20. R. Zhang, T. L. Delworth, Geophys. Res. Lett. 34, n/a(2007).

21. P. Chylek, J. D. Klett, G. Lesins, M. K. Dubey, N. Hengartner,Geophys. Res. Lett. 41, 1689–1697 (2014).

22. M. Ting, Y. Kushnir, R. Seager, C. Li, J. Clim. 22, 1469–1481(2009).

23. K. E. Trenberth, D. J. Shea, Geophys. Res. Lett. 33, L12704(2006).

24. J. R. Knight, J. Clim. 22, 1610–1625 (2009).25. L. Terray, Geophys. Res. Lett. 39, L19712 (2012).26. K. E. Taylor, R. J. Stouffer, G. A. Meehl, Bull. Am. Meteorol. Soc.

93, 485–498 (2012).27. Materials and methods are available as supplementary

materials on Science Online.28. R. L. Miller et al., J. Adv. Model. Earth Syst. 6, 441–477

(2014).29. M. Collins et al., Long-Term Climate Change: Projections,

Commitments and Irreversibility, T. F. Stocker et al., Eds.(Cambridge Univ. Press, Cambridge, 2013), pp. 1029–1136.

30. G. A. Schmidt, D. T. Shindell, K. Tsigaridis, Nat. Geosci. 7,158–160 (2014).

31. J. Hansen, R. Ruedy, M. Sato, K. Lo, Rev. Geophys. 48, RG4004(2010).

32. K. Cowtan, R. G. Way, Q. J. R. Meteorol. Soc. 140, 1935–1944(2014).

33. N. A. Rayner, J. Geophys. Res. 108 (D14), 4407 (2003).34. T. M. Smith, R. W. Reynolds, T. C. Peterson, J. Lawrimore,

J. Clim. 21, 2283–2296 (2008).35. Y. Xue, T. M. Smith, R. W. Reynolds, J. Clim. 16, 1601–1612

(2003).36. A. Kaplan et al., J. Geophys. Res. 103 (C9), 18,567–18,589

(1998).37. D. E. Parker, P. D. Jones, C. K. Folland, A. Bevan, J. Geophys.

Res. 99 (D7), 14373 (1994).38. R. W. Reynolds, T. M. Smith, J. Clim. 7, 929–948 (1994).39. The regression analyses applied to the full CMIP5 multimodel

mean yields a scaling factor (“beta”) for Northern Hemispheretemperature changes that slightly exceeds unity (beta = 1.053 T

0.0169), implying a real-world forced response that is slightlygreater than that estimated by the CMIP5 multimodel mean. Incontrast, North Atlantic mean temperatures yields a scalingfactor slightly below unity (beta = 0.916 T 0.0155), andNorth Pacific mean temperatures yield a scaling factorsubstantially below unity (beta = 0.629 T 0.0182), suggestingthat the CMIP5 multimodel mean substantially overestimatesthe amplitude of forced temperature changes over theNorth Pacific. Further details, including results for the twosubensembles (CMIP5-A1E and CMIP5-GISS), are available inthe supplementary materials (table S3).

40. S. Kravtsov, M. G. Wyatt, J. A. Curry, A. A. Tsonis, Geophys. Res.Lett. 41, 6881–6888 (2014).

41. NMO = 0.35 AMO + 0.43 PMO for CMIP5-All; NMO = 0.42 AMO +0.36 PMO for CMIP5-GISS; NMO = –0.06 AMO + 0.85 PMOfor CMIP5-AIE; AMO and PMO regression coefficients aresignificant at the P << 0.05 level based on a one-sided test.

