REPORTS'

REPORTS' Fig. 3. Drag coefficient as a function of wind speed. CD is shown for an observation- based resistance coefficient, r = 0.02 an 54. The red open circles are the eval- uated CD from the current and nind Observations, the solid red line is a fitted quadratic curve to the CD estimates, and the red dashed lines are the 95% confidence limits for this quadratic curve. The black dotted tines represent the window for CD reported in (6), whereas the blue dots represent CD reported in (4). I nos as w a0 40 Mind Wiwi (wf0, m C') speeds below 30 m are somewhat noisy as a result of measurement uncertainty and the need to calculate a velocity derivative, which tends to enhance noise. However, they consistently show a decreasing trend of Co for wind speeds greater than 32 m s-I, the lower threshold for a category I hurricane on the Saffir-Simpson Scale. It is also apparent that the CD values are weakly dependent on the choice of the resistance co- efficient and are larger for increasing values of r. The drag coefficient estimates evaluated for r 0.1 cm s n are, on average, 20% greater than those calculated for r 0.001 cm s I from Erg- 3- To produce the best representation of CD for each r, a second-order curve (a function of the wind speed) was fitted by a least-squares technique to all estimated values of CD. The curves are displayed in Figs. 2 and 3. Addition- ally, the 95% confidence limits for the fitted curve are shown in Fig. 3. The pattern of the relationship between Co and the wind speed is robust, but the curve coefficients are determined by the value chosen for r in Eq. 3. However, all curves clearly show an initial increase of the drag coefficient and monotonic decrease as found by recent studies (3—B) after reaching a maximum value at —32 m s-I. Some of these studies (3, /9) imply that the decreasing drag at high winds seems to be related to the spray, foam, and bubbles from breaking waves that reduce the drag and allow the hurricane to slip over the sea. With the nearly full water-column ocean cur- rent measurements. the only unknown term left in the simplified equation of motion is the wind stress. Thus, the behavior of the drag coefficient (CD) can easily be estimated for a range of strong winds. Despite the fact that the drag coefficient is evaluated dittbrently here, estimates of Co determined "bottom-up" reasonably replicate the values determined "top-down" in recent studies (3-7). Results from our research show that CD peaks at a wind speed near 32 in s-I and ea then steadily decreases as the wind speed continues to rise. Our values for CD are in a range of Co values found using meteorological observations (4) for wind speeds greater than 32 m 5-I but are higher for lower wind speeds. These differences may be attributed to uncertain- ties in the wind measurements and the applica- bility of the simplified ocean dynamics at the lower wind speeds. References and Notes 1. K. Emanuel, Notate 436, 686 (2005). 2. 5. E. Larsen et oL, In Mod Stress Over the Ocean, I. S. F. Hoes,Y. rota, Eds. (Cambridge UMV. Press, New Wet 200U, chap. 7. 3. M. A. Damian et of, Geophys. Rex fa 31, 118306 10.10291200461019460 (2000. 4. M. D. Powell, P. J. Vickery, 1. A. Reinhold, Nature 422, 279 (20031. 5. E. D. Fernandez et oL, J. Geophys. Res. 111, C08013 10.1029/2005)(003018 (2006). 6. I. LA:saki. Ginis,I. Hara, J. Armos. SW 61,2334 (2004). 7. J. A. 1. Bye, A. O. Jenkins, I. Geophys. Res. 111, C03024 10.1029/2005jC003110 (2006). B. K. Emanuel,?. Armes. Sri. 60, 1420 (2003). 9. M A. lAncheR, W. J. Teague, E. prow, D. W. Wang, Geophys. Rn. Lela 32, L11610 10.1029/2005GL023014 (2005). 10. 0. w. Wang, O. A. Mitchell, W I. Teague, E. faros, M. S. Hulbert, Some 309, 896 (20051. IL W. J. Teague, E. prom, 0. W. Wang, O. A. Michell, ). Phys. OreoftogL, m press. 12. W. J. league, E. Jarosz, M. R. Carnes, D. A. Mitchell, P. J. H09311, Coot Shelf Res. 26, 2559 (2006). 