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The Plant Journal (1998) 14(2), 247–257

TECHNICAL ADVANCE

Glucocorticoid-inducible expression of a bacterial avirulence gene in transgenic Arabidopsis induces hypersensitive cell death Timothy W. McNellis1, Mary Beth Mudgett1, Karen Li1, Takashi Aoyama2, Diana Horvath3, Nam-Hai Chua3 and Brian J. Staskawicz1,* 1Department of Plant and Microbial Biology, 111 Koshland Hall, University of California, Berkeley, CA 94720, USA, 2Institute for Chemical Research, Kyoto University, Uji, Kyoto 611, Japan, and 3Laboratory of Plant Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10021–6339, USA

Summary Pathogenic strains of Pseudomonas syringae pv. tomato carrying the avrRpt2 avirulence gene specifically induce a hypersensitive cell death response in Arabidopsis plants that contain the complementary RPS2 disease resistance gene. Transient expression of avrRpt2 in Arabidopsis plants having the RPS2 gene has been shown to induce hypersensitive cell death. In order to analyze the effects of conditional expression of avrRpt2 in Arabidopsis plants, transgenic lines were constructed that contained the avrRpt2 gene under the control of a tightly regulated, glucocorticoid-inducible promoter. Dexamethasoneinduced expression of avrRpt2 in transgenic lines having the RPS2 gene resulted in a specific hypersensitive cell death response that resembled a Pseudomonas syringaeinduced hypersensitive response and also induced the expression of a pathogenesis-related gene (PR1). Interestingly, high level expression of avrRpt2 in a mutant rps2– 101C background resulted in plant stress and ultimately cell death, suggesting a possible role for avrRpt2 in Pseudomonas syringae virulence. Transgenic RPS2 and rps2 plants that contain the glucocorticoid-inducible avrRpt2 gene will provide a powerful new tool for the genetic, physiological, biochemical, and molecular dissection of an avirulence gene-specified cell death response in both resistant and susceptible plants.

Received 10 November 1997; revised 15 January 1998; accepted 20 January 1998. *For correspondence (fax 11 51064 29017; e-mail [email protected]).

© 1998 Blackwell Science Ltd

Introduction The RPS2 disease resistance gene of Arabidopsis thaliana confers resistance to Pseudomonas syringae pv. tomato (P. syringae) bacteria that have the avrRpt2 avirulence gene (Kunkel et al., 1993). This pattern is known as gene-forgene disease resistance (Flor, 1971). When a plant having the RPS2 gene is challenged with P. syringae bacteria bearing avrRpt2, the plant initiates defense responses that together serve to limit the growth of the pathogen within the plant and prevent disease (Innes et al., 1993; Kunkel et al., 1993). P. syringae bacteria bearing avrRpt2 also induce a rapid cell death response at the infection site in plants having the RPS2 resistance gene. This reaction is called the hypersensitive response (HR), and it is strongly correlated with resistance to disease (Kiraly, 1980). However, it is not known if the HR plays a causal role in disease resistance, since in at least some cases the HR is not required to stop pathogen growth (Century et al., 1995). The RPS2/avrRpt2 gene-for-gene disease resistance gene pair serves as an excellent model interaction for the study of pathogen-induced defense responses and signal transduction events since both genes have been cloned and characterized at the molecular level. The RPS2 gene encodes a 909-amino acid, 105 kDa protein which has a leucine zipper domain, a nucleotide-binding site, and 14 imperfect leucine-rich repeats (Bent et al., 1994; Mindrinos et al., 1994). Both the leucine zipper (Alber, 1992) and the leucine-rich repeat (Kobe and Deisenhofer, 1994) domains have been implicated in protein–protein interactions. The leucine-rich repeat domains are also involved in ligand binding in proteins such as the gonadotropin receptors (Braun et al., 1991). The nucleotide binding site implies that the function of RPS2 may involve kinase activity (Traut, 1994). These structural features of the RPS2 protein suggest that it is involved in signal transduction processes and that it may serve as a receptor for the AvrRpt2 protein or the enzymatic product of the AvrRpt2 protein, as is implied by the gene-for-gene interaction (Staskawicz et al., 1995). The avrRpt2 gene encodes a protein of 28.2 kDa having 255 amino acids (Innes et al., 1993). AvrRpt2 has no significant homology to any other known protein, and its biochemical function is unknown. However, transient expression of avrRpt2 in the cells of plants having the RPS2 resistance gene using a particle bombardment technique causes 247

248 Timothy W. McNellis et al. hypersensitive cell death (Leister et al., 1996). This result suggests that the AvrRpt2 protein or its putative enzymatic product can act as a trigger of hypersensitive cell death in plants that have the RPS2 resistance gene and that perception of the avrRpt2 gene-derived signal can occur inside the plant cell. In addition, both transient and stable expression of the avrB avirulence gene has also been demonstrated to trigger hypersensitive cell death in plants having the corresponding RPM1 resistance gene (Gopalan et al., 1996). In this study, we confirmed and extended these findings by creating transgenic plants that had the avrRpt2 gene under the control of a newly developed, tightly regulated, glucocorticoid-inducible promoter (Aoyama and Chua, 1997). Use of the glucocorticoid-inducible promoter enabled us to analyze the effects of avrRpt2 expression in whole plants, seedlings, and individual leaves. Expression of avrRpt2 in leaves of adult plants having the RPS2 gene caused hypersensitive cell death that resembled a P. syringae-induced HR according to a number of criteria including rapid onset, appearance, electrolyte leakage and dependence on a functional RPS2 resistance gene. Furthermore, avrRpt2 expression in plants having the RPS2 resistance gene induced the expression of the PathogenesisRelated 1 gene (PR1, Uknes et al., 1992), suggesting that expression of avrRpt2 was sufficient to trigger other defense responses in addition to hypersensitive cell death. The utility of this system for genetic, biochemical, and physiological analysis of avirulence gene-specified hypersensitive cell death is discussed. Results

Production of stably transformed Arabidopsis plants having the glucocorticoid-inducible avrRpt2 gene For this study we utilized the two-component glucocorticoid-inducible transgenic system essentially as previously described (Aoyama and Chua, 1997). Figure 1(a) depicts the T-DNA region of the construct pTA7001-avrRpt2 which was used for plant transformation. A glucocorticoid-regulated transcription factor is encoded by the GVG gene (Aoyama and Chua, 1997), whose transcription is controlled by the CaMV 35S promoter (35S) (Odell et al., 1985); transcription of the avrRpt2 gene is controlled by the glucocorticoid-activated promoter (6 3 UASgal4) (Aoyama and Chua, 1997); the selection marker for plant transformation is the hygromycin phosphotransferase gene HPT, Blochlinger and Diggelmann, 1984) whose transcription is controlled by the nopaline synthetase promoter (Nos; Bevan, 1984). All of these genes are on the T-DNA segment defined by the right and left border sequences (RB and LB) of the commercially available binary plant transformation vector pBI101.

