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23. S. Carlquist, Ecological Strategies of Xylem Evolution (Univ. of California Press, Berkeley, CA, 1975). 24. A. Igersheim and P. K. Endress, Bot. J. Linn. Soc. 124, 213 (1997); P. K. Endress and A. Igersheim, Bot. J. Linn. Soc. 125, 93 (1997); A. Igersheim and P. K. Endress, Bot. J. Linn. Soc. 127, 289 (1998). 25. S. J. Weller, M. J. Donoghue, D. Charlesworth, in Experimental and Molecular Approaches to Plant Biosystematics, P. C. Hoch and A. G. Stephenson, Eds. (Missouri Botanical Garden, St. Louis, MO, 1995), pp. 355382. 26. P. K. Endress, unpublished data. 27. L. B. Thein, unpublished data. 28. E. L. Schneider and S. Carlquist, Am. J. Bot. 83, 1236 (1996). 29. S. Carlquist, Am. J. Bot. 79, 660 (1992). 30. D. A. Young, Syst. Bot. 6, 313 (1981); M. J. Donoghue, Evolution 43, 1137 (1989); T. S. Feild, M. A. Zwieniecki, M. J. Donoghue, N. M. Holbrook, Proc. Natl. Acad. Sci. U.S.A. 95, 14256 (1998). 31. M. J. Sanderson and M. J. Donoghue, Science 264, 1590 (1994). 32. M. Lavin, E. Eshbaugh, J.-M. Hu, S. Mathews, R. A. Sharrock, Am. J. Bot. 85, 412 (1998). 33. D. L. Swofford, PAUP* 4.0 (Sinauer Associates, Sunderland, MA, 1999). 34. We thank J. Doyle, P. Endress, L. Thein, P. Soltis, S. Graham, Y.-L. Qiu, J. Palmer, and C. dePamphilis for helpful discussions and for sharing unpublished data, and C. Soohoo and C. Davis for technical support. Financial support was provided by NSF grant DEB-9806937. 2 September 1999; accepted 1 October 1999

A Species of Small Antisense RNA in Posttranscriptional Gene Silencing in Plants

Andrew J. Hamilton and David C. Baulcombe*
Posttranscriptional gene silencing (PTGS) is a nucleotide sequencespecic defense mechanism that can target both cellular and viral mRNAs. Here, three types of transgene-induced PTGS and one example of virus-induced PTGS were analyzed in plants. In each case, antisense RNA complementary to the targeted mRNA was detected. These RNA molecules were of a uniform length, estimated at 25 nucleotides, and their accumulation required either transgene sense transcription or RNA virus replication. Thus, the 25-nucleotide antisense RNA is likely synthesized from an RNA template and may represent the specicity determinant of PTGS. Posttranscriptional gene silencing occurs in plants and fungi transformed with foreign or endogenous DNA and results in the reduced accumulation of RNA molecules with sequence similarity to the introduced nucleic acid (1, 2). Double-stranded RNA induces a similar effect in nematodes (3), insects (4), and protozoa (5). PTGS can be suppressed by several virus-encoded proteins (6) and is closely related to RNA-mediated virus resistance and cross-protection in plants (7, 8). Therefore, PTGS may represent a natural antiviral defense mechanism and transgenes might be targeted because they, or their RNA, are perceived as viruses. PTGS could also represent a defense system against transposable elements and may function in plant development (9 11). To account for the sequence specificity and posttranscriptional nature of PTGS, it has been proposed that antisense RNA forms a duplex with the target RNA, thereby promoting its degradation or interfering with its translation (12). If these hypothetical antisense RNA molecules are of a similar size to typical mRNAs, they would have been readily detected by routine RNA analyses. However, there have been no reports of such antisense RNA that is detected exclusively in plants or animals exhibiting PTGS. Nevertheless, PTGS-specific antisense RNA may exSainsbury Laboratory, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK. *To whom correspondence should be addressed. Email: [email protected]

was no PTGS of GUS in 6b5 271 because of the transcriptional suppression of the 35S GUS transgene (18). Hybridization with a GUS-specific probe revealed that low molecular weight GUS antisense RNA was present in T4 and 6b5 (Fig. 2, lanes 1 and 2) but absent from line 6b5 271 (Fig. 2, lane 3). The amount of antisense RNA correlated with the extent of PTGS: Line 6b5 has stronger PTGS of GUS than line T4 (18) and had more GUS antisense RNA (Fig. 2).

