The edr2-6 mutant was indistinguishable from wild type in growth and development up to ~3 weeks of age (Fig. 1A,1B). The wild type remained free of lesions whether inoculated with the powdery mildew pathogen or not (Fig. 1C). The mutant did not develop lesions spontaneously. It only became chlorotic and formed lesions at infection sites and did not support visible fungal growth (Fig. 1D,1E; see also Fig. 1a of Tang et al. 16). The lesions on edr2-6 leaves did not spread to non-infected parts of inoculated leaves or to uninoculated leaves of the same plant (Fig. 1E). At later stages, the petioles of edr2-6 leaves were slightly shorter and the leaves tended to be crinkled (Fig. 1A,1B).

The severity of the edr2-6 phenotype varied with inoculation density. Fungal growth measurements up to 5 days post-inoculation (dpi) obtained under very low inoculation densities (~1 conidium per mm²) are similar on Col-0 and edr2-6 (data not shown). Similarly, their macroscopic phenotypes were identical up to this time point (data not shown). By contrast, when fungal growth was monitored at 7 dpi under high inoculum density (~100 conidia per mm²), the mutant and wild type were clearly distinguished with wild type supporting significantly more fungal growth than edr2-6. Under these conditions, wild-type leaves had an average of 236 ± 87 conidiophores per mm², whereas the edr2-6 mutant had 49 ± 40 conidiophores per mm² (average ± standard deviation, n = 75, p ≤ 0.01 by Student's t-test).

The timing of macroscopic lesion formation in the mutant varied with inoculation density and, as also reported by Tang et al. 16, occurred relatively late in the infection cycle compared to the rapid HR (<24 hours post-inoculation [hpi]) typically elicited by incompatible interactions governed by plant resistance and pathogen avirulence genes. Macroscopically, the first lesions appeared 4 dpi at high inoculation densities and 7 dpi at low inoculation densities. The wild-type plants did not develop visible lesions upon powdery mildew infection regardless of inoculum density. Fungal infection eventually led to an apparent acceleration of senescence in a density-dependent fashion in both wild type and mutant, but the amount of chlorosis was greater in the mutant. At 7 dpi, leaves infected with ~100 conidia per mm², had 6.0% ± 6.2% chlorotic tissue in the wild type, whereas the edr2-6 leaves had 22.1% ± 13.9% chlorotic and 10.6% ± 5.6% necrotic tissue (average ± standard deviation, n = 15 leaves). The amount of necrotic tissue in the mutant also correlated with the inoculation density. Thus, at 7 dpi with ~20 conidia per mm², the edr2-6 mutant had 14.3% ± 10.7% chlorotic tissue and 2.6% ± 2.3% necrotic tissue (average ± standard deviation, n = 10 leaves).

Cell death was also monitored microscopically at various time points during the infection process under high inoculation density. In wild-type plants, no macroscopic lesions were observed upon inoculation with G. cichoracearum and only rare groups of more than three collapsed mesophyll cells were observed 7 dpi (Fig. 2A). Up to 2 dpi, edr2-6 leaves were indistinguishable from wild type, with no apparent cell death. By 3 dpi, a few isolated epidermal and mesophyll cells appeared dead in edr2-6 and were usually associated with fungal hyphae. The first small groups of dead mesophyll cells (2 to 3) also appeared 3 dpi. From 4 to 7 dpi, these lesions were more frequent, and increased in size (up to 50 to 100 cells) (Fig. 2B; see also Fig. 1D of Tang et al. 16).

The occurrences of hydrogen peroxide and callose, which typically accumulate in cells that undergo an HR, were assessed. In both wild-type and edr2-6 plants, hydrogen peroxide and callose were present at the fungal penetration sites (Fig. 3). In wild type, both compounds were found in papillae, cell wall appositions deposited by the plant at sites of attempted penetration. Both compounds also accumulated in whole cells, predominantly in edr2-6 leaves, following the pattern already observed for the appearance of dead cells. Autofluorescent compounds, believed to be antimicrobial molecules, also accumulated in whole edr2-6 cells in a similar pattern to that observed for the appearance of dead cells (data not shown).

