a growth room. Mutants of glip2-1 (SALK_025414) and glip2-4 (SALK_109449) were isolated from the SALK T-DNA insertion lines. For hormone treatments, SA (1 mM), methyl JA (100 lM), ethephon (100 lM), and 2,4-dichlorophenoxyacetic acid (2,4-D) (10 and 100 lM) were dissolved in water and applied to 2-week-old seedlings grown in liquid MS medium with continuous shaking. Tissues were harvested at the times indicated. RNA analysis. RT-PCR analysis was performed as described previously [27]. The primers used were as follows: GLIP2, 50 -GGA GGA AGG AAG TTT GGA TTC-30 and 50 -CTC TGC AAT TTG TTG ATG TGC30 ; IAA1, 50 -GAG CTT CGT TTG GGA TTA C-30 and 50 -CAT AAG GCA GTA GGA GCT TC-3’; IAA2, 50 -CGG AAG AAC AGA GAA GAT C-30 and 50 -GTC TAG AGC AGG AGC GTC-30 ; Actin, 50 -GGC GAT GAA GCT CAA TCC AAA CG-30 and 50 -GGT CAC GAC CAG CAA GAT CAA GAC G-30 . GUS staining. Whole seedlings were immersed in GUS staining solution containing 100 mM NaH2PO4 (pH 7.0), 10 mM EDTA, 0.5 mM Triton X-100, 0.5 mM K4[Fe(CN)6] and 2 mM 5-bromo-4chloro-3-indolyl-b-glucuronide, and then incubated for 16 h at 37 °C. The staining solution was removed and samples were washed with 70% ethanol. Purification of GLIP2 proteins and assays for lipase and anti-microbial activities. The full-length coding region of GLIP2 was cloned

into the pGEX 6P-1 vector (Amersham) and used for transformation of Escherichia coli strain BL21 (DE3). Purification of GLIP2 and the lipase assay were performed as described previously [1]. The antimicrobial activity of GLIP2 was determined by adding recombinant proteins (0.1–5 lg) to a 10 ll A. brassicola spore suspension (5  105 spores/ml). Samples were placed on glass slides, incubated for 18 h at 23 °C, and then observed by light microscopy. Measurement of gravitropism. Seedlings were grown vertically in Petri dishes for 3 days, gravistimulated by rotating the dishes by 90°, and then grown in darkness for a further 24 h. Images of the seedlings were then taken and the angle of gravicurvature was measured. The experiment was repeated three times. Inoculation with Erwinia carotovora SSC1. Three-week-old seedlings were inoculated with 10 ll of an E. carotovora cell suspension (1  107 cells/ml). This experiment was designed as a randomized complete block with 5 replications and one plant per replication. The experiment was repeated at least three times. Results and discussion Expression analysis of GLIP2 Previously, we performed an Arabidopsis secretome analysis and identified GLIP1 as an SA-responsive secreted protein that is involved in the incompatible interaction with A. brassicicola [1]. We

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Fig. 1. Analysis of GLIP2 gene expression. (A) Semi-quantitative RT-PCR analysis of GLIP1 and GLIP2 expression in tissues. (B) Histochemical staining of GUS expression in pGLIP2:GUS transgenic plants at different developmental stages. GUS staining was performed on three independent transformed lines and similar results were obtained. (C) Semi-quantitative RT-PCR analysis of GLIP2 expression in roots in response to hormone treatments. Two-week-old seedlings grown in liquid MS media were treated with SA (1 mM), methyl JA (100 lM) and ethephon (100 lM) and roots were harvested at the times indicated.

Fig. 2. Lipase and anti-microbial activities of recombinant GLIP2 proteins. (A) Coomassie blue-stained gel of purified recombinant GST and GST-GLIP2 proteins. (B) Lipase activity of the recombinant GLIP2 proteins. In the assays, proteins (4 lg) were incubated with two substrates, p-nitrophenyl acetate (pa3) and p-nitrophenyl butyrate (pa4). (C) Antimicrobial activity of recombinant GLIP2 proteins against A. brassicicola. Recombinant proteins (5 lg) were added to 10 ll of an A. brassicicola spore suspension (5  105 spores/ml). The samples were incubated for 18 h at 23 °C and then germination and structural damage were assessed using a microscope. GST was used as a mock control.