ist, but may be too short for easy detection. We carried out analyses specifically to detect low molecular weight antisense RNA in four classes of PTGS in plants (13). The first class tested was transgene-induced PTGS of an endogenous gene (cosuppression). We used five tomato lines (T1.1, T1.2, T5.1, T5.2, and T5.3), each transformed with a tomato 1-aminocyclopropane-1-carboxylate oxidase (ACO) cDNA sequence placed downstream of the cauliflower mosaic virus 35S promoter (35S). Two lines (T5.2 and T5.3) exhibited PTGS of the endogenous ACO mRNA (Fig. 1A). Low molecular weight nucleic acids purified from the five lines were separated by denaturing polyacrylamide gel electrophoresis, blotted, and hybridized to an ACO sense (antisense-specific) RNA probe (Fig. 1B). A discrete, ACO antisense RNA (14) of 25 nucleotides (nt) was present in both PTGS lines but absent from the nonsilencing lines. Twenty-fivenucleotide ACO RNA of sense polarity and at the same abundance as the 25-nt ACO antisense RNA was also present only in the PTGS lines (Fig. 1C). PTGS induced by transgenes can also occur when a transgene does not have homology to an endogenous gene (1). Therefore, we tested whether this type of PTGS was also associated with small antisense RNA. We analyzed three tobacco lines carrying 35S- glucuronidase (GUS) transgenes. Two of these lines, T4 (15) and 6b5 (16), exhibited PTGS of GUS. The third line (6b5 271) tested was produced by crossing 6b5 with line 271 (17), in which there is a transgene suppressor of the 35S promoter in 6b5. There

Fig. 1. Twenty-venucleotide ACO antisense and sense RNA in PTGS lines. (A) Endogenous ACO mRNA abundance in ve tomato lines containing 35S-ACO transgenes. ACO mRNA was amplied by reverse transcriptasepolymerase chain reaction and detected by hybridization with labeled ACO cDNA. (B and C) Low molecular weight RNA from the same ve lines and a 30-nt ACO antisense RNA were fractionated, blotted, and hybridized with either ACO sense RNA (B) or antisense RNA (C) transcribed from full-length ACO cDNA. The low hybridization temperature permitted some nonspecic hybridization to tRNA and small ribosomal RNA species, which constitute most of the RNA mass in these fractions. The oligonucleotide hybridized only to the antisense-specic probe (B). Twenty-venucleotide, PTGS-specic RNA is indicated.

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As for PTGS of ACO in tomato, the GUS antisense RNA was a discrete species of 25 nt. In some examples of PTGS, silencing is initiated in a localized region of the plant. A signal molecule is produced at the site of initiation and mediates systemic spread of silencing to other tissues of the plant (19, 20). We investigated whether systemic PTGS of a transgene encoding the green fluorescent protein (GFP) is associated with 25-nt GFP antisense RNA. PTGS was initiated in Nicotiana benthamiana expressing a GFP transgene by infiltration of a single leaf with Agrobacterium tumefaciens containing GFP sequences in a binary plant transformation vector (19). Two to 3 weeks after this infiltration, the GFP fluorescence disappeared owing to systemic spread of PTGS as described (11, 20). We detected 25-nt GFP antisense RNA in systemic tissues exhibiting PTGS of GFP. It was not detected in equivalent leaves of plants that had not been infiltrated or in nontransformed plants that had been infiltrated with A. tumefaciens (Fig. 3). A natural manifestation of PTGS is the RNA-mediated defense induced in virus-infected cells (8). Therefore we investigated whether virus-specific, 25-nt RNA could be detected in a virus-infected plant. Twentyfivenucleotide RNA complementary to the positive strand (genomic) of potato virus X (PVX) was detected 4 days after inoculation of N. benthamiana and continued to accumulate for at least another 6 days in the inoculated leaf (Fig. 4). Twenty-fivenucleotide PVX RNA accumulated to a similar extent in systemically infected leaves but was not detected in mock-inoculated leaves. Thus, 25-nt antisense RNA, complementary to targeted mRNAs, accumulates in four types of PTGS. We have detected 25-nt RNA in other examples of PTGS (22), and never detected 25-nt RNA in the absence of PTGS. This correlation and the properties of 25-nt RNA are consistent with a direct role for 25-nt RNA in PTGS induced by transgenes or viruses (12). Twenty-fivenucleotide RNA species also serve as molecular markers for PTGS. Their presence could be used to confirm other examples of transgene- or virusinduced PTGS and perhaps also to identify endogenous genes that are targeted by PTGS in nontransgenic plants. The 25-nt antisense RNA species are not degradation products of the target RNA because they have antisense polarity. A more likely source of these RNAs is the transcription of an RNA template. This is consistent with the presence of the 25-nt PVX RNA in PVX-infected cells that do not contain a DNA template (Fig 4, syst. leaf ). The dependency of 25-nt GUS antisense RNA accumulation on sense transcription of a GUS transgene also supports the RNA template model (Fig. 2). An RNAdependent RNA polymerase, as required by this model, is required for PTGS in Neurospora crassa (23). With the present data, we cannot distinguish whether the antisense RNA is made directly as a 25-nt species or as longer molecules that are subsequently processed. The precise role of 25-nt RNA in PTGS remains to be determined. However, because they are long enough to convey sequence specificity yet small enough to move through plasmodesmata, it is possible that they are components of the systemic signal and specificity determinants of PTGS.
References and Notes

