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Phosphoinositide-signaling is one component of a robust plant defense response - PubMed

  • ️Wed Jan 01 2014

Phosphoinositide-signaling is one component of a robust plant defense response

Chiu-Yueh Hung et al. Front Plant Sci. 2014.

Abstract

The phosphoinositide pathway and inositol-1,4,5-triphosphate (InsP3) have been implicated in plant responses to many abiotic stresses; however, their role in response to biotic stress is not well characterized. In the current study, we show that both basal defense and systemic acquired resistance responses are affected in transgenic plants constitutively expressing the human type I inositol polyphosphate 5-phosphatase (InsP 5-ptase) which have greatly reduced InsP3 levels. Flagellin induced Ca(2+)-release as well as the expressions of some flg22 responsive genes were attenuated in the InsP 5-ptase plants. Furthermore, the InsP 5-ptase plants were more susceptible to virulent and avirulent strains of Pseudomonas syringae pv. tomato (Pst) DC3000. The InsP 5-ptase plants had lower basal salicylic acid (SA) levels and the induction of SAR in systemic leaves was reduced and delayed. Reciprocal exudate experiments showed that although the InsP 5-ptase plants produced equally effective molecules that could trigger PR-1 gene expression in wild type plants, exudates collected from either wild type or InsP 5-ptase plants triggered less PR-1 gene expression in InsP 5-ptase plants. Additionally, expression profiles indicated that several defense genes including PR-1, PR-2, PR-5, and AIG1 were basally down regulated in the InsP 5-ptase plants compared with wild type. Upon pathogen attack, expression of these genes was either not induced or showed delayed induction in systemic leaves. Our study shows that phosphoinositide signaling is one component of the plant defense network and is involved in both basal and systemic responses. The dampening of InsP3-mediated signaling affects Ca(2+) release, modulates defense gene expression and compromises plant defense responses.

Keywords: Arabidopsis; Ca2+; InsP3; SAR; phosphoinositides; plant defense signaling; salicylic acid.

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Figures

Figure 1
Figure 1

The response of the InsP 5-ptase plants to flagellin. (A) One week old wild type (Wt) and InsP 5-ptase (T8) seedlings were incubated in media containing either 0 or 0.1 μM flg22. Root length was measured after 5 days. Growth inhibition was calculated as the percentage of the non-treated control. Data is the average of four independent experiments (n = 10 seedlings per experiment) ± SE. (B) Five-day old seedlings expressing aequorin were reconstituted with coelenterazine overnight and placed in the luminometer. Seedlings were treated with 10 μM flg22 after 1 min (indicated by red arrow). Ca2+ concentrations were calculated after measuring the discharged Ca2+. A representative trace is shown. (C) The table lists the average peak Ca2+ concentration. (N = independent biological replicates). The average baseline concentrations were 0.097 and 0.098 for wild type and transgenic respectively. (D) ROS production was measured in leaf discs treated with 1 μM flg22. Luminescence was measured at 14 s intervals. Data plotted is the average of five biological replicates ± SD. (E) qRT-PCR analysis of flg22 responsive gene expression at 30 and 120 min post treatment. Data plotted is the average ± SE normalized to Wt control from three independent experiments. *Indicates significant difference between Wt and T8 expression at the same time point (P < 0.05).

Figure 2
Figure 2

The response of InsP 5-ptase plants to Pseudomonas syringae. Leaves of Arabidopsis plants inoculated with Pst DC3000 at OD600 = 0.001 (7 × 105 cfu/ml) were photographed at day 2 after inoculation (A). Bacterial growth measured from plants at day 2 after inoculation with Pst DC3000 (B) or PstDC3000+avrRpt2 (C). Starting bacterial cultures are OD600 = 0.01 (8 × 106 cfu/ml), 0.001 (7 × 105 cfu/ml) or 0.0005 (3.5 × 105 cfu/ml). (D) Infected leaves used for RT-PCR were inoculated with PstDC3000+avrRpt2 (OD600 = 0.001, 7 × 105 cfu/ml). RT-PCR was carried out with gene-specific primers for PR-1 and Actin. (E) For bacterial growth counts taken at day 4 after inoculation with the two non-pathogens, PstDC3000+hrcC- (Pst hrcC-) and P. syringae pv. Phaseolicola race 6 (Psp rc6), the concentration of inoculants were OD600 = 0.001 (7 × 105 cfu/ml). Plant lines used were Wt (white), T8 (black), and the rps2/rpm1 double knock-out mutant (gray). *P < 0.05.