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also identified six additional GLIP genes in the Arabidopsis genome, which were designated GLIP2 to GLIP7. To expand our understanding on the roles of GLIPs in defense responses, GLIP2 that exhibits 81% identity to GLIP1 at the amino acid level was subjected to further studies. Microarray data available at Genevestigator (https://www. genevestigator.ethz.ch) indicate strong expression of GLIP2 in root tissue. We used RT-PCR to compare the expression of GLIP2 and GLIP1 in various tissues at different stages (Fig. 1A). GLIP2 was expressed strongly in root and stem tissues and in early seedling stages (1-week-old), whereas GLIP1 showed expression in all tissues tested and at all stages. To determine GLIP2 expression more precisely, we generated GLIP2 promoter-b-glucuronidase (GUS) reporter transgenic plants (pGLIP2:GUS) expressing a fusion construct comprising 1.9 kb of GLIP2 promoter and GUS reporter gene. Several independent transgenic lines were generated and pGLIP2:GUS expression was monitored in various tissues and at different developmental stages (Fig. 1B). Using histochemical staining, we detected GUS activity throughout stages of development with different tissue-specific expression patterns. GUS expression was observed in cotyledons, hypocotyls and the roots of young seed-

lings (5- and 9-day-old), whereas in 4-week-old plants, GUS activity was found predominantly in root and stem tissues. These GUS staining results are consistent with the RT-PCR data for GLIP2 expression (Fig. 1A). Defense responses to pathogens are regulated by networks of SA, JA, and ET signaling pathways [28]. We thus evaluated the effect of SA, methyl JA, and ethephon on GLIP2 expression in above- and underground tissues (stem and leaf, or root tissues, respectively; Fig. 1C). Hormone treatments induced GLIP2 expression in roots but not in aboveground tissues (data not shown), suggesting that GLIP2 functions specifically in root tissues. Although each hormone elicited a distinct GLIP2 expression pattern, induction was elicited by all three treatments, in contrast to GLIP1, which was induced only by ethephon [1]. The accumulation of GLIP2 transcripts peaked 12–24 h after SA treatment, and then decreased to the initial expression level. GLIP2 expression was enhanced 12–48 h after methyl JA treatment and ethephon strongly induced GLIP2 expression at 48 h. These different responses to hormones suggest that GLIP2 is involved in plant defense and that its expression is regulated by SA, JA and ET in distinct ways.

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Fig. 3. Auxin-related responses of glip2 plants. (A) Genomic organization of GLIP2 and location of T-DNA insertion in glip2 mutants. Arrows indicate the positions of T-DNA insertions (triangles). Genomic GLIP2 DNA is represented by exons (black) and introns (white). The T-DNA orientation is indicated by left (LB) and right (RB) borders. The numbers refer to nucleotides in the genomic GLIP2 DNA. Chr 1, chromosome 1. (B) RT-PCR analysis of GLIP2 expression in wild type and glip2 mutant plants. (C) Phenotypes of 10-day-old wild type and glip2 mutant seedlings. (D) RT-PCR analysis of IAA genes. Expression of IAA genes were determined in 2-week-old wild type seedlings treated with 2,4-D for 3 h (upper gels). Transcript levels of IAA genes were measured in 2-week-old wild type and glip2 mutant seedlings (lower gels). (E) Gravitropic response of wild type and glip2 mutant plants. Gravitropic curvature was observed in wild type and glip2 seedlings grown vertically for 3 days, rotated by 90°, and incubated for an additional 24 h in darkness.

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Lipase and antimicrobial activities of GLIP2 proteins

