Article Figures & Data
Figures
- Figure 1.
Compromised disease resistance to bacterial infection in cds2-1D mutant. A, Disease symptom of wild-type Col-0 (left) and mutant cds2-1D (right) plants 3 d after hand-infiltration of Pst. The arrows indicate the inoculated leaves. B, Bacterial growth of various P. syringae strains in leaves from cds2-1D mutant (gray) and wild-type Col-0 (white) plants. C, Flagellin-induced restriction of Pst growth is attenuated in cds2-1D mutant. Leaves of Col-0 and cds2-1D plants were pretreated for 24 h by infiltration with 100 nm flg22 peptide (gray) or water as control (white) before bacterial challenge. D, Bacterial growth of wild-type Pst and the TTSS-deficient hrcC strain in detached leaves from Col-0 (white) and cds2-1D (gray) plants. The hand-infiltrated leaves were excised from plants and inserted in 0.7% water-agarose plate after the excessive water had dissipated. The plates were sealed with 3M Micropore surgical tape and incubated under the same condition as the in planta bacterial growth assay. In all the above experiments, leaves were infiltrated with 105 cfu mL−1 of bacteria and were collected 2 d after inoculation for growth assay. Data shown are means ± sd, and similar results were observed in three replicate experiments. dpi, Days post inoculation.
- Figure 2.
SA induced PR1 expression and systemic acquired resistance (SAR) is suppressed in the cds2-1D mutant. Leaves were collected at 0, 1, 2, 3, and 4 d after SA treatment or bacterial challenge. A, RNA gel blot analysis of PR1 gene transcript levels in wild-type Col-0 and cds2-1D mutant plants sprayed with 1 mm SA. dpi, Days post inoculation. B, Systemic induction of PR1 expression is suppressed in the cds2-1D mutant plant. Two leaves of each plant were inoculated with 5 × 106 cfu mL−1 of Psm-avrRpm1. The inoculated local leaves and noninoculated systemic leaves were collected for RNA gel blot assay of PR1 transcript levels. C, Attenuated systemic acquired resistance in cds2-1D mutant. Treatments consist of inoculation of two leaves with 5 × 106 cfu mL−1 of avirulent Psm-avrRpm1, followed 2 d later with the second inoculation of systemic leaves with 105 cfu mL−1 of virulent Pst. Magnesium chloride (10 mm) was infiltrated as control to avirulent bacterial treatment. Growth of virulent Pst in systemic leaves was monitored 2 d after the second inoculation. Significant reduction of Pst growth in systemic leaves was observed in Col-0 plants between MgCl2 and avirulent Psm-avrRpm1 treatments (indicated by ***, t test, n = 6, P < 0.001), whereas the difference in cds2-1D plants was not statistically significant (indicated by “ns”). This experiment was repeated twice with similar results. [See online article for color version of this figure.]
- Figure 3.
Functional analysis of cds2-1D allele and the related gene family members involved in Arabidopsis-P. syringae interaction. A, Structure of T-DNA insertion in cds2-1D mutant. The detected head-to-head T-DNA left and right borders are marked as LB and RB, respectively. The uncharacterized T-DNA region is indicated by dashed line. B, CDS2 expression is massively enhanced in the cds2-1D mutant. RNA gel blot assay of CDS2 gene transcript in healthy leaves from Col-0 and cds2-1D mutant plants. The bottom panel shows ubiquitin transcript levels of the same samples. C, Arabidopsis NCED genes are induced by bacterial infection. RT-PCR analysis of NCED transcripts levels in leaves from Col-0 plants that were infiltrated with 5 × 107 cfu mL−1 of virulent Psm or avirulent Psm-avrRpm1. Water was infiltrated as mock treatment. No PCR product could be observed when nonreverse-transcribed total RNA was used as negative controls to amplify NCED genes (data not shown). D, Constitutive overexpression of NCED genes enhanced susceptibility to bacterial infection. One of each representative transgenic lines constitutively overexpressing NCED3 or NCED5 genes, together with wild-type Col-0 and cds2-1D mutant plants, was hand-infiltrated with 104 cfu mL−1 of virulent Psm, and in planta bacterial growth were determined at 3 d after inoculation. E, Conditional overexpression of NCED3 enhances susceptibility to bacterial infection. Wild-type Col-0 plants and plants transformed with empty vector pER8 or pER8-NCED3 were sprayed with 10 μm β-estrodial 1 d before hand-infiltration of virulent Pst at 105 cfu mL−1. Water was sprayed as uninduced control. The in planta bacterial growth was determined 3 d after inoculation. Significant differences in bacterial growth were only detected between control and induced pER8:NCED3 plants (indicated by *, t test, P < 0.05). dpi, Days post inoculation. Data shown in the bacterial growth assays are means ± sd, and each experiment was repeated at least twice with similar results.