Figure 1. The glucocorticoid-inducible system for expression of avrRpt2 in stably transformed Arabidopsis plants. (a) The T-DNA region of the construct used for Arabidopsis transformations. The genes necessary for glucocorticoid-inducible expression of avrRpt2 were cloned into the binary vector pBI101 as described in Aoyama and Chua (1997) and in the methods. RB, right T-DNA border; 35S, 35S promoter; GVG, glucocorticoid-inducible transcription factor; E9, pea rbcS-E9 polyadenylation sequence; Nos, nopaline synthetase promoter; HPT, hygromycin phosphotransferase; Nt, nopaline synthetase polyadenylation sequence; 63UASgal4, GVG-regulated promoter; avrRpt2, avrRpt2 coding region; 3A, pea rbcS-3 A polyadenylation sequence; LB, left T-DNA border. (b) A schematic diagram depicting the dexamethasone-inducible transcriptional activation of the avrRpt2 transgene and the subsequent induction of plant cell death. The T-DNA region is shown in the nucleus where it has integrated into the plant nuclear genome. For clarity, the HPT cassette is omitted and the genes are shown in a left-to-right orientation.

Figure 1(b) depicts how the glucocorticoid-inducible system works in the transgenic plant cell to express the avrRpt2 transgene and trigger the avrRpt2 gene-specified hypersensitive cell death response. The GVG gene is constitutively transcribed and translated due to the activity of the 35S promoter (Odell et al., 1985). The GVG protein is a tripartite fusion protein (Aoyama and Chua, 1997). Starting from the N terminus, the first domain is the DNA-binding domain from the yeast GAL4 protein (Keegan et al., 1986); the second domain is the transactivating domain from the herpes simplex virus VP16 protein (Triezenberg et al., 1988); the third domain is the hormone-binding domain of the rat glucocorticoid receptor (Picard et al., 1988; Rusconi and Yamamoto, 1987). The GVG protein is made in the cytoplasm and retained there in an inactivated state (GVGi) by interaction with heat shock proteins, including hsp70 and hsp90, which bind to the rat glucocorticoid hormone-binding domain of GVG and maintain it in a partially unfolded conformation (Picard et al., 1988). Once GVGi binds to a glucocorticoid such as dexamethasone, which is a strong synthetic glucocorticoid that readily permeates the plant cell (Aoyama and Chua, 1997; Lloyd et al., 1994), GVGi is converted into an active state (GVGa) and is targeted to the nucleus through the action of nuclear localization signals in the hormone © Blackwell Science Ltd, The Plant Journal, (1998), 14, 247–257

Glucocorticoid-inducible cell death 249 binding domain portion of the protein (Picard and Yamamoto, 1987). In the nucleus, the GAL4 DNA-binding domain of GVGa binds to the target promoter, which has six copies of the DNA binding site of the GAL4 protein (63UASgal4) (Giniger et al., 1985) as well as minimal promoter elements from the CaMV 35S promoter (Odell et al., 1985). Transcription of the target gene is then initiated by the action of the VP16 transactivating domain of GVGa (Triezenberg et al., 1988). This results in the transcription of avrRpt2 in the nucleus and the production of AvrRpt2 protein in the plant cell cytoplasm. The AvrRpt2 protein is then expected to initiate signal transduction events involving the RPS2 gene product resulting in avrRpt2 gene-specified hypersensitive cell death. Eight independent transgenic lines were produced for this study using the Agrobacterium tumefaciens vacuum infiltration transformation method (Bent et al., 1994). The Columbia-0 (Col-0) wild-type accession was chosen for transformation because it is widely used and it contains the RPS2 resistance gene (Kunkel et al., 1993). All the lines segregated for one T-DNA locus according to hygromycin resistance segregation patterns. A representative line homozygous for the avrRpt2 transgene was used for all the experiments described. Transgenic plants homozygous for the glucocorticoid-inducible avrRpt2 transgene and homozygous for the RPS2 disease resistance gene were designated as transgenic avrRpt2(RPS2) plants. A number of transgenic lines containing the pTA7001 empty vector were also generated for use as controls to show that the glucocorticoid-inducible system did not have any adverse effects on the plant. Transgenic plants homozygous for the empty pTA7001 T-DNA and homozygous for the RPS2 resistance gene were designated as transgenic empty vector (RPS2) plants.

Analysis of the accumulation of avrRpt2 message and AvrRpt2 protein in transgenic avrRpt2(RPS2) plants in response to dexamethasone treatment The kinetics of induction of avrRpt2 message accumulation and AvrRpt2 protein accumulation in transgenic avrRpt2(RPS2) plants after treatment of the plants with dexamethasone was monitored. Transgenic avrRpt2(RPS2) seedlings were grown in shaken liquid cultures for 7 days and then dexamethasone was added to the medium to a final concentration of 30 µM. Total RNA and total protein were isolated from the plants at various time points after induction and subjected to RNA gel blot and protein gel blot analysis. Figure 2(a) depicts an RNA gel blot showing the accumulation of avrRpt2 message over time in transgenic avrRpt2(RPS2) seedlings after exposure to dexamethasone. The µ1 kb avrRpt2 message was detectable within 0.5 h after induction. The level of avrRpt2 message reached a peak at the 4 h time point and remained at approximately the © Blackwell Science Ltd, The Plant Journal, (1998), 14, 247–257

