Article Figures & Data
Figures
- Figure 1.
Visible and Defense Phenotypes of acd6, npr1 acd6, and acd6–nahG Plants.
(A) Thirteen-day-old plants photographed at the same distance. nahG control plants were indistinguishable in appearance from acd6–nahG plants (not shown).
(B) First true leaf of 8-week-old aseptically grown plants of the indicated genotypes, showing enhanced chlorophyll loss and cell death in acd6.
(C) Two representative leaves of the indicated genotypes photographed 24 hr after infection with 4 × 108 cfu/mL P. s. pv maculicola ES4326/avrRpt2, showing induced hypersensitive cell death of ACD6 leaves and no cell death on acd6 leaves beyond that which was already present on the leaves prior to inoculation. ACD6 leaves treated with P. s. pv maculicola ES4326/vector control alone or mock-treated showed no visible symptoms at this time point (24 hr). acd6 leaves treated with P. s. pv maculicola ES4326 or mock treated were indistinguishable from those shown here.
(D) Four-week-old npr1 acd6 leaves. Arrow points to an abnormal growth. npr1, wild-type, and acd6 control plants showed no obvious abnormal growths.
(E) Three-week-old plants were treated as indicated and photographed 2 days later.
(F) Four-week-old plants were treated as indicated and photographed 4 days later. The middle panel shows a close-up view of BTH-induced abnormal growths that have coalesced to reveal large, disorganized unpigmented cells along the bottom surface of the leaf. Arrow points to an abnormal growth. This experiment was repeated four times with similar results. Bars = 1 mm of leaf tissue.
- Figure 2.
Microscopic Cell Death and Cell Growth Phenotypes of acd6 Plants.
Representative leaves stained with trypan blue are shown.
(A) to (C) The first true leaves from 11-day-old plants. (A) ACD6 leaf; (B) acd6 leaf; and (C) acd6/ACD6 leaf.
(D) Thirty-day-old acd6/ACD6 leaf with a small growth.
(E) and (F) Twenty-four-day-old triploid leaves. (E) ACD6 triploid leaf; (F) acd6/ACD6/ACD6 triploid leaf.
(G) and (H) Thirty-day-old acd6–nahG leaves. (G) shows a leaf 4 days after water treatment. (H) shows a leaf 4 days after treatment with 100 μM BTH. BTH did not cause any microscopic cell death or cell enlargement in the wild-type or nahG control plants (data not shown).
(I) and (J) Twenty-four-day-old leaves. (I) npr1 leaf; (J) npr1 acd6 leaf.
(K) First true leaf of an acd6 plant. Leaf was 6 mm long.
White arrows point to clusters of enlarged and dead (stained) cells. Black arrows point to small growths. These experiments were repeated twice with similar results. Bars in (A) to (J) = 1 mm.
- Figure 3.
Steady State Transcript Levels of Defense-Related Genes in acd6 Plants.
RNA gel blot analysis of total RNA extracted from leaves of 20-day-old plants is shown. The blot was probed with the defense-related genes PR-1, GST1, and AIG1. +, ACD6; +/6, acd6/ACD6; 6/6, acd6. This experiment was repeated once with similar results.
- Figure 4.
Steady State Levels of PR-1 Gene Transcripts in npr1 acd6 Plants and acd6/ACD6/ACD6 Triploids.
RNA gel blot analysis of PR-1 transcript levels determined from leaves from the indicated genotypes at 24 days after planting. +/acd6, acd6/ACD6; ++++, CS3432 (tetraploid Arabidopsis); ++/+, CS3432 × ACD6 F1; ++/acd6, CS3432 × acd6 F1; n6, npr1 acd6; n/6, npr1 acd6/ACD6. This experiment was repeated once with similar results.
- Figure 5.
Analysis of Growth of P. syringae Strains in acd6 Plants.
For all graphs, error bars indicate standard deviations.
(A) Growth of P. s. maculicola ES4326 in ACD6 and nahG plants after treatment with 100 μM BTH compared with acd6 double mutants with npr1 and nahG, respectively. Infections were done to give 3.5 × 103 cfu/cm2 leaf area 3 days after BTH treatment, and bacterial growth determinations were done 3 days after the infection. +, ACD6; +B, ACD6 with 100 μM BTH; 6/6, acd6; +/6, acd6/ACD6; n, npr1; n6, npr1 acd6; n/6, npr1 acd6/ACD6; +/n +/6, npr1/NPR1 acd6/ACD6; nG, nahG; nG B, nahG with 100 μM BTH; nG 6/6, acd6–nahG.
(B) Growth of P. s. maculicola ES4326 in triploid ACD6 (circles) and acd6/ACD6/ACD6 plants (squares). Each experiment was repeated at least once with similar results.
(C) Growth of P. s. maculicola ES4326/vector, P. s. maculicola ES4326/avrRpt2, and P. s. maculicola ES4326/avrRpm1 in ACD6 (open symbols) and acd6 (closed symbols) plants. Squares, P. s. maculicola ES4326/vector control; triangles, P. s. maculicola ES4326/avrRpm1; circles, P. s. maculicola ES4326/avrRpt2.
- Figure 6.
Camalexin Levels in P. syringae–Infected acd6 Plants and BTH-Treated Plants.
(A) Plants were treated with P. s. tomato DC3000 (vir [Pst]) or P. s. tomato/avrRpt2 (avr) for 1 day or with 10 mM MgSO4 (mock) or P. s. maculicola ES4326 (vir [Psm]) for 2 days. Six samples per treatment were used. Error bars show standard deviations. This experiment was repeated once with similar results.