13. J. F. Price, t. 8. Sanford, G. Z. Fcenstall, Phys. Oreonogr. 24, 233 (1994). 14. Matenals and methods are available as supporting material on Science Online. 15. G. T. Mitchum, W. Sturges, I. Plot Deettooge. 12,1310 (1982). 16. 5. 1. LenD, L Phys. &mow. 24, 2061 (1994). 17. 5.1. LenD, L Phys. Greasy,. 31, 2749 (2001). 1B. J. M. Mart Phys. Ocean*. 32, 3101 (2002). 19. E. L Andreas, ). Phys. °alma". 34, 1029 (2004). 20. We thank M. S. Hulbert, A. J. Quaid, and W. A. Goode for mowing support. We also thank the crews of the research vessels Seward Johnson I and II. This sot was supported by the Office of Naval Research as a part of the Naval Research Laboratory's basic research protect 'Mope to Shell Energetics and Exchange Dynamics (SEED)' under program element 0601153N, through the Minerals Management Service Environmental Studies Program Technology, and by the Minerals Management Service technology Assessment and Research Program on Nurticane Ivan. Supporting Online Material wvrtscierxemag.orgfcgikontentilull/315/5819/1707/DC1 SOM Tea Fig. S1 References 1B October 2006; accepted 10 February 2007 10.1126Ndence.1136466 CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes Rodolphe Barrangou,' Christophe Fremaux,2 Hake Deveau,3 Melissa Richards,' Patrick Boyava1,2 Sylvain Moineau,3 Dennis A. Romero,' Philippe Horvath's' Clustered regularly interspaced short palindromic repeats (CRISPR) are a distinctive feature of the genomes of most Bacteria and Archaea and are thought to be involved in resistance to bacteriophages. We found that, after viral challenge, bacteria integrated new spacers derived from phage genomic sequences. Removal or addition of particular spacers modified the phage-resistance phenotype of the cell. Thus, CRISPR, together with associated cos genes, provided resistance against phages, and resistance specificity is determined by spacer-phage sequence similarity. B acteriophages are arguably the most abundant biological entity on the planet (1) Their ubiquitous distribution and abundance have an important impact on micro- bial ecology and the evolution of bacterial genomes (2). Consequently, bacteria have devel- oped a variety of natural defense mechanisms that target diverse steps of the phage life cycle, notably blocking adsorption, preventing DNA injection, restricting the incoming DNA, and abortive infection systems. These antiviral bar- riers can also be engineered and manipulated to better control phase populations (2. 3). Numerous bacteria have been selected by humans and used extensively for lemrentation and biotechnology processes. Unfortunately. do- mesticated bacteria used in industrial applications are often susceptible to phage attack including Downloaded from www.sciencemag.org on November 2, 2008 wxfw.sciencemag.org SCIENCE VOL 315 23 MARCH 2007 1709 EFTA00610311 IREPORTS genera and species widely used as dairy cultures (4). Accordingly, the industry has devised various strategies to combat phage based on strain di- versity, bacteriophage-insensitive mutants, and plagnids bearing phage-resistance mechanisms. Streptococcus thermophilus is a low G+C Gram-positive bacterium and a key species ex- ploited in the formulation of dairy culture sys- tems for the production of yogurt and cheese. Comparative genomics analyses of closely related S. thennopinlus strains have previously revealed that genetic polymorphism primarily occurs at hypervariable loci, such as the cps and qac operons, as well as two clustered regularly interspaced short palindromic repeats (CRISPR) loci (5-7). CRISPR loci typically consist of sev- eral noncontiguous direct repeats separated by stretches of variable sequences called spacers and are oftentimes adjacent to cuts genes (CRISPR- associated). Although the function of CRISPR loci has not been established biologically, in silico analyses of the space's have revealed se- quence homology with foreign elements, includ- ing bacteriophage and plasmid sequences (7-9). Based exclusively on in silico analyses, several hypotheses have been put forward proposing roles for CRISPR and am genes, which include providing immunity against foreign genetic ele- ments via a mechanism based on RNA inter- ference (/0). We analyzed the CRISPR sequences of vari- ous S. thernwpItitus strains, including closely related industrial strains and phage-resistant var- iants (fig. SI). Differences in the number and type of spacers were observed primarily at the CRISPRI locus. Notably, phage sensitivity ap- peared to be correlated with CRISPRI spacer content. Specifically, spacer content was nearly identical between parental strains and phase- resistant derivatives, except for additional spacers present in the latter. These findings therefore suggest a potential relation between the presence of additional spacers and the differences ob- served in the phase sensitivity of a given strain. This observation prompted us to investigate the origin and Auction of additional spacers present in phage-resistant mutants. First, we tested the hypothesis that CRISPR loci are altered during the natural generation of phage-resistant mutants. A phage-host model system was selected, consisting of a phage- sensitive wild-type S. thermophilus strain widely used in the dairy industry, DGCC77I0 [wild type (WT)j and two distinct but closely related virulent bacteriophages isolated from industrial yogurt samples, phase 858 and phase 2972 (//). 'Danisco USA Inc., 3329 Agriculture Drive, Madison, WI 53716, USA. iDanisco France SAS, Bate Postale 10, F-86220 Dange.Saint.Romain. France. -tepartement de Bi3chimie et de fAiaolnolegie, Faculte des Sciences et de Genie, Groupe de Recherche en Frolcoie Buccale, lactate de Medecine Dentaire, Felix Reference Center for Bacterial Viruses, Universite Laval, G1K 7Pa Quebec, Canada. *To whom c hould be addressed. Email: Nine phage-resistant mutants were generated independently by challenging the WT strain with phage 858, phage 2972. or simultaneously with both (12), and their CRISPR loci were analyzed. Differences were consistently observed at the CRISPRI locus, where I to 4 additional spacers were inserted next to the 32 spacers present in the WT strain (Fig. l). The addition of new spacers in response to phage infection seemed to be polarized toward one end of the CRISPRI lo- cus. This is consistent with previous observations of spacer hypervariability at the leader end of the CRISPR locus in various strains (9. 13). Se- quence analysis of the additional spacers inserted in the CRISPR I locus of the various phase- msistant mutants revealed similarity to sequences found within the genomes of the phages used in the challenge (Fig. 2 and fig. S2). Interestingly. similarities were observed throughout the phage gentles. in most finictional modules, both on the coding and noneoding strands. No particular se- quence, gene. or functional gmup seemed to be targeted specifically. Them results reveal that, on becoming resistant to bacteriophages, the CRISPRI locus was modified by the integration of novel spacers, apparently derived from phage DNA. Surprisingly, we observed that some strains were resistant to both phases, whereas others caa5 cast cash cas7 repeetfspecer region ORF . '' 5 7 e 9 l0 11 12 13 14 16 16 17 It 19 20 21 22 23 24 26 26 27 26 29 30 31 31`r L 4421 WT .i412. Wrom*SIS2 LL .41C Wrats.S3 0.#141;011 Wroem'S. CL .41•2I Wroem'S5 Cc 44I Wron,7246 Wro2072' EL • I .<5*.Z.54. 1• 21 I fa.ii.;C WT4.20,2•40 WT,m6. 2977411610.611412 Wrimwte9,24.513311 Sensitivity to 0658 Sensitivity to 02972 fie'la tIO te. BP PM HO I .0,10. .0.10, I 1 1 1 I.!