Figure 2. Time course of accumulation of avrRpt2 message and AvrRpt2 protein in transgenic avrRpt2(RPS2) seedlings after dexamethasone treatment. (a) An RNA gel blot showing the accumulation of avrRpt2 message over time in transgenic avrRpt2(RPS2) seedlings after exposure of the seedlings to medium containing 30 µM dexamethasone. U, uninduced sample; avrRpt2, avrRpt2 message; rRNA, the identical RNA gel blot hybridized to a probe specific for the 18S ribosomal RNA. Thirty micrograms of total RNA were loaded in each lane. (b) Protein gel blot showing the accumulation of AvrRpt2 protein over time in transgenic avrRpt2(RPS2) seedlings after exposure of the seedlings to medium containing 30 µM dexamethasone. The locations of the 19, 28, 33, and 48 kDa molecular weight markers are shown. AvrRpt2, the processed 22 kDa AvrRpt2 protein detected by the antibody. The arrow indicates the position at which the full-length 28.2 kDa AvrRpt2 protein was expected to appear. The band appearing at about 48 kDa was detected in all plant extracts and appeared to be non-specific. Fifty micrograms of total protein were loaded in each lane.

same level throughout the remainder of the experiment. Figure 2(b) depicts a protein gel blot showing the accumulation of AvrRpt2 protein over time in transgenic avrRpt2(RPS2) seedlings after exposure to dexamethasone. The polyclonal anti-AvrRpt2 antibody detected a 22 kDa AvrRpt2 polypeptide within 2 h after dexamethasone treatment. This protein represents a processed form of AvrRpt2, whose full-length form is 28.2 kDa. Processing of AvrRpt2 occurs in plant cells expressing avrRpt2 but not in P. syringae cells expressing the same gene (M. B. Mudgett and B. J. Staskawicz, personal communication). The effect of dexamethasone dosage on the accumulation of avrRpt2 message and the accumulation of AvrRpt2 protein in transgenic avrRpt2(RPS2) seedlings was also determined. Transgenic avrRpt2(RPS2) seedlings were grown in shaken liquid cultures for 7 days and then treated with various concentrations of dexamethasone. After 48 h, total RNA

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Figure 3. Effects of dexamethasone dosage on the accumulation of avrRpt2 message and AvrRpt2 protein in transgenic avrRpt2(RPS2) seedlings. (a) An RNA gel blot showing the level of avrRpt2 message accumulation in transgenic avrRpt2(RPS2) seedlings 48 h after exposure of the seedlings to media containing the concentrations of dexamethasone indicated. [DEX], concentration of dexamethasone; avrRpt2, avrRpt2 message; rRNA, the identical RNA gel blot hybridized to a probe specific for the 18S ribosomal RNA. Thirty micrograms of total RNA were loaded in each lane. (b) Protein gel blot showing the accumulation of AvrRpt2 protein in transgenic avrRpt2(RPS2) seedlings 48 h after exposure of the seedlings to media containing the concentrations of dexamethasone indicated. The blot shows the processed 22 kDa form of AvrRpt2. Fifty micrograms of total protein were loaded in each lane.

and total protein were extracted and subjected to RNA gel blot and protein gel blot analysis, respectively. Figure 3(a) depicts an RNA gel blot showing the accumulation of avrRpt2 message in transgenic avrRpt2(RPS2) seedlings 48 h after dexamethasone treatment. A dose of 0.1 µM dexamethasone was sufficient to induce a low level of avrRpt2 expression. Maximal avrRpt2 expression was observed in plants exposed to 10 µM dexamethasone. Figure 3(b) depicts a protein gel blot showing the accumulation of AvrRpt2 protein in the same transgenic avrRpt2(RPS2) seedlings 48 h after exposure to various concentrations of dexamethasone. The 22 kDa processed AvrRpt2 protein was detectable in plants exposed to as little as 0.1 µM dexamethasone and accumulation of AvrRpt2 protein reached a maximum at about 20 µM dexamethasone.

Hypersensitive collapse of dexamethasone-treated transgenic avrRpt2(RPS2) plant leaves Figure 4 shows leaves of adult Col-0 wild-type, transgenic empty vector (RPS2), and transgenic avrRpt2(RPS2) plants 24 h after hand-inoculation with 30 µM dexamethasone. Leaves of transgenic avrRpt2(RPS2) plants inoculated with 30 µM dexamethasone exhibited rapid collapse, while Col0 wild-type and transgenic empty vector plant leaves showed no visible response. The response of transgenic avrRpt2(RPS2) plant leaves to dexamethasone inoculation

was very rapid, occurring in as little as 6 h. The same phenotype was observed in every independent transgenic avrRpt2(RPS2) line and the effect strictly cosegregated with the avrRpt2 transgene (data not shown). A dose of 90 nM dexamethasone was sufficient to induce hypersensitive collapse in transgenic avrRpt2(RPS2) plant leaves (data not shown), and this correlated with the dose of dexamethasone that induced detectable avrRpt2 message accumulation and AvrRpt2 protein accumulation (Figure 3). To test whether the response of the transgenic avrRpt2(RPS2) plants to dexamethasone inoculation depended on a functional RPS2 resistance gene, the representative transgenic avrRpt2(RPS2) line was crossed to plants homozygous for the rps2–101C mutation, which truncates the protein after amino acid 234 (Mindrinos et al., 1994). Plants homozygous for both the avrRpt2 transgene and the rps2–101C mutation were obtained, and these plants were designated as transgenic avrRpt2(rps2) plants. The inducible expression level of avrRpt2 in the transgenic avrRpt2(rps2) plants was identical to that observed in transgenic avrRpt2(RPS2) plants as indicated by RNA gel blot analysis (data not shown). Leaves of transgenic avrRpt2(rps2) plants did not exhibit collapse 24 h after inoculation with 30 µM dexamethasone (Figure 4). However, after 48 h, the 30 µM dexamethasone-treated avrRpt2(rps2) plant leaves showed extensive purple anthocyanin pigment accumulation, and treatment of such leaves with 30 µM dexamethasone for a second time caused leaf senescence and death within 96 h (data not shown). Leaves of Col-0 wild-type, transgenic empty vector, transgenic avrRpt2(RPS2), and transgenic avrRpt2(rps2) plants were also hand inoculated with 1 3 108 cells ml–1 Pseudomonas fluorescens (P. fluorescens) 55 (pHIR11) (pDSK600-avrRpt2) bacteria that are capable of delivering the avrRpt2 gene-derived signal to plants having the RPS2 resistance gene (Huang et al., 1988; M. B. Mudgett and B. J. Staskawicz, unpublished results). Col-0 wild-type, transgenic empty vector (RPS2), and transgenic avrRpt2(RPS2) plant leaves exhibited rapid HR collapse in response to P. fluorescens 55 (pHIR11) (pDSK600-avrRpt2) bacteria while the transgenic avrRpt2(rps2) plant leaves showed no response (Figure 4). It should be noted that the hypersensitive collapse induced by dexamethasone treatment of transgenic avrRpt2(RPS2) plants was much more rapid than that induced by inoculation with bacteria bearing avrRpt2. The dexamethasone-induced hypersensitive collapse occurred within 6 h, while the bacterial-induced HR took about 20 h to develop.