Blumeria graminis f.sp. hordei, the barley powdery mildew, which is not a pathogen of Arabidopsis, was also able to induce macroscopic lesion formation in edr2-6, but not Col-0 (Fig. 4A). In edr2-6 mutants, the number of dead cells was comparable to wild type until 3 dpi. By 4 dpi, the first small lesions occurred at high inoculation densities and increased in size until 7 dpi (Fig. 4A).

To ascertain whether lesion formation on edr2-6 leaves was specifically triggered by pathogen infection, plants were treated with several types of abiotic stress (mechanical, thermal, drought and light stress). No macroscopic or microscopic lesions were observed after any of these treatments as determined by visual observation and trypan blue staining (data not shown). After wounding, the amount and the localization of dead cells were comparable in Col-0 and in edr2-6, and no spreading cell death was observed in the mutant.

A number of lesion mimic mutants exhibit resistance to a broad spectrum of pathogens. To determine the resistance specificity of edr2-6, mutant plants were challenged with an oomycete pathogen, Hyaloperonospora parasitica, and a bacterial pathogen, Pseudomonas syringae pv tomato DC3000. The level of edr2-6 resistance to a biotrophic H. parasitica Emco5 was evaluated by counting the number of sporangia per cotyledon at 9 dpi. At high inoculum concentration (3 × 10⁵ sporangia per ml), the wild type had 8.4 ± 4.9 sporangia per cotyledon, whereas the edr2-6 mutant had 3.5 ± 2.3 (average ± standard deviation, n = 30, p ≤ 0.01 by Student's t-test). At lower inoculum concentrations (10⁵ sporangia per ml), wild type and mutant were indistinguishable (2.5 ± 2.4 and 2.0 ± 1.8 sporangia per cotyledon, respectively [average ± standard deviation, n = 30], p = 0.40 by Student's t-test). A second H. parasitica strain, Noco2, showed reduced growth and elicited lesions when inoculated onto the leaves of 3-week-old edr2-6 plants. The number of spores per mm² were 12.8 ± 10.5 (n = 15) and 6.0 ± 4.5 (n = 18) for wild type and edr2-6, respectively (p = 0.03 by Student's t-test).

P. syringae tomato DC3000 multiplies in both Col-0 and edr2-6 plants, eventually producing water-soaked lesions. Unlike the response to powdery mildew, the timing and extent of macroscopic and microscopic symptom development were similar for mutant and wild-type plants, regardless of the concentration of inoculum (Fig. 4B). The estimation of bacterial titers in the infected leaves did not reveal any significant difference between bacterial multiplication rates in Col-0 and edr2-6 (growth curves not shown). The edr2-6 plants, which carry the RPS2 resistance gene, mounted a normal hypersensitive necrosis response following infiltration with the avirulent bacterial strain carrying the AvrRpt2 gene (Fig. 4C). Similar results were reported by Tang et al. 16. The loss of EDR2 function did not interfere with the ability of the plants to mount a normal HR.

Some lesion mimic mutants are disease resistant because defenses, including the SA signal transduction pathway, are constitutively activated 112. Similar to the results of Tang et al. 16, PR1 transcript levels, a marker for the SA pathway, in uninfected edr2-6 plants were negligible and similar to uninfected wild-type plants, indicating that the SA pathway was not constitutively activated in the mutant. After 2 dpi, PR1 expression was induced in both wild-type and mutant plants and PR1 levels remained high up to 7 dpi (Fig. 5A). The level of PR1 induction was two-fold higher in edr2-6 relative to Col-0 plants at every time point suggesting possibly that edr2-6 mutants are predisposed to respond to a stimulus activating the SA pathway. This stimulus may be lesion formation as SA levels increase following treatments that induce lesions 12.

PR1 levels were also monitored in plants treated with SA and the SA mimic, BTH. As expected, these treatments induced PR1 expression in both Col-0 and edr2-6. However, in the mutant plants, PR1 gene expression was two-fold higher than in wild-type plants (Fig. 5B). The same trend in PR1 up-regulation occurred in edr2-6 plants infected with the virulent bacterium P. syringae tomato DC3000, in a dosage dependent manner (Fig. 5C).

The transcript levels of the PDF1.2 gene encoding an anti-microbial defensin, a marker for the jasmonate/ethylene signal transduction pathway, over the time course used for PR1 gene expression analysis were not significantly different between edr2-6 and Col-0 (data not shown).