42. M. E. Mann, B. A. Steinman, S. K. Miller, Geophys. Res. Lett. 41,3211–3219 (2014).

43. K. E. Trenberth, J. T. Fasullo, Earth’s Future 1, 19–32(2013).

44. M. H. England et al., Nature Clim. Change 4, 222–227(2014).

45. Y. Kosaka, S.-P. Xie, Nature 501, 403–407 (2013).46. S. McGregor et al., Nature Clim. Change 4, 888–892 (2014).47. M. E. Mann et al., Science 326, 1256–1260 (2009).48. M. E. Mann, K. A. Emanuel, Eos Trans. AGU 87, 233 (2006).49. E. R. Martin, C. Thorncroft, Geophys. Res. Lett. 41, 2169–2175

(2014).50. J. R. Knight, C. K. Folland, A. A. Scaife, Geophys. Res. Lett. 33,

L17706 (2006).51. Y. Kushnir, R. Seager, M. Ting, N. Naik, J. Nakamura, J. Clim.

23, 5610–5628 (2010).52. B. B. B. Booth, N. J. Dunstone, P. R. Halloran, T. Andrews,

N. Bellouin, Nature 484, 228–232 (2012).

ACKNOWLEDGMENTS

All raw data, Matlab code, and results from our analysis are availableat the supplementary website: www.meteo.psu.edu/holocene/public_html/supplements/Science2015. We acknowledge the WorldClimate Research Programme’s Working Group on CoupledModeling, which is responsible for CMIP, and we thank the climatemodeling groups for producing and making available their modeloutput. We thank K. Emanuel and G. Schmidt for helpful commentson earlier versions of the manuscript. B.A.S. acknowledges support

by the U.S. National Science Foundation Atmospheric andGeospace Sciences–Postdoctoral Research Fellowships (AGS-PRF)(AGS-1137750). Kaplan SST V2 data were provided by the NOAA/Office of Oceanic and Atmospheric Research/Earth SystemResearch Laboratory Physical Sciences Division, Boulder, Colorado,USA: www.esrl.noaa.gov/psd. HadISST data were provided by theMet Office Hadley Centre: www.metoffice.gov.uk/hadobs. ERSSTdata were provided by NOAA: www.ncdc.noaa.gov/data-access/marineocean-data/extended-reconstructed-sea-surface-temperature-ersst-v3b.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/347/6225/988/suppl/DC1Materials and MethodsSupplementary TextFigs. S1 to S7Tables S1 to S3References (53–58)

24 June 2014; accepted 26 January 201510.1126/science.1257856

PEST CONTROL

Full crop protection from aninsect pest by expression of longdouble-stranded RNAs in plastidsJiang Zhang,1 Sher Afzal Khan,2 Claudia Hasse,1 Stephanie Ruf,1

David G. Heckel,2 Ralph Bock1*

Double-stranded RNAs (dsRNAs) targeted against essential genes can trigger alethal RNA interference (RNAi) response in insect pests. The application of thisconcept in plant protection is hampered by the presence of an endogenous plantRNAi pathway that processes dsRNAs into short interfering RNAs. We found that longdsRNAs can be stably produced in chloroplasts, a cellular compartment that appears tolack an RNAi machinery.When expressed from the chloroplast genome, dsRNAs accumulatedto as much as 0.4% of the total cellular RNA.Transplastomic potato plants producing dsRNAstargeted against the b-actin gene of the Colorado potato beetle, a notorious agriculturalpest, were protected from herbivory and were lethal to its larvae. Thus, chloroplastexpression of long dsRNAs can provide crop protection without chemical pesticides.

Double-stranded RNA (dsRNA) fed to in-sects can be taken up by midgut cells andprocessed into small interfering RNAs(siRNAs) by the insect’s Dicer endoribo-nuclease (1–3). If the sequence of the fed

dsRNA matches that of an insect gene, gene silenc-ing by RNA interference (RNAi) disrupts expressionof the insect’s gene (3, 4). By targeting essentialinsect genes, dsRNAs can be developed into highlyspecies-specific insecticides (4). However, althoughexpression of dsRNAs targeted against insect genesin transgenic plants (1, 2, 5–8) has impaired growthand development, complete protection of the plantsand efficient killing of the insects have not beenachieved. dsRNAs at least 60 base pairs (bp) inlength are required for efficient uptake andbiological activity in the target insect (3), but theplant’s own system for producing small RNAs(9) prevents the accumulation of high amountsof long dsRNA. The major processing products ofdsRNA cleavage by Dicer are 21-bp siRNAs, butthese had little (10) or no effect when fed toinsects (3). Thus, rapid turnover of dsRNAs in theplant limits the efficacy of transgenic RNAi-basedanti-insect strategies.The plastids (chloroplasts) of plant cells are