• 1 1 1 1 1 Fig. 1. Streptococcus thermophilus CRISPR1 locus overview, newly acquired spacers in phage- resistant mutants, and corresponding phage sensitivity. The CRISPR1 locus of DGCC7710 (WI) is at the top. The repeat-spacer region of WT is in the middle: repeats (black diamonds), spacers (numbered gray boxes), leader (., white box), and terminal repeat (T, black diamond). (Bottom left) The spacer content on the leader side of the locus in phage-resistant mutants is detailed, with newly acquired spacers (white boxes, 51 to 514). (Bottom right) The sensitivity of each strain to phages 858 and 2972 is represented as a histogram of the efficiency of plaguing (EOP), which is the plaque count ratio of a mutant strain to that of the wild-type. 4,858 packaging 02972 S9 S11 Sti eitpsW morphogenes 49S 57 S10 Mil morphogenesis S3 S12 SS sr, noar les is 314 $7 410 42' se se 43 412 $5' 41 44 413 Se replication "riser *" ; regulation SP S41 S13 1 kb Fig. 2. S. thermophilus phase genome maps with the position of sequences similar to the acquired CRISPR1 spacers of the phage-resistant mutants. Spacers shown above and below the genome maps indicate that the spacer matches a sequence on the (+) and on the (—) strand, respectively. An asterisk indicates the existence of a SNP between the spacer sequence and that of the phage genome (fig. S1). The genome sequences of phage 2972 (accession number AY699705) and phase 858 are 93% identical. Downloaded from www.sciencemag.org on November 2, 2008 1710 23 MARCH 2007 VOL 315 SCIENCE www.sciencemag.org EFTA00610312 REPORTS' were resistant only to the phase used in the chal- lenge (Fig. I). The phage-resistance profile seemed correlated to the spacer content, such that strains with spacers showing 100% identity to sequences conserved in both phases were resistant to both phases, such as spacers 53, S6, and S7. In contra* when nucleotide polymor- phians were observed between the spacer and the phase sequence [from I to 15 single-nucleotide polymorphisms (SNPs) over 29 or 30 nucleo- tides], the spacer did not seem to provide re- sistance, such as spacers SI, S2, S4, S5, and S8 (Fig. I and fig. S2). In addition, when several spacers were inserted (S9 to 514), phase re- sistance levels were higher. These findings indi- cate that the CRISPR I locus is subject to dynamic and rapid evolutionary changes driven by phase exposure. Altogether. these results reveal that CRISPR loci can indeed be altered during the generation of phase-resistant mutants and also establish a link between CRISPR content and phase sensitivity. These findings suggest that the presence of a CRISPR spacer identical to a phase sequence provides resistance against phases containing this particular sequence. Iv• To determine whether CRISPR spacer con- tent defines phase resistance, we altered the CRISPR I locus by adding and deleting spacers (/2) and tested subsequent strain sensitivity to phases. All constructs were generated and inte- grated into the S. thermaphilas chromosome with the system developed by Russell and Klaenhanuner ( / 4). We removed the spacers and repeats in the CRISPRI locus of strain WT,/,,014-sis2 and replaced them with a single repeat without any sparer (12). The resulting strain Art,„„s- I s2ACRISPR l was sensitive to phase 858, which indicated that the phase resistance of the original phage-resistant mutant (WT.:45045152) was probably linked to the presence of SI and S2 (Fig. 3). Further, to address the critical question of whether adding spacers provides novel phase resistance, we replaced the CRISPRI locus of strain WT02972. with a version containing only spacers SI and S2 (12) and tested whether the phase sensitivity was affected. Remarkably, the resuhing strain WT,/,29724s4::pS1S2 gained re- sistance to phase 858, which suggested that these two spacers have the ability to provide phase resistance de novo (Fig. 3). Altogether, COCOS. Cast 0986 caul.. ORF the rot, ant case cas7\ ORF toss 19 cast cast cas7 poRi ORF CI cast cast ficV pau cat cast 44997.....: V. 114±. VI. C4SIC;646 4D Sensitivity to ease Sensitivity to 02972 19, 114 1,1 ir I ly .0. tO• i i i I I I i t L WLesesINN N. WiessesleteiCRISPR1 NI. WT •ets2::pR IV. WT,„ 44::pS1S2 V. WTeases122::pcas.5— vi. WT •sis2::pcas7— Fig. 3. CRISPR spacer engineering, cos gene inactivation, and corresponding phage sensi- tivity. I, mutant WTItts' s 2; II, mutan WT,pess• sis2ACRISPR1 in which CRISPR1 was deleted; Ill, mutant WToesan '::pR in which CRISPR1 was displaced and replaced with a unique repeat; IV, WT.2972' 5°::p5152, mutant of strain WT4,25,72' 5° in which CRISPR1 was displaced and re- placed with a version containing 51 and 52; V, favass.sis2::pcos5— with cosy inactivated; VI, Wfsesa' sis2::pcos7— with cas7 inactivated. pORI indicates the integrated plasmid (12). The phage sensitivity of each strain to phages 858 and 2972 is represented at the bottom as a histogram of the efficiency of plaguing (EOP). 0 ORF these observed modifications establish the link between the CRISPR spacer content and phase resistance. In the process of generating strain WTinsx' sls2ACRISPRI, we created WTosso-sis2::pR, a variant that contains the inte- gration vector with a single repeat inserted be- tween the cats genes and the native CRISPR I locus (Fig. 3). Unexpectedly, strain WT,/,1,3x' sis2::pR was sensitive to phase 858, although spacers SI and S2 remained on the chromosome (Fig. 3). Similarly, the WT029724S4::pS1 S2 construct lost the resistance to phase 2972, although spacer S4 is present in the chromosome (Fig. 3). These results indicated that spacers alone did not provide resistance, and perhaps, that they have to be in a particular genetic context to be effective. Although initial work suggested involvement in DNA repair (15), the current hypothesis is that eves genes (5, 16) are involved in CRISPR- mediated inununity (JO). Consequently, we in- activated two car genes in strain WT,t45,,,esis2 (12): cars (00G3513) and am 7. which are equiv- alent to No66571.0O657 and str06601sta0660, respectively (6, 7). The cars inactivation re- sulted in loss of the phase resistance (Fig. 3), and perhaps Cas5 acts as a nuclease, because it contains an IINII-type nuclease motif. In con- trast, inactivating clis7 did not alter the resist- ance to phase 858 (Fig. 3). Interestingly, we were repeatedly unable to generate CRISPR I phage-resistant mutants from the car? knock- out, perhaps because Cas7 is involved in the synthesis and/or insertion of new spacers and additional repeats. When we tested the sensitivity of the phage- resistant mutants, we found that plaque formation was dramatically reduced. but that a relatively small population of bacteriophage retained the ability to infect the mutants. We further analyzed phase variants derived from phage 858 that retained the ability to infect Ari.Nom S I S2. In par- ticular, we investigated the sequence of the ge- nome region corresponding to additional spacers SI and S2 in two virulent phase variants. In both cases, the genome sequence of the phase var- iant had mutated, and two distinct SNPs were identified in the sequence corresponding to spacer SI (fig. 53). Overall, prokaryotes appear to have evolved a nucleic acid-based "immunity" system where- by specificity is dictated by the CRISPR spacer content, while the resistance is provided by the Cas enzymatic machinery. Additionally, we spec- ulate that some of the cats genes not directly providing resistance are actually involved in the insertion of additional CRISPR spacers and re- peas. as part of an adaptive "immune" response. Further studies are desired to better characterize the mechanism of action and to identify the specific function of the various my genes. This nucleic acid-based system contrasts with amino acid-based counterpart in eukaryotes through which adaptative immunity is not inheritable. Downloaded from www.sciencemag.org on November 2, 2008 www.sciencemag.org SCIENCE VOL 315 23 MARCH 2007 1711 EFTA00610313 IREPORTS The inheritable nature of CRISPR spacers sup- ports the use of CRISPR loci as targets for evo- lutionary. typing, and comparative genomic studies (9, 17-19). Because this system is reactive to