Electrolyte leakage from transgenic avrRpt2(RPS2) plant leaves undergoing dexamethasone-induced collapse Electrolyte leakage has been shown to occur in tissues that are undergoing an HR induced by bacteria (Goodman, © Blackwell Science Ltd, The Plant Journal, (1998), 14, 247–257

Glucocorticoid-inducible cell death 251

Figure 4. Comparison of dexamethasoneinduced hypersensitive collapse of transgenic avrRpt2(RPS2) leaves with the hypersensitive response induced by bacteria carrying avrRpt2. Leaves from plants of the indicated genotypes were inoculated with either 30 µM dexamethasone (DEX) or 1 3 108 cells ml–1 P. fluorescens 55(pHIR11) (pDSK600-avrRpt2) bacteria (P. f. avrRpt2). Plant genotypes: Col-0 wild-type, untransformed Col-0 wild-type; ev(RPS2), transgenic empty vector (RPS2); avrRpt2(RPS2), transgenic avrRpt2(RPS2); avrRpt2(rps2), transgenic avrRpt2(rps2). Bar 5 1 cm.

Figure 7. Dexamethasone-induced death of transgenic avrRpt2(RPS2) and avrRpt2(rps2) seedlings. (a) Untransformed Col-0 wild-type, transgenic empty vector [ev(RPS2)], transgenic avrRpt2(RPS2), and transgenic avrRpt2(rps2) seedlings grown for 7 days on GM media containing the concentrations of dexamethasone indicated. [DEX], concentration of dexamethasone. Bar 5 1 cm. (b) An untransformed Col-0 wild-type seedling grown for 10 days on GM medium containing 10 nM dexamethasone. Bar 5 1 mm. (c) A transgenic avrRpt2(RPS2) seedling grown for 10 days on GM medium containing 10 nM dexamethasone. Bar 5 1 mm. (d) A transgenic avrRpt2(rps2) seedling grown for 10 days on GM medium containing 1 µM dexamethasone. The magnification is the same in panels (c) and (d).

© Blackwell Science Ltd, The Plant Journal, (1998), 14, 247–257

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Figure 5. Time course of dexamethasone-induced electrolyte leakage from transgenic avrRpt2(RPS2) leaf tissue. Leaf disks cut from leaves of transgenic avrRpt2(RPS2) plants and untransformed Col-0 wild-type plants were floated either on a solution containing 30 µM dexamethasone in 0.1% ethanol (avrRpt2(RPS2)/DEX and Col-0 wild-type/DEX, respectively), or on a control 0.1% ethanol solution (avrRpt2(RPS2)/EtOH and Col-0 wild-type/EtOH, respectively). The conductivity of the solution was measured at the time points indicated. Units of conductivity are expressed as micro-Siemens (µS). The vertical lines indicate standard error.

1972). To test if the dexamethasone-induced collapse of transgenic avrRpt2(RPS2) plant leaves also involved electrolyte leakage, leaf disks were cut from mature leaves of Col-0 wild-type and transgenic avrRpt2(RPS2) plants and floated on either a 0.1% ethanol solution containing 30 µM dexamethasone, or on 0.1% ethanol solution alone as a control. The leakage of electrolytes into the liquid medium was monitored using a conductivity meter. Figure 5 shows that significant electrolyte leakage occurred over time from transgenic avrRpt2(RPS2) leaf disks but not from Col-0 wild-type leaf disks exposed to 30 µM dexamethasone. Neither transgenic avrRpt2(RPS2) leaf disks nor Col-0 wildtype leaf disks exhibited significant electrolyte leakage when they were floated on 0.1% ethanol. After the 24 h time point, leaf disks of transgenic avrRpt2(RPS2) plants appeared water-soaked as a result of hypersensitive tissue collapse. PR1 induction in transgenic avrRpt2(RPS2) plants following glucocorticoid treatment To test whether dexamethasone treatment of transgenic avrRpt2(RPS2) plant leaves could induce the expression of PR1, Col-0 wild-type and transgenic avrRpt2(RPS2) plants were inoculated on two leaves with either 30 µM dexamethasone in 0.1% ethanol, 1 3 108 cells ml–1 of P. fluorescens 55 (pHIR11) (pDSK600-avrRpt2) bacteria, or 0.1% ethanol. The inoculated leaves were excised two days

Figure 6. Induction of PR1 message accumulation by dexamethasone in transgenic avrRpt2(RPS2) plants. An RNA gel blot showing the accumulation of PR1 message in untransformed Col-0 wild-type and transgenic avrRpt2(RPS2) plants that were inoculated on two leaves with either P. fluorescens 55 (pHIR11) (pDSK600-avrRpt2) bacteria [P. f. (avrRpt2)], 30 µM dexamethasone in 0.1% ethanol (dexamethasone), or 0.1% ethanol control solution (EtOH). PR1, PR1 gene message; rRNA, the identical blot hybridized to a probe specific for the 18S ribosomal RNA. Thirty micrograms of total RNA were loaded in each lane.

post inoculation. Four days post inoculation total RNA was isolated from the distal leaf portions of the plants, subjected to RNA gel blot analysis and then hybridized to a radiolabeled probe specific for the Arabidopsis PR1 gene message (Uknes et al., 1992). Transgenic avrRpt2(RPS2) plants inoculated with 30 µM dexamethasone displayed a large amount of PR1 message accumulation while Col-0 wildtype plants inoculated with 30 µM dexamethasone did not display any PR1 transcript accumulation, as shown in Figure 6. Both Col-0 wild-type and transgenic avrRpt2(RPS2) plants showed PR1 message accumulation in response to inoculation with P. fluorescens 55 (pHIR11) (pDSK600-avrRpt2) bacteria (Figure 6). Neither Col-0 wildtype nor transgenic avrRpt2(RPS2) plants showed PR1 message accumulation in response to inoculation with 0.1% ethanol (Figure 6). It is interesting to note that the level of PR1 induction by dexamethasone in the transgenic avrRpt2(RPS2) plants was dramatically higher than that induced by inoculation with bacteria bearing avrRpt2.