To analyze the involvement of the major defense signaling pathways, double mutants with defects in the SA or jasmonate/ethylene pathways were analyzed for their resistance and lesion phenotype. Resistance was lost in plants expressing the NahG gene and in the edr2-6 pad4-1 double mutant (Fig. 1F,1G,1H,1I; see also Fig. 3 of Tang et al. 16). Similar to edr2-6, the double mutants edr2-6 coi1-1 and edr2-6 ein2-1 did not support fungal growth and showed lesion formation (Fig. 1J,1L; see also Fig. 3 of Tang et al. 16). At the microscopic level, the lesion phenotype was not suppressed by the coi1-1 or ein2-1 mutations (Fig. 2C,2D), but was lost in edr2-6 plants expressing NahG and in the edr2-6 pad4-1 double mutant (Fig. 2E,2F). Thus, the SA pathway contributes to lesion formation and the resistance phenotype in edr2-6 mutants. Since G. cichoracearum is an obligate biotrophic pathogen, requiring living host tissue for growth, the resistance phenotype is likely a consequence in part of the inability of the chlorotic and lesioned tissue to support growth of this pathogen.

The segregation analysis of F₂ plants from a cross between edr2-6 and wild type suggested that the edr2-6 mutation was linked to a single T-DNA insertion, and that both the disease resistance and the lesion phenotypes of edr2-6 following powdery mildew infection were due to a unique recessive mutation (data not shown). The EDR2 gene was isolated by cloning the regions flanking the T-DNA insert. Sequencing revealed that the T-DNA was inserted in a predicted intron of gene At4g19040, a gene cloned previously as EDR2 16. A 12 kb fragment covering this putative gene was cloned into a binary Ti plasmid and used to transform homozygous edr2-6 plants. The wild-type phenotypes (susceptibility and no lesions following powdery mildew inoculation) were restored in 54 (98.2%) of the 55 T₁ plants tested (data not shown). The progeny of six of these T₁ plants segregated 3:1 (susceptible:resistant) for powdery mildew resistance, as expected.

A cDNA for the EDR2 gene was isolated by RT-PCR. Its sequence was identical to the NCBI deduced cDNA sequence NM_118022. The genomic sequence of EDR2, which is composed of 22 exons and 21 introns, extends 5,373 nucleotides. The coding sequence is 2,157 nucleotides long and encodes for a protein of 718 amino acids with a predicted molecular weight of 82 kD. The EDR2 protein consists of an N-terminal pleckstrin homology (PH) domain (2.6 × e^-9 confidence value), a central region with a StAR-related lipid-transfer (START) domain (1.8 × e^-8) and a C-terminal, plant-specific, domain of unknown function, DUF1336 (1.5 × e^-115) (Fig. 6) 18.

A gene on chromosome V, At5g45560, is homologous to EDR2 with >75% nucleotide identity across the entire gene and approximately 89% identity on the protein level. Two other genes in the Arabidopsis genome, At2g28320 and At3g54800, are predicted to encode proteins that display the same domain structure as EDR2 with PH, START and DUF1336 domains. These proteins show relatively little sequence similarity to EDR2 (36% and 32% amino acid sequence identity, respectively).

A survey of published gene expression profiling data showed that EDR2 is expressed in all organs 19, corroborating pEDR2:GUS expression results from Tang et al. 16. Its expression did not vary substantially during development, with the exception that in stamens and senescing leaves, EDR2 transcript levels were ~2–3-fold higher than in most other organs or developmental stages (Table 1). As observed by Tang et al. 16, EDR2 transcript levels were generally unresponsive to biotic stresses. The highest inductions (~2.2-2.3 fold) were elicited by inoculation with the necrotrophic fungal pathogen, Botrytis cinerea, at 48 hpi and by the bacterial pathogen, P. syringae tomato DC3000, at 24 hpi (Table 2). In contrast, At5g45560 transcript levels were generally ~1/3 those of EDR2 during development and in various organs, and mostly below the level of reliable detection in the biotic stress experiments. Transcript levels of At3g54800 were very low and not detected in most organs, developmental stages or under various biotic stresses, with the exception of the stamens, which expressed this gene at levels ~100-fold higher than in most other organs. At2g28320 was expressed at ~1/2 the level of EDR2 with its highest transcript levels occurring in mature flower parts. The expression of this latter gene was unresponsive to biotic stresses.

