Figure 1.
Disease development of bacterial wilt and growth of Ralstonia solanacearum in DS1 plants.
Control and DS1 plant leaves infiltrated with R. solanacearum. (A) Characteristic symptoms in control and DS1 plants. Photograph was taken 12 days after inoculation with R. solanacearum. (B) Disease development of bacterial wilt was rated daily on a 0–4 disease index in control (open squares) or DS1 (solid squares) plants. Asterisks denote values significantly different from those of control plants (*; P < 0.05, t-test). (C) Control and DS1 plant leaves infiltrated with R. solanacearum (108 CFU/ml). Bacterial population was determined by plating at specified time points. Values are means of four replicate experiments with SD. Asterisks denote values significantly different from those of empty PVX controls (*; P < 0.05, t-test). (D) Total RNA was isolated from N. benthamiana leaves inoculated with water, R. solanacearum strain (RsOE1-1), strain 8107 (Rs8107), and hrp-deficient mutant of R. solanacearum OE1-1 (RsOE1-1ΔY). Transcript levels of DS1 were estimated by qRT-PCR. Values represent mean ± SD from triplicate experiments. Asterisks denote values significantly different from those of water-inoculated controls (*; P < 0.05, t-test).
Figure 2.
Deduced amino acid sequence and phylogenic analysis of DS1.
(A) Alignment of deduced amino acid sequence of DS1 and its ortholog in N. tabacum (NtPAP), phosphatidic acid phosphatase 2-like proteins from Vitis vinifera (VvPAPLP), Thellungiella halophila (ThPAP2LP), Arabidopsis thaliana (AtPAP2LP), and Oryza sativa (OsPAP2LP). Dots show identical amino acids, bars show amino acids that are not present in sequences. Boxes show conserved PAP catalytic motifs I, II, and III essential for PAP activity. Putative transmembrane motifs are shown in italic with underline. (B) Phylogenic tree of lipid phosphatases in plants. Gray boxes show DS1 and its ortholog in N. tabacum. The scale bar represents 0.1 JTT distance matrix units.
Figure 3.
(A) Isogenic yeast strain ∆lpp1∆dpp1∆pah1 containing empty pDO105 plasmid (+pDO105) or pDO105 containing DS1 (+pDO105: DS1) were cultured on agar YPD plates and incubated at 30°C or 37°C. (B) PAP activity of DS1 and dehydrofolate reductase (DHFR; negative control) was determined in the presence or absence of Mg2+ as described in Materials and Methods. Values are means and SD from triplicate experiments. Asterisks denote values significantly different from those of control (*; P < 0.05). (C) PAP activity in control (white bar) and DS1 plants (black bar). Crude protein fractions were isolated from control and DS1 plants 0 and 24 h after inoculation with R. solanacearum. PAP activity was determined without Mg2+ as described in Materials and Methods. Values are means and SD from triplicate experiments. Asterisks denote values significantly different from those of control (*; P < 0.05). (D) Phosphatidic acid contents in control and DS1 plants. Total lipid fraction was extracted from control and DS1 plants 0 to 24 h after inoculation with R. solanacearum. PA was separated by ethyl acetate TLC as described in Materials and Methods.
Figure 4.
Up-regulation of defense-related responses in DS1 plants against R. solanacearum infection.
(A) Total RNA was isolated from control (Control) and DS1 plants (DS1) 6 to 72 h after inoculation with R. solanacearum. Expression values of DS1, PR-1a, PR-4, and NbEF-1α were analyzed by semi-quantitative RT-PCR with specific primers for NbPR-1a, NbPR-4, DS1, and NbEF-1α. (B) HR-like lesion formation in DS1 plant in response to R. solanacearum infection. Control and DS1 plants were infiltrated with R. solanacearum. Pictures of N. benthamiana leaves taken 36 hours after bacterial infiltration. (C) Cell death was determined by Evans blue staining. Values are means of four replicate experiments with SD. Asterisks denote values significantly different from those of empty PVX control (*; P < 0.05, t-test). (D) ROS production was visualized 48 h after inoculation with R. solanacearum by DAB staining as described in Materials and Methods.