derived from formerly free-living cyanobacteria,a group of prokaryotes that lack an RNAi path-

way. We reasoned that chloroplasts might becapable of stably accumulating long dsRNAs, inwhich case dsRNA expression from the plastidgenome could provide better protection againstinsect pests than dsRNA expression from thenuclear genome. To test the feasibility of stabledsRNA expression in plastids, we transformed thetobacco (Nicotiana tabacum) plastid genomewith three different types of dsRNA constructs(Fig. 1A and fig. S1). In ptDP constructs, thedsRNA is generated by transcription from twoconvergent (dual) promoters. In ptSL constructs,the dsRNA is also produced from two convergentpromoters, but each strand is additionally flankedby sequences forming stem-loop–type second-ary structures, which increase RNA stability inplastids (11). In ptHP constructs, hairpin-typedsRNA (hpRNA) is produced by transcriptionof two transgene copies arranged as an invertedrepeat (Fig. 1A). We targeted the Colorado po-tato beetle (Leptinotarsa decemlineata; CPB), anotorious insect pest of potato and other sola-naceous crops (e.g., tomato and eggplant). Bothlarvae and adults feed on foliage, skeletonizethe leaves, and, if left uncontrolled, completelydestroy the crop. In many areas of the world,the beetle has no natural enemies, and chemicalpesticides are the main method of CPB control.However, since the middle of the 20th century,CPB has developed resistance to all major insec-ticide classes (and therefore has been brandedan “international superpest”) (12).

SCIENCE sciencemag.org 27 FEBRUARY 2015 • VOL 347 ISSUE 6225 991

1Max-Planck-Institut für Molekulare Pflanzenphysiologie,D-14476 Potsdam-Golm, Germany. 2Max-Planck-Institutfür Chemische Ökologie, D-07745 Jena, Germany.*Corresponding author. E-mail: rbock@mpimp-golm.mpg.de

RESEARCH | REPORTS

on

Feb

ruar

y 26

, 201

5w

ww

.sci

ence

mag

.org

Dow

nloa

ded

from

o

n F

ebru

ary

26, 2

015

ww

w.s

cien

cem

ag.o

rgD

ownl

oade

d fr

om

on

Feb

ruar

y 26

, 201

5w

ww

.sci

ence

mag

.org

Dow

nloa

ded

from

o

n F

ebru

ary

26, 2

015

ww

w.s

cien

cem

ag.o

rgD

ownl

oade

d fr

om

As essential target genes, the CPB ACT andSHR genes were chosen. ACT encodes b-actin, anessential cytoskeletal protein, and SHR encodesShrub (also known as Vps32 or Snf7), an es-sential subunit of a protein complex involved inmembrane remodeling for vesicle transport.Disruption of these genes when the insects arefed in vitro synthesized dsRNAs induces mortal-ity with high efficacy (3, 13). To test longer dsRNAsand test for a possible synergistic action, we alsoproduced an ACT+SHR fusion gene. To confirmthe activity of these dsRNAs in the beetles, wesynthesized the dsRNAs (ACT, SHR, ACT+SHRfusion, and GFP as a control) by in vitro tran-scription, painted them onto young potato leaves,and fed the leaves to second-instar CPB larvae.All three insect gene–derived dsRNAs reducedlarval growth (fig. S2). The ACT dsRNA was moreeffective than the SHR dsRNA, and the ACT+SHRdsRNA was the least effective dsRNA (fig. S2), in-dicating that targeting two insect genes withthe same dsRNA does not necessarily enhanceinsecticidal activity.

