Biological materials and growth conditions
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 [38,39]. 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.
Staining, imaging and microscopy
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).
Abiotic treatments
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.
Double mutant analysis
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].
Nucleic acid analysis and manipulations
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 [19,56].
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 [18,58]. 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 [28,60].
Phosphoinositide binding assays
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).
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