- Figure 4.
Physiological role of ABA in bacterial susceptibility. A, The cds2-1D mutant plants have increased basal level of free ABA in comparison to wild-type Col-0 plants. B, The enhanced bacterial growth in cds2-1D mutant is dependent on ABA biosynthesis pathway. Leaves of wild-type and homozygous mutant plants were hand-infiltrated with 105 cfu mL−1 of virulent Pst, and bacterial growth was determined 2 d after inoculation. C, Exogenous ABA treatment enhances bacterial susceptibility. Leaves of wild-type Col-0 plants were hand-infiltrated with 105 cfu mL−1 of virulent Pst before they were excised from the plants with a razor blade and fed through the petiole with sterilized water (white) or 10 μm ABA (gray). Bacterial number in the inoculated leaves was determined 60 h after inoculation. D, Bacterial susceptibility is attenuated in ABA-deficient mutant plants. Leaves from wild-type Col-0 (white) and aba3-1 mutant (gray) plants were hand-infiltrated with 106 cfu mL−1 of virulent Pst, and the leaf bacterial number was determined 2 d after inoculation. E, Water restriction enhanced bacterial susceptibility in Arabidopsis. Five-week-old Col-0 plants were supplied daily with sufficient level (8 mL/plant, white) or restricted level (2 mL/plant, gray) of water for 1 week and inoculated with 105 cfu mL−1 of virulent Pst. Bacterial number in the inoculated leaves was determined 3 d after inoculation. Data shown in the above assays are means ± sd; g−1dw is per gram dry weight. All the experiments were repeated at least twice with similar results. dpi, Days post inoculation; hpi, hours post inoculation.
- Figure 5.
Accumulation of free SA, ABA, and JA in Arabidopsis leaves infected with P. syringae. Leaves of 5-week-old plants were hand-infiltrated with water or 5 × 106 cfu mL−1 of virulent Pst. The inoculated leaves were collected at 0, 6, 12, 24, 36, and 48 h post inoculation (Hpi) and the extracts of leaf tissue used for LC-MS quantification of free SA, ABA, and JA. A, SA, ABA, and JA induction by bacterial infection (solid circle) of wild-type Col-0 plants. Water was infiltrated as mock treatment (open circle). B, Comparison of SA, ABA, and JA induction in wild-type Col-0, aba3-1 (solid triangle), and cds2-1D (solid square) mutant plants in response to bacterial infection. For each data point, three biological replicates were assayed, and data shown are means ± sd; g−1dw is per gram dry weight. The experiments were repeated twice with similar outcomes.
- Figure 6.
ABA is involved in other Arabidopsis-microbial interactions. A, ABA biosynthesis is required for H. arabidopsis to attain full virulence on Arabidopsis. Three-week-old wild-type Col-0, aba3-1, and cds2-1D seedlings were sprayed with 4 × 104 spores mL−1 of H. arabidopsis, and the leaves were collected at 7 d after inoculation to determine levels of pathogen sporulation. Data shown are means ± sd; g−1fw is per gram fresh weight. B, ABA is required for resistance to A. brassicicola in Arabidopsis. Leaves of 6-week-old plants were inoculated with 5 μL of 105 spores mL−1 of A. brassicicola, and 7 d after the inoculated leaves from one representative plant of aba3-1 (top), Col-0 (middle), and cds2-1D (bottom) genotypes were excised for photography. These experiments were repeated twice with similar outcomes.
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