We first evaluated the three strategies for in vivodsRNA production (ptDP, ptSL, and ptHP con-structs; Fig. 1A and fig. S1C) with the ACT+SHRfusion gene in tobacco plants, because chloroplasttransformation is relatively routine in this species.Transplastomic tobacco lines were produced byparticle gun–mediated chloroplast transformationand purified to homoplasmy by additional roundsof regeneration and selection. Transgene integrationinto the plastid genome by homologous recombi-nation and elimination of all wild-type copies ofthe highly polyploid plastid genome were confirmedby restriction fragment length polymorphism analy-ses and inheritance assays (figs. S1E and S3A). Alltransplastomic lines (referred to as Nt-ptDP-ACT+SHR, Nt-ptSL-ACT+SHR, and Nt-ptHP-ACT+ SHR)displayed no visible phenotype and were indistin-guishable fromwild-type plants, both under in vitroculture conditions and upon growth in the green-house (fig. S3, B and D), indicating that dsRNA ex-pression in the chloroplast is phenotypically neutral.To test whether dsRNAs stably accumulate in

chloroplasts, we performed Northern blot analyses.

The results revealed that all three types of expres-sion constructs triggered production of substan-tial amounts of long dsRNAs (Fig. 1B), suggestingthe absence of efficient dsRNA-degrading mech-anisms from plastids. dsRNA accumulation levelsin Nt-ptDP and Nt-ptSL plants were very similar,indicating that the terminal stem-loop structuresadded to the ptSL constructs do not increasedsRNA stability (Fig. 1, A and B). dsRNA ac-cumulation levels in Nt-ptHP lines were evenhigher but included shorter-than-expected tran-scripts (Fig. 1B), possibly because the plastid RNApolymerase encounters difficulties transcribing se-quences containing large inverted repeats. There-fore, we used the convergent promoter approach(ptDP constructs) for dsRNA expression in all sub-sequent experiments.We next introduced the three target gene con-

structs (ACT, SHR, and ACT+SHR, integrated intothe ptDP cassette; Fig. 1A) into the plastid genomeof potato (14) (see supplementary materials), themain host of CPB, and isolated homoplasmic trans-plastomic lines (St-ptDP-ACT, St-ptDP-SHR, and

992 27 FEBRUARY 2015 • VOL 347 ISSUE 6225 sciencemag.org SCIENCE

Fig. 1. Expression of dsRNAs in plastids. (A) Map of transformation vec-tors for dsRNA expression from the plastid genome.The cassettes designed toproduce the three different types of dsRNAs (ptDP, ptSL, and ptHP) are sche-matically depicted below the map, along with the expected structures and sizesof the dsRNAs. The selectable marker gene aadA is driven by the psbA pro-moter (PpsbA) and the 3′ untranslated region (3′ UTR) of the rbcL gene(TrbcL) from Chlamydomonas reinhardtii. DNA sequences from CPB targetgenes are shown in orange. SL1 and SL2, stem-loop–encoding sequences;Prrn, tobacco rRNA operon promoter; TrrnB, rrnB terminator from E. coli;intron, first intron from the potato GA20 oxidase gene. (B) Northern blot analysisof dsRNA accumulation in transplastomic tobacco and potato lines; 5 mg of totalRNAwas loaded in each lane, and the band sizes of the RNAmarker are given atthe left. The ethidium bromide–stained gel prior to blotting is shown below eachblot. The asterisk indicates a shorter-than-expected transcript present inNt-ptHP-ACT+SHR lines. Accumulation of some larger RNA species is likelydue to read-through transcription, which is common in plastids (22, 23). Note