The PH domain of EDR2, expressed as a PH domain-GST fusion protein, had strong in vitro binding affinity for phosphatidylinositol-4-phosphate (PI-4-P) (Fig. 7). Very weak binding to phosphatidylinositol-3-phosphate or phosphatidylinositol-5-phosphate was also observed. In the course of cloning the PH domain, we fortuitously obtained a mutant PH-GST construct in which the phenylalanine in position 93 was replaced by a serine. This fusion protein completely lacked the ability to bind to PI-4-P, suggesting that this amino acid is important for the PI-4-P binding (Fig. 7).

A C-terminal eGFP fusion construct with expression driven by the native EDR2 promoter was transformed into edr2-6 and the resulting lines were analyzed for complementation of the mutant phenotype and GFP fluorescence (Fig. 8A). The construct complemented both the resistance and the lesion phenotype in five independent transgenic lines (Fig. 8B). Using a spinning disk scanning laser confocal microscope, EDR2:HA:eGFP was observed in the endoplasmic reticulum, plasma membrane and in small endosomes in young seedlings (Fig. 8C, upper panels). In young dividing cells, the expression of EDR2 seemed greatly reduced relative to levels observed in more mature cells (Fig. 8C, asterisked cells). In the rosette leaves of mature plants, EDR2:HA:eGFP was localized to the same three subcellular compartments, although to a lesser relative extent to the endoplasmic reticulum. EDR2:HA:eGFP did not co-localize with the mitochondrial dye MitoTracker (Fig. 8D).

The edr2-6 mutant is a T-DNA insertion mutant derived from Col-0 37 and was backcrossed to Col-0 once. The plants were grown in growth chambers at 22°C with a 14-h photoperiod, except for those plants to be inoculated with Hyaloperonospora parasitica, which were grown at 16°C in a 10-h photoperiod. The maintenance of the G. cichoracearum UCSC1, the production of inoculum on a secondary host, squash (variety Kuta), and the inoculation procedures were previously described 3839. The barley powdery mildew, Blumeria graminis f.sp. hordei race CR3, was maintained on barley variety CI-16138 (=AlgerianS) and inoculated onto barley or Arabidopsis plants as described 40. Maintenance and infiltration with Pseudomonas syringae pv tomato DC3000 41 and inoculation with H. parasitica Emco5 42 were performed as described by Vogel and Somerville 37. Bacterial growth curves were performed by estimating the titers of bacteria in leaves up to 4 dpi 13.

Some powdery mildew inoculations were performed at high (~100 conidia per mm²) and low (~1 conidium per mm²) densities. Inoculation densities were assessed by placing 1 cm² cover slips, coated with agar, among plants to be inoculated and then counting the number of conidia per mm² by light microscopy. Unless stated otherwise, macroscopic disease development and lesion formation was monitored at 7 dpi. The extent of G. cichoracearum growth was quantified by one of two methods. At low inoculation densities, the total hyphal length of individual fungal colonies was measured periodically up to 4 dpi and the total number of conidiophores per colony were counted at 5 dpi 37. At high inoculation densities, the number of mature conidiophores per field of view (0.16 mm²) was determined at 7 dpi. In each case, pictures of five randomly chosen fields of view per leaf and a minimum of 10 leaves per experiment were used to assess fungal growth. Growth of H. parasitica was monitored as described in 37. All experiments were conducted at least twice.

Using 3-week old plants at 7 dpi, areas of healthy, chlorotic (yellow) and dead tissues were measured on 15 leaves of each treatment from photographs taken with a Spot Camera (Diagnostic Instruments, Inc.) attached to a dissecting microscope (Leica Wild M8, Leica Instruments, Inc., Exton, PA, USA). The photographs were imported into Photoshop 5.0 (Adobe, San Jose, CA, U.S.A.) and the "magic wand tool" was used to decompose the image into three components: green tissue (healthy tissue); yellow tissue (chlorotic tissue) and brown tissue (lesions and necrotic tissue). The area of each of these three components was measured with NIH IMAGE software 1.6267 43 and used to calculate the percentage of total leaf area corresponding to each of the three components.

The staining method used to visualize fungal colonies and the software program employed to measure total hyphal length were described previously 37. Microscopic lesions and fungal structures were visualized by staining the leaves with trypan blue 37. Callose staining with aniline blue and the visualization of autofluorescent compounds were described in Adam and Somerville 44. Hydrogen peroxide was visualized with 3,3'-diamino benzidine-HCl 45.

