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1.
Figure 5

Figure 5. ROS Generation in Arabidopsis Response to Pathogen Infection. From: Arabidopsis phospholipase D?1 modulates defense responses to bacterial and fungal pathogens.

The wild-type (Col-0), pldβ1-1, RNAi1, and RNAi2 plants were inoculated with Pst DC3000 (2 × 10 7 cfu/ml) or Botrytis cinerea spores. Data are presented as means ± SD (n = 5).
(a) Nitroblue tetrazolium staining of 5-week old Arabidopsis leaves 12 h post inoculation with Pst DC3000 bacterial pathogen (2 × 10 7 cfu/ml).
(b) H2O2 quantification in Pst DC3000-infected leaves 12 h post inoculation.
(c) Nitroblue tetrazolium staining of 5-week old Arabidopsis leaves 12 h post inoculation with B. cinerea.
(d) H2O2 quantification in B. cinerea spore-inoculated leaves.
Different letters indicate sample groups with significant differences (p < 0.05) from each other.

Jian Zhao, et al. New Phytol. ;199(1):228-240.
2.
Figure 7

Figure 7. Levels of total LPC, LPE, and LPG from Leaves of Wild-type and pldβ1-1 Arabidopsis with and without Inoculation of B. cinerea, Pst DC3000, or avirulent Pst DC3000 (avrRpt2). From: Arabidopsis phospholipase D?1 modulates defense responses to bacterial and fungal pathogens.

Col-0, pldβ1-1, and pldβ1-COM plant were inoculated with control solutions (-C) or with pathogens (-T). Control solutions were water for Botrytis cinerea and 10 mM MgSO4 for virulent strain Pst DC3000 and avirulent strain Pst DC3000 (avrRpt2). Leaf samples were collected for lipid profiling 24 h post inoculation. Values are means of ±SE (n = 5). Different letters indicate values with significant differences (P < 0.05) from each other.

Jian Zhao, et al. New Phytol. ;199(1):228-240.
3.
Figure 6

Figure 6. Phospholipid and Galactolipid Content from Leaves of Wild-type and pldβ1-1 Arabidopsis as affected by B. cinerea, Pst DC3000, or Avirulent Pst DC3000 (avrRpt2) Infection. From: Arabidopsis phospholipase D?1 modulates defense responses to bacterial and fungal pathogens.

Col-0, pldβ1-1, and pldβ1 complementation (COM) plant were inoculated with control solutions (-C) or with pathogens (-T). Control solutions were water for Botrytis cinerea spores (5 × 107 spores/ml) and 10 mM MgSO4 for virulent strain Pst DC3000 and avirulent strain Pst DC3000 (avrRpt2) (2 × 10 7 cfu/ml). Leaf samples were collected for lipid profiling 24 h post inoculation. Values are means ± SE (n = 5). Different letters indicate sample groups with significant differences (p < 0.05) from each other.

Jian Zhao, et al. New Phytol. ;199(1):228-240.
4.
Figure 1

Figure 1. Production and Confirmation of PLDβ1 RNAi Knockdown and T-DNA Insertion Knockout Arabidopsis Lines. From: Arabidopsis phospholipase D?1 modulates defense responses to bacterial and fungal pathogens.

(a) Construction of PLDβ RNAi vector. Inverted repeats of exons (gray boxes) and an intron (empty boxes) with restriction sites were cloned in tandem, and their expression was driven by double CaMV 35S promoters and terminated by an NOS terminator.
(b) RNA blotting of PLDβ1 and PLDα1 expression in RNAi lines. Equal amounts of total RNA from Arabidopsis leaves were loaded and rRNA detected with ethidium bromide (EtBr) was used as loading control. PLDα1 protein levels were also used as loading controls. The density of the PLDβ1 RNA band was quantified from three experiments. Data bars represent the mean (±SD) of three repeats.
(c) Immunoblotting of PLDβ1 and PLDα1 protein levels in RNAi lines. Equal amounts of Arabidopsis leaf proteins were subjected to SDS-PAGE, followed by immunoblotting using antibodies against PLDβ1 and PLDα1. The band density of PLDβ1 was quantified from three experiments. Data bars represent the mean (±SD) of three repeats.
(d) T-DNA insertional knockout of PLDβ1. Left panel, T-DNA insertion position in PLDβ1 with exons (gray boxes) and introns (bars between exons). Right panel, northern blot showing the absence of the PLDβ1 transcript in T-DNA insertion knockout. PLDα1 expression and 28S RNA (detected with ethidium bromide) were used as loading controls.

Jian Zhao, et al. New Phytol. ;199(1):228-240.
5.
Figure 3

Figure 3. Salicylic Acid in Pathogen-Inoculated PLDβ1 Arabidopsis Mutants. From: Arabidopsis phospholipase D?1 modulates defense responses to bacterial and fungal pathogens.