that transplastomic lines generated with the same construct show identicaltransgene expression levels due to targeting by homologous recombination.(C) Comparison of dsRNA accumulation levels in leaves and tubers of tran-splastomic potato lines expressing ACTdsRNA; 5 mg of total cellular RNA wasloaded per lane. The ethidium bromide–stained gel prior to blotting is shownbelow the blot. (D) Analysis of ACT dsRNA accumulation by Northern blot-ting. The amount of total RNA loaded in each lane is given (in mg). Theethidium bromide–stained gel prior to blotting is shown below the blot as aloading control. Note that 10 times as much RNA was loaded for thenuclear transgenic lines. (E) Analysis of siRNA accumulation by Northernblotting. Note that siRNAs accumulate only in the nuclear transgenic plantsbut not in the transplastomic plants, confirming that the dsRNAs produced inthe plastid stay put. Thus, although the CPB ACT sequence used has somesimilarity to the potato ACT gene (66% over a stretch of 226 nucleotides), itcannot even theoretically silence the plant’s endogenous ACT gene becausethe chloroplast-produced dsRNAs do not leak out into the cytosol.

RESEARCH | REPORTS

St-ptDP-ACT+SHR; fig. S1, B and F). To be able tocompare the level of protection from herbivory intransplastomic and nuclear transgenic plants,we introduced the identical transgenes (as hairpinconstructs containing a spliceosomal intron thatisposttranscriptionally removed) (15) into thenucleargenomebyAgrobacterium-mediated transformation

(St-nuHP; fig. S1D). Phenotypic analyses showed thatall transplastomic and nuclear transgenic potatoplants were indistinguishable from wild-type plantswith regard to growth and tuber production (fig. S3,C and E, and figs. S4 and S5).Northern blot analyses of transplastomic po-

tato lines revealed that the accumulation levels

of ACT dsRNAs were higher than those of SHRand ACT+SHR dsRNAs (Fig. 1B). Comparison to adilution series of in vitro synthesized RNA re-vealed dsRNA accumulation levels in leaves of~0.4% of the total cellular RNA for ACT, ~0.05%for SHR, and ~0.1% for ACT+SHR (fig. S6). By con-trast, hybridization signals in the nuclear transgenic

SCIENCE sciencemag.org 27 FEBRUARY 2015 • VOL 347 ISSUE 6225 993

Fig. 2. Feeding assays of CPB larvae on trans-plastomic and nuclear transgenic potato plants.(A) Survivorship of first-instar larvae upon feedingon detached leaves of wild-type, transplastomic,and nuclear transgenic potato plants. (B) Growthof surviving larvae. The weight of survivors wasdetermined after 3, 5, 7, and 9 days of feeding.Data are means T SD (n = 30). Significant differ-ences to the wild-type control were identified byanalysis of variance. *P < 0.05, **P < 0.01, ***P <0.001. The best-performing nuclear transgeniclines were included in the assay (see figs. S7 toS10). For assays with additional transplastomiclines, see fig. S9. Note that theweight of survivors inthe assays with the transplastomic plants expres-sing ACT dsRNA (St-ptDP-ACT21) could only bemeasured until day 3, because all larvae werealready dead at day 5 [see (A)]. (C) Example of abioassay with detached leaves of wild-type potatoplants and nuclear transgenic and transplastomicleaves expressingACTdsRNA. Leaveswere exposedto first-instar CPB larvae, replaced with fresh youngleaves every day, and the photograph was taken atday 3. Note that almost no visible damage is seen inSt-ptDP-ACT21 leaves.

Fig. 3. Consumption of detached leaves ofpotato plants by adult beetles, and survivor-ship of larvae upon feeding on whole plants.(A) Leaf area consumed by freshly emerged adultbeetles fed on leaves of wild-type potato plants,nuclear transgenic plants, and transplastomic plantsexpressing ACT dsRNA. As an additional control,leaves painted with in vitro synthesized GFP-deriveddsRNA were included. Data are mean T SD (n = 24for St-wt, n = 12 for all other lines). (B) Survivorshipof second-instar CPB larvae after feeding on wholeplants at day 5 (see fig. S12).