Figure 3
Figure 3

InsP3 changes and Ca2+ release in response to avirulent Pseudomonas syringae pv tomato DC3000. (A) Wild type Arabidopsis plants were sprayed with Pst DC3000 or Pst DC3000+avrRpt2 or + avrRpm1 at a concentration of OD600 = 0.004 (2 × 106 cfu/ml). Mock spray was 10 mM MgCl2. Treated leaves were harvested at different time points and InsP3 was quantified. Data shown is the average of 5 independent experiments ± SE. (B) Wild type (Wt) or InsP 5-ptase seedlings carrying aequorin were reconstituted with coelenterazine and inoculation with Pst DC3000+avrRpm1 (OD600 = 0.5, 5 × 108 cfu/ml) or mock solution (10 mM MgCl2). A representative experiment is shown with luminescence counts taken in every 5 s. The table lists the average time of the second sustained Ca2+ peak (N = independent biological replicates).

Figure 4
Figure 4

InsP 5-ptase plants have a low basal level of SA; however the local response to exogenous SA is normal. (A) SA levels were measured in untreated leaves (basal) or in leaves harvested from plants inoculated with Pst DC3000+avrRpt2 at a concentration of OD600 = 0.1 (1 × 108 cfu/ml) at day 2 post-inoculation. Data is the average of three to five experiments ± SE. Each experiment consisted of a pool of leaves from four individual plants/line. (B) Wild type (Wt), two independent InsP 5-ptase lines (T6 and T8), and vector control (C2) plants were sprayed with 300 μM of SA or control solution (0.05% ethanol). Leaves were harvested before (0) and at 3, 8, 24, and 48 h after treatment. RT-PCR was carried out with gene-specific primers for PR-1 and Actin.

Figure 5
Figure 5

The SAR response is delayed in the InsP 5-ptase plants. The SAR assay was carried out using wild type (Wt), vector control (C2) and two independent InsP 5-ptase lines (T6 and T8). Plants were either first inoculated with Pst DC3000+avrRpt2 (OD600 = 0.001, 7 × 105 cfu/ml) (A), or 10 mM MgCl2 mock solution (B), then sprayed with PstDC3000 (OD600 = 0.004, 2 × 106 cfu/ml) at day 0, 1, 2, or 3. Systemic leaves were harvested and the bacterial growth was quantified at day 4 post spray. Data is the average of three experiments ± SD. Each experiment has three plants per line. Results from Wt and C2 (Wt/C2), and T6 and T8 (T8/T6) were pooled for analysis. *P < 0.05 (C) Systemic leaves were also harvested before (0) and after initial inoculation at 3, 8, 24, and 48 h for RT-PCR carried out with gene-specific primers for PR-1, PAD4, and UBQ10. (D) For the exudate experiment, only Wt and T8 and their reciprocal treatments are shown. Leaves were harvested before or at 48 h post exudate (ex) or mock (m) infiltration for RT-PCR carried out with gene-specific primers for PR-1 and Actin.

Figure 6
Figure 6

Summary of InsP 5-ptase plants responses to flg22 and Pseudomonas syringae. The major results described in the paper are listed. The transport of mobile signals from local to distal leaves was not investigated and is indicated by the “?.” PTI, PAMP triggered immunity; ETI, Effector triggered immunity; SA, salicylic acid; ROS, reactive oxygen species; SAR, systemic acquired resistance.

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