Dexamethasone-induced death of transgenic avrRpt2(RPS2) seedlings Col-0 wild-type, transgenic empty vector (RPS2), transgenic avrRpt2(RPS2), and transgenic avrRpt2(rps2) seeds were sown on GM medium without dexamethasone or supplemented with dexamethasone at concentrations ranging from 6 nM to 30 µM. Figure 7(a) shows the responses of these seedlings after 7 days of growth. Col-0 wild-type and © Blackwell Science Ltd, The Plant Journal, (1998), 14, 247–257

Glucocorticoid-inducible cell death 253 transgenic empty vector (RPS2) seedlings did not show any visible response to any of the concentrations of dexamethasone tested. Transgenic avrRpt2(RPS2) seedlings appeared healthy on GM medium lacking dexamethasone. However, even 6 nM dexamethasone in the growth medium caused stunting of transgenic avrRpt2(RPS2) seedlings. Transgenic avrRpt2(RPS2) seedlings were killed after 7 days of growth on media containing 8 nM dexamethasone or more. Transgenic avrRpt2(rps2) seedlings were much less sensitive to dexamethasone in the medium and they could grow normally on medium containing up to 20 nM dexamethasone (Figure 7a). Transgenic avrRpt2(rps2) seedlings grew slowly on medium containing from 50 to 100 nM dexamethasone and were stunted. Transgenic avrRpt2(rps2) seedlings were killed on medium containing 1 µM dexamethasone or more. Figure 7(b) and (c) show Col-0 wild-type and transgenic avrRpt2(RPS2) seedlings, respectively, grown for 10 days on medium containing 10 nM dexamethasone. The Col-0 wild-type seedling appears normal and unaffected by the dexamethasone, while the transgenic avrRpt2(RPS2) seedling has been killed. Transgenic avrRpt2(RPS2) seedlings growing on 10 nM dexamethasone were able to open their cotyledons and green during the first 3–4 days of growth, after which they became chlorotic. Figure 7(d) shows a transgenic avrRpt2(rps2) seedling grown for 10 days on medium containing 30 µM dexamethasone. Transgenic avrRpt2(rps2) seedlings grown on 30 µM dexamethasone rarely were able to open their cotyledons and were never able to green. Discussion In this study we have utilized a newly developed glucocorticoid-inducible gene expression system to express avrRpt2 in stably transformed Arabidopsis plants. Previous studies have demonstrated that transient expression of avrRpt2 in the cells of plants having the RPS2 disease resistance gene causes hypersensitive cell death (Leister et al., 1996). The data presented here show that the avrRpt2 gene-specified cell death response in leaves of mature transgenic avrRpt2(RPS2) plants resembles the HR induced by bacteria delivering the avrRpt2 gene-derived signal according to a number of criteria, including appearance and timing (Figure 4), and electrolyte leakage (Figure 5). The avrRpt2 gene-specified hypersensitive cell death response did not occur in transgenic avrRpt2(rps2) plant leaves, demonstrating the requirement for a functional RPS2 gene. In addition, the avrRpt2 gene-specified hypersensitive cell death response in plants having the RPS2 gene strongly induced expression of PR1. This provided further evidence that the hypersensitive cell death response caused by expression of avrRpt2 in plants having the RPS2 resistance gene reflects the HR caused by bacteria delivering the avrRpt2 gene-derived signal. © Blackwell Science Ltd, The Plant Journal, (1998), 14, 247–257

Induction of avrRpt2 gene expression in the transgenic avrRpt2(RPS2) plants appeared to result in a much stronger cell death and defense response signal than that caused by bacteria bearing avrRpt2. This would account for the observation that dexamethasone inoculation of transgenic avrRpt2(RPS2) plants caused hypersensitive cell death within 6 h, while the HR caused by inoculation with P. fluorescens bacteria bearing avrRpt2 developed after 20 h. Similarly, it would also explain the very high level of PR1 gene expression in dexamethasone-treated transgenic avrRpt2(RPS2) plants as compared to bacterial-inoculated plants. Induction of the avrRpt2 transgene by dexamethasone may result in the production of a larger amount of AvrRpt2 protein in the plant cell than is delivered by the bacteria during infection, thus triggering a stronger defense response and cell death signal. Induction of avrRpt2 expression in seedlings that had the RPS2 resistance gene also caused hypersensitive cell death (Figure 7). This cell death response occurred within the first 4 days of seedling growth, indicating that seedlings are competent to undergo the avrRpt2 gene-specified cell death response soon after germination. This suggests that Arabidopsis plants are capable of initiating disease defense responses at a very early stage in their development. Transgenic avrRpt2(RPS2) seedlings were much more sensitive to dexamethasone than were adult transgenic avrRpt2(RPS2) plants. Transgenic avrRpt2(RPS2) seedlings growing on media containing 8 nM dexamethasone were killed by a systemic cell death response, while a minimum concentration of 90 nM dexamethasone was required to elicit cell death in leaves of adult transgenic avrRpt2(RPS2) plants. Although 8 nM dexamethasone is well below the minimum concentration that induced detectable avrRpt2 message accumulation or AvrRpt2 protein accumulation, it is likely that the seedlings concentrated the dexamethasone in their tissues by uptake and transpiration. Alternatively, 8 nM dexamethasone may have induced an extremely low level of avrRpt2 expression that was sufficient to elicit cell death in seedlings but was too low to be detected by RNA gel blotting. Interestingly, transgenic avrRpt2(rps2) seedlings were killed when they were grown on medium containing 1 µM dexamethasone or more (Figure 7), suggesting that highlevel expression of avrRpt2 is lethal in rps2 mutant plants. This effect may reflect a virulence activity of the AvrRpt2 protein, although the amount of AvrRpt2 protein that may be delivered to the plant cell by P. syringae during infection is not presently known, and high-level expression of avrRpt2 may not be biologically relevant. Because the rps2–101C mutant allele is not a true null but an early truncation of the protein, the cell death observed in transgenic avrRpt2(rps2) plants could also be due to residual activity of the mutant rps2 protein, leading to a delayed cell death response. Alternatively, other disease resistance