Fig. 2. Twenty-venucleotide antisense GUS RNA is dependent on transcription from the 35S promoter. Twenty-venucleotide GUS antisense RNA was detected by hybridization with hydrolyzed GUS sense RNA transcribed from the 3 700 base pairs of the GUS cDNA.

Fig. 3. Twenty-venucleotide antisense GFP RNA in systemically silenced tissue. Lower leaves of untransformed N. benthamiana (WT) and N. benthamiana carrying an active 35S-GFP transgene (35S-GFP) were inltrated with A. tumefaciens containing the same 35S-GFP transgene in a binary vector. RNA from upper, noninltrated leaves of these plants (inf.) and from equivalent leaves of noninltrated plants ( ) was hybridized with GFP sense RNA transcribed from a full-length GFP cDNA. Only the transgenic N. benthamiana inltrated with the A. tumefaciens accumulated 25-nt GFP antisense RNA.

Fig. 4. Twenty-venucleotide antisense PVX RNA accumulates during virus replication. RNA was extracted from inoculated leaves after 2, 4, 6, and 10 days and from systemic (syst.) leaves after 6 and 10 days (d.p.i.: days post inoculation). RNA was extracted from mock-inoculated leaves after 2 days. Twenty-venucleotide PVX antisense RNA was detected by hybridization with PVX sense RNA transcribed from a full-length PVX cDNA.

1. H. Vaucheret et al., Plant J. 16, 651 (1998). 2. C. Cogoni and G. Macino, Trends Plant Sci. 2, 438 (1997). 3. A. Fire et al., Nature 391, 806 (1998). 4. J. R. Kennerdell and R. W. Carthew, Cell 95, 1017 (1998). 5. H. Ngo, C. Tschudi, K. Gull, E. Ullu, Proc. Natl. Acad. Sci. U.S.A. 95, 14687 (1998). 6. G. Pruss, X. Ge, X. M. Shi, J. C. Carrington, V. B. Vance, Plant Cell 9, 859 (1997); R. Anandalakshmi et al., Proc. Natl. Acad. Sci. U.S.A. 95, 13079 (1998); K. D. Kasschau and J. C. Carrington, Cell 95, 461 (1998); G. Brigneti et al., EMBO J. 17, 6739 (1998); C. Beclin, R. Berthome, J.-C. Palauqui, M. Tepfer, H. Vaucheret, Virology 252, 313 (1998). 7. J. A. Lindbo, L. Silva-Rosales, W. M. Proebsting, W. G. Dougherty, Plant Cell 5, 1749 (1993). 8. F. Ratcliff, B. D. Harrison, D. C. Baulcombe, Science 276, 1558 (1997); S. N. Covey, N. S. Al-Kaff, A. Langara, D. S. Turner, Nature 385, 781 (1997); F. Ratcliff, S. MacFarlane, D. C. Baulcombe, Plant Cell 11, 1207 (1999). 9. R. B. Flavell, Proc. Natl. Acad. Sci. U.S.A. 91, 3490 (1994). 10. R. A. Jorgensen, R. G. Atkinson, R. L. S. Forster, W. J. Lucas, Science 279, 1486 (1998). 11. O. Voinnet, P. Vain, S. Angell, D. C. Baulcombe, Cell 95, 177 (1998). 12. D. Grierson, R. G. Fray, A. J. Hamilton, C. J. S. Smith, C. F. Watson, Trends Biotechnol. 9, 122 (1991); W. G. Dougherty and T. D. Parks, Curr. Opin. Cell Biol. 7,