RESEARCH | REPORTS

plants could be detected only upon overload-ing of the gels and/or overexposure of the blots,consistent with efficient degradation of dsRNAsinto siRNAs by the plant’s endogenous RNAimachinery (Fig. 1D and figs. S7 and S8A). Thepresence of siRNAs in nuclear transgenic plantsand their absence from transplastomic plantswere directly confirmed by Northern blot analy-ses (Fig. 1E).Because CPB larvae and adults feed on leaves

but not on belowground potato tubers, only theleaves need to be protected from herbivory. Non-photosynthetic tissues, such as tubers, expressplastid to much lower levels than photosynthetictissues, such as leaves (16, 17). Thus, despite whole-plant transformation, dsRNA production in tubers,where accumulation of transgene-derived RNAis unnecessary and perhaps undesired by the con-sumer, was below or near the detection limit (Fig.1C and fig. S8B).Having established that long dsRNAs accu-

mulate to high levels in leaves of transplastomicpotato lines, we next tested whether dsRNA pro-duction in the chloroplast offers protection againstCPB. To this end, the mortality of first-instar CPBlarvae was determined upon feeding on detachedleaves from wild-type, transplastomic, and nu-clear transgenic potato plants for 9 consecutivedays (Fig. 2A and fig. S9A). In addition, the weightof all surviving larvae was measured to followtheir growth (Fig. 2, B and C; fig. S9, B to D; andfig. S10). The bioassays revealed that all trans-plastomic potato plants induced high mortality(Fig. 2, A and C, and fig. S9A). Consistent withthe high expression level of ACT dsRNA (Fig. 1Band fig. S6) and the high efficacy of in vitro syn-thesized ACT dsRNA (fig. S2), the most potentinsecticidal activity was conferred by the ACTdsRNA–expressing transplastomic plants thatcaused 100% mortality within 5 days. By con-trast, none of the nuclear transgenic potato plantsconferred any larval mortality (Fig. 2A and fig.S9A), in line with the earlier finding that shortsiRNAs fed to insects have only small effects ordo not induce an RNAi response at all (3). How-ever, all nuclear transgenic lines caused reducedgrowth of CPB larvae (Fig. 2B), presumably dueto the small amounts of dsRNAs the plants ac-cumulate (Fig. 1D and figs. S7 and S8). While noneof the CPB larvae survived feeding on trans-plastomic St-ptDP-ACT plants, some of the larvaesurvived for 9 days on St-ptDP-SHR and St-ptDP-ACT+SHR leaves. However, these survivors suf-fered from very strong growth retardation (Fig.2B and fig. S9, C and D).To confirm that the killing of the CPB larvae

by the transplastomic plants was due to inductionof RNAi, we determined expression of the targetgenes in the larval gut after 3 days of feeding(i.e., when the larvae fed on the transplastomicplants were still alive). Already at this early stage,expression of b-actin and Shrub was suppressedin the insects (fig. S11, A and B). As expected onthe basis of the mortality data (Fig. 2A), targetgene suppression was strongest in larvae fed onSt-ptDP-ACT plants. Moreover, accumulation ofACT-derived siRNAs was detected in gut tissue of

larvae fed with transplastomic leaves, whereas ac-cumulation in larvae fed with nuclear transgenicleaves was below the limit of reliable detection(fig. S11C).CPB resistance of transplastomic potato plants

was further assessed by determining the leaf areaconsumed by CPB larvae and adult beetles. Al-most no visible consumption of leaf biomassoccurred in St-ptDP-ACT leaves (Fig. 2C and fig.S12A), due to complete cessation of larval feed-ing after 24 hours, even prior to mortality (Fig.2A). Similarly, adult beetles caused very littleadditional damage to St-ptDP-ACT leaves after2 days (Fig. 3A). Finally, we exposed whole plantsto second-instar larvae (which are generally lesssensitive to insecticidal agents than first-instarlarvae) and scored survival. This test resulted in17% survival of the larvae after 5 days of feedingon St-ptDP-ACT plants (and 63% survival uponfeeding on St-ptDP-SHR plants after 6 days; Fig.3B, fig. S12, B to D, and fig. S13), presumably dueto the initial larval growth and development onwild-type leaves. However, the larvae grew verypoorly after transfer to the transplastomic plants,and the damage they caused to the leaves wasvery small (fig. S12, B to D). In nature, CPB larvaetypically hatch and feed on the same plant, andtherefore they would not enjoy a wild-type dietbefore feeding on the transplastomic plant.To ultimately confirm that the plastid-expressed