254 Timothy W. McNellis et al. gene products that do not normally recognize the AvrRpt2 protein might be able to recognize AvrRpt2 when the levels of AvrRpt2 protein are very high in the plant cell, thereby triggering cell death. Finally, production of high levels of AvrRpt2 protein might disrupt the homeostasis of the cell causing a non-specific cell necrosis. This study also demonstrated the utility of the twocomponent glucocorticoid-inducible gene expression system in transgenic plants. Using the glucocorticoid-inducible gene expression system to control the expression of avrRpt2 in plants having the RPS2 resistance gene served as a stringent test of the system, since the avrRpt2 gene product is an extremely potent trigger of cell death when expressed in plants having the RPS2 resistance gene (Leister et al., 1996; this work). We were successful in generating transgenic plant lines that showed no expression of avrRpt2 in the absence of glucocorticoid and that could be induced to express avrRpt2 to a high level when treated with dexamethasone. This indicates that the twocomponent glucocorticoid-inducible system developed by Aoyama and Chua (1997) should be suitable for studying the effects of expression of most genes of interest, even those that are extremely toxic or deleterious to the plant. The availability of stable transgenic glucocorticoid-inducible avrRpt2(RPS2) plant lines will allow us to perform genetic, molecular, and biochemical analyses of the avrRpt2 gene-specified cell death response. Homozygous transgenic avrRpt2(RPS2) lines have been mutagenized and are currently being screened for mutants that are defective in the avrRpt2 gene-specified cell death response. A number of potential mutants that are unable to undergo avrRpt2 gene-specified hypersensitive cell death have been isolated and are being characterized (T. W. McNellis and B. J. Staskawicz, unpublished results). These mutants may represent mutations in genes involved in the signal transduction pathway(s) leading from the perception of the avrRpt2 gene-derived signal to the plant hypersensitive cell death response. The transgenic avrRpt2(RPS2) plants will be used to generate a large amount of plant material that is synchronous in its response to the avrRpt2 genederived cell death signal. By isolating RNA at various time points after induction of the avrRpt2 transgene by dexamethasone, the temporal induction patterns of plant genes specifically expressed during the avrRpt2 genespecified cell death response can be monitored. Polyclonal antibodies recognizing the AvrRpt2 protein are currently being used to identify the subcellular localization of the AvrRpt2 protein in the plant cell. This may allow identification of the site in the plant cell where AvrRpt2 is recognized in resistant plants and also may indicate where in the cell AvrRpt2 carries out its potential virulence function. Transgenic avrRpt2(rps2) seeds have also been mutagenized and M2 seeds are being screened on a medium containing 1–30 µM dexamethasone. Any seedlings that

can survive will be selected as potential mutants that may disrupt the virulence target(s) of AvrRpt2. In summary, the transgenic glucocorticoid-inducible avrRpt2(RPS2) and transgenic avrRpt2(rps2) lines will serve as powerful new tools in the dissection of the avrRpt2 gene-specified plant cell death response and the analysis of the potential virulence activity of the avrRpt2 gene product.

Experimental procedures

Transformation construct The binary transformation plasmid pTA7001 containing the complete two-component glucocorticoid-inducible system (Aoyama and Chua, 1997) was cut with XhoI and SpeI (New England Biolabs, Beverly, MA, USA) according to the enzyme manufacturer’s instructions. The XhoI and SpeI restriction sites of pTA7001 are between the 63UASgal4 promoter and its pea rbcS-3 A polyadenylation signal, with the XhoI site closest to the promoter and the SpeI site closest to the terminator. The avrRpt2 coding region was engineered to have an XhoI site at the 59 end and an SpeI site at the 39 end using the polymerase chain reaction (PCR). The forward primer had the sequence 59-CCGCTCGAGATGAAAATTGCTCCAGTTGCC- 39; the reverse primer had the sequence 59-GACTAGTTTAGCGGTAGAGCATTGCGTG-39 (restriction sites in bold and avrRpt2-homologous sequences in italics). Using these primers, the avrRpt2 coding region was PCR amplified from the pRSRO plasmid containing avrRpt2 (Innes et al., 1993) using Pfu polymerase (Promega, Madison, WI, USA) according to the enzyme manufacturer’s instructions. PCR reactions were performed in a 25 µl volume containing 10 ng of pRSRO template DNA, 100 pmol of each primer, and 1 unit of Pfu polymerase. Fifteen amplification cycles were performed at 94°C for 1 min (denaturation), 61°C for 30 sec (annealing), and 72°C for 1 min (extension). The avrRpt2 PCR-amplified DNA fragment was digested with XhoI and SpeI, gel purified using the GeneClean protocol (Bio 101, Vista, CA, USA), and ligated into the pTA7001 plasmid using T4 DNA ligase (New England Biolabs) according to the enzyme manufacturer’s instructions. A clone of avrRpt2 in pTA7001 was selected and the integrity of the avrRpt2 insert was verified by cycle-sequencing using the Prism Ready Reaction DyeDeoxy Terminator Cycle Sequencing Kit and the ABI Prism Model 377 DNA Sequencer (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. The clone of avrRpt2 in pTA7001 was designated as pTA7001-avrRpt2.

Plant transformations Wild-type Columbia (Col-0) Arabidopsis plants were used for all transformations using the Agrobacterium vacuum infiltration procedure as described previously (Bent et al., 1994). Seeds from the vacuum infiltrated plants were surface-sterilized by treating them with a solution of 1.5% sodium hypochlorite/0.01% Tween 20 (Sigma, St. Louis, MO, USA) for 15–20 min and then washed five times with sterile distilled water. The sterilized seeds were then resuspended in 0.1% agarose and sown in Petri dishes containing GM medium (1 3 MS salts pH 5.7, 1 3 B5 vitamins, solidified with 0.8% phytagar (Gibco BRL, Grand Island, NY, USA)) containing and 20 µg ml–1 hygromycin B (Sigma). After plating, the seeds were vernalized for 4–7 days at 4°C and then moved to a growth chamber maintained at 22°C under long day (16 h light/ © Blackwell Science Ltd, The Plant Journal, (1998), 14, 247–257