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399 (1995); D. C. Baulcombe and J. J. English, Curr. Opin. Biotechnol. 7, 173 (1996). 13. Total RNA was extracted from leaves of tomato, tobacco, and N. benthamiana as described [E. Mueller, J. E. Gilbert, G. Davenport, G. Brigneti, D. C. Baulcombe, Plant J. 7, 1001 (1995)]. From these preparations, low molecular weight RNA was enriched by ion-exchange chromatography on Qiagen columns after removal of high molecular weight species by precipitation with 5% polyethylene glycol 8000 0.5 M NaCl (for tobacco and N. benthamiana) or by ltration through Centricon 100 concentrators (Amicon) (for tomato). Low molecular weight RNA was separated by electrophoresis through 15% polyacrylamide7 M urea 0.5 tris-borate EDTA gels, transferred onto Hybond Nx lters (Amersham), and xed by ultraviolet cross-linking. Prehybridization was in 45% formamide, 7% SDS, 0.3 M NaCl, 0.05 M Na2HPO4NaH2PO4 (pH 7), 1 Denhardts solution, and sheared, denatured, salmon sperm DNA (100 mg/ml) at between 30 and 40C. Hybridization was in the same solution with single-stranded RNA probes transcribed with -32P-labeled uridine triphosphate. Before addition to the lters in the prehybridization solution, probes were hydrolyzed to lengths averaging 50 nt. Hybridization was for 16 hours at 30C (ACO probes), 35C (GUS probe), or 40C (GFP and PVX probes). Sizes of RNA molecules were estimated by comparison with 33P-phosphorylated DNA oligonucleotides run on the same gels but imaged separately. Additionally, samples from different types of PTGS, including those shown, were frequently run on the same gel. Alignment of the lters after hybridization with different specic probes conrmed that the PTGS-specic signals were identical in size. The probes used are in each case sequence specic. We have observed no cross-hybridization between 25-nt signals in different PTGS systems using either lter hybridization or RNAase protection (www.sciencemag.org/feature/data/1042575.shl). We do not have an exact measurement of the amount of 25-nt RNA per cell, but given the short exposure times routinely used to detect these molecules and taking into account their size, they are likely to be abundant in cells exhibiting PTGS. The 25-nt ACO antisense signal was completely abolished by pretreatment with either RNAaseONE (Promega) or NaOH. S. L. A. Hobbs, T. D. Warkentin, C. M. O. DeLong, Plant Mol. Biol. 21, 17 (1993). T. Elmayan and H. Vaucheret, Plant J. 9, 787 (1996). H. Vaucheret, C. R. Acad. Sci. Paris 316, 1471 (1993). J. J. English, G. F. Davenport, T. Elmayan, H. Vaucheret, D. C. Baulcombe, Plant J. 12, 597 (1997). O. Voinnet and D. C. Baulcombe, Nature 389, 553 (1997). J.-C. Palauqui and S. Balzergue, Curr. Biol. 9, 59 (1999). A high-titer, synchronized PVX infection on leaves of untransformed N. benthamiana was initiated by inltration of single leaves with A. tumefaciens containing a binary plasmid incorporating a 35S-PVXGFP sequence. Once transcribed, the PVX RNA replicon is independent of the 35S-PVX-GFP DNA, replicates to high levels, and moves systemically through the plant. The A. tumefaciens does not spread beyond the inltrated patch and is not present in systemic leaves (20). The GFP reporter in the virus was used to allow visual monitoring of infection progress. We have obtained similar signals with wild-type PVX inoculated as virions in sap taken from an infected plant. 22. The other examples of PTGS tested were in N. benthamiana (spontaneous silencing of a 35S-GFP transgene), tomato (35S-ACO containing an internal direct and inverted repeat), petunia (cosuppression of chalcone synthase transgenes and endogenes), and Arabidopsis thaliana (PTGS of 35S-GFP by a 35S-PVXGFP transgene). 23. C. Cogoni and G. Macino, Nature 399, 166 (1999). 24. We thank D. Grierson, C. DeLong, H. Vaucheret, and R. Hellens for transgenic plants. We are also grateful to O. Voinnet, D. Bradley, A. Bendahmane, and F. Ratcliff for helpful comments and suggestions. This work was carried out under M.A.F.F. licence PHL 24A/2921. Funded by the Biotechnology and Biological Sciences Research Council and the Gatsby Charitable Foundation. 11 June 1999; accepted 14 September 1999

14. 15. 16. 17. 18. 19. 20. 21.