ACT dsRNA silences the actin gene in CPB larvae,we examined actin filaments in the larval mid-gut, hindgut, and Malpighian tubules by stainingwith fluorescein isothiocyanate (FITC)–labeledphalloidin. Already after 1 to 2 days of feedingon transplastomic leaves, the larvae displayeddisorganized actin filaments, which were partic-ularly obvious in the columnar cells of the midgut(fig. S14). Also, the intensity of phalloidin-FITClabeling progressively decreased with the time offeeding (fig. S14), which suggests that actin de-ficiency is the cause of death. RNA-dependent RNApolymerase (RdRP) genes are absent from the ge-nomes of insects (18). Therefore, silencing signalsare not amplified at the RNA level, and RNAi ef-fects remain restricted to those cells that havetaken up (or produced) silencing-inducing dsRNAs.Consequently, a continuous input of dsRNAs isrequired for efficient gene silencing in insects.Because of the low stability of dsRNAs expressedfrom the nuclear genome and their efficient pro-cessing by the plant’s own Dicer endoribonu-cleases, complete protection of plants from insectpests has not been accomplished (1, 2). Our find-ings underscore the importance of producinglarge amounts of long dsRNAs to achieve efficientprotection. Whereas transplastomic ACT dsRNA–expressing plants cause 100% lethality to CPB lar-vae, SHR dsRNA–expressing plants are somewhatless efficient (70% mortality after 9 days; Fig. 2A).This correlates with the factor of ~8 loweraccumulation levels of the SHR dsRNA. Becauseboth constructs are driven by the same expressionsignals,we conclude that theSHR sequence chosenis less stable in potato plastids than the ACT se-quence. Consequently, testing other fragments ofthe SHR gene seems an appropriate future strategy

to improve the insecticidal efficiency of SHR dsRNA–expressing transplastomic plants.As insect pests are developing increasing re-

sistance to chemical insecticides and Bt toxins(12, 19–21), RNAi technology is becoming a prom-ising future strategy for plant protection. RNAi-activating dsRNAs can be chosen from a vastnumber of potential target genes. Moreover, thedsRNA approach provides plant protection with-out chemicals and without synthesis of foreignproteins in the plant. We have shown that plas-tids can be engineered to produce the quantitiesof dsRNA needed to control a major agriculturalpest, the Colorado potato beetle. Shifting the tar-get of transgenesis from the nucleus to the plastidremoves the major hurdle on the way to exploit-ing transgenically delivered RNAi for efficientplant protection in the field (18).

REFERENCES AND NOTES

1. J. A. Baum et al., Nat. Biotechnol. 25, 1322–1326 (2007).2. Y.-B. Mao et al., Nat. Biotechnol. 25, 1307–1313 (2007).3. R. Bolognesi et al., PLOS ONE 7, e47534 (2012).4. S. Whyard, A. D. Singh, S. Wong, Insect Biochem. Mol. Biol. 39,

824–832 (2009).5. W. Zha et al., PLOS ONE 6, e20504 (2011).6. M. Pitino, A. D. Coleman, M. E. Maffei, C. J. Ridout,

S. A. Hogenhout, PLOS ONE 6, e25709 (2011).7. P. Kumar, S. S. Pandit, I. T. Baldwin, PLOS ONE 7, e31347 (2012).8. J.-Q. Zhu et al., PLOS ONE 7, e38572 (2012).9. F. Vazquez, S. Legrand, D. Windels, Trends Plant Sci. 15,

337–345 (2010).10. M. Kumar, G. P. Gupta, M. V. Rajam, J. Insect Physiol. 55,

273–278 (2009).11. D. B. Stern, W. Gruissem, Cell 51, 1145–1157 (1987).12. A. Alyokhin, M. Baker, D. Mota-Sanchez, G. Dively, E. Grafius,

Am. J. Potato Res. 85, 395–413 (2008).13. F. Zhu, J. Xu, R. Palli, J. Ferguson, S. R. Palli, Pest Manag. Sci.