Function of GLIP2 in auxin signaling To assess the function of GLIP2 in vivo, we obtained two T-DNA insertion mutants glip2-1 and glip2-4, in which T-DNA had inserted in the first intron and second exon, respectively (Fig. 3A). These mutants did not accumulate GLIP2 transcripts, as determined by RT-PCR analysis (Fig. 3B). Under normal growth conditions, the aboveground phenotypes of both the glip2-1 and glip2-4 mutants appeared similar to wild type plants (Fig. 3C). In contrast, we observed significantly enhanced root development in the mutants, which showed a dramatic increase in the number of lateral roots compared to wild type. Since lateral root formation is regulated by auxin [29,30], the observed changes in glip2 phenotypes suggest that auxin signaling has been de-repressed or promoted in glip2 plants. To test for the possibility that GLIP2 function is implicated in auxin response, we compared AUX/IAA gene expression in wild type and glip2 mutant plants (Fig. 3D). Increased levels of IAA1 and IAA2 transcripts were detected in glip2-1 and glip2-4 mutants, whereas only exogenously added synthetic auxin 2,4-D elevated these transcript levels in wild type plants. Next, we examined the gravitropic response, which is also regulated by auxin [31,32]. The hypocotyls of wild type seedlings bent almost vertically after 24 h gravistimulation, whereas glip2 seedlings exhibited impaired gravitropic curvature. The results together suggest that GLIP2 negatively regulates auxin signaling. Response of glip2 mutant plants to Erwinia carotovora We examined the resistance phenotypes of wild type, glip2-1 and glip2-4 plants against the necrotropic bacterial pathogen E. carotovora. Following pathogen infection, both glip2 mutants displayed enhanced disease symptoms compared to wild type plants (Fig. 4A). It was then assessed whether GLIP2 expression was induced in response to E. carotovora. We divided plants into underground (roots) and aboveground (non-roots) parts, after which GLIP2 induction was evaluated by RT-PCR analysis (Fig. 4B). Following treatment with E. carotovora, we only detected accumulation of GLIP2 transcripts in root tissue. It is intriguing to consider that disease symptoms appear in leaf tissues of pathogen-inoculated glip2 mutants, but pathogen-induced GLIP2 expression occurs in root tissue. These results suggest that GLIP2 may function systemically in pathogen resistance, possibly in a similar manner to GLIP1, which is involved in systemic resistance signaling [1]. Although GLIP2 and GLIP1 have properties in common, it is likely that they play distinct roles in the pathogen response. GLIP2 expression was only detected in root and stem tissues, and could be induced by all three defense-related hormones (SA, JA and ET). In contrast, GLIP1 expression was detected in all tissues and only increased in response to ET treatment. Our studies with glip2

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To examine whether GLIP2 possesses lipolytic activity, we expressed recombinant GST-GLIP2 fusion proteins in E. coli (Fig. 2A). Following protein purification, lipase activity was determined using p-nitrophenyl acetate and p-nitrophenyl butyrate as substrates (Fig. 2B). GST-GLIP2 displayed a large increase in lipase activity relative to the GST control. We then examined the antimicrobial activity of GLIP2 against A. brassicicola (Fig. 2C). In contrast to treatment with the GST control, we observed little germination and significant structural damage of fungal spores following incubation with GST-GLIP2 proteins. These results indicate that GLIP2 possesses both lipase/esterase and anti-microbial activities, as previously determined for GLIP1 [1].

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Fig. 4. GLIP2 functions in response to E. carotovora. (A) Disease development in glip2 plants inoculated with E. carotovora. Disease rates (0–5) were determined in 10-day-old wild type and glip2 seedlings 2 days after inoculation with a 5-ll drop of E. carotovora suspension (107 cells/ml). (B) Semi-quantitative RT-PCR analysis of GLIP2 expression in underground (roots) and aboveground (non-roots) tissues inoculated with E. carotovora. Seedlings (2-week-old) were treated with a 5-ll drop of E. carotovora suspension (107 cells/ml), incubated for 3 days, and then used for RNA extraction.

mutant plants suggest that GLIP2 is involved in auxin signaling, as supported by enhanced lateral root formation and elevated AUX/ IAA gene expression, as well as alteration in the gravitropic response. The association of GLIP1 with auxin signaling remains to be determined. Many microbes produce IAA, which regulates both microbial and host plant physiology [33]. Recent studies suggest that auxin is an important disease susceptibility factor and thus, repression of plant auxin signaling represents a critical factor in increased resistance to bacterial pathogens [24,25]. Consistent with these previous reports, activation of auxin signaling may be associated with susceptibility to E. carotovora in glip2 mutant plants. Taken together, our results suggest that GLIP2 may function in pathogen resistance via negative effect on auxin signaling in plants. Acknowledgments We thank Soon Il Kwon for technical comments. This work was supported by grants from the Plant Signaling Network Research Center (R11-2003-008-04004-0), the Biotechnology Development Program (2006-02762) funded by the Korea Science and Engineering Foundation and from the Basic Research Program (C00441) funded by the Korea Research Foundation. References [1] I.S. Oh, A.R. Park, M.S. Bae, S.J. Kwon, Y.S. Kim, J.E. Lee, N.Y. Kang, S. Lee, H. Cheong, O.K. Park, Secretome analysis reveals an Arabidopsis lipase involved in defense against alternaria brassicicola, Plant Cell 17 (2005) 2832–2847. [2] C.C. Akoh, G.C. Lee, Y.C. Liaw, T.H. Huang, J.F. Shaw, GDSL family of serine esterases/lipases, Prog. Lipid Res. 43 (2004) 534–552. [3] D.J. Brick, M.J. Brumlik, J.T. Buckley, J.X. Cao, P.C. Davies, S. Misra, T.J. Tranbarger, C. Upton, A new family of lipolytic plant enzymes with members in rice, arabidopsis and maize, FEBS Lett. 377 (1995) 475–480.