Figure 5.
Reduction of defense-related responses and disease resistance in DS1-overexpressing plants.
(A) Phosphatidic acid content in control and DS1-overexpressing plants (OX-#2 and OX-#8). Total lipid fraction was extracted from control (WT) and DS1-overexpressing plants (OX-#2 and OX-#8) 0–24 h after inoculation with R. solanacearum. PA was separated and quantified as described in Materials and Methods. Values are means of four replicate experiments with SD. Asterisks denote values significantly different from those of control (*; P < 0.05, t-test). (B) Total RNA was isolated from wild-type control (Control), DS1-overexpressing plants (OX-#2 and OX-#8) at 12 and 24 h after inoculation with R. solanacearum. Expression values of PR-4 are expressed as [Qty] after normalization to actin. Values represent means and SD from triplicate experiments. Asterisks denote values significantly different from those of control (*; P < 0.05, t-test). (C) ROS production was visualized by DAB staining as described in Materials and Methods. Bar indicates @@@. (D) Brown deposits were quantified as described in Materials and Methods. (E) Bacterial population was determined by plating at specified time points. Values are means of four replicate experiments with SD. Asterisks denote values significantly different from those of control (*; P < 0.05, t-test). (F) Disease development of bacterial wilt was rated daily on a 0–4 disease index in control (WT) and DS1-overexpressing (OX#2 and OX#8) plants. Values are means of four replicate experiments with SD. Asterisks denote values significantly different from those of empty PVX control (*; P < 0.05, t-test). (G) Characteristic symptoms in control and DS1-overexpressing (OX#2 and OX#8) plants. Photograph was taken 12 days after inoculation with R. solanacearum.
Figure 6.
Role of NbrboHB in DS1 phenotype
Control, DS1, and DS1:NbrboHB (DS1:rbohB) double-knockdown plant leaves were infiltrated with R. solanacearum. (A) Photograph of HR-like lesion was taken 36 h after inoculation Dot lined circles showed lesion developed areas. ROS production was visualized 48 h after inoculation by DAB staining as described in Materials and Methods. Bars indicated 500 nm. (B) Cell death was determined by Evans blue staining (OD600 disk-1, see Materials and Methods). (C) Bacterial population was determined by plating at specified time points. Values are means of four replicate experiments with SD. Different letters show significant differences among control, DS1, and double-knockdown plants (ANOVA test). (D) Disease development of bacterial wilt was rated daily on a 0–4 disease index in control (open circles), DS1 (solid circles), or DS1:NbrboHB (closed squares) plants. Values are means of four replicate experiments with SD. Different letters show significant differences among controls, DS1, and double-knockdown plants (p<0.05 ANOVA). (E) Characteristic symptoms in control, DS1, and DS1:rboHB plants. Photograph was taken 12 days after inoculation with R. solanacearum.
Figure 7.
Role of jasmonic acid pathway in DS1 phenotype.
Control, DS1, and DS1:Coi1 double-knockdown plant leaves were infiltrated with R. solanacearum. (A) Total RNA was isolated from control (Control), DS1 (DS1), and DS1:NbCoi1 (DS1:Coi1) double-knockdown plant 24 h after inoculation with R. solanacearum. Expression values of PR-4 are expressed as [Qty] after normalization to actin. Values represent means and SD from triplicate experiments. Different letters show significant differences among control, DS1, and double-knockdown plants (ANOVA test). (B) Bacterial population was determined by plating at specified time points. Values are means of four replicate experiments with SD. Different letters show significant differences among controls, DS1, and double-knockdown plants (p<0.05 ANOVA). (C) Disease development of bacterial wilt was rated daily on a 0–4 disease index in control (open circles), DS1 (solid circles), or DS1:Coi1 (closed squares) plants. Values are means of four replicate experiments with SD. Asterisks denote values significantly different from those of empty PVX control (*; P < 0.05, t-test). (D) Characteristic symptoms in control, DS1, and DS1:Coi1 plants. Photograph was taken 12 days after inoculation with R. solanacearum.