the phage environment, it likely plays a sig- 3. nificant role in prokaryotic evolution and ecol- ogy and provides a historical perspective of phage exposure, as well as a predictive tool for phage sensitivity. The CRISPR-cos system may accordingly be exploited as a virus defense mech- animn and also potentially used to reduce the dissemination of mobile genetic elements and the acquisition of undesirable traits such as anti- biotic resistance genes and virulence markers. From a phage evolution perspective. the inte- grated phage sequences within CRISPR loci may also provide additional anchor points to facilitate recombination during subsequent phage infec- tions, thus increasing the gene pool to which phages have access (20). Because CRISPR loci are found in the majority of bacterial genera and are ubiquitous in Archaea (5, 13,21), their study will provide new insights into the relation and codirected evolution between prokaryotes and their predators. 1. 2. 4. 5. 6. 7. 8. 9. to. 11. 12. 13. 14. 15. References and Notes M Brenban, F. Rotates, fiends Microbic[ 13, 278 (2005). S. Clbarn-Chennoull, A. &won, fa.-L Diroann, H. Bdosav, 1. Beetroot 186, 3677 (2004). ). M. Stoma, T. R. Klaenhammer, Nor. Rev. Mktobiol. 4, 395 (2006). H. Brasses, AMA Rev. Mirtabtot. 55, 283 (2001). R. Jansen, J. 0.A. van Embrien, W. Gaasva, L. M. Scholl, Mot Whatnot 43, 1565 (2002). A. gelatin er a., Not Madinat. 22, 1554 (2004). A. Solaa, B. (Moguls, A. Sorokin, S. D. Ehrlich, Mkrobiofogy 151, 2551 (2005). F. J. M. Mona, C. Ellez-VIUasencr, I. Garda-Manfnez, E. Soda, 1. Ala! for. 60, 174 (2005). C. Poured, G. Salvignol, G. Yergnaud, AlIClabiology 151, 653 (2005). K. S. Makarova, N. V. Cabin, S. A. Shabalina, Y.1. Wolf, E. V. Koomn, &of. Thera 1, 7 (2006). C. Levesque et at, Appl. fortiori. MktobioL 71, 4057 (2005). Information on matenals and methods for the generation of phage-resistant mutants, engineering of CRISPR spacers (figs. 54 and 55), and inactivation of cm genes is available on Selena Online. R. K Wiest'', P. Redder, R. A Gantt, K. flrigger, erchoro 2, 59 (2006). W. M. Russell. 1. R. Klaenhammer, Ant &Sion aunotoot 67, 4361 12001). K. S. Makarova, L marina N. V. Grishin, I. B. Rogow, E. V. KOOMII, maker ones Rel. 30, 482 (2002). 16. 0. H. Haft, J. Selengut, E. F. lIcogodin, IL E. Nelson, rad Cookout. Biol. 1, e60 (2005). 17. P. M A. Greener), A. E. Ilunuhoten, D. van Soolingen, J. O. A. van EmbOen, mat microbe:et 10, 1057 (1993). 18. E. F. mengodm at al, J. &trend. 187, 4935 (2005). 19. R. 1. Deem, E. F. Mongoohn, I. 8. Emerson, K. E. Nelson, Beetenot 188, 2360 (2006). 20. R. W. Hendrix et of., Proc. Nod. Arad. Sc!. II.S.A 96, 2192 (1999). 21. J. S. Godde, A. Bickerton, J. MaL Eva. 62, 718 (2806). 22. We thank L. Bayer, C. Vos, and A..C. Codti.Mcovoisin of Caruso) !moaner', as well as J. Laboot6 and 0. Tremblay of Universne Laval for technical supporL and E. Beth Hansen for diuussion and critical review of the manuuript. Also, we thank 1. R. Klaenhammer for providing the integration system. This work was supported by holing from Danisco erS. Also, S. AL would like to acknovAedge support from the Natural Sciences and EnMneenng Research Council of Canada (NSERC) Cnscovery Program. Sequences sere deposited in GenBank, accession numbers Et434458 to 0434500. Supporting Online Material iwm.sciencemag.orgfcgikonteouluW315/5819/1709/DC1 Materials and Methods Fags. 51 to 55 References and Nam 29 November 2006: accepted 16 Fetguany 2007 ronzurnerde.rustoo A G Protein-Coupled Receptor Is a Plasma Membrane Receptor for the Plant Hormone Abscisic Acid Xigang Liu,1*3 Yanling Yue,' Bin Li,3 Yanli Hie/ Wei Litz Wei-Hua Wu,3 Ligeng Ma" The plant hormone abscisic add (ABA) regulates many physiological and developmental processes in plants. The mechanism of ABA perception at the cell surface is not understood. Here, we report that a G protein—coupled receptor genetically and physically interacts with the G protein