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[4] C. Upton, J.T. Buckley, A new family of lipolytic enzymes?, Trends Biochem Sci. 20 (1995) 178–179. [5] K.K. Kim, K.Y. Hwang, W.H. Jang, H. Lee, O.J. Yoo, M.U. Choi, S.W. Suh, Crystallization and preliminary X-ray crystallographic analysis of carboxylesterase from pseudomonas fluorescens, Arch. Biochem. Biophys. 302 (1993) 417–419. [6] M.E. Carinato, P. Collin-Osdoby, X. Yang, T.M. Knox, C.A. Conlin, C.G. Miller, The apeE gene of Salmonella typhimurium encodes an outer membrane esterase not present in Escherichia coli, J. Bacteriol. 180 (1998) 3517–3521. [7] J. Li, U. Derewenda, Z. Dauter, S. Smith, Z.S. Derewenda, Crystal structure of the Escherichia coli thioesterase II, a homolog of the human Nef binding enzyme, Nat. Struct. Biol. 7 (2000) 555–559. [8] Y.T. Huang, Y.C. Liaw, V.Y. Gorbatyuk, T.H. Huang, Backbone dynamics of Escherichia coli thioesterase/protease I: evidence of a flexible active-site environment for a serine protease, J. Mol. Biol. 307 (2001) 1075–1090. [9] D. Vujaklija, W. Schröder, M. Abramic´, P. Zou, I. Lescic´, P. Franke, J. Pigac, A novel streptomycete lipase: cloning, sequencing and high-level expression of the Streptomyces rimosus GDS(L)-lipase gene, Arch. Microbiol. 178 (2002) 124–130. [10] K. Riedel, D. Talker-Huiber, M. Givskov, H. Schwab, L. Eberl, Identification and characterization of a GDSL esterase gene located proximal to the swr quorumsensing system of Serratia liquefaciens MG1, Appl. Environ. Microbiol. 69 (2003) 3901–3910. [11] D. Pringle, R. Dickstein, Purification of ENOD8 proteins from Medicago sativa root nodules and their characterization as esterases, Plant Physiol. Biochem. 42 (2004) 73–79. [12] H. Ling, J. Zhao, K. Zuo, C. Qiu, H. Yao, J. Qin, X. Sun, K. Tang, Isolation and expression analysis of a GDSL-like lipase gene from Brassica napus L, J. Biochem. Mol. Biol. 39 (2006) 297–303. [13] K.J. Kim, J.H. Lim, M.J. Kim, T. Kim, H.M. Chung, K.H. Paek, GDSL-lipase1 (CaGL1) contributes to wound stress resistance by modulation of CaPR-4 expression in hot pepper, Biochem. Biophys. Res. Commun. 374 (2008) 693– 698. [14] J.K. Hong, H.W. Choi, I.S. Hwang, D.S. Kim, N.H. Kim, D.S. Choi, Y.J. Kim, B.K. Hwang, Function of a novel GDSL-type pepper lipase gene, CaGLIP1, in disease susceptibility and abiotic stress tolerance, Planta 227 (2008) 539– 558. [15] L.M. Blakely, R.M. Blakely, P.M. Colowit, D.S. Elliott, Experimental studies on lateral root formation in radish seedling roots. II. Analysis of the dose-response to exogenous auxin, Plant Physiol. 87 (1988) 414–419. [16] M.J. Laskowski, M.E. Williams, H.C. Nusbaum, I.M. Sussex, Formation of lateral root meristems is a two-stage process, Development 121 (1995) 3303–3310. [17] R.C. Reed, S.R. Brady, G.K. Muday, Inhibition of auxin movement from the shoot into the root inhibits lateral root development in Arabidopsis, Plant Physiol. 118 (1998) 1369–1378.