T3 transformants of edr2-6 containing the pCH2 construct encoding the EDR:HA:GFP driven by the EDR2 promoter were germinated on nutrient agar plants containing Musashige and Skoog salts 4.3 g per L, pH 5.7,1.5% agar, and hygromycin (30 μg per mL) (Sigma, St. Louis, MO). At 1 week after germination, plants expressing GFP were observed under a Leica DMIRE2 spinning disk confocal laser scanning microscope (Leica Microsystems, Inc.). The seedlings were mounted in water and excited with an Argon laser (488 nm) for eGFP visualization. Rosette leaves from 7-week-old plants were also observed. In addition, rosette leaves of 7-week-old EDR2:HA:eGFP expressing plants were stained with 4 μM MitoTracker Red CMXRos (Molecular Probes, Eugene, OR, USA) for 90 min. Collected image data sets were subsequently analyzed with the digital image analysis programs Image J (v. 1.30, N.I.H., USA) and Adobe Photoshop (v. 7.0).

Mechanical stresses were inflicted by wounding (e.g., cutting, puncturing, infiltrating with water or folding) mature leaves. Temperature stresses were performed at 4°C for 8 weeks or at 37°C for 6 or 15 h. The influence of the length of the day was tested by growing plants either in continuous light, or in conditions where the photoperiod was 14 or 10 h. The consequences of hydric stresses (drought, water saturated atmosphere for 24 h) were also recorded. Trypan blue staining of dead cells was performed at 1, 3 and 7 days after each treatment and visualized by light microscopy as described above. All tests were performed on 3-week-old plants and were repeated at least three times.

Standard genetic crosses were used to make double mutant lines of edr2-6 with pad4-1 21, ein2-1 46, coi1-1 47 and the transgene NahG 48. F2 plants homozygous for edr2-6 and pad4-1 or edr2-6 and NahG were identified by PCR 49. The ein2-1 mutation was identified in plants by their lack of root growth inhibition when grown on Murashige and Skoog medium supplemented with 10 μM 1-aminocyclopropane-1-carboxylic acid. The coi1-1 mutation was identified in plants that did not exhibit a stunted growth habit when grown in Murashige and Skoog medium containing 20 μM methyl-jasmonate 50.

The edr2-6 mutation was generated by inserting a 5.8 kb T-DNA fragment containing a right border (RB) and a left border (LB), the BAR gene, the NPTII gene and a fragment of an leucine-rich repeat gene driven by the 35S promoter. Given both that the phenotype of edr2-6 resembles that of the edr2-1 (a point mutation leading to the change, W256STOP) 16 and that the rescue experiment with the cloned EDR2 gene restored the wild-type phenotype to the edr2-6 mutant, we feel that the additional sequences in this construct did not contribute to the phenotypes described in the text. To test cosegregation of the edr2-6 mutation with the T-DNA, edr2-6 was crossed to Col-0 and 250 F₂ plants were first analyzed for disease phenotypes 7 dpi with G. cichoracearum 37, and then scored for resistance to BASTA (25 μL glufonsinate ammonium per L) (Bayer Crop Sciences).

To clone EDR2, genomic regions flanking the T-DNA insert were amplified by PCR using the Universal Genome Walker Kit (Clontech, Mountain View, CA). The T-DNA insert-specific primers used were: LB 5'-AAC TTG ATT TGG GTG ATG GTT CAC GTA GTG-3', LB nested 5'-GCC CTG ATA GAC GGT TTT TCG CCC TTT GAC-3', RB 5'-CAA TCC ATC TTG TTC AAT CAT GCG AAA CGA-3' and RB nested 5'-CGA CTT TTG AAC GCG CAA TAA TGG TTT CTG-3'. Two BAC clones (F13C5 and F16M6) encompassing the region of interest were used to subclone a wild-type copy of the gene that was disrupted by the T-DNA insert in the edr2-6 mutant. A 12 kb NcoI fragment encompassing the gene was cloned into the pCAMBIA1380 51. The construct was introduced into Agrobacterium tumefaciens and subsequently into edr2-6 plants 52. Transgenic plants were tested in the T1 and T2 generations for powdery mildew resistance.