67, 175–182 (2011).14. V. T. Valkov et al., Transgenic Res. 20, 137–151 (2011).15. N. A. Smith et al., Nature 407, 319–320 (2000).16. S. Kahlau, R. Bock, Plant Cell 20, 856–874 (2008).17. V. T. Valkov et al., Plant Physiol. 150, 2030–2044 (2009).18. D. R. Price, J. A. Gatehouse, Trends Biotechnol. 26, 393–400

(2008).19. L. J. Gahan, F. Gould, D. G. Heckel, Science 293, 857–860

(2001).20. J. S. Griffitts, J. L. Whitacre, D. E. Stevens, R. V. Aroian,

Science 293, 860–864 (2001).21. A. J. Gassmann et al., Proc. Natl. Acad. Sci. U.S.A. 111,

5141–5146 (2014).22. F. Zhou, D. Karcher, R. Bock, Plant J. 52, 961–972 (2007).23. M. Oey, M. Lohse, B. Kreikemeyer, R. Bock, Plant J. 57,

436–445 (2009).

ACKNOWLEDGMENTS

We thank P. Endries for help with plant transformation,E. Maximova for help with microscopy, Y. Zhang for providingphalloidin-FITC, the MPI-MP Greenteam for plant cultivation,M. Kaltenpoth and B. Weiss for help in insect tissue preparation,and T. Krügel and the MPI-CE greenhouse team. This researchwas financed by the Max Planck Society. Plastid transformationvectors and transplastomic plants are available under a materialtransfer agreement. J.Z., S.A.K., D.G.H., R.B., and the Max PlanckSociety (MPG) have filed a patent application (EP14199415) thatrelates to the transplastomic production of dsRNAs for plantprotection. Supplement contains additional data.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/347/6225/991/suppl/DC1Materials and MethodsFigs. S1 to S14Table S1References (24–37)

24 September 2014; accepted 12 January 201510.1126/science.1261680

994 27 FEBRUARY 2015 • VOL 347 ISSUE 6225 sciencemag.org SCIENCE

RESEARCH | REPORTS

DOI: 10.1126/science.1261680, 991 (2015);347 Science et al.Jiang Zhang

double-stranded RNAs in plastidsFull crop protection from an insect pest by expression of long

This copy is for your personal, non-commercial use only.

clicking here.colleagues, clients, or customers by , you can order high-quality copies for yourIf you wish to distribute this article to others

  here.following the guidelines

can be obtained byPermission to republish or repurpose articles or portions of articles

  ): February 26, 2015 www.sciencemag.org (this information is current as of

The following resources related to this article are available online at

http://www.sciencemag.org/content/347/6225/991.full.htmlversion of this article at:

including high-resolution figures, can be found in the onlineUpdated information and services,

http://www.sciencemag.org/content/suppl/2015/02/25/347.6225.991.DC1.html can be found at: Supporting Online Material

http://www.sciencemag.org/content/347/6225/991.full.html#relatedfound at:

can berelated to this article A list of selected additional articles on the Science Web sites

http://www.sciencemag.org/content/347/6225/991.full.html#ref-list-1, 8 of which can be accessed free:cites 37 articlesThis article

http://www.sciencemag.org/content/347/6225/991.full.html#related-urls1 articles hosted by HighWire Press; see:cited by This article has been

http://www.sciencemag.org/cgi/collection/botanyBotany

subject collections:This article appears in the following

registered trademark of AAAS. is aScience2015 by the American Association for the Advancement of Science; all rights reserved. The title

CopyrightAmerican Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by theScience

on

Feb

ruar

y 26

, 201

5w

ww

.sci

ence

mag

.org

Dow

nloa

ded

from