Glucocorticoid-inducible cell death 255 8 h dark) conditions. Hygromycin-resistant T1 seedlings were selected after 2–3 weeks. Transformation was confirmed by Southern blot analysis. Genomic DNA was isolated from hygromycin-resistant plants using a modification of the method of Shure et al. (1983). Two young leaves were homogenized in a microcentrifuge tube containing 150 µl of extraction buffer (0.3 M NaCl, 50 mM Tris–Cl pH 7.5, 20 mM EDTA, 2% sarkosyl, 0.5% SDS, 5 M urea, and 5% phenol added just before use) using a Kontes pestle. An equal volume of phenol/chloroform (1:1) was added, and the sample was mixed for 5 min by gentle shaking and then centrifuged for 5 min at 14 000 g. The supernatant was transferred to a new tube and the DNA recovered by adding an equal volume of isopropanol, mixing gently, and centrifuging for 5 min at 14 000 g. The DNA pellet was washed twice with 70% ethanol, air-dried, and resuspended in 20 µl dH2O. Two micrograms of genomic DNA were digested with SpeI and/or XhoI (New England Biolabs), electrophoresed on a 1% agarose gel in 1 3 TAE buffer and transferred to a Hybond N1 (Amersham, Arlington Heights, IL, USA) membrane under alkaline conditions according to the membrane manufacturer’s instructions. The avrRpt2 coding region was amplified from the pRSRO plasmid using the primers and conditions described above, except that 30 amplification cycles were used, and the avrRpt2 gene DNA fragment was gel purified using the GeneClean kit (Bio 101). The avrRpt2 DNA fragment was labeled with 32P-dCTP by random primer labeling using chemically synthesized random octamer primers (Sambrook et al., 1989) and Klenow enzyme (New England Biolabs). The probe was hybridized to the blot for 16 h at 65°C in 5 3 SSC, 0.5% SDS, 5 3 Denhardt’s solution, and 100 µg ml–1 of sonicated herring sperm DNA. Blots were washed once in 2 3 SSC, 0.1% SDS at room temperature for 15 min, once in 1 3 SSC, 0.1% SDS at 65°C for 15 min, and once in 0.1 3 SSC, 0.1% SDS at 65°C for 15 min and then exposed to x-ray film for 2 days. 1 3 SSC is 0.15 M NaCl, 0.015 M sodium citrate.

Glucocorticoid treatments Dexamethasone (Sigma) was dissolved in 100% ethanol to make a 30 mM dexamethasone stock solution which was stored at –20°C in a light-tight vial. For the time course experiments monitoring avrRpt2 message accumulation and AvrRpt2 protein accumulation in response to dexamethasone treatment, transgenic avrRpt2(RPS2) seeds were surface sterilized as described above and vernalized for 24 h in a microcentrifuge tube at 4°C. They were then added to a 250 ml Erlenmeyer flask containing 50 ml of sterile liquid GM medium supplemented with 1% sucrose. One flask with about 150 seeds was prepared for each time point. The plants were grown with gentle shaking in a growth chamber at 22°C under long day growth conditions (16 h light/8 h dark). After 1 week’s growth, dexamethasone was added to the medium to a final concentration of 30 µM. At 0, 0.25, 0.5, 1, 2, 4, 8, 24, and 48 h time points after addition of dexamethasone, the seedlings from a flask were removed, rinsed in distilled water, and immediately frozen in liquid nitrogen. One control flask was not treated with dexamethasone and the tissue was frozen at the beginning of the experiment. For the dexamethasone dosage experiments monitoring avrRpt2 message accumulation and AvrRpt2 protein accumulation in response to different concentrations of dexamethasone, transgenic avrRpt2(RPS2) seeds were surface sterilized, vernalized at 4°C for 24 h in a microcentrifuge tube and grown in shaken liquid cultures as described above. One flask was prepared for each concentration of dexamethasone to be tested. After one week of growth, dexamethasone was added to the medium to a final © Blackwell Science Ltd, The Plant Journal, (1998), 14, 247–257

concentration of 0, 0.01, 0.1, 1, 10, 20, or 30 µM. After two additional days of growth, the seedlings were removed from the flasks, rinsed with distilled water, and frozen in liquid nitrogen. For leaf inoculations, Col-0 wild-type, transgenic empty vector (RPS2), transgenic avrRpt2(RPS2), and transgenic avrRpt2(rps2) plants were grown for 3 or 4 weeks in soil at 22°C under short day (8 h light/16 h dark) conditions in a growth chamber to enhance leaf expansion and delay flowering. The underside of the leaf to be inoculated was nicked with a razor blade and 30 µM dexamethasone in 0.1% ethanol was forced into the intercellular spaces of the leaf through the wound using a 1cm3 syringe without a needle. Plant responses were scored 24 h post inoculation. GM medium for plates containing dexamethasone was made as described above except that 1% sucrose was added. Dexamethasone was added to the medium after autoclaving. Seeds of Col-0 wild-type, transgenic empty vector (RPS2), transgenic avrRpt2(RPS2), and transgenic avrRpt2(rps2) plants were surface sterilized and sown onto the plates as described above. The plates were vernalized for 24 h at 4°C and then moved to a long day (16 h light/8 h dark) growth chamber at 22°C. The plants were photographed after either 7 or 10 days of growth.

RNA isolation and RNA blot analysis Total RNA was isolated from seedlings and adult leaves using Trizol reagent (Gibco BRL, Grand Island, NY, USA) according to the manufacturer’s instructions. For RNA gel blot hybridization, 30 µg of total RNA for each sample was denatured and separated on 1.0% agarose-formaldehyde gels (Sambrook et al., 1989). RNA was transferred onto a Hybond N membrane (Amersham) and crosslinked to the membrane using a Stratalinker UV crosslinking apparatus (Stratagene, La Jolla, CA, USA). The RNA blots were hybridized to their respective 32P-labeled probes for 16 h at 65°C in 5 3 SSC, 0.5% SDS, 5 3 Denhardt’s solution, and 100 µg ml–1 of sonicated herring sperm DNA. Blots were washed once in 2 3 SSC, 0.1% SDS at room temperature for 15 min, once in 1 3 SSC, 0.1% SDS at 65°C for 15 min, and twice in 0.1 3 SSC, 0.1% SDS at 65°C for 15 min and then exposed to X-ray film for 1–7 days. The avrRpt2 DNA fragment was obtained by PCR amplification as described for Southern blot analysis. The 18S rDNA fragment was obtained by digesting the pHA1 plasmid containing a pea ribosomal DNA clone (Jorgensen et al., 1987) with XmnI and EcoRI (New England Biolabs) and gel purification of the 1.5 kb 18S rDNA fragment in a 1% agarose TAE gel using the GeneClean kit (Bio 101). The PR1 DNA fragment was obtained by digesting a plasmid containing the cloned Arabidopsis PR1 cDNA (Uknes et al., 1992) with EcoRI and XhoI (New England Biolabs) and gel purification of the 1 kb PR1 DNA fragment using the GeneClean kit (Bio 101). All the DNA fragments were labeled with 32P-dCTP by random priming as described above.