A fragment of EDR2 cDNA was amplified via RT-PCR, cloned, and sequenced. The primers used were complementary to the poly-A tail (5'-GGC CAC GCG TCG ACT AGT ACT TTT TTT TTT TTT TTT T-3') and a region about 50 nucleotides before the predicted EDR2 start codon (5'-CCG TGG GGA AGT TTT GTG-3').

An EDR2:HA:GFP fusion construct under the control of the EDR2 native promoter was created. A fragment containing 1.4 kb upstream of the predicted translational start of EDR2 and 2.1 kb EDR2:HA cDNA were PCR amplified or RT-PCR amplified, respectively and fused together via two-template PCR using primers 5'-GCA GTC GAC GGT ACC AAT TCT GAC AGG TGC AGC TTT TCC-3' and 5'-CGG TCG AGA CCC GGG GAG CAT AAT CTG GAA CAT CGT ATG GAT AGC CTC CTG ACT CCA GAT TCG GAA C-3' 53. The two templates were generated with primers 5'-GCA GTC GAC GGT ACC AAT TCT GAC AGG TGC AGC TTT TCC -3' and 5'-GAT CTT CCT CCT TCC ATA CCT AA-3' (promoter) and 5'-AAA TCT TCG CTA ATC GCA GAG AC-3' and 5'-CGG TCG AGA CCC GGG GAG CAT AAT CTG GAA CAT CGT ATG GAT AGC CTC CTG ACT CCA GAT TCG GAA C-3' (EDR2 cDNA with HA tag). The promoter (EDR2):EDR2 cDNA:HA construct was subsequently cloned into the KpnI/SmaI sites of pSK001H to obtain pCH2. The plasmid pSK001H, which contained the gene for eGFP, was derived from pEZR(H)-NL by removing a SacI fragment containing the CaMV 35S promoter. Plasmid pZEZR(H)-NL was provided by Dave Ehrhardt (Carnegie Institution, Stanford, CA) 54. The plasmid pCH2 was introduced into edr2-6 mutants via Agrobacterium-mediated transformation 52 and T1 transformants were selected on hygromycin plates.

RNA extraction and northern blot analysis, using 15 μg of total RNA, were performed as described 55. Signals were detected using a PhosphorImager (Typhoon 8600) and quantified using the ImagQuant program (Molecular Dynamics, Sunnyvale, CA). Additional information about the expression of EDR2 and related genes was recovered from Genevestigator 1956.

Information about the EDR2 protein structure was recovered from TAIR, the Arabidopsis Information Resource 57, and from SUBA, the Subcellular Location of Proteins in Arabidopsis database 1858. Domains present in EDR2 were identified with the Hidden Markov Model program 59. Proteins from other organisms that contained the same domains as EDR2 (PH, START, DUF1336 or PH, START) were identified using the Conserved Domain search provided by NCBI 2860.

A fragment encoding the first 191 amino acids of EDR2 was amplified via PCR using the primers 5'-GCG GGA TCC ATG TCT AAG GTA GTG TAC GAA-3' and 5'-CCG GAA TTC TGG TTC GCC AAC TCT GCA TCA A-3'. This fragment was cloned into the BamHI and EcoRI sites of the vector pGEX-2TK (Amersham Biosciences; Piscataway, NJ) and transformed into the E. coli strain BL21-CodonPlus(DE3)-RP (Stratagene; La Jolla, CA). Protein expression was induced with 1 mM isopropyl-β-D-thiogalactoside for 3 h. The purification of the glutathione-S-transferase (GST)-fusion protein was done according to the method described by Smith and Johnson 61 except that 1 M urea was included in the elution buffer.

PIP strips from Echelon Biosciences Inc. (Salt Lake City, UT) were blocked in TBS+M (10 mM Tris, HCl pH 7.0, 150 mM NaCl, 5% (w/v) milk powder) for 1 h and incubated with 0.05 mg/ml of the GST fusion protein in TBS+M for 1.5 h. The membranes were washed 4 times for 5 min with TBS + 0.05% (v/v) Tween20 and 2 times for 5 min with TBS. Incubation with the anti-GST (diluted 1:1000) and the anti-mouse antibody (1:7500) (both from Sigma, St. Louis, MO) were made for 1 h each in TBS+M with washing steps after each incubation step as described. Signals were detected with the SuperSignal West Pico Chemiluminescent Substrate from Pierce (Rockford, IL).