(B) Plants were treated with the indicated doses of BTH, and camalexin was extracted and quantitated after 2 days. Four samples were used for each treatment. Error bars show standard deviations. This experiment was repeated twice with similar results.
- Figure 7.
Transient Assay of avrRpm1 and β-Glucuronidase (GUS) Expression in Four Different Plant Genotypes.
Three sets of DNA constructs (pKEx4tr-GUS [carries 35S-GUS]; pKEx4tr-GUS plus pKEx4tr [vector control]; and pKEx4tr-GUS plus pKEx4tr-avrRpm1) were biolistically transformed or cotransformed into plant leaves, and GUS activity was assayed by counting the number of leaf cells staining blue. Two replicate experiments were performed except in the case of transformation into the acd6/ACD6 heterozygote. In each case, the box at left is from experiment 1 and the box at right is from experiment 2. In the case of acd6/ACD6, data were obtained in experiment 1. The box plots show the mean (+ in the center of the box) and the second and third quartiles, which indicate the dispersion of 50% of the data points (open boxes) and the range (vertical lines above and below the boxes). Statistical outliers are indicated by open circles.
- Figure 8.
Time Course of the Steady State Levels of Defense-Related Transcripts in BTH-Treated Plants.
(A) Three-week-old plants were untreated (lane 1) or treated with water (lane 2), 1 μM BTH (lane 3), 10 μM BTH (lane 4), 100 μM BTH (lane 5), or 300 μM BTH (lane 6) for 24 hr and then harvested. acd6nG, acd6–nahG.
(B) Three-week-old plants were treated with water (lane 1), 1 μM BTH (lane 2), 10 μM BTH (lane 3), 100 μM BTH (lane 4), or 300 μM BTH (lane 5) for 48 hr and then harvested. This experiment was repeated three times with similar results.
- Figure 9.
A Model for How SA Acts to Activate Cell Death, Cell Growth, and Defenses in acd6 Plants.
The suppression of all the acd6-conferred phenotypes by nahG suggests that SA is required for all the acd6-conferred phenotypes. Only some phenotypes of acd6 are suppressed by npr1; therefore, acd6 acts through the NPR1-dependent pathway 1 and the NPR1-independent pathway 2. Because all acd6-conferred phenotypes require SA, although SA is not sufficient to induce these phenotypes when applied to wild-type plants, we hypothesize that SA acts together with a second unknown signal (?) to induce the pathway 2 phenotypes. The second signal thus requires activation by acd6 and the presence of SA to activate pathway 2. Furthermore, because acd6–nahG plants treated with BTH showed hyperactivation of the acd6-conferred phenotypes, we hypothesize that the second signal is negatively regulated by SA in addition to acting with SA. The unknown second signal might be two signals.
Tables
Phenotype Cross (Recipient × Pollen Donor) Type Healthy Mild Cell Deatha Cell Deathb χ2 Hypothesisc Fit? acd6/ACD6(M2)d Self 22 43 19 0.262e 1:2:1 Yes ACD6 × acd6/ACD6(M2) F1 39 37 0 0.053e 1:1:0 Yes ACD6 × acd6/ACD6(M2) F2f 28 63 30 0.149e 1:2:1 Yes ACD6 × acd6/ACD6(M2) F2g 79 0 0 1:0:0 acd6/ACD6 × ACD6 F1 16 18 0 0.118e 1:1:0 Yes acd6/ACD6 Self 36 65 30 0.561e 1:2:1 Yes acd6 × ACD6 F1 0 29 0 0:1:0 ACD6 × acd6 F1 0 17 0 0:1:0 ACD6 × acd6/ACD6 F1 37 40 0 0.117e 1:1:0 Yes CS3432h × ACD6 F1 46 0 0 1:0:0 ACD6 × CS3432 F1 59 0 0 1:0:0 CS3432 × acd6 F1 0 53 0 0:1:0 acd6 × CS3432 F1 0 67 0 0:1:0 -
↵a Plants in this phenotypic class had slightly yellowed leaves and microscopic cell death and were of medium stature.
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↵b b Plants in this phenotypic class had many visible punctate cell death patches and were of reduced stature.
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↵c The hypothesis is that acd6 segregates as a semidominant trait with respect to cell death.
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↵d M2 designates the original mutant plant.
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↵e Not significantly different from the expected value (P > 0.7 to 0.9 in each of these cases).
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↵f This is the progeny of a mild cell death and medium stature F1 plant.
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↵g This is the progeny of a healthy, normal stature F1 plant.
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↵h CS3432 is a gi-2 co-1 tetraploid Arabidopsis in the Col-0 background (acd6 is from the same background).
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Genotype/Phenotypea μg Camalexin cm–2 (sd) ACD6 <0.001 acd6 0.086 (0.040) acd6/ACD6 0.089 (0.040) acd6/nahG F1 <0.001 acd6-nahG <0.001 nahG <0.001 npr1 0.001 npr1 acd6 F3b 0.207 npr1 acd6 F3b 0.207 npr1 acd6 F3b 0.107 npr1 acd6 F3b 0.089 ACD6/ACD6/ACD6 0.003 (0.001) acd6/ACD6/ACD6 0.157 (0.049) -
↵a Four to six replicates were assayed for each genotype.
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↵b An npr1 acd6/ACD6 F2 individual was self-fertilized. Individual F3 progeny that showed slightly yellowed leaves were assayed. These plants may have been homozygous or heterozygous for acd6 (see text). This experiment was repeated twice with similar results.
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