Bacterial inoculations Reactions of wild-type and transgenic avrRpt2 plants to inoculation with the P. fluorescens 55 (pHIR11) (pDSK600- avrRpt2) (Huang et al., 1988; M. B. Mudgett and B. J. Staskawicz, unpublished results) transconjugant were determined as described previously (Whalen et al., 1991). The P. fluorescens 55 (pHIR11) (pDSK600avrRpt2) strain was cultured at 28°C on King’s Medium B (King et al., 1954) supplemented with antibiotics (Sigma) at the following concentrations (µg ml–1): nalidixic acid, 20; tetracycline, 10; spectinomycin, 50; and streptomycin, 25. Bacterial suspensions were

256 Timothy W. McNellis et al. hand-infiltrated at 1 3 108 cells ml–1 in 10 mM MgCl2. Reactions were scored at 24 h post inoculation. Cosmid pHIR11 contains the hrp gene cluster from P. syringae pv. syringae 61 necessary for bacterial elicitation of the HR (Huang et al., 1988). The fragment containing the avrRpt2 gene from pRSRO (Innes et al., 1993) was subcloned into the broad-host range expression vector pDSK600 (Murillo et al., 1994) to make the pDSK600-avrRpt2 plasmid.

Production of antibodies To construct a 63His epitope tag at the N-terminus of AvrRpt2, the coding sequence of avrRpt2 was subcloned into the pRSET vector (Invitrogen). N-63His-AvrRpt2 protein was overexpressed in E. coli BL21 cells and purified using Ni-NTA agarose (Qiagen, Chatsworth, CA, USA) under denaturing conditions, eluting bound protein with pH 4.5 buffer as described by the manufacturer. Polyclonal antiserum was raised against the purified N-63HisAvrRpt2 fusion protein in rabbits (Babco, Berkeley, CA, USA).

Protein gel blot analysis Protein was extracted from liquid nitrogen-frozen plant tissue by homogenization using a Kontes pestle in a microcentrifuge tube. Ground tissue was then resuspended with sample buffer (180 mM Tris–HCl, pH 6.8, 6% SDS, 2.1 M 2-mercaptoethanol, 35.5% glycerol, and 0.004% bromophenol blue). Soluble protein was collected following centrifugation at 14 000 g at 4°C for 15 min. A modification of the Lowry procedure (Bailey, 1967) was used to determine the concentration of protein after precipitation with 1 ml of 10% trichloroacetic acid. Protein samples (50 µg) were analyzed by SDS-polyacrylamide slab gel electrophoresis using the buffer system described by Laemmli (1970) and then transferred to MSI nitrocellulose membranes (Micron Separations Inc., Westborough, MA, USA) by electroblotting in transfer buffer containing 25 mM Tris–HCl, pH 8.3, 192 mM glycine, and 28% (v/v) methanol at 0.5 A for 2 h. AvrRpt2 protein was detected with a rabbit polyclonal antibody (1:1000) raised against N-6XHis-AvrRpt2 fusion protein using the ECL Western blotting kit (Amersham).

Crossing the avrRpt2 transgene into the rps2–101C mutant background Homozygous transgenic avrRpt2(RPS2) plants were crossed with homozygous rps2–101C mutant plants (Mindrinos et al., 1994) both as pollen donors and as pollen receivers. Three week-old soil-grown F2 plants were challenged with P. fluorescens 55 (pHIR11) (pDSK600-avrRpt2) bacteria by inoculation of at least 3 leaves. Plants that did not exhibit an HR in any of their inoculated leaves were selected as homozygous rps2–101C mutants. Genomic DNA was isolated from these plants as described above and plants that were at least hemizygous for the avrRpt2 transgene were identified by PCR. The avrRpt2 transgene was amplified from plant genomic DNA using the same conditions described above, except that 100 ng of DNA was used as the template and 30 amplification cycles were performed. F3 seeds from each of these plants were harvested separately, surface sterilized, and sown on GM medium containing 20 µg ml–1 hygromycin plus 1% sucrose as described above. Families of F3 seed that segregated 100% hygromycin resistant were selected as homozygous for both the avrRpt2 transgene and the rps2–101C mutation. The presence of the rps2–101C mutation in these plants was confirmed by DNA

sequencing (Bent et al., 1994). These plants were designated as transgenic avrRpt2(rps2) plants.

Electrolyte leakage measurements Leaf disks were cut from leaves of 3 to 4-week-old plants grown under short day (8 h light/16 h dark) conditions in a growth chamber at 22°C. A total area of 2 cm2 of leaf tissue was used for each condition at each time point. The leaf disks were floated abaxial side up on 2 ml of either 30 µM dexamethasone in 0.1% ethanol, or 0.1% ethanol in distilled water in small Petri dishes. The leaf disks were maintained in growth chamber at 22°C with continual light for the duration of the experiment. Electrolyte leakage from the leaf disks into the liquid medium was measured by drawing 1 ml of the liquid into the conductivity cell of a type CDM3 conductivity meter (Radiometer, Copenhagen, Denmark). At least three independent samples were measured for each time point for each plant type.

PR1 induction Col-0 wild-type and transgenic avrRpt2(RPS2) plants were grown for 4 weeks in a growth chamber under short day conditions (8 h light/16 h dark) at 22°C. Two leaves of each plant were inoculated with either P. syringae 55 (pHIR11) (pDSK600-avrRpt2), 30 µM dexamethasone in 0.1% ethanol, or 0.1% ethanol as described above. Two days post inoculation the inoculated leaves were removed from the plants. Four days post inoculation the uninoculated leaves of the plants were harvested and frozen in liquid nitrogen. At least three plants were treated with each inoculum, and their tissues were pooled for RNA isolation.

Acknowledgements We thank the members of the laboratory of Brian Staskawicz for helpful discussions and Dr Fumiaki Katagiri for kindly providing the rps2–101C mutant. We also thank Paul Rangel for technical assistance. T.W.M is supported by NIH NRSA postdoctoral training grant 5 F32 GM17610–03 and M.B.M. is supported by NIH NRSA postdoctoral training grant 1 F32 GM18414–01. This work is supported by Department of Energy grant DE-FG03–88ER13917 to B.J.S. Work in N.-H.C.’s laboratory was supported by a DOE grant(DOE 94ER 20143).

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