[18] I. Casimiro, A. Marchant, R.P. Bhalerao, T. Beeckman, S. Dhooge, R. Swarup, N. Graham, D. Inze, G. Sandberg, P.J. Casero, M. Bennett, Auxin transport promotes Arabidopsis lateral root initiation, Plant Cell 13 (2001) 843–852. [19] S. Abel, A. Theologis, Early genes and auxin action, Plant Physiol. 111 (1996) 9– 17. [20] G. Hagen, T. Guilfoyle, Auxin-responsive gene expression genes, promoters and regulatory factors, Plant Mol. Biol. 49 (2002) 373–385. [21] T. Ulmasov, J. Murfett, G. Hagen, T.J. Guilfoyle, Aux/IAA proteins repress expression of reporter genes containing natural and highly active synthetic auxin response elements, Plant Cell 9 (1997) 1963–1971. [22] E. Liscum, J.W. Reed, Genetics of Aux/IAA and ARF action in plant growth and development, Plant Mol. Biol. 49 (2002) 387–400. [23] P.J. O’Donnell, E.A. Schmelz, P. Moussatche, S.T. Lund, J.B. Jones, H.J. Klee, Susceptible to intolerance—a range of hormonal actions in a susceptible Arabidopsis pathogen response, Plant J. 33 (2003) 245–257. [24] Z. Chen, J.L. Agnew, J.D. Cohen, P. He, L. Shan, J. Sheen, B.N. Kunkel, Pseudomonas syringae type III effector AvrRpt2 alters Arabidopsis thaliana auxin physiology, Proc. Natl. Acad. Sci. USA 104 (2007) 20131–20136. [25] L. Navarro, P. Dunoyer, F. Jay, B. Arnold, N. Dharmasiri, M. Estelle, O. Voinnet, J.D. Jones, A plant miRNA contributes to antibacterial resistance by repressing auxin signaling, Science 312 (2006) 436–439. [26] D. Wang, K. Pajerowska-Mukhtar, A.H. Culler, X. Dong, Salicylic acid inhibits pathogen growth in plants through repression of the auxin signaling pathway, Curr. Biol. 17 (2007) 1784–1790. [27] S. Lee, E.J. Lee, E.J. Yang, J.E. Lee, A.R. Park, W.H. Song, O.K. Park, Proteomic identification of annexins, calcium-dependent membrane binding proteins that mediate osmotic stress and abscisic acid signal transduction in Arabidopsis, Plant Cell 16 (2004) 1378–1391. [28] P.M. Schenk, K. Kazan, I. Wilson, J.P. Anderson, T. Richmond, S.C. Somerville, J.M. Manners, Coordinated plant defense responses in Arabidopsis revealed by microarray analysis, Proc. Natl. Acad. Sci. USA 97 (2000) 11655–11660. [29] I.D. Smet, S. Vanneste, D. Inze, T. Beeckman, Lateral root initiation or the birth of a new meristem, Plant Mol. Biol. 60 (2006) 871–887. [30] H. Fukaki, Y. Okushima, M. Tasaka, Auxin-mediated lateral root formation in higher plants, Int. Rev. Cytol. 256 (2007) 111–137. [31] P. Ottenschläger, C. Wolff, R.P. Wolverton, G. Bhalerao, H. Sandberg, M. Ishikawa, K. Evans, Palme, gravity-regulated differential auxin transport from columella to lateral root cap cells, Proc. Natl. Acad. Sci. USA 100 (2003) 2987– 2991. [32] R. Swarup, E.M. Kramer, P. Perry, K. Knox, H.M. Leyser, J. Haseloff, G.T. Beemster, R. Bhalerao, M.J. Bennett, Root gravitropism requires lateral root cap and epidermal cells for transport and response to a mobile auxin signal, Nat. Cell Biol. 7 (2005) 1057–1065. [33] S. Spaepen, J. Vanderleyden, R. Remans, Indole-3-acetic acid in microbial and microorganism-plant signaling, FEMS Microbiol. Rev. 31 (2007) 425–448.