TCR-Mediated Internalization of Peptide-MHC Complexes Acquired by T Cells

Jing-Feng Huang,1 Young Yang,1 Homero Sepulveda,1 Weixing Shi,1 Inkyu Hwang,2 Per A. Peterson,1 Michael R. Jackson,1 Jonathan Sprent,2 Zeling Cai1*
Peptidemajor histocompatibility complex protein complexes (pMHCs) on antigen-presenting cells (APCs) are central to T cell activation. Within minutes of peptide-specic T cells interacting with APCs, pMHCs on APCs formed clusters at the site of T cell contact. Thereafter, these clusters were acquired by T cells and internalized through T cell receptormediated endocytosis. During this process, T cells became sensitive to peptide-specic lysis by neighboring T cells (fratricide). This form of immunoregulation could explain the exhaustion of T cell responses that is induced by high viral loads and may serve to downregulate immune responses. T cell responses are initiated by T cell receptor (TCR) recognition of pMHCs on APCs (1). Upon specific interaction of T cells with APCs, TCR and MHC molecules are assembled at the center of supramolecular activation clusters (2). The fate of these TCR-MHC clusters at the T cellAPC contact site is unclear. However, it is known that interaction of TCRs with pMHC complexes is followed by TCR down-regulation (3) and that T cell APC interaction can cause APC-derived sur1 R. W. Johnson Pharmaceutical Research Institute, 3210 Merryeld Row, San Diego, CA 92121, USA. 2 Department of Immunology, IMM4, Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

*To whom correspondence should be addressed. Email: [email protected]

face molecules to adhere to the surface of T cells (4). To investigate the fate of MHC clusters at the T cellAPC contact sites, we used Drosophila, RMA-S, and dendritic cell lines expressing MHC class I (Ld)green fluorescent protein fusion molecules (Ld-GFP) (5) as APCs to activate CD8 T cells from the 2C TCR transgenic mouse line (6, 7). 2C T cells recognize Ld plus either QL9 peptide or a closely related peptide, p2Ca (8). Empty Ld-GFP molecules expressed on the surface of RMA-S and Drosophila cells can be efficiently loaded with exogenous peptides (7, 9). Within 5 min of 2C T cells interacting with either Drosophila cells (Fly.LdGFP) or RMA-S cells (RMA-S.Ld-GFP) plus the QL9 peptide, Ld-GFP molecules formed large clusters at the T cellAPC contact site [Fig. 1A and Web figure 1 (10)]. After 30 min,

the Ld-GFP clusters at the T cellAPC interface decreased in size, and small aggregates of LdGFP appeared concomitantly within 2C T cells at sites distal to the contact site [Fig. 1, B and C, Web figure 2 (10)]. This process was not seen with P1A (11) (Fig. 1D), a control peptide that binds to Ld but is not recognized by 2C TCR (7). Aggregates of Ld-GFP were also detected inside 2C T cells after interaction with the Ld-GFPtransfected dendritic cell line DC2.4 (12) (Fig. 1E), which presents endogenous p2Ca peptide. To exclude the possibility that GFP itself could mediate the acquisition of Ld by T cells, we used Drosophila cells expressing untagged Ld (no GFP) loaded with QL9 peptide as APCs. After culture for 1 hour, multiple aggregates of Ld molecules were detected in 2C T cells by intracellular staining with monoclonal antibody (mAb) to Ld (13) (Fig. 1F). Thus, in the presence of antigenic peptides, Ld molecules can be acquired from APCs by T cells, and this acquisition is independent of GFP. Acquisition of APC-derived Ld molecules by 2C T cells was further demonstrated by immunoprecipitation (Fig. 1, G and H). After culture with 35S-methioninelabeled fibroblasts (L cells) transfected with Ld (L-Ld) (14), 2C T cells were highly purified and immunoprecipitation was performed with mAbs to class I molecules. Immunoprecipitation of Ld from 2C T cells cultured with L-Ld plus QL9 peptide was prominent (Fig. 1G). In the presence of the control P1A peptide (11), precipitation of Ld was limited, but clearly detectable, presumably due to the presentation of endogenous p2Ca peptide in L-Ld cells. In contrast, other MHC class I molecules (Kk and Dk) expressed by L cells were not detectable in 2C T cells by immunoprecipitation (Fig. 1G). The peptide-

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