Total SA (free SA + SA Glycosides) in the wild-type, pldβ1-1, RNAi1, and RNAi2 were determined by HPLC. Data are presented as means ± SD (n = 3). Different letters indicate sample groups with significant differences (p < 0.05) from each other.
(a) SA levels in inoculated leaves as a function of time post Pst DC3000 inoculation (2 × 10 7 cfu/ml).
(b) SA levels in leaves from local and distal leaves from Pst DC3000-inoculated plants for 12 h.
(c) Expression of defense-related genes in Col-0, pldβ1-1, RNAi1, RNAi2, and pldβ1-1- complementation (COM) plants infected with Pst DC3000 pathogens (pst) or Pst DC3000(Avr Rpt2) (Rpt) pathogens (2 × 107 cfu/mL) for 12 h. Leaves treated with 10 mM MgSO4 (m) were used as a control. The relative expression level of PR-1 was analyzed by quantitative PCR. Expression levels were normalized with respect to the housekeeping genes. Data bars represent the mean (±SD) of three repeats.

Jian Zhao, et al. New Phytol. ;199(1):228-240.
6.
Figure 2

Figure 2. Responses of PLDβ1 Arabidopsis Mutants to Bacterial Pathogens. From: Arabidopsis phospholipase D?1 modulates defense responses to bacterial and fungal pathogens.

Fully expanded leaves of Col-0, pldβ1-1, RNAi mutants, and pldβ1-1 complementation (COM) plants were infiltrated with a suspension containing Pst DC3000 with or without avrRpt2.
(a) Expression of PLDβ1 and PLDβ2 in Col-0 plants infected with Pst DC3000 pathogens or Pst DC3000(avr Rpt2) pathogens (2 × 107 cfu/mL) for 12 h. Leaves treated with 10 mM MgSO4 were used as a control (mock). The relative expression level of PLDβ1 and PLDβ2 was analyzed by quantitative PCR. Expression levels were normalized with respect to the housekeeping genes UBQ and Tubulin. Data bars represent the mean (±SD) of three repeats.
(b) Defensive response of Arabidopsis plants with altered PLDβ1 expression to virulent and avirulent Pst DC3000 (2 × 107 cfu/mL). The photographs show representative leaves inoculated with bacterial pathogen for 12 h.
(c) Proliferation of the virulent Pst DC3000 (left panel) and avirulent Pst DC3000 (avrRpt2) (2 × 105 cfu/mL) (right panel) pathogens in Col-0, pldβ1-1, RNAi plants. Bacterial growth in five leaf discs was measured. The bacterial numbers expressed as the increase of colony-forming units (cfu) per leaf disc after bacterial inoculation (i.e. Log cfu post inoculation-Log cfu at time zero). Data represent the average of three samples ± SD. Different letters indicate sample groups with significant differences (p < 0.05) from each other.

Jian Zhao, et al. New Phytol. ;199(1):228-240.
7.
Figure 4

Figure 4. Responses of PLDβ1 Arabidopsis Mutants to B. cinerea. From: Arabidopsis phospholipase D?1 modulates defense responses to bacterial and fungal pathogens.

(a) Leaves of three week-old Col-0, pldβ1-1, RNAi, and pldβ1-1-complementation (COM) plants were inoculated with B. cinerea spores (5 × 107 spores/ml) or water (control). The photographs show representative leaves 5 days post-inoculation.
(b) Lesions scored at 5 days post inoculation. The lesion developing rate was the number of leaves with a necrotic area among the inoculated leaves in each line. Data are presented as mean ± SD (n = 25). Different letters indicate sample groups with significant differences (p < 0.05) from each other.
(c) Expression of defense-related genes in Col-0 and PLDβ1 mutants infected with fungal pathogens by quantitative PCR. Expression levels were normalized with respect to ubiquitin 5 (UBQ5). Data bars represent the mean (±SD) of three repeats. Leaves treated with water were used as control.
(d) JA levels in wild-type (Col-0), pldβ1-1, and pldβ1-complementation (COM) plants at various hours post inoculation (hpi) with B. cinerea. C indicates control (i.e. mock-inoculated) and T indicated treated (i.e. pathogen-inoculated). Three week-old soil-grown seedlings were treated with B. cinerea spores. Values are means ± SE of three independent replicate experiments.
(e) Salicylic acid in Botrytis cinerea spore-inoculated PLDβ1 mutants. Samples were harvested after inoculation Botrytis cinerea spores (5 × 107 cfu/m1) for different time. Total SA (free SA + SA Glycosides) in the wild-type, pldβ1-1, RNAi1, RNAi2, and pldβ1-1 complementation plants were determined by HPLC. Data are presented as means ± SD (n = 3).

Jian Zhao, et al. New Phytol. ;199(1):228-240.

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