Article Figures & Data
Figures
- Figure 1.
Analysis of Arabidopsis Seedlings Harboring the MPK6ihp Construct.
(A) RT-PCR analysis of 2-week-old seedlings from Col-0 and MPK6ihp lines for expression of MPK3, MPK4, MPK6, and β-TUB. For each line (L1 to L6) and the Col-0 parent, RNA was extracted from two individual plants as described.
(B) Kinase activity in response to wounding. Leaves of 4-week-old Col-0 and MPK6ihp-expressing plants were wounded, and protein extracts prepared from samples harvested at the indicated times. Kinase activity was monitored with an in-gel kinase assay using MBP as the substrate. Molecular size markers are indicated at right. These experiments were independently performed twice with two control and two MPK6 silenced lines; similar results were observed.
- Figure 2.
Specificity of Arabidopsis MAPK Antibodies.
(A) Crude extracts (5 μg/lane) of E. coli-expressed His-tagged versions of MPK3, MPK4, and MPK6 were loaded onto duplicate blots and subjected to protein gel blot analysis with antibodies raised against MPK3 (α-C-3), MPK4 (α-C-4), and MPK6 (α-N-6). To ensure the recombinant proteins were loaded evenly on the gels, the blots were stripped and reprobed with a T7 antibody (α-T7-tag).
(B) Protein gel blot analysis of Arabidopsis MAPKs. Crude protein extracts (20 μg/lane) from Col-0 (Con) or MPK6-silenced (Sil) plants were prepared, loaded onto duplicate blots, and subjected to protein gel blot analysis with the α-C-3, α-C-4, or α-N-6 antibodies. Molecular size markers are indicated at left of the panels in (A) and (B).
- Figure 3.
MPK6-Silenced Lines Exhibit Reduced Resistance to P. parasitica.
(A) Seven- to ten-day-old seedlings were inoculated with P. parasitica Emwa1 (105 spores/mL). At 6 to 8 dpi, single cotyledons from 35 to 40 seedlings were analyzed under the microscope and categorized into one of six categories (0, 1 to 5, 6 to 10, 11 to 15, 16 to 20, or >20) depending on the number of sporangiophores observed. Col-0 is mostly resistant to this isolate, whereas Ws-0 is highly susceptible. MPK3ihp L5 is the nonsilenced control. Experiments were repeated three times with similar results.
(B) Inoculation of Col-0, MPK3ihp L5, and MPK6 silenced lines with P. parasitica Hiks1, which is virulent on the Keswick (Ksk) accession. Inoculation and analysis of 40 to 44 seedlings was done as described in (A).
(C) Trypan blue staining of P. parasitica Emwa1-inoculated cotyledons at 96 hpi. Trypan blue stains dead leaf cells (including xylem cells) and oomycete hyphae.
- Figure 4.
MPK6-Silenced Lines Display Enhanced Susceptibility to P. s. tomato.
(A) Four-week-old plants were inoculated with P. s. tomato, and four leaf disks per sample were harvested at 0 dpi and 3 dpi. The number of colony forming units (cfu) per sample was calculated and expressed as cfu/mL. Col-0 and MPK3ihp L5 were used as controls, along with the MPK6ihp L1, L4, and L7 lines. Experiments were done twice with similar results.
(B) Four-week-old plants were inoculated with P. s. tomato expressing avrRpm1, and four leaf disks per sample were harvested at 0 dpi and 3 dpi.
(C) Four-week-old plants were inoculated with P. s. tomato expressing avrRpt2, and four leaf disks per sample were harvested at 0, 1, and 2 dpi.
(D) Four-week-old plants were inoculated with P. s. tomato expressing avrRps4, and four leaf disks per sample were harvested at 0, 2, and 3 dpi.
- Figure 5.
Pathogen-Induced Gene Expression in MPK6-Silenced Plants.
Four-week-old MPK6-silenced and control plants were dipped in virulent P. s. tomato suspension (108 cfu/mL), and samples were taken at selected time points between 0 hpi and 24 hpi. Samples were snap frozen in liquid nitrogen, and RNA extraction and Q-PCR were performed as described in Methods. Equal amounts of RT reaction products were used as templates to amplify selected regions of the indicated genes with specific primer pairs. Expression levels are represented by arbitrary units, which were set at 100 for control line at t = 0. The expression level of all other samples were related to the control sample t = 0. These experiments were done with two controls (Col-0 and MPK3ihp L5) and two MPK6-silenced lines (MPK6ihp L1 and L4), and averages of both lines are shown.
- Figure 6.
Induced/Acquired Resistance Is Intact in MPK6-Silenced Lines.
Percentage diseased leaves at 3 dpi with virulent P. s. tomato in ISR- and SAR-expressing plants and control plants. The effectiveness of ISR and SAR expression was tested in MPK6-silenced lines (MPK6ihp4 and MPK6ihp7) and compared with nonsilenced transgenic lines (MPK3ihp4 and MPK3ihp5) and the parent Columbia (Col-0). Error bars indicated SE (n = 18).
- Figure 7.
Ethylene Responses in MPK6-Silenced Lines.
(A) and (B) Seeds for MPK6-silenced lines and control lines were germinated in the dark on MS medium containing the indicated amount of 1-aminocyclopropane-1-carboxylic acid (ACC). Four days after germination, the hypocotyl and root lengths of the etiolated seedlings was measured.
(C) Q-PCR analysis of ethylene-inducible gene expression in control and MPK6-silenced lines. Two-week-old plants were exposed to 5 ppm ethylene, and samples harvested and snap frozen in liquid nitrogen at indicated times. Q-PCR was performed as described in Figure 5. b-CHI, basic Chitinase; B-TUB, β-Tubulin.
