edr2 exhibits a late acting resistance phenotype associated with cell death
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 (2, 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).
Lesion formation in edr2-6 is only induced by biotic stresses
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.
edr2-6 mutants are resistant to some but not all pathogens
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.
edr2-6 plants do not exhibit constitutively active defense responses
Some lesion mimic mutants are disease resistant because defenses, including the SA signal transduction pathway, are constitutively activated [1,12]. 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).
Both the resistance and lesion phenotypes are dependent on the SA pathway
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.
EDR2 encodes a novel, ubiquitously expressed protein
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 specifically binds to phosphatidylinositol-4-phosphate in vitro
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).
EDR2 is localized to multiple compartments
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).
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