a subunit GPA1 to mediate all known ABA responses in Arobidopsis. Overexpressing this receptor results in an ABA-hypersensitive phenotype. This receptor binds ABA with high affinity at physiological concentration with expected kinetics and stereospecifidty. The binding of ABA to the receptor leads to the dissociation of the receptor-GPA1 complex in yeast. Our results demonstrate that this G protein—coupled receptor is a plasma membrane ABA receptor. A bscisic acid (ABA) is an important hormone that mediates many aspects of plant growth and development, particu- larly in response to the environmental stresses (1-3). Several components involved in the ABA signaling pathway have been identified (4). Two recent reports have shown that the nuclear RNA binding protein flowering time control protein (FCA) (5) and the chloroplast protein Mg chelatase II subunit (6) are ABA receptors (6). 'National Institute of Biological Sciences, 7 Science Park Road, 2.tiongguancun Life Science Park, Beijing 102206, China. /Laboratory of Molecular and Cellular Biology, Hebei Normal University, Shipathuang, Hebei 050016, China. 'State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences. China Agricultural University, Beijing 100094, China. 'To whom coat nOence should be addressed. Email: In contrast, several earlier experiments had sug- gested that extracellular perception is critical for ABA to achieve its functions (7-9). Thus, other ABA receptors, especially plasma membrane- localized receptors. may be the major players for perceiving extracellular ABA and mediating the classic ABA signaling responses. Ligand-mediated signaling through G protein- coupled receptors (GPCR5) is a conserved mechanism for the extracellular signal percep- tion at the plasma membrane in entrap/one organisms (10). The GPCR-mediated signaling pathway plays a central role in vital processes such as vision, taste, and olfaction in animals (11). IlOwever. the higher plant Arubidopsir thulium has only one canonical Ga (GPAI) subunit, one G0 subunit, and two Gy subunits (12-16). The significance of these subunits in plant systems is poorly understood; only one Arubidoµsis putative GPCR protein (GCRI ) has been characterized in plants (17-20), and no ligand has been defined for any plant GPCR. To identify previously unrecognized GPCR proteins in Arubidopsir, we started by searching the Arabidopuis genome and found a gene (GCR2, GenBank accession code At Ig52920) encoding a putative GPCR. Transmembrane structure prediction suggests that GCR2 is a membrane protein with seven transmembrane helices (fig. SI, A and B). The subsequent cel- lular localization analysis confimied its plasma membrane localization in the transgenic plant root (fig. SIC). GCR2—yellow fluorescent pro- tein (YFP) is detected in the membrane fraction isolated from the GCR2-YFP transgenic plant. Similar to GCRI (19), GCR2 is mostly asso- ciated with the membrane fraction (fig. SID). Furthermore. even after washing with detergent or a higher pH buffer, GCR2 is retained with the membrane fraction, suggesting that GCR2 is an integral membrane protein (fig. SID). One feature of the GPCR is its ability to interact with G protein to form a complex. To confirm the physical interaction between GCR2 and Ger. we used four different approaches to detect their interaction. We first used surface plasmon resonance spectroscopy to investigate the interaction between GCR2 and GPAI. For this purpose. we expressed and purified recom- binant GCR2 and GPAI proteins in bacteria (fig. S2). This in vitro assay clearly indicated that GPAI is capable of binding to GCR2, where- as no binding activity was detected between GPAI and bovine serum albumin (BSA) (fig. S3, A and B). The dissociation binding con- stant (Kd) for GCR2 and GPA I is 2.1 x 10-9 M (fig. 53C). Downloaded from www.sciencemag.org on November 2, 2008 1712 23 MARCH 2007 VOL 315 SCIENCE www.sciencemag.org EFTA00610314